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Provide examples of how molecular transporters contribute to multi-drug resistance in bacteria. | MDR efflux pumps began causing clinical problems relatively recently, in parallel with the extensive use of antibiotics in medicine and as supplements in animal feeds. However, our analyses indicate that these MDR efflux pumps did not arise through recent mutations in genes encoding transporters that changed their substrate specificities.
Instead, such MDR pumps are encoded within the genomes of virtually all microorganisms, so these genes are present and thus need only to be activated to become problematic. Moreover, lateral transfer of genes among bacteria has occurred frequently, particularly for plasmid-encoded systems, suggesting that such genes can be acquired fairly readily even if they are not initially present. Finally, although mutations that enable transporters to act on different types of substrate are rare, experiments and phylogenetic analyses indicate that simple point mutations can readily narrow or broaden a particular transporter's specificity toward a single class of compounds (e.g., sugars, amino acids, or drugs). | The efflux systems of bacteria protect cells from antibiotics and biocides by
actively transporting compounds out of the cytoplasm and/or periplasm and
thereby limit their steady-state accumulation at their site(s) of action. The
impact of efflux systems on the efficacy of antibiotics used in human medicine
and animal husbandry is becoming increasingly apparent from the characterization
of drug-resistant strains with altered drug efflux properties. In most
instances, efflux-mediated antibiotic resistance arises from mutational events
that result in their elevated expression and, in the case of efflux pumps with
broad substrate specificity, can confer multi-drug resistance (MDR) to
structurally unrelated antibiotics. Knowledge of the role of efflux systems in
conferring antibiotic resistance has now been successfully exploited in the
pharmaceutical industry and contributed, in part, to the development of new
members of the macrolide and tetracycline classes of antibiotics that circumvent
the efflux-based resistance mechanisms that have limited the clinical utility of
their progenitors. The therapeutic utility of compounds that inhibit bacterial
drug efflux pumps and therein potentiate the activity of a co-administered
antibiotic agent remains to be validated in the clinical setting, but the
approach holds promise for the future in improving the efficacy and/or extending
the clinical utility of existing antibiotics. This review discusses the
potential of further exploiting the knowledge of efflux-mediated antibiotic
resistance in bacteria toward the discovery and development of new
chemotherapeutic agents. Antibiotic-resistant bacteria, particularly gram negative species, present
significant health care challenges. The permeation of antibiotics through the
outer membrane is largely effected by the porin superfamily, changes in which
contribute to antibiotic resistance. A series of antibiotic resistant E. coli
isolates were obtained from a patient during serial treatment with various
antibiotics. The sequence of OmpC changed at three positions during treatment
giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and
OmpC33, in which OmpC20 was derived from the first clinical isolate). We
demonstrate that expression of the OmpC K12 porin in the clinical isolates
lowers the MIC, consistent with modified porin function contributing to drug
resistance. By a range of assays we have established that the three mutations
that occur between OmpC20 and OmpC33 modify transport of both small molecules
and antibiotics across the outer membrane. This results in the modulation of
resistance to antibiotics, particularly cefotaxime. Small ion unitary
conductance measurements of the isolated porins do not show significant
differences between isolates. Thus, resistance does not appear to arise from
major changes in pore size. Crystal structures of all four OmpC clinical mutants
and molecular dynamics simulations also show that the pore size is essentially
unchanged. Molecular dynamics simulations suggest that perturbation of the
transverse electrostatic field at the constriction zone reduces cefotaxime
passage through the pore, consistent with laboratory and clinical data. This
subtle modification of the transverse electric field is a very different source
of resistance than occlusion of the pore or wholesale destruction of the
transverse field and points to a new mechanism by which porins may modulate
antibiotic passage through the outer membrane. OBJECTIVES: To study the potential factors include gene mutation, efflux pump
and alteration of permeability associated with quinolone-resistance of
Salmonella enterica strains isolated from patients with acute gastroenteritis
and to evaluate the degree of synergistic activity of efflux pump inhibitors
when combined with ciprofloxacin against resistant isolates.
METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates
were tested. Five isolates were selected to study the mechanism of resistance
associated with quinolone group, including mutation in topoisomerase-encoding
gene, altered cell permeability, and expression of an active efflux system. In
addition, the combination between antibiotics and efflux pump inhibitors to
overcome the microbial resistance was evaluated.
RESULTS: Five Salmonella isolates totally resistant to all quinolones were
studied. All isolates showed alterations in outer membrane proteins including
disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F).
Minimum inhibitory concentration values of ciprofloxacin were determined in the
presence/absence of the efflux pump inhibitors: carbonyl cyanide
m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory
concentration values for two of the isolates were 2-4 fold lower with the
addition of efflux pump inhibitors. All five Salmonella isolates were amplified
for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22
had double mutations at codon 83 and 87 in addition to three mutations at parC
at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons
83, 87 and 119, but no mutations at parC.
CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in
Salmonella efflux systems which are the major efflux pumps responsible for
multidrug resistance in Gram-negative clinical isolates. To determine the association of tetracycline resistance determits with
tigecycline sensitivity, tetracycline-resistant Salmonella spp. isolated from
clinical and food samples were tested for the presence of tetracycline
resistance determits, tigecycline sensitivity, and the impact of tetA on
tigecycline resistance. In addition, the impacts of multiple resistance
mechanisms on tigecycline resistance were determined using an isolate with ramR
mutation. Of the 49 tetracycline-resistant Salmonella isolates screened, 32 were
positive for tetA, 13 were positive for tetB, 2 were positive for tetC and 1
isolate was positive for both tetA and tetB. The minimum inhibitory
concentration (MIC) of tigecycline for tetA-carrying isolates ranged from 0.19
mg/L to 3mg/L (mode 0.75 mg/L), whereas the MIC of tigecycline for tetB-carrying
isolates ranged from 0.064 mg/L to 0.5mg/L (modes 0.25mg/L and 0.38 mg/L,
excluding the isolate with both tetA and tetB). Double frameshift mutations in
codons 201, 202 and 203 were observed in partial sequences of the tetA genes in
these strains and the majority of published tetA gene sequences. Curing of the
tetA genes from three isolates reduced the tigecycline MICs, whilst deletion of
ramR increased tigecycline MICs. This study indicates that the tetA gene
decreases sensitivity to tigecycline in Salmonella spp. at a low level. With
additional resistance mechanisms, tetA-carrying strains can reach the breakpoint
for tigecycline resistance. Antibiotic resistance is an ancient problem, owing to the co-evolution of
antibiotic-producing and target organisms in the soil and other environments
over millennia. The environmental "resistome" is the collection of all genes
that directly or indirectly contribute to antibiotic resistance. Many of these
resistance determits originate in antibiotic-producing organisms (where they
serve to mediate self-immunity), while others become resistance determits
only when mobilized and over-expressed in non-native hosts (like plasmid-encoded
β-lactamases). The modern environmental resistome is under selective pressure
from human activities such as agriculture, which may influence the composition
of the local resistome and lead to gene transfer events. Beyond the environment,
we are challenged in the clinic by the rise in both frequency and diversity of
antibiotic resistant pathogens. We assume that clinical resistance originated in
the environment, but few examples of direct gene exchange between the
environmental resistome and the clinical resistome have been documented. Strong
evidence exists to suggest that clinical aminoglycoside and vancomycin
resistance enzymes, the extended-spectrum β-lactamase CTX-M and the quinolone
resistance gene qnr have direct links to the environmental resistome. In this
review, we highlight recent advances in our understanding of horizontal gene
transfer of antibiotic resistance genes from the environment to the clinic.
Improvements in sequencing technologies coupled with functional metagenomic
studies have revealed previously underappreciated diversity in the environmental
resistome, and also established novel genetic links to the clinic. Understanding
mechanisms of gene exchange becomes vital in controlling the future
dissemination of antibiotic resistance. Latium, a region in central Italy, is known for its extensive volcanic areas
that make a significant contribution to the arsenic (As) contamination of
freshwater environments, even though some degree of As water pollution may be
caused by human activities. The information available on indigenous As-resistant
prokaryotes in aquatic environments of Latium is, however, still limited. In
this study, we describe new bacteria that are resistant to arsenic toxicity and
were isolated from the surface waters of Lake Vico and the Sacco River, two
groundwater systems in Latium, as well as from bottled natural mineral water
from the same region. The 16S rRNA gene sequence analysis for the As-resistant
strains in lake and river waters points to a prevalence of β- and
γ-Proteobacteria, while α-Proteobacteria, Firmicutes and Bacteroidetes are
represented to a lesser extent. By contrast, solely γ-Proteobacteria were
isolated from groundwater samples. The presence of Actinobacteria was documented
exclusively in bottled mineral water. In addition, we conducted a DNA
sequence-based study on the gene codifying arsB, an As(III) efflux membrane
protein pump related to arsenic resistance, for all the As-resistant bacterial
isolates. A phylogenetic analysis was carried out on the newly sequenced 16S
rRNA genes and arsB in the present study as well as on an additional 16S
rRNA/arsB dataset we obtained previously from Lake Albano, from the Tiber and
from a well in Bassano Romano located in Latium (Davolos and Pietrangeli, 2011).
Overall, the phylogenetic diversity of As-resistant bacteria in underground
water was very limited if compared with lentic and lotic waters. Lastly, our
molecular data support the hypothesis that the horizontal gene transfer of ars
in As-containing freshwater environments is not limited to closely-related
genomes, but also occurs between bacteria that are distant from an evolutionary
viewpoint, thereby indicating that such genetic events may be considered a
source of microbial resistance to arsenic-toxicity. Resistance nodulation cell division (RND)-type efflux transporters play the main
role in intrinsic resistance to various antimicrobial agents in many
gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes
in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we
cloned the other 11 RND-type efflux transporter genes and characterized them in
Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing
either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations
(MICs) for several antimicrobial agents. Additional four RND-type transporters
were functional as efflux pumps only when co-expressed with VpoC, an outer
membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ
co-expressed with VpoC exhibited a broader substrate specificity and conferred
higher resistance than that with TolC of E. coli. Deletion mutants of these
transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and
ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the
number of viable cells after exposure to deoxycholate were markedly reduced.
Strains in which 12 operons were all disrupted had very low MICs and much lower
fluid accumulation in rabbit ileal loops. These results indicate that resistance
nodulation cell division-type efflux transporters contribute not only to
intrinsic resistance but also to exerting the virulence of V. parahaemolyticus. The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium
Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems
spanning the inner and outer membranes and is involved in secretion of various
compounds, including lipids, metabolites, antibiotics, and proteins. Several
components of HgdD-dependent tripartite transport systems have been identified,
but the diversity of inner membrane energizing systems is still unknown. Here we
identified six putative resistance-nodulation-cell division (RND) type factors.
Four of them are expressed during late exponential and stationary growth phase
under normal growth conditions, whereas the other two are induced upon
incubation with erythromycin or ethidium bromide. The constitutively expressed
RND component Alr4267 has an atypical predicted topology, and a mutant strain
(I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as
well as an altered filament shape. An insertion mutant of the ethidium
bromide-induced all7631 did not show any significant phenotypic alteration under
the conditions tested. Mutants of the constitutively expressed all3143 and
alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant
I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic
sensitivity, lipid profile, and ethidium efflux. In addition, expression of the
RND genes all3143 and all3144 partially complements the capability of
Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND
transporter All3143 and the predicted membrane fusion protein All3144, as
homologs of E. coli AcrB and AcrA, respectively, are major players for
antibiotic resistance in Anabaena sp. PCC 7120. The intracellular bacterial pathogen Listeria monocytogenes activates a robust
type I interferon response upon infection. This response is partially dependent
on the multidrug resistance (MDR) transporter MdrM and relies on cyclic-di-AMP
(c-di-AMP) secretion, yet the functions of MdrM and cyclic-di-AMP that lead to
this response are unknown. Here we report that it is not MdrM alone but a cohort
of MDR transporters that together contribute to type I interferon induction
during infection. In a search for a physiological function of these
transporters, we revealed that they play a role in cell wall stress responses. A
mutant with deletion of four transporter genes (ΔmdrMTAC) was found to be
sensitive to sublethal concentrations of vancomycin due to an inability to
produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP is
involved in this phenotype, as overexpression of the c-di-AMP phosphodiesterase
(PdeA) resulted in increased susceptibility of the ΔmdrMTAC mutant to
vancomycin, whereas overexpression of the c-di-AMP diadenylate cyclase (DacA)
reduced susceptibility to this drug. These observations suggest a physiological
association between c-di-AMP and the MDR transporters and support the model that
MDR transporters mediate c-di-AMP secretion to regulate peptidoglycan synthesis
in response to cell wall stress. Streptococcus mutans, a dental pathogen, secretes different kinds of lantibiotic
and nonlantibiotic bacteriocins. For self-protection, a bacteriocin producer
strain must possess one or more cognate immunity mechanisms. We report here the
identification of one such immunity complex in S. mutans strain GS-5 that
confers protection against Smb, a two-component lantibiotic. The immunity
complex that we identified is an ABC transporter composed of two proteins: SmbF
(the ATPase component) and SmbT (the permease component). Both of the
protein-encoding genes are located within the smb locus. We show that GS-5
becomes sensitized to Smb upon deletion of smbT, which makes the ABC transporter
nonfunctional. To establish the role SmbFT in providing immunity, we
heterologously expressed this ABC transporter complex in four different
sensitive streptococcal species and demonstrated that it can confer resistance
against Smb. To explore the specificity of SmbFT in conferring resistance, we
tested mutacin IV (a nonlantibiotic), nisin (a single peptide lantibiotics), and
three peptide antibiotics (bacitracin, polymyxin B, and vancomycin). We found
that SmbFT does not recognize these structurally different peptides. We then
tested whether SmbFT can confer protection against haloduracin, another
two-component lantibiotic that is structurally similar to Smb; SmbFT indeed
conferred protection against haloduracin. SmbFT can also confer protection
against an uncharacterized but structurally similar lantibiotic produced by
Streptococcus gallolyticus. Our data suggest that SmbFT truly displays immunity
function and confer protection against Smb and structurally similar
lantibiotics. Pleiotropic drug resistance is a complex phenomenon that involves many proteins
that together create a network. One of the common mechanisms of multidrug
resistance in eukaryotic cells is the active efflux of a broad range of
xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces
cerevisiae is often used as a model to study such activity because of the
functional and structural similarities of its ABC transporters to mammalian
ones. Numerous ABC transporters are found in humans and some are associated with
the resistance of tumors to chemotherapeutics. Efflux pump modulators that
change the activity of ABC proteins are the most promising candidate drugs to
overcome such resistance. These modulators can be chemically synthesized or
isolated from natural sources (e.g., plant alkaloids) and might also be used in
the treatment of fungal infections. There are several generations of synthetic
modulators that differ in specificity, toxicity and effectiveness, and are often
used for other clinical effects. |
What is the minimal genome build? | The identification of the essential genes of bacteria and the minimal genome for the free-living cellular life could provide insights into the origin, evolution, and essence of life forms. The field of Synthetic Biology seeks to apply engineering principles to biology in order to produce novel biological systems. One approach to accomplish this goal is the genome-driven cell engineering approach, which searches for functioning minimal genomes in naturally occurring microorganisms, which can then be used as a template for future systems. Currently a prototypical minimal genome has not been discovered. | A central undertaking in synthetic biology (SB) is the quest for the 'minimal
genome'. However, 'minimal sets' of essential genes are strongly
context-dependent and, in all prokaryotic genomes sequenced to date, not a
single protein-coding gene is entirely conserved. Furthermore, a lack of
consensus in the field as to what attributes make a gene truly essential adds
another aspect of variation. Thus, a universal minimal genome remains elusive.
Here, as an alternative to defining a minimal genome, we propose that the
concept of gene persistence can be used to classify genes needed for robust
long-term survival. Persistent genes, although not ubiquitous, are conserved in
a majority of genomes, tend to be expressed at high levels, and are frequently
located on the leading DNA strand. These criteria impose constraints on genome
organization, and these are important considerations for engineering cells and
for creating cellular life-like forms in SB. We constructed and simulated a "minimal proteome" model using Langevin dynamics.
It contains 206 essential protein types that were compiled from the literature.
For comparison, we generated six proteomes with randomized concentrations. We
found that the net charges and molecular weights of the proteins in the minimal
genome are not random. The net charge of a protein decreases linearly with
molecular weight, with small proteins being mostly positively charged and large
proteins negatively charged. The protein copy numbers in the minimal genome have
the tendency to maximize the number of protein-protein interactions in the
network. Negatively charged proteins that tend to have larger sizes can provide
a large collision cross-section allowing them to interact with other proteins;
on the other hand, the smaller positively charged proteins could have higher
diffusion speed and are more likely to collide with other proteins. Proteomes
with random charge/mass populations form less stable clusters than those with
experimental protein copy numbers. Our study suggests that "proper" populations
of negatively and positively charged proteins are important for maintaining a
protein-protein interaction network in a proteome. It is interesting to note
that the minimal genome model based on the charge and mass of Escherichia coli
may have a larger protein-protein interaction network than that based on the
lower organism Mycoplasma pneumoniae. Minimal bacterial gene set comprises the genetic elements needed for survival of
engineered bacterium on a rich medium. This set is estimated to include 300-350
protein-coding genes. One way of simplifying an organism with such a minimal
genome even further is to constrain the amino acid content of its proteins. In
this study, comparative genomics approaches and the results of gene knockout
experiments were used to extrapolate the minimal gene set of mollicutes, and
bioinformatics combined with the knowledge-based analysis of the
structure-function relationships in these proteins and their orthologs, paralogs
and analogs was applied to examine the challenges of completely replacing the
rarest residue, cysteine. Among several known functions of cysteine residues,
their roles in the active centers of the enzymes responsible for
deoxyribonucleoside synthesis and transfer RNA modification appear to be
crucial, as no alternative chemistry is known for these reactions. Thus, drastic
reduction of the content of the rarest amino acid in a minimal proteome appears
to be possible, but its complete elimination is challenging. The minimal cell concept represents a pragmatic approach to the question of how
few genes are required to run a cell. This is a helpful way to build a
parts-list, and has been more successful than attempts to deduce a minimal gene
set for life by inferring the gene repertoire of the last universal common
ancestor, as few genes trace back to this hypothetical ancestral state. However,
the study of minimal cellular systems is the study of biological outliers where,
by practical necessity, coevolutionary interactions are minimized or ignored. In
this paper, we consider the biological context from which minimal genomes have
been removed. For instance, some of the most reduced genomes are from
endosymbionts and are the result of coevolutionary interactions with a host; few
such organisms are "free-living." As few, if any, biological systems exist in
complete isolation, we expect that, as with modern life, early biological
systems were part of an ecosystem, replete with organismal interactions. We
favor refocusing discussions of the evolution of cellular systems on processes
rather than gene counts. We therefore draw a distinction between a pragmatic
minimal cell (an interesting engineering problem), a distributed genome (a
system resulting from an evolutionary transition involving more than one cell)
and the looser coevolutionary interactions that are ubiquitous in ecosystems.
Finally, we consider the distributed genome and coevolutionary interactions
between genomic entities in the context of early evolution. |
Is cocaine use associated with increased risk for intracerebral hemorrhage? | Cocaine use is associated with increased incidence of intracranial hemorrhages most likely due to pressure increases. | Medical problems related to cocaine consumption are not rare in our environment;
however, to this respect, descriptions in the spanish literature are scarce. We
present three cases of intracerebral hemorrhage which occurred after cocaine
consumption (intranasal route in two cases and intravenous route in one case).
In the three cases, another associated risk factor was observed (alcohol in one
case, hypertension in other case, arteriovenous malformation in the third case).
We comment on the pathophysiological theories of the cause of bleeding,
high-lighting the routine search of this association. An increase in cocaine abuse by pregt women has been associated with a range
of maternal/fetal cardiovascular complications. Intracerebral hemorrhage has
been reported as a cocaine-related complication, but has not previously been
associated with pregcy. We report a case of cocaine-associated intracerebral
hemorrhage in the postpartum period which complicated the differential diagnosis
and management of hypertension and seizures. In a review of the records of 3,712 drug abusers, 13 patients were identified
with neurologic deficits attributable to the use of cocaine. Ischemic
manifestations were the most frequent, occurring in seven (54%) patients, with a
mean age of 34.2 years. Three (23%) patients had subarachnoid hemorrhage, and
three (23%) had intracerebral hemorrhage. Three patients from other institutions
were included in the analysis, for a total of 16 patients, (eight with cerebral
ischemia, four with subarachnoid hemorrhage, and four with intracerebral
hemorrhage). Of the six patients with head computed tomographic findings of
cerebral infarction, five had subcortical infarcts. Two of the four patients who
presented with a subarachnoid hemorrhage had a congenital intracranial aneurysm.
One of the four patients with an intracerebral hemorrhage had an underlying
arteriovenous malformation. The mechanism through which these complications
occur is not completely understood. Factors that may play a role include the
acute hypertensive response that occurs with cocaine use as well as disordered
neurovascular control. BACKGROUND AND PURPOSE: Stroke subtypes and prognosis differ among older black
patients compared with whites; however, few data are available regarding stroke
among young black patients.
METHODS: To determine the risk factors for stroke, stroke subtype, and prognosis
among young black patients, we retrospectively reviewed the medical records of
all 15- to 44-year-old patients admitted with stroke to a university-affiliated
public hospital from January 1990 through June 1994.
RESULTS: Of the 248 eligible patients admitted with stroke, 219 were blacks.
Hypertension was more frequently associated with stroke in young black than in
non-black patients (55% versus 24%, P = .003). Cocaine abuse was frequent among
both black and non-black patients (27% versus 38%, P = NS). Hypertensive
intracerebral hemorrhage (64%) was the most common subtype of intracerebral
hemorrhage (n = 67), and lacunar infarction (21%) was the most common subtype of
cerebral infarction (n = 112) in young black patients. Outcome in black patients
with stroke at discharge was 69% independent, 8% dependent, and 23% dead.
CONCLUSIONS: The high frequency of hypertension, hypertensive intracerebral
hemorrhage, and lacunar infarction among young black patients with stroke
suggests accelerated hypertensive arteriolar damage, possibly due to poor
control of hypertension. We analyzed 26 autopsy cases of cocaine induced intracerebral hemorrhage and
compared those findings with those of 26 autopsy cases of cocaine induced
cerebral aneurysm rupture. The incidence of hypertensive cardiovascular disease
(HCVD) was significantly higher in persons with intracerebral hemorrhage than in
those with aneurysm rupture. Our findings suggest that HCVD predisposes to
cocaine induced intracerebral hemorrhage. We propose that the foregoing
relationship results from a cocaine induced alteration of cerebral
autoregulation in the context of increased cerebral blood flow. Use of cocaine in the USA, has reached epidemic proportions since 1983, when
"crack" was introduced, its higher potency compared with cocaine HCl has been
associated with a tremendous increase in the incidence of strokes. This study
reports our experience with 55 cases of neurovascular events (25 ischemic and 30
hemorrhagic) related to cocaine use in 54 patients. Only 15 patients had other
risk factors for stroke. Twenty six patients smoked "crack", 10 snorted cocaine
and 12 injected it intravenously. Strokes occurred within 3 h of cocaine use in
15 patients with infarcts and 17 with hemorrhages. Ten infarcts occurred after
an overnight binge. Of the hemorrhage group 9 were subarachnoid, 16
intracerebral (8 basal ganglia, 7 hemispheric and one brain stem) and 5
intraventricular. Computerized tomography (CT) showed an aneurysm of the
anterior communicating artery, as well as one of the vein of Galen. Four
aneurysms and 3 AVMs were identified on angiography. CT revealed 15 infarcts; it
was normal in 7 patients with pure motor hemiparesis and in 3 with findings
consistent with anterior spinal artery infarction. Several mechanisms may be
responsible for the cerebrovascular complications. A sudden rise in systemic
arterial pressure may cause hemorrhages, frequently in association with an
underlying aneurysm or AVM. Vasospasm, arteritis, myocardial infarction with
cardiac arrhythmias and increased platelet aggregation may provoke infarcts. BACKGROUND: Cocaine use has been temporally associated with neurovascular
complications, including the rupture of intracerebral aneurysms. The purpose of
the current study was to determine the type of neurovascular complications
associated with cocaine use in our patient population, the temporal relationship
between cocaine use and their onset, and whether cocaine users with subarachnoid
hemorrhage (SAH) presented with smaller aneurysms at an earlier age than a
control group of noncocaine users with SAH.
METHODS: Thirty-three patients who presented to the Detroit Medical Center with
neurovascular sequelae associated with cocaine use were identified. All patients
were chronic cocaine users who related a history of recent use confirmed by a
drug screen. Cocaine users with SAH were compared to a control group of 44
patients with SAH who presented without evidence of cocaine use.
RESULTS: Sixteen patients presented with SAH. Twelve patients subsequently
underwent four-vessel cerebral arteriogram revealing 14 aneurysms; six patients
presented with intracerebral hemorrhage (ICH) and seven patients with evidence
of ischemic stroke. Eighteen (54.5%) patients noted onset of their symptoms
while using cocaine, 87.9% noted onset within 6 hours of use. Delayed
presentation occurred predomitly in patients who suffered ischemic strokes.
The average age of patients who used cocaine and presented with SAH secondary to
a ruptured intracerebral aneurysm was 32.8 years with an average aneurysm
diameter of 4.9 mm versus an average age of 52.2 years with an average aneurysm
diameter of approximately 11.0 mm in noncocaine users. Population differences
were statistically significant at the p < 0.05 level. Mortality was 27.3% for
patients who presented with neurovascular sequelae of their cocaine use, with
77.8% of deaths occurring in patients who presented with SAH.
CONCLUSIONS: Chronic cocaine use appears to predispose patients who harbor
incidental neurovascular anomalies to present at an earlier point in their
natural history than similar non-cocaine users. INTRODUCTION AND OBJECTIVE: The use of cocaine has been increasingly associated
with cerebrovascular disease specially in young adults. We review the cases of
stroke related to cocaine abuse in this group.
PATIENTS AND METHODS: We performed a retrospective study between 1989-1998. Data
were obtained from the Young Adults Stroke Registry. To investigate the etiology
of stroke all patients underwent cardiologic examination, coagulation and
neuroimaging tests.
RESULTS: We identified 13 patients under 45 years of age with stroke related to
cocaine abuse (0.39% of all strokes and 7.60% of the ones in young adults). Mean
age in this group was 28.30 years. Eight developed ischemic manifestations (5
infarcts, 2 TIAs and 1 encephalopathy with multiple ischemic lesions), 4 had
intraparenchymal hemorrhages and 1 had a subarachnoid hemorrhage. The principal
route of administration was intranasal and the time course from cocaine use to
stroke ranged from several hours to several years. 61.53% had history of other
drug abuse and in 84.61% other risk factors were identified. Angiographic
studies demonstrated: arterial occlusions (3 cases), changes consistent with
vasospasm (1), segmental narrowing (1) and arterial wall irregularities (1). No
aneurysms or arterio-venous malformations were found. The frequency of
cocaine-related stroke in young adults has decreased from 8.33% in 1989 to 5% in
1998.
CONCLUSIONS: Cocaine is a well known cause of stroke, specially in young adults.
In most cases other risk factors can be identified. Multiple overlapping
mechanisms may be involved (vasospasm-thrombosis, high blood pressure, embolism. OBJECTIVE: To determine the risk factors for intracerebral hemorrhage (ICH) in
African Americans aged 18 to 45 years. African Americans are at a higher risk
for ICH than Whites, particularly in the younger age groups. However, few data
are available regarding the factors that contribute to the high risk of ICH
among younger African Americans.
DESIGN: A case-control study.
SETTINGS: A university-affiliated public hospital.
PARTICIPANTS: One hundred and twenty-two African-American patients admitted with
non-traumatic ICH to Grady Memorial Hospital (Atlanta, Ga.) and 366 age- and sex
matched African-American controls selected from a nationally representative
sample of the civilian, non-institutionalized US population.
MAIN OUTCOME MEASURE: Association between ICH and various demographic and
clinical factors determined by stepwise logistic regression.
RESULTS: Cocaine use (OR 6.1, 95% CI 3.3-11.8), hypertension (OR 5.2, 95% CI
3.2-8.7) and alcohol use (OR 1.9, 95% CI 1.1-3.3) were independently associated
with increased risk for ICH.
CONCLUSIONS: Cocaine use, hypertension and alcohol use contributed to the high
risk of ICH observed in younger African Americans. Primary preventive strategies
are required to reduce the high frequency of modifiable risk factors
predisposing younger African Americans to ICH. BACKGROUND AND PURPOSE: To identify risk factors for subarachnoid hemorrhage
(SAH) and intracerebral hemorrhage, we designed a case-control study of men and
women 18 to 49 years of age (the Hemorrhagic Stroke Project [HSP]). This report
focuses on SAH.
METHODS: Patients were recruited from 44 hospitals in the United States. Cases
with SAH must have had a ruptured aneurysm documented by angiography or surgery.
Two controls, identified by random digit dialing and matched to each patient for
age, sex, race, and telephone exchange, were sought for each case subject.
RESULTS: Between 1994 and 1999, 425 patients with SAH were enrolled in HSP, and
312 cases met the criteria for aneurysmal SAH. The present analyses also
included 618 matched controls. Of the 312 cases, 66% were current cigarette
smokers compared with 30% of controls (adjusted odds ratio [OR], 3.73; 95% CI,
2.67 to 5.21). Cocaine use within the previous 3-day period was reported by 3%
of cases and no controls (bivariate exact OR, 24.97; 95% exact CI, 3.95 to
infinity; adjusted estimate not calculable). Other independent risk factors in
the multivariable model included hypertension (adjusted OR, 2.21; 95% CI, 1.48
to 3.29), low body mass index (OR, 1.59; 95% CI, 1.08 to 2.35), primary family
history of hemorrhagic stroke (OR, 3.83; 95% CI, 1.73 to 8.46), caffeine in
pharmaceutical products (OR, 2.48; 95% CI, 1.19 to 5.20), lower educational
achievement (OR, 2.36; 95% CI, 1.44 to 3.87), and nicotine in pharmaceutical
products (adjusted estimate not calculable).
CONCLUSIONS: Aneurysmal SAH may be largely a preventable disease among the young
and middle-aged because several prevalent risk factors can be modified by
medication (eg, hypertension) or behavioral change (eg, cigarette smoking,
cocaine use). The association of caffeine and nicotine in pharmaceutical
products and aneurysmal SAH warrants further study. Thromboaspiration was performed in a young adult in a coma because of acute
basilar artery occlusion associated with cocaine and ecstasy abuse 30 hours
after symptom onset. There was complete recanalization of the basilar artery and
favorable recovery. Because cocaine and ecstasy abuse has been reported to be a
risk factor for ischemic stroke and fatal brain hemorrhage, thromboaspiration
may be an alternative therapy to thrombolysis. BACKGROUND AND PURPOSE: Cocaine is a cause of intracerebral hemorrhage (ICH),
but there are no large studies that have characterized the location, pathology,
and outcome of patients with cocaine-associated ICH.
METHODS: We performed a retrospective analysis of all patients admitted to our
stroke service from 2004 to 2007 who had nontraumatic ICH and urine drug screens
positive for cocaine and compared them with similar patients who had negative
drug screens for cocaine.
RESULTS: We identified 45 patients with cocaine-associated ICH and 105 patients
with cocaine-negative ICH. There were no significant differences in age or
gender, but there was a significantly higher incidence of black patients in the
cocaine-positive group. Cocaine-associated ICH patients had higher admission
blood pressures, significantly more subcortical hemorrhages, and higher rates of
intraventricular hemorrhage compared to patients with cocaine-negative ICH.
Cocaine-positive patients had worse functional outcome, defined as modified
Rankin Scale score >3 at the time of discharge (OR, 4.90; 95% CI, 2.19-10.97),
and were less likely to be discharged home or to inpatient rehabilitation.
Patients with cocaine-associated ICH were nearly 3-times more likely to die
during their acute hospitalization when compared to cocaine-negative patients.
CONCLUSION: Recent cocaine ingestion is associated with hemorrhages that occur
more frequently in subcortical locations, have a higher risk of intraventricular
hemorrhage, and have a poor prognosis compared to patients with
cocaine-negative, spontaneous ICH. Intracerebral hemorrhage (ICH) is a well-recognized complication of recreational
cocaine use. The precise mechanism of the cocaine-induced hemorrhagic event is
unclear, although multiple factors have been implicated. We report a case of a
62-year-old woman who suffered left parieto-occipital ICH with herniation and
death, following a cocaine binge. Microscopic examination also revealed
extensive cerebral amyloid angiopathy (CAA) in the vicinity of the hemorrhage.
We additionally studied brain tissue in eight subjects between ages of 60 and 80
who were positive for cocaine metabolites at autopsy; of these, none had
vascular amyloid-β deposits by immunohistochemistry. Whereas we found no
evidence that chronic cocaine use is a risk factor for CAA, given the
age-associated nature of CAA and the aging population using cocaine, CAA-induced
hemorrhage in the setting of cocaine use may be more common than recognized.
This is the first reported case of CAA-associated ICH precipitated by cocaine. BACKGROUND: Stroke in crack-cocaine abusers is increasingly recognized. We aimed
to identify significant differences in stroke risk factors, characteristics, and
outcomes between hospitalized stroke patients with and without crack-cocaine
abuse.
METHODS: This was a retrospective study that compared stroke patients with
crack-cocaine abuse (cases) to stroke patients without crack-cocaine (controls).
RESULTS: We identified 93 crack-cocaine cases and 93 controls admitted between
January 2004 and May 2006 to one teaching hospital. There were significant
differences between crack-cocaine cases and controls in age (48.7 years vs. 55
years) (P = 0.0001), male gender (65.6% vs. 40.9%) (odds ratios, OR = 1.64, 95%
CI 1.22-2.21), arterial hypertension (61.1% vs. 83.9%) (OR = 0.30, 95% CI
0.15-0.60), hypercholesterolemia (18.7% vs. 68.5%) (OR = 0.10, 95% CI
0.05-0.21), diabetes (20.9% vs. 41.9%) (OR = 0.36, 95% CI 0.19-0.70), cigarette
smoking (70.6% vs. 29%) (OR = 5.86, 95% CI 3.07-11.20), ischemic stroke (61.3%
vs. 79.6%) (OR = 0.40, 95% CI 0.21-0.78), and intracerebral hemorrhage (33.3%
vs. 17.2%) (OR = 3.03, 95% CI 1.53-6.00). Also, there were significant
differences in National Institutes of Health Stroke Scale scores (3.3 vs. 7) (P
< 0.0001), and MRS scores (1.8 vs. 2.5) (P = 0.0022) at hospital discharge.
Using univariable and multivariable logistic regression, we found that
crack-cocaine abusers had 2.28 higher odds of having a favorable functional
outcome (MRS score ≤ 2) at hospital discharge, after adjusting for stroke risk
factors and characteristics.
CONCLUSIONS: Our study suggests that crack-cocaine abusers with stroke had fewer
traditional risk factors, and more favorable functional outcome as compared to
non-crack-cocaine abusers. |
Is miR-21 related to carcinogenesis? | Yes. It has been demonstrated in several experimental studies that miR-21 has oncogenic potential, and is significantly dysregulated in numerous types of cancer. Therefore, miR-21 is closely related to carcinogenesis. | Expression levels of microRNAs (miRNAs) are globally reduced in cancer compared
with matched normal tissues, and miRNA function has recently been implicated in
tumorigenesis. To test whether epigenetic silencing contributes to miRNA
suppression in tumors, lung cancer cells were treated with inhibitors of DNA
methylation or histone deacetylation. No significant alteration in miRNA
expression was detected using microarray profiling. To search for
tumor-associated mutations that could affect processing and expression of mature
miRNAs, a panel of 91 cancer-derived cell lines was analyzed for sequence
variations in 15 miRNAs implicated in tumorigenesis by virtue of their known
target transcripts (let-7 family targeting oncogenic Ras) or their localization
to sites of frequent chromosomal instability (miR-143, miR-145, miR-26a-1, and
miR-21). No mutations were detected within any of the short mature miRNA
sequences. In addition to previously reported polymorphisms, 1 sequence variant
in a precursor miRNA and 15 variants in primary miRNA (pri-miRNA) transcripts
were identified. Despite pri-miRNAs having dramatic changes in the predicted
secondary folding structure flanking putative cleavage sites, processing and
miRNA maturation were not affected in vivo. Thus, genetic variants in miRNA
precursors are common in cancer cells but are unlikely to have physiologic
significance. PURPOSE: We investigated the global microRNA expression patterns in normal
pancreas, pancreatic endocrine tumors and acinar carcinomas to evaluate their
involvement in transformation and maligt progression of these tumor types.
MicroRNAs are small noncoding RNAs that regulate gene expression by targeting
specific mRNAs for degradation or translation inhibition. Recent evidence
indicates that microRNAs can contribute to tumor development and progression and
may have diagnostic and prognostic value in several human maligcies.
MATERIALS AND METHODS: Using a custom microarray, we studied the global microRNA
expression in 12 nontumor pancreas and 44 pancreatic primary tumors, including
12 insulinomas, 28 nonfunctioning endocrine tumors, and four acinar carcinomas.
RESULTS: Our data showed that a common pattern of microRNA expression
distinguishes any tumor type from normal pancreas, suggesting that this set of
microRNAs might be involved in pancreatic tumorigenesis; the expression of
miR-103 and miR-107, associated with lack of expression of miR-155,
discriminates tumors from normal; a set of 10 microRNAs distinguishes endocrine
from acinar tumors and is possibly associated with either normal endocrine
differentiation or endocrine tumorigenesis; miR-204 is primarily expressed in
insulinomas and correlates with immunohistochemical expression of insulin; and
the overexpression of miR-21 is strongly associated with both a high Ki67
proliferation index and presence of liver metastasis.
CONCLUSION: These results suggest that alteration in microRNA expression is
related to endocrine and acinar neoplastic transformation and progression of
maligcy, and might prove useful in distinguishing tumors with different
clinical behavior. Non-coding RNA molecules such as microRNAs (miRNAs) may play an important role
in human carcinogenesis. Their expression has been profiled in many human
cancers but there are few published studies in head and neck cancer. In this
study, the relative expression of 261 mature miRNA genes was determined in nine
head and neck cancer cell lines using an oligonucleotide array platform.
Thirty-three miRNAs in the array were found to be highly expressed and 22 showed
low levels of expression in all cell lines. Notable was the high expression of
miR-21 and miR-205. Expression of several miRNAs was validated using Northern
blot analysis. Potential targets of validated miRNAs included tumor suppressor
genes, kinesin family member 1B isoform alpha (KIF1B), and hypermethylated in
cancer 2 (HIC2), and pleomorphic adenoma gene 1 (PLAG1). This study provides the
largest genomewide survey of mature miRNA transcripts in head and neck cancer
cell lines. Noncoding microRNAs act as posttranscriptional repressors of gene function and
are often deregulated in cancers and other diseases. Here we review recent
findings on microRNA roles in tumorigenesis and report a microRNA profiling
screen in transforming growth factor-beta1 (TGF-beta)-induced
epithelial-mesenchymal transition (EMT) in human keratinocytes, a model of
epithelial cell plasticity underlying epidermal injury and skin carcinogenesis.
We describe a novel EMT-specific microRNA signature that includes induction of
miR-21, a candidate oncogenic microRNA associated with carcinogenesis. By
integrating the microRNA screen results with target prediction algorithms and
gene expression profiling data, we outline a framework for TGF-beta-directed
microRNA:messenger RNA (mRNA) regulatory circuitry and discuss its biological
relevance for tumor progression. MicroRNAs (miRNAs) are a new class of short noncoding regulatory RNAs (18-25
nucleotides) that are involved in diverse developmental and pathologic
processes. Altered miRNA expression has been associated with several types of
human cancer. However, most studies did not establish whether miRNA expression
changes occurred within cells undergoing maligt transformation. To obtain
insight into miRNA deregulation in breast cancer, we implemented an in situ
hybridization (ISH) method to reveal the spatial distribution of miRNA
expression in archived formalin-fixed, paraffin-embedded specimens representing
normal and tumor tissue from >100 patient cases. Here, we report that expression
of miR-145 and miR-205 was restricted to the myoepithelial/basal cell
compartment of normal mammary ducts and lobules, whereas their accumulation was
reduced or completely eliminated in matching tumor specimens. Conversely,
expression of other miRNAs was detected at varying levels predomitly within
luminal epithelial cells in normal tissue; expression of miR-21 was frequently
increased, whereas that of let-7a was decreased in maligt cells. We also
analyzed the association of miRNA expression with that of epithelial markers;
prognostic indicators such as estrogen receptor, progesterone receptor, and
HER2; as well as clinical outcome data. This ISH approach provides a more direct
and informative assessment of how altered miRNA expression contributes to breast
carcinogenesis compared with miRNA expression profiling in gross tissue
biopsies. Most significantly, early manifestation of altered miR-145 expression
in atypical hyperplasia and carcinoma in situ lesions suggests that this miRNA
may have a potential clinical application as a novel biomarker for early
detection. CONTEXT: MicroRNAs have potential as diagnostic biomarkers and therapeutic
targets in cancer. No study has evaluated the association between microRNA
expression patterns and colon cancer prognosis or therapeutic outcome.
OBJECTIVE: To identify microRNA expression patterns associated with colon
adenocarcinomas, prognosis, or therapeutic outcome.
DESIGN, SETTING, AND PATIENTS: MicroRNA microarray expression profiling of
tumors and paired nontumorous tissues was performed on a US test cohort of 84
patients with incident colon adenocarcinoma, recruited between 1993 and 2002. We
evaluated associations with tumor status, TNM staging, survival prognosis, and
response to adjuvant chemotherapy. Associations were validated in a second,
independent Chinese cohort of 113 patients recruited between 1991 and 2000,
using quantitative reverse transcription polymerase chain reaction assays. The
final date of follow-up was December 31, 2005, for the Maryland cohort and
August 16, 2004, for the Hong Kong cohort.
MAIN OUTCOME MEASURES: MicroRNAs that were differentially expressed in tumors
and microRNA expression patterns associated with survival using cancer-specific
death as the end point. RESULTS Thirty-seven microRNAs were differentially
expressed in tumors from the test cohort. Selected for validation were miR-20a,
miR-21, miR-106a, miR-181b, and miR-203, and all 5 were enriched in tumors from
the validation cohort (P < .001). Higher miR-21 expression was present in
adenomas (P = .006) and in tumors with more advanced TNM staging (P < .001). In
situ hybridization demonstrated miR-21 to be expressed at high levels in colonic
carcinoma cells. The 5-year cancer-specific survival rate was 57.5% for the
Maryland cohort and was 49.5% for the Hong Kong cohort. High miR-21 expression
was associated with poor survival in both the training (hazard ratio, 2.5; 95%
confidence interval, 1.2-5.2) and validation cohorts (hazard ratio, 2.4; 95%
confidence interval, 1.4-3.9), independent of clinical covariates, including TNM
staging, and was associated with a poor therapeutic outcome.
CONCLUSIONS: Expression patterns of microRNAs are systematically altered in
colon adenocarcinomas. High miR-21 expression is associated with poor survival
and poor therapeutic outcome. MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively control
expression of target genes in animals and plants. The microRNA-21 gene (mir-21)
has been identified as the only miRNA commonly overexpressed in solid tumors of
the lung, breast, stomach, prostate, colon, brain, head and neck, esophagus and
pancreas. We initiated a screen to identify miR-21 target genes using a reporter
assay and identified a potential miR-21 target in the 3'-UTR of the programmed
cell death 4 (PDCD4) gene. We cloned the full-length 3'-UTR of human PDCD4
downstream of a reporter and found that mir-21 downregulated, whereas a modified
antisense RNA to miR-21 upregulated reporter activity. Moreover, deletion of the
putative miR-21-binding site (miRNA regulatory element, MRE) from the 3'-UTR of
PDCD4, or mutations in the MRE abolished the ability of miR-21 to inhibit
reporter activity, indicating that this MRE is a critical regulatory region.
Western blotting showed that Pdcd4 protein levels were reduced by miR-21 in
human and mouse cells, whereas quantitative real-time PCR revealed little
difference at the mRNA level, suggesting translational regulation. Finally,
overexpression of mir-21 in MCF-7 human breast cancer cells and mouse epidermal
JB6 cells promoted soft agar colony formation by downregulating Pdcd4 protein
levels. The demonstration that miR-21 promotes cell transformation supports the
concept that mir-21 functions as an oncogene by a mechanism that involves
translational repression of the tumor suppressor Pdcd4. BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs
that have been shown to be aberrantly expressed in many human carcinomas. Of
these miRNAs, miR-21 appears to be important in tumorigenesis given its
up-regulation in almost all types of human cancer examined. However, its
association with the clinicopathological features of human gastric cancer has
yet to be addressed.
PATIENTS AND METHODS: Cancer tissues and corresponding normal tissues from 37
patients with gastric cancer were examined for the expression level of miR-21
using quantitative PCR and the clinical relevance of miR-21 was statistically
analyzed.
RESULTS: miR-21 was overexpressed in 92% (34/37) of the gastric cancer samples
examined. However, the patients with higher miR-21 expression did not have a
worse prognosis.
CONCLUSION: miR-21 could serve as an efficient diagnostic marker for gastric
cancer, but does not affect the clinical prognosis of gastric cancer patients. BACKGROUND AND AIM: MicroRNAs (miRNAs) play important roles in carcinogenesis.
The global miRNA expression profile of gastric cancer has not been reported. The
purpose of the present study was to determine the miRNA expression profile of
gastric cancer.
METHODS: Total RNA were first extracted from primary gastric cancer tissues and
adjacent non-tumorous tissues and then small isolated RNAs (< 300 nt) were
3'-extended with a poly(A) tail. Hybridization was carried out on a microParaflo
microfluidic chip (LC Sciences, Houston, TX, USA). After hybridization detection
by fluorescence labeling using tag-specific Cy3 and Cy5 dyes, hybridization
images were collected using a laser scanner and digitized using Array-Pro image
analysis software (Media Cybernetics, Silver Spring, MD, USA). To validate the
results and investigate the biological meaning of differential expressed miRNAs,
immunohistochemistry was used to detect the differential expression of target
genes.
RESULTS: The most highly expressed miRNAs in non-tumorous tissues were
miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b. Three of
them, miR-139-5p, miR-497 and miR-768-3p, were first found in non-tumorous
tissues. The most highly expressed miRNAs in gastric cancer tissues were
miR-20b, miR-20a, miR-17, miR-106a, miR-18a, miR-21, miR-106b, miR-18b, miR-421,
miR-340*, miR-19a and miR-658. Among them, miR-340*, miR-421 and miR-658 were
first found highly expressed in cancer cells. The expression of some target
genes (such as Rb and PTEN) in cancer tissues was found to be decreased.
CONCLUSION: To our knowledge, this is the first report about these miRNAs
associated with gastric cancer. This new information may suggest the potential
roles of these miRNAs in the diagnosis of gastric cancer. BACKGROUND: Non-coding RNA molecules, such as microRNAs, may play an important
role in carcinogenesis. Recent studies have indicated that microRNAs are
involved in initiation and progression of various maligcies. However, little
work has been done to compare the microRNA expression patterns in oral cancer.
In this study, we constructed an animal model of oral squamous cell carcinoma to
investigate expression profiles of microRNAs in oral carcinogenesis.
METHODS: The animal model of oral squamous cell carcinoma was conducted by
tri-weekly (Monday, Wednesday, and Friday) painting with 5% DMBA in acetone. Six
Syrian hamsters, including three from the treated group and three from the
control group, were used as a training group for microRNA microarray analysis.
All microarray data were analyzed by Significance Analysis of Microarrays (SAM)
and CLUSTER 3.0 software, and this result was further confirmed by qRT-PCR
assay.
RESULTS: Seventeen microRNAs were differentially expressed in oral squamous cell
carcinoma. Five microRNAs (hsa-miR-21, hsa-miR-200b, hsa-miR-221, hsa-miR-338,
and mmu-miR-762) were significantly upregulated and twelve microRNAs
(hsa-miR-16, hsa-miR-26a, hsa-miR-29a, hsa-miR-124a, hsa-miR-125b,
mmu-miR-126-5p, hsa-miR-143, hsa-miR-145, hsa-miR-148b, hsa-miR-155,
hsa-miR-199a, and hsa-miR-203) were down-regulated in cancer tissues. The
expression levels of hsa-miR-21 and hsa-miR-16 seen with Stem-loop qRT-PCR were
also seen in microarray analysis in all samples.
CONCLUSION: Our findings identified specific microRNA expression in oral
squamous cell carcinoma and suggested that microRNAs have a role in oral
carcinogenesis. BACKGROUND: Flat epithelial atypia (FEA) of the breast is characterised by a few
layers of mildly atypical luminal epithelial cells. Genetic changes found in
ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also
found in FEA, albeit at a lower concentration. So far, miRNA expression changes
associated with invasive breast cancer, like miR-21, have not been studied in
FEA.
METHODS: We performed miRNA in-situ hybridization (ISH) on 15 cases with
simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional
cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was
investigated by immunohistochemistry.
RESULTS: Two out of fifteen cases showed positive staining for miR-21 in normal
breast ductal epithelium, seven out of fifteen cases were positive in the FEA
component and nine out of twelve cases were positive in the DCIS component. A
positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all
three components were present in one tissue block and an increase of miR-21 from
normal breast to FEA and to DCIS was observed in five cases. In three cases the
FEA component was negative, whereas the DCIS component was positive for miR-21.
In three other cases, normal, FEA and DCIS components were negative for miR-21
and in the last case all three components were positive. Overall we observed a
gradual increase in percentage of miR-21 positive cases from normal, to FEA,
DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes
in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of
PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1
staining decreased from positive in normal breast to negative in most DCIS and
IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast
tissue.
CONCLUSION: Upregulation of miR-21 from normal ductal epithelial cells of the
breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No
clear relation was observed between the staining pattern of miR-21 and its
previously reported target genes. MicroRNAs are short regulatory RNAs that negatively modulate gene expression at
the post-transcriptional level, and are deeply involved in the pathogenesis of
several types of cancers. To investigate whether specific miRNAs and their
target genes participate in the molecular pathogenesis of laryngeal carcinoma,
oligonucleotide microarrays were used to assess the differential expression
profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with
normal tissues. The oncogenic miRNA, microRNA-21 (miR-21), was found to be
upregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific
antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells,
whereas overexpression of miR-21 elevated growth activity of the cells, as
detected by the colony formation assay. The cell number reduction caused by
miR-21 inhibition was due to the loss of control of the G1-S phase transition,
instead of a noticeable increase in apoptosis. Subsequently, a new target gene
of miR-21, BTG2, was found to be downregulated in laryngeal carcinoma tissues.
BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These
findings indicate that aberrant expression of miR-21 may contribute to the
maligt phenotype of laryngeal carcinoma by maintaining a low level of BTG2.
The identification of the oncogenic miR-21 and its target gene, BTG2, in
laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy. Fifteen percent of lung cancer cases occur in never-smokers and show
characteristics that are molecularly and clinically distinct from those in
smokers. Epidermal growth factor receptor (EGFR) gene mutations, which are
correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), are
more frequent in never-smoker lung cancers. In this study, microRNA (miRNA)
expression profiling of 28 cases of never-smoker lung cancer identified
aberrantly expressed miRNAs, which were much fewer than in lung cancers of
smokers and included miRNAs previously identified (e.g., up-regulated miR-21)
and unidentified (e.g., down-regulated miR-138) in those smoker cases. The
changes in expression of some of these miRNAs, including miR-21, were more
remarkable in cases with EGFR mutations than in those without these mutations. A
significant correlation between phosphorylated-EGFR (p-EGFR) and miR-21 levels
in lung carcinoma cell lines and the suppression of miR-21 by an EGFR-TKI,
AG1478, suggest that the EGFR signaling is a pathway positively regulating
miR-21 expression. In the never-smoker-derived lung adenocarcinoma cell line
H3255 with mutant EGFR and high levels of p-EGFR and miR-21, antisense
inhibition of miR-21 enhanced AG1478-induced apoptosis. In a
never-smoker-derived adenocarcinoma cell line H441 with wild-type EGFR, the
antisense miR-21 not only showed the additive effect with AG1478 but also
induced apoptosis by itself. These results suggest that aberrantly increased
expression of miR-21, which is enhanced further by the activated EGFR signaling
pathway, plays a significant role in lung carcinogenesis in never-smokers, as
well as in smokers, and is a potential therapeutic target in both EGFR-mutant
and wild-type cases. MicroRNAs are involved in cancer-related processes. The microRNA-21(miR-21) has
been identified as the only miRNA over-expressed in a wide variety of cancers,
including cervical cancer. However, the function of miR-21 is unknown in
cervical carcinomas. In this study, we found that the inhibition of miR-21 in
HeLa cervical cancer cells caused profound suppression of cell proliferation,
and up-regulated the expression of the tumor suppressor gene PDCD4. We also
provide direct evidence that PDCD4-3'UTR is a functional target of miR-21 and
that the 18bp putative target site can function as the sole regulatory element
in HeLa cells. These results suggest that miR-21 may play an oncogenic role in
the cellular processes of cervical cancer and may serve as a target for
effective therapies. OBJECTIVES: The contribution of overexpressed microRNA-21 and -221 (miR-21 and
miR-221) to the maligt phenotype was determined by inhibiting these miRNAs
using antisense oligonucleotides.
METHODS: The effects of antisense to miR-21 and miR-221 on cell proliferation,
cell cycle arrest, induction of apoptosis, combinatorial effects with
gemcitabine, and effects on target protein levels were studied.
RESULTS: Low omolar concentrations of both antisense oligonucleotides reduced
proliferation of pancreatic cancer cell lines. Reduced proliferation was less
pronounced in the normal ductal epithelial cell line human pancreatic
Nestin-expressing cell or in pancreatic cancer cell lines exposed to an
irrelevant control oligonucleotide. Inhibition of miR-21 and miR-221 increased
the amount of apoptosis in HS766T cells by 3- to 6-fold compared with the
control oligonucleotide. HS766T cells exposed to miR-21 antisense resulted in
cell cycle arrest (G1 phase). Protein levels of tumor suppressor targets of the
miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27).
Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine, and the
antisense-gemcitabine combinations were synergistic at high fraction affected.
CONCLUSIONS: We demonstrate that antisense to miR-21 and miR-221 results in
significant cell killing under various conditions and that antisense
oligonucleotides targeted to miRNA represents a potential new therapy for
pancreatic cancer. PURPOSE: Inflammatory genes and microRNAs have roles in colon carcinogenesis;
therefore, they may provide useful biomarkers for colon cancer. This study
examines the potential clinical utility of an inflammatory gene expression
signature as a prognostic biomarker for colon cancer in addition to previously
examined miR-21 expression.
EXPERIMENTAL DESIGN: Quantitative reverse transcriptase-PCR. was used to measure
the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent
noncancerous tissues from 196 patients. These data were used to develop models
for cancer-specific mortality on a training cohort (n = 57), and this model was
tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data
for miR-21 were available for these patients and were compared and combined with
inflammatory gene expression.
RESULTS: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous
tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor
tissues were associated with poor prognosis based on Cox regression (/Z-score/
>1.5) and were used to generate the inflammatory risk score (IRS). IRS was
associated with cancer-specific mortality in the training, test (P = 0.01), and
validation (P = 0.02) cohorts. This association was strong for stage II cases (P
= 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a,
and NOS2a, providing evidence that the function of this microRNA and these
inflammatory genes are linked. Both IRS and miR-21 expression were independently
associated with cancer-specific mortality, including stage II patients alone.
CONCLUSION: IRS and miR-21 expression are independent predictors of colon cancer
prognosis and may provide a clinically useful tool to identify high-risk
patients. MicroRNAs (miRNAs) are small, non-protein-coding RNAs that can function as tumor
suppressors or oncogenes. Deregulation of miRNA expression has been reported in
lung cancer. However, modulation of miRNA expression by chemopreventive agents
remains to be defined. In the present study, we examined if the chemopreventive
agent indole-3-carbinol (I3C) reversed vinyl carbamate (VC)-induced deregulation
of miRNA levels in lung tissues of female A/J mice. Lung tissues were obtained
from a previous chemoprevention study, in which mice were treated with VC and
given I3C in the diet for 15 weeks. Microarray studies revealed alterations in
the expression of a number of miRNAs in lung tumors relative to that of normal
lungs. miR-21, mir-31, miR-130a, miR-146b and miR-377 were consistently
upregulated, whereas miR-1 and miR-143 were downregulated in lung tumors
relative to normal lungs. In mice treated with VC and given I3C in the diet,
levels of miR-21, mir-31, miR-130a, miR-146b and miR-377 were reduced relative
to the level in mice treated with the carcinogen only. The results of the
microarray study were confirmed by quantitative reverse transcription-polymerase
chain reaction and gel analysis of polymerase chain reaction products. Further
studies with miR-21 indicated that phosphatase and tensin homolog, programmed
cell death 4 and rich protein with Kazal motifs are potential targets for the
oncogenic effect of miR-21 and the chemopreventive activity of I3C. Taken
together, we showed here that miRNAs are deregulated during VC-induced mouse
lung tumorigenesis and their levels are modulated by I3C. Therefore, miRNAs and
their target genes are promising biomarkers for the diagnosis of lung cancer and
efficacy of chemopreventive/chemotherapeutic agents. MicroRNAs (miRNAs) are small non-coding RNA molecules that are widely involved
in cancer-related processes. The microRNA-21 (miR-21) has been identified as the
only miRNA overexpressed in a variety of cancers, including leukemia. However,
the function of miR-21 is yet unknown in chronic myelogenous leukemia (CML).
Antisense oligonucleotides (ASOs), as inhibitors of miRNAs, have already been
applied to therapeutic development and functional identification in miRNA
research. In this study, we found that the antisense inhibition of miR-21 in
K562 cells suppressed cell migration, promoted cell apoptosis, and inhibited
cell growth, and up-regulated the expression of the tumor suppressor gene PDCD4.
Meanwhile, pre-miRNA-21 increased migration and decreased cell apoptosis without
affecting proliferation. We also validated that PDCD4 is a functional target of
miR-21 in K562 cells. These effects of miR-21 might be partially due to its
regulation of PDCD4. Our data suggest that miR-21 may play an oncogenic role in
the cellular processes of CML, and antisense inhibition of miR-21 may therefore
be useful as CML therapy. MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small
non-coding RNAs that are endogenously expressed with crucial functions in
fundamental cellular processes such as cell cycle, apoptosis and
differentiation. Disturbance of miRNA expression and function leads to
deregulation of basic cellular processes leading to tumorigenesis. A growing
body of experimental evidence suggests that human tumors have deregulated
expression of microRNAs, which have been proposed as novel oncogenes or tumor
suppressors. Recent studies have shown that microRNA expression patterns serve
as phenotypic signatures of different cancers and could be used as diagnostic,
prognostic and therapeutic tools. A few studies have analyzed global microRNA
expression profiles or the functional role of microRNAs in prostate cancer. Here
we have reviewed the role of microRNAs in prostate carcinogenesis by summarizing
the findings from such studies. In addition, recent evidence indicates that
dietary factors play an important role in the process of carcinogenesis through
modulation of miRNA expression, though such studies are lacking in regards to
prostate cancer. It has been proposed that dietary modulation of miRNA
expression may contribute to the cancer-protective effects of dietary
components. In this review, we have summarized findings from studies on the
effect of dietary agents on miRNA expression and function. This symposium presented recent progress of the pathogenesis and treatment of
lung cancer. Aberrantly increased expression of miR-21 plays a significant role
in lung carcinogenesis and is a potential therapeutic target in both epidermal
growth factor receptor-mutant and wild-type cases. miR-34 may be necessary for
the radiation-induced DNA damage response. Detailed expression profiling
analyses of transcriptome have potential to provide increased understanding of
the molecular biology of lung cancer. An embryonic signature is present in lung
adenocarcinoma only, associated with a worse clinical outcome. Cytoplasmic
expression of caveolin and membranous expression of CD26 are specific to
mesothelioma. Nectin-4 is a new candidate for serum and tissue biomarker as well
as a therapeutic target for lung cancer. Clinical presentations have provided us
a great deal information on epidermal growth factor receptor mutations for
personalized therapy, combination therapy with inhibitors of the tyrosine kinase
activity of epidermal growth factor receptor and cytotoxic agents,
antibody-dependent cellular cytotoxicity activity, and current management of
lung cancer depending on both the extent of the disease and the treatment
approach. BACKGROUND: MicroRNAs are non-coding RNA molecules that posttranscriptionally
regulate expression of target genes and have been implicated in the progress of
cancer proliferation, differentiation and apoptosis. The aim of this study was
to determine whether microRNA-21 (miR-21), a specific microRNA implicated in
multiple aspects of carcinogenesis, impacts breast cancer invasion by regulating
the tissue inhibitor of metalloproteinase 3 (TIMP3) gene.
METHODS: miR-21 expression was investigated in 32 matched breast cancer and
normal breast tissues, and in four human breast cancer cell lines, by Taqman
quantitative real-time PCR. Cell invasive ability was determined by matrigel
invasion assay in vitro, in cells transfected with miR-21 or anti-miR-21
oligonucleotides. In addition, the regulation of tissue inhibitor of
metalloproteinase 3 (TIMP3) by miR-21 was evaluated by western blotting and
luciferase assays.
RESULTS: Of the 32 paired samples analyzed, 25 breast cancer tissues displayed
overexpression of miR-21 in comparison with matched normal breast epithelium.
Additionally, incidence of lymph node metastasis closely correlated with miR-21
expression, suggesting a role for miR-21 in metastasis. Similarly, each of the
four breast cancer cell lines analyzed overexpressed miR-21, to varied levels.
Further, cells transfected with miR-21 showed significantly increased matrigel
invasion compared with control cells, whereas transfection with anti-miR-21
significantly decreased cell invasion. Evaluation of TIMP3 protein levels, a
peptidase involved in extarcellular matrix degredation, inversely correlated
with miR-21 expression.
CONCLUSION: As knockdown of miR-21 increased TIMP3 protein expression and
luciferase reporter activity, our data suggests that miR-21 could promote
invasion in breast cancer cells via its regulation of TIMP3. Human glioblastomas often develop resistance to radiation therapy. The molecular
details of this phenomenon are not completely understood. Recent studies have
suggested that deficiency in DNA repair pathways may alter the resistance to
ionizing radiation in gliobastomas. The human glioma cell line M059J is
deficient in DNA-dependent protein kinase (DNA-PK), whereas cell line M059K,
isolated from the same maligt tumor, has normal DNA-PK activity. DNA-PK plays
a central role in the repair of ionizing-radiation-induced double-strand break
repair, and its deficiency has been correlated with ionizing radiation
sensitivity in these glioblastoma cells. We argued that other cellular pathways
could also play a role in the resistance to radiation therapy in gliomas. We
hypothesized that micro-RNAs (miRNAs) are differentially modulated in M059J and
M059K cells exposed to ionizing radiation and that the miRNA modulation
contributes to the resistance to ionizing radiation. miRNAs are small nonprotein
coding single-stranded RNA molecules, which are crucial posttranscriptional
regulators of gene expression. Numerous studies have documented the
participation of miRNAs in a wide range of biological processes. The
contribution of miRNAs in mediating resistance of glioblastoma cell to ionizing
radiation treatment has not been elucidated. To test this hypothesis, we
examined the expression patterns of a number of miRNAs involved in
carcinogenesis in irradiated M059J and M059K cells. The relative expression
level as determined by real-time quantitative PCR for miRNAs belonging to the
let-7 family indicated an upregulation in irradiated M059K cells. On the
contrary, the analysis of irradiated M059J cells for the modulation of let-7
family of miRNAs revealed an overall downregulation. The miR-17-3p, miR-17-5p,
miR-19a, miR-19b, miR-142-3p, and miR-142-5p were upregulated in both M059K and
M059J cells. The miR-15a, miR-16, miR-143, miR-155, and miR-21 were upregulated
in M059K, and the modulation of these miRNAs fluctuated in M059J cells in a
time-dependent manner. These results indicate the involvement of miRNAs in the
differential response of glioblastoma cells to ionizing radiation treatment. MicroRNAs are involved in different cancer-related processes. MicroRNA-21
(miR-21), as an oncomiR, is overexpressed in all kinds of tumors and the role of
miR-21 in carcinogenesis is elucidated in many cancers gradually. However, the
function of miR-21 in osteosarcoma is still unclear. In our study, we found that
miR-21 was significantly overexpressed in osteosarcoma tissues. More
importantly, we confirmed that knockdown of miR-21 greatly decreased cell
invasion and migration of MG-63. Furthermore, we identified that RECK
(reversion-inducing-cysteine-rich protein with kazal motifs), a tumor suppressor
gene, was a direct target of miR-21. Finally, the expression of RECK protein
negatively correlated with the expression of miR-21 in human osteosarcoma
tissues, indicating the potential regulation of RECK by miR-21. Our results
suggest that miR-21 expression has a key role in regulating cellular processes
in osteosarcoma, likely through regulating RECK and may serve as a therapeutic
target. Systemic lupus erythematosus is a complex autoimmune disease caused by genetic
and epigenetic alterations. DNA methylation abnormalities play an important role
in systemic lupus erythematosus disease processes. MicroRNAs (miRNAs) have been
implicated as fine-tuning regulators controlling diverse biological processes at
the level of posttranscriptional repression. Dysregulation of miRNAs has been
described in various disease states, including human lupus. Whereas previous
studies have shown miRNAs can regulate DNA methylation by targeting the DNA
methylation machinery, the role of miRNAs in aberrant CD4+ T cell DNA
hypomethylation of lupus is unclear. In this study, by using high-throughput
microRNA profiling, we identified that two miRNAs (miR-21 and miR-148a)
overexpressed in CD4+ T cells from both patients with lupus and lupus-prone
MRL/lpr mice, which promote cell hypomethylation by repressing DNA
methyltransferase 1 (DNMT1) expression. This in turn leads to the overexpression
of autoimmune-associated methylation-sensitive genes, such as CD70 and LFA-1,
via promoter demethylation. Further experiments revealed that miR-21 indirectly
downregulated DNMT1 expression by targeting an important autoimmune gene,
RASGRP1, which mediated the Ras-MAPK pathway upstream of DNMT1; miR-148a
directly downregulated DNMT1 expression by targeting the protein coding region
of its transcript. Additionally, inhibition of miR-21 and miR-148a expression in
CD4+ T cells from patients with lupus could increase DNMT1 expression and
attenuate DNA hypomethylation. Together, our data demonstrated a critical
functional link between miRNAs and the aberrant DNA hypomethylation in lupus
CD4+ T cells and could help to develop new therapeutic approaches. MicroRNAs, which are endogenously expressed regulatory noncoding RNAs, have an
altered expression in colorectal cancer. The aim of our study was to assess the
relationship of miR-21 and miR-143 expression to the
prognostic/clinicopathological features of colorectal carcinoma (CRC) and
colorectal liver metastases (CLM). The estimation was performed in 46 paired
(tumor and control) tissue samples of CRC. Further, we studied 30 tissue samples
of CLM. MiR-21 and miR-143 expressions were quantified by using the quantitative
reverse transcription polymerase chain reaction method. Relation of miR-21 and
miR-143 expression to disease-free interval (DFI) (Wilcoxon; P = 0.0026 and P =
0.0191, respectively) was recorded. There was shorter DFI in patients with a
higher expression of miR-21 and, surprisingly, also in patients with a higher
expression of miR-143, which is a putative tumor suppressor. There was a higher
expression of miR-21 and lower expression of miR-143 in CRC tissue in comparison
with adjacent normal colon tissue (P < 0.0001; P < 0.0001, respectively).
Similarly, we observed a higher expression of miR-21 and a lower expression of
miR-143 in CLM in comparison with normal colon tissue (P < 0.0001; P < 0.0001,
respectively). Our results support the hypothesis about oncogenic function of
miR-21 and show its relation to DFI. The role of miR-143 in carcinogenesis seems
to be more complex. MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein
expression by cleaving or repressing the translation of target mRNAs. In mammal
animals, their function mainly represses the target mRNAs transcripts via
imperfectly complementary to the 3'UTR of target mRNAs. Several miRNAs have been
recently reported to be involved in modulation of glioma development, especially
some up-regulated miRNAs, such as microRNA-21 (miR-21), which has been found to
function as an oncogene in cultured glioblastoma multiforme cells. Temozolomide
(TMZ), an alkylating agent, is a promising chemotherapeutic agent for treating
glioblastoma. However, resistance develops quickly and with high frequency. To
explore the mechanism of resistance, we found that miR-21 could protect human
glioblastoma U87MG cells from TMZ induced apoptosis. Our studies showed that TMZ
markedly enhanced apoptosis in U87MG cells compared with untreated cells
(P<0.05). However, over-express miR-21 in U87MG cells could significantly reduce
TMZ-induced apoptosis (P<0.05). Pro-apoptotic Bax and anti-apoptotic Bcl-2
proteins are known to regulate the apoptosis of glioma cells. Bcl-2, resistance
to induction of apoptosis, constitutes one major obstacle to chemotherapy in
many cancer cells. Bax is shown to correlate with an increased survival of
glioblastoma multiforme patients. Further research demonstrated that the
mechanism was associated with a shift in Bax/Bcl-2 ratio and change in caspase-3
activity. Compared to control cells, cells treated with TMZ showed a significant
increase in the Bax/Bcl-2 ratio and caspase-3 activity (P<0.01). However, such
effect was partly prevented by treatment of cells with miR-21 overexpression
before, which appeared to downregulate the Bax expression, upregulate the Bcl-2
expression and decrease caspase-3 activity. Taken together, these results
suggested that over-express miR-21 could inhibit TMZ-induced apoptosis in U87MG
cells, at least in part, by decreasing Bax/Bcl-2 ratio and caspase-3 activity,
which highlighted the possibility of miR-21 overexpression in the clinical
resistance to chemotherapeutic therapy of TMZ. Tamoxifen is the most commonly prescribed therapy for patients with estrogen
receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a
serious clinical problem especially in patients with tumors that also
overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical
models of HER2 overexpression fail to recapitulate the clinical spectrum of
endocrine resistance associated with HER2/ER-positive tumors. Here, we show that
ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16,
which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen
resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells
evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of
BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family
pharmacological inhibitor ABT-737 restores tamoxifen sensitivity.
Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in
response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen
treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in
suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of
miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized
MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in
tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen
resistance. Our results suggest that HER2Δ16 expression promotes
endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type
HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple
phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16
therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and
suppression of miR-15a/16, provides a template for unique therapeutic
interventions combining tamoxifen with modulation of microRNAs and/or
ABT-737-mediated BCL-2 inhibition and apoptosis. This study determined whether expression levels of a panel of biologically
relevant microRNAs can be used as prognostic or predictive biomarkers in
patients who participated in the International Adjuvant Lung Cancer Trial
(IALT), the largest randomized study conducted to date of adjuvant chemotherapy
in patients with radically resected non-small cell lung carcinoma (NSCLC).
Expression of miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a was determined
by quantitative real-time PCR in formalin-fixed paraffin-embedded tumor
specimens from 639 IALT patients. The prognostic and predictive values of
microRNA expression for survival were studied using a Cox model, which included
every factor used in the stratified randomization, clinicopathologic prognostic
factors, and other factors statistically related to microRNA expression.
Investigation of the expression pattern of microRNAs in situ was performed. We
also analyzed the association of TP53 mutation status and miR-34a/b/c
expression, epidermal growth factor receptor and KRAS mutation status, and
miR-21 and Let-7a expression. Finally, the association of p16 and miR-29b
expression was assessed. Overall, no significant association was found between
any of the tested microRNAs and survival, with the exception of miR-21 for which
a deleterious prognostic effect of lowered expression was suggested. Otherwise,
no single or combinatorial microRNA expression profile predicted response to
adjuvant cisplatin-based chemotherapy. Together, our results indicate that the
microRNA expression patterns examined were neither predictive nor prognostic in
a large patient cohort with radically resected NSCLC, randomized to receive
adjuvant cisplatin-based chemotherapy versus follow-up only. MicroRNAs (miRNAs) play important roles in regulating a plethora of
physiological and pathophysiogical processes including neurodegeneration. In
both HIV associated dementia in humans and its monkey model SIV encephalitis we
find miR-21, a miRNA largely known for its link to oncogenesis, to be
significantly upregulated in the brain. In situ hybridization of the diseased
brain sections revealed induction of miR-21 in neurons. MiR-21 can be induced in
neurons by prolonged N-methyl-D-aspartic acid receptor stimulation, an
excitotoxic process active in HIV and other neurodegenerative diseases.
Introduction of miR-21 into human neurons leads to pathological functional
defects. Furthermore, we show that miR-21 specifically targets the mRNA of
myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal
function, and reduces its expression. MEF2C is dramatically downregulated in
neurons of HIV-associated dementia patients as well as monkeys with SIVE.
Together, this study elucidates a novel role for miR-21 in the brain, not only
as a potential signature of neurological disease but also as a crucial effector
of HIV induced neuronal dysfunction and neurodegeneration. Programmed cell death 4 (PDCD4) has recently been demonstrated to be a new tumor
suppressor gene involved in colon carcinogenesis. PDCD4 immunohistochemical
expression was assessed in 300 polypoid lesions of the colon mucosa (50
hyperplastic polyps [HP], 50 serrated adenomas [SA], 50 tubular adenomas with
low-grade-intraepithelial neoplasia [LG-IEN], 50 tubular adenomas with
high-grade-IEN [HG-IEN]), and in 50 colon adenocarcinomas (CRC). As normal
controls, we considered 50 biopsy samples obtained from patients with irritable
bowel syndrome (N). We further investigated PDCD4 messenger RNA (mRNA) levels by
quantitative real-time polymerase chain reaction (PCR) in a different series of
N, LG-IEN, HG-IEN, and CRC biopsy samples. miR-21 expression (an important
PDCD4-expression regulator) was also determined by quantitative real-time PCR
and in situ hybridization. Normal colocytes and HP featured strong PDCD4 nuclear
immunostaining whereas a significantly lower PDCD4 nuclear expression was
observed in dysplasia (low- and high-grade adenomas and SA) and invasive CRC.
PDCD4 immunostaining and mRNA levels decreased significantly as the phenotypic
changes occurring during colon carcinogenesis progressively increased
(p < 0.001). As expected, miR-21 expression was significantly upregulated in
preneoplastic/neoplastic samples, consistent with PDCD4 downregulation. These
results consistently support the use of nuclear PDCD4 immunohistochemical
downregulation as a novel biomarker for the diagnosis of dysplastic/neoplastic
lesions in colon biopsy samples. We have recently demonstrated that nutritional bioactives (fish oil and pectin)
modulate microRNA molecular switches in the colon. Since integrated analysis of
microRNA and mRNA expression at an early stage of colon cancer development is
lacking, in this study, four computational approaches were utilized to test the
hypothesis that microRNAs and their posttranscriptionally regulated mRNA
targets, i.e., both total mRNAs and actively translated mRNA transcripts, are
differentially modulated by carcinogen and diet treatment. Sprague-Dawley rats
were fed diets containing corn oil ± fish oil with pectin ± cellulose and
injected with azoxymethane or saline (control). Colonic mucosa was assayed at an
early time of cancer progression, and global gene set enrichment analysis was
used to obtain those microRNAs significantly enriched by the change in
expression of their putative target genes. In addition, cumulative distribution
function plots and functional network analyses were used to evaluate the impact
of diet and carcinogen combination on mRNA levels induced via microRNA
alterations. Finally, linear discrimit analysis was used to identify the best
single-, two-, and three-microRNA combinations for classifying dietary effects
and colon tumor development. We demonstrate that polysomal profiling is tightly
related to microRNA changes when compared with total mRNA profiling. In
addition, diet and carcinogen exposure modulated a number of microRNAs (miR-16,
miR-19b, miR-21, miR26b, miR27b, miR-93, and miR-203) linked to canonical
oncogenic signaling pathways. Complementary gene expression analyses showed that
oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at
both the mRNA and protein levels. Oncocytoma, chromophobe renal cell carcinoma (chRCC), and the eosinophilic
variant of clear cell RCC (ccRCC) are morphologically similar tumors with
significantly different clinical courses. These renal tumor subtypes show
characteristic structural genetic changes; however, the mRNA expression patterns
of oncocytoma and chRCC are strikingly similar. MicroRNAs (miRNA) are small RNA
molecules that regulate the expression of many genes and have been shown to be
useful for tumor classification and identification. The miRNA expression was
analyzed from formalin-fixed paraffin-embedded tissue in 5 cases each of
oncocytoma, ccRCC, papillary RCC, chRCC, and 4 normal kidney tissues using
microarrays. Affymetrix single-nucleotide polymorphism arrays were used to
detect chromosomal imbalances in each of the tumors. Eighteen miRNAs were
significantly different among the 4 tumor types. The microRNA miR-21, a known
oncogenic miRNA, was found to be upregulated in papillary and clear cell
carcinomas. Four miRNAs could differentiate oncocytomas from chRCCs and the 3
could differentiate papillary RCC from ccRCC, including miR-126, a known
vasculogenic miRNA. Of the 18 differentially expressed miRNAs, only 2 correlated
with copy number changes in the chromosomal region harboring these genes. One
tumor, originally diagnosed as an oncocytoma by morphology, showed a virtual
karyotype and miRNA expression pattern consistent with chromophobe carcinoma.
Further investigation of the tumor showed vascular invasion. Our study suggests
that miRNA expression can be used to differentiate the common subtypes of renal
epithelial neoplasms but further validation is necessary. In addition, the lack
of correlation between miRNA expression and virtual karyotype suggests a
non-copy-number-related mechanism for miRNA gene expression regulation in renal
neoplasia. MicroRNA 21 (miR-21) is overexpressed in virtually all types of carcinomas and
various types of hematological maligcies. To determine whether miR-21
promotes tumor development in vivo, we knocked out the miR-21 allele in mice. In
response to the 7,12-dimethylbenz[a]anthracene
(DMBA)/12-O-tetradecanoylphorbol-13-acetate mouse skin carcinogenesis protocol,
miR-21-null mice showed a significant reduction in papilloma formation compared
with wild-type mice. We revealed that cellular apoptosis was elevated and cell
proliferation was decreased in mice deficient of miR-21 compared to wild-type
animals. In addition, we found that a large number of validated or predicted
miR-21 target genes were up-regulated in miR-21-null keratinocytes, which are
precursor cells to skin papillomas. Specifically, up-regulation of Spry1, Pten,
and Pdcd4 when miR-21 was ablated coincided with reduced phosphorylation of ERK,
AKT, and JNK, three major downstream effectors of Ras activation that plays a
predomit role in DMBA-initiated skin carcinogenesis. These results provide in
vivo evidence that miR-21 exerts its oncogenic function through negatively
regulating its target genes. Maligt gliomas are the most common and lethal primary intracranial tumors. To
date, no reliable biomarkers for the detection and risk stratification of
gliomas have been identified. Recently, we demonstrated significant levels of
microRNAs (miRNAs) to be present in cerebrospinal fluid (CSF) samples from
patients with primary CNS lymphoma. Because of the involvement of miRNA in
carcinogenesis, miRNAs in CSF may serve as unique biomarkers for minimally
invasive diagnosis of glioma. The objective of this pilot study was to identify
differentially expressed microRNAs in CSF samples from patients with glioma as
potential novel glioma biomarkers. With use of a candidate approach of miRNA
quantification by reverse-transcriptase polymerase chain reaction (qRT-PCR),
miRNAs with significant levels in CSF samples from patients with gliomas were
identified. MiR-15b and miR-21 were differentially expressed in CSF samples from
patients with gliomas, compared to control subjects with various neurologic
disorders, including patients with primary CNS lymphoma and carcinomatous brain
metastases. Receiver-operating characteristic analysis of miR-15b level revealed
an area under the curve of 0.96 in discriminating patients with glioma from
patients without glioma. Moreover, inclusion of miR-15b and miR-21 in combined
expression analyses resulted in an increased diagnostic accuracy with 90%
sensitivity and 100% specificity to distinguish patients with glioma from
control subjects and patients with primary CNS lymphoma. In conclusion, the
results of this pilot study demonstrate that miR-15b and miR-21 are markers for
gliomas, which can be assessed in the CSF by means of qRT-PCR. Accordingly,
miRNAs in the CSF have the potential to serve as novel biomarkers for the
detection of gliomas. Although microRNA-21 (miR-21) is emerging as an oncogene and has been shown to
target several tumor suppressor genes, including programmed cell death 4
(PDCD4), its precise mechanism of action on cancer stem cells (CSCs) is unclear.
Herein, we report that FOLFOX-resistant HCT-116 and HT-29 cells that are
enriched in CSCs show a 3- to 7-fold upregulation of pre- and mature miR-21 and
downregulation of PDCD4. Likewise, overexpression of miR-21 in HCT-116 cells,
achieved through stable transfection, led to the downregulation of PDCD4 and
transforming growth factor beta receptor 2 (TGFβR2). In contrast, the levels of
β-catenin, TCF/LEF activity and the expression of c-Myc, Cyclin-D, which are
increased in CSCs, are also augmented in miR-21 overexpressing colon cancer
cells, accompanied by an increased sphere forming ability in vitro and tumor
formation in SCID mice. Downregulation of TGFβR2 could be attributed to
decreased expression of the receptor as evidenced by reduction in the activity
of the luciferase gene construct comprising TGFβR2-3' untranslated region (UTR)
sequence that binds to miR-21. Moreover, we observed that downregulation of
miR-21 enhances luciferase-TGFβR2-3' UTR activity suggesting TGFβR2 as being one
of the direct targets of miR-21. Further support is provided by the observation
that transfection of TGFβR2 in HCT-116 cells attenuates TCF/LEF luciferase
activity, accompanied by decreased expression of β-catenin, c-Myc and Cyclin-D1.
Our current data suggest that miR-21 plays an important role in regulating
stemness by modulating TGFβR2 signaling in colon cancer cells. Gastric carcinogenesis is a multistep process involving genetic and epigenetic
alteration of protein-coding proto-oncogenes and tumor-suppressor genes.
Microribonucleic acids (miR) are a recently-described class of genes encoding
small non-coding RNA molecules, which primarily act by downregulating the
translation of target mRNA. It has become apparent that miR are also key factors
in cancer, playing both oncogenic and tumor-suppressing roles in gastric cancer.
Recent studies have shown that a substantial number of miR show differential
expression in gastric cancer tissues, and they are turning out to be just like
any other regulatory gene. In this connection, miR dysregulation are reported to
be associated with incidence, early diagnosis and prognosis of gastric cancer.
Therefore, investigation of the biological aspects of miR dysregulation might
help us better understand the pathogenesis of gastric cancer and promote the
development of miR-directed therapeutics against this deadly disease. The aim of
the present review was to describe the mechanisms of several known miR,
summarize recent studies on oncogenic miR (e.g. miR-21, miR-106a and miR-17),
tumor suppressor miR (e.g. miR-101, miR-181, miR-449, miR-486, let-7a) and
controversial roles of miR (e.g. miR-107, miR-126) for gastric cancer. In
addition, their potential clinical applications and prospects in gastric cancer,
such as biomarkers and clinical therapy tools, are also briefly discussed. Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of
B cell cancers and lymphoproliferative disorders. During latent infection, EBV
expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific
host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral
oncogenesis. To date, only a limited number of EBV miRNA targets have been
identified; thus, the role of EBV miRNAs in viral pathogenesis and/or
lymphomagenesis is not well defined. Here, we used photoactivatable
ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined
with deep sequencing and computational analysis to comprehensively examine the
viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid
cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular
3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24
PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays.
Our results reveal that EBV miRNAs predomitly target cellular transcripts
during latent infection, thereby manipulating the host environment. Furthermore,
targets of EBV miRNAs are involved in multiple cellular processes that are
directly relevant to viral infection, including innate immunity, cell survival,
and cell proliferation. Finally, we present evidence that myc-regulated host
miRNAs from the miR-17/92 cluster can regulate latent viral gene expression.
This comprehensive survey of the miRNA targetome in EBV-infected B cells
represents a key step towards defining the functions of EBV-encoded miRNAs, and
potentially, identifying novel therapeutic targets for EBV-associated
maligcies. Substantial evidence indicates that microRNA-21 (miR-21) is a key oncomiR in
carcinogenesis and is significantly elevated in multiple myeloma (MM). In this
study, we explored the role of miR-21 in human MM cells and searched for miR-21
targets. By knocking down the expression of endogenous miR-21 in U266 myeloma
cells, we observed reduced growth, an arrested cell cycle, and increased
apoptosis. To further understand its molecular mechanism in the pathogenesis of
MM, we employed a SILAC (stable isotope labeling by amino acids in cell
culture)-based quantitative proteomic strategy to systematically identify
potential targets of miR-21. In total, we found that the expression of 178
proteins was up-regulated significantly by miR-21 inhibition, implying that they
could be potential targets of miR-21. Among these, the protein inhibitor of
activated STAT3 (PIAS3) was confirmed as a direct miR-21 target by Western
blotting and reporter gene assays. We further demonstrated that miR-21 enhances
the STAT3-dependent signal pathway by inhibiting the function of PIAS3 and that
down-regulation of PIAS3 contributes to the oncogenic function of miR-21. This
elucidation of the role of PIAS3 in the miR-21-STAT3 positive regulatory loop
not only may shed light on the molecular basis of the biological effects of
miR-21 observed in MM cells but also has direct implications for the development
of novel anti-MM therapeutic strategies. Sprouty2 (Spry2) was identified recently as a tumor suppressor gene in cancer
cells which inhibits the activation of receptor tyrosine kinases (RTKs). The
present study explored the effect of Spry2 in colon cancer cells in order to
assess its potential use in the treatment of colon cancer. Expression of Spry2
inhibited the growth of a colon cancer cell line, HCT116, and induced
sensitization to fluorouracil (5-FU) and metformin. Spry2 promoted apoptosis of
cancer cells in association with activation of the phosphatase and tensin
homolog deleted on chromosome 10 (PTEN) pathway and the blockade of Ras-Raf-Erk
signaling. Treatment of Spry2-HCT116 cells with metformin resulted in a more
prominent effect on the inhibition of cell migration. Inhibition of microRNA-21
(mir‑21) induced upregulation of Spry2 and PTEN which underscores the importance
of mir-21 in Spry2-associated tumorigenesis of the colon. These results point
toward a potential strategy for colon cancer treatment worthy of further
investigation. The detection of circulating tumor cells (CTCs) has received great attention.
MicroRNA-21 (miR-21) plays crucial roles in carcinogenesis and is considered as
one of the most studied oncomiRNAs. We determined if miR-21 could be used a
marker for the detection of CTCs in gastric cancer patients. Peripheral blood
samples were collected from 53 preoperative patients with gastric cancer and 20
healthy volunteers. Real-time reverse transcription-polymerase chain reaction
was used to detect the level of miR-21. Receiver operator characteristic curves
(ROC) were constructed. Patients with gastric cancer display a significantly
higher level of miR-21 in peripheral blood than those from controls. The miR-21
level was associated with the tumor node metastasis (TNM) stage, tumor size and
tissue categories. The area under ROC curve was up to 0.853 ± 0.086. This study
highlights the potential of the detection of miR-21 in peripheral blood as a
novel tool for monitoring CTCs in gastric cancer patients. PURPOSE: MicroRNA-21 (miR-21) is one of the miRNAs that are frequently and
highly overexpressed in tumor tissue of colorectal cancer (CRC) patients;
however, only a little is known about its functional role in CRC.
METHODS: We examined the expression level of miR-21 in 44 paired samples of
tumoral and non-tumoral colon tissues diagnosed for CRC using TaqMan real-time
PCR method. Furthermore, we used miR-21 inhibitor (anti-miR-21) to transient
knockdown of miR-21 in DLD-1 colon cancer cells and examined the effects of
miR-21 silencing on viability, apoptosis, chemosensitivity, cell cycle, and
migration of DLD1 cells.
RESULTS: The expression levels of miR-21 were significantly increased in CRC
tumor tissue (P < 0.0001). Significant differences in miR-21 levels were
observed also between CRC tissues of patients with CRC in different clinical
stages: I vs. II (P = 0.033) and I vs. IV (P = 0.021). Kaplan-Meier analysis
proved that the miR-21 expression levels are correlated to shorter overall
survival of CRC patients (P = 0.0341). MiR-21 silencing in DLD1 cell line had no
effect on the cell viability; however, when combined with chemotherapeutics
(5-FU, L-OHP, and SN38), it contributed to the decrease of cell viability.
Suppression of miR-21 decreased cell migration ability of DLD-1 cells by nearly
30 % (P = 0.016).
CONCLUSION: We have confirmed the overexpression of miR-21 in CRC samples and
its correlation with advanced disease and shorter overall survival. These
findings could be described in part by the fact that CRC cells with increased
expression of miR-21 have higher migration ability. 1. MicroRNAs (miRNAs) have been shown to play critical roles in regulating the
progress of leukemia. We performed miRNA expression profile in six Chinese
patients with chronic lymphocytic leukemia (CLL), and in peripheral B cells from
pooled 30 healthy donors, using a platform containing 866 human miRNAs. The most
frequent changes in miRNAs in CLL cells included downregulation of miR-126,
miR-572, miR-494, miR-923, miR-638, miR-130a, miR-181a and miR-181b and
up-regulation of miR-29a, miR-660, miR-20a, miR-106b, miR-142-5p, miR-101,
miR-30b, miR-34a, miR-let-7f, miR-21 and miR-155. Among the miRNAs
down-regulated in CLL cells, we showed that miR-181a/b expression levels were
significantly lower in poor prognostic subgroups defined by unmutated
immunoglobulin heavy chain variable status and p53 aberrations. Furthermore,
under-expression of miR-181a and miR-181b was associated with shorter overall
survival and treatment-free survival in CLL patients. We further evaluated
fludarabine-induced apoptosis after transfection of primary CLL cells from 40
patients with miR-15a, miR-16-1, miR-34a, miR-181a and miR-181b mimics.
Transfection of miR-34a, miR-181a and miR-181b mimics into CLL cells from p53
wild-type patients led to significant increase in apoptosis compared with miRNA
control. However, enforced expression of these miRNAs had no effect on B-CLL
cells from p53-attenuated patients. We further demonstrated that miR-181a and
miR-181b inhibiting BCL-2, MCL-1 and X-linked inhibitor of apoptosis protein by
direct binding to 3'UTR. Thus, these results suggest that miR-181a/b may play
important roles in the pathogenesis of CLL and may provide a possible
therapeutic avenue and a sensitive indicator of the activity of the p53 axis in
CLL. Mutant KRAS in lung cancers induces molecular pathways that regulate cellular
proliferation, survival and inflammation, which enhance tumorigenesis. Inducible
nitric oxide synthase (NOS2) upregulation and sustained nitric oxide generation
are induced during the inflammatory response and correlate positively with lung
tumorigenesis. To explore the mechanistic contribution of NOS2 to KRAS-induced
lung tumorigenesis and inflammation, we used a genetic strategy of crossing NOS2
knockout (NOS2KO) C57BL6 inbred mice with a KRAS(G12D)-driven mouse lung cancer
model. KRAS(G12D);NOS2KO mice exhibited delayed lung tumorigenesis and a longer
overall survival time compared to that of KRAS(G12D);NOS2WT (wild-type)
controls. Correspondingly, tumors in KRAS(G12D);NOS2KO mice had reduced tumor
cell proliferation in adenomas and carcinomas. NOS2 deficiency also led to
markedly suppressed inflammatory response by attenuation of macrophage
recruitment into alveoli and within tumor foci. In contrast, FOXP3+ regulatory T
cells were increased in tumors from KRAS(G12D) ;NOS2KO mice. We further analyzed
the expression of microRNA-21 (miR-21), an oncogenic noncoding RNA involved in
oncogenic Ras signaling, by quantitative reverse-transcription polymerase chain
reaction and in situ hybridization. Lung carcinomas dissected from
KRAS(G12D);NOS2KO mice showed a significantly reduced miR-21 expression along
with decreased tumor cell proliferation, suggesting that NOS2 deficiency could
attenuate RAS signaling pathways that transactivate miR-21 expression.
Therefore, deletion of NOS2 decreases lung tumor growth as well as inflammatory
responses initiated by oncogenic KRAS, suggesting that both KRAS and NOS2
cooperate in driving lung tumorigenesis and inflammation. Inhibition of NOS2 may
have a therapeutic value in lung cancers with oncogenic KRAS mutations. Increasing evidence shows the beneficial effects of fish oil on breast cancer
growth and invasion in vitro and in animal models. Expression of CSF-1 (colony
stimulating factor-1) by breast cancer cells acts as potent activator of
maligcy and metastasis. In this report, we used two human breast cancer cell
lines, MDA-MB-231 and MCF-7, to show that the bioactive fish oil component DHA
(docosahexaenoic acid) inhibits expression of CSF-1 and its secretion from these
cancer cells. We found that the tumor suppressor protein PTEN regulates CSF-1
expression through PI 3 kinase/Akt signaling via a transcriptional mechanism.
The enhanced abundance of microRNA-21 (miR-21) in breast cancer cells
contributes to the growth and metastasis. Interestingly, DHA significantly
inhibited expression of miR-21. miR-21 Sponge, which derepresses the miR-21
targets, markedly decreased expression of CSF-1 and its secretion. Furthermore,
miR-21-induced upregulation of CSF-1 mRNA and its transcription were prevented
by expression of PTEN mRNA lacking 3'-untranslated region (UTR) and miR-21
recognition sequence. Strikingly, miR-21 reversed DHA-forced reduction of CSF-1
expression and secretion. Finally, we found that expression of miR-21 as well as
CSF-1 was significantly attenuated in breast tumors of mice receiving a diet
supplemented with fish oil. Our results reveal a novel mechanism for the
therapeutic function of fish oil diet that blocks miR-21, thereby increasing
PTEN levels to prevent expression of CSF-1 in breast cancer. Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma
(ESCC). In a rat model, chronic ZD induces an inflammatory gene signature that
fuels ESCC development. microRNAs regulate gene expression and are aberrantly
expressed in cancers. Here we investigated whether chronic ZD (23 weeks) also
induces a protumorigenic microRNA signature. Using the oString technology, we
evaluated microRNA profiles in ZD esophagus and six additional tissues (skin,
lung, pancreas, liver, prostate and peripheral blood mononuclear cells [PBMC]).
ZD caused overexpression of inflammation genes and altered microRNA expression
across all tissues analyzed, predictive of disease development. Importantly, the
inflammatory ZD esophagus had a distinct microRNA signature resembling human
ESCC or tongue SCC miRNAomes with miR-31 and miR-21 as the top-up-regulated
species. Circulating miR-31 was also the top-up-regulated species in PBMCs. In
ZD esophagus and tongue, oncogenic miR-31 and miR-21 overexpression was
accompanied by down-regulation of their respective tumor-suppressor targets
PPP2R2A and PDCD4. Importantly, esophageal miR-31 and miR-21 levels were
directly associated with the appearance of ESCC in ZD rats, as compared with
their cancer-free Zn-sufficient or Zn-replenished counterparts. In situ
hybridization analysis in rat and human tongue SCCs localized miR-31 to tumor
cells and miR-21 to stromal cells. In regressing tongue SCCs from
Zn-supplemented rats, miR-31 and miR-21 expression was concomitantly reduced,
establishing their responsiveness to Zn therapy. A search for putative microRNA
targets revealed a bias toward genes in inflammatory pathways. Our finding that
ZD causes miR-31 and miR-21 dysregulation associated with inflammation provides
insight into mechanisms whereby ZD promotes ESCC. BACKGROUND: MicroRNAs (miRNAs) are involved in carcinogenesis and tumor
progression by regulating post-transcriptional gene expression. However, the
miRNA-mRNA regulatory network is far from being fully understood. The objective
of this study is to identify the colorectal cancer (CRC) specific miRNAs and
their target mRNAs using a multi-step approach.
RESULTS: A multi-step approach combining microarray miRNA and mRNA expression
profile and bioinformatics analysis was adopted to identify the CRC specific
miRNA-mRNA regulatory network. First, 32 differentially expressed miRNAs and
2916 mRNAs from CRC samples and their corresponding normal epithelial tissues
were identified by miRNA and mRNA microarray, respectively. Secondly, 22
dysregulated miRNAs and their 58 target mRNAs (72 miRNA-mRNA pairs) were
identified by a combination of Pearson's correlation analysis and prediction by
databases TargetScan and miRanda. Bioinformatics analysis revealed that these
miRNA-mRNAs pairs were involved in Wnt signaling pathway. Additionally, 6
up-regulated miRNAs (mir-21, mir-223, mir-224, mir-29a, mir-29b, and mir-27a)
and 4 down-regulated predicted target mRNAs (SFRP1, SFRP2, RNF138, and KLF4)
were selected to validate the expression level and their anti-correlationship in
an extended cohort of CRC patients by qRT-PCR. Except for mir-27a, the
differential expression and their anti-correlationship were proven. Finally, a
transfection assay was performed to validate a regulatory relationship between
mir-29a and KLF4 at both RNA and protein levels.
CONCLUSIONS: Seventy-two miRNA-mRNA pairs combined by 22 dysregulated miRNAs and
their 58 target mRNAs identified by the multi-step approach appear to be
involved in CRC tumorigenesis. The results in our study were worthwhile to
further investigation via a functional study to fully understand the underlying
regulatory mechanisms of miRNA in CRC. BACKGROUND AND PURPOSE: As an important oncogenic miRNA, miR-21 has been
reported to play crucial roles in glioblastoma (GBM) carcinogenesis. However,
the precise biological function and molecular mechanism of miR-21 in GBM remain
elusive. This study is designed to explore the mechanism of miR-21 involved in
the control of GBM cell growth.
METHODS AND RESULTS: MTT assay, cell cycle analysis, and apoptosis analysis
showed that reduction of miR-21 inhibited cell growth in U87 and LN229 GBM
cells. Further, reduction of miR-21 decreased the expression of human telomerase
reverse transcriptase (hTERT) and repressed STAT3 expression and STAT3
phosphorylation. STAT3 inhibition led to a remarkable depletion of hTERT at both
mRNA and protein levels by binding to the hTERT gene promoter by performing
luciferase reporter assay and chromatin Immunoprecipitation PCR. Finally,
knockdown of miR-21 considerably inhibited tumor growth and diminished the
expression of STAT3 and hTERT in xenograft model.
CONCLUSION: Our findings indicate that miR-21 regulates hTERT expression
mediated by STAT3, therefore controlling GBM cell growth. BACKGROUND: MicroRNAs (miRNAs) are involved in the pathogenesis of human
cancers, including medullary thyroid carcinoma (MTC). The aim of this study was
to test the hypothesis that different miRNA profiles are related to RET status
and prognosis in patients with hereditary MTC (hMTC) and sporadic MTC (sMTC).
METHODS: We analyzed the expression of nine miRNAs (miR-21, miR-127, miR-154,
miR-224, miR-323, miR-370, miR-9*, miR-183, and miR-375) by quantitative
real-time-polymerase chain reaction in 34 cases of sMTC, 6 cases of hMTC, and 2
cases of C-cell hyperplasia (CCH). We also analyzed the immunohistochemical
expression of PDCD4, an miR-21 gene target. sMTC (n=34) was genotyped for
somatic RET and RAS mutations. Disease status was defined on the basis of the
concentration of serum calcitonin at the latest follow-up and other parameters
as indicated in the results.
RESULTS: MTC and CCH were both characterized by a significant overexpression of
the whole set of miRNAs (the increase being 4.2-fold for miR-21, 6.7-fold for
miR-127, 8.8-fold for miR-154, 6.6-fold for miR-224, 5.8-fold for miR-323,
6.1-fold for miR-370, 13-fold for miR-9*, 6.7-fold for miR-183, and 10.1 for
miR-375, p<0.0001). PDCD4 expression was significantly downregulated in MTC
samples, consistent with miR-21 upregulation. Significantly lower miR-127 levels
were observed in sMTC carrying somatic RET mutations in comparison to sMTC
carrying a wild-type RET. In sMTC and familial MTC, the miR-224 upregulation
correlated with the absence of node metastases, lower stages at diagnosis, and
with biochemical cure during follow-up.
CONCLUSIONS: miRNAs are significantly dysregulated in MTC, and this
dysregulation is probably an early event in C-cell carcinogenesis. miR-224
upregulation could represent a prognostic biomarker associated with a better
outcome in MTC patients. BACKGROUND: MicroRNAs (miRNAs), endogenous small non-coding RNAs, are stably
detected in human plasma. Early diagnosis of gastric cancer (GC) is very
important to improve the therapy effect and prolong the survival of patients. We
aimed to identify whether four miRNAs (miR-223, miR-21, miR-218 and miR-25)
closely associated with the tumorigenesis or metastasis of GC can serve as novel
potential biomarkers for GC detection.
METHODOLOGY: We initially measured the plasma levels of the four miRNAs in 10 GC
patients and 10 healthy control subjects by quantitative reverse transcription
polymerase chain reaction (qRT-PCR), and then compared plasma miRNA results with
the expressions in cancer tissues from eight GC patients. Finally, the presence
of miR-223, miR-21 and miR-218 in the plasma was validated in 60 GC patients and
60 healthy control subjects, and the areas under the receiver operating
characteristic (ROC) curves of these miRNAs were analyzed.
RESULTS: We found that the plasma levels of miR-223 (P<0.001) and miR-21
(P<0.001) were significantly higher in GC patients than in healthy controls,
while miR-218 (P<0.001) was significantly lower. The ROC analyses yielded the
AUC values of 0.9089 for miR-223, 0.7944 for miR-21 and 0.7432 for miR-218, and
combined ROC analysis revealed the highest AUC value of 0.9531 in discriminating
GC patients from healthy controls. Moreover, the plasma levels of miR-223
(P<0.001) and miR-21 (P = 0.003) were significantly higher in GC patients with
stage I than in healthy controls. Furthermore, the plasma levels of miR-223 were
significantly higher in GC patients with helicobacter pylori (Hp) infection than
those without (P = 0.014), and significantly higher in healthy control subjects
with Hp infection than those without (P = 0.016).
CONCLUSIONS: Plasma miR-223, miR-21 and miR-218 are novel potential biomarkers
for GC detection. BACKGROUND: Cellular senescence can be a functional barrier to carcinogenesis.
We hypothesized that inflammation modulates carcinogenesis through senescence
and DNA damage response (DDR). We examined the association between senescence
and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro
experiments tested the ability of macrophages to induce senescence in primary
cells. Inflammation modulating microRNAs were identified in senescence colon
tissue for further investigation.
METHODOLOGY/PRINCIPAL FINDINGS: Quantitative immunohistochemistry identified
protein expression by colon cell type. Increased cellular senescence (HP1γ; P =
0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with
high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced
senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that
macrophages induce senescence. Interestingly, macrophage-induced senescence was
partly dependent on nitric oxide synthase, and clinically relevant NO• levels
alone induced senescence. NO• induced DDR in vitro, as detected by
immunofluorescence. In contrast to UC, we noted in Crohn's disease (CD) that
senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were
higher, and macrophages were not associated with senescence. We hypothesize that
nitric oxide may modulate senescence in CD; epithelial cells of CD had higher
levels of NOS2 expression than in UC (P = 0.001). Microarrays and
quantitative-PCR identified miR-21 expression associated with macrophage
infiltration and NOS2 expression.
CONCLUSIONS: Senescence was observed in IBD with senescence-associated
β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in
UC, and in vitro experiments with primary human cells showed that macrophages
induce senescence, partly through NO•, and that NO• can induce DDR associated
with senescence. Future experiments will investigate the role of NO• and miR-21
in senescence. This is the first study to implicate macrophages and nitrosative
stress in a direct effect on senescence and DDR, which is relevant to many
diseases of inflammation, cancer, and aging. Considering the high mortality rates and the unfavorable prognosis of gastric
cancer (GC) as well as the lack of a clinical predictive marker, which is
sufficiently sensitive to GC, it is of great significance to investigate new
sensitive and specific markers for GC diagnosis. MicroRNAs (miRNAs) could be a
practical form of potential biomarkers in the diagnosis of human disease, and
they are confirmed to be closely associated with GC. In this review, we discuss
the recent research results that indicate the feasibility and clinical
applications of miRNAs in GC. Although several challenges remain to be
addressed, miRNAs have the potential to be applied in the diagnosis of GC. Both the morphogenesis and the molecular pathways of thyroid cancers are
controversial. Programmed cell death 4 (PDCD4) is a tumor suppressor gene whose
expression is controlled by miR-21. By applying immunohistochemistry for PDCD4
and both quantitative real-time PCR (qRT-PCR) and in situ hybridization for
miR-21, this study explored PDCD4 expression in human follicular-cell-derived
thyroid neoplastic lesions. PDCD4 protein expression was semiquantitatively
assessed in 100 consecutive thyroid tumors (25 follicular adenomas (FA), 25
follicular carcinomas (FC), 25 papillary carcinomas (PC), and 25
poorly-differentiated/anaplastic cancers (PD-AC)). Twenty-five additional
nonneoplastic thyroid tissue samples were included as controls. To further
support the data, miR-21 expression was tested (by qRT-PCR and in situ
hybridization) in a different series of 75 cases (15 FAs, 15 FCs, 15 PCs, 15
PD-ACs, and 15 controls). Nonneoplastic thyrocytes consistently featured a
strong nuclear PDCD4 expression, while the protein's expression was
significantly downregulated in neoplastic epithelia. PDCD4 downregulation was
significantly associated with less well-differentiated cancer phenotypes
(p < 0.001) and more advanced tumor stages (p < 0.001). Consistently with PDCD4
downregulation, miR-21 was upregulated in neoplastic by comparison with
nonneoplastic tissue samples. The present results provide evidence of PDCD4
having a role in thyroid carcinogenesis; further studies should investigate the
diagnostic value and the prognostic impact of PDCD4 in thyroid neoplasia. Inflammatory bowel diseases (IBDs; both ulcerative colitis [UC] and Crohn's
colitis [CC]) are well-established predisposing pathological conditions for
colorectal cancer (CRC) development. In IBDs, both the endoscopy and the
histology assessment of CRC precursors (i.e., dysplasia, also defined as
intraepithelial neoplasia) are associated with low interobserver consistency,
and no reliable dysplasia-specific biomarker is available. The programmed cell
death 4 (PDCD4) tumor suppressor gene is involved in sporadic colorectal
oncogenesis, but scanty information is available on its involvement in
IBD-associated colorectal oncogenesis. One hundred twenty tissue samples
representative of active and inactive IBD and of flat dysplasia were obtained
from 30 cases of UC and 30 of CC who undergone colectomy. Twenty additional
biopsy samples obtained from patients with irritable bowel syndrome acted as
normal controls. PDCD4 expression was assessed by immunohistochemistry; the
expression of miR-21 (a major PDCD4 regulator) was investigated by quantitative
real-time PCR and in situ hybridization in different series of a hundred
samples. Tissue specimens from both controls and inactive IBD consistently
featured strong PDCD4 nuclear immunostain; conversely, lower PDCD4 nuclear
expression was featured by both active IBD and IBD-associated dysplastic
lesions. Significant PDCD4 down-regulation distinguished IBD-associated
dysplasia (p < 0.001) versus active IBD. In both active IBD and dysplasia, PDCD4
down-regulation was significantly associated with miR-21 up-regulation. PDCD4
nuclear down-regulation (which parallels miR-21 up-regulation) is involved in
the molecular pathway of IBD-associated carcinogenesis. PDCD4 nuclear expression
may be usefully applied as ancillary maker in the histological assessment of
IBD-associated dysplastic lesions. BACKGROUND: We investigated the effect of corn-derived biodiesel glycerol on
microRNAs (miRNAs) and mRNAs, which play a central role in regulating cell
survival, apoptosis and carcinogenesis.
MATERIALS AND METHODS: Inbred Balb/c mice were treated with purified glycerol
from biodiesel for 24 hours. After administration, we determined the expressions
of miR-21, miR-27a, miR-34a, miR-93, miR-143, miR-146a, miR-148a, miR-155,
miR-196a, miR-203, miR-205, miR-221 and nuclear factor kappa-light-chain
enhancer of activated B-cells-1 (Nfκb1), mitogen-activated protein kinase-8
(Mapk8) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-ras) genes
in the liver of mice.
RESULTS: We found a parallel altered expression of miRNAs and mRNAs in animals
consuming biodiesel glycerol that compared to control mice; these alterations
reached significant levels only in few cases.
CONCLUSION: Biodiesel glycerol presents no higher risk for carcinogenicity or
toxicity. BACKGROUND: microRNA expression profile analysis provides evaluation of the
early stages of carcinogenesis. This study focuses on early alteration of miRNA
expression after treatment with different carcinogens.
MATERIALS AND METHODS: Mice were intraperitoneally injected with one dose of
7,12-dimethylbenz(α)anthracene (DMBA) and N-methyl-N-nitrosourea (MNU). The
expression of miRNAs were analyzed 3 and 6 hours after the treatment, using
quantitative real-time-polymerase chain reaction.
RESULTS: Underexpression of miR-34a and miR-155 were detected in the liver,
spleen and kidneys at 3 and 6 hours after MNU treatment. In the lungs and
kidneys, the expression of miR-21 was significantly elevated 6 hours after DMBA
treatment, while in the liver, MNU induced higher expression levels of miR-21 at
3 and 6 hours compared to treatment with DMBA.
CONCLUSION: The different response of miRNAs to carcinogens emphasizes their
possible role as potential epidemiological biomarkers in early phases of
environmental tumorigenesis. The molecular mechanisms by which arsenic (As ( 3+) ) causes human cancers
remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the
catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes
trimethylation of lysine 27 of histone H3, leading to altered expression of
tumor suppressors or oncogenes. In the present study, we determined the effect
of As ( 3+) on EZH2 phosphorylation and the signaling pathways important for As
( 3+) -induced EZH2 phosphorylation in human bronchial epithelial cell line
BEAS-2B. The involvement of kinases in As ( 3+) -induced EZH2 phosphorylation
was validated by siRNA-based gene silencing. The data showed that As ( 3+) can
induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells
and that the phosphorylation of EZH2 requires an As ( 3+) -activated signaling
cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific
for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3,
Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream
kinase involved in As ( 3+) -induced EZH2 phosphorylation. Because As ( 3+) is
capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses
protein translation of PTEN or Spry2, we also tested the role of STAT3 and
miR-21 in As ( 3+) -induced EZH2 phosphorylation. Ectopic overexpression of
miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting
miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and
EZH2 phosphorylation. Taken together, these results demonstrate a contribution
of the JNK, STAT3 and Akt signaling axis to As ( 3+) -induced EZH2
phosphorylation. Importantly, these findings may reveal new molecular mechanisms
underlying As ( 3+) -induced carcinogenesis. Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency is
reported in 5-10% of colorectal cancers (CRCs) complicating inflammatory bowel
diseases (IBD). The molecular mechanisms underlying MMR deficiency may be
different in IBD CRCs, and in sporadic and hereditary MSI tumors. Here, we
hypothesize that overexpression of miR-155 and miR-21, two inflammation-related
microRNAs that target core MMR proteins, may constitute a pre-neoplastic event
for the development of MSI IBD CRCs. We studied miR-155 and miR-21 expression
using real-time quantitative PCR in MSI (n = 10) and microsatellite stable (n =
10) IBD CRCs, and in MSI (n = 32) and microsatellite stable (n = 30) non-IBD
CRCs. We also screened colonic samples from IBD patients without cancer (n = 18)
and used healthy colonic mucosa as controls (n = 20). MiR-155 and miR-21
appeared significantly overexpressed not only in the colonic mucosa of IBD
subjects without CRC but also in neoplastic tissues of IBD patients compared
with non-IBD controls (P < 0.001). Importantly, in patients with IBD CRCs,
miR-155 and miR-21 overexpression extended to the distant non-neoplastic mucosa
(P < 0.001). Ratios of expressions in tumors versus matched distant mucosa
revealed a nearly significant association between miR-155 overexpression and MSI
in IBDs (P = 0.057). These results show a strong deregulation of both
MMR-targeting microRNAs in IBD subjects with or without cancer. MiR-155
overexpression being particularly associated to MSI IBD CRCs and extending to
distant non-neoplastic mucosa, strongly suggests that a pre-neoplastic miR-155
field defect may promote MSI-driven transformation of the colonic mucosa. The
detection and monitoring of miR-155 field defect may, therefore, have
implications for the prevention and treatment of MSI IBD CRCs. BACKGROUND: MicroRNAs (miRNAs) play a crucial role in carcinogenesis; however,
it largely remains unclear whether miRNAs in gastric juice, which is specific
for gastric tissues, can be used as biomarkers for gastric cancer. The objective
of the current study was to investigate the feasibility of using gastric juice
miRNAs as potential biomarkers to assist in screening for gastric cancer.
METHODS: Gastric juice samples were collected from 141 patients who underwent
upper gastrointestinal endoscopy examination between September 2010 and December
2011. Gastric cancer and adjacent normal biopsy specimens also were collected.
The existence and stability of miRNAs in gastric juices were determined by
real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)
and sequencing. miRNA levels in tissues and gastric juices were detected by
RT-qPCR. A receiver operating characteristic (ROC) curve was constructed for
differentiating gastric cancer from benign gastric diseases.
RESULTS: Levels of miRNA-21 (miR-21) and miR-106a in gastric cancer tissues were
significantly higher compared with the levels in adjacent tissues (P = .006 and
P = .001, respectively). Patients who had gastric cancer had significantly
different levels of gastric juice miR-21 and miR-106a compared with patients who
had benign gastric diseases (both P < .001). There were significant correlations
between miR-21/miR-106a levels and Borrmann types. miR-21 levels in intestinal
type gastric cancer specimens were higher than that in diffuse (P = .003) or
mixed (P < .001) gastric cancer types. The area under the ROC curve was up to
0.969 for miR-21 and 0.871 for miR-106a.
CONCLUSIONS: The current results indicated that certain miRNAs in gastric juice
are potential biomarkers that can assist in screening for gastric cancer. OBJECTIVE: Alterations in microRNA (miRNA) expression have been described in
thyroid tumors, suggesting a role for miRNAs in thyroid carcinogenesis.
BRAF(V600E) is the most frequently identified genetic alteration in papillary
thyroid carcinoma (PTC). We investigated the link between BRAF(V600E) status and
the expression of miRNAs in PTC and analyzed the associations of these factors
with clinicopathological characteristics.
DESIGN AND METHODS: Prospective study of patients who underwent thyroid surgery
between October 8, 2008 and November 1, 2010. BRAF(V600E) status was determined
by mutant allele-specific amplification PCR and direct sequencing of exon 15 of
the BRAF gene in 69 PTC tissues and 69 respective paracancerous normal thyroid
tissues. Initially, miRNA expression was analyzed in 12 PTC tissues and three
associated paracancerous tissues using a miRNA microarray. miRNAs differentially
expressed between BRAF(V600E)-positive and -negative PTC tissues were then
validated by real-time quantitative PCR on 69 PTC tissues and 69 paracancerous
tissues. We also explored the associations between BRAF(V600E) status or
differential miRNA expression and clinicopathological characteristics.
RESULTS: The mutation rate of BRAF(V600E) in PTC was 47.8%. Twelve miRNAs were
upregulated and six were downregulated in PTC tissues, among which miR-15a,
15a*, 34a*, 34b*, 551b, 873, 876-3p, and 1274a were first identified. miR-21*
and 203 were significantly dysregulated (P<0.05) in PTC tissues with
BRAF(V600E). Additionally, there were significant associations (P<0.05) between
BRAF(V600E) and a higher tumor-node-metastasis staging (III/IV), and between
miR-21* over-expression and lymph node metastasis.
CONCLUSIONS: We identified two miRNAs that are differentially expressed in PTC
tissues with BRAF(V600E) and revealed their associations with
clinicopathological features. These findings may lead to the development of a
potential diagnostic biomarker or prognostic indicator of PTC. MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key
molecule that by its dysregulation plays roles in inflammatory-driven
carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and
its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis
of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA).
The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA
expression assay and immunohistochemistry in liver tissues of both O. viverrini
plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n=23
cases). The functional assay for miR-21 was performed in CCA cell lines by the
anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels
were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini
plus NDMA-induced group and had a reverse response with its target PDCD4
proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared
with nontumor tissues (P=0.0034) and had a negative correlation with PDCD4
protein (P=0.026). It was also found that high expression of miR-21 was
significantly correlated with shorter survival (P<0.05) and lymph node
metastasis (P=0.037) of CCA patients. Transient transfection of pre-miR-21
reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and
wound-induced migration ability. These results indicated that miR-21 plays a
role in the carcinogenesis and metastasis of O. viverrini-associated CCA by
suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may
be a useful strategy to inhibit tumor cell phenotypes or improve response to
chemotherapy. Barrett's esophagus is the precursor lesion of esophageal adenocarcinoma, whose
progression follows sequential stages. However, the low progression rate and the
inadequacy and subjective interpretation of histologic grading in predicting
Barrett's esophagus progression call for more objective biomarkers that can
improve risk prediction. We conducted a genome-wide profiling of 754 human
microRNAs (miRNA) in 35 normal epithelium, 34 Barrett's esophagus, and 36
esophageal adenocarcinoma tissues using TaqMan real-time PCR-based profiling.
Unsupervised hierarchical clustering using 294 modestly to highly expressed
miRNAs showed clear clustering of two groups: normal epithelium versus Barrett's
esophagus/esophageal adenocarcinoma tissues. Moreover, there was an excellent
clustering of Barrett's metaplasia (without dysplasia) tissues from normal
epithelium tissues. However, Barrett's esophagus tissues of different stages and
esophageal adenocarcinoma tissues were interspersed. There were differentially
expressed miRNAs at different stages. The majority of miRNA aberrations involved
upregulation of expression in Barrett's esophagus and esophageal adenocarcinoma
tissues, with the most dramatic alterations occurring at the Barrett's
metaplasia stage. Known oncomiRs, such as miR-21, miR-25, and miR-223, and tumor
suppressor miRNAs, including miR-205, miR-203, let-7c, and miR-133a, showed
progressively altered expression from Barrett's esophagus to esophageal
adenocarcinoma. We also identified a number of novel miRNAs that showed
progressively altered expression, including miR-301b, miR-618, and miR-23b. The
significant miRNA alterations that were exclusive to esophageal adenocarcinoma
but not Barrett's esophagus included miR-375 downregulation and upregulation of
five members of the miR-17-92 and its homologue clusters, which may become
promising biomarkers for esophageal adenocarcinoma development. Despite tremendous efforts worldwide from clinicians and cancer scientists,
pancreatic ductal adenocarcinoma (PDA) remains a deadly disease for which no
cure is available. Recently, microRNAs (miRNAs) have emerged as key actors in
carcinogenesis and we demonstrated that microRNA-21 (miR-21), oncomiR is
expressed early during PDA. In the present study, we asked whether targeting
miR-21 in human PDA-derived cell lines using lentiviral vectors (LVs) may impede
tumor growth. We demonstrated that LVs-transduced human PDA efficiently
downregulated miR-21 expression, both in vitro and in vivo. Consequently, cell
proliferation was strongly inhibited and PDA-derived cell lines died by
apoptosis through the mitochondrial pathway. In vivo, miR-21 depletion stopped
the progression of a very aggressive model of PDA, to induce cell death by
apoptosis; furthermore, combining miR-21 targeting and chemotherapeutic
treatment provoked tumor regression. We demonstrate herein for the first time
that targeting oncogenic miRNA strongly inhibit pancreatic cancer tumor growth
both in vitro and in vivo. Because miR-21 is overexpressed in most human tumors;
therapeutic delivery of miR-21 antagonists may still be beneficial for a large
number of cancers for which no cure is available. Epithelial-mesenchymal transition (EMT) is an essential step for cancer
metastasis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate
target-mRNAs post-transcriptionally. The expression and function of miRNAs in
EMT of HT-29 colonic cells remain elusive. This study looks at expression of
miRNAs in EMT and explores the effects of miRNAs on EMT in HT-29 cell line.
HT-29 was treated with TGF β to establish an EMT model, in which a collection of
miRNAs was dynamically regulated by real-time PCR (qPCR) analysis. Among them,
miR-21 and miR-27 were significantly upregulated, while miR-22, miR-26, miR-30,
miR-181, miR-200b, miR-200c and miR-214 were markedly downregulated.
MiRNA-inhibitors were used to knockdown miRNAs in HT-29 and EMT markers were
determined by qPCR to monitor the effects of miRNAs on EMT process. Results
showed that miR-22 could not alter the expression of EMT markers, while
knockdown of miR-200b could significantly increase that of epithelial markers,
N-cadherin, Vimentin, α-Sma and Twist1 and decrease that of mesenchymal marker,
E-cadherin. Bioinformatic analysis and Western blot showed that ZEB1 was
directly suppressed by miR-200b. In conclusion, miRNAs are dynamically regulated
in TGF β-induced EMT of HT-29 and miR-200b was essential for EMT by suppressing
the expression of ZEB1 in HT-29. |
Which is the most prevalent form of arrhythmia worldwide? | Atrial fibrillation (AF) is the most common arrhythmia worldwide, and it has a significant effect on morbidity and mortality. It is a significant risk factor for stroke and peripheral embolization, and it has an effect on cardiac function. Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting up to 1-1.5% of the population. | Cardiac arrhythmias continue to pose a major medical challenge and significant
public health burden. Atrial fibrillation, the most prevalent arrhythmia,
affects more than two million Americans annually and is associated with a
twofold increase in mortality. In addition, more than 250,000 Americans each
year suffer ventricular arrhythmias, often resulting in sudden cardiac death.
Despite the high incidence and societal impact of cardiac arrhythmias, presently
there are insufficient insights into the molecular mechanisms involved in
arrhythmia generation, propagation, and/or maintece or into the molecular
determits of disease risk, prognosis, and progression. In addition, present
therapeutic strategies for arrhythmia abatement often are ineffective or require
palliative device therapy after persistent changes in the electrical and
conduction characteristics of the heart have occurred, changes that appear to
increase the risk for arrhythmia progression. This article reviews our present
understanding of the complexity of mechanisms that regulate cardiac membrane
excitability and cardiac function and explores the role of derangements in these
mechanisms that interact to induce arrhythmogenic substrates. Approaches are
recommended for future investigations focused on providing new mechanistic
insights and therapeutic interventions. Atrial fibrillation (AF) is associated with increased morbidity and is in
addition the most prevalent cardiac arrhythmia. Compounds used in
pharmacological treatment has traditionally been divided into Na(+) channel
inhibitors, β-blockers, K(+) channel inhibitors, and Ca(2+) channel inhibitors,
whereas newer multichannel blockers such as amiodarone and ranolazine have been
introduced later. This study was devoted to the evaluation of an acute
pacing-induced in vivo model of AF in rats. Antiarrhythmic effects of well-known
compounds such as lidocaine, dofetilide, and ranolazine were confirmed in this
model. In addition, antiarrhythmic effects of different inhibitors of
Ca(2+)-activated small conductance K(+) (SK) channels were demonstrated.
Intravenous application of 5 mg/kg of the negative SK channel modulator NS8593
reduced AF duration by 64.5%, and the lowest significantly effective dose was
1.5 mg/kg. A dose-effect relationship was established based on 6 different dose
groups. Furthermore, it was demonstrated that the antiarrhythmic effect of
NS8593 and other tested drugs was associated with an increase in atrial
effective refractory period. The functional role of SK channels was confirmed by
2 other SK channel inhibitors, UCL1684 and apamin, thereby confirming the
hypothesis that these channels might constitute a new promising target for
antiarrhythmic treatment. Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and has a
significant impact on morbidity and mortality. Current antiarrhythmic drugs for
AF suffer from limited safety and efficacy, probably because they were not
designed based on specific pathological mechanisms. Recent research has provided
important insights into the mechanisms contributing to AF and highlighted
several potential novel antiarrhythmic strategies. In this review, we highlight
the main pathological mechanisms of AF, discuss traditional and novel aspects of
atrial antiarrhythmic drugs in relation to these pathological mechanisms, and
present potential novel therapeutic approaches including structure-based
modulation of atrial-specific cardiac ion channels, restoring abnormal Ca(2+)
handling in AF and targeting atrial remodeling. Atrial fibrillation (AF) is the most common arrhythmia worldwide, and it has a
significant effect on morbidity and mortality. It is a significant risk factor
for stroke and peripheral embolization, and it has an effect on cardiac
function. Despite widespread interest and extensive research on this topic, our
understanding of the etiology and pathogenesis of this disease process is still
incomplete. As a result, there are no set primary preventive strategies in place
apart from general cardiology risk factor prevention goals. It seems intuitive
that a better understanding of the risk factors for AF would better prepare
medical professionals to initially prevent or subsequently treat these patients.
In this article, we discuss widely established risk factors for AF and explore
newer risk factors currently being investigated that may have implications in
the primary prevention of AF. For this review, we conducted a search of PubMed
and used the following search terms (or a combination of terms): atrial
fibrillation, metabolic syndrome, obesity, dyslipidemia, hypertension, type 2
diabetes mellitus, omega-3 fatty acids, vitamin D, exercise toxicity, alcohol
abuse, and treatment. We also used additional articles that were identified from
the bibliographies of the retrieved articles to examine the published evidence
for the risk factors of AF. Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia in
clinical practice associated with significant morbidity and mortality. With the
growing number of the affected individuals, the development of safe and
effective treatment options for AF has become a worldwide priority. Currently
available antiarrhythmic medications for the restoration and maintece of
sinus rhythm have limitations due to the modest efficacy and a potential for
adverseeffects. Although substantial progress has been made in AF-ablation
techniques, broad application of these nonpharmacological treatment modalities
is limited and antiarrhythmic drug treatment is still the cornerstone and the
first-line therapy for the majority of AF patients. Improvements in the
understanding of the principal pathophysiological mechanisms of AF obtained in
the last several years have provided promising treatment opportunities. New
therapeutic options are based on the more selective targeting of ion channels
and intercellular connection proteins predomitly expressed in the atria, the
restoration of intracellular Ca(2+) homeostasis and the prevention of
AF-associated electrical and structural remodeling. In this review, we provide a
highlight of the most important pathophysiological mechanisms in AF with a
relation to the potential therapeutic interventions, and discuss novel findings
regarding the current and future pharmacological AF management and recent
patents. Atrial fibrillation remains the most prevalent cardiac arrhythmia, and its
incidence is increasing as the population ages. Common conditions associated
with an increased incidence include advanced age, hypertension, heart failure,
and valvular heart disease. Patients with atrial fibrillation may complain of
palpitations, fatigue, and decreased exercise tolerance or may be completely
asymptomatic. Options for treating patients who experience atrial fibrillation
include rate-controlling drugs such as digoxin, β-blockers, and calcium channel
blockers or a rhythm-controlling strategy with agents such as sodium channel
blockers and potassium channel blockers. Atrial fibrillation increases the risk
of stroke due to atrial thrombus formation and embolization. Anticoagulation
with the vitamin K antagonist, warfarin, remains the most widely prescribed
treatment option to decrease stroke risk. Several other antithrombotic agents
have recently become available and offer excellent alternatives to warfarin.
Catheter ablation can be undertaken as a nonpharmacologic rhythm control option
with varying degrees of success depending on duration of atrial fibrillation and
follow-up time from the procedure. This review article further describes the
management options for patients presenting with atrial fibrillation. Atrial fibrillation is the most common heart rhythm disorder in the world, with
major public health impact especially due to increased risk of stroke and
hospitalizations. The recently published results on epidemiology of atrial
fibrillation from the Global Burden of Diseases, Injuries, and Risk Factors
Study confirm the existence of a significant and progressive worldwide increase
in the burden of atrial fibrillation. However, there appears to be regional
variation in both the burden of atrial fibrillation and availability of
epidemiological data regarding this condition. In this review, the authors
identify issues that are unique to the developed versus developing regions and
outline a road map for possible approaches to surveillance, management, and
prevention of atrial fibrillation at the global level. |
Are thyroid hormone receptor alpha1 mutations implicated in thyroid hormone resistance syndrome? | thyroid hormone receptor alpha1 mutations are implicated in thyroid hormone resistance syndrome | Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid
hormone receptor beta (TR beta) gene. Almost all RTH patients are heterozygous
with an autosomal domit pattern of inheritance. That most are clinically
euthyroid suggests a compensatory role of the TR alpha1 isoform in maintaining
the normal functions of thyroid hormone (T3) in these patients. To understand
the role of TR alpha1 in the manifestation of RTH, we compared the phenotypes of
mice with a targeted domitly negative mutant TR beta (TR betaPV) with or
without TR alpha1. TR betaPV mice faithfully recapitulate RTH in humans in that
these mice demonstrate abnormalities in the pituitary-thyroid axis and
impairment in growth. Here we show that the dysregulation of the
pituitary-thyroid axis was worsened by the lack of TR alpha1 in TR betaPV mice,
and severe impairment of postnatal growth was manifested in TR betaPV mice
deficient in TR alpha1. Furthermore, abnormal expression patterns of T3-target
genes in TR betaPV mice were altered by the lack of TR alpha1. These results
demonstrate that the lack of TR alpha1 exacerbates the manifestation of RTH in
TR betaPV mice. Therefore, TR alpha1 could play a compensatory role in mediating
the functions of T3 in heterozygous patients with RTH. This compensatory role
may be especially crucial for postnatal growth. BACKGROUND: Thyroid hormone acts via receptor subtypes (TRα1, TRβ1, TRβ2) with
differing tissue distributions, encoded by distinct genes (THRA, THRB). THRB
mutations cause a disorder with central (hypothalamic-pituitary) resistance to
thyroid hormone action with markedly elevated thyroid hormone and normal TSH
levels.
SCOPE OF REVIEW: This review describes the clinical features, genetic and
molecular pathogenesis of a homologous human disorder mediated by defective
THRA. Clinical features include growth retardation, skeletal dysplasia and
constipation associated with low-normal T4 and high-normal T3 levels and a low
T4/T3 ratio, together with subnormal reverse T3 levels. Heterozygous TRa1
mutations in affected individuals generate defective mutant receptors which
inhibit wild-type receptor action in a domit negative manner.
MAJOR CONCLUSIONS: Mutations in human TRα1 mediate RTH with features of
hypothyroidism in particular tissues (e.g. skeleton, gastrointestinal tract),
but are not associated with a markedly dysregulated pituitary-thyroid axis.
GENERAL SIGNIFICANCE: Human THRA mutations could be more common but may have
eluded discovery due to the absence of overt thyroid dysfunction. Nevertheless,
in the appropriate clinical context, a thyroid biochemical signature (low T4/T3
ratio, subnormal reverse T3 levels), may enable future identification of cases.
This article is part of a Special Issue entitled Thyroid hormone signalling. CONTEXT: Recently the first patients with inactivating mutations in T₃ receptor
(TR)-α1 have been identified. These patients have low free T₄, low T₄, high T₃,
low rT₃, and normal TSH serum levels, in combination with growth retardation,
delayed bone development, and constipation.
OBJECTIVE: The aim of the current study was to report the effects of
levothyroxine (LT4) treatment on the clinical phenotype of 2 patients (father
and daughter) with a heterozygous inactivating mutation in TRα1.
SETTING AND PARTICIPANTS: Both patients were treated with LT4 for the last 5
years. To evaluate the effect of LT4 treatment, LT4 was withdrawn for 35 days
and subsequently reinitiated. Data were collected from medical records, by
reanalysis of serum collected over the last 6 years, and by a detailed clinical
evaluation.
RESULTS: Treatment with LT4 resulted in a suppression of serum TSH and
normalization of serum free T₄ and rT₃, whereas T₃ levels remained elevated in
both patients. In addition, there was a normalization of the dyslipidemia as
well as a response in serum IGF-I, SHBG, and creatine kinase in the index
patient. All these parameters returned to pretreatment values when LT4 was
briefly stopped. LT4 also resulted in an improvement of certain clinical
features, such as constipation and nerve conductance. However, cognitive and
fine motor skill defects remained.
CONCLUSION: This study reports the consequences of LT4 treatment over a
prolonged period of time in 2 of the first patients with a heterozygous mutation
in TRα1. LT4 therapy leads to an improvement of certain but not all features of
the clinical phenotype. |
Which agents are included in the FLAMSA chemotherapy regimen? | Fludarabine, cytarabine and amsacrine are included in the FLAMSA chemotherapy regimen. | The goal of this analysis was to define the role of the moderate-intensity
fludarabin Ara-C amsacrin (FLAMSA)-reduced intensity conditioning (RIC) regimen
for patients with high-risk AML undergoing allogeneic SCT (alloSCT) in first
CR1. High-risk was defined by (1) AML secondary to MDS or radio/chemotherapy,
(2) unfavorable cytogenetics or (3) delayed response to induction chemotherapy.
A total of 23 of 44 AML patients referred to the University of Munich for
alloSCT in CR1 between 1999 and 2006 fulfilled these criteria and received
FLAMSA chemotherapy, followed by RIC (4 Gy TBI/cyclophosphamide/ATG) for
alloSCT. Twenty-two patients engrafted, one died in aplasia. Two-year cumulative
incidences for relapse and nonrelapse mortality (NRM) were 4.6 and 22.5%,
respectively. Four-year overall and leukemia-free survival was 72.7% (median
follow-up among survivors: 35 months). The results of this high-risk cohort were
compared to the outcome of 21 consecutive standard-risk patients <55 years, who
had received standard, myeloablative sibling SCT in CR1 AML within the same
center and time period. Survival and cumulative incidences of relapse and NRM
were identical in both groups. In conclusion, the FLAMSA-RIC regimen produces
long-term remission in a high proportion of patients with high-risk AML
transplanted in CR1. In this cohort, FLAMSA-RIC showed equivalent antileukemic
activity as compared to the standard protocols. In this prospective study, we examined the toxicity and efficacy of an
intensified conditioning regimen for treatment of patients with relapsed or
high-risk acute lymphoblastic leukemia who undergo allogeneic hematopoietic stem
cell transplantation. Fifteen patients received fludarabine 30 mg/m(2),
cytarabine 2000 mg/m(2), amsacrine 100 mg/m(2) on days -10, -9, -8 and -7,
anti-thymocyte globulin (ATG-Fresenius) 20 mg/kg body weight on days -6, -5 and
-4 and fractionated total body irradiation 2 x 2 Gy on days -3, -2 and -1
(FLAMSA-ATG-TBI) before allogeneic hematopoietic stem cell transplantation. At
the time of hematopoietic stem cell transplantation, 10 patients were in
complete remission (8 CR1; 2 CR2), 3 with primary refractory and 2 suffered from
refractory relapse. All patients achieved a complete remission after
hematopoietic stem cell transplantation; and after a median follow-up time of
1091 days (range, 334-1554 days), nine patients (60%) are alive and free from
disease, including three patients with prior refractory disease. Three patients
died due to treatment-related mortality. The most frequent and severe
conditioning-related toxicities observed in 9 out of 15 patients were grade
III/IV infections according to common toxicity criteria. Thus, conditioning with
the FLAMSA-ATG-TBI regimen is a feasible and effective alternative for patients
with relapsed or high-risk acute lymphoblastic leukemia. Patients suffering from high-risk myelodysplastic syndrome (MDS) or acute
myelogenous leukemia (AML) secondary to MDS (sAML) are characterized by poor
response to conventional cytotoxic chemotherapy. The purpose of our prospective
single-center study was to examine the safety and efficacy of an allogeneic
hematopoietic stem cell transplantation (HSCT) following a sequential
conditioning regimen as first-line therapy for previously untreated patients
with high-risk MDS or sAML. Between November 2003 and June 2010, 30 patients (20
high-risk MDS, 10 sAML) received fludarabine (4 × 30 mg/m(2)), amsacrine (4 ×
100 mg/m(2)), and Ara-C (4 × 2 g/m(2), FLAMSA). After 2 to 3 days of rest,
patients received high-dose melphalan alone (200 mg/m(2) for patients with an
age <50 years, 150 mg/m(2) for patients with an age between 50 and 60 years, and
100 mg/m(2) for patients with an age >60 years; n = 24) or melphalan and
thiotepa (10 mg/kg, Mel/Thio, n = 6). Following these high-dose conditioning
regimens, a median number of 7.7 × 10(6) CD34(+) cells/kg body weight (range:
2.9 × 10(6)-17.2 × 10(6)) were transplanted from 13 related or 17 unrelated
donors. Antithymocyte globulin (Fresenius 30-60 mg/kg) as well as tacrolimus and
mycophenolate mofetil were used for graft-versus-host disease (GVHD)
prophylaxis. All patients except 1 with primary graft failure achieved complete
remission after HSCT. After a median follow-up time of 28 months (range: 7-81),
21 patients (70%) were alive and free of disease. Overall, 4 patients relapsed.
At 2 years, overall survival, event-free survival, and treatment-related
mortality were 70%, 63%, and 30%, respectively. Because of undue toxicity,
thiotepa is no longer part of the conditioning regimen. Our results add to the
body of evidence that a FLAMSA-based sequential conditioning therapy is
effective for previously untreated patients with high-risk MDS or sAML. Based on molecular aberrations, in particular the NPM1 mutation (NPM1(mut)) and
the FLT3 internal tandem duplication (Flt3-ITD), prognostic subgroups have been
defined among patients with acute myeloid leukemia with normal karyotype.
Whereas these subgroups are known to play an important role in outcome in first
complete remission, and also in the indication for allogeneic stem cell
transplantation, data are limited on their role after transplantation in
advanced disease. To evaluate the role of molecular subgroups of acute myeloid
leukemia with normal karyotype after allogeneic stem cell transplantation beyond
first complete remission, we analyzed the data from 141 consecutive adults
(median age: 51.0 years, range 18.4-69.3 years) who had received an allogeneic
transplant either in primary induction failure or beyond first complete
remission. A sequential regimen of cytoreductive chemotherapy (fludarabine,
high-dose AraC, amsacrine) followed by reduced intensity conditioning
(FLAMSA-RIC), was uniformly used for conditioning. After a median follow up of
three years, overall survival from transplantation was 64 ± 4%, 53 ± 4% and 44 ±
5% at one, two and four years, respectively. Forty patients transplanted in
primary induction failure achieved an encouraging 2-year survival of 69%. Among
101 patients transplanted beyond first complete remission, 2-year survival was
81% among patients with the NPM1(mut)/FLT3(wt) genotype in contrast to 43% in
other genotypes. Higher numbers of transfused CD34(+) cells (hazard ratio 2.155,
95% confidence interval 0.263-0.964, P=0.039) and favorable genotype (hazard
ratio 0.142, 95% confidence interval: 0.19-0.898, P=0.048) were associated with
superior overall survival in multivariate analysis. In conclusion, patients with
acute myeloid leukemia with normal karyotype can frequently be rescued after
primary induction failure by allogeneic transplantation following FLAMSA-RIC.
The prognostic role of NPM1(mut)/FLT3-ITD based subgroups was carried through
after allogeneic stem cell transplantation beyond first complete remission. Patients with primary refractory or relapsed acute myeloid leukemia (AML) have a
dismal prognosis. We report a retrospective single center analysis of
aplasia-inducing chemotherapy using fludarabine, cytarabine, and amsacrine
(FLAMSA) followed by reduced-intensity conditioning (RIC) for allogeneic
hematopoietic cell transplantation (HCT) in 62 consecutive primary refractory or
relapsed AML patients. Two-year event-free survival and overall survival (OS)
were 26 and 39%, respectively. Risk stratification according to cytogenetic and
molecular genetic markers showed superior survival in patients in the
intermediate-1 risk group (2-year OS 70%) compared to the intermediate-2 risk
(2-year OS 34%, p = 0.03) and adverse risk (2-year OS 38%, p = 0.06) group. The
use of HLA-matched versus HLA-mismatched donors had no significant influence on
survival (p = 0.98). Two-year OS in the elderly subgroup defined by age ≥60
years was 31% compared to 46% in the group of younger patients <60 years (p =
0.19). Cumulative incidence of non-relapse mortality at 2 years adjusted for
relapse as competing risk was 20% for patients <60 years and 26% for older
patients (p = 0.55). Chronic graft-versus-host disease was associated with a
statistically significant superior survival (p < 0.01). FLAMSA-RIC followed by
allogeneic HCT enables long-term disease-free survival in primary refractory or
relapsed AML even in the elderly patient population. Sequential use of chemotherapy and reduced-intensity conditioning (RIC) with
allogeneic stem cell transplantation (SCT) has been proposed to improve the
treatment outcomes in patients with high-risk acute myeloid leukemia (AML).
Here, we present our experience with this procedure in a cohort of 60 AML
patients with primary induction failure (n = 9); early, refractory, or ≥ second
relapse (n = 41); or unfavorable cytogenetics (n = 10). A combination of
fludarabine (30 mg/m²/day), cytarabine (2 g/m²/day), and amsacrine (100
mg/m²/day) for 4 days was used. After 3 days of rest, RIC was carried out,
consisting of 4 Gy total body irradiation, antithymocyte globulin
(ATG-Fresenius), and cyclophosphamide (fludarabine, amsacrine, and cytarabine
(FLAMSA)-RIC protocol). Prophylactic donor lymphocyte infusions (pDLIs) were
given in patients with complete remission (CR) and without evidence of
graft-versus-host disease ≥120 days after SCT. The median time of neutrophil
engraftment was 17 days. CR was achieved in 47 of 60 patients (78%). Eleven
patients received pDLIs resulting in long-term CR in eight of them. Non-relapse
mortality after 1 and 3 years was 25 and 28%, respectively. With a median
follow-up of 37 months (range, 10-69), 3-year overall survival and 3-year
progression-free survival were 42 and 33%, respectively. In a multivariate
analysis, dose of CD34(+) cells >5 × 10⁶/kg (p = 0.005; hazard ratio (HR) =
0.276), remission of AML before SCT (p = 0.044; HR = 0.421), and achievement of
complete chimerism after SCT (p = 0.001; HR = 0.205) were significant factors of
better overall survival. The use of the FLAMSA-RIC protocol in suitable
high-risk AML patients results in a long-term survival rate of over 40%. |
Which are the subunits of the IkB protein kinase (IKK)? | Proinflammatory NF-kappaB activation requires the IkappaB (inhibitor of NF-kappaB) kinase (IKK) complex that contains two catalytic subunits named IKKalpha and IKKbeta and a regulatory subunit named NF-kappaB essential modulator (NEMO).Additional components may exist, transiently or permanently, but their characterization is still unsure. | The IkappaB-Kinase (IKK) complex is a multisubunit protein complex crucial for
signal-induced phosphorylation of the IkappaB proteins and thus controls the
activity of the transcription factor NF-kappaB. Besides the two kinases IKKalpha
and IKKbeta, the IKK complex contains NEMO/IKKgamma, an additional subunit with
regulatory and adaptor functions. NEMO not only confers structural stability to
the IKK complex but also participates in the activation process of the IKK
complex by linking the IKK subunits to upstream activators. In this study we
analyze the IKKbeta-mediated phosphorylation of the IKK-binding domain of NEMO.
In vitro, IKKbeta phosphorylates three serine residues in the domain of NEMO at
positions 43, 68, and 85. However, mutational analysis revealed that only the
phosphorylation of serine 68 in the center of the IKK-binding domain plays an
essential role for the formation and the function of the IKK complex. Thus,
Ser(68) phosphorylation attenuates the amino-terminal dimerization of NEMO as
well as the IKKbeta-NEMO interaction. In contrast, the NEMO-IKKalpha interaction
was only mildly affected by the phosphorylation of Ser(68). However, functional
analysis revealed that Ser(68) phosphorylation primarily affects the activity of
IKKalpha. Furthermore, in complementation experiments of NEMO-deficient murine
embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant
decreased, TNF-alpha-induced NF-kappaB activity, thus emphasizing the inhibitory
role of the Ser(68) phosphorylation on the signal-induced NF-kappaB activity.
Finally, we provide evidence that the protein phosphatase PP2A is involved in
the regulation of the Ser(68)-based mechanism. In summary, we provide evidence
for a signal-induced phosphorylation-dependent alteration of the IKK complex
emphasizing the dynamic nature of this multisubunit kinase complex. The anti-apoptotic transcription factor nuclear factor-kappaB (NF-kappaB) is
constitutively activated in CD34(+) myeloblasts from high-risk myelodysplastic
syndrome (MDS) and acute myeloid leukemia (AML) patients. Inhibition of
NF-kappaB by suppressing the canonical NF-kappaB activation pathway, for
instance by knockdown of the three subunits of the inhibitor of NF-kappaB (I
kappaB) kinase (IKK) complex (IKK1, IKK2 and NEMO) triggers apoptosis in such
cells. Here, we show that an MDS/AML model cell line exhibits a constitutive
interaction, within the nucleus, of activated, S1981-phosphorylated ataxia
telangiectasia mutated (ATM) with NEMO. Inhibition of ATM with two distinct
pharmacological inhibitors suppressed the activating autophosphorylation of ATM,
blocked the interaction of ATM and NEMO, delocalized NEMO as well as another
putative NF-kappaB activator, PIDD, from the nucleus, abolished the activating
phosphorylation of the catalytic proteins of the IKK complex (IKK1/2 on serines
176/180), enhanced the expression of I kappaB alpha and caused the
relocalization of NF-kappaB from the nucleus to the cytoplasm, followed by
apoptosis. Knockdown of ATM with small-interfering RNAs had a similar effect
that could not be enhanced by knockdown of NEMO, PIDD and the p65 NF-kappaB
subunit, suggesting that an ATM inhibition/depletion truly induced apoptosis
through inhibition of the NF-kappaB system. Pharmacological inhibition of ATM
also induced the nucleocytoplasmic relocalization of p65 in maligt
myeloblasts purified from patients with high-risk MDS or AML, correlating with
the induction of apoptosis. Altogether, these results support the contention
that constitutively active ATM accounts for the activation of NF-kappaB in
high-risk MDS and AML. Proinflammatory NF-kappaB activation requires the IkappaB (inhibitor of
NF-kappaB) kinase (IKK) complex that contains two catalytic subunits named
IKKalpha and IKKbeta and a regulatory subunit named NF-kappaB essential
modulator (NEMO). NEMO and IKKbeta are essential for tumor necrosis factor
(TNF)-induced NF-kappaB activation, and we recently demonstrated that NEMO and
IKKalpha are sufficient for interleukin (IL)-1-induced signaling. IKKalpha and
IKKbeta both contain a functional NEMO-binding domain (NBD); however, the role
of NEMO association with each kinase in NF-kappaB signaling and IKK complex
formation remains unclear. To address this question, we stably reconstituted
IKKalpha(-/-) and IKKbeta(-/-) murine embryonic fibroblasts (MEFs) with
wild-type (WT) or NBD-deficient (DeltaNBD) versions of IKKalpha and IKKbeta,
respectively. TNF-induced classical NF-kappaB activation in IKKbeta(-/-) MEFs
was rescued by IKKbeta(WT) but not IKKbeta(DeltaNBD), whereas neither
IKKbeta(WT) nor IKKbeta(DeltaNBD) affected IL-1-induced NF-kappaB signaling. As
previously described, classical NF-kappaB transcriptional activity was absent in
IKKalpha(-/-) cells. Reconstitution with either IKKalpha(WT) or
IKKalpha(DeltaNBD) rescued both IL-1 and TNF-induced transcription,
demonstrating that NEMO association is not required for IKKalpha-dependent
regulation of NF-kappaB-dependent transcription. Stably expressed IKKalpha(WT)
or IKKbeta(WT) associated with endogenous IKKs and NEMO in IKKalpha(-/-) or
IKKbeta(-/-) MEFs, respectively, resulting in formation of the heterotrimeric
IKKalpha-IKKbeta-NEMO complex. In contrast, although the IKKalpha(DeltaNBD) and
IKKbeta(DeltaNBD) mutants associated with endogenous IKKs containing an NBD,
these dimeric endogenous IKK-IKK(DeltaNBD) complexes did not associate with
NEMO. These findings therefore demonstrate that formation of the heterotrimeric
IKKalpha-IKKbeta-NEMO holocomplex absolutely requires two intact NEMO-binding
domains. The transcription factor NF-kappaB is essential for osteoclastogenesis and is
considered an immune-modulator of rheumatoid arthritis and inflammatory
osteolysis. Activation of NF-kappaB subunits is regulated by the upstream
IkappaB kinase (IKK) complex which contains IKKalpha, IKKbeta, and IKKgamma; the
latter also known as NF-kappaB essential modulator (NEMO). The role of IKKalpha
and IKKbeta in the skeletal development and inflammatory osteolysis has been
described, whereas little is known regarding the role of NEMO in this setting.
Typically, signals induced by RANK ligand (RANKL) or TNF prompt oligomerization
of NEMO monomers through the coiled-coil-2 (CC2) and leucine zipper (LZ) motifs.
This step facilitates binding to IKKs and further relaying signal transduction.
Given the central role of NF-kappaB in osteoclastogenesis, we asked whether NEMO
is essential for osteoclastogenesis and whether interruption of NEMO
oligomerization impedes osteoclast differentiation in vitro and in vivo. Using
cell-permeable short peptides overlapping the CC2 and LZ motifs we show that
these peptides specifically bind to NEMO monomers, prevent trimer formation, and
render NEMO monomers susceptible for ubiquitin-mediated degradation. Further,
CC2 and LZ peptides attenuate RANKL- and TNF-induced NF-kappaB signaling in bone
marrow-derived osteoclast precursors (OCPs). More importantly, these peptides
potently inhibit osteoclastogenesis, in vitro, and arrest RANKL-induced
osteolysis, in mice. To further ascertain its role in osteoclastogenesis, we
were able to block osteoclastogenesis using NEMO siRNA knockdown approach.
Collectively, our data establish that obstruction of NEMO oligomerization
destabilizes NEMO monomers, inhibits NF-kappaB activation, impedes
osteoclastogenesis and arrests inflammatory osteolysis. Thus, NEMO presents
itself as a promising target for anti-osteolytic intervention. Helicobacter pylori, the etiological agent of several human gastric diseases,
induces the transcription factor nuclear factor-κB (NF-κB) in colonized
epithelial cells leading to the release of proinflammatory mediators. Activation
of NF-κB involves the IκB kinase (IKK)-complex composed of two catalytic
subunits, IKKα and IKKβ, and a regulatory scaffold protein, IKKγ. IKKβ was shown
to be essential for NF-κB activation in response to a variety of stimuli
including H. pylori. In addition to the phosphorylation of serine residues,
tyrosine phosphorylation could be crucial for IKKβ activation. Here we provide
evidence that IKKβ phosphorylation is induced in lipid rafts (DRM fractions) of
H. pylori-infected cells, but not TNFα-stimulated cells. Furthermore, H. pylori
transiently induces binding of IKKβ to c-Src kinase. Inhibition of c-Src by
specific inhibitors as well as knockdown of c-Src by small interfering RNA
reduced phosphorylation of IκBα as well as of p65. Thus, tyrosine-phosphorylated
IKKβ contributes at least in part to NF-κB activation in response to H. pylori
infection. INTRODUCTION: NF-kB is a key regulator of inflammation and immunity in cancer
development. The IkB kinase (IKK) is a multisubunit complex containing catalytic
subunits termed IKK-α, -β and -γ. It is well known that many pro-inflammatory
stimuli require the IKK-β subunit for NF-kB activation.
AREAS COVERED: NF-kB affects the progression of inflammation-related
diseases,such as myocardial ischemia, bronchial asthma, arthritis, cancer and
other diseases. We review the characteristics and effects of these inhibitors on
inflammatory and other diseases.
EXPERT OPINION: Various synthesized IKK inhibitors have been developed and they
will be used clinically in the near future. NF-κB transcription factors are pivotal players in controlling inflammatory and
immune responses, as well as cell proliferation and apoptosis. Aberrant
regulation of NF-κB and the signaling pathways that regulate its activity have
been involved in various pathologies, particularly cancers, as well as
inflammatory and autoimmune diseases. NF-κB activation is tightly regulated by
the IκB kinase (IKK) complex, which is composed of two catalytic subunits IKKα
and IKKβ, and a regulatory subunit IKKγ/NEMO. Although IKKα and IKKβ share
structural similarities, IKKα has been shown to have distinct biological
functions. However, the molecular mechanisms that modulate IKKα activity have
not yet been fully elucidated. To understand better the regulation of IKKα
activity, we purified IKKα-associated proteins and identified ABIN-2. Here, we
demonstrate that IKKα and IKKβ both interact with ABIN-2 and impair its
constitutive degradation by the proteasome. Nonetheless, ABIN-2 enhances IKKα-
but not IKKβ-mediated NF-κB activation by specifically inducing IKKα
autophosphorylation and kinase activity. Furthermore, we found that ABIN-2
serine 146 is critical for the ABIN-2-dependent IKKα transcriptional
up-regulation of specific NF-κB target genes. These results imply that ABIN-2
acts as a positive regulator of NF-κB-dependent transcription by activating
IKKα. General (macro)autophagy and the activation of NFκB constitute prominent
responses to a large array of intracellular and extracellular stress conditions.
The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase
(IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB
activation pathway, limits autophagy induction by physiological or
pharmacological triggers, while constitutive active IKK subunits suffice to
stimulate autophagy. The activation of IKK usually relies on TGFβ-activated
kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy
in multiple settings. TAK1 interacts with two structurally similar
co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in
resting conditions both TAB2 and TAB3 bind the essential autophagic factor
Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3
dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The
inhibitory interaction between TABs and Beclin 1 is mediated by their
coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or
TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point
to the existence of a direct molecular crosstalk between the canonical NFκB
activation pathway and the autophagic core machinery that guarantees the
coordinated induction of these processes in response to stress. The IκB kinase (IKK) complex plays a crucial role in the activation of the
transcription factor NF-κB by phosphorylating an inhibitory molecule IκBα.
Recently, we showed that IKK2 (also called IKKβ), a catalytic subunit of the IKK
complex, induces immunoglobulin E-mediated degranulation in mast cells by
phosphorylating SNAP-23, the target-membrane soluble N-ethylmaleimide-sensitive
fusion factor attachment protein receptor (SNARE). In addition to IKK2, a recent
study has shown that ELKS, a regulatory subunit of the IKK complex, also induces
the degranulation of rat basophilic leukemia cells. These findings indicate that
the two subunits of the IKK complex, IKK2 and ELKS, function not only in
NF-κB-dependent transcriptional activation but also in NF-κB-independent
pathways. This review focuses on the functions of IKK2 and ELKS in mast cell
degranulation. NEMO (NF-κB essential modulator) associates with catalytic subunits IKKα and
IKKβ to form the IκB kinase (IKK) complex and is a key regulator of NF-κB
pathway signaling. Biochemical and structural characterization of NEMO has been
challenging, however, leading to conflicting data about basic biochemical
properties such as the oligomeric state of active NEMO and its binding affinity
for IKKβ. We show that up to seven of NEMO's 11 cysteine residues can be mutated
to generate recombit full-length NEMO that is highly soluble and active.
Using a fluorescence anisotropy binding assay, we show that full-length NEMO
binds a 44-mer peptide encompassing residues 701-745 of IKKβ with a K(D) of 2.2
± 0.8 nM. The IKKβ binding affinities of mutants with five and seven Cys-to-Ala
substitutions are indistinguishable from that of wild-type NEMO. Moreover, when
expressed in NEMO -/- fibroblasts, the five-Ala and seven-Ala NEMO mutants can
interact with cellular IKKβ and restore NF-κB signaling to provide protection
against tumor necrosis factor α-induced cell death. Treatment of the
NEMO-reconstituted cells with H₂O₂ led to the formation of covalent dimers for
wild-type NEMO and the five-Ala mutant, but not for the seven-Ala mutant,
confirming that Cys54 and/or Cys347 can mediate interchain disulfide bonding.
However, the IKKβ binding affinity of NEMO is unaffected by the presence or
absence of interchain disulfide bonding at Cys54, which lies within the IKKβ
binding domain of NEMO, or at Cys347, indicating that NEMO exists as a
noncovalent dimer independent of the redox state of its cysteines. This
conclusion was corroborated by the observation that the secondary structure
content of NEMO and its thermal stability were independent of the presence or
absence of interchain disulfide bonds. |
Is PLK2 involved in alpha-synuclein phosphorylation in Parkinson disease? | PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assayThese results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system. | BACKGROUND: α-Synuclein aggregates in Lewy bodies and plays a central role in
the pathogenesis of a group of neurodegenerative disorders, known as
"Synucleinopathies", including Parkinson's disease. Parkin mutations result in
loss of parkin E3-ubiquitin ligase activity and cause autosomal recessive early
onset parkinsonism.
RESULTS: We tested how these two genes interact by examining the effects of
parkin on post-translational modification of α-Synuclein in gene transfer animal
models, using a lentiviral gene delivery system into the striatum of 2-month old
male Sprague Dawley rats.Viral expression of wild type α-Synuclein caused
accumulation of α-Synuclein and was associated with increased cell death and
inflammation. α-Synuclein increased PLK2 levels and GSK-3β activity and
increased the levels of phosphorylated α-Synuclein and Tau. Parkin co-expression
reduced the levels of phosphorylated α-Synuclein and attenuated cell death and
inflammation. Parkin reduced PLK2 levels and increased PP2A activation.
CONCLUSIONS: These data suggest that parkin reduces α-Synuclein levels and
alters the balance between phosphatase and kinase activities that affect the
levels of phosphorylated α-Synuclein. These results indicate novel mechanisms
for parkin protection against α-Synuclein-induced toxicity in PD. |
Which glands are subject to attack by lymphocytes in Sjogren's syndrome? | Sjögren's syndrome (SjS) is a human autoimmune disease characterized by exocrine dysfunction resulting from chronic autoimmune attack primarily against the lacrimal and/or salivary glands. | NOD mice manifest many features of autoimmune exocrinopathy (Sjögren's
syndrome), a disease generally characterized by a chronic, progressive
immunological attack against the exocrine tissues of the salivary and lacrimal
glands. Previous studies using the NOD congenic partner strain, NOD.Igmu(null),
defined an important role for B lymphocytes in the development of xerostomia,
implicating autoantibodies reactive with the acetylcholine muscarinic receptor
(M3R) as the possible effector mechanism. In the present study, we have examined
the impact of the cytokine, interleukin (IL)-4, on autoimmune exocrinopathy by
using the IL-4 gene knockout (KO) NOD mouse strain, NOD.IL-4-/-. Despite
manifesting the physiological aberrations and marked leukocytic infiltration of
the salivary glands characteristic of autoimmune xerostomia in NOD mice, the
NOD.IL-4-/- mice do not develop xerostomia. However, NOD.IL-4-/- mice that
received adoptively transferred T lymphocytes derived from NOD.Igmu-/- mice
progress to xerostomia, thereby reversing the defect. While progression or lack
of progression to xerostomia correlated with the ability of the NOD.IL-4-/- mice
to express detectable anti-M3R autoantibodies, the precise mechanism of how IL-4
influences the development of autoimmune xerostomia remains speculative. The purpose of this qualitative research was to explore the lived experiences of
women with Sjogren's Syndrome (SS), a chronic autoimmune syndrome in which
invading lymphocytes attack moisture-producing glands of the body. This
syndrome, affecting 4 million Americans, involves extreme dryness of the eyes
and mouth and other systemic complications such as fatigue and muscle and joint
pain. Ten women, ranging in age from 27 to 83, with SS were interviewed to
understand their coping strategies and attitudes. This study used Strauss'
framework to view the quality of life of the chronically ill-specifically, those
with SS. Nine questions were asked to elicit information on limitations with
daily living, relationships, emotions about lifestyle changes, and comparisons
with others living with chronic disease. In addition to SS, 8 of the 10 women
also had fibromyalgia, and 4 had other conditions such as lupus and rheumatoid
arthritis (secondary Sjogren's). Their responses were categorized into four
groups: helping, hindering, hoping, and hurting. This led to the development of
the 4H Model of Chronic Autoimmune Disease with subcategories such as:
supporting, joining, comparing, coping, health promoting, fearing, becoming a
chronic sufferer, feeling, wishing, appearing, impeding, restricting, isolating,
suffering, doctoring, and diagnosing. J Prof Nurs 17:194-202, 2001. Sjögren's syndrome (SjS) is a systemic autoimmune disease in which an
immunological attack against the salivary and lacrimal glands results,
respectively, in severe dry mouth and dry eye diseases. Although a CD4+ T
lymphocyte population is an integral component in the pathogenesis of SjS,
recent studies have focused on the importance the B lymphocyte plays in both the
pre-clinical and clinical phases of the disease process. To understand the
molecular and cellular mechanisms involved in SjS, numerous mouse models that
mimic major clinical manifestations of the human disease have been developed.
Studies have begun to define the genetics, the nature of the autoimmune response
towards the salivary and lacrimal glands, as well as the possible mechanisms for
effecting glandular dysfunction, thereby establishing insights to new
intervention therapies. Not surprising, the B cell is taking center stage. Here,
we present an in-depth discussion of how B cell populations may be involved in
orchestrating or determining exocrine gland dysfunction. Sjögren's syndrome is an autoimmune disease in which immune cells chronically
attack the lachrymal and salivary glands. The Id3 knockout mouse is a newly
established animal model for primary Sjögren's syndrome. To address the role of
B cells in Sjögren's syndrome and autoimmune disease, we studied the effect of
CD20 monoclonal antibody treatment on the disease in Id3 knockout mice. Antibody
treatment at 2-month intervals led to efficient and sustained B-cell depletion
in Id3 knockout mice. A significant improvement of histopathology was observed
accompanied by the recovery of saliva secretory function after CD20 antibody
treatment. We further show that serum immunoglobulin G3, which is abnormally
high in untreated Id3 knockout mice, was reduced after CD20 antibody treatment.
This study establishes a new animal model for immunotherapy of Sjögren's
symptoms and suggests a possible link between immunoglobulin G3 and disease
pathology in Id3 knockout mice. In Sjögren's syndrome, like in most other autoimmune diseases, the enigma
leading to a pathogenic attack against self has not yet been solved. By
definition, the disease must be mediated by specific immune reactions against
endogenous tissues to qualify as an autoimmune disease. In Sjögren's syndrome
the autoimmune response is directed against the exocrine glands, which, as
histopathological hallmark of the disease, display persistent and progressive
focal mononuclear cell infiltrates. Clinically, the disease in most patients is
manifested by two severe symptoms: dryness of the mouth (xerostomia) and the
eyes (keratoconjunctivitis sicca). A number of systemic features have also been
described and the presence of autoantibodies against the ubiquitously expressed
ribonucleoprotein particles Ro (Sjögren's-syndrome-related antigen A - SSA) and
La (SSB) further underline the systemic nature of Sjögren's syndrome. The
original explanatory concept for the pathogenesis of Sjögren's syndrome proposed
a specific, self-perpetuating, immune mediated loss of acinar and ductal cells
as the principal cause of salivary gland hypofunction. Although straightforward
and plausible, the hypothesis, however, falls short of accommodating several
Sjögren's syndrome-related phenomena and experimental findings. Consequently,
researchers considered immune-mediated salivary gland dysfunction prior to
glandular destruction and atrophy as potential molecular mechanisms underlying
the symptoms of dryness in Sjögren's syndrome. Accordingly, apoptosis, fibrosis
and atrophy of the salivary glands would represent consequences of salivary
gland hypofunction. The emergence of advanced bio-analytical platforms further
enabled the identification of potential biomarkers with the intent to improve
Sjögren's syndrome diagnosis, promote the development of prognostic tools for
Sjögren's syndrome and the long-term goal to identify possible processes for
therapeutic treatment interventions. In addition, such approaches allowed us to
glimpse at the apparent complexity of Sjögren's syndrome. |
Describe Malgaigne fracture. | Bilateral pubic rami fractures are characteristic to Malgaigne fractures. Patients with Malgaigne fractures are particularly prone to additional injuries. | An external fixator has been designed that is rigid enough to eliminate the need
for skeletal traction in patients with unstable pelvic-ring fractures. This
Wichita frame is similar to the Pittsburgh frame but is stiffened by the use of
locked crossbars connecting the side triangles. The frame was tested in
cadaveric specimens by techniques previously reported. In addition,
finite-element modeling of the various frame designs was performed to ensure
that the frame configuration was optimal and to supplement in vitro test
results. Multiple variables that can influence frame failure loads were
examined. Pelvic girdle injuries leading to anterior and posterior disruption are rarely
seen in children. External fixation is useful to reduce and stabilize the pelvis
in order to control massive blood loss due to severe venous bleeding. The
anterior tension band element of the external fixator can successfully maintain
reduction of the pelvic girdle. Dislocation or fracture dislocation of the
sacroiliacal joint cannot be reduced with simple traction techniques. Leg length
discrepancy is to be expected when the hemipelvis is displaced. Seven cases of
this rare injury in children are presented together with the late outcome.
Treatment modalities are discussed. Fractures of the sacrum and lesions of the intervertebral disks are seldom
reported in childhood and adolescence. In our report, illustrated by three
representative cases that we treated during the past six months, we show that
sacrum fractures, not an easy diagnosis, may be more frequent than currently
assumed, and that in quite a number of children and adolescents an anterior
pelvic fracture may in fact be an unrecognized Malgaigne type pelvic fracture
with a posterior fracture plane cutting through the sacrum. They may be
accompanied by herniations and/or lacerations of intervertebral disks. Eleven cases of sacroiliac dislocation and/or fracture (Malgaigne pattern) were
successfully reduced and stabilized using two threaded compression rods. The
mean follow-up period was 26.1 months (range, seven to 45 months). None of the
implants failed and there was no subsequent displacement. Two patients had mild
residual lower back pain, and one was treated with implant removal without
subsequent relief of pain. One patient, in whom the operation was done 110 days
after dislocation, had extension of an incomplete preoperative peroneal nerve
palsy. After anterior pelvic ring stabilization has been performed, two threaded
3/16-inch diameter rods are driven from the normal posterior iliac wing
superficial to the sacrum and through the reduced opposite iliac wing.
Compression is obtained with washers and nuts. This procedure can be performed
safely and effectively, providing stable fixation and allowing early
mobilization to help lessen or prevent the complications associated with
prolonged bed rest. We compared the mechanical performance of the Orthofix pelvic external fixator
with that of the Pittsburgh triangular frame (PTF) on eight fresh-frozen cadaver
pelves with experimentally created Malgaigne (double-vertical)
fracture/dislocations. The pelves were quasi-statically loaded in longitudinal
compression and loss of reduction (i.e., failure) was defined as 1.5 cm of
diastasis at either of the fracture/dislocation sites. The Orthofix frame-pelvis
complex (four 6-mm half-pins) was comparable to the PTF-pelvis complex (eight
5-mm half-pins) in terms of load to failure and overall stiffness, but was 4-7
times stiffer at lower load levels. Both frame-and-pin complexes deformed
moderately at the pin-bone interface, but, while the PTF also showed moderate
displacement between frame components, the Orthofix had almost no frame
intercomponent motion. Threaded compression rods were placed between the posterior-superior spines as a
means of posterior stabilization of pelvic fractures. To document the increase
in sacroiliac stability afforded by this technique, biomechanical testing was
performed. Malgaigne-type fractures with sacroiliac disruptions were created in
four cadaver pelvises. The fractures were stabilized with anterior frames of the
Slatis or Pittsburgh type and subjected to longitudinal and torsional loading
patterns on an Instron machine. The anterior fixation was then augmented with
threaded compression rods placed between the posterior-superior spines to
compress the disrupted sacroiliac joints, and repeat testing was conducted.
Anterior frames alone were found to provide little stabilization of the
disrupted sacroiliac joints with either longitudinal or torsional loading.
Markedly improved stabilization in both loading modes was achieved with
posterior augmentation. Two typical cases are presented to demonstrate that
posterior stabilization is as efficacious in clinical practice as in the
biomechanics laboratory. Seventy consecutive surviving patients with pelvic fractures were reviewed to
define those at high risk for intra-abdominal injury. Eleven (16%) had serious
associated visceral injuries. Initial hypotension, Malgaigne and bilateral pubic
rami fractures, and blood requirements exceeding 2,000 cc were suggestive of
abdominal trauma. These high-risk patients should undergo peritoneal lavage as
an integral part of their initial management. If the lavage is negative,
continued hemorrhage should be evaluated with angiography and nonoperative means
of vascular control attempted before resorting to laparotomy. The paper deals with the displacement of the uterus observed during management
of Malgaigne type pelvic fracture in a 33 years old female. An intrauterine
device seen on the radiographs revealed displacement of the uterus towards
contralateral side of the pelvis. The reduction of the fracture stabilized with
Molski metal clamp reconstituted normal anatomy. Traumatic avulsion of the right diaphragm from the lumbocostal arch is a very
rare lesion. The authours report the case of a 27-year-old man who had suffered
a severe polytrauma with blunt thoracic injury, fracture of the lumbar spine,
Malgaigne-type fracture of the pelvis and fracture of the femoral shaft on the
right side, 10 years before. At the time of injury the lesion of the diaphragm
went unnoticed. The diagnosis was made 10 years later when the patient was
referred for chronic right thoracic pain combined with postprandial abdominal
distension and crampy pain in the abdomen. The chest radiogram and CT-scan
showed displacement of the right kidney and most of the right colon into the
thorax due to avulsion of the diaphragm from its dorsal insertion on the
lumbocostal arch. Surgical repair was necessary to obtain relief from pain and
to prevent intestinal obstruction. Reduction of the hernia, reinsertion of the
diaphragm to the lumbocostal arch and reinforcement of the repair with a prolene
mesh prosthesis was performed through a right thoracophrenolumbotomy incision. RATIONALE AND OBJECTIVES: The purpose of this study was to determine the
percentage of patients with known pelvic fractures who have additional findings
of intraabdominal injury, as diagnosed at abdominal computed tomography (CT),
and to determine if patients with specific types or patterns of fractures are
more likely to have additional injuries.
MATERIALS AND METHODS: The authors reviewed the medical records of 200
consecutive patients (125 women, 75 men; age range, 4-86 years) who had been
admitted to a level 1 trauma center with osseous pelvic injury secondary to
blunt trauma and who had undergone abdominal CT examinations. Abdominal CT
findings in these patients were classified as negative, positive, or minimal and
correlated with mechanism of pelvic fracture.
RESULTS: Sixty-five (32%) of the 200 patients had negative CT findings, 43 (22%)
had findings attributable to the trauma but required no follow-up, and 92 (46%)
had positive findings that required nonsurgical management or exploratory
laparotomy. Additional pelvic fractures were identified in 63 (32%) patients.
The highest prevalence of additional injuries was in patients with Malgaigne
fractures (four of 15, 27%) or bilateral pubic rami fractures (six of 18, 33%).
CONCLUSION: CT examinations revealed that 135 (68%) of 200 patients with pelvic
fractures secondary to blunt trauma had concomitant internal or skeletal
injuries and that 92 (46%) patients had injuries severe enough to require
nonsurgical management or exploratory laparotomy. Patients with bilateral pubic
rami fractures or Malgaigne fractures were particularly prone to additional
injuries; therefore, abdominal CT examinations are recommended in these
patients. |
Name five popular computer programs used to identify genes in genomic sequences | Any five from the following not exhaustive list: AUGUSTUS, MGENE, CRAIG, Agene, EUGENE, Fgenesh++C, Fgenesh++, Fgenesh, GeneID, GeneMark.hmm, GENOMIX, GESECA, GLEAN, GlimmerHMM, Gramene,JIGSAW, MAKER, ,MGENE, N-SCAN, SGP2, SNAP, ExonHunter, Evigan, Genescan, HMMGene, MZEF, Genie, Twinscan, SLAM, GRAIL. | Sequencing centers such as the Human Genome Center at LBNL are producing an
ever-increasing flood of genetic data. Annotation can greatly enhance the
biological value of these sequences. Useful annotations include possible gene
locations, homologies to known genes, and gene signal such as promoters and
splice sites. Genotator is a workbench for automated sequence annotation and
annotation browsing. The back end runs a series of sequence analysis tools on a
DNA sequence, handling the various input and output formats required by the
tools. Genotator currently runs five different gene-finding programs, three
homology searches, and searches for promoters, splice sites, and ORFs. The
results of the analyses run by Genotator can be viewed with the interactive
graphical browser. The browser displays color-coded sequence annotations on a
canvas that can be scrolled and zoomed, allowing the annotated sequence to be
explored at multiple levels of detail. The user can view the actual DNA sequence
in a separate window; when a region is selected in the map display, it is
highlighted automatically in the sequence display, and vice versa. By displaying
the output of all of the sequence analyses, Genotator provides an intuitive way
to identify the significant regions (for example, probable exons) in a sequence.
Users can interactively add personal annotations to label regions of interest.
Additional capabilities of Genotator include primer design and pattern
searching. MOTIVATION: A number of programs have been developed to predict the eukaryotic
gene structures in DNA sequences. However, gene finding is still a challenging
problem.
RESULTS: We have explored the effectiveness when the results of several
gene-finding programs were re-analyzed and combined. We studied several methods
with four programs (FEXH, GeneParser3, GEN-SCAN and GRAIL2). By HIGHEST-policy
combination method or BOUNDARY method, approximate correlation (AC) improved by
3-5% in comparison with the best single gene-finding program. From another
viewpoint, OR-based combination of the four programs is the most reliable to
know whether a candidate exon overlaps with the real exon or not, although it is
less sensitive than GENSCAN for exon-intron boundaries. Our methods can easily
be extended to combine other programs.
AVAILABILITY: We have developed a server program (Shirokane System) and a client
program (GeneScope) to use the methods. GeneScope is available through a WWW
site (http://gf.genome.ad.jp/).
CONTACT: (katsu,takagi)@ims.u-tokyo.ac.jp Computational methods for automated genome annotation are critical to our
community's ability to make full use of the large volume of genomic sequence
being generated and released. To explore the accuracy of these automated feature
prediction tools in the genomes of higher organisms, we evaluated their
performance on a large, well-characterized sequence contig from the Adh region
of Drosophila melanogaster. This experiment, known as the Genome Annotation
Assessment Project (GASP), was launched in May 1999. Twelve groups, applying
state-of-the-art tools, contributed predictions for features including gene
structure, protein homologies, promoter sites, and repeat elements. We evaluated
these predictions using two standards, one based on previously unreleased
high-quality full-length cDNA sequences and a second based on the set of
annotations generated as part of an in-depth study of the region by a group of
Drosophila experts. Although these standard sets only approximate the unknown
distribution of features in this region, we believe that when taken in context
the results of an evaluation based on them are meaningful. The results were
presented as a tutorial at the conference on Intelligent Systems in Molecular
Biology (ISMB-99) in August 1999. Over 95% of the coding nucleotides in the
region were correctly identified by the majority of the gene finders, and the
correct intron/exon structures were predicted for >40% of the genes.
Homology-based annotation techniques recognized and associated functions with
almost half of the genes in the region; the remainder were only identified by
the ab initio techniques. This experiment also presents the first assessment of
promoter prediction techniques for a significant number of genes in a large
contiguous region. We discovered that the promoter predictors' high
false-positive rates make their predictions difficult to use. Integrating gene
finding and cDNA/EST alignments with promoter predictions decreases the number
of false-positive classifications but discovers less than one-third of the
promoters in the region. We believe that by establishing standards for
evaluating genomic annotations and by assessing the performance of existing
automated genome annotation tools, this experiment establishes a baseline that
contributes to the value of ongoing large-scale annotation projects and should
guide further research in genome informatics. We present an independent comparative analysis of seven recently developed
gene-finding programs: FGENES, GeneMark.hmm, Genie, Genescan, HMMgene, Morgan,
and MZEF. For evaluation purposes we developed a new, thoroughly filtered, and
biologically validated dataset of mammalian genomic sequences that does not
overlap with the training sets of the programs analyzed. Our analysis shows that
the new generation of programs has substantially better results than the
programs analyzed in previous studies. The accuracy of the programs was also
examined as a function of various sequence and prediction features, such as G +
C content of the sequence, length and type of exons, signal type, and score of
the exon prediction. This approach pinpoints the strengths and weaknesses of
each individual program as well as those of computational gene-finding in
general. The dataset used in this analysis (HMR195) as well as the tables with
the complete results are available at http://www.cs.ubc.ca/~rogic/evaluation/. MOTIVATION: Computational gene identification plays an important role in genome
projects. The approaches used in gene identification programs are often tuned to
one particular organism, and accuracy for one organism or class of organism does
not necessarily translate to accurate predictions for other organisms. In this
paper we evaluate five computer programs on their ability to locate coding
regions and to predict gene structure in Neurospora crassa. One of these
programs (FFG) was designed specifically for gene-finding in N.crassa, but the
model parameters have not yet been fully 'tuned', and the program should thus be
viewed as an initial prototype. The other four programs were neither designed
nor tuned for N.crassa.
RESULTS: We describe the data sets on which the experiments were performed, the
approaches employed by the five algorithms: GenScan, HMMGene, GeneMark, Pombe
and FFG, the methodology of our evaluation, and the results of the experiments.
Our results show that, while none of the programs consistently performs well,
overall the GenScan program has the best performance on sensitivity and Missing
Exons (ME) while the HMMGene and FFG programs have good performance in locating
the exons roughly. Additional work motivated by this study includes the creation
of a tool for the automated evaluation of gene-finding programs, the collection
of larger and more reliable data sets for N.crassa, parameterization of the
model used in FFG to produce a more accurate gene-finding program for this
species, and a more in-depth evaluation of the reasons that existing programs
generally fail for N.crassa.
AVAILABILITY: Data sets, the FFG program source code, and links to the other
programs analyzed are available at http://jerry.cs.uga.edu/~wang/genefind.html.
CONTACT: [email protected]. We describe and assess the performance of the gene finding program pretty handy
annotation tool (Phat) on sequence from the malaria parasite Plasmodium
falciparum. Phat is based on a generalized hidden Markov model (GHMM) similar to
the models used in GENSCAN, Genie and HMMgene. In a test set of 44 confirmed
gene structures Phat achieves nucleotide-level sensitivity and specificity of
greater than 95%, performing as well as the other P. falciparum gene finding
programs Hexamer and GlimmerM. Phat is particularly useful for P. falciparum and
other eukaryotes for which there are few gene finding programs available as it
is distributed with code for retraining it on new organisms. Moreover, the full
source code is freely available under the GNU General Public License, allowing
for users to further develop and customize it. MOTIVATION: A growing number of genomes are sequenced. The differences in
evolutionary pattern between functional regions can thus be observed genome-wide
in a whole set of organisms. The diverse evolutionary pattern of different
functional regions can be exploited in the process of genomic annotation. The
modelling of evolution by the existing comparative gene finders leaves room for
improvement.
RESULTS: A probabilistic model of both genome structure and evolution is
designed. This type of model is called an Evolutionary Hidden Markov Model
(EHMM), being composed of an HMM and a set of region-specific evolutionary
models based on a phylogenetic tree. All parameters can be estimated by maximum
likelihood, including the phylogenetic tree. It can handle any number of aligned
genomes, using their phylogenetic tree to model the evolutionary correlations.
The time complexity of all algorithms used for handling the model are linear in
alignment length and genome number. The model is applied to the problem of gene
finding. The benefit of modelling sequence evolution is demonstrated both in a
range of simulations and on a set of orthologous human/mouse gene pairs.
AVAILABILITY: Free availability over the Internet on www server:
http://www.birc.dk/Software/evogene. MOTIVATION: Since the early 1980s of the twentieth century, there has been great
progress in the development of computational gene-finding algorithms. Some
problems, however, have not yet been solved currently. Recognizing short genes
in prokaryotes and short exons in eukaryotes is one of such problems. The paper
is devoted to assessing various algorithms, including those currently available
and the new ones proposed here, in order to find the best algorithm to solve the
issue.
RESULTS: The databases consisting of phase-specific coding and non-coding
sequences of human genes with length of 192, 162, 129, 108, 87, 63 and 42 bp,
respectively, have been established. Based on the databases and a standard
benchmark, 19 algorithms were evaluated, which include the methods of Markov
models with orders of 1 through 5, codon usage, hexamer usage, codon preference,
amino acid usage, codon prototype, Fourier transform and 8 Z curve methods with
various numbers of parameters. Consequently, the Z curve methods with 69 and 189
parameters are the best ones among them, based on the databases constructed
here. In addition to the highest recognition accuracy confirmed by 10-fold
cross-validation tests, the Z curve methods are much simpler computationally
than the second best one, the fifth-order Markov chain model, in which 12 288
parameters are used. We hope that the Z curve methods presented in this paper
would be beneficial to the further development of gene-finding algorithms.
AVAILABILITY: The programs of various Z curve methods are available on request. The performance of gene-predicting tools varies considerably if evaluated with
respect to the parameters sensitivity and specificity or their capability to
identify the correct start codon. We were interested to validate tools for gene
prediction and to implement a metatool named YACOP, which combines existing
tools and has a higher performance. YACOP parses and combines the output of the
three gene-predicting systems Criticia, Glimmer and ZCURVE. It outperforms each
of the programs tested with its high sensitivity and specificity values combined
with a larger number of correctly predicted gene starts. Performance of YACOP
and the gene-finding programs was tested by comparing their output with a
carefully selected set of annotated genomes. We found that the problem of
identifying genes in prokaryotic genomes by means of computational analysis was
solved satisfactorily. In contrast, the correct localization of the start codon
still appeared to be a problem, as in all cases under test at least 7.8% and up
to 32.3% of the positions given in the annotations differed from the locus
predicted by any of the programs tested. YACOP can be downloaded from
http://www.g2l.bio.uni-goettingen.de. BACKGROUND: Generalized hidden Markov models (GHMMs) appear to be approaching
acceptance as a de facto standard for state-of-the-art ab initio gene finding,
as evidenced by the recent proliferation of GHMM implementations. While
prevailing methods for modeling and parsing genes using GHMMs have been
described in the literature, little attention has been paid as of yet to their
proper training. The few hints available in the literature together with
anecdotal observations suggest that most practitioners perform maximum
likelihood parameter estimation only at the local submodel level, and then
attend to the optimization of global parameter structure using some form of ad
hoc manual tuning of individual parameters.
RESULTS: We decided to investigate the utility of applying a more systematic
optimization approach to the tuning of global parameter structure by
implementing a global discriminative training procedure for our GHMM-based gene
finder. Our results show that significant improvement in prediction accuracy can
be achieved by this method.
CONCLUSIONS: We conclude that training of GHMM-based gene finders is best
performed using some form of discriminative training rather than simple maximum
likelihood estimation at the submodel level, and that generalized gradient
ascent methods are suitable for this task. We also conclude that partitioning of
training data for the twin purposes of maximum likelihood initialization and
gradient ascent optimization appears to be unnecessary, but that strict
segregation of test data must be enforced during final gene finder evaluation to
avoid artificially inflated accuracy measurements. BACKGROUND: Despite the continuous production of genome sequence for a number of
organisms, reliable, comprehensive, and cost effective gene prediction remains
problematic. This is particularly true for genomes for which there is not a
large collection of known gene sequences, such as the recently published chicken
genome. We used the chicken sequence to test comparative and homology-based
gene-finding methods followed by experimental validation as an effective genome
annotation method.
RESULTS: We performed experimental evaluation by RT-PCR of three different
computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken
genome. A Venn diagram was computed and each component of it was evaluated. The
results showed that de novo comparative methods can identify up to about 700
chicken genes with no previous evidence of expression, and can correctly extend
about 40% of homology-based predictions at the 5' end.
CONCLUSIONS: De novo comparative gene prediction followed by experimental
verification is effective at enhancing the annotation of the newly sequenced
genomes provided by standard homology-based methods. Finding new protein-coding genes is one of the most important goals of
eukaryotic genome sequencing projects. However, genomic organization of novel
eukaryotic genomes is diverse and ab initio gene finding tools tuned up for
previously studied species are rarely suitable for efficacious gene hunting in
DNA sequences of a new genome. Gene identification methods based on cDNA and
expressed sequence tag (EST) mapping to genomic DNA or those using alignments to
closely related genomes rely either on existence of abundant cDNA and EST data
and/or availability on reference genomes. Conventional statistical ab initio
methods require large training sets of validated genes for estimating gene model
parameters. In practice, neither one of these types of data may be available in
sufficient amount until rather late stages of the novel genome sequencing.
Nevertheless, we have shown that gene finding in eukaryotic genomes could be
carried out in parallel with statistical models estimation directly from yet
anonymous genomic DNA. The suggested method of parallelization of gene
prediction with the model parameters estimation follows the path of the
iterative Viterbi training. Rounds of genomic sequence labeling into coding and
non-coding regions are followed by the rounds of model parameters estimation.
Several dynamically changing restrictions on the possible range of model
parameters are added to filter out fluctuations in the initial steps of the
algorithm that could redirect the iteration process away from the biologically
relevant point in parameter space. Tests on well-studied eukaryotic genomes have
shown that the new method performs comparably or better than conventional
methods where the supervised model training precedes the gene prediction step.
Several novel genomes have been analyzed and biologically interesting findings
are discussed. Thus, a self-training algorithm that had been assumed feasible
only for prokaryotic genomes has now been developed for ab initio eukaryotic
gene identification. BACKGROUND: The ENCODE gene prediction workshop (EGASP) has been organized to
evaluate how well state-of-the-art automatic gene finding methods are able to
reproduce the manual and experimental gene annotation of the human genome. We
have used Softberry gene finding software to predict genes, pseudogenes and
promoters in 44 selected ENCODE sequences representing approximately 1% (30 Mb)
of the human genome. Predictions of gene finding programs were evaluated in
terms of their ability to reproduce the ENCODE-HAVANA annotation.
RESULTS: The Fgenesh++ gene prediction pipeline can identify 91% of coding
nucleotides with a specificity of 90%. Our automatic pseudogene finder (PSF
program) found 90% of the manually annotated pseudogenes and some new ones. The
Fprom promoter prediction program identifies 80% of TATA promoters sequences
with one false positive prediction per 2,000 base-pairs (bp) and 50% of
TATA-less promoters with one false positive prediction per 650 bp. It can be
used to identify transcription start sites upstream of annotated coding parts of
genes found by gene prediction software.
CONCLUSION: We review our software and underlying methods for identifying these
three important structural and functional genome components and discuss the
accuracy of predictions, recent advances and open problems in annotating genomic
sequences. We have demonstrated that our methods can be effectively used for
initial automatic annotation of the eukaryotic genome. BACKGROUND: A large number of gene prediction programs for the human genome
exist. These annotation tools use a variety of methods and data sources. In the
recent ENCODE genome annotation assessment project (EGASP), some of the most
commonly used and recently developed gene-prediction programs were
systematically evaluated and compared on test data from the human genome.
AUGUSTUS was among the tools that were tested in this project.
RESULTS: AUGUSTUS can be used as an ab initio program, that is, as a program
that uses only one single genomic sequence as input information. In addition, it
is able to combine information from the genomic sequence under study with
external hints from various sources of information. For EGASP, we used genomic
sequence alignments as well as alignments to expressed sequence tags (ESTs) and
protein sequences as additional sources of information. Within the category of
ab initio programs AUGUSTUS predicted significantly more genes correctly than
any other ab initio program. At the same time it predicted the smallest number
of false positive genes and the smallest number of false positive exons among
all ab initio programs. The accuracy of AUGUSTUS could be further improved when
additional extrinsic data, such as alignments to EST, protein and/or genomic
sequences, was taken into account.
CONCLUSION: AUGUSTUS turned out to be the most accurate ab initio gene finder
among the tested tools. Moreover it is very flexible because it can take
information from several sources simultaneously into consideration. BACKGROUND: Predicting complete protein-coding genes in human DNA remains a
significant challenge. Though a number of promising approaches have been
investigated, an ideal suite of tools has yet to emerge that can provide near
perfect levels of sensitivity and specificity at the level of whole genes. As an
incremental step in this direction, it is hoped that controlled gene finding
experiments in the ENCODE regions will provide a more accurate view of the
relative benefits of different strategies for modeling and predicting gene
structures.
RESULTS: Here we describe our general-purpose eukaryotic gene finding pipeline
and its major components, as well as the methodological adaptations that we
found necessary in accommodating human DNA in our pipeline, noting that a
similar level of effort may be necessary by ourselves and others with similar
pipelines whenever a new class of genomes is presented to the community for
analysis. We also describe a number of controlled experiments involving the
differential inclusion of various types of evidence and feature states into our
models and the resulting impact these variations have had on predictive
accuracy.
CONCLUSION: While in the case of the non-comparative gene finders we found that
adding model states to represent specific biological features did little to
enhance predictive accuracy, for our evidence-based 'combiner' program the
incorporation of additional evidence tracks tended to produce significant gains
in accuracy for most evidence types, suggesting that improved modeling efforts
at the hidden Markov model level are of relatively little value. We relate these
findings to our current plans for future research. MOTIVATION: The numbers of finished and ongoing genome projects are increasing
at a rapid rate, and providing the catalog of genes for these new genomes is a
key challenge. Obtaining a set of well-characterized genes is a basic
requirement in the initial steps of any genome annotation process. An accurate
set of genes is needed in order to learn about species-specific properties, to
train gene-finding programs, and to validate automatic predictions.
Unfortunately, many new genome projects lack comprehensive experimental data to
derive a reliable initial set of genes.
RESULTS: In this study, we report a computational method, CEGMA (Core Eukaryotic
Genes Mapping Approach), for building a highly reliable set of gene annotations
in the absence of experimental data. We define a set of conserved protein
families that occur in a wide range of eukaryotes, and present a mapping
procedure that accurately identifies their exon-intron structures in a novel
genomic sequence. CEGMA includes the use of profile-hidden Markov models to
ensure the reliability of the gene structures. Our procedure allows one to build
an initial set of reliable gene annotations in potentially any eukaryotic
genome, even those in draft stages.
AVAILABILITY: Software and data sets are available online at
http://korflab.ucdavis.edu/Datasets. Predicting protein-coding genes still remains a significant challenge. Although
a variety of computational programs that use commonly machine learning methods
have emerged, the accuracy of predictions remains a low level when implementing
in large genomic sequences. Moreover, computational gene finding in newly
sequenced genomes is especially a difficult task due to the absence of a
training set of abundant validated genes. Here we present a new gene-finding
program, SCGPred, to improve the accuracy of prediction by combining multiple
sources of evidence. SCGPred can perform both supervised method in previously
well-studied genomes and unsupervised one in novel genomes. By testing with
datasets composed of large DNA sequences from human and a novel genome of
Ustilago maydi, SCG-Pred gains a significant improvement in comparison to the
popular ab initio gene predictors. We also demonstrate that SCGPred can
significantly improve prediction in novel genomes by combining several foreign
gene finders with similarity alignments, which is superior to other unsupervised
methods. Therefore, SCG-Pred can serve as an alternative gene-finding tool for
newly sequenced eukaryotic genomes. The program is freely available at
http://bio.scu.edu.cn/SCGPred/. We present a highly accurate gene-prediction system for eukaryotic genomes,
called mGene. It combines in an unprecedented manner the flexibility of
generalized hidden Markov models (gHMMs) with the predictive power of modern
machine learning methods, such as Support Vector Machines (SVMs). Its excellent
performance was proved in an objective competition based on the genome of the
nematode Caenorhabditis elegans. Considering the average of sensitivity and
specificity, the developmental version of mGene exhibited the best prediction
performance on nucleotide, exon, and transcript level for ab initio and
multiple-genome gene-prediction tasks. The fully developed version shows
superior performance in 10 out of 12 evaluation criteria compared with the other
participating gene finders, including Fgenesh++ and Augustus. An in-depth
analysis of mGene's genome-wide predictions revealed that approximately 2200
predicted genes were not contained in the current genome annotation. Testing a
subset of 57 of these genes by RT-PCR and sequencing, we confirmed expression
for 24 (42%) of them. mGene missed 300 annotated genes, out of which 205 were
unconfirmed. RT-PCR testing of 24 of these genes resulted in a success rate of
merely 8%. These findings suggest that even the gene catalog of a well-studied
organism such as C. elegans can be substantially improved by mGene's
predictions. We also provide gene predictions for the four nematodes C.
briggsae, C. brenneri, C. japonica, and C. remanei. Comparing the resulting
proteomes among these organisms and to the known protein universe, we identified
many species-specific gene inventions. In a quality assessment of several
available annotations for these genomes, we find that mGene's predictions are
most accurate. This unit describes how to use several gene-finding programs from the GeneMark
line developed for finding protein-coding ORFs in genomic DNA of prokaryotic
species, in genomic DNA of eukaryotic species with intronless genes, in genomes
of viruses and phages, and in prokaryotic metagenomic sequences, as well as in
EST sequences with spliced-out introns. These bioinformatics tools were
demonstrated to have state-of-the-art accuracy and have been frequently used for
gene annotation in novel nucleotide sequences. An additional advantage of these
sequence-analysis tools is that the problem of algorithm parameterization is
solved automatically, with parameters estimated by iterative self-training
(unsupervised training). This unit describes how to use the gene-finding programs GeneMark.hmm-E and
GeneMark-ES for finding protein-coding genes in the genomic DNA of eukaryotic
organisms. These bioinformatics tools have been demonstrated to have
state-of-the-art accuracy for many fungal, plant, and animal genomes, and have
frequently been used for gene annotation in novel genomic sequences. An
additional advantage of GeneMark-ES is that the problem of algorithm
parameterization is solved automatically, with parameters estimated by iterative
self-training (unsupervised training). |
Which are the major transcription factors regulating glycolysis in mammals? | The main positive transcriptional regulator of Glycolysis in mammals is HIF1-alpha (Hypoxia Inducible Factor 1a). HIF1-alpha is upregulated by the oncogenes c-Myc and Src, which therefore also positively regulate glycolysis. Several reports have linked HIF-1α induction with STAT3 activation. SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha. NF-κB also stimulates glycolysis through the inhibition of PDK4 expression. Loss of the widely expressed transcription factor Oct1 induces a coordinated metabolic shift: mitochondrial activity and amino acid oxidation are increased, while glucose metabolism is reduced. HNF4alpha is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis. | The product of the c-myc proto-oncogene (c-Myc) is involved in the control of
cell proliferation, differentiation, and apoptosis. It acts as a transcription
factor that recognizes the CACGTG motif. This sequence has also been found in
the glucose-responsive elements of genes involved in the control of liver
glycolysis and lipogenesis. To determine whether c-Myc can regulate hepatic
carbohydrate metabolism in vivo, transgenic mice that overexpress c-myc under
control of the P-enolpyruvate carboxykinase (PEPCK) gene promoter have been
generated. These mice showed a threefold increase in c-Myc protein in liver
nuclei. Hepatocytes from transgenic mice were normal and did not acquire the
fetal phenotype. However, transgenic mice showed higher levels (threefold) of
L-type pyruvate kinase mRNA and enzyme activity than control mice. The increase
in pyruvate kinase activity led to a three- to fivefold increase in liver
lactate content and a fivefold induction of lactate production by hepatocytes in
primary culture. The expression of the 6-phosphofructo-2-kinase gene was also
increased in the liver of these transgenic mice. The induction of hepatic
glycolysis was related with an increase in the expression (about fourfold) and
activity (about threefold) of liver glucokinase, whereas no change was noted in
hexokinase-I. This change in glucokinase activity led to an increase in both
glucose 6-phosphate and glycogen contents in the liver of transgenic mice. The
expression of the liver-specific glucose transporter GLUT2 was also increased in
transgenic mice, whereas no change was noted in the mRNA concentration of GLUT1.
Furthermore, the changes of liver glucose metabolism led to a marked reduction
of blood glucose (25%) and insulin (40%) concentrations in starvation, whereas
the fall in both was only 10% in fed mice. Thus, liver glucose metabolism could
determine the blood glucose and insulin set points in the transgenic mice. All
these results indicated that the increase in c-Myc protein was able to induce
liver glucose utilization and accumulation, and suggested that c-Myc
transcription factor is involved in the control in vivo of liver carbohydrate
metabolism. Hepatocyte nuclear factor 4alpha (HNF4alpha) plays a critical role in regulating
the expression of many genes essential for normal functioning of liver, gut,
kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the
HNF4alpha gene is responsible for an autosomal domit, early-onset form of
non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young;
gene named MODY1). Although this mutation is predicted to delete 187 C-terminal
amino acids of the HNF4alpha protein the molecular mechanism by which it causes
diabetes is unknown. To address this, we first studied the functional properties
of the MODY1 mutant protein. We show that it has lost its transcriptional
transactivation activity, fails to dimerize and bind DNA, implying that the
MODY1 phenotype is because of a loss of HNF4alpha function. The effect of loss
of function on HNF4alpha target gene expression was investigated further in
embryonic stem cells, which are amenable to genetic manipulation and can be
induced to form visceral endoderm. Because the visceral endoderm shares many
properties with the liver and pancreatic beta-cells, including expression of
genes for glucose transport and metabolism, it offers an ideal system to
investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting
this system we have identified several genes encoding components of the
glucose-dependent insulin secretion pathway whose expression is dependent upon
HNF4alpha. These include glucose transporter 2, and the glycolytic enzymes
aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate
kinase. In addition we have found that expression of the fatty acid binding
proteins and cellular retinol binding protein also are down-regulated in the
absence of HNF4alpha. These data provide direct evidence that HNF4alpha is
critical for regulating glucose transport and glycolysis and in doing so is
crucial for maintaining glucose homeostasis. Unlike normal mammalian cells, which use oxygen to generate energy, cancer cells
rely on glycolysis for energy and are therefore less dependent on oxygen. We
previously observed that the c-Myc oncogenic transcription factor regulates
lactate dehydrogenase A and induces lactate overproduction. We, therefore,
sought to determine whether c-Myc controls other genes regulating glucose
metabolism. In Rat1a fibroblasts and murine livers overexpressing c-Myc, the
mRNA levels of the glucose transporter GLUT1, phosphoglucose isomerase,
phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate
kinase, and enolase were elevated. c-Myc directly transactivates genes encoding
GLUT1, phosphofructokinase, and enolase and increases glucose uptake in Rat1
fibroblasts. Nuclear run-on studies confirmed that the GLUT1 transcriptional
rate is elevated by c-Myc. Our findings suggest that overexpression of the c-Myc
oncoprotein deregulates glycolysis through the activation of several components
of the glucose metabolic pathway. Hypoxia-inducible factor (HIF)-1 is a master transcription factor, which
up-regulates glycolysis, erythropoiesis, and angiogenesis under hypoxia.
HIF-1alpha accumulates in normoxic tumor cells, leading to glycolysis under
aerobic conditions. This phenomenon, known as the "Warburg effect," is caused by
a yet unknown mechanism. Here we show that transformed cells that express
constitutively active pp60(c-Src) (Src) express HIF-1alpha protein under
normoxia, which results in the expression of multiple HIF-1alpha target genes.
We show that this occurrence is due to an enhanced rate of HIF-1alpha protein
synthesis and not due to reduced HIF-1alpha degradation. Furthermore, we show
that the Src-induced increase in protein synthesis is due to the global increase
in the rate of cap-dependent translation and does not involve inhibition of
HIF-1alpha degradation. Enzymes required for de novo lipogenesis are induced in mammalian liver after a
meal high in carbohydrates. In addition to insulin, increased glucose metabolism
initiates an intracellular signaling pathway that transcriptionally regulates
genes encoding lipogenic enzymes. A cis-acting sequence, the carbohydrate
response element (ChoRE), has been found in the promoter region of several of
these genes. ChREBP (carbohydrate response element-binding protein) was recently
identified as a candidate transcription factor in the glucose-signaling pathway.
We reported that ChREBP requires the heterodimeric partner Max-like factor X
(Mlx) to bind to ChoRE sequences. In this study we provide further evidence to
support a direct role of Mlx in glucose signaling in the liver. We constructed
two different domit negative forms of Mlx that could dimerize with ChREBP but
block its binding to DNA. When introduced into hepatocytes, both domit
negative forms of Mlx inhibited the glucose response of a transfected
ChoRE-containing promoter. The glucose response was rescued by adding exogenous
wild type Mlx or ChREBP, but not MondoA, a paralog of ChREBP that can also form
a heterodimer with Mlx. Furthermore, domit negative Mlx blocked the induction
of glucose-responsive genes from their natural chromosomal context under high
glucose conditions. In contrast, genes induced by the insulin and thyroid
hormone-signaling pathways were unaffected by domit negative Mlx. Mlx was
present in the glucose-responsive complex of liver nuclear extract from which
ChREBP was purified. In conclusion, Mlx is an obligatory partner of ChREBP in
regulating lipogenic enzyme genes in liver. The nucleus pulposus is an aggrecan-rich, avascular tissue that permits the
intervertebral disk to resist compressive loads. In the disk, nucleus pulposus
cells express hypoxia-inducible factor (HIF)-1alpha, a transcription factor that
responds to oxygen tension and regulates glycolysis. The goal of the present
study was to examine the importance of HIF-1alpha in rat nucleus pulposus cells
and to probe the function of this transcription factor in terms of regulating
aggrecan gene expression. We found that HIF-1alpha protein levels and mRNA
stability were similar at 20 and 2% O(2); there was a small, but significant
increase in HIF-1alpha transactivation domain activity in hypoxia. With respect
to HIF-1alpha target genes GAPDH, GLUT-1, and GLUT-3, mRNA and protein levels
were independent of the oxygen tension. Other than a modest increase in
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase reporter activity, the
oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities.
Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted
in a significant suppression in ATP synthesis in normoxia, whereas treatment
with mitochondrial inhibitors did not affect ATP production and cell viability.
However, measurement of the rate of fatty acid oxidation indicated that these
cells contained functioning mitochondria. Finally, we showed that when
HIF-1alpha was suppressed, irrespective of the oxemic state, there was a partial
loss of aggrecan expression and promoter activity. Moreover, when cells were
treated with 2-DG, there was inhibition in aggrecan promoter activity. Results
of this study indicate that oxygen-independent stabilization of HIF-1alpha in
nucleus pulposus cells is a metabolic adaptation that drives glycolysis and
aggrecan expression. Cancer cells use aerobic glycolysis preferentially for energy provision and this
metabolic change is important for tumour growth. Here, we have found a link
between the tumour suppressor p53, the transcription factor NF-kappaB and
glycolysis. In p53-deficient primary cultured cells, kinase activities of
IKKalpha and IKKbeta and subsequent NF-kappaB activity were enhanced. Activation
of NF-kappaB, by loss of p53, caused an increase in the rate of aerobic
glycolysis and upregulation of Glut3. Oncogenic Ras-induced cell transformation
and acceleration of aerobic glycolysis in p53-deficient cells were suppressed in
the absence of p65/NF-kappaB expression, and were restored by GLUT3 expression.
It was also shown that a glycolytic inhibitor diminished the enhanced IKK
activity in p53-deficient cells. Moreover, in Ras-expressing p53-deficient
cells, IKK activity was suppressed by p65 deficiency and restored by GLUT3
expression. Taken together, these data indicate that p53 restricts activation of
the IKK-NF-kappaB pathway through suppression of glycolysis. These results
suggest that a positive-feedback loop exists, whereby glycolysis drives
IKK-NF-kappaB activation, and that hyperactivation of this loop by loss of p53
is important in oncogene-induced cell transformation. Insulin secretion from pancreatic beta cells is stimulated by glucose
metabolism. However, the relative importance of metabolizing glucose via
mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion
remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL,
negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor
implicated in promoting a glycolytic form of metabolism. Here we report a
central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose
utilization, insulin secretion, and glucose homeostasis. Conditional
inactivation of Vhlh in beta cells promoted a diversion of glucose away from
mitochondria into lactate production, causing cells to produce high levels of
glycolytically derived ATP and to secrete elevated levels of insulin at low
glucose concentrations. Vhlh-deficient mice exhibited diminished
glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical
activity, and insulin secretion, which culminate in impaired systemic glucose
tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these
phenotypes, implying that they are the result of HIF1alpha activation. Together,
these results identify pVHL and HIF1alpha as key regulators of insulin secretion
from pancreatic beta cells. They further suggest that changes in the metabolic
strategy of glucose metabolism in beta cells have profound effects on whole-body
glucose homeostasis. Cancer cells frequently undergo a shift from oxidative to glycolytic metabolism.
Although there is interest in targeting metabolism as a form of cancer therapy,
this area still remains in its infancy. Using cells, embryos and adult animals,
we show here that loss of the widely expressed transcription factor Oct1 induces
a coordinated metabolic shift: mitochondrial activity and amino acid oxidation
are increased, while glucose metabolism is reduced. Altered expression of direct
Oct1 targets encoding metabolic regulators provides a mechanistic underpinning
to these results. We show that these metabolic changes directly oppose
tumorigenicity. Collectively, our findings show that Oct1, the genes it
regulates and the pathways these genes affect could be used as targets for new
modes of cancer therapy. Although cancers have altered glucose metabolism, termed the Warburg effect,
which describes the increased uptake and conversion of glucose to lactate by
cancer cells under adequate oxygen tension, changes in the metabolism of
glutamine and fatty acid have also been documented. The MYC oncogene, which
contributes to the genesis of many human cancers, encodes a transcription factor
c-Myc, which links altered cellular metabolism to tumorigenesis. c-Myc regulates
genes involved in the biogenesis of ribosomes and mitochondria, and regulation
of glucose and glutamine metabolism. With E2F1, c-Myc induces genes involved in
nucleotide metabolism and DNA replication, and microRNAs that homeostatically
attenuate E2F1 expression. With the hypoxia inducible transcription factor
HIF-1, ectopic c-Myc cooperatively induces a transcriptional program for hypoxic
adaptation. Myc regulates gene expression either directly, such as glycolytic
genes including lactate dehydrogenase A (LDHA), or indirectly, such as
repression of microRNAs miR-23a/b to increase glutaminase (GLS) protein
expression and glutamine metabolism. Ectopic MYC expression in cancers,
therefore, could concurrently drive aerobic glycolysis and/or oxidative
phosphorylation to provide sufficient energy and anabolic substrates for cell
growth and proliferation in the context of the tumor microenvironment.
Collectively, these studies indicate that Myc-mediated altered cancer cell
energy metabolism could be translated for the development of new anticancer
therapies. SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases
with various roles in metabolism, stress resistance, and life span.
SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early
in life; however, the mechanism underlying this hypoglycemia remained unclear.
Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to
control the expression of multiple glycolytic genes. Specifically, SIRT6 appears
to function as a corepressor of the transcription factor Hif1alpha, a critical
regulator of nutrient stress responses. Consistent with this notion,
SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased
glucose uptake with upregulation of glycolysis and diminished mitochondrial
respiration. Our studies uncover a role for the chromatin factor SIRT6 as a
master regulator of glucose homeostasis and may provide the basis for novel
therapeutic approaches against metabolic diseases, such as diabetes and obesity. Cancer cells predomitly utilize glycolysis for ATP production even in the
presence of abundant oxygen, an environment that would normally result in energy
production through oxidative phosphorylation. Although the molecular mechanism
for this metabolic switch to aerobic glycolysis has not been fully elucidated,
it is likely that mitochondrial damage to the electron transport chain and the
resulting increased production of reactive oxygen species are significant
driving forces. In this study, we have investigated the role of the
transcription factor Ets-1 in the regulation of mitochondrial function and
metabolism. Ets-1 was over-expressed using a stably-incorporated
tetracycline-inducible expression vector in the ovarian cancer cell line 2008,
which does not express detectable basal levels of Ets-1 protein. Microarray
analysis of the effects of Ets-1 over-expression in these ovarian cancer cells
shows that Ets-1 up-regulates key enzymes involved in glycolysis and associated
feeder pathways, fatty acid metabolism, and antioxidant defense. In contrast,
Ets-1 down-regulates genes involved in the citric acid cycle, electron transport
chain, and mitochondrial proteins. At the functional level, we have found that
Ets-1 expression is directly correlated with cellular oxygen consumption whereby
increased expression causes decreased oxygen consumption. Ets-1 over-expression
also caused increased sensitivity to glycolytic inhibitors, as well as growth
inhibition in a glucose-depleted culture environment. Collectively our findings
demonstrate that Ets-1 is involved in the regulation of cellular metabolism and
response to oxidative stress in ovarian cancer cells. CRP (cAMP receptor protein), the global regulator of genes for carbon source
utilization in the absence of glucose, is the best-studied prokaryotic
transcription factor. A total of 195 target promoters on the Escherichia coli
genome have been proposed to be under the control of cAMP-bound CRP. Using the
newly developed Genomic SELEX screening system of transcription factor-binding
sequences, however, we have identified a total of at least 254 CRP-binding
sites. Based on their location on the E. coli genome, we predict a total of at
least 183 novel regulation target operons, altogether with the 195 hitherto
known targets, reaching to the minimum of 378 promoters as the regulation
targets of cAMP-CRP. All the promoters selected from the newly identified
targets and examined by using the lacZ reporter assay were found to be under the
control of CRP, indicating that the Genomic SELEX screening allowed to identify
the CRP targets with high accuracy. Based on the functions of novel target
genes, we conclude that CRP plays a key regulatory role in the whole processes
from the selective transport of carbon sources, the glycolysis-gluconeogenesis
switching to the metabolisms downstream of glycolysis, including tricarboxylic
acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration.
One unique regulation mode is that a single and the same CRP molecule bound
within intergenic regions often regulates both of divergently transcribed
operons. Hypoxia-inducible factor-1α (HIF-1α), which is a transcription factor that
enhances glycolysis in cells in response to hypoxia, is induced in hypertrophied
adipocytes in obesity. Recent studies have shown that growth factors are able to
induce HIF-1α by mechanisms independent of hypoxia. Since basic fibroblast
growth factor (bFGF), an angiogenic factor, is concentrated in expanding adipose
tissue, the possible effects of bFGF on regulation of HIF-1α in adipocytes were
investigated. Treatment of differentiated 3T3-L1 adipocytes with bFGF induced
HIF-1α. Concomitantly, glucose transporter 1 (GLUT1), which is a target gene of
HIF-1α, was induced at both mRNA and protein levels and was translocated to the
plasma membrane. A chromatin immunoprecipitation assay and an RNA interference
study indicated that bFGF-induced HIF-1α directly upregulates GLUT1. In
addition, it was observed that bFGF increases lactate production of adipocytes.
This result indicates that bFGF reprograms the metabolism toward glycolysis.
Intraperitoneal injection of bFGF into mice upregulated HIF-1α and GLUT1 in
adipose tissues, suggesting that bFGF regulates the metabolism of adipocytes via
HIF-1α-GLUT1 regulation in vivo. We also found that bFGF inhibits
insulin-induced phosphorylation of insulin receptor substrate-1 and Akt,
suggesting that bFGF attenuates the insulin signal in adipocytes. Taken
together, the findings suggest that bFGF has a harmful effect on the development
of type 2 diabetes through metabolism reprogramming and attenuation of the
insulin signal. Cell proliferation is a metabolically demanding process. It requires active
reprogramming of cellular bioenergetic pathways towards glucose metabolism to
support anabolic growth. NF-κB/Rel transcription factors coordinate many of the
signals that drive proliferation during immunity, inflammation and oncogenesis,
but whether NF-κB regulates the metabolic reprogramming required for cell
division during these processes is unknown. Here, we report that NF-κB organizes
energy metabolism networks by controlling the balance between the utilization of
glycolysis and mitochondrial respiration. NF-κB inhibition causes cellular
reprogramming to aerobic glycolysis under basal conditions and induces necrosis
on glucose starvation. The metabolic reorganization that results from NF-κB
inhibition overcomes the requirement for tumour suppressor mutation in oncogenic
transformation and impairs metabolic adaptation in cancer in vivo. This
NF-κB-dependent metabolic pathway involves stimulation of oxidative
phosphorylation through upregulation of mitochondrial synthesis of cytochrome c
oxidase 2 (SCO2; ref. ). Our findings identify NF-κB as a physiological
regulator of mitochondrial respiration and establish a role for NF-κB in
metabolic adaptation in normal cells and cancer. The concept that invasive cancer is associated with increased levels of reactive
oxygen species (ROS) generated by mitochondria is consistent with an
ROS-mediated signaling mechanism. As a tumor grows, it encounters adverse
microenvironments, one of which is low oxygen (hypoxia), which selects tumor
cells with characteristics of increased invasiveness. Hypoxic environments
select for tumor cells with stabilized HIF1 apha, a transcription factor that
regulates genes coding for pro-tumor cytokines that signal stromal cells such as
macrophages and fibroblasts to support an invasive tumor cell phenotype. HIF1
alpha-mediated switches in the energy production of tumor cells from OXPHOS to
glycolysis, as well as age-associated decreases in the metabolic rate of the
host, enhance invasive qualities of tumor cells. An increase in environmental
oxygen in combination with a mitochondrial targeted catalase mimetic and a
metabolism booster may be of interest to investigate as a treatment strategy for
invasive cancer. Although tyrosine-phosphorylated or activated STAT3 (pY-STAT3) is a
well-described mediator of tumorigenesis, its role in thyroid cancer has not
been investigated. We observed that 63 of 110 (57%) human primary papillary
thyroid carcinoma (PTC) cases expressed nuclear pY-STAT3 in tumor cells,
preferentially in association with the tumor stroma. An inverse relationship
between pY-STAT3 expression with tumor size and the presence of distant
metastases was observed. Using human thyroid cancer-derived cell lines
[harboring rearranged during transfection (RET)/PTC, v-RAF murine sarcoma viral
oncogene homolog B (BRAF), or rat sarcoma virus oncogene (RAS) alterations], we
determined that IL-6/gp130/JAK signaling is responsible for STAT3 activation.
STAT3 knockdown by shRNA in representative thyroid cancer cell lines that
express high levels of pY-STAT3 had no effect on in vitro growth. However,
xenografted short hairpin STAT3 cells generated larger tumors than control
cells. Similarly, STAT3 deficiency in a murine model of BRAFV600E-induced PTC
led to thyroid tumors that were more proliferative and larger than those tumors
expressing STAT3wt. Genome expression analysis revealed that STAT3 knockdown
resulted in the down-regulation of multiple transcripts, including the tumor
suppressor insulin-like growth factor binding protein 7. Furthermore, STAT3
knockdown led to an increase in glucose consumption, lactate production, and
expression of Hypoxia-inducible factor 1 (HIF1α) target genes, suggesting that
STAT3 is a negative regulator of aerobic glycolysis. Our studies show that, in
the context of thyroid cancer, STAT3 is paradoxically a negative regulator of
tumor growth. These findings suggest that targeting STAT3 in these cancers could
enhance tumor size and highlight the complexities of the role of STAT3 in
tumorigenesis. As a hallmark of tumor cells, metabolic alterations play a critical role in
tumor development and could be targeted for tumor therapy. Tumor suppressor p53
plays a central role in tumor prevention. As a transcription factor, p53 mainly
exerts its function in tumor suppression through its transcriptional regulation
of its target genes to initiate various cellular responses. Cell cycle arrest,
apoptosis and senescence are most well-understood functions of p53, and are
traditionally accepted as the major mechanisms for p53 in tumor suppression.
Recent studies have revealed a novel function of p53 in regulation of cellular
metabolism. p53 regulates mitochondrial oxidative phosphorylation, glycolysis,
glutamine metabolism, lipid metabolism, and antioxidant defense. Through the
regulation of these metabolic processes, p53 maintains the homeostasis of
cellular metabolism and redox balance in cells, which contributes significantly
to the role of p53 as a tumor suppressor. Further understanding of the role and
molecular mechanism of p53 in cellular metabolism could lead to the
identification of novel targets and development of novel strategies for tumor
therapy. |
Mutations in which gene determine response to both erlotinib and gefitinib? | Patients who carry somatic activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene, respond well to erlotinib and gefitinib. | BACKGROUND: Lung adenocarcinomas from patients who respond to the tyrosine
kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor
somatic gain-of-function mutations in exons encoding the kinase domain of the
epidermal growth factor receptor (EGFR). Despite initial responses, patients
eventually progress by unknown mechanisms of "acquired" resistance.
METHODS AND FINDINGS: We show that in two of five patients with acquired
resistance to gefitinib or erlotinib, progressing tumors contain, in addition to
a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20,
which leads to substitution of methionine for threonine at position 790 (T790M)
in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive
EGFR mutation whose tumor progressed on adjuvant gefitinib after complete
resection also contained the T790M mutation. This mutation was not detected in
untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS
mutations, which have been associated with primary resistance to these drugs.
Biochemical analyses of transfected cells and growth inhibition studies with
lung cancer cell lines demonstrate that the T790M mutation confers resistance to
EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly,
a mutation analogous to T790M has been observed in other kinases with acquired
resistance to another kinase inhibitor, imatinib (Gleevec).
CONCLUSION: In patients with tumors bearing gefitinib- or erlotinib-sensitive
EGFR mutations, resistant subclones containing an additional EGFR mutation
emerge in the presence of drug. This observation should help guide the search
for more effective therapy against a specific subset of lung cancers. Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor
receptor (EGFR) gene in non-small cell lung cancers are associated with
increased sensitivity of these cancers to drugs that inhibit EGFR kinase
activity such as gefitinib and erlotinib. Responses to TK inhibitors in the
absence of EGFR gene mutation for BAC patients have not been reported. A case of
a patient with BAC refractory to chemotherapy who responded to gefitinib in the
absence of EGFR gene mutations is reported. Tyrosine kinase inhibitors may have
a role in BAC in the absence of EGFR gene mutations. Additional studies on other
molecular alterations of the EGFR family members are needed to better predict
response to these agents. Somatic mutations in the tyrosine kinase domain of the epidermal growth factor
receptor (EGFR) gene are present in lung adenocarcinomas that respond to the
EGFR inhibitors gefitinib and erlotinib. Two types of mutations account for
approximately 90% of mutated cases: short in-frame deletions in exon 19 and a
specific point mutation in exon 21 at codon 858 (L858R). Screening for these
mutations has been based mainly on direct sequencing. We report here the
development and validation of polymerase chain reaction-based assays for these
two predomit types of EGFR mutations. The assay for exon 19 mutations is
based on length analysis of fluorescently labeled polymerase chain reaction
products, and the assay for the exon 21 L858R mutation is based on a new Sau96I
restriction site created by this mutation. Using serial dilutions of DNAs from
lung cancer cell lines harboring either exon 19 or 21 mutations, we detected
these mutations in the presence of up to approximately 90% normal DNA. In a test
set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases
whereas our assays were positive in 29 cases, including 4 cases in which
mutations were not apparent by sequencing. These assays offer higher sensitivity
and ease of scoring and eliminate the need for sequencing, providing a robust
and accessible approach to the rapid identification of most lung cancer patients
likely to respond to EGFR inhibitors. Epidermal growth factor receptor (EGFR) gene mutations have been found in a
subset of non-small cell lung cancer (NSCLC) with good clinical response to
gefitinib therapy. A quick and sensitive method with large throughput is
required to utilize the information to determine whether the molecular targeted
therapy should be applied for the particular NSCLC patients. Using probes for
the 13 different mutations including 11 that have already been reported, we have
genotyped the EGFR mutation status in 94 NSCLC patients using the TaqMan PCR
assay. We have also genotyped the EGFR mutations status in additional 182 NSCLC
patients, as well as 63 gastric, 95 esophagus and 70 colon carcinoma patients.
In 94 NSCLC samples, the result of the TaqMan PCR assay perfectly matched with
that of the sequencing excluding one patient. In one sample in which no EGFR
mutation was detected by direct sequencing, the TaqMan PCR assay detected a
mutation. This patient was a gefitinib responder. In a serial dilution study,
the assay could detect a mutant sample diluted in 1/10 with a wild-type sample.
Of 182 NSCLC samples, 46 mutations were detected. EGFR mutation was
significantly correlated with gender, smoking status, pathological subtypes, and
differentiation of lung cancers. There was no mutation detected by the TaqMan
PCR assay in gastric, esophagus and colon carcinomas. TaqMan PCR assay is a
rapid and sensitive method of detection of EGFR mutations with high throughput,
and may be useful to determine whether gefitinib should be offered for the
treatment of NSCLC patients. The TaqMan PCR assay can offer us a complementary
and confirmative test. Somatic mutations in exons encoding the tyrosine kinase domain of the epidermal
growth factor receptor (EGFR) gene are found in human lung adenocarcinomas and
are associated with sensitivity to the tyrosine kinase inhibitors gefitinib and
erlotinib. Nearly 90% of the EGFR mutations are either short, in-frame deletions
in exon 19 or point mutations that result in substitution of arginine for
leucine at amino acid 858 (L858R). To study further the role of these mutations
in the initiation and maintece of lung cancer, we have developed transgenic
mice that express an exon 19 deletion mutant (EGFR(DeltaL747-S752)) or the L858R
mutant (EGFR(L858R)) in type II pneumocytes under the control of doxycycline.
Expression of either EGFR mutant leads to the development of lung
adenocarcinomas. Two weeks after induction with doxycycline, mice that express
the EGFR(L858R) allele show diffuse lung cancer highly reminiscent of human
bronchioloalveolar carcinoma and later develop interspersed multifocal
adenocarcinomas. In contrast, mice expressing EGFR(DeltaL747-S752) develop
multifocal tumors embedded in normal lung parenchyma with a longer latency. With
mice carrying either EGFR allele, withdrawal of doxycycline (to reduce
expression of the transgene) or treatment with erlotinib (to inhibit kinase
activity) causes rapid tumor regression, as assessed by magnetic resoce
imaging and histopathology, demonstrating that mutant EGFR is required for tumor
maintece. These models may be useful for developing improved therapies for
patients with lung cancers bearing EGFR mutations. Mutations of the epidermal growth factor receptor (EGFR) gene have been reported
in non-small-cell lung cancer (NSCLC), especially in patients with
adenocarcinoma and never smokers. Some common somatic mutations in EGFR,
including deletion mutations in exon 19 and leucine-to-arginine substitution at
amino acid position 858 (L858R) in exon 21, have been examined for their ability
to predict sensitivity to gefitinib or erlotinib, which are selective EGFR
tyrosine kinase inhibitors (EGFR-TKIs). On the other hand, reports have shown
that the threonine-to-methionine substitution at amino acid position 790 (T790M)
in exon 20 is related to gefitinib resistance. Some studies have indicated that
high copy numbers of the EGFR gene may be a more effective molecular predictor
to responsiveness and prolonged survival in patients treated with EGFR-TKIs.
Here, we describe two NSCLC patients with the L858R mutation who did not respond
to gefitinib. Case 1 harbored both the T790M and L858R mutations, and
fluorescence in situ hybridization showed EGFR gene amplification. Case 2
harbored both the L858R and aspartic acid-to-tyrosine substitution at amino acid
position 761 in exon 19 of EGFR mutations and had a high polysomy status for
EGFR. In these two cases, tumors showed resistance to gefitinib treatment
despite the presence of EGFR L858R mutation and increased copy number. Our
findings encourage further molecular analysis to elucidate the relationship
between the EGFR status, including mutations and amplifications, and the
responsiveness of NSCLC to gefitinib. PURPOSE: Agents inhibiting the epidermal growth factor receptor (EGFR) have
shown clinical benefit in a subset of non-small cell lung cancer patients
expressing amplified or mutationally activated EGFR. However, responsive
patients can relapse as a result of selection for EGFR gene mutations that
confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and
gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel
spectrum-selective kinase inhibitor with potent activity against the EGF and
vascular endothelial growth factor receptor tyrosine kinase families, against
both wild-type (WT) and mutant EGFR in vitro and in vivo.
EXPERIMENTAL DESIGN: The activity of EGFR inhibitors against WT and mutant EGFRs
and their effect on downstream signal transduction was examined in cellular
assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and
T790M mutant EGFR) xenograft tumors.
RESULTS: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in
vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway
activation in the erlotinib-resistant H1975 cell line that harbors a double
mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that
EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and
reduced both tumor EGFR signaling and tumor vessel density. Additionally,
EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and
vascularization of MDA-MB-231 xenografts, a model which is more reliant on
signaling through vascular endothelial growth factor receptors.
CONCLUSIONS: These studies provide a preclinical basis for clinical trials of
XL647 in solid tumors and in patients bearing tumors that are resistant to
existing EGFR-targeted therapies. Combination therapy with platinum preparations still occupies a central position
in chemotherapy for lung cancer. Third-generation regimens that combine an
anticancer drug and a platinum preparation that were published in the 1990s
remain standard therapy for untreated non-small-cell lung cancer today.
Cisplatin or carboplatin is used as the platinum preparation, but combination
therapy with cisplatin has been found to have a greater antitumor effect than
combination therapy with carboplatin. However, there is very little difference
between them, and on balance, when adverse reactions, etc. are taken into
consideration, we do not think that it makes much difference which one of them
is used. Clinical studies of combinations between platinum preparations and
pemetrexed and S-1, which have been developed since 2000, have been conducted
recently. Docetaxel has been established as standard therapy for recurrent
cases, but based on the results of recent comparative studies, a
survival-prolonging effect has been shown for pemetrexed and for EGFR tyrosine
kinase inhibitors (gefitinib, erlotinib), which are molecularly targeted drugs,
and it has now become possible to select treatment methods by choosing from a
number of anticancer drugs. EGFR tyrosine kinase inhibitors have been
demonstrated to have a very high cytoreductive effect on lung cancers that have
EGFR gene mutations. The frequency of EGFR gene mutations is high in East Asia,
including Japan, whereas it is very low in Western countries. Thus, the future
course of development of chemotherapy for non-small-cell lung cancer may differ
in Western countries and Asia, and the method of using EGFR tyrosine kinase
inhibitors is expected to have great implications in Asia. BACKGROUND: Somatic mutations in the epidermal growth factor receptor (EGFR)
gene are associated with the responses to the tyrosine kinase inhibitors
gefitinib and erlotinib in patients with non-small-cell lung cancer (NSCLC).
Although various methods for detecting EGFR gene mutations have been developed,
they have several disadvantages. We attempted to establish a new method for the
detection of EGFR gene mutations with the use of paraffin-embedded samples.
METHODS: The detections of T790M mutations in exon 20 and L858R mutations in
exon 21 are based on the principle of allele-specific oligonucleotide polymerase
chain reaction (PCR). We also designed PCR primers that enable to detect all
types of deletions in exon 19. We assessed the basic performance efficiency of
this method, and to confirm its clinical applicability, we performed PCR using
DNA extracted from 66 tissue sections that were obtained from patients with
NSCLC and embedded in paraffin.
RESULTS: The sensitivity of this method for the detection of deletions or
mutations was as low as 0.5%. In the 66 subjects whose samples were analyzed, we
detected the following deletions and mutations in the EGFR gene: 11 deletions in
exon 19, 8 L858R mutations, and 1 double mutation of L858R and T790M.
CONCLUSION: The present method is sensitive and specific for the detection of
deletions and mutations in the EGFR gene and is thus suitable for use in
laboratory tests. The development of resistance to epidermal growth factor receptor tyrosine
kinase inhibitors (EGFR-TKI) seems almost inevitable, even in patients with lung
cancer that initially respond well to EGFR-TKIs. MET amplification was recently
found to be a mechanism of escape from the anticancer effect of EGFR inhibitors.
In the present study, we investigated the means whereby MET affects sensitivity
to EGFR-TKIs in PC-9 cells. Gefitinib- or erlotinib-resistant sublines were
established by exposing the parental PC-9 cell line to chronic, repeated
treatments with these drugs. These resistant sublines showed more than 100-fold
more resistance to gefitinib and erlotinib and acquired cross-resistance to
other EGFR-TKIs. The T790M EGFR mutation was found by pyrosequencing, and this
seemed to be the cause of drug resistance. Resistant cells also showed MET
activation, although gene amplification was not detected. Furthermore, the
induction of MET activity was not found to be associated with sensitivity to
EGFR-TKIs. Interestingly, increased passage number without exposure to gefitinib
or erlotinib caused MET activation, but this did not affect sensitivity to
EGFR-TKIs. In addition, hepatocyte growth factor was found to block the ability
of EGFR-TKIs to inhibit MET activation. However, sustained MET activation by
hepatocyte growth factor did not modulate the cellular effects of gefitinib or
erlotinib. Rather, activated MET enhanced migration and invasion abilities.
Summarizing, MET activation may be acquired during cancer cell proliferation and
enhances migratory and invasive abilities without affecting cellular sensitivity
to EGFR-TKIs. Accordingly, the present study suggests that MET activation caused
by factors other than MET gene amplification is not a suitable surrogate marker
of resistance to EGFR-TKIs. Seventy-five percent of lung adenocarcinomas with epidermal growth factor
receptor (EGFR) mutations respond to treatment with the tyrosine kinase
inhibitors (TKIs) gefitinib and erlotinib; however, drug-resistant tumors
eventually emerge. In 60% of cases, resistant tumors carry a secondary mutation
in EGFR (T790M), amplification of MET, or both. Here, we describe the
establishment of erlotinib resistance in lung tumors, which were induced by
mutant EGFR, in transgenic mice after multiple cycles of drug treatment; we
detect the T790M mutation in five out of 24 tumors or Met amplification in one
out of 11 tumors in these mice. This preclinical mouse model, therefore,
recapitulates the molecular changes responsible for resistance to TKIs in human
tumors and holds promise for the discovery of additional mechanisms of drug
resistance in lung cancer. PURPOSE: The tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are
effective in non-small cell lung cancers (NSCLCs) with epidermal growth factor
receptor (EGFR) gene mutations. The usual clinical dose of gefitinib (250 mg/d)
is only one third of its maximum tolerated dose, whereas the dose of erlotinib
(150 mg/d) is at its maximum tolerated dose. In NSCLC cell lines, both TKIs have
similar micromolar inhibitory concentrations. We explored whether erlotinib at
25 mg/d (trough serum concentration similar to gefitinib 250 mg/d) would be
efficacious in EGFR-mutated NSCLC.
METHODS: To study the inhibitory concentrations of gefitinib and erlotinib, we
exposed EGFR-mutated cell lines (HCC827, H3255, PC-9, and H1975) to increasing
concentrations of these TKIs. Further on, we performed a retrospective
evaluation of seven patients with advanced EGFR-mutated (exon 19 deletions and
L858R) NSCLC that were given erlotinib at 25 mg/d as their first EGFR TKI.
RESULTS: Gefitinib and erlotinib generated similar inhibitory curves across our
panel of EGFR-mutated NSCLC cell lines with overlapping mean 50% inhibitory
concentration 95% confidence intervals for HCC827, PC-9, and H1975. Both drugs
also displayed a high degree of correlation in mean 50% inhibitory concentration
(Pearson's r = 0.99, p = 0.0417). Of the seven patients, five patients (71.5%)
had partial responses to erlotinib 25 mg/d. Median progression-free survival was
17 months (95% confidence interval, 6-35 months). Toxicities were minimal, with
only two (28.5%) patients having a rash and none experiencing (0%) diarrhea.
CONCLUSIONS: In NSCLC cell lines, gefitinib and erlotinib have similar
inhibitory profiles. In patients with NSCLC and EGFR-activating mutations, a
dose of erlotinib 25 mg/d (equivalent to gefitinib 250 mg/d) leads to impressive
response rates and progression-free survival similar to the growing experience
with the approved doses of gefitinib (250 mg/d) and erlotinib (150 mg/d).
Identifying prospectively the lowest and clinically active dose ranges of
erlotinib and gefitinib will help further to personalize care for patients with
tumors harboring EGFR mutations. Postoperative adjuvant chemotherapy with tegafur-uracil has become the standard
of care for patients with completely resected stage I adenocarcinoma according
to the positive result from phase III trial conducted in Japan. The
effectiveness of tegafur-gimeracil-oteracil potassium in advanced non-small cell
lung cancer (NSCLC) is now under investigation in a phase III trial in the
first-line treatment in combination with platinum agent as well as in
second-line treatment as monotherapy. The evidence of epidermal growth factor
receptor (EGFR)-tyrosine kinase inhibitor (TKI) such as gefitinib and erlotinib
was first confirmed as the second-line treatment for non-selective patients with
advanced NSCLC. However, now it is essential to examine some active EGFR gene
mutations such as exon 19 deletion or L858R in exon 21, the novel predictive
factor for high efficacy of EGFR-TKI, in terms of the risk-benefit balance. For
patients with EGFR-mutated advanced NSCLC, the first-line treatment with
gefitinib had achieved a significant prolongation of progression-free survival
compared with standard platinum doublet chemotherapy in a few phase III trials,
and became a new standard of care. Gefitinib is highly effective for patients
with advanced NSCLC with EGFR mutation even if their performance status is poor,
although one should always pay careful attention to fatal interstitial lung
disease. Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR)
respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably
emerges. To elucidate mechanisms of acquired drug resistance, we performed
systematic genetic and histological analyses of tumor biopsies from 37 patients
with drug-resistant non-small cell lung cancers (NSCLCs) carrying EGFR
mutations. All drug-resistant tumors retained their original activating EGFR
mutations, and some acquired known mechanisms of resistance including the EGFR
T790M mutation or MET gene amplification. Some resistant cancers showed
unexpected genetic changes including EGFR amplification and mutations in the
PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal
transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC
into small cell lung cancer (SCLC) and were sensitive to standard SCLC
treatments. In three patients, serial biopsies revealed that genetic mechanisms
of resistance were lost in the absence of the continued selective pressure of
EGFR inhibitor treatment, and such cancers were sensitive to a second round of
treatment with EGFR inhibitors. Collectively, these results deepen our
understanding of resistance to EGFR inhibitors and underscore the importance of
repeatedly assessing cancers throughout the course of the disease. Patients presenting with non-small cell lung cancer (NSCLC) and active EGFR
mutation have a high response rate (60-70%) to EGFR tyrosine kinase inhibitors
(TKI) with little immediate progression (primary resistance). However,
progression on this treatment (secondary resistance) is inevitable even for
those who responded initially. These two situations are distinct in terms of
management. In case of primary resistance, screening for other associated
molecular abnormalities (tumour heterogeneity) should be done, even resulting in
a false positive in the initial screening of EGFR mutation. In case of secondary
resistance, a new pathology sample should be taken insofar as is possible to
determine the presence of an acquired mutation of EGFR resistance (T790M in 60%
of cases) or c-Met amplification (20% of cases). The presence of a T790M
mutation could respond to irreversible EGFR-TKI, while a c-Met amplification
could be managed with a targeted anti-Met therapy. However, the gold standard is
still cytotoxic chemotherapy at present if a clinical trial based on a targeted
therapy is not possible. Non-small cell lung cancers (NSCLCs) are heterogeneous cancers. In 2004, the
identification of epidermal growth factor receptor (EGFR) somatic mutations
provided the first glimpse of a clinically relevant NSCLC oncogene.
Approximately 70% of NSCLCs with EGFR mutations (exon 19 deletions or the exon
21 L858R) attain responses to EGFR tyrosine kinase inhibitors (TKIs) gefitinib
and erlotinib, with improved response rate (RR), progression-free survival (PFS)
and in some reports overall survival (OS) when compared with EGFR wildtype (WT)
cases. Three randomized trials of gefitinib versus chemotherapy (IPASS,
WJTOG3405, NEJ002) in stage IV NSCLC have consistently demonstrated better RR
and PFS (hazard ratios of 0.48 [IPASS], 0.49 [WJTOG3405] and 0.30 [NEJ002]) for
EGFR-mutated NSCLCs treated with gefitinib. Novel irreversible EGFR TKIs
(afatinib, XL647, PF00299804) show similar activity in EGFR-mutated patients. A
translocation involving the anaplastic lymphoma kinase (ALK) gene with EML4,
identified in 2007, is the most recent oncogene found in NSCLC. Crizotinib
(PF02341066), an ALK TKI, has shown impressive activity against ALK translocated
NSCLC in an expanded cohort of a phase I trial (NCT00585195). Over 80 patients
have been treated and the RR is ∼60% with the 6-month PFS rate exceeding 70%. A
registration phase III trial of crizotinib versus second-line chemotherapy
(pemetrexed/docetaxel) is underway (PROFILE 1007, NCT00932893). KRAS, EGFR
mutations and ALK translocations are mutually exclusive and few EGFR WT NSCLCs
respond to EGFR TKIs. The promising results of EGFR and ALK TKIs in molecular
subgroups of NSCLCs herald a new age of drug and clinical trial development for
patients with NSCLC. Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most
common histologic subtype of non-small cell lung cancer (NSCLC), are frequently
associated with activating mutations in the epidermal growth factor receptor
(EGFR) gene. Although these patients often respond clinically to the EGFR
tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs,
suggesting the development of escape mechanisms that promote cell survival.
Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we
identified that the canonical Wnt pathway contributes to the maintece of
NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating
enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling.
Inhibition of tankyrase and various other components of the Wnt pathway with
shRNAs or small molecules significantly increased the efficacy of EGFR
inhibitors both in vitro and in vivo. Our findings therefore reveal a critical
role for tankyrase and the canonical Wnt pathway in maintaining lung cancer
cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway
together with EGFR inhibition may improve clinical outcome in patients with
NSCLC. Mutations in the epidermal growth factor receptor gene (EGFR) are frequently
observed in non-small-cell lung cancer (NSCLC), occurring in about 40% to 60% of
never-smokers and in about 17% of patients with adenocarcinomas. EGFR tyrosine
kinase inhibitors (TKIs), such as gefitinib and erlotinib, have transformed
therapy for patients with EGFR-mutant NSCLC and have proved superior to
chemotherapy as first-line treatment for this patient group. Despite these
benefits, there are currently 2 key challenges associated with EGFR inhibitor
therapy for patients with NSCLC. First, only 85% to 90% of patients with the
EGFR mutation derive clinical benefit from EGFR TKIs, with the remainder
demonstrating innate resistance to therapy. Second, acquired resistance to EGFR
TKIs inevitably occurs in patients who initially respond to therapy, with a
median duration of response of about 10 months. Mutant EGFR activates various
subcellular signaling cascades, including the phosphatidylinositol 3-kinase
(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which demonstrates
maintained activity in a variety of TKI-resistant cancers. Given the fundamental
role of the PI3K/Akt/mTOR pathway in tumor oncogenesis, proliferation, and
survival, PI3K pathway inhibitors have emerged as a possible solution to the
problem of EGFR TKI resistance. However resistance to EGFR TKIs is associated
with considerable heterogeneity and complexity. Preclinical experiments
investigating these phenomena suggest that in some patients, PI3K inhibitors
will have to be paired with other targeted agents if they are to be effective.
This review discusses the preclinical data supporting PI3K/Akt/mTOR pathway
inhibitor combinations in EGFR TKI-resistant NSCLC from the perspective of the
various agents currently being investigated in clinical trials. Although cancers may have many genetic alterations, there are only a few
mutations actually associated with essential traits of cancer cells such as cell
proliferation or evasion from apoptosis. Because cancer cells are "addicted" to
these "drive genes" , pharmacologic inhibition of these gene function is highly
effective. Epidermal growth factor receptor(EGFR)-tyrosine kinase
inhibitor(TKI)(such as gefitinib or erlotinib)treatment of lung cancer harboring
EGFR gene mutation is one of the prototypes of such therapies. Several clinical
trials clearly demonstrated that progression-free survival of patients treated
with EGFR-TKI is significantly longer than that of those treated by conventional
platinum doublet chemotherapy. EGFR-TKI therapy dramatically changed the
paradigm of lung cancer treatment. Furthermore, in 2012, crizotinib was approved
for lung cancer treatment with anaplastic lymphoma kinase(ALK)gene
translocation. Targeted therapies for lung cancers "addicted" to other driver
gene mutations including ROS1, RET or HER2 are also under development. Through
these personalized approaches, lung cancer is changing from an acute fatal
disease to a more chronic disease, and eventually we might be able to cure it. |
Which protein complexes contain mitofilin? | mitochondrial inter-membrane space bridging (MIB) complex
mitochondrial inner membrane organizing system (MINOS)
MitOS for mitochondrial organizing structure | The mitochondrial inner membrane consists of two domains, inner boundary
membrane and cristae membrane that are connected by crista junctions.
Mitofilin/Fcj1 was reported to be involved in formation of crista junctions,
however, different views exist on its function and possible partner proteins. We
report that mitofilin plays a dual role. Mitofilin is part of a large inner
membrane complex, and we identify five partner proteins as constituents of the
mitochondrial inner membrane organizing system (MINOS) that is required for
keeping cristae membranes connected to the inner boundary membrane.
Additionally, mitofilin is coupled to the outer membrane and promotes protein
import via the mitochondrial intermembrane space assembly pathway. Our findings
indicate that mitofilin is a central component of MINOS and functions as a
multifunctional regulator of mitochondrial architecture and protein biogenesis. Crista junctions (CJs) are tubular invaginations of the inner membrane of
mitochondria that connect the inner boundary with the cristae membrane. These
architectural elements are critical for mitochondrial function. The yeast inner
membrane protein Fcj1, called mitofilin in mammals, was reported to be
preferentially located at CJs and crucial for their formation. Here we
investigate the functional roles of individual domains of Fcj1. The most
conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function.
In its absence, formation of CJ is strongly impaired and irregular, and stacked
cristae are present. This domain interacts with full-length Fcj1, suggesting a
role in oligomer formation. It also interacts with Tob55 of the translocase of
outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM)
complex, which is required for the insertion of β-barrel proteins into the outer
membrane. The association of the TOB/SAM complex with contact sites depends on
the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly
affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex
leads to altered cristae morphology and a moderate reduction in the number of
CJs. We propose that the C-terminal domain of Fcj1 is critical for the
interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in
close proximity to the outer membrane. These results assign novel functions to
both the C-terminal domain of Fcj1 and the TOB/SAM complex. The mitochondrial inner membrane contains a large protein complex crucial for
membrane architecture, the mitochondrial inner membrane organizing system
(MINOS). MINOS is required for keeping cristae membranes attached to the inner
boundary membrane via crista junctions and interacts with protein complexes of
the mitochondrial outer membrane. To study if outer membrane interactions and
maintece of cristae morphology are directly coupled, we generated mutant
forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core
component of MINOS. Mitofilin consists of a transmembrane anchor in the inner
membrane and intermembrane space domains, including a coiled-coil domain and a
conserved C-terminal domain. Deletion of the C-terminal domain disrupted the
MINOS complex and led to release of cristae membranes from the inner boundary
membrane, whereas the interaction of mitofilin with the translocase of the outer
membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced.
Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae
morphology; however, the interactions of mitofilin with TOM and SAM were
differentially affected. Finally, deletion of both intermembrane space domains
disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the
intermembrane space domains of mitofilin play distinct roles in interactions
with outer membrane complexes and maintece of MINOS and cristae morphology,
demonstrating that MINOS contacts to TOM and SAM are not sufficient for the
maintece of inner membrane architecture. The mitochondrial inner membrane organizing system (MINOS) is a conserved large
hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial
for the maintece of cristae morphology. MINOS has been suggested to represent
the core of an extended protein network that controls mitochondrial function and
structure, and has been linked to several human diseases. The spatial
arrangement of MINOS within mitochondria is ill-defined, however. Using
super-resolution stimulated emission depletion (STED) microscopy and immunogold
electron microscopy, we determined the distribution of three known human MINOS
subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution
microscopy revealed that all three subunits form similar clusters within
mitochondria, and that MINOS is more abundant in mitochondria around the nucleus
than in peripheral mitochondria. At the submitochondrial level, mitofilin, a
core MINOS subunit, is preferentially localized at cristae junctions. In primary
human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of
clusters arranged in parallel to the cell growth surfaces. We suggest that this
array of MINOS complexes might explain the observed phenomenon of largely
horizontally arranged cristae junctions that connect the inner boundary membrane
to lamellar cristae. The super-resolution images demonstrate an unexpectedly
high level of regularity in the oscale distribution of the MINOS complex in
human mitochondria, supporting an integrating role of MINOS in the structural
organization of the organelle. Insulin resistance plays a major role in the development of type 2 diabetes and
obesity and affects a number of biological processes such as mitochondrial
biogenesis. Though mitochondrial dysfunction has been linked to the development
of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism
linking the two is not well understood. We used high fat diet (HFD)-induced
obesity dependent diabetes mouse models to gain insight into the potential
pathways altered with metabolic disease, and carried out quantitative proteomic
analysis of liver mitochondria. As previously reported, proteins involved in
fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid
cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of
HFD fed mice compared with that of normal diet. Further, our studies revealed
that retinol metabolism is distinctly down-regulated and the mitochondrial
structural proteins-components of mitochondrial inter-membrane space bridging
(MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins-essential for
protein import, are significantly up-regulated in HFD fed mice. Structural and
functional studies on HFD and normal diet liver mitochondria revealed remodeling
of HFD mitochondria to a more condensed form with increased respiratory capacity
and higher ATP levels compared with normal diet mitochondria. Thus, it is likely
that the structural remodeling is essential to accommodate the increased protein
content in presence of HFD: the mechanism could be through the MIB complex
promoting contact site and crista junction formation and in turn facilitating
the lipid and protein uptake. |
Define marine metaproteomics | Marine metaproteomics is the study of the activities of whole marine microbial communities. The proteomic analyses are applied directly without the need for prior microbial culturing. The samples can be sediments,seawater, etc. | BACKGROUND: Natural microbial communities are extremely complex and dynamic
systems in terms of their population structure and functions. However, little is
known about the in situ functions of the microbial communities.
RESULTS: This study describes the application of proteomic approaches
(metaproteomics) to observe expressed protein profiles of natural microbial
communities (metaproteomes). The technique was validated using a constructed
community and subsequently used to analyze Chesapeake Bay microbial community
(0.2 to 3.0 microm) metaproteomes. Chesapeake Bay metaproteomes contained
proteins from pI 4-8 with apparent molecular masses between 10-80 kDa.
Replicated middle Bay metaproteomes shared approximately 92% of all detected
spots, but only shared 30% and 70% of common protein spots with upper and lower
Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no
significant matches to known proteins. Three Chesapeake Bay proteins were
tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching.
The proteins identified were of marine microbial origin and correlated with
abundant Chesapeake Bay microbial lineages, Bacteroides and
alpha-proteobacteria.
CONCLUSION: Our results represent the first metaproteomic study of aquatic
microbial assemblages and demonstrate the potential of metaproteomic approaches
to link metagenomic data, taxonomic diversity, functional diversity and
biological processes in natural environments. Metagenomics has revolutionized microbiology by paving the way for a
cultivation-independent assessment and exploitation of microbial communities
present in complex ecosystems. Metagenomics comprising construction and
screening of metagenomic DNA libraries has proven to be a powerful tool to
isolate new enzymes and drugs of industrial importance. So far, the majority of
the metagenomically exploited habitats comprised temperate environments, such as
soil and marine environments. Recently, metagenomes of extreme environments have
also been used as sources of novel biocatalysts. The employment of
next-generation sequencing techniques for metagenomics resulted in the
generation of large sequence data sets derived from various environments, such
as soil, the human body, and ocean water. Analyses of these data sets opened a
window into the enormous taxonomic and functional diversity of environmental
microbial communities. To assess the functional dynamics of microbial
communities, metatranscriptomics and metaproteomics have been developed. The
combination of DNA-based, mRNA-based, and protein-based analyses of microbial
communities present in different environments is a way to elucidate the
compositions, functions, and interactions of microbial communities and to link
these to environmental processes. System approaches to elucidate ecosystem functioning constitute an emerging area
of research within microbial ecology. Such approaches aim at investigating all
levels of biological information (DNA, RNA, proteins and metabolites) to capture
the functional interactions occurring in a given ecosystem and track down
characteristics that could not be accessed by the study of isolated components.
In this context, the study of the proteins collectively expressed by all the
microorganisms present within an ecosystem (metaproteomics) is not only crucial
but can also provide insights into microbial functionality. Overall, the success
of metaproteomics is closely linked to metagenomics, and with the exponential
increase in the availability of metagenome sequences, this field of research is
starting to experience generation of an overwhelming amount of data, which
requires systematic analysis. Metaproteomics has been employed in very diverse
environments, and this review discusses the recent advances achieved in the
context of human biology, soil, marine and freshwater environments as well as
natural and bioengineered systems. Heterotrophic marine bacteria play key roles in remineralizing organic matter
generated from primary production. However, far more is known about which groups
are domit than about the cellular processes they perform in order to become
domit. In the Southern Ocean, eukaryotic phytoplankton are the domit
primary producers. In this study we used metagenomics and metaproteomics to
determine how the domit bacterial and archaeal plankton processed bloom
material. We examined the microbial community composition in 14 metagenomes and
found that the relative abundance of Flavobacteria (dominated by Polaribacter)
was positively correlated with chlorophyll a fluorescence, and the relative
abundance of SAR11 was inversely correlated with both fluorescence and
Flavobacteria abundance. By performing metaproteomics on the sample with the
highest relative abundance of Flavobacteria (Newcomb Bay, East Antarctica) we
defined how Flavobacteria attach to and degrade diverse complex organic
material, how they make labile compounds available to Alphaproteobacteria
(especially SAR11) and Gammaproteobacteria, and how these heterotrophic
Proteobacteria target and utilize these nutrients. The presence of
methylotrophic proteins for archaea and bacteria also indicated the importance
of metabolic specialists. Overall, the study provides functional data for the
microbial mechanisms of nutrient cycling at the surface of the coastal Southern
Ocean. Current knowledge of the physiology and phylogeny of polycyclic aromatic
hydrocarbon (PAH) degrading bacteria often relies on laboratory enrichments and
isolations. In the present study, in situ microcosms consisting of activated
carbon pellets (BACTRAP®s) were loaded with either (13) C-naphthalene or (13)
C-fluorene and were subsequently exposed in the contamit source and plume
fringe region of a PAH-contaminated aquifer. Metaproteomic analysis and
protein-stable isotope probing revealed Burkholderiales, Actinomycetales, and
Rhizobiales as the most active microorganisms in the groundwater communities.
Proteins identified of the naphthalene degradation pathway showed a relative
(13) C isotope abundance of approximately 50 atom% demonstrating that the
identified naphthalene-degrading bacteria gained at least 80% of their carbon by
PAH degradation. Although the microbial community grown on the fluorene-BACTRAPs
showed a structure similar to the naphthalene-BACTRAPs, the identification of
fluorene degraders and degradation pathways failed in situ. In complementary
laboratory microcosms, a clear enrichment in proteins related to Rhodococcus and
possible fluorene degradation enzymes was observed. This result demonstrates the
impact of laboratory conditions on microbial community structure and activity of
certain species and underlines the need on in situ exploration of microbial
community functions. In situ microcosms in combination with protein-stable
isotope probing may be a significant tool for in situ identification of
metabolic key players as well as degradation pathways. Metaproteomics is a new field within the 'omics' science which investigates
protein expression from a complex biological system and provides direct evidence
of physiological and metabolic activities. Characterization of the metaproteome
will enhance our understanding of the microbial world and link microbial
communities to ecological functions. Recently, the availability of extensive
metagenomic sequences from various marine microbial communities has extended the
postgenomic era to the field of oceanography. Although still in its infancy,
metaproteomics has shown its powerful potential with regard to functional gene
expression within microbial habitats and their interactions with the ambient
environment as well as their biogeochemical functions. However, the application
of metaproteomic approaches to complex marine samples still faces considerable
challenges. This review summarizes the recent progress in marine metaproteomics
and discusses the limitations of and perspectives for this approach in the study
of the marine ecosystem. This article is part of a Special Issue entitled:
Trends in Microbial Proteomics. Advances in tandem mass spectrometry (tandem MS) and sequencing have enabled the
field of community proteomics, which seeks to identify expressed proteins, their
sequence variability, and the physiological responses of organisms to variable
environmental conditions. Bottom-up tandem MS-based community proteomic
approaches generate fragmentation spectra from peptides. Fragmentation spectra
are then searched against genomic or metagenomic databases to deduce the amino
acid sequences of peptides, providing positive identifications for proteins.
Marine community proteomic studies have verified the importance of nutrient
transport, energy generation, and carbon fixation functions in bacteria and
archaea and revealed spatial and temporal shifts in the expressed functions of
communities. Here, we discuss sample collection, preparation, and processing
methods for planktonic tandem MS-based community proteomics. Metaproteomic studies of whole microbial communities from environmental samples
(e.g., soil, sediments, freshwater, seawater, etc.) have rapidly increased in
recent years due to many technological advances in mass spectrometry (MS). A
single 24-h liquid chromatograph-tandem mass spectrometry (LC-MS/MS) measurement
can potentially detect and quantify thousands of proteins from many domit and
subdomit naturally occurring microbial populations. Importantly, amino acid
sequences and relative abundance information for detected peptides are
determined, which allows for the characterization of expressed protein functions
within communities and specific matches to be made to microbial lineages, with
potential subspecies resolution. Continued optimization of protein extraction
and fractionation protocols, development of quantification methods, and advances
in mass spectrometry instrumentation are enabling more accurate and
comprehensive peptide detection within samples, leading to wider research
applicability, greater ease of use, and overall accessibility. This chapter
provides a brief overview of metaproteomics experimental options, including a
general protocol for sample handling and LC-MS/MS measurement. |
Mutation of which gene and which chromosome cause Neurofibromatosis type I? | Neurofibromatosis Type I is an autosomal dominant condition associated with NF-1 gene mutation that is located in the long arm of chromosome 17 (17q11.2). In the majority (95%) of Neurofibromatosis Type I individuals, the mutation is found in the NF1 gene, while the remaining 5% of the patients have different types of genetic abnormalities.
| The von Recklinghausen neurofibromatosis (NF1) gene has been localized to the
pericentromeric region of chromosome 17. We have screened six multigenerational
families with multiple, tightly linked markers to aid in mapping this region of
the chromosome. More than 150 members in six families were typed with probes
including HHH202, D17Z1, EW203, EW206, EW207, EW301, pA10-41, D17S37, and
D17S36. Two-point lod scores for NF1 versus all markers were calculated. HHH202
demonstrated the tightest linkage to NF1 with theta = .0, z = 3.86 (95%
confidence limits [CL] of theta = .0-.13), suggesting that HHH202 be considered
as a potential candidate marker for use in carrier detection and prenatal
diagnosis. Pairwise marker-to-marker lod scores were used in examining the most
likely order of subsets of the markers. Of those tested, the most likely order
was (pter)-pA10-41-EW301-D17Z1-HHH202-NF1-E W206-EW207-EW203-(qter). In
addition, we have ascertained an NF1 x NF1 half-cousin mating in which there are
four affected family members who are potentially homozygous for the disease
gene. Two of these four individuals have been sampled and typed for marker loci.
When their D17Z1 genotypes are considered, the probability that both these
individuals are heterozygous is 85%. Nine markers from the pericentromeric region of chromosome 17 were typed in 16
British and five South African families with neurofibromatosis type 1 (NF1). The
markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination
fractions less than 1%. No evidence of locus heterogeneity was detected.
Inspection of recombit events in families informative for several markers
suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and
EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12). The gene for von Recklinghausen neurofibromatosis type 1 (NF1) has recently been
mapped to the pericentromeric region of human chromosome 17. To further localize
the NF1 gene, linkage analysis using chromosome 17 DNA markers was performed on
11 multigeneration families with 175 individuals, 57 of whom were affected. The
markers used were D17Z1 (p17H8), D17S58 (EW301), D17S54 (EW203), D17S57 (EW206),
D17S73 (EW207), CRI-L946, HOX-2, and growth hormone. Tight linkage was found
between NF1 and D17Z1, D17S58, and D17S57 with a recombination fraction of zero.
One recombit was detected between NF1 and D17S73, showing linkage with a 10%
recombination fraction. No linkage was detected between NF1 and CRI-L946 or
between HOX-2 and growth hormone. Our data are consistent with the proposed gene
order pter D17S58-D17Z1-NF1-D17S57-D17S73 qter. In addition to reporting, in accompanying papers, their individual analyses of
mapping the neurofibromatosis type 1 (NF1) gene on chromosome 17, members of the
International Consortium for NF1 Linkage contributed their data for our joint
analysis to determine the exact sequence of flanking markers and to obtain
precise estimates and confidence limits of the recombination fractions for the
closest markers, in anticipation of clinical use. With specimens from 142
families and more than 700 affected persons, eight teams used 31 markers in the
pericentric region of chromosome 17 to perform 13,838 genotypings. With the
combined data, we used the computer program CRI-MAP to build the most likely
sequence of loci by sequentially adding single loci to a fixed pair of loci and
separately calculating the likelihood of all permutations of four consecutive
loci. The best order is pter-pA10-41-EW301-centromere
(p17H8)-pHHH202-NF1-EW206-EW207-EW203++ +-CRI-L581-CRI-L946-HOX2-NGFR-qter. The
total genetic distance from pA10-41 to NGFR is 26 cM in males and 56 cM in
females, and the overall difference in sex-specific maps is statistically
significant (P = .006). The upper 99% confidence limits of the recombination
fraction of the closest proximal marker, pHHH202, is 4%, and that for the
closest distal marker, EW206, is 9%. These limits should decrease with the use
of additional probes and the further evaluation of DNA from the six persons
showing multiple recombinations within short genetic distances. Clinical
application is technically feasible with currently available markers, although
its appropriate use for prenatal and presymptomatic diagnosis requires further
discussion and evaluation. To better map the location of the von Recklinghausen neurofibromatosis (NF1)
gene, we have characterized a somatic cell hybrid designated 7AE-11. This
microcell-mediated, chromosome-transfer construct harbors a centromeric segment
and a neo-marked segment from the distal long arm of human chromosome 17. We
have identified 269 cosmid clones with human sequences from a 7AE-11 library
and, using a panel of somatic cell hybrids with a total of six chromosome 17q
breakpoints, have mapped 240 of these clones on chromosome 17q. The panel
included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an
NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three
of the cosmids map into a region spanning the NF13 breakpoint, as defined by the
two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a
subset of these cosmids have been mapped by linkage analysis in normal reference
families, to localize the NF1 gene more precisely and to enhance the potential
for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an
important resource for testing DNA blots of large-fragment restriction-enzyme
digests from NF1 patient cell lines, to detect rearrangements in patients' DNA
and to identify the 17;22 NF1 translocation breakpoint. The neurofibromatosis type I gene encodes a protein, neurofibromin, which may
function as a tumor suppressor gene product. Recent studies have demonstrated
loss of neurofibromin in tumors from NF1 and non-NF1 patients, including
neurofibrosarcomas, neuroblastomas and maligt melanomas. Since neurofibromin
is expressed in the adrenal gland, six pheochromocytomas and one adrenal
cortical tumor were examined for neurofibromin expression. In all seven tumors,
no neurofibromin could be detected. Furthermore, loss of heterozygosity (LOH)
analysis demonstrated that in one of the pheochromocytomas, reduction to
homozygosity was observed for both 17p and 17q markers while the adrenal
cortical tumor demonstrated LOH for only 17q markers. The frequent LOH
surrounding the NF1 locus and lack of neurofibromin expression in these tumors
suggest that NF1 gene mutations may contribute to the development of adrenal
gland neoplasms in patients with NF1. Neurofibromatosis type I (NFI) is a common autosomal domit disorder with an
increased risk for developing benign and maligt tumors. The NFI gene has been
cloned and maps to 17q11.2, and the gene product acts as a tumor suppressor
gene. Here we analyzed the role of mutations in TP53 in four maligt NFI
tumors. Mutations were found in 3 out of 4 tumors. One of these mutations is a
common missense mutation in codon 278 in one of the previously identified hot
spots for mutations. The two other are hitherto unreported mutations, including
a splice mutation of exon 3 and a nonsense mutation in exon 4. In addition,
these four tumors also showed loss of heterozygosity (LOH) for markers on
chromosome 17 in the region of TP53. Maligt NFI tumors are initiated by a
somatic inactivation of the second NFI allele. Tumor progression, however,
occurs by accumulation of additional genetic abnormalities, such as homozygous
inactivation of TP53, as demonstrated in this paper. Neurofibromatosis type I (NF1) is an autosomal domit disorder affecting 1 in
3000 people. The NF1 gene is located on chromosome 17q11.2, spans 350 kb of
genomic DNA, and contains 60 exons. A major phenotypic feature of the disease is
the widespread occurrence of benign dermal and plexiform neurofibromas. Genetic
and biochemical data support the hypothesis that NF1 acts as a tumour suppressor
gene. Molecular analysis of a number of NF1 specific tumours has shown the
inactivation of both NF1 alleles during tumourigenesis, in accordance with
Knudson's "two hit" hypothesis. We have studied 82 tumours from 45 NF1 patients.
Two separate strategies were used in this study to search for the somatic
changes involved in the formation of NF1 tumours. First, evidence of loss of
heterozygosity (LOH) of the NF1 gene region was investigated, and, second, a
screen for the presence of sequence alterations was conducted on a large panel
of DNA derived from matched blood/tumour pairs. In this study, the largest of
its kind to date, we found that 12% of the tumours (10/82) exhibited LOH;
previous studies have detected LOH in 3-36% of the neurofibromas examined. In
addition, an SSCP/HA mutation screen identified five novel NF1 germline and two
somatic mutations. In a plexiform neurofibroma from an NF1 patient, mutations in
both NF1 alleles have been characterised. The Miller-Dieker syndrome (type I lissencephaly) is a neuronal migration
disorder which is associated with microdeletions in the short arm of chromosome
17. Neurofibromatosis type I (NF1) is an autosomal domit condition associated
with mutations in the long arm of chromosome 17, and characterised by
neurofibromas, café-au-lait spots and axillary freckling. The neonatal period
for a female infant born at 39 weeks gestation by emergency Caesarean section
was complicated by frequent epileptic seizures as well as hypotonia. A computed
tomography scan revealed evidence of lissencephaly, and chromosomal analysis
showed a microdeletion on the short arm of chromosome 17 (17p13.3), confirming
the diagnosis as Miller-Dieker syndrome. The child died at the age of 4 years
and examination of the brain confirmed lissencephaly with a thickened cortex,
deficient white matter, and grey matter heteropias. The mother had café-au-lait
spots, and axillary freckling. In addition, the mother's and maternal
grandmother's genetic analysis showed identical mutations in the
neurofibromatosis I gene on the long arm of chromosome 17, confirming the
diagnosis of NF1. The child did not possess the mutation. This case illustrates
a rare neuronal migration disorder appearing in a child from a family with a
history of NF1. Neurofibromatosis Type 1 (NF1) is one of the most common inherited diseases in
humans. It is caused by a mutation in the NF1 gene on chromosome 17, and is
associated with numerous central and peripheral nervous system manifestations.
Children with NF1 are at high risk of harboring numerous lesions that may
require the attention of a neurosurgeon. Some of these include optic nerve
gliomas, hydrocephalus, intraspinal tumors, and peripheral nerve tumors.
Although most of the neoplasms that affect the brain, spine, and peripheral
nerves of children are low-grade lesions, there is a small but real risk that
some of these lesions may become high grade over time, requiring other forms of
therapy than surgery alone. Other associated disorders that may result from NF1
in childhood include Chiari malformation Type I, scoliosis, and pulsating
exophthalmos from the absence of the sphenoid wing. In this review, the major
lesions that are found in children with NF1 are reviewed as well as the types of
treatment that are offered by neurosurgeons and other members of the treating
team. Today, optimum care of the child with NF1 is provided by a
multidisciplinary team comprising neurosurgeons, neurologists, ophthalmologists,
radiologists, orthopedic surgeons, and plastic surgeons. Neurofibromatosis type I (NF1) is an autosomal domit familial tumor syndrome
characterized by the presence of multiple benign neurofibromas. In 95% of NF1
individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the
germline mutation consists of a microdeletion that includes the NF1 gene and
several flanking genes. We studied the frequency of loss of heterozygosity (LOH)
in the NF1 region as a mechanism of somatic NF1 inactivation in neurofibromas
from NF1 patients with and without a microdeletion. There was a statistically
significant difference between these two patient groups in the proportion of
neurofibromas with LOH. None of the 40 neurofibromas from six different NF1
microdeletion patients showed LOH, whereas LOH was observed in 6/28
neurofibromas from five patients with an intragenic NF1 mutation (P = 0.0034,
Fisher's exact). LOH of the NF1 microdeletion region in NF1 microdeletion
patients would de facto lead to a nullizygous state of the genes located in the
deletion region and might be lethal. The mechanisms leading to LOH were further
analyzed in six neurofibromas. In two out of six neurofibromas, a chromosomal
microdeletion was found; in three, a mitotic recombination was responsible for
the observed LOH; and in one, a chromosome loss with reduplication was present.
These data show an important difference in the mechanisms of second hit
formation in the 2 NF1 patient groups. We conclude that NF1 is a familial tumor
syndrome in which the type of germline mutation influences the type of second
hit in the tumors. We present monozygotic twins discordant for the autosomal domit disorder
neurofibromatosis type 1 (NF1). The affected twin was diagnosed with NF1 at age
12, based upon accepted clinical criteria for the disorder. Both twins were
re-examined at ages 35 and 57, at which times the unaffected twin continued to
show no clinical manifestations of NF1. Short tandem repeat marker (STR)
genotyping at 10 loci on chromosome 17 and 10 additional loci dispersed across
the genome revealed identical genotypes for the twins, confirming their
monozygosity. The affected twin has three children, two of whom also have NF1,
while the unaffected twin has two children, both unaffected. Using
lymphoblastoid, fibroblast, and buccal cell samples collected from both twins
and from other family members in three generations, we discovered a pathogenic
nonsense mutation in exon 40 of the NF1 gene. This mutation was found in all
cell samples from the affected twin and her affected daughter, and in
lymphoblastoid and buccal cells but not fibroblasts from the unaffected twin. We
also found a novel non-synonymous change in exon 16 of the NF1 gene that was
transmitted from the unaffected mother to both twins and co-segregated with the
pathogenic mutation in the ensuing generation. All cells from the twins were
heterozygous for this apparent exon 16 polymorphism and for single nucleotide
polymorphisms (SNPs) within 2.5 kb flanking the site of the exon 40 nonsense
mutation. This suggests that the NF1 gene of the unaffected twin differed in the
respective lymphoblastoid cells and fibroblasts only at the mutation site
itself, making post-zygotic mutation leading to mosaicism the most likely
mechanism of phenotypic discordance. Although the unaffected twin is a mosaic,
the distribution of the mutant allele among different cells and tissues appears
to be insufficient to cause overt clinical manifestations of NF1. Astroblastoma is a distinctive brain tumor when its histologic features occur in
pure form. More often, the tumor pattern is seen to emerge in infiltrative
astrocytic tumors. The former are rare. Astroblastoma as a de novo component of
gliosarcoma has not previously been described. Furthermore, astroblastoma has
only once been reported to occur in the setting of neurofibromatosis Type I
(NF1), a condition more often associated with pilocytic and diffuse or
infiltrative astrocytic tumors. Herein, we describe a unique case of anaplastic
de novo astroblastoma-sarcoma, in essence a variant of gliosarcoma, occurring in
a 50-year-old female with documented NF1. Genetic study (fluorescence in situ
hybridization) demonstrated no chromosomal losses or gains. Testing for
abnormalities of chromosomes 7, 9, 10, 12, 17, 19 and 20, including the EGFR,
p16, PTEN, MDM2 and NF1 gene regions, we found the tumor to exhibit a deletion
of PTEN, monosomy 17 and gains of chromosomes 19 and 20q. The latter
alterations, having been reported in astroblastoma, were noted in both tumor
components, thus confirming the common origin of the glial and sarcomatous
elements. PURPOSE: Neurofibromatosis type 1 (NF1) is a common autosomal domit disorder
with an estimated incidence of one in 3,500 births. Clinically, NF1 is
characterized by café-au-lait (CAL) spots, neurofibromas, freckling of the
axillary or inguinal region, Lisch nodules, optic nerve glioma, and bone
dysplasias. NF1 is caused by inactivating mutations of the 17q11.2-located NF1
gene. We present a clinical and molecular study of an Italian family with NF1.
METHODS: The proband, a 10-year-old boy, showed large CAL spots and freckling on
the axillary region and plexiform neurofibromas on the right side only. His
father (47 years old) showed, in addition to the similar signs, numerous
neurofibromas of various sizes on his thorax, abdomen, back, and shoulder. Two
additional family members (a brother and a sister of the proband) presented only
small CAL spots. The coding exons of NF1 gene were analyzed for mutations by
denaturing high-performance liquid chromatography and sequencing in all family
members.
RESULTS: The mutational analysis of the NF1 gene revealed a novel frameshift
insertion mutation in exon 4c (c.654 ins A) in all affected family members. This
novel mutation creates a shift on the reading frame starting at codon 218 and
leads to the introduction of a premature stop at codon 227.
CONCLUSIONS: The segregation of the mutation with the affected phenotype and its
absence in the 200 normal chromosomes suggest that it is responsible for the NF1
phenotype. Introduction. This is the case of a young male patient who presented to his
family physician with atypical left foot pain, which was extremely resistant to
analgesia and caused significant disability. Despite extensive investigations,
the cause of his pain was not identified until 18 months after his initial
symptoms, when the official diagnosis of maligt peripheral nerve sheath
tumour (MPNST) was made. Detailed review of the patient's past history
established the diagnosis of type I neurofibromatosis (NF-1), previously
undetected. Discussion. NF-1 is an autosomal domit genetic disorder caused by
loss of function mutations of the NF1 gene in chromosome 17. Patients with this
condition are at increased risk for developing MPNSTs which, however, are
treatable only in early stages. Conclusion. Although monitoring NF-1 patients
for the development of MPNSTs is common practice, the index of clinical
suspicion in patients without an established NF-1 diagnosis is low. Any atypical
pain in young adults should raise the possibility of this maligcy, and this
case illustrates the fact that MPNSTs can be the first manifestation of NF-1 in
patients previously undiagnosed with the disease. A Thai woman, who was affected with neurofibromatosis type 1, was followed up
and re-evaluated at ages 45, 61, and 67 years. Her mother and her three brothers
were also affected. The proposita was very severely affected. She was born blind
with underdeveloped eyeglobes and had large plexiform neurofibromas on her face.
Her eyelids were gigantic and tears drained from the orifice between them.
Cutaneous neurofibromas were observed all over her body. A novel mutation
c.4821delA was identified in NF1 gene, which predicted truncation of
neurofibromin (p.Leu1607fs). Germline mutations in the RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, MAX, TMEM127, NF1
or VHL genes are identified in about 30% of patients with pheochromocytoma or
paraganglioma and somatic mutations in RET, VHL or MAX genes are reported in 17%
of sporadic tumors. In the present study, using mutation screening of the NF1
gene, mapping of chromosome aberrations by single nucleotide polymorphism (SNP)
array, microarray-based expression profiling and immunohistochemistry (IHC), we
addressed the implication of NF1 somatic alterations in pheochromocytomas and
paragangliomas. We studied 53 sporadic tumors, selected because of their
classification with RET/NF1/TMEM127-related tumors by genome wide expression
studies, as well as a second set of 11 independent tumors selected on their low
individual levels of NF1 expression evaluated by microarray. Direct sequencing
of the NF1 gene in tumor DNA identified the presence of an inactivating NF1
somatic mutation in 41% (25/61) of analyzed sporadic tumors, associated with
loss of the wild-type allele in 84% (21/25) of cases. Gene expression signature
of NF1-related tumors highlighted the downregulation of NF1 and the major
overexpression of SOX9. Among the second set of 11 tumors, two sporadic tumors
carried somatic mutations in NF1 as well as in another susceptibility gene.
These new findings suggest that NF1 loss of function is a frequent event in the
tumorigenesis of sporadic pheochromocytoma and strengthen the new concept of
molecular-based targeted therapy for pheochromocytoma or paraganglioma. Neurofibromatosis is a collective name for a group of genetic conditions in
which benign tumours affect the nervous system. Type 1 is caused by a genetic
mutation in the NF1 gene (OMIM 613113) and symptoms can vary dramatically
between individuals, even within the same family. Some people have very mild
skin changes, whereas others suffer severe medical complications. The condition
usually appears in childhood and is diagnosed if two of the following are
present: six or more café-au-lait patches larger than 1.5 cm in diameter,
axillary or groin freckling, 2 or more Lisch nodules (small pigmented areas in
the iris of the eye), 2 or more neurofibromas, optic pathway gliomas, bone
dysplasia, and a first-degree family relative with Neurofibromatosis type 1. The
pattern of inheritance is autosomal domit, however, half of all NF1 cases are
'sporadic' and there is no family history. Neurofibromatosis type 1 is an
extremely variable condition whose morbidity and mortality is largely dictated
by the occurrence of the many complications that may involve any of the body
systems. We describe a family affected by NF1 in whom genetic molecular analysis
identified the same mutation in the son and father. Routine MRI showed pontine
focal lesions in the eight-year-old son, though not in the father. We performed
a four years follow-up study and at follow-up pontine hamartoma size remained
unchanged in the son, and the father showed still no brain lesions, confirming
thus an intra-familial phenotype variability. |
Which genes does thyroid hormone receptor alpha1 regulate in the heart? | β-myosin heavy chain, alpha-myosin heavy chain, SR(Ca)ATPase, phospholamban, nucleotide-gated potassium channel 2, KCNE1, HCN2, HCN4, KCND2, KCND3, KCNA4 | Physiological and pathological cardiac hypertrophy have directionally opposite
changes in transcription of thyroid hormone (TH)-responsive genes, including
alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum
Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional
abnormalities in pathological hypertrophy, such as pressure overload. These
findings suggest relative hypothyroidism in pathological hypertrophy, but serum
levels of TH are usually normal. We studied the regulation of TH receptors (TRs)
beta1, alpha1, and alpha2 in pathological and physiological rat cardiac
hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH
target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were
downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype,
phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was
upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine,
T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or
excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and
promoters. In addition, TR cotransfection and treatment with the
TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR
isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and
TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude
that TR isoforms have distinct regulation and function in rat cardiac myocytes.
Changes in myocyte TR levels can explain in part the characteristic molecular
phenotypes in physiological and pathological cardiac hypertrophy. Exercise training improves the aging-induced downregulation of myosin heavy
chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in
the regulation of cardiac contraction and relaxation. Thyroid hormone receptor
(TR), a transcriptional activator, affected the regulation of gene expression of
MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling
contributes to a molecular mechanism of exercise training-induced improvement of
MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated
whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged
heart are affected by exercise training, using the hearts of sedentary young
rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo
old, swimming training for 8 wk). Trained aged rats showed improvement in
cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart
were significantly lower in sedentary aged rats than in sedentary young rats and
were significantly higher in trained aged rats than in sedentary aged rats. The
activity of TR DNA binding to the transcriptional regulatory region in the
alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of
alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and
thyroxine levels were altered in association with changes in the myocardial TR
protein levels. These findings suggest that exercise training improves the
aging-induced downregulation of myocardial TR signaling-mediated transcription
of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of
cardiac function in trained aged hearts. Hypothyroid heart displays a phenotype of cardioprotection against ischemia and
this study investigated whether administration of dronedarone, an
amiodarone-like compound that has been shown to preferentially antagonize
thyroid hormone binding to thyroid hormone receptor alpha1 (TRalpha1), results
in a similar effect. Dronedarone was given in Wistar rats (90 mg/kg, once daily
(od) for 2 weeks) (DRON), while untreated animals served as controls (CONT).
Hypothyroidism (HYPO) was induced by propylthiouracil administration. Isolated
rat hearts were perfused in Langendorff mode and subjected to 20 minutes of
zero-flow global ischemia (I) followed by 45 minutes of reperfusion (R). 3,5,3'
Triiodothyronine remained unchanged while body weight and food intake were
reduced. alpha-Myosin heavy chain (alpha-MHC) decreased in DRON while
beta-myosin heavy chain (beta-MHC) and sarcoplasmic reticulum Ca2+ adenosine
triphosphatase (ATPase) expression (SERCA) was similar to CONT. In HYPO,
alpha-MHC and SERCA were decreased while beta-MHC was increased. Myocardial
glycogen content was increased in both DRON and HYPO. In DRON, resting heart
rate and contractility were reduced and ischemic contracture was significantly
suppressed while postischemic left ventricular end-diastolic pressure and
lactate dehydrogenase release (IU/L min) after I/R were significantly decreased.
In conclusion, dronedarone treatment results in cardioprotection by selectively
mimicking hypothyroidism. This is accompanied by a reduction in body weight
because of the suppression of food intake. TRs might prove novel pharmacologic
targets for the treatment of cardiovascular illnesses. The profound effects of thyroid hormone (TH) on heart development and function
are mediated by the thyroid hormone receptors (TR) alpha(1) and beta(1). While
numerous patients with TRbeta(1) mutations have been identified, patients with
similar mutations in TRalpha(1) are yet to be discovered. Recently generated
heterozygous mice with a domit negative mutation in TRalpha(1) (TRalpha(1)+/m
mice) have normal TH levels, which may have hampered the discovery of patients
with such mutations. We now measure intracellular Ca(2+) and contraction in
cardiomyocytes isolated from TRalpha(1)+/m mice and wildtype littermates (WT).
TRalpha(1)+/m cardiomyocytes showed a phenotype similar to that in
hypothyroidism with significant slowing of voltage-activated Ca(2+) transients
and contractions. Increased stimulation frequency (from 0.5 to 3 Hz) or
beta-adrenergic stimulation reduced the differences between TRalpha(1)+/m and WT
cardiomyocytes. However, in TRalpha(1)+/m cells stimulation at 3 Hz gave a
marked increase in diastolic Ca(2+) and beta-adrenergic stimulation triggered
spontaneous Ca(2+) release events during relaxation. Both TRalpha(1)+/m and WT
cardiomyocytes responded to TH treatment by displaying a "hyperthyroid"
phenotype with faster and larger Ca(2+) transients and contractions. Excised
TRalpha(1)+/m hearts showed an increased expression of phospholamban (PLB). In
conclusion, isolated TRalpha(1)+/m cardiomyocytes display major dysfunctions
with marked slowing of the Ca(2+) transients and contractions. Maladaptive cardiac hypertrophy results in phenotypic changes in several genes
that are thyroid hormone responsive, suggesting that thyroid hormone receptor
(TR) function may be altered by cellular kinases, including protein kinase C
(PKC) isozymes that are activated in pathological hypertrophy. To investigate
the role of PKC signaling in regulating TR function, cultured neonatal rat
ventricular myocytes were transduced with adenovirus (Ad) expressing wild-type
(wt) or kinase-inactive (dn) PKC alpha or constitutively active (ca) PKC delta
and PKC epsilon. Overexpression of wtPKC alpha, but not caPKC delta or caPKC
epsilon, induced a 28-fold increase (P < 0.001) in TR alpha1 protein in the
nuclear compartment and a smaller increase in the cytosol. Furthermore, TR
alpha1 mRNA was increased 55-fold (P < 0.001). This effect of PKC alpha was
dependent on its kinase activity because dnPKC alpha was without effect. Phorbol
12-myristate 13-acetate (PMA) induced nuclear translocation of endogenous PKC
alpha and Ad-wtPKC alpha concomitantly with an increase in nuclear TR alpha1
protein. In contrast, PMA-induced nuclear translocation of dnPKC alpha resulted
in a decrease of TR alpha1. The increase in TR alpha1 protein in Ad-wtPKC
alpha-transduced cardiomyocytes was not the result of a reduced rate of protein
degradation, nor was the half-life of TR alpha1 mRNA prolonged, suggesting a PKC
alpha-mediated effect on TR alpha transcription. Although phosphorylation of
ERK1/2 was increased in Ad-wtPKC alpha-transduced cells, inhibition of
phospho-ERK did not change TR alpha1 expression. PKC alpha overexpression in
cardiomyocytes caused marked repression of triiodothyronine (T3)-responsive
genes, alpha-myosin heavy chain, and the sarcoplasmic reticulum
calcium-activated adenosinetriphosphatase SERCA2. Treatment with T3 for 4 h
resulted in significant reductions of PKC alpha in nuclear and cytosolic
compartments, and decreased TR alpha1 mRNA and protein, with normalization of
phenotype. These results implicate PKC alpha as a regulator of TR function and
suggest that nuclear localization of PKC alpha may control transcription of the
TR alpha gene, and consequently, affect cardiac phenotype. Pressure overload-induced cardiac hypertrophy leads to decreased contractile
performance, frequently progressing to heart failure. Cardiac hypertrophy and
heart failure can be accompanied by the so-called sick thyroid syndrome,
resulting in decreased serum T(3) levels along with decreased expression of
thyroid hormone receptors (TRalpha1 and TRbeta1) and sarco(endo)plasmic
reticulum Ca-ATPase (SERCA). Because the binding of T(3) occupied receptors to
the thyroid response elements in the SERCA promotor can increase gene
expression, we wanted to determine whether increasing TR expression in the
hypertrophied heart could also improve SERCA expression and cardiac function.
Mice subjected to aortic constriction to generate pressure overload-induced
hypertrophy were also subjected to gene therapy using adeno-associated virus
(AAV) expressing either TRalpha1 or TRbeta1, with LacZ expressing AAV serving as
control. After 8 wk of aortic constriction, a similar degree of hypertrophy was
observed in all three groups; however, mice treated with TRalpha1 or TRbeta1
showed improved contractile function. Administration of a physiological dose of
T(3) increased serum T(3) levels only into the lower range of normal. This T(3)
dose, with or without AAV TR treatment, did not result in any significant
increase in contractile performance. Calcium transients measured in isolated
myocytes also exhibited an enhanced rate of decay associated with TRalpha1 or
TRbeta1 treatment. Western blot analysis showed increased SERCA expression in
the TRalpha1- or TRbeta1-treated groups relative to the LacZ-treated control
group. These results demonstrate that increasing TR expression in the
hypertrophied heart is associated with an improvement in contractile function
and increased SERCA expression. Thyroid hormone receptor alpha1 (TRalpha1) is predomitly expressed in the
myocardium but its biological function under physiological or pathological
conditions remains largely unknown. The present study investigated possible
interactions between alpha1 adrenergic and thyroid hormone signaling at the
level of TRalpha1, potential underlying mechanisms and physiological
consequences, as well as the role of TRalpha1 in cell differentiation. This may
be of physiological relevance since both thyroid hormone and adrenergic
signalling are implicated in the pathophysiology of cardiac remodelling.
Neonatal cardiomyocytes obtained from newborn rats (2-3 days) were exposed to
phenylephrine (PE, an alpha1 adrenergic agonist) for 5 days, in the absence or
excess of T3 in the culture medium. PE, in the absence of T3, resulted in 5.0
fold increase in TRalpha1 expression in nucleus and 2.0 fold decrease in
TRalpha1 expression in cytosol, P<0.05. As a result, a fetal pattern of myosin
isoform expression with marked expression of beta-MHC was observed in PE treated
vs the untreated cells, P<0.05. PD98059 (an ERK signalling inhibitor) abrogated
this response. In the presence of T3 in the culture medium, TRalpha1 expression
was increased 1.6 fold in nucleus and 2.0 fold in cytosol in PE-T3 vs PE treated
cells, P<0.05, and the fetal pattern of myosin isoform expression was prevented.
Parallel studies with H9c2 myoblasts showed that reduction of T3 binding to
TRalpha1 receptor delayed cardiac myoblasts differentiation without affecting
proliferation. In conclusion, in neonatal cardiomyocytes, nuclear TRalpha1 is
overexpressed after prolonged activation of the alpha1- adrenergic signalling by
PE. This response seems to be an ERK kinase dependent process. Over-expression
of TRalpha1 may lead to fetal cardiac phenotype in the absence of thyroid
hormone availability. Furthermore, TRalpha1 seems to be critical in cardiac
myoblast differentiation. The cardiac transient outward current I(to) is regulated by thyroid hormone
(T3). However, it remains unclear whether T3 directly modulates underlying gene
transcription and which thyroid receptor (TR) isoform might be responsible for
gene transactivation. To clarify this situation, we analysed the role of T3 and
its receptors alpha1 (TRalpha1) and beta1 (TRbeta1) in regulation of KCNA4,
KCND2, KCND3 and KCNIP2 transcription in rat cardiomyocytes. Initial results
demonstrated a T3-mediated increase of I(to) current density. T3 stimulation
enhanced KCND2 and KCND3 expression and decreased KCNA4 transcription, while
KCNIP2 remained unaffected. To dissect the role of TRalpha1 and TRbeta1 in
T3-dependent I(to) modulation, TRalpha1 and TRbeta1 were overexpressed in
cardiomyocytes by adenovirus-mediated gene transfer. TRalpha1 increased I(to),
while TRbeta1 significantly reduced I(to) in size, which was associated with
TRalpha1-mediated increase and TRbeta1-mediated reduction of KCND2/3
transcription. To further evaluate a possible direct interaction of TRalpha1 and
TRbeta1 with the KCND3 promoter, TR expression vectors were cotransfected with a
construct containing 2335 bp of the KCND3 5'-flanking sequence linked to a
luciferase reporter into ventricular myocytes. While the TRalpha1 aporeceptor
enhanced KCND3 transcription, the TRbeta1 aporeceptor suppressed KCND3
expression, with both effects exhibiting ligand-dependent amplification upon T3
stimulation. Deletion of the KCND3 5'-flanking region localized the suppressible
promoter sequence for TRbeta1 to within -293 bp and the activating promoter
sequence for TRalpha1 to within -2335 to -1654 bp of the transcription start
site. Disruption of putative TR binding sites by mutagenesis abolished the
TRalpha1- (G-1651T) and TRbeta1- (G-73T) mediated effects, indicating that
TRalpha1 and TRbeta1 response elements map to different regions of the KCND3
promoter. Thus, I(to) is modulated by diverse T3-dependent regulation of
underlying gene transcription. TRalpha1 and TRbeta1 exhibit distinct effects on
KCND3 transactivation with TRalpha1 enhancing and TRbeta1 suppressing KCND3
transcription. AIMS: The reduced heart rate and prolonged QT(end) duration in mice deficient in
thyroid hormone receptor (TR) alpha1 may involve aberrant expression of the K(+)
channel alpha-subunit KCNQ1 and its regulatory beta-subunit KCNE1. Here we focus
on KCNE1 and study whether increased KCNE1 expression can explain changes in
cardiac function observed in TRalpha1-deficient mice.
METHODS: TR-deficient, KCNE1-overexpressing and their respective wildtype (wt)
mice were used. mRNA and protein expression were assessed with Northern and
Western blot respectively. Telemetry was used to record electrocardiogram and
temperature in freely moving mice. Patch-clamp was used to measure action
potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster
ovary (CHO) cells.
RESULTS: KCNE1 was four to 10-fold overexpressed in mice deficient in TRalpha1.
Overexpression of KCNE1 with a heart-specific promoter in transgenic mice
resulted in a cardiac phenotype similar to that in TRalpha1-deficient mice,
including a lower heart rate and prolonged QT(end) time. Cardiomyocytes from
KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected
with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying
current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at
higher KCNE1 levels.
CONCLUSION: The bradycardia and prolonged QT(end) time in hypothyroid states can
be explained by altered K(+) channel function due to decreased
TRalpha1-dependent repression of KCNE1 expression. Thyroid hormone has profound direct effects on cardiac function, but the
hormonal interactions with the autonomic control of heart rate are unclear.
Because thyroid hormone receptor (TR)-alpha1 has been implicated in the
autonomic control of brown adipose energy metabolism, it might also play an
important role in the central autonomic control of heart rate. Thus, we aimed to
analyze the role of TRalpha1 signaling in the autonomic control of heart rate
using an implantable radio telemetry system. We identified that mice expressing
the mutant TRalpha1R384C (TRalpha1+m mice) displayed a mild bradycardia, which
becomes more pronounced during night activity or on stress and is accompanied by
a reduced expression of nucleotide-gated potassium channel 2 mRNA in the heart.
Pharmacological blockage with scopolamine and the beta-adrenergic receptor
antagonist timolol revealed that the autonomic control of cardiac activity was
similar to that in wild-type mice at room temperature. However, at
thermoneutrality, in which the regulation of heart rate switches from
sympathetic to parasympathetic in wild-type mice, TRalpha1+m mice maintained
sympathetic stimulation and failed to activate parasympathetic signaling. Our
findings demonstrate a novel role for TRalpha1 in the adaptation of cardiac
activity by the autonomic nervous system and suggest that human patients with a
similar mutation in TRalpha1 might exhibit a deficit in cardiac adaptation to
stress or physical activity and an increased sensitivity to beta-blockers. Thyroid hormone receptor α1 (TRα1) is shown to be critical for the maturation of
cardiomyocytes and for the cellular response to stress. TRα1 is altered during
post ischemic cardiac remodeling but the physiological significance of this
response is not fully understood. Thus, the present study explored the potential
consequences of selective pharmacological inhibition of TRα1 on the mechanical
performance of the post-infarcted heart. Acute myocardial infarction was induced
in mice (AMI), while sham operated animals served as controls (SHAM). A group of
mice was treated with debutyl-dronedarone (DBD), a selective TRα1 inhibitor
(AMI-DBD). AMI resulted in low T3 levels in plasma and in down-regulation of
TRα1 and TRβ1 expression. Left ventricular ejection fraction (LVEF%) was
significantly reduced in AMI [33 (SEM 2.1) vs 79(2.5) in SHAM, p < 0.05] and was
further declined in AMI-DBD [22(1.1) vs 33(2.1), respectively, p < 0.05].
Cardiac mass was increased in AMI but not in AMI-DBD hearts, resulting in
significant increase in wall tension index. This increase in wall stress was
accompanied by marked activation of p38 MAPK, a kinase that is sensitive to
mechanical stretch and exerts negative inotropic effect. Furthermore, AMI
resulted in β-myosin heavy chain overexpression and reduction in the ratio of
SR(Ca)ATPase to phospholamban (PLB). The latter further declined in AMI-DBD
mainly due to increased expression of PLB. AMI induces downregulation of thyroid
hormone signaling and pharmacological inhibition of TRα1 further depresses
post-ischemic cardiac function. p38 MAPK and PLB may, at least in part, be
involved in this response. |
What is the content of the METLIN database? | METLIN is a metabolite database containing tandem mass spectrometry data for each metabolite. | Metabolites in islets of Langerhans and Escherichia coli strain DH5-alpha were
analyzed using negative-mode, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS). For analysis of anionic
metabolites by MALDI, 9-aminoacridine as the matrix yielded a far superior
signal in comparison to alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic
acid, 2,4,6,-trihydroxyacetophenone, and 3-hydroxypicolinic acid. Limits of
detection for metabolite standards were as low as 15 nM for GDP, GTP, ADP, and
ATP and as high as 1 muM for succinate in 1-muL samples. Analysis of islet
extracts allowed detection of 44 metabolites, 29 of which were tentatively
identified by matching molecular weight to compounds in METLIN and KEGG
databases. Relative quantification was demonstrated by comparing the ratio of
selected di- and triphosphorylated nucleotides for islets incubated with
different concentrations of glucose. For islets at 3 mM glucose, concentration
ratios of ATP/ADP, GTP/GDP, and UTP/UDP were 1.9 +/- 1.39, 1.12 +/- 0.50, and
0.79 +/- 0.35 respectively, and at 20 mM glucose stimulation, the ratios
increased to 4.13 +/- 1.89, 5.62 +/-4.48, and 4.30 +/- 4.07 (n = 3). Analysis
was also performed by placing individual, intact islets on a MALDI target plate
with matrix and impinging the laser directly on the dried islet. Direct analysis
of single islets allowed detection of 43 metabolites, 28 of which were database
identifiable. A total of 43% of detected metabolites from direct islet analysis
were different from those detected in islet extracts. The method was extended to
prokaryotic cells by analysis of extracts from E. coli. Sixty metabolites were
detected, 39 of which matched compounds in the MetaCyc database. A total of 27%
of the metabolites detected from prokaryotes overlapped those found in islets.
These results show that MALDI can be used for detection of metabolites in
complex biological samples. Endogenous metabolites have gained increasing interest over the past 5 years
largely for their implications in diagnostic and pharmaceutical biomarker
discovery. METLIN (http://metlin.scripps.edu), a freely accessible web-based
data repository, has been developed to assist in a broad array of metabolite
research and to facilitate metabolite identification through mass analysis.
METLINincludes an annotated list of known metabolite structural information that
is easily cross-correlated with its catalogue of high-resolution Fourier
transform mass spectrometry (FTMS) spectra, tandem mass spectrometry (MS/MS)
spectra, and LC/MS data. Mass spectrometry based metabolomics represents a new area for bioinformatics
technology development. While the computational tools currently available such
as XCMS statistically assess and rank LC-MS features, they do not provide
information about their structural identity. XCMS(2) is an open source software
package which has been developed to automatically search tandem mass
spectrometry (MS/MS) data against high quality experimental MS/MS data from
known metabolites contained in a reference library (METLIN). Scoring of hits is
based on a "shared peak count" method that identifies masses of fragment ions
shared between the analytical and reference MS/MS spectra. Another functional
component of XCMS(2) is the capability of providing structural information for
unknown metabolites, which are not in the METLIN database. This "similarity
search" algorithm has been developed to detect possible structural motifs in the
unknown metabolite which may produce characteristic fragment ions and neutral
losses to related reference compounds contained in METLIN, even if the precursor
masses are not the same. In an effort to simplify and streamline compound identification from
metabolomics data generated by liquid chromatography time-of-flight mass
spectrometry, we have created software for constructing Personalized Metabolite
Databases with content from over 15,000 compounds pulled from the public METLIN
database (http://metlin.scripps.edu/). Moreover, we have added extra
functionalities to the database that (a) permit the addition of user-defined
retention times as an orthogonal searchable parameter to complement accurate
mass data; and (b) allow interfacing to separate software, a Molecular Formula
Generator (MFG), that facilitates reliable interpretation of any database
matches from the accurate mass spectral data. To test the utility of this
identification strategy, we added retention times to a subset of masses in this
database, representing a mixture of 78 synthetic urine standards. The synthetic
mixture was analyzed and screened against this METLIN urine database, resulting
in 46 accurate mass and retention time matches. Human urine samples were
subsequently analyzed under the same analytical conditions and screened against
this database. A total of 1387 ions were detected in human urine; 16 of these
ions matched both accurate mass and retention time parameters for the 78 urine
standards in the database. Another 374 had only an accurate mass match to the
database, with 163 of those masses also having the highest MFG score.
Furthermore, MFG calculated a formula for a further 849 ions that had no match
to the database. Taken together, these results suggest that the METLIN Personal
Metabolite database and MFG software offer a robust strategy for confirming the
formula of database matches. In the event of no database match, it also suggests
possible formulas that may be helpful in interpreting the experimental results. A rapid, sensitive and versatile liquid chromatography/electrospray ionization
tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the
comprehensive analyses of the chemical constituents contained in the Chinese
medicine-Venenum Bufonis (VB, Chan' Su in Chinese). LC analysis was carried out
on an Agilent Eclipse plus C₁₈ RRHD column (2.1 × 150 mm, 1.8 μm) with a linear
gradient solvent system of water (0.1% formic acid) and acetonitrile (0.1%
formic acid) as mobile phase. Detection and quantification were performed by
multiple reaction monitoring (MRM) transitions via electrospray ionization (ESI)
source operating in the positive ionization mode. Through "Molecular Feature
Extraction" (MFE), more than 900 features were detected from VB extracts. Among
them, a total of 97 components were identified using the Agilent METLIN accurate
mass matching database (DB) established according to those reported in the
literatures. Further more, 30 high quality matches were obtained by comparisons
of their accurate mass and retention times (AMRT) with those imported out in the
developed personal database (METLIN DB with AMRT). The characteristic
fragmentation pathways were proposed for the tentative characterization of four
representative types of bufadienolides in the present work. The targeted MS/MS
experiment of the 30 major compounds was performed for their quantification and
semi-quantification. And 7 of them were quantified over the assaying
concentration range of 5.0-500 pg/μL. The lowest limit of detection and
quantification of them were 0.25-0.50 and 1.25-0.25 pg/μL, respectively. The
recoveries varied from 83 to 106% depending on the chemical types and different
extraction solvents. The remained 23 bufosteroids were simultaneously
semi-quantified using three representative standard compounds as their standard
references, respectively. Untargeted metabolomics provides a comprehensive platform for identifying
metabolites whose levels are altered between two or more populations. By using
liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS),
hundreds to thousands of peaks with a unique m/z ratio and retention time are
routinely detected from most biological samples in an untargeted profiling
experiment. Each peak, termed a metabolomic feature, can be characterized on the
basis of its accurate mass, retention time and tandem mass spectral
fragmentation pattern. Here a seven-step protocol is suggested for such a
characterization by using the METLIN metabolite database. The protocol starts
from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the
bioinformatics program XCMS, and it describes a strategy for selecting
interesting features as well as performing subsequent targeted tandem MS. The
seven steps described will require 2-4 h to complete per feature, depending on
the compound. Malaria is a global infectious disease that threatens the lives of millions of
people. Transcriptomics, proteomics and functional genomics studies, as well as
sequencing of the Plasmodium falciparum and Homo sapiens genomes, have shed new
light on this host-parasite relationship. Recent advances in accurate mass
measurement mass spectrometry, sophisticated data analysis software, and
availability of biological pathway databases, have converged to facilitate our
global, untargeted biochemical profiling study of in vitro P.
falciparum-infected (IRBC) and uninfected (NRBC) erythrocytes. In order to
expand the number of detectable metabolites, several key analytical steps in our
workflows were optimized. Untargeted and targeted data mining resulted in
detection of over one thousand features or chemical entities. Untargeted
features were annotated via matching to the METLIN metabolite database. For
targeted data mining, we queried the data using a compound database derived from
a metabolic reconstruction of the P. falciparum genome. In total, over one
hundred and fifty differential annotated metabolites were observed. To
corroborate the representation of known biochemical pathways from our data, an
inferential pathway analysis strategy was used to map annotated metabolites onto
the BioCyc pathway collection. This hypothesis-generating approach resulted in
over-representation of many metabolites onto several IRBC pathways, most
prominently glycolysis. In addition, components of the "branched" TCA cycle,
partial urea cycle, and nucleotide, amino acid, chorismate, sphingolipid and
fatty acid metabolism were found to be altered in IRBCs. Interestingly, we
detected and confirmed elevated levels for cyclic ADP ribose and phosphoribosyl
AMP in IRBCs, a novel observation. These metabolites may play a role in
regulating the release of intracellular Ca(2+) during P. falciparum infection.
Our results support a strategy of global metabolite profiling by untargeted data
acquisition. Untargeted and targeted data mining workflows, when used together
to perform pathway-inferred metabolomics, have the benefit of obviating MS/MS
confirmation for every detected compound. Reversed-phase gradient LC-MS was used to perform untargeted metabonomic
analysis on extracts of human colorectal cancer (CRC) cell lines (COLO 205,
HT-29, HCT 116 and SW620) subcutaneously implanted into age-matched athymic nude
male mice to study small molecule metabolic profiles and examine possible
correlations with human cancer biopsies. Following high mass accuracy data
analysis using MS and MS/MS, metabolites were identified by searching against
major metabolite databases including METLIN, MASSBANK, The Human Metabolome
Database, PubChem, Biospider, LipidMaps and KEGG. HT-29 and COLO 205 tumor
xenografts showed a distribution of metabolites that differed from SW620 and HCT
116 xenografts (predomitly on the basis of relative differences in the
amounts of amino acids and lipids detected). This finding is consistent with
NMR-based analysis of human colorectal tissue, where the metabolite profiles of
HT-29 tumors exhibit the greatest similarity to human rectal cancer tissue with
respect to changes in the relative amounts of lipids and choline-containing
compounds. As the metabolic signatures of cancer cells result from
oncogene-directed metabolic reprogramming, the HT-29 xenografts in mice may
prove to be a useful model to further study the tumor microenvironment and
cancer biology. PURPOSE: To determine if plasma metabolic profiles can detect differences
between patients with neovascular age-related macular degeneration (NVAMD) and
similarly-aged controls.
METHODS: Metabolomic analysis using liquid chromatography with Fourier-transform
mass spectrometry (LC-FTMS) was performed on plasma samples from 26 NVAMD
patients and 19 controls. Data were collected from mass/charge ratio (m/z) 85 to
850 on a Thermo LTQ-FT mass spectrometer, and metabolic features were extracted
using an adaptive processing software package. Both non-transformed and log2
transformed data were corrected using Benjamini and Hochberg False Discovery
Rate (FDR) to account for multiple testing. Orthogonal Partial Least
Squares-Discrimit Analysis was performed to determine metabolic features that
distinguished NVAMD patients from controls. Individual m/z features were matched
to the Kyoto Encyclopedia of Genes and Genomes database and the Metlin
metabolomics database, and metabolic pathways associated with NVAMD were
identified using MetScape.
RESULTS: Of the 1680 total m/z features detected by LC-FTMS, 94 unique m/z
features were significantly different between NVAMD patients and controls using
FDR (q = 0.05). A comparison of these features to those found with log2
transformed data (n = 132, q = 0.2) revealed 40 features in common, reaffirming
the involvement of certain metabolites. Such metabolites included di- and
tripeptides, covalently modified amino acids, bile acids, and vitamin D-related
metabolites. Correlation analysis revealed associations among certain
significant features, and pathway analysis demonstrated broader changes in
tyrosine metabolism, sulfur amino acid metabolism, and amino acids related to
urea metabolism.
CONCLUSIONS: These data suggest that metabolomic analysis can identify a panel
of individual metabolites that differ between NVAMD cases and controls. Pathway
analysis can assess the involvement of certain metabolic pathways, such as
tyrosine and urea metabolism, and can provide further insight into the
pathophysiology of AMD. BACKGROUND: Metabolomics is a real challenge owing to the complexity and
chemical heterogeneity of biological samples. In this study, a comparative
analysis of metabolite profiling using ten metabolite extraction techniques were
investigated on a pooled plasma sample followed by direct infusion ESI-MS
analysis on both positive and negative modes. Methodology & Results: Metabolites
from a pooled sample of 50 healthy volunteers were separated one- and
two-dimensionally utilizing solvent precipitation, solid-phase extraction and
molecular weight fractionation. Numbers of unique metabolites that are specific
to a particular sample preparation approach were also identified by online
available database, Metlin. The (1)H NMR study of different extraction
procedures were also recorded to elaborate the comparative profiling of the
various fractionation procedures.
CONCLUSION: The results of this study suggest that the metabolite extraction
procedures based on 1- and 2-dimensions, followed by direct infusion ESI-MS
analysis were able to detect endogenous metabolites belonging to different
chemical classes. RATIONALE: Data analysis is a key step in mass spectrometry based untargeted
metabolomics, starting with the generation of generic peak lists from raw liquid
chromatography/mass spectrometry (LC/MS) data. Due to the use of various
algorithms by different workflows, the results of different peak-picking
strategies often differ widely.
METHODS: Raw LC/HRMS data from two types of biological samples (bile and urine),
as well as a standard mixture of 84 metabolites, were processed with four
peak-picking softwares: Peakview®, Markerview™, MetabolitePilot™ and XCMS
Online. The overlaps between the results of each peak-generating method were
then investigated. To gauge the relevance of peak lists, a database search using
the METLIN online database was performed to determine which features had
accurate masses matching known metabolites as well as a secondary filtering
based on MS/MS spectral matching.
RESULTS: In this study, only a small proportion of all peaks (less than 10%)
were common to all four software programs. Comparison of database searching
results showed peaks found uniquely by one workflow have less chance of being
found in the METLIN metabolomics database and are even less likely to be
confirmed by MS/MS.
CONCLUSIONS: It was shown that the performance of peak-generating workflows has
a direct impact on untargeted metabolomics results. As it was demonstrated that
the peaks found in more than one peak detection workflow have higher potential
to be identified by accurate mass as well as MS/MS spectrum matching, it is
suggested to use the overlap of different peak-picking workflows as preliminary
peak lists for more rugged statistical analysis in global metabolomics
investigations. An autonomous metabolomic workflow combining mass spectrometry analysis with
tandem mass spectrometry data acquisition was designed to allow for simultaneous
data processing and metabolite characterization. Although previously tandem mass
spectrometry data have been generated on the fly, the experiments described
herein combine this technology with the bioinformatic resources of XCMS and
METLIN. As a result of this unique integration, we can analyze large profiling
datasets and simultaneously obtain structural identifications. Validation of the
workflow on bacterial samples allowed the profiling on the order of a thousand
metabolite features with simultaneous tandem mass spectra data acquisition. The
tandem mass spectrometry data acquisition enabled automatic search and matching
against the METLIN tandem mass spectrometry database, shortening the current
workflow from days to hours. Overall, the autonomous approach to untargeted
metabolomics provides an efficient means of metabolomic profiling, and will
ultimately allow the more rapid integration of comparative analyses, metabolite
identification, and data analysis at a systems biology level. |
Which is the RNA sequence of the canonical polyadenylation signal? | A polyadenylation signal (AAUAAA) nearby the 3' end of pre-mRNA is required for poly(A) synthesis. | It is well known that nearly all eukaryotic mRNAs contain a 3' poly(A) tail. A
polyadenylation signal (AAUAAA) nearby the 3' end of pre-mRNA is required for
poly(A) synthesis. The protein complex involved in the pre-mRNA polyadenylation
is coupled with RNA polymerase II during the transcription of a gene. According
to the commonly accepted view, only RNAs synthesized by RNA polymerase II can be
polyadenylated in an AAUAAA-dependent manner. Here we report the polyadenylation
of short interspersed elements (SINEs) B2 and VES transcripts generated by RNA
polymerase III. HeLa cells were transfected with SINE constructs with or without
polyadenylation signals. The analyses of the SINE transcripts showed that only
the RNAs with the AAUAAA-signal contained poly(A) tails. Polyadenylated B2 RNA
was found to be much more stable in cells than B2 RNA without a poly(A) tail. A large-scale analysis of human polyadenylation signals was carried out in
silico. The most canonical AAUAAA hexamer and its 11 single-nucleotide variants
that are most frequent in human genes were used to search for polyadenylation
signals in the terminal sequences. Out of 18,277 poly(A) sites that were
identified from 26,414 human genes, 82.5% of the sites were found to contain at
least one of these 12 hexamers as a polyadenylation signal within 40 nucleotides
upstream of the poly(A) site. The rest (17.5%) did not contain any of these
hexamers, which suggests the existence of yet unknown signals. A total of 20,347
terminal sequences in close proximity to 12 polyadenylation signals were
collected using modified EST clustering technique to establish a large-scale
database of polyadenylation signals. To characterize the 12 hexamers, the
locations of polyadenylation signals that were identified as "authentic" and the
uracil contents of the downstream region of the signal were examined. Based on
this analysis, the 11 variants of the canonical AAUAAA were identified as
possibly forming "functional" signals as AAUAAA. Moreover, the observed
frequency of 41.9% for AAUAAA was significantly lower than those of other
reports, suggesting that the non-canonical variants are more important in the
polyadenylation process than frequently recognized. Since the poly(A) sites
processed by those non-canonical variants have not been generally annotated in
major gene databases, it is important to determine whether the variant hexamers
could work as polyadenylation signals that may be responsible for generating
heterogeneity of mRNAs by alternative polyadenylation. Proteinase inhibitors play important roles in host defence systems involving
blood coagulation and pathogen digestion. We isolated and characterized a cDNA
clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library
of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of
three exons and two introns. KPI cDNA contains an open reading frame of 396 bp,
a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes
a polypeptide of 131 amino acids with a putative signal peptide of 21 amino
acids. The deduced amino acid sequence of KPI contains two homologous Kazal
domains, each with six conserved cysteine residues. The mRNA of KPI is expressed
in the hemocytes of healthy shrimp, and the higher expression of KPI transcript
is observed in shrimp infected with the white spot syndrome virus (WSSV),
suggesting a potential role for KPI in host defence mechanisms. Sox genes share a highly conserved DNA-binding motif, the HMG (high mobility
group)-box domain, and have diverse roles in vertebrate embryonic development. A
novel SRY-related cDNA (temporarily called Sox33) isolated from the Chinese
alligator (Alligator sinensis) is 1,819 bp in length, with an open reading frame
from 220 to 1113 bp, encoding a protein of 298 amino acids. Two putative
polyadenylation signal sequences (AATAAA) are present upstream of the poly(A)
tail in the 3' UTR (at 1255-1260 and 1774-1779). The putative protein contains
an HMG-box domain most closely related to hSox12, mSox4, rtSox11, and mSox11
homologs, indicating that alligator Sox33 belongs to group C in the Sox gene
family. Alligator adult and developing tissues were tested for Sox33 mRNA by
independent Northern blots using a 336-bp probe (at 907-1243) between the
HMG-box and the poly(A) site I and a 277-bp probe (at 1477-1754) between the two
polyadenylation sites. Two transcripts (1.3 kb and 1.8 kb) in developing brain
and one (1.8 kb) in adult brain were identified by the 336-bp probe; only one
transcript (1.8 kb) in developing and adult brains was detected by the 277-bp
probe. The results suggest that alligator Sox33 may use a different
polyadenylation mechanism in the developing brain and play a role in the
development and maintece of the nervous system. Polyadenylation (poly(A)) of eukaryotic mRNA is a critical step for gene
expression. In plants, poly(A) signals leading to the formation of polyadenosine
tails after mRNAs include the far upstream elements, the AAUAAA-like signals,
and the mRNA cleavage sites for poly(A). Multiple AAUAAA signals leading to
alternative polyadenosine formation have been found in many genes, but the
effects of each AAUAAA signal on gene expression remain to be uncovered. A DNA
fragment, whose transcript contains two canonical AAUAAA signals from the
3'-untranslation region of endochitinase gene of tobacco (Nicotiana tabacum L.
cv. W38), was mutated and constructed into the downstream of beta-glucuronidase
(GUS) coding region. Transient expression of GUS gene from these constructs
indicated that the distal AAUAAA signal from the stop codon was more important
than the proximal one in stimulating gene expression. Also, the sequence rather
than the distance between the stop codon and the AAUAAA signal region was
critical for gene expression. Transgenic tobaccos with these constructs were
also generated, and the position of the polyadenosine tail formation in this
region was mapped. Results revealed that both AAUAAA signals were functional,
and that polyadenosine tails of most transcripts were directed by the distal
AAUAAA signal. Finally, the RNA stabilities of these variants in transgenic
plants were measured. RNAs from the variants with the functional distal AAUAAA
signal were more stable than those with the functional proximal one only. The
possible secondary structure in this poly(A) signal region was predicted and
discussed. BACKGROUND: Revolver is a newly discovered multi-gene family of transposable
elements in the Triticeae genome. Revolver encompasses 2929 to 3041 bp, has 20
bp of terminal inverted repeated sequences at both ends, and contains a
transcriptionally active gene encoding a DNA-binding-like protein. A putative
TATA box is located at base 221, with a cap site at base 261 and a possible
polyadenylation signal AATAAA at base 2918. Revolver shows considerable
quantitative variation in wheat and its relatives.
RESULTS: Revolver cDNAs varied between 395 and 2,182 bp in length. The first
exon exhibited length variation, but the second and third exons were almost
identical. These variants in the Revolver family shared the downstream region of
the second intron, but varied structurally at the 5' first exon. There were 58
clones, which showed partial homology to Revolver, among 440,000 expressed
sequence tagged (EST) clones sourced from Triticeae. In these Revolver
homologues with lengths of 360-744 bp, the portion after the 2nd exon was
conserved (65-79% homology), but the 1st exon sequences had mutually low
homology, with mutations classified into 12 types, and did not have EST
sequences with open reading frames (ORFs). By PCR with the 3'-flanking region of
a typical genomic clone of Revolver-2 used as a single primer, rye chromosomes
1R and 5R could be simultaneously identified. Extensive eco-geographic diversity
and divergence was observed among 161 genotypes of the single species Triticum
dicoccoides collected from 18 populations in Israel with varying exposures to
abiotic and biotic stresses (soil, temperature, altitude, water availability,
and pathogens).
CONCLUSIONS: On the base of existing differences between Revolver variants, the
molecular markers that can distinguish different rye chromosomes were developed.
Eco-geographic diversification of wild emmer T. dicoccoides in Israel and high
Revolver copy numbers are associated with higher rainfall and biotic stresses.
The remarkable quantitative differences among copy numbers of Revolver in the
same species from different ecosystems suggest strong amplification activity
within the last 10,000 years. It is the interesting finding because the majority
of Triticeae high-copy transposable elements seem to be inactive at the recent
time except for BARE-1 element in Hordeum and the fact might be interesting to
perceive the processes of plant adaptive evolution. We characterized thioredoxin reductase 1 (TrxR1) from Chironomus riparius
(CrTrxR1) and studied its expression under oxidative stress. The full-length
cDNA is 1820bp long and contains an open reading frame (ORF) of 1488bp. The
deduced CrTrxR1 protein has 495 amino acids and a calculated molecular mass of
54.41kDa and an isoelectric point of 6.15. There was a 71bp 5' and a 261bp 3'
untranslated region with a polyadenylation signal site (AATAAA). Homologous
alignments showed the presence of conserved catalytic domain
Cys-Val-Asn-Val-Gly-Cys (CVNVGC), the C-terminal amino acids 'CCS' and conserved
amino acids required in catalysis. The expression of CrTrxR1 is measured using
quantitative real-time PCR after exposure to 50 and 100mg/L of paraquat (PQ) and
2, 10 and 20mg/L of cadmium chloride (Cd). CrTrxR1 mRNA was upregulated after PQ
exposure at all conditions tested. The highest level of CrTrxR1 expression was
observed after exposure to 10mg/L of Cd for 24h followed by 20mg/L for 48h.
Significant downregulation of CrTrxR1 was observed after exposure to 10 and
20mg/L of Cd for 72h. This study shows that the CrTrxR1 could be potentially
used as a biomarker of oxidative stress inducing environmental contamits. SV40 PolyA (Simian virus 40 PolyA, also called PolyA) sequence is DNA sequence
(240 bp) that possesses the activity of transcription termination and can add
PolyA tail to mRNA. PolyA contains AATAAA hexanucleotide polyadenylation signal.
Fourteen copies of Alu in sense orientation (Alu14) were inserted downstream of
GFP in pEGFP-C1 to construct pAlu14 plasmid, and then HeLa cells were
transiently transfected with pAlu14. Northern blot and fluorescence microscope
were used to observe GFP RNA and protein expressions. Our results found that Alu
tandem sequence inhibited remarkably GFP gene expression, but produced
higher-molecular-mass GFP fusion RNA. PolyA and its sequence that was deleted
AATAAA signal in sense or antisense orientation were inserted between GFP and
Alu tandem sequence in pAlu14. The results showed that all the inserted PolyA
sequences partly eliminated the inhibition induced by Alu14. PolyA sequences
without AATAAA signal in sense or antisense orientation still induced
transcription termination. Antisense PolyA (PolyAas) was divided into four
fragments that all are 60 bp long and the middle two fragments were named 2F2R
and 3F3R. 2F2R or 3F3R was inserted upstream of Alu tandem sequence in pAlu14.
The molecular mass of GFP fusion RNA increased when the copy number of 2F2R
increased. 2F2R can support transcription elongation when 2F2R is located
upstream of other 2F2R. Nevertheless, 2F2R located upstream of Alu tandem
sequence can induce transcription termination. Inserting one copy or 64 copies
of 3F3R in upstream of Alu tandem sequence caused the production of
lower-molecular-mass GFP RNA. Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is
mediated by cis elements collectively called the polyadenylation signal.
Genome-wide analysis of such polyadenylation signals was missing in fission
yeast, even though it is an important model organism. We demonstrate that the
canonical AATAAA motif is the most frequent and functional polyadenylation
signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from
cells grown under various physiological conditions, we identify 3' UTRs for
nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is
alternative polyadenylation within and between conditions. We validated the
computationally identified sequence elements likely to promote polyadenylation
by functional assays, including qRT-PCR and 3'RACE analysis. The biological
importance of the AATAAA motif is underlined by functional analysis of the genes
containing it. Furthermore, it has been shown that convergent genes require
trans elements, like cohesin for efficient transcription termination. Here we
show that convergent genes lacking cohesin (on chromosome 2) are generally
associated with longer overlapping mRNA transcripts. Our bioinformatic and
experimental genome-wide results are summarized and can be accessed and
customized in a user-friendly database Pomb(A). We identified and characterized a CYP9 family gene, CrCYP9AT2, from Chironomus
riparius, an eco-toxicologically important model organism. The 1978 base pair
(bp) length CrCYP9AT2 cDNA has an open reading frame of 1587 bp encoding a
putative 528 amino acid protein. There was 267 bp 5' and 123 bp 3' untranslated
region with a polyadenylation signal site (AATAAA). The putative heme-binding
cysteine at position 471 and the typical p450 signature sequence of
463-FGIGPRNCIG-473 were also present. The CrCYP9AT2 transcript was present in
all life stages with the highest expression in larvae. The modulation of
CrCYP9AT2 was studied using real-time polymerase chain reaction after 24h
exposure to cadmium chloride, benzo(a)pyrene; bisphenol A; nonylphenol;
chlorpyrifos and ethinylestradiol. Significant up-regulation of CrCYP9AT2 gene
was observed after exposure to Cd, B(a)P and CP. However, CrCYP9AT2 was
significantly down-regulated after exposure to BPA, NP and EE. |
Which computational frameworks are available for predicting enhancers? | DEEP integrates three components with diverse characteristics that streamline the analysis of enhancer's properties in a great variety of cellular conditions. DEEP uses features derived from histone modification marks or attributes coming from sequence characteristics. Experimental results indicate that DEEP performs better than four state-of-the-art methods on the ENCODE data. The PRISM (predicting regulatory information from single motifs) approach obtains 2543 TF function predictions in a large variety of contexts, at a false discovery rate of 16%. The predictions are highly enriched for validated TF roles, and 45 of 67 (67%) tested binding site regions in five different contexts act as enhancers in functionally matched cells. | The human genome encodes 1500-2000 different transcription factors (TFs).
ChIP-seq is revealing the global binding profiles of a fraction of TFs in a
fraction of their biological contexts. These data show that the majority of TFs
bind directly next to a large number of context-relevant target genes, that most
binding is distal, and that binding is context specific. Because of the effort
and cost involved, ChIP-seq is seldom used in search of novel TF function. Such
exploration is instead done using expression perturbation and genetic screens.
Here we propose a comprehensive computational framework for transcription factor
function prediction. We curate 332 high-quality nonredundant TF binding motifs
that represent all major DNA binding domains, and improve cross-species
conserved binding site prediction to obtain 3.3 million conserved, mostly
distal, binding site predictions. We combine these with 2.4 million facts about
all human and mouse gene functions, in a novel statistical framework, in search
of enrichments of particular motifs next to groups of target genes of particular
functions. Rigorous parameter tuning and a harsh null are used to minimize false
positives. Our novel PRISM (predicting regulatory information from single
motifs) approach obtains 2543 TF function predictions in a large variety of
contexts, at a false discovery rate of 16%. The predictions are highly enriched
for validated TF roles, and 45 of 67 (67%) tested binding site regions in five
different contexts act as enhancers in functionally matched cells. Transcription regulation in multicellular eukaryotes is orchestrated by a number
of DNA functional elements located at gene regulatory regions. Some regulatory
regions (e.g. enhancers) are located far away from the gene they affect.
Identification of distal regulatory elements is a challenge for the
bioinformatics research. Although existing methodologies increased the number of
computationally predicted enhancers, performance inconsistency of computational
models across different cell-lines, class imbalance within the learning sets and
ad hoc rules for selecting enhancer candidates for supervised learning, are some
key questions that require further examination. In this study we developed DEEP,
a novel ensemble prediction framework. DEEP integrates three components with
diverse characteristics that streamline the analysis of enhancer's properties in
a great variety of cellular conditions. In our method we train many individual
classification models that we combine to classify DNA regions as enhancers or
non-enhancers. DEEP uses features derived from histone modification marks or
attributes coming from sequence characteristics. Experimental results indicate
that DEEP performs better than four state-of-the-art methods on the ENCODE data.
We report the first computational enhancer prediction results on FANTOM5 data
where DEEP achieves 90.2% accuracy and 90% geometric mean (GM) of specificity
and sensitivity across 36 different tissues. We further present results derived
using in vivo-derived enhancer data from VISTA database. DEEP-VISTA, when tested
on an independent test set, achieved GM of 80.1% and accuracy of 89.64%. DEEP
framework is publicly available at http://cbrc.kaust.edu.sa/deep/. |
Which proteins in the cerebro-spinal fluid can be used for early diagnosis of Alzheimer's disease? | CSF is a clear fluid that bathes and cushions the brain and spinal cord. Adults have about 1 pint of CSF, which physicians can sample through a minimally invasive procedure called a lumbar puncture, or spinal tap. Research suggests that Alzheimer's disease in its earliest stages may cause changes in CSF levels of tau and beta-amyloid, two proteins that form abnormal brain deposits strongly linked to the disease. | The purpose of the study was to determine whether oxiracetam crosses the human
blood-brain barrier and to evaluate its comparative kinetics in serum and in
cerebro-spinal fluid (CSF). Six DAT patients, undergoing CSF collection for
diagnostic purposes, received 2 g oxiracetam daily, by a 60 min i.v. infusion,
for 7 days. On the last day, in four patients blood samples were collected at
time 0, 30, 60 and 120 min, and lumbar drainage was performed at the end of
infusion: at this time mean CSF concentration was 3.5 micrograms/ml, i.e. 4.0%
of the serum one, demonstrating that oxiracetam crosses the blood-brain barrier.
In two patients, blood samples were collected at time 0, 60, 120 and 240 min,
and lumbar drainage was performed 60 min after the end of infusion: at this time
mean CSF concentration was 2.8 micrograms/ml, i.e. 5.3% of the serum one,
indicating a persistence of oxiracetam within this deep compartment. These
results provide the first evidence in humans that oxiracetam penetrates the
central nervous system and contribute to the understanding of its long-lasting
pharmacodynamic effect in man. Neurofibrillary degeneration is one of the histopathological hallmarks of
Alzheimer disease (AD). Previous studies have shown an association of ubiquitin
with the cytoskeletal protein pathology in AD. In the present study, we report
(i) the measurement of ubiquitin levels in cerebrospinal fluid (CSF) from
histopathologically confirmed AD and control cases, using a new rapid
immunoassay, the competitive enzyme-linked immunoflow assay (CELIFA), (ii) the
determination of ubiquitin levels in brain tissue taken from the same cases,
using a competitive enzyme-linked immunosorbent assay (ELISA), and (iii) an
evaluation of the correlation between levels of ubiquitin in CSF and in brain
tissue. Ubiquitin levels in CSF of AD and neurological control groups are
significantly higher than those of non-neurological aged controls. Ubiquitin
levels in brain homogenates of the AD group are significantly higher than those
of both non-neurological aged and neurological control groups. The source of
this increase in brain ubiquitin in AD is the particulate fraction, because
ubiquitin levels in the brain cytosol fraction are the same among the three
groups. In AD and non-neurological aged controls, there is a significant
positive correlation between ubiquitin levels in CSF and in homogenate of
cerebral white matter. In contrast, the correlation in the non-AD neurological
control group has a negative tendency. These studies suggest that in AD,
elevated levels of ubiquitin in the CSF reflect the increased amount of the
protein in the brain and, therefore, can serve as a biomarker of the
neuropathology in this disease. It is now known that possession of one of the three common forms of the
apolipoprotein E gene (allele epsilon 4) confers an increased risk for
Alzheimer's disease (AD), both familial and sporadic, and that this risk is
dose-dependent. Other genes that may play a role in AD, either through
independent association with the disease or through modification of, or
interaction with, the existing apolipoprotein E (APOE) risk, are now under
investigation including the alpha-1-antichymotrypsin (ACT) gene, the very low
density lipoprotein receptor (VLDL-R) gene, and the presenilin-1 (PS-1) gene.
Kamboh et al. [1995] reported that a polymorphism in the
alpha-1-antichymotrypsin gene could modify the risk for AD conferred by the APOE
locus, specifically by increasing the risk for AD among epsilon 4 homozygotes.
The ACT gene, which is found on chromosome 14, has previously been proposed as a
candidate for AD due to the presence of the ACT protein in senile plaques and
the reported elevation of the protein in the cerebro-spinal fluid (CSF) and
serum of AD cases. We have investigated this reported association within our
familial and sporadic AD dataset, where we find no independent association
between ACT and the occurrence of AD. Logistic regression analysis excludes ACT
or the interaction between ACT and APOE as significant contributors in the
prediction of disease status. By this analysis, ACT genotyping does not provide
additional information about an individual's risk of Alzheimer's disease beyond
the risk information conferred by APOE genotype alone. We present a 61-year-old male patient with progressive dementia. A brain SPECT
with Tc-99m-bicisate was performed for confirmation of clinically suspected
Alzheimer-dementia. At the time of the SPECT-investigation marked apraxia and
aphasia besides severe dementia were present. Electrophysiological as well as
anatomical neuroimaging findings showed non-diagnostic alterations. SPECT
revealed distinct perfusion defects, which made Alzheimer Dementia unlikely. The
further course of the patient was determined by rapidly progressive
deterioration with development of akinetic mutism. Thereafter, increased levels
of neuron-specific enolase as well as 14-3-3 proteins were found in the
cerebro-spinal fluid (CSF). The patient finally died with signs of cerebral
decortication. Due to the clinical course and the CSF-findings the patient's
final diagnosis was Creutzfeldt-Jakob-disease, nevertheless no autopsy was
performed. The presented case report underscores the clinical utility of
perfusion brain SPECT in the differential diagnosis of dementias. In 45 patients meeting NINCDS-ARDRA criteria for probably diagnosis of Alzheimer
disease (AD), ApoE genotype and tau protein level in cerebro-spinal fluid (CSF)
were determined. Frequency of e4 allele occurrence in group of AD patients was
73.3% and showed high statistic significance in comparison with control group.
Increase of tau protein level in CSF was also statistically significant. No
correlation in ApoE allele and tau protein level in CSF was revealed. The
authors emphasize the usefulness of tau protein level measurement and
determination of ApoE allele in diagnosis of Alzheimer disease. Subjects with mild cognitive impairment (MCI) are at a high risk of developing
clinical Alzheimer's disease (AD). We asked to what extent the core biomarker
candidates cerebro-spinal fluid (CSF) beta-amyloid(1-42) (Abeta(1-42)) and CSF
tau protein concentrations predict conversion from MCI to AD. We studied 52
patients with MCI, 93 AD patients, and 10 healthy controls (HC). The MCI group
was composed of 29 patients who had converted to AD during follow-up, and of 23
patients who showed no cognitive decline. CSF Abeta(1-42) and tau protein levels
were assessed at baseline in all subjects, using enzyme-linked immunosorbent
assays. For assessment of sensitivity and specificity, we used independently
established reference values for CSF Abeta(1-42) and CSF tau. The levels of CSF
tau were increased, whereas levels of Abeta(1-42) were decreased in MCI
subjects. Abeta(1-42) predicted AD in converted MCI with a sensitivity of 59%
and a specificity of 100% compared to HC. Tau yielded a greater sensitivity of
83% and a specificity of 90%. In a multiple Cox regression analysis within the
MCI group, low baseline levels of Abeta(1-42), but not other predictor variables
(tau protein, gender, age, apolipoprotein E epsilon4 carrier status, Mini Mental
Status Examination score, observation time, antidementia therapy), correlated
with conversion status (P<0.05). Our findings support the notion that CSF tau
and Abeta(1-42) may be useful biomarkers in the early identification of AD in
MCI subjects. The in vivo diagnosis of Alzheimer's disease (AD) may be facilitated by
cerebro-spinal fluid (CSF) biomarkers in combination with imaging and clinical
assessments. By determining the concentration of beta amyloid fragments, total
tau (t-tau) and phospho-tau (p-tau), it is possible to detect the conversion of
mild cognitive impairment (MCI) to AD or distinguish AD vs. pseudo-dementia.
However, these markers are poorly sensitive to the progressive disease stages.
And far from clear is their role in "mixed" forms of dementia, as far as
hemodynamic deficits complicate the clinical history. We have studied cerebral
hemodynamic impairment in AD patients, relative to control subjects. Mean flow
velocity (MFV), pulsatility index (PI) and cerebrovascular reactivity (assayed
as breath-holding index, BHI) were evaluated by bilateral transcranial Doppler
(TCD) monitoring of middle cerebral arteries. MFV and BHI were significantly
lower and PI was significantly higher in AD patients with respect to control
subjects. The presence of white-matter changes (WMC) in the AD cases did not
influence any of the hemodynamic variables. Noticeably, MMSE score was
correlated to BHI reduction (P<0.005). Our results, consistent with the recent
literature indicate that hemodynamic impairment is a critical marker of
cognitive decline and supports once more the hypothesis of a significant
pathigenic role of vascular damage in AD. Similar functional alterations might
be early hallmarks in a variety of dementia subtypes, including "mixed"
dementia, whose prevalence is undoubtedly increased. Assessment of hemodynamic
reactivity could provide valuable correlations with individual patient's
cognitive profile, which in turn would assist in the identification of critical
steps in disease progression and the validation of effective therapies. The diagnosis of dementing disorders is severely hampered by the absence of
reliable biomarkers that can be measured in body fluids such as blood, urine and
cerebro-spinal fluid (CSF). Searching for biomarkers is hampered by the huge
variability between individuals; the use of autopsy specimens induces
significant data fluctuation due to rapid post-mortem changes in the specimens.
The search for biomarkers obtained from living donors has contributed already a
vast amount of data. The role of amyloid and tau as early diagnostic markers in
the pathology of dementia has been reported in differential involvement in
Alzheimer's disease (AD), late onset Alzheimer disease (LOAD), Lewy Body
dementia (DLBD), Vascular dementia, fronto-temporal lobar degeneration (FTLD),
Mild cognitive impairment (MCI) and non neurological controls. In the coming
decennia, brain/tissue/biobanks (BTB-banks) will have a major role in
identifying the relevant biomarkers and will collect, preserve and type RNA and
DNA extracted from brain/tissue/body fluids in order to update the pathological
hallmarks of dementing disorders. The present paper reviews and compares the
currently known/clinically applied biomarkers in dementia which can be
identified and incorporated into clinical drug trials and elucidate proposed
mechanisms of disease and drug action. Furthermore, the review screens a panel
of biomarkers used for early and differential diagnosis and comments on the
validity of these biomarkers in reflecting the typical hallmarks of neurological
disorders. The aim of this study was to investigate the dynamics of four of the most
validated biomarkers for Alzheimer's disease (AD), cerebro-spinal fluid (CSF)
Abeta 1-42, tau, hippocampal volume, and FDG-PET, in patients at different stage
of AD. Two hundred twenty-nine cognitively healthy subjects, 154 mild cognitive
impairment (MCI) patients converted to AD, and 193 (95 early and 98 late) AD
patients were selected from the Alzheimer's Disease Neuroimaging Initiative
(ADNI) database. For each biomarker, individual values were Z-transformed and
plotted against ADAS-cog scores, and sigmoid and linear fits were compared. For
most biomarkers the sigmoid model fitted data significantly better than the
linear model. Abeta 1-42 time course followed a steep curve, stabilizing early
in the disease course. CSF tau and hippocampal volume changed later showing
similar monotonous trends, reflecting disease progression. Hippocampal loss
trend was steeper and occurred earlier in time in APOE epsilon4 carriers than in
non-carriers. FDG-PET started changing early in time and likely followed a
linear decline. In conclusion, this study provides the first evidence in favor
of the dynamic biomarker model which has recently been proposed. PURPOSE: The enzyme cholesterol 24S-hydroxylase (Cyp46A1) is responsible for the
conversion of cholesterol to its more polar metabolite 24S-hydroxycholesterol,
thereby enabling the intracerebral elimination of cholesterol. An intronic
single nucleotide polymorphism in the gene CYP46A1 (IVS2 -150 T>C; rs754203) has
recently been associated with primary open angle glaucoma (POAG). This
association, however, lacks confirmation in other studies. The purpose of the
present study was to investigate a hypothesized association between rs754203 and
the presence of POAG in a Central European population of Caucasian descent.
METHODS: The present institutional study comprised a total of 581 unrelated
subjects: 330 patients with POAG, and 251 control subjects. Main outcome
measures are genotype distributions and allelic frequencies determined by
polymerase chain reaction.
RESULTS: No significant differences in either genotype distribution or allelic
frequencies were found between patients with POAG and control subjects (p>0.05).
The presence of the rs754203 T-allele was associated with a nonsignificant odds
ratio of 0.81 (95% CI: 0.63-1.04; p=0.11) for POAG.
CONCLUSIONS: Our data suggest that the rs754203 polymorphism itself is unlikely
a genetic risk factor for POAG in Caucasian individuals. The European Medicines Agency (EMA) in London is responsible for the Regulatory
review of new medicinal products for Marketing Authorisation, through which
pharmaceutical companies may obtain first Marketing Authorisation and subsequent
Variations valid throughout the EU and EFTA. The qualification opinion of novel
methodologies is a new procedure where applicants can obtain scientific advice
on new methodologies for regulatory clinical trials of efficacy of new
compounds. It will help benefit/risk assessment of the CHMP. The definition of
prodromal AD is acceptable. The "Dubois Criteria" as criteria to define the
population must be validated in full at the time of the submission of the
dossiers. Including a positive CSF biomarker profile is considered predictive
for the evaluation of the AD-dementia type. However, although high CSF tau and
low CSF Aβ42 are predictive of Alzheimer's disease, the criterion "positive CSF
tau/Aβ42 ratio" is not well defined. The qualification of biomarkers in the
pre-dementia stage of Alzheimer's disease will allow better inclusion criteria
of patients in pre-dementia trials in which the benefit/risk is higher for
treatment with these novel compounds. INTRODUCTION: Corticobasal syndrome (CBS) has a heterogeneous clinical
presentation with no specific pathologic substratum. Its accurate diagnosis is a
challenge for neurologists; in order to establish CBS definitively, postmortem
confirmation is required. Some clinical and radiological features can help to
distinguish it from other neurodegenerative conditions, such as
Creutzfeldt-Jakob disease (CJD).
CLINICAL CASE: A 74-year-old woman presented with language impairment,
difficulty in walking and poor attentiveness that had begun 10 days before.
Other symptoms, such as asymmetrical extra-pyramidal dysfunction, limb dystonia
and 'alien limb' phenomena, were established over the next 2 months, with rapid
progression. Death occurred 3 months after symptom onset. Laboratory results
were normal. Initially, imaging only showed restricted diffusion with bilateral
parieto-occipital gyri involvement on DWI-MRI, with unspecific EEG changes. An
autopsy was performed. Brain neuropathology confirmed sporadic CJD (sCJD).
CONCLUSIONS: CBS is a heterogeneous clinical syndrome whose differential
diagnosis is extensive. CJD can occasionally present with clinical
characteristics resembling CBS. MRI detection of abnormalities in some sequences
(FLAIR, DWI), as previously reported, has high diagnostic utility for sCJD
diagnosis - especially in early stages - when other tests can still appear
normal. Abnormalities on DWI sequencing may not correlate with neuropathological
findings, suggesting a functional basis to explain the changes found. Previously it was reported that Alzheimer's disease (AD) patients have reduced
amyloid (Aβ 1-42) and elevated total tau (t-tau) and phosphorylated tau (p-tau
181p) in the cerebro-spinal fluid (CSF), suggesting that these same measures
could be used to detect early AD pathology in healthy elderly (CN) and mild
cognitive impairment (MCI). In this study, we tested the hypothesis that there
would be an association among rates of regional brain atrophy, the CSF
biomarkers Aβ 1-42, t-tau, and p-tau 181p and ApoE ε4 status, and that the
pattern of this association would be diagnosis specific. Our findings primarily
showed that lower CSF Aβ 1-42 and higher tau concentrations were associated with
increased rates of regional brain tissue loss and the patterns varied across the
clinical groups. Taken together, these findings demonstrate that CSF biomarker
concentrations are associated with the characteristic patterns of structural
brain changes in CN and MCI that resemble to a large extent the pathology seen
in AD. Therefore, the finding of faster progression of brain atrophy in the
presence of lower Aβ 1-42 levels and higher p-tau levels supports the hypothesis
that CSF Aβ 1-42 and tau are measures of early AD pathology. Moreover, the
relationship among CSF biomarkers, ApoE ε4 status, and brain atrophy rates are
regionally varying, supporting the view that the genetic predisposition of the
brain to amyloid and tau mediated pathology is regional and disease stage
specific. Mexico City Metropolitan Area children chronically exposed to high
concentrations of air pollutants exhibit an early brain imbalance in genes
involved in oxidative stress, inflammation, innate and adaptive immune responses
along with accumulation of misfolded proteins observed in the early stages of
Alzheimer and Parkinson's diseases. A complex modulation of serum cytokines and
chemokines influences children's brain structural and gray/white matter
volumetric responses to air pollution. The search for biomarkers associating
systemic and CNS inflammation to brain growth and cognitive deficits in the
short term and neurodegeneration in the long-term is our principal aim. We
explored and compared a profile of cytokines, chemokines (Multiplexing LASER
Bead Technology) and Cellular prion protein (PrP(C)) in normal
cerebro-spinal-fluid (CSF) of urban children with high vs. low air pollution
exposures. PrP(C) and macrophage inhibitory factor (MIF) were also measured in
serum. Samples from 139 children ages 11.91 ± 4.2 years were measured. Highly
exposed children exhibited significant increases in CSF MIF (p = 0.002), IL6 (p
= 0.006), IL1ra (p = 0.014), IL-2 (p = 0.04), and PrP(C) (p = 0.039) vs.
controls. MIF serum concentrations were higher in exposed children (p = 0.009).
Our results suggest CSF as a MIF, IL6, IL1Ra, IL-2, and PrP(C) compartment that
can possibly differentiate air pollution exposures in children. MIF, a key
neuro-immune mediator, is a potential biomarker bridge to identify children with
CNS inflammation. Fine tuning of immune-to-brain communication is crucial to
neural networks appropriate functioning, thus the short and long term effects of
systemic inflammation and dysregulated neural immune responses are of deep
concern for millions of exposed children. Defining the linkage and the health
consequences of the brain / immune system interactions in the developing brain
chronically exposed to air pollutants ought to be of pressing importance for
public health. |
Is there evidence for somatic mosaicism in Tuberous Sclerosis? | Yes, somatic mosaicism in Tuberous Sclerosis has been documented with the use of mutation identification, and subsequent linkage analysis in the affected families. In a large family with both parents unaffected, 3 affected children and 5 unaffected siblings, a 4 bp insertion in TSC2 gene was shown to be inherited from the mother. | We have investigated a family in which three siblings with the autosomal
domit disorder tuberous sclerosis had unaffected parents. The family were
typed for polymorphic markers spanning the two genes known to cause tuberous
sclerosis located at 9q34 (TSC1) and 16p13.3 (TSC2). TSC1 markers showed
different maternal and paternal haplotypes in affected children, excluding a
mutation in TSC1 as the cause of the disease. For the TSC2 markers all the
affected children had the same maternal and paternal haplotypes, as did three of
their unaffected siblings. Mutation screening by RT-PCR and direct sequencing of
the TSC2 gene identified a 4 bp insertion TACT following nucleotide 2077 in exon
18 which was present in the three affected children but not in five unaffected
siblings or the parents. This mutation would cause a frameshift and premature
termination at codon 703. Absence of the mutation in lymphocyte DNA from the
parents was consistent with germline mosaicism and this was confirmed by our
finding of identical chromosome 16 haplotypes in affected and unaffected
siblings, providing unequivocal evidence of two different cell lines in the
gametes. Molecular analysis of the TSC2 alleles present in the affected subjects
showed that the mutation had been inherited from the mother. This is the first
case of germline mosaicism in tuberous sclerosis proven by molecular genetic
analysis and also the first example of female germline mosaicism for a
characterized autosomal domit gene mutation apparently not associated with
somatic mosaicism. |
What is GRO-seq? | Global run-on sequencing (Gro-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. | RNA polymerases are highly regulated molecular machines. We present a method
(global run-on sequencing, GRO-seq) that maps the position, amount, and
orientation of transcriptionally engaged RNA polymerases genome-wide. In this
method, nuclear run-on RNA molecules are subjected to large-scale parallel
sequencing and mapped to the genome. We show that peaks of promoter-proximal
polymerase reside on approximately 30% of human genes, transcription extends
beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent.
Additionally, most promoters have an engaged polymerase upstream and in an
orientation opposite to the annotated gene. This divergent polymerase is
associated with active genes but does not elongate effectively beyond the
promoter. These results imply that the interplay between polymerases and
regulators over broad promoter regions dictates the orientation and efficiency
of productive transcription. We report the immediate effects of estrogen signaling on the transcriptome of
breast cancer cells using global run-on and sequencing (GRO-seq). The data were
analyzed using a new bioinformatic approach that allowed us to identify
transcripts directly from the GRO-seq data. We found that estrogen signaling
directly regulates a strikingly large fraction of the transcriptome in a rapid,
robust, and unexpectedly transient manner. In addition to protein-coding genes,
estrogen regulates the distribution and activity of all three RNA polymerases
and virtually every class of noncoding RNA that has been described to date. We
also identified a large number of previously undetected estrogen-regulated
intergenic transcripts, many of which are found proximal to estrogen receptor
binding sites. Collectively, our results provide the most comprehensive
measurement of the primary and immediate estrogen effects to date and a resource
for understanding rapid signal-dependent transcription in other systems. Global run-on sequencing (GRO-seq) is a recent addition to the series of
high-throughput sequencing methods that enables new insights into
transcriptional dynamics within a cell. However, GRO-sequencing presents new
algorithmic challenges, as existing analysis platforms for ChIP-seq and RNA-seq
do not address the unique problem of identifying transcriptional units de novo
from short reads located all across the genome. Here, we present a novel
algorithm for de novo transcript identification from GRO-sequencing data, along
with a system that determines transcript regions, stores them in a relational
database and associates them with known reference annotations. We use this
method to analyze GRO-sequencing data from primary mouse macrophages and derive
novel quantitative insights into the extent and characteristics of non-coding
transcription in mammalian cells. In doing so, we demonstrate that Vespucci
expands existing annotations for mRNAs and lincRNAs by defining the primary
transcript beyond the polyadenylation site. In addition, Vespucci generates
assemblies for un-annotated non-coding RNAs such as those transcribed from
enhancer-like elements. Vespucci thereby provides a robust system for defining,
storing and analyzing diverse classes of primary RNA transcripts that are of
increasing biological interest. |
What is the role of the RUNX1-MYEF2 complex? | A novel complex, RUNX1-MYEF2, represses hematopoietic genes in erythroid cells. | |
Does smoking increase risk for glioblastoma? | No. Smoking does not increase risk for glioblastoma. | A case-control study of brain tumor was conducted in collaborating hospitals in
Boston, Providence, and Baltimore. Cases were 160 consecutive patients being
treated for glioblastoma, grade 3 or 4 astrocytoma, or anaplastic astrocytoma.
Controls were 128 healthy persons identified among the case's friends. A complex
self-administered questionnaire was used to assess exposure to factors of
interest. There was some evidence that glioblastoma is associated with a
decreased susceptibility to allergies, a finding that may call attention to the
involvement of immunologic disturbances in brain tumors. Our data are not
supportive of previous reports of an association between brain tumors and
exposure to pets or farm environment, family history of CNS maligcies or
other neurologic conditions, or irradiation to the head. We did not find any
evidence for an association with life-style characteristics such as cigarette
smoking, alcohol consumption, use of drugs of any kind, or dietary intake of
cured or smoked meat or fish. Gliomas of astrocytic, oligodendroglial and ependymal origin account for more
than 70% of all brain tumors. The most frequent (65%) and most maligt
histological type is the glioblastoma. Since the introduction of computerized
tomography and magnetic resoce imaging, the incidence rates of brain tumors
have been rather stable, with a tendency of higher rates in highly developed,
industrialized countries. Some reports indicate that Caucasians have higher
incidence than black or Asian populations, but to some extent, this may reflect
socio-economic differences and under-ascertainment in some regions, rather than
a significant difference in genetic susceptibility. With the exception of
pilocytic astrocytomas, the prognosis of glioma patients is still poor. Less
than 3% of glioblastoma patients are still alive at 5 years after diagnosis,
higher age being the most significant predictor of poor outcome. Brain tumors
are a component of several inherited tumor syndromes, but the prevalence of
these syndromes is very low. Several occupations, environmental carcinogens, and
diet (N-nitroso compounds) have been reported to be associated with an elevated
glioma risk, but the only environmental factor unequivocally associated with an
increased risk of brain tumors, including gliomas, is therapeutic X-irradiation.
In particular, children treated with X-irradiation for acute lymphoblastic
leukemia show a significantly elevated risk of developing gliomas and primitive
neuroectodermal tumor (PNET), often within 10 years after therapy. TP53
mutations are frequent in low-grade gliomas and secondary glioblastomas derived
therefrom. Approximately 60% of mutations are located in the hot spot codons 248
and 273, and the majority of these are G:C-->A:T transitions at CpG sites. TP53
mutations are significantly more frequent in low-grade astrocytomas with
promoter methylation of the O(6)-methylguanine-DNA methyltransferase repair
gene, suggesting that, in addition to deamination of 5-methylcytosine, exogenous
or endogenous alkylation in the O(6) position of guanine may contribute to the
formation of these mutations. BACKGROUND: Glutathione transferases (GST) detoxify environmental and endogenous
compounds and levels of two polymorphic GST proteins, GSTM3 and GSTP1, are high
in the brain. Previous studies of GSTM3 and GSTP1 polymorphisms and adult brain
tumor risk have produced inconsistent results, whereas the GSTM3 -63 variant is
newly identified and, therefore, has not yet been studied in this context. We
therefore examined associations between GSTM3 -63, GSTM3 *A/*B, GSTP1 105, and
GSTP1 114 variants and adult brain tumor risk and the interaction of the effects
of these same polymorphisms with cigarette smoking. In addition, the enzymes
NQO1 and CYP1A1 alter susceptibility to oxidative brain damage. Because there is
less previous evidence for a role of NQO1, CYP1A1, GSTM1, and GSTT1 variants, we
restricted analysis of these variants to a small preliminary study.
METHODS: We genotyped DNA collected for an international population-based
case-control study of 725 glioma cases, 329 of which were glioblastoma cases,
546 meningioma cases and 1,612 controls. Study participants were residents of
Sweden, southeast England, Denmark, and Finland.
RESULTS: We found no associations between the GSTM3, GSTP1, NQO1, CYP1A1, GSTM1,
or GSTT1 polymorphisms and adult brain tumor risk with the possible exception of
a weak association between the G-C (Val-Ala) GSTP1 105/114 haplotype and glioma
[odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.54, 0.99], nor was
there an interaction between the effects of the GSTM3 or GSTP1 polymorphisms and
cigarette smoking.
CONCLUSIONS: Overall, we observed no strong evidence for an association between
GST or related enzyme polymorphisms and adult brain tumor risk. The aim of this study was to compare the locomotor functional recovery of
patients with brain tumor and patients with stroke. Each patient with a brain
tumor was matched to a patient with stroke according to the lesion side.
Twenty-one patients operated for intracranial tumors and 21 patients with stroke
were studied. The mean ages (+/- standard deviation) of patients were 52.5+/-16
years for patients with brain tumor and 56.7+/-11 years for patients with
stroke. For each patient, the age, gender, presence of aphasia, smoking habit,
co-morbidities, lesion origin and lesion size (for the brain tumor group) were
recorded. Locomotor and functional recovery were evaluated by using the Postural
Assessment Scale for Stroke, the Berg Balance Scale, Motor Assessment Scale and
the mobility section of the Functional Impairment Measure. There was no
difference regarding demographic characteristics between the two groups. After
rehabilitation both groups had significantly improved in terms of all
parameters, but the extent of improvement did not differ between the two groups. OBJECTIVE: Although causal relationships between smoking and cancer risk have
been established for many sites, most studies of brain cancer have not supported
an association. However, two recent cohort studies showed increased risks of
glioma among smokers. We quantified the association between smoking and glioma
through a meta-analysis of the literature.
METHODS: Of 20 eligible studies, 17 (6 cohort and 11 case–control) were included
in an analysis of ever versus never smoking. Multivariate-adjusted risk
estimates in the papers were pooled to calculate cumulative risk.
RESULTS: The cumulative estimated risk associated with ever smoking was 1.06
(95% CI: 0.97–1.15), for all, 1.10 (95% CI: 1.01–1.20) for cohort, and 1.00 (95%
CI: 0.88–1.15) for case–control studies. A significantly increased risk
associated with past smoking was noted for cohort studies, OR = 1.16 (p =
0.007), while an increased risk of borderline significance was seen for all
studies, OR = 1.10 (p = 0.08). In general, dose–response analysis did not
support an association and was limited because very few studies included these
variables and could be pooled.
CONCLUSION: Overall, results of pooling of all studies suggested that smoking is
not associated with risk of glioma. However, the small but significant increased
risk seen for cohort studies remains to be clarified. BACKGROUND: Epidermal growth factor (EGF) is critical in cancer process. EGF and
EGF receptor (EGFR) interaction plays a pivotal role in cell proliferation,
differentiation, and tumorigenesis of epithelial tissues. Variations of the EGF
+61G/A (rs4444903) may lead to an alteration in EGF production and/or activity,
which can result in individual susceptibility to brain glioma. The purpose of
this study was to investigate the potential association between EGF +61G/A and
brain glioma in a Chinese population.
METHODS: In this study, we analyzed single nucleotide polymorphism of EGF +61G/A
in 677 patients with glioma and 698 gender- and age-matched controls. Genotyping
was performed by polymerase chain reaction-ligation detection reaction (PCR-LDR)
method.
RESULTS: The A allele (minor Allele) was 33.0% in cases and 27.3% in controls.
The additive model was more powerful to reveal the association in our study than
that of recessive and domit model. Our data showed the genotype G/A and A/A
was associated with increased risk for glioma (adjusted OR = 1.48, 95%CI:
1.17-1.87, p = 0.001 for G/A, adjusted OR = 1.81, 95%CI: 1.20-2.72, p = 0.005
for A/A, respectively), and for glioblastoma (adjusted OR = 1.51, 95%CI:
1.06-2.17, p = 0.024 and adjusted OR = 2.35, 95%CI: 1.34-4.15, p = 0.003,
respectively). The A allele significantly increased glioma risk (OR = 1.31,
95%CI: 1.11-1.55, p = 0.001). The additive model (G/G vs G/A vs A/A) showed that
both G/A and A/A genotype increased glioma risk (adjusted OR = 1.40, 95% CI:
1.17-1.66, p = 0.0002).G/A and A/A genotypes or EGF +61 A allele increased risk
in both low and high WHO grade glioma. Non-smokers with G/A and A/A genotype
showed increased glioma risk compared with G/G genotype (adjusted OR = 1.72,
95%CI: 1.29-2.30, p = 0.0002 and adjusted OR = 1.81, 95%CI: 1.10-2.99, p =
0.020, respectively). This association was not found in ever- or
current-smokers.
CONCLUSIONS: Our study indicated that G/A and A/A genotypes or EGF +61 A allele
were associated with higher glioma risk in Chinese. This is in contrast with
previous studies which reported G allele as a risk factor of glioma in
Caucasian. The role of EGF +61 A/G polymorphism in glioma susceptibility needs
further investigation. |
Which genes are implicated in short QT syndrome? | The genes that are implicated in short QT syndrome are KCNJ2, KCNH2, CACNA2D1 and KCNQ1. | BACKGROUND: The electrocardiographic short QT-interval syndrome forms a distinct
clinical entity presenting with a high rate of sudden death and exceptionally
short QT intervals. The disorder has recently been linked to gain-of-function
mutation in KCNH2. The present study demonstrates that this disorder is
genetically heterogeneous and can also be caused by mutation in the KCNQ1 gene.
METHODS AND RESULTS: A 70-year man presented with idiopathic ventricular
fibrillation. Both immediately after the episode and much later, his QT interval
was abnormally short without any other physical or electrophysiological
anomalies. Analysis of candidate genes identified a g919c substitution in KCNQ1
encoding the K+ channel KvLQT1. Functional studies of the KvLQT1 V307L mutant
(alone or coexpressed with the wild-type channel, in the presence of IsK)
revealed a pronounced shift of the half-activation potential and an acceleration
of the activation kinetics leading to a gain of function in I(Ks). When
introduced in a human action potential computer model, the modified biophysical
parameters predicted repolarization shortening.
CONCLUSIONS: We present an alternative molecular mechanism for the short
QT-interval syndrome. Functional and computational studies of the KCNQ1 V307L
mutation identified in a patient with this disorder favor the association of
short QT with mutation in KCNQ1. The short QT syndrome constitutes a new clinical entity that is associated with
a high incidence of sudden cardiac death, syncope, and/or atrial fibrillation
even in young patients and newborns. Patients with this congenital electrical
abnormality are characterized by rate-corrected QT intervals<320 ms. Missense
mutations in KCNH2 (HERG) linked to a gain-of-function of the rapidly activating
delayed-rectifier current I(Kr) have been identified in the first two reported
families with familial sudden cardiac death. Recently, two further
gain-of-function mutations in the KCNQ1 gene encoding the alpha-subunit of the
KvLQT1 (I(Ks)) channel and in the KCNJ2 gene encoding the strong inwardly
rectifying channel protein Kir2.1 confirmed a genetically heterogeneous disease.
The possible substrate for the development of ventricular tachyarrhythmias may
be a significant transmural dispersion of the repolarisation due to a
heterogeneous abbreviation of the action potential duration. The implantable
cardioverter defibrillator is the therapy of choice in patients with syncope and
a positive family history of sudden cardiac death. However, ICD therapy in
patients with a short QT syndrome has an increased risk for inappropriate shock
therapies due to possible T wave oversensing. The impact of sotalol, ibutilide,
flecainide, and quinidine on QT prolongation has been evaluated, but only
quinidine effectively suppressed gain-of-function in I(Kr) with prolongation of
the QT interval. In patients with a mutation in HERG, it rendered ventricular
tachycardias/ventricular fibrillation non-inducible and restored the QT
interval/heart rate relationship towards a normal range. It may serve as an
adjunct to ICD therapy or as a possible alternative treatment, especially for
children and newborns. Short QT syndrome is a new inherited disorder associated with familial atrial
fibrillation and/or sudden death or syncope. To date, three different mutations
in genes encoding cardiac ion channels (KCNH2, KCNQ1 and KCNJ2) have been
identified as causing short QT syndrome. All mutations lead to a gain in
function of the affected current (IK(r), IK(s )and IK(1)). The syndrome is
characterized in the few patients identified so far by a shortened QT interval
of less than 300-325 ms after correction for heart rate at rates below 80 beats
per minute. However, no boundary or limit for the QT interval can yet be
determined, as more knowledge about this disease is still restricted to a small
patient population. Furthermore, the QT interval lacks adaptation to heart rate.
The majority of patients exhibit shortened atrial and ventricular effective
refractory periods and inducibility of ventricular fibrillation. Death already
occurs in newborns, so the short QT syndrome may also account for deaths
classified as sudden infant death syndrome. The therapy of choice in families
with a history of sudden death or syncope seems to be the implantable
cardioverter-defibrillator. Whether patients without a family history of sudden
death or symptoms need a defibrillator cannot yet be answered, and requires
further investigation. Pharmacologic treatment has only been investigated in
patients with a mutation in KCNH2 (HERG), and it could be demonstrated that the
mutant currents may be insufficiently suppressed by drugs that are targeted to
block the specific current (e.g., sotalol or ibutilide) in patients with a
mutation in the IK(r-)coding gene KCNH2 (HERG). Interestingly, in this specific
patient population, quinidine proved to be efficient in prolonging the QT
interval and normalizing the effective refractory periods. Implantable
cardioverter-defibrillator therapy is associated with an increased risk of
inappropriate therapies for T-wave oversensing, although this risk can be
resolved by reprogramming implantable cardioverter-defibrillator detection
algorithms. Short QT syndrome is a new genetic disorder associated with familial atrial
fibrillation and/or sudden death or syncope. To date, different mutations in
genes encoding for cardiac ion channels (KCNH2, KCNQ1, and KCNJ2) have been
identified to cause the short QT syndrome. The mutations lead to a gain of
function of the affected current (IKr, IKs, and IK1). The phenotype is
characterized by a shortened QT interval<335 ms after correction for heart rate
at rates<80 beats/min. Furthermore, the QT interval poorly adapts to heart rate.
Patients exhibit shortened atrial and ventricular effective refractory periods
and, in the majority, inducibility of ventricular fibrillation. Death occurs
already in newborns. Therapy of choice seems to be the implantable cardioverter
defibrillator because of the high incidence of sudden death. Pharmacological
treatment has been studied and it could be demonstrated, that some mutant
currents may be insufficiently suppressed by drugs targeted to block the
specific current such as, e.g., sotalol or ibutilide in patients with a mutation
in the IKr-coding gene KCNH2 (HERG). Quinidine proved to be efficient in
prolonging the QT interval and normalizing the effective refractory periods in
some patients. PURPOSE OF REVIEW: Sudden cardiac death in patients without structural heart
disease remains a challenge in diagnostics and risk stratification. Genetically
determined arrhythmias are a potential cause for a primary electrical disease. A
recently discovered primary electrical disease is discussed.
RECENT FINDINGS: The inherited short QT syndrome is a recently recognized
genetic condition, which is associated with atrial fibrillation, syncope and/or
sudden cardiac death. Attention has been focused on diagnostic ECG features, the
identification of underlying mutations and mechanisms of arrhythmogenesis.
SUMMARY: The short QT syndrome is clinically associated with atrial
fibrillation, syncope and sudden cardiac death. A shortened QT interval (QTc
<360 ms) and reduced ventricular refractory period together with an increased
dispersion of repolarization constitute the potential substrate for reentry and
life-threatening ventricular tachyarrhythmia. To date, gain-of-function
mutations in KCNH2, KCNQ1, KCNJ2, encoding potassium channels and
loss-of-function mutations in CACNA1C and CACNB2b, encoding L-type calcium
channel subunits have been identified. The therapy of choice is the implantable
cardioverter defibrillator in symptomatic patients. Quinidine has been shown to
prolong the QT interval and to normalize the effective refractory periods of the
atrium and ventricle in patients with short QT-1 syndrome. Short QT syndrome (SQTS) is a gene-related arrhythmogenic syndrome harbouring a
large spectrum of symptoms ranging from mild palpitations to sudden cardiac
death.The mutation of genes (KCNH2, KCNQ1, and KCNJ2) encoding for cardiac
potassium channels plays a central role in SQTS. Electrocardiography is the
primary important step in the diagnosis (short QT interval along with T wave
changes), but ECG findings may be easily ignored. Treatment of the syndrome is
still controversial. Some specific antiarrhythmic drugs and an implantable
converter/defibrillator (ICD) have been considered as main therapeutic
strategies. ICD implantation may be a life-saving procedure due to the presence
of sudden cardiac death risk in patients with SQTS, but ICD-related problems
such as inappropriate shock deliveries due to oversensing of prominent T waves
have made medical therapy an alternative option. Notwithstanding the scarcity of
cases, clinicians should keep this syndrome in mind, and be familiar with its
clinical findings particularly when evaluating patients with palpitation,
syncope or a history of sudden cardiac death.We present a brief review of the
literature concerning the aetiology, clinical findings and therapeutic approach
to this rare entity. Le syndrome du QT court (SQTC) est une maladie génétique rare qui s’accompagne
d’un risque de mort subite d’origine cardiaque. Le présent rapport décrit, chez
un jeune homme, une syncope ayant occasionné un accident de la route.
L’électrocardiogramme et les analyses initiales n’ont révélé aucune
particularité, mais l’épreuve d’effort a montré une absence d’adaptation de
l’intervalle QT qui a été décrite dans le SQTC. Pour évaluer un diagnostic
possible de SQTC, le séquençage de l’ADN des gènes reconnus pour leur lien avec
le SQTC a permis de reconnaître une nouvelle mutation au niveau du gène KCNH2.
Par conséquent, le patient a reçu un diagnostic de SQTC et il a accepté de
suivre notre recommandation de se faire implanter un
cardioverseur-défibrillateur avant de quitter l’hôpital. The idiopathic short QT syndrome (SQTS) is a recently identified condition
characterized by abbreviated QT intervals (typically 300 ms or less) and in
affected families is associated with an increased incidence of atrial and
ventricular arrhythmias and sudden cardiac death. Genetic analysis has, to date,
identified three distinct forms of the condition, involving gain-of-function
mutations to three different cardiac potassium channel genes: KCNH2 (SQT1),
KCNQ1 (SQT2) and KCNJ2 (SQT3). This article reviews recent advances in
understanding this syndrome, discussing the basis of QT interval shortening,
possible mechanisms for the associated arrhythmogenic risk in SQT1, current
approaches to treatment of the SQTS (focusing on SQT1) and avenues for future
investigation. The Short QT Syndrome is a recently described new genetic disorder,
characterized by abnormally short QT interval, paroxysmal atrial fibrillation
and life threatening ventricular arrhythmias. This autosomal domit syndrome
can afflict infants, children, or young adults; often a remarkable family
background of cardiac sudden death is elucidated. At electrophysiological study,
short atrial and ventricular refractory periods are found, with atrial
fibrillation and polymorphic ventricular tachycardia easily induced by
programmed electrical stimulation. Gain of function mutations in three genes
encoding K(+) channels have been identified, explaining the abbreviated
repolarization seen in this condition: KCNH2 for I(kr) (SQT1), KCNQ1 for I(ks)
(SQT2) and KCNJ2 for I(k1) (SQT3). The currently suggested therapeutic strategy
is an ICD implantation, although many concerns exist for asymptomatic patients,
especially in pediatric age. Pharmacological treatment is still under
evaluation; quinidine has shown to prolong QT and reduce the inducibility of
ventricular arrhythmias, but awaits additional confirmatory clinical data. A gain of function mutation N588K in the KCNH2 gene that encodes HERG channels
has been shown to underlie the SQT1 form of short QT syndrome (SQTS). We
describe a different mutation in the KCNH2 gene in a Chinese family with
clinical evidence of SQTS. A Chinese family with a markedly short QT interval
(QTc=316 ± 9 ms, n=4) and a strong family history of sudden death was
investigated. Analysis of candidate genes contributing to ventricular
repolarization identified a C1853T mutation in the KCNH2 gene coding for the
HERG channel, resulting in an amino acid change (T618I) that was found to 100%
co-segregate with the SQTS phenotype (n=4). Whole cell voltage clamp studies of
the T618I mutation in HEK-cells demonstrated a 6-fold increase in maximum steady
state current (146.1 ± 16.7 vs 23.8 ± 5.5 pA/pF) that occurred at a 20 mV more
positive potential compared to the wild type channels. The voltage dependence of
inactivation was significantly shifted in the positive voltage direction (WT
-78.6 ± 6.8 vs T618I -29.3 ± 1.7 mV). Kinetic analysis revealed slower
inactivation rates of T618I but faster rates of recovery from inactivation.
Quinidine (5 μM) and sotalol (500 μM) had similar inhibitory effects on steady
currents measured at +20 mV in WT and T618I but were less effective in
inhibiting tail currents of mutant channels. The altered function of T618I-HERG
channels suggests that this mutation in the KCNH2 gene is responsible for the
SQTS phenotype in this family. Both quinidine and sotalol may be therapeutic
options for patients with the T618I HERG mutation. AIMS: Short QT syndrome (SQTS) is a genetically determined ion-channel disorder,
which may cause maligt tachyarrhythmias and sudden cardiac death. Thus far,
mutations in five different genes encoding potassium and calcium channel
subunits have been reported. We present, for the first time, a novel
loss-of-function mutation coding for an L-type calcium channel subunit.
METHODS AND RESULTS: The electrocardiogram of the affected member of a single
family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and
symmetrical T-waves. Invasive electrophysiological testing showed short
ventricular refractory periods and increased vulnerability to induce ventricular
fibrillation. DNA screening of the patient identified no mutation in previously
known SQTS genes; however, a new variant at a heterozygous state was identified
in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding
for the Ca(v)α(2)δ-1 subunit of the L-type calcium channel. The pathogenic role
of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using
co-expression of the two other L-type calcium channel subunits, Ca(v)1.2α1 and
Ca(v)β(2b), in HEK-293 cells. Barium currents (I(Ba)) were recorded in these
cells under voltage-clamp conditions using the whole-cell configuration.
Co-expression of the p.Ser755Thr Ca(v)α(2)δ-1 subunit strongly reduced the I(Ba)
by more than 70% when compared with the co-expression of the wild-type (WT)
variant. Protein expression of the three subunits was verified by performing
western blots of total lysates and cell membrane fractions of HEK-293 cells. The
p.Ser755Thr variant of the Ca(v)α(2)δ-1 subunit was expressed at a similar level
compared with the WT subunit in both fractions. Since the mutant Ca(v)α(2)δ-1
subunit did not modify the expression of the pore-forming subunit of the L-type
calcium channel, Ca(v)1.2α1, it suggests that single channel biophysical
properties of the L-type channel are altered by this variant.
CONCLUSION: In the present study, we report the first pathogenic mutation in the
CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be
determined whether mutations in this gene lead to other manifestations of the
J-wave syndrome. OBJECTIVES: The aim of this study was to investigate the clinical
characteristics and the long-term course of a large cohort of patients with
short QT syndrome (SQTS).
BACKGROUND: SQTS is a rare channelopathy characterized by an increased risk of
sudden death. Data on the long-term outcome of SQTS patients are not available.
METHODS: Fifty-three patients from the European Short QT Registry (75% males;
median age: 26 years) were followed up for 64 ± 27 months.
RESULTS: A familial or personal history of cardiac arrest was present in 89%.
Sudden death was the clinical presentation in 32%. The average QTc was 314 ± 23
ms. A mutation in genes related to SQTS was found in 23% of the probands; most
of them had a gain of function mutation in HERG (SQTS1). Twenty-four patients
received an implantable cardioverter defibrillator, and 12 patients received
long-term prophylaxis with hydroquinidine (HQ), which was effective in
preventing the induction of ventricular arrhythmias. Patients with a HERG
mutation had shorter QTc at baseline and a greater QTc prolongation after
treatment with HQ. During follow-up, 2 already symptomatic patients received
appropriate implantable cardioverter defibrillator shocks and 1 had syncope.
Nonsustained polymorphic ventricular tachycardia was recorded in 3 patients. The
event rate was 4.9% per year in the patients without antiarrhythmic therapy. No
arrhythmic events occurred in patients receiving HQ.
CONCLUSIONS: SQTS carries a high risk of sudden death in all age groups.
Symptomatic patients have a high risk of recurrent arrhythmic events. HQ is
effective in preventing ventricular tachyarrhythmia induction and arrhythmic
events during long-term follow-up. AIMS: Short-QT syndrome (SQTS) is a recently recognized disorder associated with
atrial fibrillation (AF) and sudden death due to ventricular arrhythmias.
Mutations in several ion channel genes have been linked to SQTS; however, the
mechanism remains unclear. This study describes a novel heterozygous
gain-of-function mutation in the inward rectifier potassium channel gene, KCNJ2,
identified in SQTS.
METHODS AND RESULTS: We studied an 8-year-old girl with a markedly short-QT
interval (QT = 172 ms, QTc = 194 ms) who suffered from paroxysmal AF. Mutational
analysis identified a novel heterozygous KCNJ2 mutation, M301K. Functional
assays displayed no Kir2.1 currents when M301K channels were expressed alone.
However, co-expression of wild-type (WT) with M301K resulted in larger outward
currents than the WT at more than -30 mV. These results suggest a
gain-of-function type modulation due to decreased inward rectification.
Furthermore, we analysed the functional significance of the amino acid charge at
M301 (neutral) by changing the residue. As with M301K, in M301R (positive), the
homozygous channels were non-functional, whereas the heterozygous channels
demonstrated decreased inward rectification. Meanwhile, the currents recorded in
M301A (neutral) showed normal inward rectification under both homo- and
heterozygous conditions. Heterozygous overexpression of WT and M301K in neonatal
rat ventricular myocytes exhibited markedly shorter action potential durations
than the WT alone.
CONCLUSION: In this study, we identified a novel KCNJ2 gain-of-function
mutation, M301K, associated with SQTS. Functional assays revealed no functional
currents in the homozygous channels, whereas impaired inward rectification
demonstrated under the heterozygous condition resulted in larger outward
currents, which is a novel mechanism predisposing SQTS. |
What is SCENAR therapy used for? | all patients experienced substantial relief of pain from the first treatment. an electronic biofeedback device (scenar) may be successfully utilized in the management of post-herpetic neuralgia. scenar) as effective in the treatment of neurogenic dysfunction of the bladder in children with nocturnal enuresis. post-herpetic neuralgia using a bioelectronical device (scenar). addition of scenar therapy to the complex conventional pharmacotherapy fastened ulcer healing, increased the effectiveness of helicobacter pylori eradication, and improved the condition of the gastroduodenal mucosa. scenar therapy to patients with localized suppurative peritonitis in the postoperative period. a new technique of low-frequency modulated electric current therapy, scenar therapy, was used in treatment of 103 patients with duodenal ulcer (du). | A new technique of low-frequency modulated electric current therapy, SCENAR
therapy, was used in treatment of 103 patients with duodenal ulcer (DU). The
influence of SCENAR therapy on the main clinical and functional indices of a DU
relapse was studied. It was shown that SCENAR therapy, which influences
disturbed mechanisms of adaptive regulation and self-regulation, led to positive
changes in most of the parameters under study. Addition of SCENAR therapy to the
complex conventional pharmacotherapy fastened ulcer healing, increased the
effectiveness of Helicobacter pylori eradication, and improved the condition of
the gastroduodenal mucosa. Administration of artrofoon in combination with SCENAR therapy to patients with
localized suppurative peritonitis in the postoperative period considerably
reduced plasma MDA level, stabilized ceruloplasmin activity, and increased
catalase activity in erythrocytes compared to the corresponding parameters in
patients receiving standard treatment in combination with SCENAR therapy. The purpose of these case reports is to describe treatment of three consecutive
patients with post-herpetic neuralgia using a bioelectronical device (SCENAR).
The instrument is approved as a Class II device in the United States. The
electrode of the device was stroked gently over the involved skin area for up to
15 minutes per session. No more than 5 sessions over a 3-week period was
required. All patients experienced substantial relief of pain from the first
treatment. One patient required only 1 treatment lasting 10 minutes. The other 2
patients required 4 to 5 treatments over a 3-week period. One patient required a
treatment for skin itch after one year with a follow up period of 6 months to 24
months. An electronic biofeedback device (SCENAR) may be successfully utilized
in the management of post-herpetic neuralgia. |
List symptoms of Gradenigo's syndrome. | Gradenigo's syndrome is a rare but life threatening complication of acute otitis media, which includes a classic triad of otitis media, deep facial pain and ipsilateral abducens nerve paralysis. | Gradenigo's syndrome, the triad of suppurative otitis media, abducens nerve
palsy and pain in the ophthalmic division of the trigeminal nerve, remains a
rare complication of otitis media. A case in a paediatric patient is described,
successfully managed conservatively. There is little evidence to support
increased use of antibiotics in acute otitis media to prevent this complication. BACKGROUND: Gradenigo's syndrome is a rare disease, which is characterized by
the triad of the following conditions: suppurative otitis media, pain in the
distribution of the first and the second division of trigeminal nerve, and
abducens nerve palsy. The full triad may often not be present, but can develop
if the condition is not treated correctly.
CASE PRESENTATION: We report a case of a 3-year-old girl, who presented with
fever and left-sided acute otitis media. She developed acute mastoiditis, which
was initially treated by intravenous antibiotics, ventilation tube insertion and
cortical mastoidectomy. After 6 days the clinical picture was complicated by
development of left-sided abducens palsy. MRI-scanning showed osteomyelitis
within the petro-mastoid complex, and a hyper intense signal of the adjacent
meninges. Microbiological investigations showed Staphylococcus aureus and
Fusobacterium necrophorum. She was treated successfully with intravenous
broad-spectrum antibiotic therapy with anaerobic coverage. After 8 weeks of
follow-up there was no sign of recurrent infection or abducens palsy.
CONCLUSION: Gradenigo's syndrome is a rare, but life-threatening complication to
middle ear infection. It is most commonly caused by aerobic microorganisms, but
anaerobic microorganisms may also be found why anaerobic coverage should be
considered when determining the antibiotic treatment. BACKGROUND: Giuseppe Gradenigo (1859-1926), a legendary figure of Otology, was
born in Venice, Italy. He soon became a pupil to Adam Politzer and Samuel
Leopold Schenk in Vienna, demonstrating genuine interest in the embryology,
morphology, physiopathology, as well as the clinical manifestations of ear
diseases. In this paper, the authors attempt to highlight the major landmarks
during Gradenigo's career and outline his contributions to neurosciences, which
have been viewed as looking forward to the 20(th) century rather than awkward
missteps at the end of the 19(th).
METHODS: Several rare photographs along with many non-English, more than a
century old articles have been meticulously selected to enrich this historical
journey in time.
RESULTS: It was after Gradenigo that the well-known syndrome consisting of
diplopia and facial pain due to a middle ear infection was named. However,
Gradenigo was much more than a syndrome. Surprisingly, despite the fact that he
is considered a pioneer of the Italian Otology of the late 19(th) and early
20(th) century, little is written of his life and his notable achievements in
the English literature.
CONCLUSIONS: Even though his name lives on nowadays only in the eponym
"Gradenigo's syndrome," his accomplishments are much wider and cast him among
the emblematic figures of science. His inherent tendency for discovering the
underlying mechanisms of diseases and his vision of guaranteeing quality of
services, professional proficiency, respect, and dedication toward the patients
is in fact what constitutes his true legacy to the next generations. Gradenigo's syndrome is a rare but life threatening complication of acute otitis
media (AOM), which includes a classic triad of otitis media, deep facial pain
and ipsilateral abducens nerve paralysis. The incidence of Fusobacterium
necrophorum infections has increased in recent years. We describe two cases of
Gradenigo's syndrome caused by F. necrophorum. Additional four cases were
identified in a review of the literature. Gradenigo's syndrome as well as other
neurologic complications should be considered in cases of complicated acute
otitis media. F. necrophorum should be empirically treated while awaiting
culture results. INTRODUCTION: Gradenigo's syndrome is nowadays a rare condition characterized by
a triad of otorrhea, facial pain with trigeminal nerve involvement and abducens
nerve palsy. Most cases are caused by medial extension of acute otitis media
into a pneumatized petrous apex and surgical drainage is usually the treatment
of choice. We present a case highlighting the pathological mechanism of this
disease, demonstrate rare radiological findings associated with this patient,
and showcase successful medical treatment without surgical intervention.
CASE PRESENTATION: A 63-year-old Thai man presented with complete Gradenigo
triad as a complication of chronic otomastoiditis in spite of clinical history
of previous radical mastoidectomy and a nonpneumatization of the petrous apex.
Magnetic resoce imaging showed abnormal prominent enhancement at the roof of
his right temporal bone, and the dura overlying the floor of right middle
cranial fossa and right cavernous sinus. Magnetic resoce imaging also
detected right petrous apicitis. With the use of intravenous antibiotics and
topical antibiotic eardrops, recovery was observed within 5 days with complete
resolution within 2 months.
CONCLUSIONS: Although there is little evidence to support the use of medical
therapy in the treatment of Gradenigo's syndrome resulting from chronic ear
disease, we here demonstrate successful conservative treatment of Gradenigo's
syndrome following chronic otitis media in a patient who underwent previous
radical mastoidectomy. Plasma cell tumors of the skull base are rare in neurosurgical practice. True
solitary osseous plasmacytoma of the skull base without development of multiple
myeloma is extremely rare. We report a case of typical Gradenigo's syndrome,
including left abducens nerve palsy, left facial pain and paresthesia in V1 and
V2 distribution of trigeminal nerve caused by solitary osseous plasmacytoma of
the left petrous apex. The patient was a 46-year-old man who presented with
diplopia for two days. Magnetic resoce imaging (MRI) of the brain showed a
hyperintense mass on T1-weighted images and slightly hypointense mass on
T2-weighted images in the left petrous apex and left parasellar area. Through a
left subtemporal middle fossa approach, subtotal resection of the lesion was
performed. Histopathological examination of the lesion revealed plasmacytoma.
The patient received 54 Gy radiation for the local tumor. Four months after
radiation, the abducens palsy improved. Four years after treatment, the patient
remained well with no symptoms or signs of local recurrence or progression to
multiple myeloma. INTRODUCTION: In 1904, Giuseppe Gradenigo published his case series on the triad
of ipsilateral abducens nerve palsy, facial pain in the trigeminal nerve
distribution, and suppurative otitis media, which would subsequently be referred
to as Gradenigo syndrome.
CASE REPORT: Our patient was a 36-year-old female, 23 weeks pregt, with a
6-day history of right-sided otalgia and hearing loss and a 4-day history of
purulent otorrhea, who presented with severe, holocephalic headache, meningeal
signs, fever, photophobia, and mental status decline. Lumbar puncture yielded a
white blood cell count of 1,559 cells/mm(3) with 95% polymorphonuclear
leukocytes, a red blood cell count of 111 cells/mm(3), a protein level of 61
mg/dl, and a glucose level of <40 mg/dl. Cerebrospinal fluid Gram stain showed
Gram-positive diplococci, which were subsequently identified as Streptococcus
pneumoniae and treated with ceftriaxone. On the second hospital day, she
developed horizontal diplopia due to right abducens nerve palsy and right
mydriasis. Both symptoms resolved on the third hospital day. Erosion of temporal
bone and opacification of mastoid air cells was shown on CT scan. A CT venogram
showed an irregularity of the left transverse and superior sagittal sinuses. She
was treated with enoxaparin for possible sinus thrombosis.
DISCUSSION: This case demonstrates rare but serious sequelae of otitis media and
Gradenigo syndrome. Holocephalic headache from meningitis masked trigeminal
pain. Involvement of the ipsilateral petrous apex and surrounding structures on
imaging and clinical improvement with antibiotic treatment supports Gradenigo
syndrome over intracranial hypertension due to venous sinus thrombosis as the
cause of the abducens nerve palsy. |
Is Wnt16b secreted in response to chemotherapy? | Yes, WNT16B is secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. | Acquired resistance to anticancer treatments is a substantial barrier to
reducing the morbidity and mortality that is attributable to maligt tumors.
Components of tissue microenvironments are recognized to profoundly influence
cellular phenotypes, including susceptibilities to toxic insults. Using a
genome-wide analysis of transcriptional responses to genotoxic stress induced by
cancer therapeutics, we identified a spectrum of secreted proteins derived from
the tumor microenvironment that includes the Wnt family member wingless-type
MMTV integration site family member 16B (WNT16B). We determined that WNT16B
expression is regulated by nuclear factor of κ light polypeptide gene enhancer
in B cells 1 (NF-κB) after DNA damage and subsequently signals in a paracrine
manner to activate the canonical Wnt program in tumor cells. The expression of
WNT16B in the prostate tumor microenvironment attenuated the effects of
cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease
progression. These results delineate a mechanism by which genotoxic therapies
given in a cyclical manner can enhance subsequent treatment resistance through
cell nonautonomous effects that are contributed by the tumor microenvironment. Innate or acquired resistance to chemotherapy presents an important and
predictable challenge in cancer therapy. Maligt tumors consist of both
neoplastic and benign cells such as stromal fibroblasts, which can influence the
tumor's response to cytotoxic therapy. In a recent article in Nature Medicine,
Sun et al. show that increased expression of Wnt family member wingless-type
MMTV integration site family member 16B (WNT16B) by the tumor microenvironment
in response to cytotoxic damage and signals through the canonical Wnt pathway to
promote tumor growth and chemotherapy resistance. Such findings outline a
mechanism by which cytotoxic therapies given in cyclical doses can actually
augment later treatment resistance and may open the door to new areas of
research and to the development of new therapeutic targets that block the DNA
damage response program. Author information:
(1)State Key Laboratory of Biotherapy/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
[email protected].
(2)State Key Laboratory of Biotherapy/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
[email protected].
(3)Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China.
[email protected].
(4)State Key Laboratory of Biotherapy/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
[email protected].
(5)Department of Medical Oncology, the Fifth People's Hospital of Chengdu,
Chengdu 611130, China. [email protected].
(6)State Key Laboratory of Biotherapy/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
[email protected].
(7)State Key Laboratory of Biotherapy/Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
[email protected]. |
Do DNA double-strand breaks play a causal role in carcinogenesis? | Yes. It has been demonstrated that induction of DNA double-strand breaks (DSBs) and defects in overall DSBs repair capacity can lead to an accumulation of mutations, resulting in genomic instability of cells. Given that genomic instability is the hallmark of cancer, DSBs play a causal role in carcinogenesis. | In order to study the role of DNA damage processing in the development of
cutaneous squamous cell carcinoma (SCC), we assessed the ability of six
keratinocyte cell lines from a multistage-tumor progression model to repair
three types of DNA damage: pyrimidine dimers, oxidative DNA lesions and DNA
double strand breaks (DSB). The model comprised the spontaneously immortalized,
non-tumorigenic human keratinocyte cell line HaCaT, four different c-Ha-ras
transfectants of HaCaT (non-, benign- and two maligt-tumorigenic) and a
SCC-derived cell line. Host cell reactivation assays with UVB-treated plasmid
vectors pRSVcat showed no significantly altered repair of UVB-induced pyrimidine
dimers in the tumorigenic cell lines, compared with the non-tumorigenic lines.
Using the singlet oxygen-treated plasmids pRSVcat the Ha-ras-HaCaT-clones and
the SCC-cells, exerted a DNA repair efficiency that was not significantly
different from HaCaT cells. In order to assess the ability of the cells to
ligate free DNA ends (repair of DSB), we used a plasmid shuttle vector assay
with linearized plasmid pZ189. We found a significant increase of DNA end
joining ability in the non-tumorigenic, the benign and in one of the maligt
HaCaT-clones II-4. The maligt HaCaT-clone II-3, however, exerted a
significantly lower rate of rejoining the linearized plasmid. This cell line
also showed a highly and significantly elevated rate of micronuclei, which
reflects a pronounced chromosomal instability. The SCC-cells exhibited a more
efficient repair of DNA DSB than the HaCaT cells. We conclude that in the
examined model, progression of human keratinocytes from the non-tumorigenic to
the highly tumorigenic phenotype, is not accompanied by a decrease in the cell's
capacity to repair UVB- and singlet oxygen-induced DNA lesions. However, an
acquired deficiency in repairing DNA double strand breaks can be one mechanism
promoting progression towards maligcy, possibly through impairing chromosomal
stability. Repair of DNA double-strand breaks (DSB) is essential for cell viability and
genome stability. Homologous recombination repair plays an important role in DSB
repair and impairment of this repair mechanism may lead to loss of genomic
integrity, which is one of the hallmarks of cancer. Recent research has shown
that the tumor suppressor genes p53 and BRCA1 and -2 are involved in the proper
control of homologous recombination, suggesting a role of this type of repair in
human cancer. We developed a novel assay based on recombination between two
Green Fluorescent Protein (GFP) sequences in transiently transfected plasmid
DNA. The plasmid construct contains an intact, emission-shifted, "blue" variant
of GFP (BFP), with a 300 nucleotide stretch of homology to a nonfunctional copy
of GFP. In the absence of homologous recombination only BFP is present, but
homologous recombination can create a functional GFP. The homologous regions in
the plasmid were constructed in both the direct and the inverted orientation of
transcription to detect possible differences in the recombination mechanisms
involved. A panel of human tumor cell lines was chosen on the basis of genetic
background and chromosome integrity and tested for homologous recombination
using this assay. The panel included cell lines with varying levels of
karyotypic abnormalities, isogenic cell lines with normal and mutant p53,
isogenic cell lines with or without DNA mismatch repair, BRCA1 and -2 mutant
cell lines, and the lymphoma cell line DT40. With this assay, the observed
differences between cell lines with the lowest and highest levels of
recombination were about 100-fold. Increased levels of recombination were
associated with mutant p53, whereas a low level of recombination was found in
the BRCA1 mutant cell line. In the cell line HT1080TG, a mutagenized derivative
of HT1080 with two mutant alleles of p53, high levels of recombination were
found with the direct orientation but not with the inverted orientation plasmid.
No difference in recombination was detected between two isogenic cell lines that
only differed in DNA mismatch repair capability. We conclude that this assay can
detect differences in homologous recombination capacity in cultured cell lines
and that these differences follow the patterns that would be expected from the
different genotypes of these cell lines. Future application in normal cells may
be useful to identify genetic determits controlling genomic integrity or to
detect differences in DNA repair capacity in individuals. DNA double-strand breaks (DSBs) are induced by ionizing radiation (IR) and
various radiomimetic agents directly, or indirectly as a consequence of DNA
repair, recombination and replication of damaged DNA. They are ultimately
involved in the generation of chromosomal aberrations and were reported to cause
genomic instability, gene amplification and reproductive cell death. To address
the question of whether DSBs act as a trigger of apoptosis, we induced DSBs by
means of restriction enzyme electroporation and compared the effect with IR in
mouse fibroblasts that differ in p53 status [wild-type (+/+) versus
p53-deficient (-/-) cells]. We show that (i) electroporation of PVU:II is highly
efficient in the induction of DSBs, (ii) electroporation of PVU:II increases the
rate of apoptosis, but not of necrosis in p53-/- cells, (iii) treatment with
gamma-rays induces both apoptosis and necrosis in p53-/- cells, (iv) the
frequency of DSBs correlates with the yield of apoptosis and (v) both PVU:II and
gamma-ray treatment reduce the level of anti-apoptotic Bcl-2 protein in p53-/-
cells whereas the level of Bax remains unaltered. Cells expressing wild-type p53
were more resistant than p53-deficient cells as to the induction of apoptosis
and did not show Bcl-2 decline upon treatment with PVU:II and gamma-rays. The
data provide evidence that blunt-ended DSBs induced by restriction enzyme PVU:II
act as a highly efficient trigger of apoptosis, but not of necrosis. This
process is related to Bcl-2 decline and does not require p53. Cancer develops when cells no longer follow their normal pattern of controlled
growth. In the absence or disregard of such regulation, resulting from changes
in their genetic makeup, these errant cells acquire a growth advantage,
expanding into precancerous clones. Over the last decade, many studies have
revealed the relevance of genomic mutation in this process, be it by
misreplication, environmental damage, or a deficiency in repairing endogenous
and exogenous damage. Here, we discuss homologous recombination as another
mechanism that can result in a loss of heterozygosity or genetic rearrangements.
Some of these genetic alterations may play a primary role in carcinogenesis, but
they are more likely to be involved in secondary and subsequent steps of
carcinogenesis by which recessive oncogenic mutations are revealed. Patients,
whose cells display an increased frequency of recombination, also have an
elevated frequency of cancer, further supporting the link between recombination
and carcinogenesis. Combined effect of vanadium and beta-carotene on rat liver DNA-chain break and
Proton induced X-ray emission (PIXE) analysis was studied during a necrogenic
dose (200 mg/kg of body weight) of Diethyl Nitrosamine (DENA) induced rat liver
carcinogenesis. Morphological and histopathological changes were observed as an
end point biomarker. Supplementation of vanadium (0.5 ppm ad libitum) in
drinking water and beta-carotene in the basal diet (120 mg/Kg of body weight)
were performed four weeks before DENA treatment and continued till the end of
the experiment (16 weeks). PIXE analysis revealed the restoration of near normal
value of zinc, copper, and iron, which were substantially altered when compared
to carcinogen treated groups. Supplementation of both vanadium and beta-carotene
four weeks before DENA injection was found to offer significant (64.73%, P <
0.001) protection against generation of single-strand breaks when compared with
the carcinogen control counter parts. A significant stabilization of hepatic
architecture of the cells was observed as compared to carcinogen control in
vanadium plus beta-carotene treated group. This study thus suggests that
vanadium, a prooxidant but potential therapeutic agent yield safe and effective
pharmacological formulation with beta-carotene, an antioxidant, in the
inhibition of experimental rat hepatocarcinogenesis. In human cells, DNA double-strand breaks (DSBs) are repaired primarily by the
DNA end-joining (EJ) process and thus, abnormal DNA EJ activities lead to an
accumulation of mutations and/or aneuploidy, resulting in genetic instability of
cells. Since genetic instability is the hallmark of cancer cells, we studied the
DNA EJ activities of normal, non-maligt immortalized and maligt human
epithelial cells to investigate the association between DNA EJ and
carcinogenesis. We found a significant diminution of precise (error-free) DNA EJ
activities in non-maligt immortalized human oral keratinocytes (HOK-16B) and
human head and neck squamous cell carcinoma (HNSCC) cells compared to that in
normal human oral keratinocytes (NHOK). Moreover, abnormal DNA EJ activities
were detected exclusively in HOK-16B and HNSCC cells due to
microhomology-mediated and non-microhomology-mediated end-joining activities in
these cells. These data indicated that aberrant DNA EJ activity may be partly
responsible for genetic instability and oncogenic transformation. BACKGROUND: Induction of DNA double strand breaks and alterations in the repair
of these breaks is implicated in breast carcinogenesis. Prior studies have
demonstrated that peripheral blood mononuclear cells (PBMC) from breast cancer
patients exhibit increased numbers of DNA strand breaks after exposure to
ionizing radiation, but these studies did not specifically measure DNA double
strand breaks and it is not known whether chemical carcinogens produce similar
effects.
MATERIALS AND METHODS: PBMC from 32 women undergoing breast surgery were
genotyped at nine loci of seven DNA repair genes. DNA double strand break repair
was measured using the neutral comet assay after exposure to ionizing radiation
(0.5 Gy) or bioactivated benzo[a]pyrene (B[a]P, 5 microM.
RESULTS: PBMC from breast cancer patients showed higher levels of residual DNA
double strand breaks 30 min after exposure to radiation than PBMC from patients
with benign breast disease (1.40 times baseline [95% confidence intervals [CI]
1.29-1.51] versus 1.24 times baseline [95% CI 1.15-1.33], respectively, P =
0.04). The response to B[a]P trended in the same direction, but did not reach
statistical significance. The MGMT K178R variant genotype was associated with
improved DNA double strand break repair in PBMC exposed to B[a]P.
CONCLUSIONS: Reduced repair of radiation-induced DNA double strand breaks in
PBMC is a robust biomarker of breast cancer risk. Reduced DNA repair capacity
may have a genetic component even in sporadic breast cancer. A tumorigenic role of the non-homologous end-joining (NHEJ) pathway for the
repair of DNA double-strand breaks (DSBs) has been suggested by our finding of a
significant association between increased breast cancer risk and a cooperative
effect of single-nucleotide polymorphisms in NHEJ genes. To confirm this
finding, this case-control study detected both in vivo and in vitro DNA
end-joining (EJ) capacities in Epstein-Barr virus-immortalized peripheral blood
mononuclear cells (PBMCs) of 112 breast cancer patients and 108 healthy controls
to identify individual differences in EJ capacity to repair DSB as a risk factor
predisposing women to breast cancer. PBMCs from breast cancer patients
consistently showed lower values of in vivo and in vitro EJ capacities than
those from healthy women (P < 0.05). Logistic regression, simultaneously
considering the effect of known risk factors of breast cancer, shows that the in
vitro EJ capacity above the median of control subjects was associated with
nearly 3-fold increased risks for breast cancer (adjusted odds ratio, 2.98; 95%
confidence interval, 1.64-5.43). Furthermore, a dose-response relationship was
evident between risk for breast cancer and EJ capacity, which was analyzed as a
continuous variable (every unit decrease of EJ capacity being associated with an
1.09-fold increase of breast cancer risk) and was divided into tertiles based on
the EJ capacity values of the controls (P for trend < 0.01). The findings
support the conclusion that NHEJ may play a role in susceptibility to breast
cancer. Defective DNA damage response (DDR) can result in genomic instability (GIN) and
lead to the transformation into cancer. P53-binding protein 1 (53BP1) belongs to
a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules
localize at the sites of DNA double strand breaks (DSBs) and rapidly form
nuclear foci, the presence of 53BP1 foci can be considered as a cytologic marker
for endogenous DSBs reflecting GIN. Although it has been proposed that GIN has a
crucial role in the progression of thyroid neoplasms, the significance of GIN
during thyroid tumorigenesis remains unclear, particularly in patients. We
analyzed, therefore, the level of GIN, as detected with immunofluorescence of
53BP1, in 40 cases of resected thyroid tissues. This study demonstrated a number
of nuclear 53BP1 foci in thyroid cancers, suggesting a constitutive activation
of DDR in thyroid cancer cells. Because follicular adenoma also showed a few
53BP1 nuclear foci, GIN might be induced at a precancerous stage of thyroid
tumorigenesis. Furthermore, high-grade thyroid cancers prominently exhibited an
intense and heterogeneous nuclear staining of 53BP1 immunoreactivity, which was
also observed in radiation-associated cancers and in mouse colonic crypts as a
delayed response to a high dose ionizing radiation, suggesting increased GIN
with progression of cancer. Thus, the present study demonstrated a difference in
the staining pattern of 53BP1 during thyroid carcinogenesis. We propose that
immunofluorescence analysis of 53BP1 expression can be a useful tool to estimate
the level of GIN and, simultaneously, the maligt potency of human thyroid
tumors. Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with
solubility playing an important role in its carcinogenic potential. Zinc
chromate, a water insoluble or 'particulate' Cr(VI) compound, has been shown to
be carcinogenic in epidemiology studies and to induce tumors in experimental
animals, but its genotoxicity is poorly understood. Our study shows that zinc
chromate induced concentration-dependent increases in cytotoxicity, chromosome
damage and DNA double strand breaks in human lung cells. In response to zinc
chromate-induced breaks, MRE11 expression was increased and ATM and ATR were
phosphorylated, indicating that the DNA double strand break repair system was
initiated in the cells. In addition, our data show that zinc chromate-induced
double strand breaks were only observed in the G2/M phase population, with no
significant amount of double strand breaks observed in G1 and S phase cells.
These data will aid in understanding the mechanisms of zinc chromate toxicity
and carcinogenesis. The protein kinase Aurora-A is a major regulator of the cell cycle that
orchestrates mitotic entry and is required for the assembly of a functional
mitotic spindle. Overexpression of Aurora-A has been strongly linked with
oncogenesis and this has led to considerable efforts at therapeutic targeting of
the kinase activity of this protein. However, the exact mechanism by which
Aurora-A promotes oncogenesis remains unclear. Here, we show that Aurora-A
modulates the repair of DNA double-strand breaks (DSBs). Aurora-A expression
inhibits RAD51 recruitment to DNA DSBs, decreases DSB repair by homologous
recombination and sensitizes cancer cells to PARP inhibition. This impairment of
RAD51 function requires inhibition of CHK1 by Polo-like kinase 1 (PLK1). These
results identify a novel function of Aurora-A in modulating the response to DNA
DSB that likely contributes to carcinogenesis and suggest a novel therapeutic
approach to the treatment of cancers overexpressing this protein. DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by
ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear
whether complex DSBs that are refractory to repair are more potently tumorigenic
compared with simple breaks that can be rapidly repaired, correctly or
incorrectly, by mammalian cells. We previously demonstrated that complex DSBs
induced by high-linear energy transfer (LET) Fe ions are repaired slowly and
incompletely, whereas those induced by low-LET gamma rays are repaired
efficiently by mammalian cells. To determine whether Fe-induced DSBs are more
potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized'
murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and
injected the surviving cells subcutaneously into nude mice. Using this model
system, we find that Fe ions are potently tumorigenic, generating tumors with
significantly higher frequency and shorter latency compared with tumors
generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied
by rampant genomic instability and multiple genomic changes, the most
interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion
of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional
loss of p15/Ink4b in tumors derived from cells that are already deficient in
p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor
suppression, especially in the absence of p16. Indeed, we find that reexpression
of p15 in tumor-derived cells significantly attenuates the tumorigenic potential
of these cells, indicating that p15 loss may be a critical event in
tumorigenesis triggered by complex DSBs. Comet assay under neutral conditions allows detection of DNA double-strand
breaks (DSBs), which has consequence to genome instability and carcinogenesis.
The present study aims to validate the neutral Comet assay for genotoxicity
assessment in Drosophila melanogaster (Oregon R(+)) with three well known DSBs
inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and
subsequently its efficacy in detecting DSBs in the organism exposed to a well
known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D.
melanogaster were fed different concentrations of BLM, CPT and CP
(50.0-200.0μg/ml) or Cr(VI) (5.0-20.0μg/ml) mixed standard Drosophila food for
48h. Neutral Comet assay was performed in cells of mid gut and brain from
control and treated larvae. Our results show a dose-dependent increase in the
migration of DNA in cells of the exposed organisms. A comparison among DNA
lesions per mole number of the test chemical in the exposed groups showed that
both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0μg/ml
was found to induce significantly increased (p<0.001) DSBs in the exposed
organism as compared to the control. The study while validating neutral Comet
assay in D. melanogaster suggests its use for in vivo assessment of
environmental chemical induced DSBs. DNA damage and consequent mutations initiate the multistep carcinogenic process.
Differentiated cells have a reduced capacity to repair DNA lesions, but the
biological impact of unrepaired DNA lesions in differentiated lung epithelial
cells is unclear. Here, we used a novel organotypic human lung three-dimensional
(3D) model to investigate the biological significance of unrepaired DNA lesions
in differentiated lung epithelial cells. We showed, consistent with existing
notions that the kinetics of loss of simple double-strand breaks (DSBs) were
significantly reduced in organotypic 3D culture compared to kinetics of repair
in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs,
a majority of complex DNA lesions were irreparable in organotypic 3D culture.
Levels of expression of multiple DNA damage repair pathway genes were
significantly reduced in the organotypic 3D culture compared with those in 2D
culture providing molecular evidence for the defective DNA damage repair in
organotypic culture. Further, when differentiated cells with unrepaired DNA
lesions re-entered the cell cycle, they manifested a spectrum of
gross-chromosomal aberrations in mitosis. Our data suggest that downregulation
of multiple DNA repair pathway genes in differentiated cells renders them
vulnerable to DSBs, promoting genome instability that may lead to
carcinogenesis. DNA double-strand breaks (DSBs) can induce chromosomal aberrations and
carcinogenesis and their correct repair is crucial for genetic stability. The
cellular response to DSBs depends on damage signaling including the
phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation
in heterochromatin (HC) is generally observed after DNA damage induction. Here,
we examine γH2AX and repair protein foci along linear ion tracks traversing
heterochromatic regions in human or murine cells and find the DSBs and damage
signal streaks bending around highly compacted DNA. Given the linear particle
path, such bending indicates a relocation of damage from the initial induction
site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1
confirms fast recruitment to heterochromatic lesions inside murine
chromocenters. Using single-ion microirradiation to induce localized DSBs
directly within chromocenters, we demonstrate that H2AX is early phosphorylated
within HC, but the damage site is subsequently expelled from the center to the
periphery of chromocenters within ∼ 20 min. While this process can occur in the
absence of ATM kinase, the repair of DSBs bordering HC requires the protein.
Finally, we describe a local decondensation of HC at the sites of ion hits,
potentially allowing for DSB movement via physical forces. AIMS: A defective DNA damage response can result in genomic instability (GIN)
and lead to transformation to cancer. As p53-binding protein 1 (53BP1) localizes
at the sites of DNA double strand breaks (DSBs) and rapidly forms nuclear foci
(NF), the presence of 53BP1 NF can be considered to be an indicator of
endogenous DSBs reflecting GIN. Our aim was to analyse the presence of DSBs by
immunofluorescence for 53BP1 expression in a series of cervical lesions, to
evaluate the significance of GIN during carcinogenesis.
METHODS AND RESULTS: A total of 80 archival cervical tissue samples, including
11 normal, 16 cervical intraepithelial neoplasia (CIN)1, 15 CIN2, 24 CIN3 and 14
squamous cell carcinoma samples, were analysed for 53BP1 NF, human
papillomavirus (HPV) infection, and p16(INK4a) overexpression. The number of
53BP1 NF in cervical cells appeared to increase with progression during
carcinogenesis. The distribution of 53BP1 NF was similar to that of the punctate
HPV signals as determined by in-situ hybridization and also to p16(INK4a)
overexpression in CIN, suggesting an association with viral infection and
replication stress.
CONCLUSIONS: Immunofluorescence analysis of 53BP1 expression can be a useful
tool with which to estimate the level of GIN. During cervical carcinogenesis,
GIN may allow further accumulation of genomic alterations, causing progression
to invasive cancer. The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our
cells from genomic instability in part by facilitating the efficient repair of
DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs
through homologous recombination and is also implicated in the regulation of
nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role
of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency
of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair
relative to BRCA1 protein levels in both control and tumorigenic breast
epithelial cells. In addition to altered BRCA1 protein levels, we tested the
effects of cellular exposure to mirin, an inhibitor of meiotic recombination
enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein
resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ
following a DSB. Inhibition of Mre11-exonuclease activity with mirin
significantly decreased the occurrence of MMEJ, but did not considerably affect
the overall mutagenic frequency of plasmid DSB repair. The results suggest that
BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in
plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair
in the absence of BRCA1 suggests a potential mechanism for carcinogenesis. Chronic inflammation induced by biological, chemical, and physical factors has
been found to be associated with the increased risk of cancer in various organs.
We revealed that infectious agents including liver fluke, Helicobacter pylori,
and human papilloma virus and noninfectious agents such as asbestos fiber
induced iNOS-dependent formation of 8-nitroguanine and 8-oxo-7,
8-dihydro-2'-deoxyguanosine (8-oxodG) in cancer tissues and precancerous
regions. Our results with the colocalization of phosphorylated ATM and γ-H2AX
with 8-oxodG and 8-nitroguanine in inflammation-related cancer tissues suggest
that DNA base damage leads to double-stranded breaks. It is interesting from the
aspect of genetic instability. We also demonstrated IL-6-modulated iNOS
expression via STAT3 and EGFR in Epstein-Barr-virus-associated nasopharyngeal
carcinoma and found promoter hypermethylation in several tumor suppressor genes.
Such epigenetic alteration may occur by controlling the DNA methylation through
IL-6-mediated JAK/STAT3 pathways. Collectively, 8-nitroguanine would be a useful
biomarker for predicting the risk of inflammation-related cancers. When cells encounter genotoxic stress, sensors for DNA lesions stabilize and
activate p53; the signals involved, however, are largely unclear. Inorganic
arsenite is a ubiquitous environmental contamit associated with an increased
risk of lung and skin damage and cancer. Although DNA double-strand breaks and
apoptosis may relate to arsenite-induced damage and carcinogenesis, the
mechanism of action remains obscure. Here, we find that, in human embryo lung
fibroblast (HELF) cells, arsenite induces the activation of dependent protein
kinase catalytic subunit (DNA-PKcs), which then phosphorylates and activates
c-Jun N-terminal kinases 2 (JNK2), but not JNK1. As a positive regulator of p53,
JNK2 binds to p53 and prevents p53 from murine double minute 2 (mdm2)-mediated,
ubiquitin-proteasome-dependent degradation. Knockdown of DNA-PKcs/JNK2 signal
pathway or p53 reduces apoptosis but elevates the DNA damage induced by a high
level of arsenite. These results suggest that DNA-PKcs-mediated stabilization of
p53 by JNK2 is involved in arsenite-induced DNA damage and apoptosis. BACKGROUND: The genome is continuously attacked by a variety of agents that
cause DNA damage. Recognition of DNA lesions activates the cellular DNA damage
response (DDR), which comprises a network of signal transduction pathways to
maintain genome integrity. In response to severe DNA damage, cells undergo
apoptosis to avoid transformation into tumour cells, or alternatively, the cells
enter permanent cell cycle arrest, called senescence. Most tumour cells have
defects in pathways leading to DNA repair or apoptosis. In addition, apoptosis
could be counteracted by nuclear factor kappa B (NF-κB), the main anti-apoptotic
transcription factor in the DDR. Despite the high clinical relevance, the
interplay of the DDR pathways is poorly understood. For therapeutic purposes DNA
damage signalling processes are induced to induce apoptosis in tumour cells.
However, the efficiency of radio- and chemotherapy is strongly hampered by cell
survival pathways in tumour cells. In this study logical modelling was performed
to facilitate understanding of the complexity of the signal transduction
networks in the DDR and to provide cancer treatment options.
RESULTS: Our comprehensive discrete logical model provided new insights into the
dynamics of the DDR in human epithelial tumours. We identified new mechanisms by
which the cell regulates the dynamics of the activation of the tumour suppressor
p53 and NF-κB. Simulating therapeutic intervention by agents causing DNA
single-strand breaks (SSBs) or DNA double-strand breaks (DSBs) we identified
candidate target proteins for sensitization of carcinomas to therapeutic
intervention. Further, we enlightened the DDR in different genetic diseases, and
by failure mode analysis we defined molecular defects putatively contributing to
carcinogenesis.
CONCLUSION: By logic modelling we identified candidate target proteins that
could be suitable for radio- and chemotherapy, and contributes to the design of
more effective therapies. BACKGROUND: Hepatocellular carcinoma (HCC) is a neoplasm for which the
prevalence and mortality rates are very high in Taiwan. The DNA non-homologous
end-joining repair gene XRCC6/Ku70 plays an important role in the repair of DNA
double-strand breaks (DSBs) induced by both exogenous and endogenous
DNA-damaging agents. Defects in overall DSB repair capacity can lead to genomic
instability and carcinogenesis. In this study, we investigated the contribution
of variant XRCC6 in relation to the risk of HCC, from the levels of DNA, RNA and
protein.
MATERIALS AND METHODS: In this hospital-based case-control study, we collected
298 patients with HCC and 298 cancer-free controls, with frequency matched by
age and gender. Firstly, the associations of XRCC6 promoter T-991C (rs5751129),
promoter G-57C (rs2267437), promoter A-31G (rs132770), and intron-3 (rs132774)
polymorphisms with HCC risk in this Taiwanese population were evaluated.
Secondly, 30 HCC tissue samples with variant genotypes were tested to estimate
the XRCC6 mRNA expression by real-time quantitative reverse transcription.
Finally, the HCC tissue samples of variant genotypes were examined by
immunohistochemistry and western blotting to estimate their XRCC6 protein
expression levels.
RESULTS: Compared with the TT genotype, the TC and CC genotypes conferred a
significantly increased risk of HCC [adjusted odds ratio (aOR)=2.43 and 3.52,
95% confidence interval (CI)=1.52-4.03 and 1.18-13.36, p=0.0003 and 0.0385,
respectively]. The mRNA and protein expression levels in HCC tissues revealed
statistically significantly lower XRCC6 mRNA and protein expressions in the HCC
samples with TC/CC genotypes compared with those with the TT genotype (p=0.0037
and 0.0003, respectively).
CONCLUSION: Our multi-approach findings at the DNA, RNA and protein levels
suggested that XRCC6 may play an important role in HCC carcinogenesis in the
Taiwanese population. The response of cells to ionizing radiation-induced DNA double-strand breaks
(DSB) is determined by the activation of multiple pathways aimed at repairing
the injury and maintaining genomic integrity. Densely ionizing radiation induces
complex damage consisting of different types of DNA lesions in close proximity
that are difficult to repair and may promote carcinogenesis. Little is known
about the dynamic behavior of repair proteins on complex lesions. In this study
we use live-cell imaging for the spatio-temporal characterization of early
protein interactions at damage sites of increasing complexity. Beamline
microscopy was used to image living cells expressing fluorescently-tagged
proteins during and immediately after charged particle irradiation to reveal
protein accumulation at damaged sites in real time. Information on the mobility
and binding rates of the recruited proteins was obtained from fluorescence
recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor
protein NBS1 accelerates with increasing lesion density and saturates at very
high damage levels. FRAP measurements revealed two different binding modalities
of NBS1 to damage sites and a direct impact of lesion complexity on the binding.
Faster recruitment with increasing lesion complexity was also observed for the
mediator MDC1, but mobility was limited at very high damage densities due to
nuclear-wide binding. We constructed a minimal computer model of the initial
response to DSB based on known protein interactions only. By fitting all
measured data using the same set of parameters, we can reproduce the
experimentally characterized steps of the DNA damage response over a wide range
of damage densities. The model suggests that the influence of increasing lesion
density accelerating NBS1 recruitment is only dependent on the different binding
modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This
elucidates an impact of damage clustering on repair without the need of invoking
extra processing steps. |
Do conserved noncoding elements co-occur with matrix-attachment regions? | Yes. It is estimated that 11% of the conserved noncoding DNA consists of predicted MARs. Conversely, more than half of the predicted MARs co-occur with one or more independently identified conserved sequence blocks. An excess of conserved predicted MARs is seen in intergenic regions preceding 5' ends of genes, suggesting that these MARs are primarily involved in transcriptional control. | Noncoding DNA in the human-mouse orthologous intergenic regions contains
"islands" of conserved sequences, the functions of which remain largely unknown.
We hypothesized that some of these regions might be matrix-scaffold attachment
regions, MARs (or S/MARs). MARs comprise one of the few classes of eukaryotic
noncoding DNA with an experimentally characterized function, being involved in
the attachment of chromatin to the nuclear matrix, chromatin remodeling and
transcription regulation. To test our hypothesis, we analyzed the co-occurrence
of predicted MARs with highly conserved noncoding DNA regions in human-mouse
genomic alignments. We found that 11% of the conserved noncoding DNA consists of
predicted MARs. Conversely, more than half of the predicted MARs co-occur with
one or more independently identified conserved sequence blocks. An excess of
conserved predicted MARs is seen in intergenic regions preceding 5' ends of
genes, suggesting that these MARs are primarily involved in transcriptional
control. |
What is the relationship between thyroid hormone and inflammatory markers in heart failure patients? | There is an inverse correlation between inflammatory markers (IL-6 and TNF alfa and PCR) and FT3 levels in patients with heart failure | To investigate relationships between thyroid states and the cardiac endocrine
system, we analyzed thyrotropin (TSH), thyroid hormone, plasma levels of
interleukin-6 (IL-6) and brain natriuretic peptide (BNP) in 50 patients with
chronic heart failure (CHF), in 30 patients with heart failure from acute
myocardial infarction (AMI), and in 15 controls. Plasma levels of IL-6 and BNP
in both CHF and AMI were significantly elevated, while free triiodothyronine
(FT3) was significantly decreased compared to controls. FT3/free thyroxine (FT4)
ratio was significantly decreased in CHF but not in AMI compared to controls. In
CHF, diuretic treatment diminished circulating BNP but not IL-6, while diuretic
treatment increased FT3/FT4 ratio. In AMI, FT3/FT4 ratio was significantly
decreased 72 h compared to 12 h after the onset of AMI, while BNP and IL-6 were
significantly increased 72 h compared to 12 h after the onset of AMI. In both
CHF and AMI, BNP significantly correlated with FT4. On the other hand,
significant correlations between IL-6 and FT3, and between IL-6 and FT3/FT4
ratio were detected in AMI but not in CHF. This preliminary study suggests that
IL-6, BNP and thyroid hormone reflect ventricular dysfunction in both acute and
chronic heart failure, and that IL-6 significantly relates to circulating
thyroid hormone in AMI but not in CHF. The derangement of neuro-endocrine control of circulation influences both
disease evolution and response to treatment in patients with heart failure, but
little data are available about the complex relationships between the degree of
neuro-hormonal activation and clinical severity. We studied the relationships
between cardiac natriuretic hormones (CNHs) and several neuro-hormones and
immunological markers in a prospective cohort of 105 consecutive patients with
cardiomyopathy (77 men and 28 women, mean age 66.7+/-12.4 years, range 33-89
years). We assayed the circulating levels of CNHs (atrial natriuretic peptide
(ANP) and brain natriuretic peptide (BNP)), plasma renin activity (PRA),
aldosterone, cortisol, adrenaline, noradrenaline, thyroid hormones and thyroid
stimulating hormone (TSH), tumour necrosis factor-alpha (TNF-alpha) and
interleukin-6 (IL-6). The concentrations of all CNHs and neurohormones were
higher in patients with heart failure compared to normal subjects, except for
free triiodothyronine (FT3), which was below normal values. ANP was positively
related to NYHA class, IL-6, adrenaline, noradrenaline and cortisol, while
negatively with ejection fraction and FT3. BNP was positively related to age,
NYHA class, IL-6, TNF-alpha, adrenaline, noradrenaline and cortisol, while
negatively with ejection fraction and FT3. A stepwise multiple linear regression
indicated that plasma ANP depended only on ejection fraction, adrenaline and
noradrenaline values, while for plasma BNP variation NYHA class contributed too.
Our data confirm a progressive activation of hormonal and immunological systems
in patients with heart failure. Furthermore, CNH circulating levels in heart
failure are affected not only by cardiac function and disease severity, but also
by activation of neuro-hormonal and stress-related cytokine systems, as well as
by the thyroid hormones, even on usual medical treatment. Effects of thyroid gland hormones on cardiovascular system have been known for
many years. Thyroid gland hormones deficiency is connected with a range of
metabolic and hemodynamic changes which can contribute to a genesis of heart
failure. Recent works refer to an importance of connection of thyroid gland
hormones metabolism with heart insufficiency pathophysiology. That is especially
a syndrome of a low trijodthyronin level marked as euthyroid sick syndrome which
is significantly more frequent in patients with chronic heart failure compared
to a population of healthy individuals. Recent clinical works proved that
treatment administration of thyroid gland hormones to patients with heart
failure is connected with favourable hemodynamic changes and increased working
capacity if the treatment is well tolerated. Heart failure is a leading cause of cardiovascular mortality and morbidity. It
has been shown that inflammatory markers may play a role in the pathogenesis of
heart failure. Therefore, inflammatory markers and anticytokine therapy in heart
failure have become the attractive subjects in the literature. There are new
studies regarding this issue in the literature. In this article, we reviewed
inflammatory markers in heart failure and their prognostic significance based on
the literature data. Cytokines and thyroid hormones are involved in the biochemical changes
associated to heart failure (HF).
AIM: Aims of the study were to investigate: plasma circulating levels of the
cytokines Interleukine-6 (IL-6) TNF alpha and C reactive protein (CRP) in
patients with stable HF in relation to the severity of left ventricular
dysfunction; the relationship between these inflammatory markers and thyroid
hormones.
METHODS: One-hundred and sixty-six patients (121 males, age 64+/-12), with
non-ischemic cardiomyopathy, were admitted to the Institute of Clinical
Physiology for progressive deterioration of symptoms. Forty-eight healthy
subjects (30 males, age range 26-75 years) were also enrolled as control group
(Group N). High sensitivity (hs)-IL-6 and hs-TNFalpha were quantified using
solid phase sandwich ELISA kits. Hs-CRP was measured by Immulite System.
RESULTS: In the whole population (HF and N), the association between
inflammatory markers and age resulted statistically significant only for IL-6
serum concentration (p<0.001) but not for TNFalpha and CRP. IL-6 and TNFalpha
were strongly higher in the HF in comparison with N (p<0.001) while CRP showed a
less significant difference (p<0.05). Whole population showed a negative
association between IL-6 and EF% and between CRP and EF% (respectively p<0.01,
r=-0.23; p<0.05, r=0.19). Comparing normal subjects with two classes of
patients, respectively with EF>35% and EF<35%, we clearly observed the
progressive enhancement of the inflammatory markers. Considering normal
subjects, patients without and with low T3 syndrome, IL-6 and TNFalpha increased
progressively from normal to patients with fT3<2 pg/ml (p<0.01 and p<0.01) while
CRP only respect to the group with low T3 syndrome (p<0.01). The inflammatory
markers were all inversely correlated with FT3 levels.
CONCLUSION: Because low FT3 serum concentration represents a negative prognostic
index, it is likely that impairment of T3 production and enhanced inflammation
represent pathogenic mechanisms linked to HF progression. |
Which variables are included in the SPAN-100 score? | SPAN-100 score includes patient's age and NIH Stroke Scale score. SPAN-100 is used for prognostication of stroke patients. | BACKGROUND: To evaluate if plasma levels of midregional pro-adrenomedullin
(MR-proADM) improve prediction of functional outcome in ischemic stroke.
METHODS: In 168 consecutive ischemic stroke patients, plasma levels of MR-proADM
were measured within 24 hours from symptom onset. Functional outcome was
assessed by the modified Rankin Scale (mRS) at 90 days following stroke.
Logistic regression, receiver operating characteristics (ROC) curve analysis,
net reclassification improvement (NRI), and Kaplan-Meier survival analysis were
applied.
RESULTS: Plasma MR-proADM levels were found significantly higher in patients
with unfavourable (mRS 3-6) compared to favourable (mRS 0-2) outcomes. MR-proADM
levels were entered into a predictive model including the patients' age,
National Institutes of Health Stroke Scale (NIHSS), and the use of
recanalization therapy. The area under the ROC curve did not increase
significantly. However, category-free NRI of 0.577 (p<0.001) indicated a
significant improvement in reclassification of patients. Furthermore, MR-proADM
levels significantly improved reclassification of patients in the prediction of
outcome by the Stroke Prognostication using Age and NIHSS-100 (SPAN-100;
NRI = 0.175; p = 0.04). Kaplan-Meier survival analysis showed a rising risk of
death with increasing MR-proADM quintiles.
CONCLUSIONS: Plasma MR-proADM levels improve prediction of functional outcome in
ischemic stroke when added to the patients' age, NIHSS on admission, and the use
of recanalization therapy. Levels of MR-proADM in peripheral blood improve
reclassification of patients when the SPAN-100 is used to predict the patients'
functional outcome. BACKGROUND AND PURPOSE: Age and stroke severity are inversely correlated with
the odds of favorable outcome after ischemic stroke. A previously proposed score
for Stroke Prognostication Using Age and NIHSS Stroke Scale (SPAN) indicated
that SPAN-100-positive patients (ie, age + NIHSS score = 100 or more) do not
benefit from IV-tPA. If this finding holds true for endovascular therapy, this
score can impact patient selection for such interventions. This study
investigated whether a score combining age and NIHSS score can improve patients'
selection for endovascular stroke therapy.
MATERIALS AND METHODS: The SPAN index was calculated for patients in the
prospective Solitaire FR Thrombectomy for Acute Revascularization study: an
international single-arm multicenter cohort for anterior circulation stroke
treatment by using the Solitaire FR. The proportion with favorable outcome
(90-day mRS score ≤2) was compared between SPAN-100-positive versus-negative
patients.
RESULTS: Of the 202 patients enrolled, 196 had baseline NIHSS scores. Fifteen
(7.7%) patients were SPAN-100-positive. There was no difference in the rate of
successful reperfusion (Thrombolysis In Cerebral Infarction 2b or 3) between
SPAN-100-positive versus -negative groups (93.3% versus 82.8%, respectively; P =
.3). Stroke SPAN-100-positive patients had a significantly lower proportion of
favorable clinical outcomes (26.7% versus 60.8% in SPAN-100-negative, P = .01).
In a multivariable analysis, SPAN-100-positive status was associated with lower
odds of favorable outcome (OR, 0.3; 95% CI, 0.1-0.9; P = .04). A higher baseline
Alberta Stroke Program Early CT Score and a short onset to revascularization
time also predicted favorable outcome in the multivariable analysis.
CONCLUSIONS: A significantly lower proportion of patients with a positive
SPAN-100 achieved favorable outcome in this cohort. SPAN-100 was an independent
predictor of favorable outcome after adjusting for time to treatment and the
extent of preintervention tissue damage according to the Alberta Stroke Program
Early CT Score. IMPORTANCE: The Stroke Prognostication using Age and the NIH Stroke Scale index,
created by combining age in years plus a National Institutes of Health (NIH)
Stroke Scale score of 100 or higher (and hereafter referred to as the SPAN-100
index), is a simple risk score for estimating clinical outcomes for patients
with acute ischemic stroke (AIS). The association between this index and
response to intravenous thrombolysis for AIS has not been properly evaluated.
OBJECTIVE: To assess the relationship between SPAN-100 index status and outcome
following treatment with intravenous thrombolysis for AIS.
DESIGN, SETTING, AND PARTICIPANTS: Using the Virtual International Stroke Trials
Archive (VISTA) database, an international repository of clinical trials data,
we assessed the SPAN-100 index among 7093 patients with AIS who participated in
4 clinical trials from 2000 to 2006. The SPAN-100 index is considered positive
if the sum of the age and the NIH Stroke Scale (a 15-item neurological
examination scale with scores ranging from 0 to 42, with higher scores
indicating more severe strokes) score is greater than or equal to 100.
Multivariable logistic regression analyses were used to determine the
independent association between SPAN-100 index status and 90-day outcomes.
MAIN OUTCOMES AND MEASURES: The primary outcome was a composite of severe
disability or death measured 90 days after stroke, and the secondary outcomes
were death alone and a composite of no disability/modest disability.
RESULTS: Of 7093 patients, 743 (10.5%) were SPAN-100 positive, and 2731 (38.5%)
received intravenous thrombolysis. Compared with SPAN-100-negative patients,
SPAN-100-positive patients were more likely to experience a catastrophic outcome
(adjusted odds ratio [AOR], 9.03 [95% CI, 6.68-12.21]) or death alone (AOR, 5.03
[95% CI, 4.06-6.23]) and less likely to experience a favorable outcome (AOR,
0.08 [95% CI, 0.06-0.13]). However, there was an interaction between SPAN-100
index status and thrombolysis treatment (P < .001) revealing a reduction in the
likelihood of severe disability/death with thrombolytic treatment for
SPAN-100-positive (AOR, 0.46 [95% CI, 0.29-0.71]) but not SPAN-100-negative
patients (AOR, 0.96 [95% CI, 0.85-1.07]). Similar interactions between SPAN-100
index status and thrombolysis treatment were observed for the 2 secondary
outcomes.
CONCLUSION AND RELEVANCE: Compared with the SPAN-100-negative patients with AIS,
the SPAN-100-positive patients with AIS seem to have poorer 3-month outcomes but
may derive greater benefit when treated with intravenous thrombolysis. The
SPAN-100-positive patients are often excluded from AIS clinical trials but
should probably not be denied thrombolysis treatment on the basis of such a
profile alone. 1. BACKGROUND: Stroke Prognostication using Age and NIH (National Institutes of
Health) Stroke Scale (SPAN)-100 is a simple and easy-to-use tool for assessing
the outcomes of ischemic stroke after thrombolysis. To explore its application,
we evaluated SPAN-100's prognostic value in predicting 3- and 12-month outcomes
in general ischemic stroke patients.
METHODS: We applied the SPAN-100 to ischemic stroke patients from the China
National Stroke Registry. Poor outcome was defined as a modified Rankin Scale of
2-6. Discrimination of SPAN-100 was assessed by the area under the
receiver-operator curves (AUC) and 95% confidence intervals (CI). We also
performed an exploratory post hoc analysis of the performance of the SPAN index
score using 80 as the cutoff point.
RESULTS: Among 11,894 ischemic stroke patients, 479 (4.0%) patients were
SPAN-100 positive. The AUC of SPAN-100 for poor outcome was .54 (95% CI,
.54-.54) at 3 months and .54 (95% CI, .54-.55) at 12 months, respectively. In
the exploratory analysis, when 80 was used as the cutoff point of SPAN index
score, the AUC for poor outcome was .66 (95% CI, .66-.67) at 3 months and .68
(95% CI, .67-.68) at 12 months, respectively.
CONCLUSIONS: SPAN-100 suffered from low prediction power for 3- and 12-month
outcomes of ischemic stroke in Chinese population. A cutoff point of 80 may
improve the performance, but none of them had an AUC above the threshold of .8
required for use in individuals. |
Are Drosophila ultraconserved elements candidate ncRNAs? | Yes. Highly constrained intergenic Drosophila ultraconserved elements are candidate ncRNAs. | Eukaryotes contain short (∼80-200 bp) regions that have few or no substitutions
among species that represent hundreds of millions of years of evolutionary
divergence. These ultraconserved elements (UCEs) are candidates for containing
essential functions, but their biological roles remain largely unknown. Here, we
report the discovery and characterization of UCEs from 12 sequenced Drosophila
species. We identified 98 elements ≥80 bp long with very high conservation
across the Drosophila phylogeny. Population genetic analyses reveal that these
UCEs are not present in mutational cold spots. Instead we infer that they
experience a level of selective constraint almost 10-fold higher compared with
missense mutations in protein-coding sequences, which is substantially higher
than that observed previously for human UCEs. About one-half of these Drosophila
UCEs overlap the transcribed portion of genes, with many of those that are
within coding sequences likely to correspond to sites of ADAR-dependent RNA
editing. For the remaining UCEs that are in nongenic regions, we find that many
are potentially capable of forming RNA secondary structures. Among ten chosen
for further analysis, we discovered that the majority are transcribed in
multiple tissues of Drosophila melanogaster. We conclude that Drosophila species
are rich with UCEs and that many of them may correspond to novel noncoding RNAs. |
Which genes does thyroid hormone receptor alpha1 regulate in the liver? | phosphoenolpyruvate-carboxykinase"//
"pyruvate kinase"//
"D1", "deiodinase 1"// | Type 1 deiodinase (D1) metabolizes different forms of thyroid hormones to
control levels of T3, the active ligand for thyroid hormone receptors (TR). The
D1 gene is itself T3-inducible and here, the regulation of D1 expression by
TRalpha1 and TRbeta, which act as T3-dependent transcription factors, was
investigated in receptor-deficient mice. Liver and kidney D1 mRNA and activity
levels were reduced in TRbeta(-/-) but not TRalpha1(-/-) mice. Liver D1 remained
weakly T3 inducible in TRbeta(-/-) mice whereas induction was abolished in
double mutant TRalpha1(-/-)TRbeta(-/-) mice. This indicates that TRbeta is
primarily responsible for regulating D1 expression whereas TRalpha1 has only a
minor role. In kidney, despite the expression of both TRalpha1 and TRbeta,
regulation relied solely on TRbeta, thus revealing a marked tissue restriction
in TR isotype utilization. Although TRbeta and TRalpha1 mediate similar
functions in vitro, these results demonstrate differential roles in regulating
D1 expression in vivo and suggest that tissue-specific factors and structural
distinctions between TR isotypes contribute to functional specificity.
Remarkably, there was an obligatory requirement for a TR, whether TRbeta or
TRalpha1, for any detectable D1 expression in liver. This suggests a novel
paradigm of gene regulation in which the TR sets both basal expression and the
spectrum of induced states. Physiologically, these findings suggest a critical
role for TRbeta in regulating the thyroid hormone status through D1-mediated
metabolism. Mice expressing the mutant thyroid hormone receptor TRalpha1R384C, which has a
10-fold reduced affinity to the ligand T(3), exhibit hypermetabolism due to an
overactivation of the sympathetic nervous system. To define the consequences in
the liver, we analyzed hepatic metabolism and the regulation of liver genes in
the mutant mice. Our results showed that hepatic
phosphoenolpyruvate-carboxykinase was up-regulated and pyruvate kinase mRNA
down-regulated, contrary to what observed after T(3) treatment. In contrast,
mice expressing a mutant TRalpha1L400R specifically in the liver did not show a
dysregulation of these genes; however, when the TRalpha1L400R was expressed
ubiquitously, the hepatic phenotype differed from TRalpha1R384C animals,
suggesting that the localization of the mutation plays an important role for its
consequences on glucose metabolism. Furthermore, we observed that glycogen
stores were completely depleted in TRalpha1R384C animals, despite increased
gluconeogenesis and decreased glycolysis. Exposure of the mutant mice to high
maternal levels of thyroid hormone during fetal development leads to a normal
liver phenotype in the adult. Our results show how genetic and maternal factors
interact to determine the metabolic setpoint of the offspring and indicate an
important role for maternal thyroid hormone in the susceptibility to metabolic
disorders in adulthood. |
What is the color of the protein Ranasmurfin? | Ranasmurfin is a blue protein. | Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein
found in the nests of the frog Polypedates leucomystax, has been purified and
crystallized. The crystals are an intense blue colour and diffract to 1.51 A
with P2(1) symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 A,
beta = 93.3 degrees . Self-rotation function analysis indicates the presence of
a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of
the protein is related to dimer formation. Sequence data for the protein are
incomplete, but thus far have identified no model for molecular replacement. A
fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds
zinc and suggesting a route for structure solution. Naturally occurring foam constituent and surfactant proteins with intriguing
structures and functions are now being identified from a variety of biological
sources. The ranaspumins from tropical frog foam nests comprise a range of
proteins with a mixture of surfactant, carbohydrate binding and antimicrobial
activities that together provide a stable, biocompatible, protective foam
environment for developing eggs and embryos. Ranasmurfin, a blue protein from a
different species of frog, displays a novel structure with a unique chromophoric
crosslink. Latherin, primarily from horse sweat, but with similarities to
salivary, oral and upper respiratory tract proteins, illustrates several
potential roles for surfactant proteins in mammalian systems. These proteins,
together with the previously discovered hydrophobins of fungi, throw new light
on biomolecular processes at air-water and other interfaces. This review
provides a perspective on these recent findings, focussing on structure and
biophysical properties. |
What is the role of Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) in development? | Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism. HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. In addition, HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. Furthermore, it is required for the formation of a functional germline and for the development of the vulva by acting in an Rb-related pathway. | Proteins of the highly conserved heterochromatin protein 1 (HP1) family have
been found to function in the dynamic organization of nuclear architecture and
in gene regulation throughout the eukaryotic kingdom. In addition to being key
players in heterochromatin-mediated gene silencing, HP1 proteins may also
contribute to the transcriptional repression of euchromatic genes via the
recruitment to specific promoters. To investigate the role played by these
different activities in specific developmental pathways, we identified HP1
homologues in the genome of Caenorhabditis elegans and used RNA-mediated
interference to study their function. We show that one of the homologues, HPL-2,
is required for the formation of a functional germline and for the development
of the vulva by acting in an Rb-related pathway. We suggest that, by acting as
repressors of gene expression, HP1 proteins may fulfil specific functions in
both somatic and germline differentiation processes throughout development. Specific nuclear domains are nonrandomly positioned within the nuclear space,
and this preferential positioning has been shown to play an important role in
genome activity and stability. Well-known examples include the organization of
repetitive DNA in telomere clusters or in the chromocenter of Drosophila and
mammalian cells, which may provide a means to control the availability of
general repressors, such as the heterochromatin protein 1 (HP1). We have
specifically characterized the intranuclear positioning of in vivo fluorescence
of the Caenorhabditis elegans HP1 homologue HPL-2 as a marker for
heterochromatin domains in developing embryos. For this purpose, the wavelet
transform modulus maxima (WTMM) segmentation method was generalized and adapted
to segment the small embryonic cell nuclei in three dimensions. The
implementation of a radial distribution algorithm revealed a preferential
perinuclear positioning of HPL-2 fluorescence in wild-type embryos compared with
the diffuse and homogeneous nuclear fluorescence observed in the lin-13 mutants.
For all other genotypes analyzed, the quantitative analysis highlighted various
degrees of preferential HPL-2 positioning at the nuclear periphery, which
directly correlates with the number of HPL-2 foci previously counted on 2D
projections. Using a probabilistic 3D cell nuclear model, we found that any two
nuclei having the same number of foci, but with a different 3D probabilistic
positioning scheme, can have significantly different counts in the 2D maximum
projection, thus showing the deceptive limitations of using techniques of 2D
maximum projection foci counts. By this approach, a strong perinuclear
positioning of HPL-2 foci was brought into light upon inactivation of conserved
chromatin-associated proteins, including the HAT cofactor TRAPP. MBT domain proteins are involved in developmental processes and tumorigenesis.
In vitro binding and mutagenesis studies have shown that individual MBT domains
within clustered MBT repeat regions bind mono- and dimethylated histone lysine
residues with little to no sequence specificity but discriminate against the
tri- and unmethylated states. However, the exact function of promiscuous histone
methyl-lysine binding in the biology of MBT domain proteins has not been
elucidated. Here, we show that the Caenorhabditis elegans four MBT domain
protein LIN-61, in contrast to other MBT repeat factors, specifically interacts
with histone H3 when methylated on lysine 9, displaying a strong preference for
di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is
implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats
two to four. Further, mutagenesis of residues conserved with other methyl-lysine
binding MBT regions in the fourth MBT repeat does not abolish interaction,
implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is
required for C. elegans vulva development within the synMuvB pathway. Mutant
LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB
function. Also, previously identified point mutant synMuvB alleles are deficient
in H3K9me2/3 interaction although these target residues that are outside of the
fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other
synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a
SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans
vulva development and fertility. Besides identifying the first sequence specific
and di-/trimethylation binding MBT domain protein, our studies imply complex
multi-domain regulation of ligand interaction of MBT domains. Our results also
introduce a mechanistic link between LIN-61 function and biology, and they
establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well
as the H3K9MT MET-2 in distinct developmental pathways. BACKGROUND: Heterochromatin protein 1 (HP1) family proteins have a
well-characterized role in heterochromatin packaging and gene regulation. Their
function in organismal development, however, is less well understood. Here we
used genome-wide expression profiling to assess novel functions of the
Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages.
RESULTS: We show that HPL-2 regulates the expression of germline genes,
extracellular matrix components and genes involved in lipid metabolism.
Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that
a significant number of genes up- and down-regulated in the absence of HPL-2 are
bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants,
consistent with the function of HPL-2 as a repressor of ectopic germ cell fate.
In addition, microarray results and phenotypic analysis suggest that HPL-2
regulates the dauer developmental decision, a striking example of phenotypic
plasticity in which environmental conditions determine developmental fate. HPL-2
acts in dauer at least partly through modulation of daf-2/IIS and TGF-β
signaling pathways, major determits of the dauer program. hpl-2 mutants also
show increased longevity and altered lipid metabolism, hallmarks of the
long-lived, stress resistant dauers.
CONCLUSIONS: Our results suggest that the worm HP1 homologue HPL-2 may
coordinately regulate dauer diapause, longevity and lipid metabolism, three
processes dependent on developmental input and environmental conditions. Our
findings are of general interest as a paradigm of how chromatin factors can both
stabilize development by buffering environmental variation, and guide the
organism through remodeling events that require plasticity of cell fate
regulation. |
Does surgery for ovarian endometriomas improve fertility? | Yes, endometrioma surgery seems to improve the success rates of fertility treatment. | Seventy-nine cases of ovarian endometrioma were treated via the laparoscope. The
surgical technique gradually evolved from drainage alone to fulguration and
finally to excision of the endometrioma cyst wall. Indications for laparoscopy
were infertility (35 cases), pain (33), hypermenorrhea (5) and pelvic mass (4);
the condition was an incidental finding in 2 cases. There were no intraoperative
or postoperative complications. The long-term results, especially the fertility
outcome, have been promising: 12 of 20 women (60%) achieved a term pregcy
following a laparoscopic endometrioma procedure alone. Of the eight women with
endometriomas who did not conceive, six had well-documented male factor
infertility. Thirty-six women with ultrasonographic diagnosis of ovarian endometrioma
(bilateral in nine of them), have been treated laparoscopically. After the
surgical procedure the patients were assigned to one of the following regimes:
Gn-RH-a for 3 months, oral contraceptives if they wanted to avoid pregcy, or
nothing. The follow-up consisted in 1-3-6-12 months ultrasound. The first
recurrences were observed at the 6-month ultrasound with an overall recurrence
rate after 12 months of 11%. Improvement of pain symptoms occurred in 87% of the
patients and fertility rate was 45%. OBJECTIVE: To investigate whether conservative surgery on ovarian endometriomas
before an IVF cycle improves fertility outcomes.
DESIGN: Retrospective, matched case-control study.
SETTING: Two academic IVF programs.
PATIENT(S): One hundred eighty-nine women with endometriomas who underwent IVF
treatment: 56 women proceeded directly to IVF, and 133 first underwent
conservative ovarian surgery.
INTERVENTION(S): Controlled ovarian hyperstimulation and IVF-ET.
MAIN OUTCOME MEASURE(S): Response to gonadotropins, fertilization, implantation,
and pregcy rates.
RESULT(S): Aside from lower peak E(2) levels on the day of hCG and a higher
total FSH dose in women previously operated for an endometrioma, no significant
differences were found between the two groups in the different IVF variables
analyzed.
CONCLUSION(S): Laparoscopic cystectomy for endometriomas before commencing an
IVF cycle does not improve fertility outcomes. Proceeding directly to controlled
ovarian hyperstimulation in women with asymptomatic ovarian endometriomas might
reduce the time to pregcy, the costs of treatment, and the hypothetical
complications of laparoscopic surgery. Conversely, conservative surgical
treatment of ovarian endometriomas in symptomatic women does not impair IVF or
intracytoplasmic sperm injection success rates. BACKGROUND: Endometriomata are endometriotic deposits within the ovary. The
surgical management of these blood filled cysts is controversial. The
laparoscopic approach to the management of endometriomata is favoured for as it
offers the advantage of a shorter hospital stay, faster patient recovery and
decreased hospital costs. Currently the commonest procedures for the treatment
of ovarian endometriomata are either excision of the cyst capsule or drainage
and electrocoagulation of the cyst wall.
OBJECTIVES: The objective of this review was to determine the most effective
technique of treating an ovarian endometrioma; either excision of the cyst
capsule or drainage and electrocoagulation of the cyst wall, with regard to
relief of pain, recurrence of the endometrioma, recurrence of symptoms and the
subsequent spontaneous pregcy rate.
SEARCH STRATEGY: The reviewers searched the Cochrane Menstrual Disorders and
Subfertility Group specialised register of trials (searched 15 Nov 2004), the
Cochrane Register of Controlled Trials (The Cochrane Library, Issue 4, 2004),
MEDLINE (1966-Nov 2004), EMBASE (1980- Nov 2004) and reference lists of
articles, the handsearching of relevant journals and conference proceedings and
by contacting leaders in the field of endoscopic surgery throughout the world.
SELECTION CRITERIA: Randomised controlled trials of excision of the cyst capsule
versus drainage and electrocoagulation of the cyst in the management of ovarian
endometriomata.
DATA COLLECTION AND ANALYSIS: Reviewers assessed eligibility and trial quality.
MAIN RESULTS: No randomised studies of the management of endometriomata by
laparotomy were found. Two randomised studies of the laparoscopic management of
ovarian endometriomata of greater than 3cm in size were included. Laparoscopic
excision of the cyst wall of the endometrioma was associated with a reduced rate
of recurrence of the endometrioma (OR 0.41 CI 0.18-0.93), reduced requirement
for further surgery (OR 0.21 CI 0.05-0.79), reduced recurrence rate of the
symptoms of dysmenorrhoea (OR 0.15 CI 0.06-0.38), dyspareunia OR 0.08 CI
0.01-0.51) and non-menstrual pelvic pain (OR 0.10 CI 0.02-0.56). It was also
associated with a subsequent increased rate of spontaneous pregcy women who
had documented prior sub-fertility (OR 5.21 CI 2.04-13.29).
AUTHORS' CONCLUSIONS: There is some evidence that excisional surgery for
endometriomata provides for a more favourable outcome than drainage and
ablation, with regard to the recurrence of the endometrioma, recurrence of
symptoms and subsequent spontaneous pregcy in women who were previously
subfertile. Consequently this approach should be the favoured surgical approach.
However we found no data as to the effect of either approach in women who
subsequently undergo assisted reproductive techniques. BACKGROUND: Endometriomata are endometriotic deposits within the ovary. The
surgical management of these blood filled cysts is controversial. The
laparoscopic approach to the management of endometriomata is favoured over a
laparotomy approach as it offers the advantage of a shorter hospital stay,
faster patient recovery and decreased hospital costs. Currently the commonest
procedures for the treatment of ovarian endometriomata are either excision of
the cyst capsule or drainage and electrocoagulation of the cyst wall.
OBJECTIVES: The objective of this review was to determine the most effective
technique of treating an ovarian endometrioma; either excision of the cyst
capsule or drainage and electrocoagulation of the cyst wall. The end-points
assessed were the relief of pain, recurrence of the endometrioma, recurrence of
symptoms and in women desiring to conceive the subsequent pregcy rate, either
spontaneous or as part of fertility treatment.
SEARCH STRATEGY: The reviewers searched the Cochrane Menstrual Disorders and
Subfertility Group specialised register of trials (searched 3rd March 2007), the
Cochrane Register of Controlled Trials (The Cochrane Library, Issue 3, 2007),
MEDLINE (1966-August 2007), EMBASE (1980- March 2007) and reference lists of
articles, the handsearching of relevant journals and conference proceedings and
by contacting leaders in the field of endoscopic surgery throughout the world.
The Cochrane Menstrual Disorders and Subfertility Group Trials Register is based
on regular searches of MEDLINE, EMBASE, CINHAL and CENTRAL.
SELECTION CRITERIA: Randomised controlled trials of excision of the cyst capsule
versus drainage and electrocoagulation of the cyst in the management of ovarian
endometriomata.
DATA COLLECTION AND ANALYSIS: Reviewers assessed eligibility and trial quality.
MAIN RESULTS: No randomised studies of the management of endometriomata by
laparotomy were found. Two randomised studies of the laparoscopic management of
ovarian endometriomata of greater than 3cm in size, for the primary symptom of
pain were included. Laparoscopic excision of the cyst wall of the endometrioma
was associated with a reduced recurrence rate of the symptoms of dysmenorrhea
(OR 0.15 CI 0.06-0.38), dyspareunia (OR 0.08 CI 0.01-0.51) and non-menstrual
pelvic pain (OR 0.10 CI 0.02-0.56), a reduced rate of recurrence of the
endometrioma (OR 0.41 CI 0.18-0.93) and with a reduced requirement for further
surgery (OR 0.21 CI 0.05-0.79) than surgery to ablate the endometrioma. For
those women subsequently attempting to conceive it was also associated with a
subsequent increased spontaneous pregcy rate in women who had documented
prior sub-fertility (OR 5.21 CI 2.04-13.29). A further randomised study was
identified that demonstrated an increased ovarian follicular response to
gonadotrophin stimulation for women who had undergone excsional surgery when
compared to ablative surgery (WMD 0.6 CI 0.04-1.16). There is insufficient
evidence to favour excisional surgery over ablative surgery with respect to the
chance of pregcy after controlled ovarian stimulation and intra-uterine
insemination (OR 1.40 CI 0.47-4.15) .
AUTHORS' CONCLUSIONS: There is good evidence that excisional surgery for
endometriomata provides for a more favourable outcome than drainage and ablation
with regard to the recurrence of the endometrioma, recurrence of pain symptoms,
and in women who were previously subfertile, subsequent spontaneous pregcy .
Consequently this approach should be the favoured surgical approach. However in
women who may subsequently may undergo fertility treatment insufficient evidence
exists to determine the favoured surgical approach. Endometrioma is commonly seen in women of reproductive age who may wish to
preserve their ovarian function. Surgical treatment is associated with a high
recurrence rate and its employment for women undergoing assisted conception has
recently been challenged. Medical treatment has not been shown to be effective
in controlling symptoms or improving fertility potential. The results of
retrospective and non-randomized studies have been inconsistent and created an
ongoing debate between gynaecologists and fertility specialists. This manuscript
reviews and critically appraises the evidence for management of endometrioma in
women of reproductive age. In asymptomatic women, surgical treatment is usually
recommended for women above the age of 40 and for large endometriomas. Except
for pelvic clearance, there is insufficient evidence to suggest that surgical
treatment of endometrioma is better than medical treatment with respect to the
long-term relief of symptoms and quality of life. Laparoscopic excision of
ovarian endometrioma prior to IVF does not offer any additional benefit over
expectant management. A large, well-designed, adequately powered randomized
controlled study that compares the effects of surgical removal versus expectant
management of endometrioma on ovarian performance and pregcy outcomes in
women undergoing IVF is warranted. We performed this retrospective case-control study analyzing 428 first-attempt
in vitro fertilization (IVF) cycles, among which 254 involved women with a
previous or present diagnosis of ovarian endometriosis. First, the results of
these 254 cycles were compared with 174 cycles involving patients with proven
non-endometriotic tubal infertility having similar age and body mass index.
Women with ovarian endometriosis had a significantly higher cancellation rate,
but similar pregcy, implantation and delivery rates as patients with tubal
infertility. Second, among the women with ovarian endometriosis, the women with
a history of laparoscopic surgery for ovarian endometriomas prior to IVF and no
visual endometriosis at ovum pick-up (n = 112) were compared with the
non-operated women and visual endometriomas at ovum pick-up (n = 142). Patients
who underwent ovarian surgery before IVF had significantly shorter period, lower
antral follicle count and required higher gonadotropin doses than patients with
non-operated endometriomas. The two groups of women with a previous or present
ovarian endometriosis did, however, have similar pregcy, implantation and
live birth rates. In conclusion, ovarian endometriosis does not reduce IVF
outcome compared with tubal factor. Furthermore, laparoscopic removal of
endometriomas does not improve IVF results, but may cause a decrease of ovarian
responsiveness to gonadotropins. The precise relationship between endometriosis and infertility is debated.
Surgery is considered to play a role within the framework of the therapeutic
options to cure infertile women with the disease even though its effectiveness
is generally modest. In fact, there is unquestionably the need to improve
surgical techniques in this area. Specifically, two main aspects require
optimization: 1) preventing the injury to the follicular reserve that follows
surgical excision of ovarian endometriomas and 2) preventing post-surgical
formation and re-formation of adhesions. The comparison between the
excision/stripping and the vaporization/coagulation techniques represents the
main point of debate on what is the best procedure to remove ovarian
endometrioma. Randomized controlled trials showed that the excision technique is
associated with a higher pregcy rate and a lower rate of recurrence although
it may determine severe injury to the ovarian reserve. Improvements to this
latter aspect may be represented by a combined excisional-vaporization technique
or by replacing diathermy coagulation with surgical ovarian suture. Barrier
agents reduce but not eliminate the post-surgical adhesion formation in women
with endometriosis. Encouraging evidence has been reported with Interceed,
Oxiplex/AP gel and Adept solution. However, available studies are mainly based
on II look laparoscopies performed few weeks after the intervention and data on
fertility is lacking. Clinical trials including pregcy rate as a specific
outcome are warranted. STUDY QUESTION: What is the current management of women with ovarian
endometriomas undergoing assisted reproductive treatment (ART) in the UK?
SUMMARY ANSWER: It appears that the majority of gynaecologists in the UK offer
surgery (mostly cystectomy) for endometriomas prior to ART, regardless of the
presence of symptoms.
WHAT IS KNOWN ALREADY: The ideal management of endometriomas in women undergoing
ART remains controversial and presents a dilemma to reproductive specialists.
STUDY DESIGN, SIZE AND DURATION: This was a national cross-sectional survey. A
total of 388 gynaecologists completed the questionnaire.
PARTICIPANTS, SETTINGS AND METHODS: All clinicians fully registered with the
Royal College of Obstetricians and Gynaecologists were contacted. An 11-item
survey was administered electronically using Survey Monkey software.
Quantitative data were analysed using descriptive and comparative statistics.
MAIN RESULTS AND THE ROLE OF CHANCE: The majority of responders were consultants
(65%), 25% practiced ART and 65% performed laparoscopic surgery. Overall, 95% of
responders would offer surgery for endometriomas in women undergoing ART, either
on the basis of the size (>3-5 cm) of the endometrioma (52%), the presence of
symptoms (16%), the presence of multiple/bilateral endometriomas (2%),
regardless of the size and symptoms (19%) or only to women undergoing IVF (6%).
The remaining 5% of responders would not offer surgery before ART. Excision was
the most common surgical modality (68%), followed by ablation (25%).
Laparoscopic surgeons were almost twice as likely to 'offer surgery to all
patients with endometriomas prior to ART' compared with clinicians performing
laparotomy (22 versus 12%, P < 0.001).
LIMITATIONS, REASONS FOR CAUTION: Our overall response rate, with answers to the
questionnaire, was low (15%). However, the response rate amongst reproductive
specialists was estimated at 60%. It is possible that there might have been an
element of bias towards over-representation of responders who are more concerned
about 'normalization' of the pelvic anatomy. Furthermore, our survey relied on
self-reporting of practice and it is possible that being presented with a list
of 'ideal' options may have resulted in respondent bias.
WIDER IMPLICATIONS OF THE FINDINGS: Despite the available evidence that surgery
for endometriomas does not improve the outcome of ART and may damage ovarian
reserve, it seems that the majority of gynaecologists in the UK offer ovarian
cystectomy to their patients. Laparoscopic excision is considered as the 'gold standard' treatment of ovarian
endometrioma. However, a frustrating aspect is that disease can recur. While
laparoscopic excision is known to improve fertility, recurrence can cause
significant ovarian damage and adverse affects on fertility. It is therefore
crucial to prevent recurrence in order to conserve 'improved' fertility.
Recurrence rates for endometrioma are reported from 11 to 32% within 1-5 years
after excision. The recurrence rate is higher in patients with advanced
endometriosis at surgery and in younger patients. Previous medical treatment for
endometriosis prior laparoscopy is a risk factor for recurrence. Pregcy soon
after surgery has a protective effect for recurrence. The accumulating evidence
suggests that the administration of oral contraceptives (OC),
levonorgestrel-releasing intrauterine system (LNG-IUS) and a combination of
gonadotropin releasing hormone (GnRH) analogue and OC may also have therapeutic
benefits. Collectively, we propose that women should be well informed about the
risks of endometrioma recurrence. We recommend that women who wish pregcy
should try conception as soon as possible. Further, we strongly advise hormonal
therapy for patients, who do not want to conceive immediately, and until
pregcy is desired. STUDY OBJECTIVE: To assess recurrence and pregcy rates in women with ovarian
endometrioma treated via ablation using plasma energy.
DESIGN: Retrospective non-comparative pilot study including 55 patients treated
during 28 months, with prospective recording of data (Canadian Task Force
classification II-2).
SETTING: Tertiary referral center.
PATIENTS: Fifty-five consecutive women with pelvic endometriosis in whom ovarian
endometriomas were managed solely via ablation using plasma energy. The minimum
follow-up was 1 year.
INTERVENTION: Endometrioma ablation using plasma energy.
MEASUREMENTS AND MAIN RESULTS: Information was obtained from the database of the
North-West Inter Regional Female Cohort for Patients with Endometriosis, based
on self-questionnaires completed before surgery, surgical and histologic data,
and systematic recording of recurrences, pregcy, and symptoms. Recurrences
were assessed using pelvic ultrasound examination. Mean (SD) follow-up was 20.6
(7.2) months (range, 12-39 months). In 75% of patients, deep infiltrating
endometriosis was treated, and 40% had colorectal involvement. Preoperative
infertility was recorded in 42% of patients. The rate of postoperative
recurrence was 10.9% for the entire series. Of 33 women who wished to conceive,
67% became pregt, spontaneously in 59%. Time from surgery to the first
pregcy was 7.6 (4.3) months. After discontinuation of postoperative hormone
therapy, the probability of not conceiving at 12 months was 0.36 (95% confidence
interval, 0.19-0.53), and at 24 months was 0.27 (95% confidence interval,
0.12-0.44).
CONCLUSIONS: Recurrence and pregcy rates are encouraging in that they seem
comparable to the best reported results after endometrioma cystectomy. Plasma
energy may have an important role in the management of ovarian endometrioma in
women seeking to conceive. Patients most in need of surgical procedures that can
spare ovarian parenchyma, such as those with bilateral endometriomas or a
history of ovarian surgery, may particularly benefit from ablation using plasma
energy. |
Are ACTA1 (alpha actin) and NEB (nebulin) genes related to nemaline myopathy? | Yes, most nemaline myopathy patients have mutations in the nebulin (NEB) or skeletal muscle alpha-actin (ACTA1) genes. | Nemaline myopathy is a clinically and genetically heterogeneous condition. The
clinical spectrum ranges from severe cases with antenatal or neonatal onset and
early death to late onset cases with only slow progression. Three genes are
known to cause nemaline myopathy: the genes for nebulin (NEB) on chromosome
2q22, slow alpha-tropomyosin (TPM3) on chromosome 1q21 and skeletal muscle
alpha-actin (ACTA1) on chromosome 1q42. We present a 39-year-old lady with a
mild form of nemaline myopathy, whom we have followed over a period of 25 years.
She presented at the age of 7 years with symptoms of mild axial and proximal
muscle weakness. The overall course was essentially static, but at 36 years, she
went into life-threatening respiratory failure, for which she is currently
treated with night-time ventilation. Muscle biopsies at 12, 17 and 39 years of
age showed typical nemaline rods, particularly in type 1 fibres. Areas with
unevenness of oxidative stain were present in the second and third biopsies. The
presence of rods and core-like areas was confirmed on electron microscopy. There
was no detectable alteration in actin expression immunocytochemically. A
domit missense mutation in the skeletal muscle alpha-actin gene (ACTA1) was
found. This case illustrates the clinical and genetic heterogeneity of nemaline
myopathy, and one phenotype of the wide spectrum of severity caused by mutations
in the skeletal muscle alpha-actin (ACTA1) gene. In addition, it shows the
diversity of pathological features that can occur in congenital myopathies due
to mutations in the same gene. Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle
weakness and the presence of nemaline rods. Five genes have now been associated
with nemaline myopathy: alpha-tropomyosin-3 (TPM3), alpha-actin (ACTA1), nebulin
(NEB), beta-tropomysin (TPM2) and troponin T (TNNT1). In addition, mutations in
the ryanodine receptor gene (RYR1) have been associated with core-rod myopathy.
Here we report linkage in two unrelated families, with a variant of nemaline
myopathy, with associated core-like lesions. The clinical phenotype consists of
muscle weakness in addition to a peculiar kind of muscle slowness. A genome-wide
scan revealed a locus for nemaline myopathy with core-like lesions on chromosome
15q21-q23 for both families. Combining the two families gave a two-point LOD
score of 10.65 for D15S993. The alpha-tropomyosin-1 gene (TPM1) located within
this region is the strongest candidate gene. However, no mutations were found in
the protein-coding region of TPM1, although small deletions or mutations in an
intron cannot be excluded. The critical region contains few other candidate
genes coding for muscle proteins and several genes of unknown function, and has
not yet been sequenced completely. The novel phenotype of nemaline myopathy in
the two presented families corresponds to an also novel, as yet uncharacterized,
genotype. Congenital myopathies are clinical and genetic heterogeneous disorders
characterized by skeletal muscle weakness ranging in severity. Three major forms
have been identified: actin myopathy, intranuclear rod myopathy, and nemaline
myopathy. Nemaline myopathy is the most common of these myopathies and is
further subdivided into seven groups according to severity, progressiveness, and
age of onset. At present, five genes have been linked to congenital myopathies.
These include alpha-actin (ACTA1), alpha- and beta-tropomyosin (TPM3 and TPM2),
troponin T (TNNT1), and nebulin (NEB). Their protein products are all components
of the thin filament of the sarcomere. The mutations identified within these
genes have varying impacts on protein structure and give rise to different forms
of congenital myopathies. Greater understanding of muscle formation and cause of
disease can be established through the study of the effect of mutations on the
functional proteins. However, a major limitation in the understanding of
congenital myopathies is the lack of correlation between the degree of
sarcomeric disruption and disease severity. Consequently, great difficulty may
be encountered when diagnosing patients and predicting the progression of the
disorders. There are no existing cures for congenital myopathies, although
improvements can be made to both the standard of living and the life expectancy
of the patient through various therapies. Most nemaline myopathy patients have mutations in the nebulin (NEB) or skeletal
muscle alpha-actin (ACTA1) genes. Here we report for the first time three
patients with severe nemaline myopathy and mutations of the ACTA1 stop codon:
TAG>TAT (tyrosine), TAG>CAG (glutamine) and TAG>TGG (tryptophan). All three
mutations will cause inclusion of an additional 47 amino acids, translated from
the 3' UTR of the gene, into the mature actin protein. Western blotting of one
patient's muscle demonstrated the presence of the larger protein, while
expression of one of the other mutant proteins fused to EGFP in C2C12 cells
demonstrated the formation of rod bodies. Nemaline myopathy (NM) is a genetically and clinically heterogenous muscle
disorder, which is myopathologically characterized by nemaline bodies. Mutations
in six genes have been reported to cause NM: Nebulin (NEB Pelin 1999),
alpha-skeletal muscle actin (ACTA1 Nowak 1999), alpha-slow tropomyosin (TPM3
Laing 1995), beta-tropomyosin (TPM2 Donner 2002), slow troponin T (TNNT1
Johnston 2000) and cofilin 2 (CFL2 Agrawal 2007). The majority of cases are due
to mutation in NEB and ACTA1. We report on the clinical, myopathological and
muscle MRI findings in a German family with autosomal domit NM due to a novel
pathogenic TPM3 mutation (p.Ala156Thr). Nemaline myopathy (NM) is the most common congenital myopathy and is caused by
mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin),
TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases
carry ACTA1 defects and these particular mutations usually induce substitutions
of single residues in the actin protein. Despite increasing clinical and
scientific interest, the contractile consequences of these subtle amino acid
substitutions remain obscure. To decipher them, in the present study, we
originally recorded and analysed the mechanics as well as the X-ray diffraction
patterns of human membrane-permeabilized single muscle fibres with a particular
peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an
unexpected cascade of molecular and cellular events. During contraction,
p.Phe352Ser greatly enhances the strain of individual cross-bridges.
Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by
altering the rate of myosin head attachment to actin monomers. Overall, at the
cell level, these divergent mechanisms conduct to an improved steady-state force
production. Such results provide new surprising scientific insights and crucial
information for future therapeutic strategies. |
What are the pregnancy outcomes in rheumatoid arthritis? | There is increased obstetrical and neonatal morbidity. Women with RA had an increased risk of LBW, SGA babies, preeclampsia and CS compared with unaffected women.
Women with RA appear to have a higher age-adjusted risk of adverse outcomes of pregnancy and longer hospital stays than do pregnant women in the general population, and careful antenatal monitoring should be performed.Patients with rheumatic disease can have successful pregnancy outcomes, particularly when a collaborative approach between the rheumatologist and obstetrician is applied.
In general, active inflammation from rheumatic diseases poses a stronger threat to the well-being of both mother and foetus than many immunosuppressant medications. Therefore, continued immunosuppression with the least risky medications will allow for the most optimal pregnancy outcomes. | OBJECTIVE: To determine whether rheumatoid arthritis (RA) is associated with
increased adverse obstetric or neonatal outcomes.
STUDY DESIGN AND SETTING: Washington State birth records and hospital discharge
data between 1987 and 2001 identified a cohort of women with rheumatoid
arthritis and a comparison group of women without rheumatoid arthritis.
Pregcy and neonatal outcomes were compared using general linear models for
common outcomes, calculating approximate relative risks and 95% confidence
intervals.
RESULTS: There were 243 women with rheumatoid arthritis and 2,559 controls.
Infants of women with rheumatoid arthritis had increased risk of cesarean
delivery (adjusted approximate relative risk, aRR=1.66, 95% CI (1.22, 2.26)),
prematurity (aRR=1.78, 95% CI (1.21, 2.60)), and longer birth hospitalization
(aRR=1.86, 95% CI (1.32, 2.60)) compared to those born to women without
rheumatoid arthritis.
CONCLUSIONS: We speculate that the increased risks for cesarean delivery,
prematurity, and longer hospitalization at birth among infants born to women
with rheumatoid arthritis may be due to the pathophysiologic changes associated
with rheumatoid arthritis or medications used to treat the disease. OBJECTIVE: Using a 3-year nationwide population-based database (2001-3), this
study aims to examine the relationship between rheumatoid arthritis (RA) and
adverse pregcy outcomes.
METHODS: The study used the Taiwan National Health Insurance Research Dataset
and birth certificate registry. In total, 1912 mothers with RA and 9560 matched
comparison mothers were included. Separate conditional logistic regression
analyses were carried out to explore the risk of low birthweight (LBW), preterm
births, small for gestational age (SGA) infants, preeclampsia and delivery mode
(vaginal vs caesarean section (CS)) for the study and comparison groups.
RESULTS: Regression analyses showed that the adjusted odds of LBW, SGA infants,
preeclampsia and CS for women with RA were 1.47 (95% CI 1.22 to 1.78), 1.20 (95%
CI 1.05 to 1.38), 2.22 (95% CI 1.59 to 3.11) and 1.19 (95% CI 1.07 to 1.31)
times, respectively, that of comparison mothers.
CONCLUSION: Women with RA had an increased risk of LBW, SGA babies, preeclampsia
and CS compared with unaffected women. Systemic lupus erythematosus (SLE) can affect the menstruation, fertility, and
pregcy outcomes of the affected subjects. There is very little data on this
aspect of the disease in Indian patients. Our aim was to study the menstrual,
fertility, and pregcy outcomes in these patients in comparison with patients
of rheumatoid arthritis (RA) and also to study the effect of cyclophosphamide
therapy on menstrual cycles in patients with SLE. Four hundred and twenty
patients of SLE (210) and RA (210) were interviewed using a standard
questionnaire and available medical records used. After disease-onset, the
chances of adverse pregcy outcomes were significantly more in patients with
SLE compared to RA [OR = 5.17 (2.13-12.52); p ≤ 0.001]. Compared to the National
average in India, the average number of living children is lesser in patients
with RA (2.39 ± 1.39, p = 0.002), but more so in patients with SLE (1.44 ± 1.35,
p = 0.001). A younger age at diagnosis and cyclophosphamide therapy was found to
be independently associated with menstrual irregularities after disease-onset.
We conclude that pregcy outcome in patients with SLE in India is worse in
comparison to patients with RA. Average family size of patients with SLE and RA
is less when compared to National average in India. Patients with SLE are more
prone for menstrual irregularities, especially those who receive
cyclophosphamide treatment. OBJECTIVE: In preclinical reproductive studies, leflunomide was found to be
embryotoxic and teratogenic. Women treated with leflunomide are advised to avoid
pregcy; those who become pregt are advised to reduce fetal exposure
through a cholestyramine drug elimination procedure. The present study was
undertaken to investigate pregcy outcomes in women who received leflunomide
and were treated with cholestyramine during pregcy.
METHODS: Sixty-four pregt women with rheumatoid arthritis (RA) who were
treated with leflunomide during pregcy (95.3% of whom received
cholestyramine), 108 pregt women with RA not treated with leflunomide, and 78
healthy pregt women were enrolled in a prospective cohort study between 1999
and 2009. Information was collected via interview of the mothers, review of
medical records, and specialized physical examination of infants.
RESULTS: There were no significant differences in the overall rate of major
structural defects in the exposed group (3 of 56 live births [5.4%]) relative to
either comparison group (each 4.2%)(P = 0.13). The rate was similar to the 3-4%
expected in the general population. There was no specific pattern of major or
minor anomalies. Infants in both the leflunomide-exposed and
non-leflunomide-exposed RA groups were born smaller and earlier relative to
infants of healthy mothers; however, after adjustment for confounding factors,
there were no significant differences between the leflunomide-exposed and
non-leflunomide-exposed RA groups.
CONCLUSION: Although the sample size is small, these data do not support the
notion that there is a substantial increased risk of adverse pregcy outcomes
due to leflunomide exposure among women who undergo cholestyramine elimination
procedure early in pregcy. These findings can provide some reassurance to
women who inadvertently become pregt while taking leflunomide and undergo the
washout procedure. Pregcy can create a challenge for physicians caring for women with rheumatic
diseases. For many women with rheumatoid arthritis (RA), pregcy can provide a
reprieve from long-term joint pain and inflammation, but others will not
experience remission and will continue to need medication. Systemic lupus
erythematosus (SLE) may remain quiet in some women, but in others may become
more aggressive during pregcy, putting both mother and foetus at risk. Women
with limited scleroderma can do remarkably well, but scleroderma renal crises
can be difficult to manage. A third of pregcies in women with
antiphospholipid syndrome (APS) may be refractory to our best therapy. In
general, active inflammation from rheumatic diseases poses a stronger threat to
the well-being of both mother and foetus than many immunosuppressant
medications. Therefore, continued immunosuppression with the least risky
medications will allow for the most optimal pregcy outcomes. Most pregcies in women with rheumatologic disease will result in the delivery
of a healthy baby. Pregcy can be particularly risky in women with active
disease or on teratogenic medications, making contraception an important issue
for these women. All women with rheumatologic disease have contraceptive
options, including barrier methods, the intra-uterine device and
progesterone-only medications. Active inflammatory disease, whether in the form
of lupus, systemic vasculitis or myositis, places the pregcy at increased
risk. Pre-eclampsia is a particular risk for women with lupus or
antiphospholipid syndrome and may be decreased by daily low-dose aspirin.
Rheumatoid arthritis typically improves and does not have a major impact on
pregcy outcomes. The expected post-partum arthritis flare may be avoided by
restarting medications soon after delivery. Judicious use of medication and
close observation may be the keys to successful pregcy in women with
rheumatologic disease. Objective. To describe obstetrical and neonatal outcomes in Canadian women with
rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Methods. An
administrative database of hospitalizations for neonatal delivery (1998-2009)
from Calgary, Alberta was searched to identify women with RA (38 pregcies) or
SLE (95 pregcies), and women from the general population matched on maternal
age and year of delivery (150 and 375 pregcies, resp.). Conditional logistic
regression was used to calculate odds ratios (OR) for maternal and neonatal
outcomes, adjusting for parity. Results. Women with SLE had increased odds for
preeclampsia or eclampsia (SLE OR 2.16 (95% CI 1.10-4.21; P = 0.024); RA OR 2.33
(95% CI 0.76-7.14; P = 0.138)). Women with SLE had increased odds for cesarean
section after adjustment for dysfunctional labour, instrumentation and previous
cesarean section (OR 3.47 (95% CI 1.67-7.22; P < 0.001)). Neonates born to women
with SLE had increased odds of prematurity (SLE OR 6.17 (95% CI 3.28-11.58; P <
0.001); RA OR 2.66 (95% CI 0.90-7.84; P = 0.076)) and of SGA (SLE OR 2.54 (95%
CI 1.42-4.55; P = 0.002); RA OR 2.18 (95% CI 0.84-5.66; P = 0.108)) after
adjusting for maternal hypertension. There was no excess risk of congenital
defects in neonates. Conclusions. There is increased obstetrical and neonatal
morbidity in Canadian women with RA or SLE. OBJECTIVE: to compare the obstetric prognosis before and after the onset of
rheumatoid arthritis (RA).
METHODS: a survey in RA women patients who become sexually active before disease
onset, and who tried or got pregt after diagnosis was performed. Obstetrical
features such as: number of pregcies, threat of abortion, abortion, early
natal death, congenital abnormalities and preeclampsia-eclampsia syndrome,
before and after RA onset were compared.
RESULTS: only 47 women were eligible (age: 40.5 ± 11.0 years; time of evolution:
15.9 ± 11.5 years; positive rheumatoid factor: 93.6 %) from 700 screened. After
RA onset there were 63 gestations in 36 patients who got pregt (secondary
infertility rate: 21.3 %). A significant increase in frequency and number of
cesarean deliveries, besides a higher number of pregcies with preeclampsia,
were found after RA onset. Additionally, four newborns with congenital anomalies
were reported after the disease onset compared to none before RA onset.
CONCLUSIONS: compared to pre-RA obstetric events, a higher frequency and number
of adverse outcomes was found in pregcies that occurred after RA onset. OBJECTIVE: To describe the outcomes of pregcies complicated by rheumatoid
arthritis (RA) and to estimate potential associations between disease
characteristics and pregcy outcomes.
STUDY DESIGN: We reviewed all pregcies complicated by RA delivered at our
institution from June 2001 through June 2009. Fisher exact tests were used to
calculate odds ratios. Univariable regression was performed using STATA 10.1
(StataCorp, College Station, TX). A p value of ≤ 0.05 was considered
statistically significant.
RESULTS: Forty-six pregcies in 40 women were reviewed. Sixty percent of
pregcies had evidence of disease flare and 28% delivered prior to 37 weeks.
We did not identify associations between preterm birth and active disease at
conception or during pregcy. In univariate analysis, discontinuation of
medication because of pregcy was associated with a significantly earlier
gestational age at delivery (362/7 versus 383/7 weeks, p = 0.022).
CONCLUSION: Women with RA may be at higher risk for preterm delivery. OBJECTIVE: The aim of this study is to describe pregcy outcomes among women
with JIA.
METHODS: Women who gave birth in New South Wales (NSW), Australia, were linked
to hospital discharge records from 2000 to 2010. Women with an ICD-10-AM code of
M08 or M09 in the hospital records were considered to have JIA. Logistic
regression was used to calculate odds ratios for pregcy outcomes and the lack
of independence in study outcomes for multiple pregcies in the same woman was
taken into account using generalized estimating equations.
RESULTS: During the study period, 601,659 women had 941,496 births. Of these
births, 78 births could be attributed to 50 women with JIA. Of 78 JIA
pregcies, 53 (68%) were delivered by either Caesarean section (n = 40, 51%)
or instrumental delivery (n = 13, 17%); compared with other women, those with
JIA had significantly higher rates of pre-eclampsia, postpartum haemorrhage and
severe maternal morbidity. Compared with other infants, those with mothers with
JIA were more likely to be born prematurely, but were not at increased risk of
being small for gestational age, requiring neonatal intensive care, having a low
Apgar score at 5 min or severe neonatal morbidity.
CONCLUSION: Infants of women with JIA did not have an increased risk of adverse
neonatal outcomes. Intensive obstetric care might be required during pregcy
for women with JIA given the increased risk of maternal morbidity. Women of childbearing age are at risk for developing systemic rheumatic
diseases. Pregcy can be challenging to manage in patients with rheumatic
diseases for a variety of reasons including the impact of physiological and
immunological changes of pregcy on underlying disease activity, the varied
presentation of rheumatic disease during pregcy, and the limited treatment
options. Previously, patients with rheumatic disease were often advised against
pregcy due to concerns of increased maternal and fetal morbidity and
mortality. However, recent advancements in the understanding of the interaction
between pregcy and rheumatic disease have changed how we counsel patients.
Patients with rheumatic disease can have successful pregcy outcomes,
particularly when a collaborative approach between the rheumatologist and
obstetrician is applied. This review aims to discuss the effect of pregcy on
patients with the most common rheumatic diseases, the effect of these diseases
on the pregcy itself, and the management of these patients during pregcy. |
What is DNAshape? | DNAshape is a method and web server for predicting DNA structural features in a high-throughput (HT) manner for massive sequence data. This approach provides the framework for the integration of DNA sequence and shape analyses in genome-wide studies. The HT methodology uses a sliding-window approach to mine DNA structural information obtained from Monte Carlo simulations. It requires only nucleotide sequence as input and instantly predicts multiple structural features of DNA (minor groove width, roll, propeller twist and helix twist). The results of rigorous validations of the HT predictions based on DNA structures solved by X-ray crystallography and NMR spectroscopy, hydroxyl radical cleavage data, statistical analysis and cross-validation, and molecular dynamics simulations provide strong confidence in this approach. The DNAshape web server is freely available at http://rohslab.cmb.usc.edu/DNAshape/. | |
How are thyroid hormones involved in the development of diabetic cardiomyopathy? | The diabetic state is associated with lowered T3 and T4 levels. Thyroid hormone treatment in diabetic cardiomyopathy may partially reverse cardiac dysfunction | The isolated working heart preparation was used to investigate the effect of
continuous triiodothyronine (T3) administration on cardiac function and
metabolism of rats rendered diabetic for a period of 4 wk with streptozocin
(STZ). T3 controlled-release pellets were implanted 1 wk after STZ (70 mg/kg)
injection. Rats injected with citrate buffer without STZ received T3 pellets 1
and/or 2 wk later. A comparable number of rats received placebo pellets.
Untreated diabetic rats exhibited a decrease in spontaneous heart rate and
myocardial cytochrome c concentrations concurrent with depressed plasma T3
values compared with untreated controls. T3 treatment did not improve in vitro
cardiac performance (assessed as cardiac output times peak systolic pressure per
gram dry heart weight) in hearts from diabetic rats perfused with glucose alone.
Addition of octanoate reversed this depression and improved cardiac function to
a greater extent in treated than in untreated diabetic animals. However, these
differences between treated and untreated diabetic animals disappeared when
heart rate was controlled by cardiac pacing. Furthermore, T3 treatment of
controls and diabetics did not alter the oxidation of octanoate or the cardiac
responsiveness to isoproterenol. These results suggest that experimental
diabetic cardiomyopathy is partly attributable to a substrate deficiency and is
not due entirely to hypothyroidism. Diabetes results in myocardial functional alterations which are accompanied by a
depression of biochemical parameters such as myosin ATPase and calcium uptake in
the sarcoplasmic reticulum. Methyl palmoxirate, a fatty acid analog, is reported
to decrease circulating glucose levels by inhibiting fatty acid metabolism, thus
forcing carbohydrate utilization. In the present study, we attempted to prevent
streptozotocin diabetes-induced myocardial alterations in the rat. Using the
isolated working heart preparation, we observed a depression of myocardial
function in rats 6 weeks after the induction of diabetes, which was
characterized by the inability of these hearts to develop left ventricular
pressures and rates of ventricular contraction and relaxation as well as control
hearts at higher left atrial filling pressures. Methyl palmoxirate treatment (25
mg kg-1 day-1 po daily) was unable to control diabetes-induced changes in plasma
glucose, triglycerides, insulin, and total lipids. Also, the functional
depression seen in diabetic rat hearts was present despite the treatment.
However, depression of calcium uptake and elevation of long chain acyl
carnitines seen in sarcoplasmic reticulum (SR) prepared from diabetic rat hearts
could be prevented by the treatment. As triiodothyronine (T3) treatment has been
shown to normalize depression of cardiac myosin ATPase in diabetic rats, we
repeated the study using a combination of T3 (30 micrograms kg-1 day-1 sc daily)
and methyl palmoxirate. While diabetic rats treated with T3 alone did not show
significant improvement of myocardial function when compared with untreated
diabetics, the function of those treated with both T3 and methyl palmoxirate was
not significantly different from that in control rat hearts.(ABSTRACT TRUNCATED
AT 250 WORDS) Numerous experimental studies have implied a link between diabetes-induced
abnormal lipid buildup in the myocardium and the development of cardiomyopathy.
Because the diabetic state in rats is associated with lowered T3
(triiodothyronine) and T4 levels and because diabetic patients excrete large
amounts of myo-inositol, a lipotropic agent, we investigated the effects of
myo-inositol and T3 on the elevated myocardial lipid levels and depressed
cardiac performance of streptozocin (55 mg/kg i.v.)-induced diabetic (STZ-D)
rats. myo-inositol (2.5 g.kg-1.day-1 in the drinking water) and T3 (30
micrograms.kg-1.day-1 s.c.) were given for an 8-wk period 3 days after diabetes
induction. Untreated diabetic rats were characterized by a decreased rate of
body weight gain, hyperglycemia, and hypoinsulinemia, which were not altered
after myo-inositol and/or T3 treatment. Thyroid status of diabetic animals was
normalized by T3 alone or in combination with myo-inositol but not by
myo-inositol alone. The elevations in plasma and myocardial lipids associated
with the diabetic state were prevented by myo-inositol treatment. However, the
plasma lipid and myocardial cholesterol levels in diabetic rats remained
elevated or were further increased with treatment with T3 or myo-inositol plus
T3. myo-inositol treatment partially improved cardiac performance in STZ-D rats.
T3 treatment alone did not prevent cardiac dysfunction in diabetic rats. There
was, however, some improvement in heart function in the groups treated with both
myo-inositol and T3, coinciding with a significant decrease in the myocardial
triacylglycerol level. The data indicate that a possible correlation may exist
between elevated myocardial triacylglycerol levels and cardiac dysfunction in
diabetic rats. In order to elucidate further the abnormal myocardial Ca2+ metabolism in
diabetes mellitus, voltage-gated Ca2+ channels and beta-receptors were
quantified in myocardial membranes of short- and long-term diabetic rats.
Diabetes was induced by an injection of streptozotocin (STZ). Animals were
killed 2, 4, 7, 90 and 200 days after STZ. A group of diabetic animals were
treated with insulin for 20 days following 180 days of untreated diabetes.
Diabetic animals developed low triiodothyronine syndrome. During short-term
diabetes, the maximum binding capacity (MBC) for Ca2+ channels was reduced by
25% at day 4 (p < 0.05) and the beta-receptor MBC was reduced by 48% (p < 0.05).
A normalizing tendency was observed at day 7 for both receptor types;
insulin-treated rats did not differ from controls at that time. After 90 and 200
days of untreated diabetes the Ca2+ channel MBC had increased by 36% and 27%,
respectively (p < 0.05). Twenty days of strictly regulated blood glucose
following 180 days of untreated diabetes totally normalized the Ca2+ channel
MBC. This is in contrast to a previous report where insulin treatment did not
normalize the Ca2+ channel MBC. Total beta-receptor MBCs did not differ from
control values 90 and 200 days after STZ. In conclusion, an increase in rat
myocardial Ca2+ channel MBC during long-term diabetes was fully normalized by
short-term insulin treatment. The increase in sarcolemmal Ca2+ channels could
serve to compensate for a defect coupling of the beta-receptor to adenylate
cyclase. An elevated Ca2+ channel number may, at least theoretically, lead to
increased Ca2+ flow across the cardiac sarcolemma and in this way contribute to
the diabetic cardiomyopathy by increasing the intracellular Ca2+ concentration. |
By which methods can we evaluate the reliability of a phylogenetic tree? | The methods for assessing the robustness/reliability of the topology of the inferred phylogenetic trees are: the widely used bootstrap method and the jackknife method. | Comparisons of pol gene nucleotide and reverse transcriptase (RT) amino acid
sequences of 47 retroviruses, 3 caulimoviruses, and 5 hepadnaviruses showed that
approximately one-third of the gene at the 5' end is much more conserved than
other pol regions. The most conserved regions on both the nucleotide and amino
acid sequences were chosen for construction of phylogenetic trees. The
maximum-parsimony and distance-matrix methods were used for analyses of aligned
amino acid sequences; these two methods, and the compatibility method, were used
to analyze the aligned nucleotide sequences. Essentially identical majority-rule
consensus trees were produced by these different methods from both the pol gene
nucleotide and RT amino acid sequences, which divided the 55 retroelements into
six major groups. The reliability of the phylogenetic trees was probed with the
bootstrapping of 100 replicates of the original sequence alignments. The
grouping results were shown to be statistically significant by multiple
comparisons with the least-significant-difference procedure. The Win95/98/NT program FreeTree for computation of distance matrices and
construction of phylogenetic or phenetic trees on the basis of random amplified
polymorphic DNA (RAPD), RFLP and allozyme data is presented. In contrast to
other similar software, the program FreeTree (available at
http://www.natur.cuni.cz/~flegr/programs/freetree or
http://ijs.sgmjournals.org/content/vol51/issue3/) can also assess the robustness
of the tree topology by bootstrap, jackknife or operational taxonomic
unit-jackknife analysis. Moreover, the program can be also used for the analysis
of data obtained in several independent experiments performed with non-identical
subsets of taxa. The function of the program was demonstrated by an analysis of
RAPD data from 42 strains of 10 species of trichomonads. On the phylogenetic
tree constructed using FreeTree, the high bootstrap values and short terminal
branches for the Tritrichomonas foetus/suis 14-strain branch suggested
relatively recent and probably clonal radiation of this species. At the same
time, the relatively lower bootstrap values and long terminal branches for the
Trichomonas vaginalis 20-strain branch suggested more ancient radiation of this
species and the possible existence of genetic recombination (sexual
reproduction) in this human pathogen. The low bootstrap values and the star-like
topology of the whole Trichomonadidae tree confirm that the RAPD method is not
suitable for phylogenetic analysis of protozoa at the level of higher taxa. It
is proposed that the repeated bootstrap analysis should be an obligatory part of
any RAPD study. It makes it possible to assess the reliability of the tree
obtained and to adjust the amount of collected data (the number of random
primers) to the amount of phylogenetic signals in the RAPD data of the taxon
analysed. The FreeTree program makes such analysis possible. Assessment of the reliability of a given phylogenetic hypothesis is an important
step in phylogenetic analysis. Historically, the nonparametric bootstrap
procedure has been the most frequently used method for assessing the support for
specific phylogenetic relationships. The recent employment of Bayesian methods
for phylogenetic inference problems has resulted in clade support being
expressed in terms of posterior probabilities. We used simulated data and the
four-taxon case to explore the relationship between nonparametric bootstrap
values (as inferred by maximum likelihood) and posterior probabilities (as
inferred by Bayesian analysis). The results suggest a complex association
between the two measures. Three general regions of tree space can be identified:
(1) the neutral zone, where differences between mean bootstrap and mean
posterior probability values are not significant, (2) near the two-branch
corner, and (3) deep in the two-branch corner. In the last two regions,
significant differences occur between mean bootstrap and mean posterior
probability values. Whether bootstrap or posterior probability values are higher
depends on the data in support of alternative topologies. Examination of star
topologies revealed that both bootstrap and posterior probability values differ
significantly from theoretical expectations; in particular, there are more
posterior probability values in the range 0.85-1 than expected by theory.
Therefore, our results corroborate the findings of others that posterior
probability values are excessively high. Our results also suggest that
extrapolations from single topology branch-length studies are unlikely to
provide any general conclusions regarding the relationship between bootstrap and
posterior probability values. Functional evolution is often driven by positive natural selection. Although it
is thought to be rare in evolution at the molecular level, its effects may be
observed as the accelerated evolutionary rates. Therefore one of the effective
ways to identify functional evolution is to identify accelerated evolution. Many
methods have been developed to test the statistical significance of the
accelerated evolutionary rate by comparison with the appropriate reference rate.
The rates of synonymous substitution are one of the most useful and popular
references, especially for large-scale analyses. On the other hand, these rates
are applicable only to a limited evolutionary time period because they saturate
quickly--i.e., multiple substitutions happen frequently because of the lower
functional constraint. The relative rate test is an alternative method. This
technique has an advantage in terms of the saturation effect but is not
sufficiently powerful when the evolutionary rate differs considerably among
phylogenetic lineages. For the aim to provide a universal reference tree, we
propose a method to construct a standardized tree which serves as the reference
for accelerated evolutionary rate. The method is based upon multiple molecular
phylogenies of single genes with the aim of providing higher reliability. The
tree has averaged and normalized branch lengths with standard deviations for
statistical neutrality limits. The standard deviation also suggests the
reliability level of the branch order. The resulting tree serves as a reference
tree for the reliability level of the branch order and the test of evolutionary
rate acceleration even when some of the species lineages show an accelerated
evolutionary rate for most of their genes due to bottlenecking and other
effects. Many methods exist for reconstructing phylogenies from molecular sequence data,
but few phylogenies are known and can be used to check their efficacy.
Simulation remains the most important approach to testing the accuracy and
robustness of phylogenetic inference methods. However, current simulation
programs are limited, especially concerning realistic models for simulating
insertions and deletions. We implement a portable and flexible application,
named INDELible, for generating nucleotide, amino acid and codon sequence data
by simulating insertions and deletions (indels) as well as substitutions. Indels
are simulated under several models of indel-length distribution. The program
implements a rich repertoire of substitution models, including the general
unrestricted model and nonstationary nonhomogeneous models of nucleotide
substitution, mixture, and partition models that account for heterogeneity among
sites, and codon models that allow the nonsynonymous/synonymous substitution
rate ratio to vary among sites and branches. With its many unique features,
INDELible should be useful for evaluating the performance of many inference
methods, including those for multiple sequence alignment, phylogenetic tree
inference, and ancestral sequence, or genome reconstruction. Supermatrix and supertree are two methods for constructing a phylogenetic tree
by using multiple data sets. However, these methods are not a panacea, as
conflicting signals between data sets can lead to misinterpret the evolutionary
history of taxa. In particular, the supermatrix approach is expected to be
misleading if the species-tree signal is not domit after the combination of
the data sets. Moreover, most current supertree methods suffer from two
limitations: (i) they ignore or misinterpret secondary (non-domit)
phylogenetic signals of the different data sets; and (ii) the logical basis of
node robustness measures is unclear. To overcome these limitations, we propose a
new approach, called SuperTRI, which is based on the branch support analyses of
the independent data sets, and where the reliability of the nodes is assessed
using three measures: the supertree Bootstrap percentage and two other values
calculated from the separate analyses: the mean branch support (mean Bootstrap
percentage or mean posterior probability) and the reproducibility index. The
SuperTRI approach is tested on a data matrix including seven genes for 82 taxa
of the family Bovidae (Mammalia, Rumitia), and the results are compared to
those found with the supermatrix approach. The phylogenetic analyses of the
supermatrix and independent data sets were done using four methods of tree
reconstruction: Bayesian inference, maximum likelihood, and unweighted and
weighted maximum parsimony. The results indicate, firstly, that the SuperTRI
approach shows less sensitivity to the four phylogenetic methods, secondly, that
it is more accurate to interpret the relationships among taxa, and thirdly, that
interesting conclusions on introgression and radiation can be drawn from the
comparisons between SuperTRI and supermatrix analyses. Multiple sequence alignment (MSA) is the basis for a wide range of comparative
sequence analyses from molecular phylogenetics to 3D structure prediction.
Sophisticated algorithms have been developed for sequence alignment, but in
practice, many errors can be expected and extensive portions of the MSA are
unreliable. Hence, it is imperative to understand and characterize the various
sources of errors in MSAs and to quantify site-specific alignment confidence. In
this paper, we show that uncertainties in the guide tree used by progressive
alignment methods are a major source of alignment uncertainty. We use this
insight to develop a novel method for quantifying the robustness of each
alignment column to guide tree uncertainty. We build on the widely used
bootstrap method for perturbing the phylogenetic tree. Specifically, we generate
a collection of trees and use each as a guide tree in the alignment algorithm,
thus producing a set of MSAs. We next test the consistency of every column of
the MSA obtained from the unperturbed guide tree with respect to the set of
MSAs. We name this measure the "GUIDe tree based AligNment ConfidencE"
(GUIDANCE) score. Using the Benchmark Alignment data BASE benchmark as well as
simulation studies, we show that GUIDANCE scores accurately identify errors in
MSAs. Additionally, we compare our results with the previously published
Heads-or-Tails score and show that the GUIDANCE score is a better predictor of
unreliably aligned regions. The rapid accumulation of whole-genome data has renewed interest in the study of
genomic rearrangements. Comparative genomics, evolutionary biology, and cancer
research all require models and algorithms to elucidate the mechanisms, history,
and consequences of these rearrangements. However, even simple models lead to
NP-hard problems, particularly in the area of phylogenetic analysis. Current
approaches are limited to small collections of genomes and low-resolution data
(typically a few hundred syntenic blocks). Moreover, whereas phylogenetic
analyses from sequence data are deemed incomplete unless bootstrapping scores (a
measure of confidence) are given for each tree edge, no equivalent to
bootstrapping exists for rearrangement-based phylogenetic analysis. We describe
a fast and accurate algorithm for rearrangement analysis that scales up, in both
time and accuracy, to modern high-resolution genomic data. We also describe a
novel approach to estimate the robustness of results-an equivalent to the
bootstrapping analysis used in sequence-based phylogenetic reconstruction. We
present the results of extensive testing on both simulated and real data showing
that our algorithm returns very accurate results, while scaling linearly with
the size of the genomes and cubically with their number. We also present
extensive experimental results showing that our approach to robustness testing
provides excellent estimates of confidence, which, moreover, can be tuned to
trade off thresholds between false positives and false negatives. Together,
these two novel approaches enable us to attack heretofore intractable problems,
such as phylogenetic inference for high-resolution vertebrate genomes, as we
demonstrate on a set of six vertebrate genomes with 8,380 syntenic blocks. A
copy of the software is available on demand. Among the criteria to evaluate the performance of a phylogenetic method,
robustness to model violation is of particular practical importance as complete
a priori knowledge of evolutionary processes is typically unavailable. For
studies of robustness in phylogenetic inference, a utility to add well-defined
model violations to the simulated data would be helpful. We therefore introduce
ImOSM, a tool to imbed intermittent evolution as model violation into an
alignment. Intermittent evolution refers to extra substitutions occurring
randomly on branches of a tree, thus changing alignment site patterns. This
means that the extra substitutions are placed on the tree after the typical
process of sequence evolution is completed. We then study the robustness of
widely used phylogenetic methods: maximum likelihood (ML), maximum parsimony
(MP), and a distance-based method (BIONJ) to various scenarios of model
violation. Violation of rates across sites (RaS) heterogeneity and simultaneous
violation of RaS and the transition/transversion ratio on two nonadjacent
external branches hinder all the methods recovery of the true topology for a
four-taxon tree. For an eight-taxon balanced tree, the violations cause each of
the three methods to infer a different topology. Both ML and MP fail, whereas
BIONJ, which calculates the distances based on the ML estimated parameters,
reconstructs the true tree. Finally, we report that a test of model homogeneity
and goodness of fit tests have enough power to detect such model violations. The
outcome of the tests can help to actually gain confidence in the inferred trees.
Therefore, we recommend using these tests in practical phylogenetic analyses. Boolean analysis (or BOOL-AN; Jakó et al., 2009. BOOL-AN: A method for
comparative sequence analysis and phylogenetic reconstruction. Mol. Phylogenet.
Evol. 52, 887-97.), a recently developed method for sequence comparison uses the
Iterative Canonical Form of Boolean functions. It considers sequence information
in a way entirely different from standard phylogenetic methods (i.e. Maximum
Parsimony, Maximum-Likelihood, Neighbor-Joining, and Bayesian analysis). The
performance and reliability of Boolean analysis were tested and compared with
the standard phylogenetic methods, using artificially evolved - simulated -
nucleotide sequences and the 22 mitochondrial tRNA genes of the great apes. At
the outset, we assumed that the phylogeny of Hominidae is generally well
established, and the guide tree of artificial sequence evolution can also be
used as a benchmark. These offer a possibility to compare and test the
performance of different phylogenetic methods. Trees were reconstructed by each
method from 2500 simulated sequences and 22 mitochondrial tRNA sequences. We
also introduced a special re-sampling method for Boolean analysis on permuted
sequence sites, the P-BOOL-AN procedure. Considering the reliability values
(branch support values of consensus trees and Robinson-Foulds distances) we used
for simulated sequence trees produced by different phylogenetic methods, BOOL-AN
appeared as the most reliable method. Although the mitochondrial tRNA sequences
of great apes are relatively short (59-75 bases long) and the ratio of their
constant characters is about 75%, BOOL-AN, P-BOOL-AN and the Bayesian approach
produced the same tree-topology as the established phylogeny, while the outcomes
of Maximum Parsimony, Maximum-Likelihood and Neighbor-Joining methods were
equivocal. We conclude that Boolean analysis is a promising alternative to
existing methods of sequence comparison for phylogenetic reconstruction and
congruence analysis. In this paper, we propose a method to classify prokaryotic genomes using the
agglomerative information bottleneck method for unsupervised clustering.
Although the method we present here is closely related to a group of methods
based on detecting the presence or absence of genes, our method is different
because it uses gene lengths as well. We show that this amended method is
reliable. For robustness evaluation, we apply bootstrap and jackknife techniques
to input data. As a result, we are able to propose an approach to determine the
stability level of a cladogram. We demonstrate that the genome tree produced for
a selected small group of genomes looks a lot like a phylogenetic tree of this
group. |
Which histone modification is primarily linked to elongating transcription? | Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Recent studies reviewed here demonstrate that histone deacetylation on the body of a transcribed gene is regulated via Set2-mediated methylation of histone H3-K36. | Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol
II elongation machinery to a highly localized domain at the 5' portion of active
mRNA coding regions. Set1 association depends upon the TFIIH-associated kinase
that phosphorylates the Pol II C-terminal domain (CTD) and mediates the
transition between initiation and elongation, and Set1 interacts with the form
of Pol II whose CTD is phosphorylated at serine 5 but not serine 2. The Rtf1 and
Paf1 components of the Pol II-associated Paf1 complex are also important for
Set1 recruitment. Although the level of dimethylated H3-K4 is fairly uniform
throughout the genome, the pattern of trimethylated H3-K4 strongly correlates
with Set1 occupancy. Hypermethylated H3-K4 within the mRNA coding region
persists for considerable time after transcriptional inactivation and Set1
dissociation from the chromatin, indicating that H3-K4 hypermethylation provides
a molecular memory of recent transcriptional activity. Histone acetylation has been shown to be required for the proper regulation of
many cellular processes including transcription, DNA repair, and chromatin
assembly. Acetylation of histone H3 on lysine 56 (H3K56) occurs both during the
premeiotic and mitotic S phase and persists throughout DNA damage repair. To
learn more about the molecular mechanism of H3K56 acetylation and factors
required for this process, we surveyed the genome of the yeast Saccharomyces
cerevisiae to identify genes necessary for this process. A comparative global
proteomic screen identified several factors required for global H3K56
acetylation, which included histone chaperone Asf1 and a protein of an unknown
function Rtt109 but not Spt10. Our results indicate that the loss of Rtt109
results in the loss of H3K56 acetylation, both on bulk histone and on chromatin,
similar to that of asf1Delta or the K56Q mutation. RTT109 deletion exhibits
sensitivity to DNA damaging agents similar to that of asf1Delta and H3K56Q
mutants. Furthermore, Rtt109 and H3K56 acetylation appear to correlate with
actively transcribed genes and associate with the elongating form of polymerase
II in yeast. This histone modification is also associated with some of the
transcriptionally active puff sites in Drosophila. Our results indicate a new
role for the Rtt109 protein in the proper regulation of H3K56 acetylation. Chromatin structure exerts vital control over gene expression, DNA replication,
recombination, and repair. In addition to altering RNA polymerase II's (Pol II)
accessibility to DNA, histones are involved in the recruitment of activator and
repressor complex(es) to regulate gene expression. Histone deacetylase Rpd3
exists in two distinct forms, Rpd3S and Rpd3L. Several recent studies
demonstrated that the Eaf3 chromodomain, an Rpd3S subunit, recognizes
Set2-methylated histone H3K36, initiating Rpd3 deacetylase activity in the wake
of transcribing Pol II. Eaf3 and Set2 inhibit internal initiation within mRNA
coding regions, similar to the transcription elongation factor and histone
chaperone, FACT. Recent studies reviewed here demonstrate that histone
deacetylation on the body of a transcribed gene is regulated via Set2-mediated
methylation of histone H3-K36. These modifications provide restoration of normal
chromatin structure in the wake of elongating Pol II and prevent inappropriate
initiation within protein-coding regions masked by chromatin. Nucleosomes must be deacetylated behind elongating RNA polymerase II to prevent
cryptic initiation of transcription within the coding region. RNA polymerase II
signals for deacetylation through the methylation of histone H3 lysine 36
(H3K36), which provides the recruitment signal for the Rpd3S histone deacetylase
complex (HDAC). The recognition of methyl H3K36 by Rpd3S requires the
chromodomain of its Eaf3 subunit. Paradoxically, Eaf3 is also a subunit of the
NuA4 acetyltransferase complex, yet NuA4 does not recognize methyl H3K36
nucleosomes. In Saccharomyces cerevisiae, we found that methyl H3K36 nucleosome
recognition by Rpd3S also requires the plant homeobox domain (PHD) of its Rco1
subunit. Thus, the coupled chromo and PHD domains of Rpd3S specify recognition
of the methyl H3K36 mark, demonstrating the first combinatorial domain
requirement within a protein complex to read a specific histone code. The ubiquitous tandem kinase JIL-1 is essential for Drosophila development. Its
role in defining decondensed domains of larval polytene chromosomes is well
established, but its involvement in transcription regulation has remained
controversial. For a first comprehensive molecular characterisation of JIL-1, we
generated a high-resolution, chromosome-wide interaction profile of the kinase
in Drosophila cells and determined its role in transcription. JIL-1 binds active
genes along their entire length. The presence of the kinase is not proportional
to average transcription levels or polymerase density. Comparison of JIL-1
association with elongating RNA polymerase and a variety of histone
modifications suggests two distinct targeting principles. A basal level of JIL-1
binding can be defined that correlates best with the methylation of histone H3
at lysine 36, a mark that is placed co-transcriptionally. The additional
acetylation of H4K16 defines a second state characterised by approximately
twofold elevated JIL-1 levels, which is particularly prominent on the
dosage-compensated male X chromosome. Phosphorylation of the histone H3
N-terminus by JIL-1 in vitro is compatible with other tail modifications. In
vivo, phosphorylation of H3 at serine 10, together with acetylation at lysine
14, creates a composite histone mark that is enriched at JIL-1 binding regions.
Its depletion by RNA interference leads to a modest, but significant, decrease
of transcription from the male X chromosome. Collectively, the results suggest
that JIL-1 participates in a complex histone modification network that
characterises active, decondensed chromatin. We hypothesise that one specific
role of JIL-1 may be to reinforce, rather than to establish, the status of
active chromatin through the phosphorylation of histone H3 at serine 10. Myc family members are critical to maintain embryonic stem cells (ESC) in the
undifferentiated state. However, the mechanism by which they perform this task
has not yet been elucidated. Here we show that Myc directly upregulates the
transcription of all core components of the Polycomb repressive complex 2 (PRC2)
as well as the ESC-specific PRC2-associated factors. By expressing Myc protein
fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc,
binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the
acetylation of histones H3 and H4 and the recruitment of elongating RNA
polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC
results in reduced expression of PRC2 and H3K27me3 at Polycomb target
developmental regulators and upregulation of genes involved in primitive
endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced
for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor
(LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with
bivalent histone modifications and keep repressed the genes involved in ESC
differentiation. Thus, Myc proteins control the expression of developmental
regulators via the upregulation of the Polycomb PRC2 complex. The expansion of CAG/CTG repeats is responsible for many diseases, including
Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are
unstable in selective somatic tissues, which accelerates disease progression.
The mechanisms underlying repeat instability are complex, and it remains unclear
whether chromatin structure and/or transcription contribute to somatic CAG/CTG
instability in vivo. To address these issues, we investigated the relationship
between CAG instability, chromatin structure, and transcription at the HD locus
using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar
transgene, albeit integrated at a different site, and recapitulate HD
tissue-specific instability. We show that instability rates are increased in
R6/2 tissues as compared to R6/1 matched-samples. High transgene expression
levels and chromatin accessibility correlated with the increased CAG instability
of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were
increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to
R6/1 matched-tissues. However, the levels of transgene expression and these
specific histone marks were similar in the striatum and cerebellum, two tissues
showing very different CAG instability levels, irrespective of mouse line.
Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the
initiating form of RNA Pol II, were tissue-specific and correlated with CAG
instability levels. Similarly, H3K36 trimethylation, a mark associated with
transcription elongation, was specifically increased at the HD locus in the
striatum and not in the cerebellum. Together, our data support the view that
transcription modulates somatic CAG instability in vivo. More specifically, our
results suggest for the first time that transcription elongation is regulated in
a tissue-dependent manner, contributing to tissue-selective CAG instability. |
Could RG7112 be used as cancer therapy? | Yes, RG7112 has shown promising results in early phases of trials in cancer patients. | Increasing knowledge of the relationship between p53 and MDM2 has led to
development of potential small molecule inhibitors useful for clinical studies.
Herein, we discuss the patented (2006-2010) inhibitors of p53-MDM2 interaction.
The anticancer agents discussed in this review belong to several different
chemical classes including benzodiazepinediones, cis-imidazolines, oxindoles,
spiro-oxindoles, and numerous miscellaneous groups. This review also provides
comprehensive information on inhibitors of p53-MDM2 interaction that are
currently being tested in clinical trials. It is important to note that many of
the disclosed inhibitors need further validation to be considered as bona fide
inhibitors of p53-MDM2 interaction and some will not be further considered for
future studies. On the other hand, JNJ-26854165, a novel tryptamine derivative
and RG7112, a cis-imidazoline representative have shown promising results in
early phases of trials in cancer patients. AT-219, a spiroindolinone in late
stage preclinical studies is a likely candidate to proceed into clinical trials.
It remains to be seen how these inhibitors will perform in future clinical
studies as single agents and in combination with the currently approved
chemotherapeutic agents. BACKGROUND: RG7112 is a selective inhibitor of p53-MDM2 binding that frees p53
from negative control, activating the p53 pathway in cancer cells leading to
cell cycle arrest and apoptosis. RG7112 was selected for evaluation by the
Pediatric Preclinical Testing Program (PPTP) due to the relatively low incidence
of p53 mutations in pediatric cancers compared with adult maligcies.
PROCEDURES: RG7112 and its inactive etiomer RG7112i were evaluated against
the 23 cell lines of the PPTP in vitro panel using 96 hours exposure (1 nM to 10
µM). It was tested against the PPTP in vivo panel focusing on p53 wild-type (WT)
xenografts at a dose of 100 mg/kg daily for 14 days followed by 4 weeks of
observation. Response outcomes were related to MDM2 and p53 expression datasets
(http://pptp.nchresearch.org/data.html).
RESULTS: RG7112 demonstrated cytotoxic activity with a lower median IC(50) for
p53 WT versus p53 mutant cell lines (approximately 0.4 µM vs. >10 µM,
respectively). RG7112 induced tumor growth inhibition meeting criteria for
intermediate activity (EFS T/C > 2) in 10 of 26 (38%) solid tumor xenografts.
Objective responses included medulloblastoma, alveolar rhabdomyosarcoma, Wilms,
rhabdoid and Ewing sarcoma xenografts. For the ALL panel, there was one partial
response, five complete responses and one maintained complete response. The ALL
xenografts expressed the highest levels of p53 among the PPTP panels.
CONCLUSIONS: RG7112 induced tumor regressions in solid tumors from different
histotype panels, and exhibited consistent high-level activity against ALL
xenografts. This high level of activity supports prioritization of RG7112 for
further evaluation. BACKGROUND: We report a proof-of-mechanism study of RG7112, a small-molecule
MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed
well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were
eligible for resection.
METHODS: Patients with well-differentiated or dedifferentiated liposarcoma were
enrolled at four centres in France. Patients received up to three 28-day
neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a
patient progressed at any point after the first cycle, the lesion was resected
or, if unresectable, an end-of-study biopsy was done. The primary endpoint was
to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation
(P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and
apoptosis). All analyses were per protocol. This trial is registered with
EudraCT, number 2009-015522-10.
RESULTS: Between June 3, and Dec 14, 2010, 20 patients were enrolled and
completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type
tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had
MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations,
assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR
4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day
8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression
had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline.
The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR
-12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of
MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed
partial response and 14 had stable disease. All patients experienced at least
one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia
(nine patients), diarrhoea (nine patients), and thrombocytopenia (eight
patients). There were 12 serious adverse events in eight patients, the most
common of which were neutropenia (six patients) and thrombocytopenia (three
patients).
DISCUSSION: MDM2 inhibition activates the P53 pathway and decreases cell
proliferation in MDM2-amplified liposarcoma. This study suggests that it is
feasible to undertake neoadjuvant biopsy-driven biomarker studies in
liposarcoma.
FUNDING: F Hoffmann-La Roche. MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2
as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding
that can reactivate p53 in cancer cells may offer an effective approach for
cancer therapy. RG7112 is a potent and selective member of the nutlin family of
MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with
high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A
crystal structure of the RG7112-MDM2 complex revealed that the small molecule
binds in the p53 pocket of MDM2, mimicking the interactions of critical p53
amino acid residues. Treatment of cancer cells expressing wild-type p53 with
RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis.
RG7112 showed potent antitumor activity against a panel of solid tumor cell
lines. However, its apoptotic activity varied widely with the best response
observed in osteosarcoma cells with MDM2 gene amplification. Interestingly,
inhibition of caspase activity did not change the kinetics of p53-induced cell
death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic
concentrations caused dose-dependent changes in proliferation/apoptosis
biomarkers as well as tumor inhibition and regression. Notably, RG7112 was
highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our
findings offer a preclinical proof-of-concept that RG7112 is effective in
treatment of solid tumors expressing wild-type p53. INTRODUCTION: The majority of human sarcomas, particularly soft tissue sarcomas,
are relatively resistant to traditional cytotoxic therapies. The
proof-of-concept study by Ray-Coquard et al., using the Nutlin human double
minute (HDM)2-binding antagonist RG7112, has recently opened a new chapter in
the molecular targeting of human sarcomas.
AREAS COVERED: In this review, the authors discuss the challenges and
prospective remedies for minimizing the significant haematological toxicities of
the cis-imidazole Nutlin HDM2-binding antagonists. Furthermore, they also chart
the future direction of the development of p53-reactivating (p53-RA) drugs in
12q13-15 amplicon sarcomas and as potential chemopreventative therapies against
sarcomagenesis in germ line mutated TP53 carriers. Drawing lessons from the
therapeutic use of Imatinib in gastrointestinal tumours, the authors predict the
potential pitfalls, which may lie in ahead for the future clinical development
of p53-RA agents, as well as discussing potential non-invasive methods to
identify the development of drug resistance.
EXPERT OPINION: Medicinal chemistry strategies, based on structure-based drug
design, are required to re-engineer cis-imidazoline Nutlin HDM2-binding
antagonists into less haematologically toxic drugs. In silico modelling is also
required to predict toxicities of other p53-RA drugs at a much earlier stage in
drug development. Whether p53-RA drugs will be therapeutically effective as a
monotherapy remains to be determined. PURPOSE: Antitumor clinical activity has been demonstrated for the MDM2
antagonist RG7112, but patient tolerability for the necessary daily dosing was
poor. Here, utilizing RG7388, a second-generation nutlin with superior
selectivity and potency, we determine the feasibility of intermittent dosing to
guide the selection of initial phase I scheduling regimens.
EXPERIMENTAL DESIGN: A pharmacokinetic-pharmacodynamic (PKPD) model was
developed on the basis of preclinical data to determine alternative dosing
schedule requirements for optimal RG7388-induced antitumor activity. This PKPD
model was used to investigate the pharmacokinetics of RG7388 linked to the
time-course of the antitumor effect in an osteosarcoma xenograft model in mice.
These data were used to prospectively predict intermittent and continuous dosing
regimens, resulting in tumor stasis in the same model system.
RESULTS: RG7388-induced apoptosis was delayed relative to drug exposure with
continuous treatment not required. In initial efficacy testing, daily dosing at
30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically
equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was
equivalent to 10 mg/kg given daily. The implementation of modeling and
simulation on these data suggested several possible intermittent clinical dosing
schedules. Further preclinical analyses confirmed these schedules as viable
options.
CONCLUSION: Besides chronic administration, antitumor activity can be achieved
with intermittent schedules of RG7388, as predicted through modeling and
simulation. These alternative regimens may potentially ameliorate tolerability
issues seen with chronic administration of RG7112, while providing clinical
benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd)
schedules were selected for RG7388 clinical testing. |
Which interleukin is blocked by Siltuximab? | Siltuximab is a monoclonal antibody that binds to interleukin-6 with high affinity and specificity. | PURPOSE: Interleukin-6 (IL-6) has emerged as a key factor in the pathogenesis of
the atypical lymphoproliferative disorder Castleman's disease (CD). Siltuximab
is a new anti-IL-6, chimeric monoclonal antibody with potential therapeutic
benefit in patients with CD.
METHODS: We report interim results from an open-label, dose-finding,
seven-cohort, phase I study in which patients with symptomatic, multicentric or
unresectable, unicentric CD received siltuximab at 1-, 2-, or 3-week intervals.
The main efficacy end point of clinical benefit response (CBR) was defined as a
composite of clinical and laboratory measures relevant to the management of CD.
In addition, radiologic response was independently assessed by using modified
Cheson criteria.
RESULTS: Eighteen (78%) of 23 patients (95% CI, 56% to 93%) achieved CBR, and 12
patients (52%) demonstrated objective tumor response. All 11 patients (95% CI,
72% to 100%) treated with the highest dose of 12 mg/kg achieved CBR, and eight
patients (73%) achieved objective tumor response. Overall objective-response
duration ranged from 44 to > or = 889 days, and one patient had complete
response for > or = 318 days. Hemoglobin increased markedly in 19 patients
(median increase, 2.1 g/dL; range, 0.2 to 4.7 g/dL) in the absence of
transfusion or erythropoiesis-stimulating agents. No dose-limiting toxicity was
reported, and only three patients had grade 3 or higher adverse events after a
median exposure of 331 days (range, 1 to 1,148 days).
CONCLUSION: These interim results strongly suggest that siltuximab is an
effective treatment with favorable safety for the management of CD. An
additional study is planned to fully evaluate safety and efficacy at the
recommended dose of 12 mg/kg every 3 weeks. Signalling through the interleukin (IL)-6 pathway induces proliferation and drug
resistance of multiple myeloma cells. We therefore sought to determine whether
the IL-6-neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could
enhance the activity of melphalan, and to examine some of the mechanisms
underlying this interaction. Siltuximab increased the cytotoxicity of melphalan
in KAS-6/1, INA-6, ANBL-6, and RPMI 8226 human myeloma cell lines (HMCLs) in an
additive-to-synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to
melphalan. These anti-proliferative effects were accompanied by enhanced
activation of drug-specific apoptosis in HMCLs grown in suspension, and in HMCLs
co-cultured with a human-derived stromal cell line. Siltuximab with melphalan
enhanced activation of caspase-8, caspase-9, and the downstream effector
caspase-3 compared with either of the single agents. This increased induction of
cell death occurred in association with enhanced Bak activation. Neutralization
of IL-6 also suppressed signalling through the phosphoinositide 3-kinase/Akt
pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and
4E-BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced
anti-proliferative effects against primary plasma cells derived from patients
with myeloma, monoclonal gammopathy of undetermined significance, and
amyloidosis. These studies provide a rationale for translation of siltuximab
into the clinic in combination with melphalan-based therapies. BACKGROUND: Interleukin-6 (IL-6) is associated with prostate cancer morbidity.
In several experimental models, IL-6 has been reported to have anti-apoptotic
and pro-angiogenic effects. Siltuximab (CNTO 328) is a monoclonal anti-IL-6
antibody which has been successfully applied in several models representing
prostate cancer. This study was designed to assess preliminary safety of
siltuximab in patients with early prostate cancer.
PATIENTS AND METHODS: Twenty patients scheduled to undergo radical prostatectomy
received either no drug or siltuximab (6 mg/kg, five patients per group with
administration once, two times, and three times prior to surgery). Blood samples
were collected for pharmacokinetic and pharmacodynamic analyses. Expression of
elements of IL-6 signaling pathways was analyzed in tumor tissue by
immunohistochemistry. Gene analysis in tumor specimens was performed with the
DASL array.
RESULTS: No adverse events related to siltuximab were observed. Patients treated
with siltuximab presented with higher levels of proliferation and apoptosis
markers. Following a single dose, serum concentrations of siltuximab declined in
a biexponential manner. This study revealed a decrease in phosphorylation of
Stat3 and p44/p42 mitogen-activated protein kinases. In addition, gene
expression analyses indicate down-regulation of genes immediately downstream of
the IL-6 signaling pathway and key enzymes of the androgen signaling pathway.
CONCLUSIONS: Preliminary safety of siltuximab is favorable. Future studies in
which siltuximab could be combined with androgen-deprivation therapy and
experimental therapies in advanced prostate cancer are justified. PURPOSE: We investigated whether inhibition of interleukin 6 (IL-6) has
therapeutic activity in ovarian cancer via abrogation of a tumor-promoting
cytokine network.
EXPERIMENTAL DESIGN: We combined preclinical and in silico experiments with a
phase 2 clinical trial of the anti-IL-6 antibody siltuximab in patients with
platinum-resistant ovarian cancer.
RESULTS: Automated immunohistochemistry on tissue microarrays from 221 ovarian
cancer cases showed that intensity of IL-6 staining in maligt cells
significantly associated with poor prognosis. Treatment of ovarian cancer cells
with siltuximab reduced constitutive cytokine and chemokine production and also
inhibited IL-6 signaling, tumor growth, the tumor-associated macrophage
infiltrate and angiogenesis in IL-6-producing intraperitoneal ovarian cancer
xenografts. In the clinical trial, the primary endpoint was response rate as
assessed by combined RECIST and CA125 criteria. One patient of eighteen
evaluable had a partial response, while seven others had periods of disease
stabilization. In patients treated for 6 months, there was a significant decline
in plasma levels of IL-6-regulated CCL2, CXCL12, and VEGF. Gene expression
levels of factors that were reduced by siltuximab treatment in the patients
significantly correlated with high IL-6 pathway gene expression and macrophage
markers in microarray analyses of ovarian cancer biopsies.
CONCLUSION: IL-6 stimulates inflammatory cytokine production, tumor
angiogenesis, and the tumor macrophage infiltrate in ovarian cancer and these
actions can be inhibited by a neutralizing anti-IL-6 antibody in preclinical and
clinical studies. PURPOSE: Siltuximab is a chimeric, anti-interleukin-6 monoclonal antibody with
potential therapeutic benefit in castration-resistant prostate cancer (CRPC)
patients. We assessed the safety and tolerability of siltuximab in combination
with docetaxel, the pharmacokinetics of docetaxel alone and with siltuximab, and
the efficacy and pharmacodynamics of siltuximab plus docetaxel.
PATIENTS AND METHODS: In an open-label, dose-escalation, multicenter, phase 1
study, patients with metastatic, progressive CRPC received docetaxel 75 mg/m(2)
q3w plus siltuximab 6 mg/kg q2w (n=12), 9 mg/kg q3w (n=12), or 12 mg/kg q3w
(n=15). Dose-limiting toxicity (DLT), PSA, and radiologic response according to
WHO criteria were evaluated.
RESULTS: DLT was reported in 1 of 11 patients receiving 6 mg/kg, 1 of 12
receiving 9 mg/kg, and in 1 of 14 receiving 12 mg/kg. Common Grade ≥ 3 adverse
events were neutropenia (73 %), leukopenia (60 %), lymphopenia (30 %), dyspnea
(19 %), and fatigue (14 %). Toxicities were not dose dependent. Siltuximab did
not affect docetaxel pharmacokinetics. The pharmacokinetic profile for
siltuximab in combination was similar to single-agent siltuximab
pharmacokinetics. Twenty-three (62 %; 95 % CI 45 %, 78 %) of 37
combination-treated patients achieved a confirmed ≥ 50 % PSA decline. Of 17
patients with measurable disease at baseline, 2 confirmed and 2 unconfirmed
radiologic partial responses ranging 190 to 193 days were achieved with 9- and
12-mg/kg siltuximab. C-reactive protein concentrations were suppressed
throughout treatment in all patients.
CONCLUSION: These results suggest that siltuximab in combination with docetaxel
is safe and shows preliminary efficacy in patients with CRPC, although
alternative siltuximab schedules may be better tolerated for future studies. Development and progression of prostate cancer (PCa) are associated with chronic
inflammation. The cytokine interleukin 6 (IL6) can influence progression,
differentiation, survival, and angiogenesis of PCa. To identify novel pathways
that are triggered by IL6, we performed a gene expression profiling of two PCa
cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN)
regulatory factor 9 (IRF9) was identified as one of the most prevalent
IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN
signaling and acts together with STAT1 and 2 to activate transcription of
IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and
protein levels by quantitative real-time PCR and western blot respectively in
both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three
PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop
displayed high expression of IRF9. A tissue microarray with 36 PCa tissues
showed that IRF9 protein expression is moderately elevated in maligt areas
and positively correlates with the tissue expression of IL6. Downregulation and
overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in
cellular proliferation of different PCa cell lines. Furthermore, expression of
IRF9 was essential to mediate the antiproliferative effects of IFNα2. We
concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for
the antiproliferative effects of IFNα2. INTRODUCTION: Interleukin-6 (IL-6) can activate downstream signaling pathways in
lung cancer cells, such as the STAT3 pathway, and is reported to be produced by
tumor cells with activating EGFR mutations. We examined IL-6/STAT3 in lung
cancer tumor tissues and the effects of siltuximab, a neutralizing antibody to
human IL-6, in mouse models of lung cancer.
METHODS: IL-6 and STAT3 activation levels were compared with tumor histology and
presence of KRAS mutations in snap-frozen, non-small-cell lung cancer tumors.
The effects of siltuximab alone or in combination with erlotinib were examined
in mouse xenograft models constructed using three cell line xenograft models and
one primary explant mouse model. We examined the influence of cancer-associated
fibroblasts (CAFs) on tumor growth and siltuximab effects.
RESULTS: IL-6 levels were higher in tumors of squamous cell versus
adenocarcinoma histology and were not associated with presence of KRAS
mutations. Tyrosine phosphorylation status of STAT3 did not correlate with tumor
IL-6 levels. Serine phosphorylation of STAT3 was correlated with KRAS mutation
status. Both tumor and stromal cells contributed to total IL-6 within tumors.
Siltuximab had minimal effect as a single agent in xenografts with tumor cells
alone; however, in models coadministered with CAFs, siltuximab had more potent
effects on tumor inhibition. We observed no effects of combined erlotinib and
siltuximab.
CONCLUSIONS: IL-6 is elevated in subsets of human NSCLCs, especially with
squamous cell histology. Tumors supported by stromal production of IL-6 seem to
be the most vulnerable to tumor growth inhibition by siltuximab. BACKGROUND: Multicentric Castleman's disease is a rare lymphoproliferative
disorder driven by dysregulated production of interleukin 6. No randomised
trials have been done to establish the best treatment for the disease. We
assessed the safety and efficacy of siltuximab-a chimeric monoclonal antibody
against interleukin 6-in HIV-negative patients with multicentric Castleman's
disease.
METHODS: We did this randomised, double-blind, placebo-controlled study at 38
hospitals in 19 countries worldwide. We enrolled HIV-negative and human
herpesvirus-8-seronegative patients with symptomatic multicentric Castleman's
disease. Treatment allocation was randomised with a computer-generated list,
with block size six, and stratification by baseline corticosteroid use. Patients
and investigators were masked to treatment allocation. Patients were randomly
assigned (2:1) to siltuximab (11 mg/kg intravenous infusion every 3 weeks) or
placebo; all patients also received best supportive care. Patients continued
treatment until treatment failure. The primary endpoint was durable tumour and
symptomatic response for at least 18 weeks for the intention-to-treat
population. Enrolment has been completed. The study is registered with
ClinicalTrials.gov, number NCT01024036.
FINDINGS: We screened 140 patients, 79 of whom were randomly assigned to
siltuximab (n=53) or placebo (n=26). Durable tumour and symptomatic responses
occurred in 18 (34%) of 53 patients in the siltuximab group and none of 26 in
the placebo group (difference 34·0%, 95% CI 11·1-54·8, p=0·0012). The incidence
of grade 3 or more adverse events (25 [47%] vs 14 [54%]) and serious adverse
events (12 [23%] vs five [19%]) was similar in each group despite longer median
treatment duration with siltuximab than with placebo (375 days [range 1-1031] vs
152 days [23-666]). The most common grade 3 or higher were fatigue (five vs
one), night sweats (four vs one), and anaemia (one vs three). Three (6%) of 53
patients had serious adverse events judged reasonably related to siltuximab
(lower respiratory tract infection, anaphylactic reaction, sepsis).
INTERPRETATION: Siltuximab plus best supportive care was superior to best
supportive care alone for patients with symptomatic multicentric Castleman's
disease and well tolerated with prolonged exposure. Siltuximab is an important
new treatment option for this disease.
FUNDING: Janssen Research & Development. We describe a case of multicentric Castleman disease with generalized
lymphadenopathy and splenomegaly, accompanied by typical B symptoms - loss of 15
kg, fever of non-infectious origin, night sweats, symptoms of anemia.
Histological examination of the nodes with the highest accumulation of
fluorodeoxyglucose, taken from mediastinum by thoracoscopy, revealed
plasmocellular type of Castleman disease. Tests for HIV and human herpesvirus 8
(HHV-8) were negative. Three recurrences of herpes zoster indicating an
alteration of immunity preceded the dia-gnosis of disease. Treatment was
initiated with combination of thalidomide, dexamethasone, and cyclophosphamide.
The response after 2 months therapy was not clear and patient doesn't tolerated
the therapy well. Therefore, this treatment was terminated and R-CHOP (Mabthera
- rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone) was
selected as a second-line therapy. Lymphadenopathy and splenomegaly were reduced
during the 2 cycles of treatment, however, serious infectious complications
accompanied the therapy. Therefore, only use of Mabthera monotherapy 375 mg /m2
was administered in 28-day intervals. This treatment has shown efficacy and
tolerability. PET-CT scan has demonstrated disappearance of lymphadenopathy and
splenomegaly, in addition, normalized accumulation of fluorodeoxyglucose.
Monotherapy with Mabthera has proved to be effective and well tolerated drug in
this case. Currently, there are more effective therapeutic alternatives in
multicentric Castleman disease: treatment with monotherapy of rituximab or in
combination therapy with immunomodulatory drugs (thalidomide or lenalidomide,
treatment with anti-IL-6 (siltuximab) or against its receptor (tocilizumab). In
the case of ineffectiveness of one treatment option must be tested other
alternative. In this case the therapy based on thalidomide wasn't successful,
whereas the treatment with Mabthera has achieved disappearance of disease
symptoms. Interleukin-6 (IL-6) is a pleiotropic cytokine implicated in the pathogenesis of
many immune-mediated disorders including several types of non-infectious
uveitis. These uveitic conditions include Vogt-Koyanagi-Harada syndrome, uveitis
associated with Behçet disease, and sarcoidosis. This review summarizes the role
of IL-6 in immunity, highlighting its effect on Th17, Th1, and plasmablast
differentiation. It reviews the downstream mediators activated in the process of
IL-6 binding to its receptor complex. This review also summarizes the biologics
targeting either IL-6 or the IL-6 receptor, including tocilizumab, sarilumab,
sirukumab, olokizumab, clazakizumab, and siltuximab. The target, dosage,
potential side effects, and potential uses of these biologics are summarized in
this article based on the existing literature. In summary, anti-IL-6 therapy for
non-infectious uveitis shows promise in terms of efficacy and side effect
profile. |
ROSIER scale is used for which disorder? | ROSIER (Recognition of Stroke in the Emergency Room) scale was developed as a stroke recognition tool on suspected patients in the prehospital setting. | OBJECTIVE: To compare the sensitivity and specificity of bedside diagnostic
stroke scales in patients with suspected stroke.
DESIGN: A cross-sectional observational study of patients with suspected acute
stroke in an emergency department in a UK hospital. DIAGNOSTIC SCALES: The
results of an assessment with the Recognition of Stroke in the Emergency Room
(ROSIER) scale, the Face Arm Speech Test (FAST) scale and the diagnosis of
definite or probable stroke by an emergency department. Reference standard A
consensus diagnosis of stroke or transient ischaemic attack (TIA) made after
discussion by an expert panel (members included stroke physicians, neurologists
and neuroradiologists), who had access to the clinical findings, imaging and
subsequent clinical course, but were blinded to the results of the assessments
by emergency-department staff.
RESULTS: In 356 patients with complete data, the expert panel assigned a
diagnosis of acute stroke or TIA in 246 and a diagnosis of mimic in 110. The
ROSIER had a sensitivity of 83% (95% CI 78 to 87) and specificity of 44% (95% CI
34 to 53), and the FAST had a sensitivity of 81% (95% CI 76 to 86) and a
specificity of 39% (95% CI 30 to 48). There was no detectable difference between
the scales in sensitivity (p = 0.39) or specificity (p = 0.30).
CONCLUSIONS: The simpler FAST scale could replace the more complex ROSIER for
the initial assessment of patients with suspected acute stroke in the emergency
department. BACKGROUND AND PURPOSE: U.K ambulance services assess patients with suspected
stroke using the Face Arm Speech Test (FAST). The Recognition Of Stroke In the
Emergency Room (ROSIER) tool has been shown superior to the FAST in identifying
strokes in emergency departments but has not previously been tested in the
ambulance setting. We investigated whether ROSIER use by ambulance clinicians
can improve stroke recognition.
METHODS: Ambulance clinicians used the ROSIER in place of the FAST to assess
patients with suspected stroke. As the ROSIER includes all FAST elements, we
calculated a FAST score from the ROSIER to enable comparisons between the two
tools. Ambulance clinicians' provisional stroke diagnoses using the ROSIER and
calculated FAST were compared with stroke consultants' diagnosis. We used
stepwise logistic regression to compare the contribution of individual ROSIER
and FAST items and patient demographics to the prediction of consultants'
diagnoses.
RESULTS: Sixty-four percent of strokes and 78% of nonstrokes identified by
ambulance clinicians using the ROSIER were subsequently confirmed by a stroke
consultant. There was no difference in the proportion of strokes correctly
detected by the ROSIER or FAST with both displaying excellent levels of
sensitivity. The ROSIER detected marginally more nonstroke cases than the FAST,
but both demonstrated poor specificity. Facial weakness, arm weakness, seizure
activity, age, and sex predicted consultants' diagnosis of stroke.
CONCLUSIONS: The ROSIER was not better than the FAST for prehospital recognition
of stroke. A revised version of the FAST incorporating assessment of seizure
activity may improve stroke identification and decision making by ambulance
clinicians. OBJECTIVE: To identify and compare the operating characteristics of existing
prehospital stroke scales to predict true strokes in the hospital.
METHODS: We searched MEDLINE, EMBASE, and CINAHL databases for articles that
evaluated the performance of prehospital stroke scales. Quality of the included
studies was assessed using the Quality Assessment of Diagnostic Accuracy
Studies-2 tool. We abstracted the operating characteristics of published
prehospital stroke scales and compared them statistically and graphically.
RESULTS: We retrieved 254 articles from MEDLINE, 66 articles from EMBASE, and 32
articles from CINAHL Plus database. Of these, 8 studies met all our inclusion
criteria, and they studied Cincinnati Pre-Hospital Stroke Scale (CPSS), Los
Angeles Pre-Hospital Stroke Screen (LAPSS), Melbourne Ambulance Stroke Screen
(MASS), Medic Prehospital Assessment for Code Stroke (Med PACS), Ontario
Prehospital Stroke Screening Tool (OPSS), Recognition of Stroke in the Emergency
Room (ROSIER), and Face Arm Speech Test (FAST). Although the point estimates for
LAPSS accuracy were better than CPSS, they had overlapping confidence intervals
on the symmetric summary receiver operating characteristic curve. OPSS performed
similar to LAPSS whereas MASS, Med PACS, ROSIER, and FAST had less favorable
overall operating characteristics.
CONCLUSIONS: Prehospital stroke scales varied in their accuracy and missed up to
30% of acute strokes in the field. Inconsistencies in performance may be due to
sample size disparity, variability in stroke scale training, and divergent
provider educational standards. Although LAPSS performed more consistently,
visual comparison of graphical analysis revealed that LAPSS and CPSS had similar
diagnostic capabilities. BACKGROUND AND PURPOSE: The objective of this study was to determine the
performance of the Recognition Of Stroke In the Emergency Room (ROSIER) scale in
risk-stratifying Chinese patients with suspected stroke in Hong Kong.
METHODS: This was a prospective cohort study in an urban academic emergency
department (ED) over a 7-month period. Patients over 18 years of age with
suspected stroke were recruited between June 2011 and December 2011. ROSIER
scale assessment was performed in the ED triage area. Logistic regression
analysis was used to estimate the impacts of diagnostic variables, including
ROSIER scale, past history and ED characteristics.
FINDINGS: 715 suspected stroke patients were recruited for assessment, of whom
371 (52%) had acute cerebrovascular disease (302 ischaemic strokes, 24 transient
ischaemic attacks (TIA), 45 intracerebral haemorrhages), and 344 (48%) had other
illnesses i.e. stroke mimics. Common stroke mimics were spinal neuropathy,
dementia, labyrinthitis and sepsis. The suggested cut-off score of>0 for the
ROSIER scale for stroke diagnosis gave a sensitivity of 87% (95%CI 83-90), a
specificity of 41% (95%CI 36-47), a positive predictive value of 62% (95%CI
57-66), and a negative predictive value of 75% (95%CI 68-81), and the AUC was
0.723. The overall accuracy at cut off>0 was 65% i.e. (323+141)/715.
INTERPRETATION: The ROSIER scale was not as effective at differentiating acute
stroke from stroke mimics in Chinese patients in Hong Kong as it was in the
original studies, primarily due to a much lower specificity. If the ROSIER scale
is to be clinically useful in Chinese suspected stroke patients, it requires
further refinement. |
How histone deacetylation causes transcriptional gene silencing? | Histone deacetylation, catalyzed by the histone deacetylase (HDAC) enzymes, is an epigenetic modification. Histone deacetylation leads to the formation of a condensed and transcriptionally repressive chromatin structure which inhibits gene transcription. | Epigenetic gene regulation is a key determit of heritable gene expression
patterns and is critical for normal cellular function. Dysregulation of
epigenetic transcriptional control is a fundamental feature of cancer,
particularly manifesting as increased promoter DNA methylation with associated
aberrant gene silencing, which plays a significant role in tumor progression. We
now globally map key chromatin parameters for genes with promoter CpG island DNA
hypermethylation in colon cancer cells by combining microarray gene expression
analyses with chromatin immunoprecipitation-on-chip technology. We first show
that the silent state of such genes universally correlates with a broad
distribution of a low but distinct level of the PcG-mediated histone
modification, methylation of lysine 27 of histone 3 (H3K27me), and a very low
level of the active mark H3K4me2. This chromatin pattern, and particularly
H3K4me2 levels, crisply separates DNA-hypermethylated genes from those where
histone deacetylation is responsible for transcriptional silencing. Moreover,
the chromatin pattern can markedly enhance identification of truly silent and
DNA-hypermethylated genes. We additionally find that when DNA-hypermethylated
genes are demethylated and reexpressed, they adopt a bivalent chromatin pattern,
which is associated with the poised gene expression state of a large group of
embryonic stem cell genes and is characterized by an increase in levels of both
the H3K27me3 and H3K4me2 marks. Our data have great relevance for the increasing
interest in reexpression of DNA-hypermethylated genes for the treatment of
cancer. Estrogen receptor alpha (ER)-negative human breast cancer cells frequently
overexpress epidermal growth factor receptor (EGFR) and respond poorly to
endocrine therapies. Our previous studies demonstrate that histone deacetylation
plays a key role in ER gene silencing, and ER expression can be restored with
histone deacetylase (HDAC) inhibitors in ER-negative human breast cancer cells.
Whether inhibition of HDAC also alters epidermal growth factor (EGF) signaling
pathways is not defined. Here we present evidence that reexpression of ER
protein by a clinically available HDAC inhibitor, suberoylanilide hydroxamic
acid (SAHA or vorinostat), is coupled with loss of EGFR in ER-negative human
breast cancer cells. Consistent with this observation, MDA-MB-231 cells, which
are ER-negative and overexpress EGFR, that are engineered to express ER show a
decrease in EGFR protein expression. Down-regulation of EGFR by SAHA results
from attenuation of its mRNA stability. We also confirm that new protein
synthesis is required for maintaining EGFR mRNA stability. Further experiments
indicate that a decrease in EGFR abolished EGF-initiated signaling pathways
including phosphorylated PAK1, p38MAPK and AKT. Thus, SAHA may not only
reactivate silenced ER, but also simultaneously deplete EGFR expression. These
data suggest that inhibition of HDAC is a promising epigenetic therapy for
ER-negative human breast cancer. The aim of the present study was to analyse the molecular mechanisms involved in
the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our
results demonstrate that TNF-alpha, a major IL-6 inducer, is able to induce IL-6
only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but
not trichostatin A (TSA), activates the expression of IL-6 in all cell lines,
indicating that DNA methylation, but not histone deacetylation, plays an
essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a
methylation status that correlates with IL-6 expression and binds MeCP2 and
H3meK9 only in the non-expressing cell lines. Our results suggest that critical
methylations located from positions -666 to -426 relative to the transcription
start site of IL-6 may act as binding sites for MeCP2. BACKGROUND: Silencing of normal gene expression occurs early in the apoptosis of
neurons, well before the cell is committed to the death pathway, and has been
extensively characterized in injured retinal ganglion cells. The causative
mechanism of this widespread change in gene expression is unknown. We
investigated whether an epigenetic change in active chromatin, specifically
histone H4 deacetylation, was an underlying mechanism of gene silencing in
apoptotic retinal ganglion cells (RGCs) following an acute injury to the optic
nerve.
RESULTS: Histone deacetylase 3 (HDAC3) translocates to the nuclei of dying cells
shortly after lesion of the optic nerve and is associated with an increase in
nuclear HDAC activity and widespread histone deacetylation. H4 in promoters of
representative genes was rapidly and indiscriminately deacetylated, regardless
of the gene examined. As apoptosis progressed, H4 of silenced genes remained
deacetylated, while H4 of newly activated genes regained, or even increased, its
acetylated state. Inhibition of retinal HDAC activity with trichostatin A (TSA)
was able to both preserve the expression of a representative RGC-specific gene
and attenuate cell loss in response to optic nerve damage.
CONCLUSIONS: These data indicate that histone deacetylation plays a central role
in transcriptional dysregulation in dying RGCs. The data also suggests that
HDAC3, in particular, may feature heavily in apoptotic gene silencing. The Arabidopsis histone deacetylase HDA6 is required to silence transgenes,
transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar domice in
genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S
rRNA gene variants that is normally inactivated during development fails to be
silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG
and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription
occurs throughout the intergenic spacers. The resulting sense and antisense
spacer transcripts facilitate a massive overproduction of siRNAs that, in turn,
direct de novo cytosine methylation of corresponding gene sequences. However,
the resulting de novo DNA methylation fails to suppress Pol I or Pol II
transcription in the absence of HDA6 activity; instead, euchromatic histone
modifications typical of active genes accumulate. Collectively, the data reveal
a futile cycle of unregulated transcription, siRNA production, and
siRNA-directed DNA methylation in the absence of HDA6-mediated histone
deacetylation. We propose that spurious Pol II transcription throughout the
intergenic spacers in hda6 mutants, combined with losses of histone deacetylase
activity and/or maintece DNA methylation, eliminates repressive chromatin
modifications needed for developmental rRNA gene dosage control. DNA methylation and histone acetylation are major epigenetic modifications in
gene silencing. In our previous research, we found that the methylated
oligonucleotide (SurKex) complementary to a region of promoter of survivin could
induce DNA methylation in a site-specific manner leading to survivin silencing.
Here, we further studied the role of histone acetylation in survivin silencing
and the relationship between histone acetylation and DNA methylation. First we
observed the levels of histone H4 and H4K16 acetylation that were decreased
after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay.
Next, we investigated the roles of histone acetylation and DNA methylation in
survivin silencing after blockade of histone deacetylation with Trichostatin A
(TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin
promoter methylation by bisulfite sequencing and examined the level of histone
acetylation by the ChIP assay. The results showed that histone deacetylation
blocked by TSA reversed the effects of SurKex on inhibiting the expression of
survivin mRNA, inducing a site-specific methylation on survivin promoter and
decreasing the level of histone acetylation. Finally, we examined the role of
histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA.
The results showed that histone deacetylation blocked by TSA reversed the
increasing effect of histone deacetylation on the expression of survivin mRNA.
This study suggests that histone deacetylation guides SurKex-induced DNA
methylation in survivin silencing possibly through increasing the expression of
DNMT1 mRNA. Overexposure of the human skin to solar ultraviolet (UV) radiation is the major
etiologic factor for development of skin cancers. Here, we report the results of
epigenetic modifications in UV-exposed skin and skin tumors in a systematic
manner. The skin and tumor samples were collected after chronic exposure of the
skin of SKH-1 hairless mice to UVB radiation using a well-established
photocarcinogenesis protocol. We found a distinct DNA hypermethylation pattern
in the UVB-exposed epidermal skin and UVB-induced skin tumors that was
associated with the elevated expression and activity of the DNA
methyltransferases (Dnmt) 1, Dnmt3a and Dnmt3b. To explore the role of
hypermethylation in skin photocarcinogenesis, we focused on the p16(INK4a) and
RASSF1A tumor suppressor genes, which are transcriptionally silenced on
methylation. We established that the silencing of these genes in UVB-exposed
epidermis and UVB-induced skin tumors is associated with a network of epigenetic
modifications, including hypoacetylation of histone H3 and H4 and increased
histone deacetylation, as well as recruitment of methyl-binding proteins,
including MeCP2 and MBD1, to the methylated CpGs. Higher levels of DNA
methylation and DNMT activity in human squamous cell carcinoma specimens than in
normal human skin suggest that the data are relevant clinically. Our data
indicate for the first time that UVB-induced DNA hypermethylation, enhanced Dnmt
activity and histone modifications occur in UVB-exposed skin and UVB-induced
skin tumors and suggest that these events are involved in the silencing of tumor
suppressor genes and in skin tumor development. Loss of E-cadherin is associated with acquisition of metastatic capacity.
Numerous studies suggest that histone deacetylation and/or hypermethylation of
CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for
E-cadherin silencing in different tumors and cancer cell lines. The hepatitis B
virus (HBV)-encoded X antigen, HBx, contributes importantly to the development
of hepatocellular carcinoma using multiple mechanisms. Experiments were designed
to test if in addition to CDH1 hypermethylation HBx promotes epigenetic
modulation of E-cadherin transcriptional activity through histone deacetylation
and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and
miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells
by western blotting, immunoprecipitation (IP), chromatin IP as well as by
immunohistochemical staining of liver and tumor tissue sections from
HBV-infected patients. In HepG2X cells, decreased levels of E-cadherin and
elevated levels of mSin3A and Snail-1 were detected. Reciprocal IP with anti-HBx
and anti-mSin3A demonstrated mutual binding. Furthermore, HBx-mSin3A
colocalization was detected by immunofluorescent staining. HBx downregulated
E-cadherin expression by the recruitment of the mSin3A/histone deacetylase
complex to the Snail-binding sites in human CDH1. Histone deacetylation
inhibition by Trichostatin-A treatment restored E-cadherin expression. Mir-373,
a positive regulator of E-cadherin expression, was downregulated by HBx in
HepG2X cells and tissue sections from HBV-infected patients. Thus, histone
deacetylation of CDH1 and downregulation of miR-373, together with the
previously demonstrated hypermethylation of CDH1 by HBx, may be important for
the understanding of HBV-related carcinogenesis. Some genetic studies indicate that plant homologues of proteins involved in
chromatin modification and remodeling in other organisms may regulate plant
development. Previously, we described an Arabidopsis mutant with altered
cold-responsive gene expression (acg1) displaying a late flowering phenotype, a
null allele of fve. FVE is a homologue of the mammalian
retinoblastoma-associated protein (RbAp), one component of a histone deacetylase
(HDAC) complex involved in transcriptional repression, and has been shown to be
involved in the deacetylation of the FLOWERING LOCUS C (FLC) chromatin encoding
for a repressor of flowering. In an effort to gain insight into the biochemical
functions of FVE, we overexpressed FVE tagged with the hemagglutinin (HA) and
FLAG epitope at the N-terminus in acg1 mutants. The results of physiological and
molecular analyses demonstrated that FVE overexpression in acg1 rescued the
mutant phenotypes, including late flowering and alterations in floral pathway
gene expression such as FLC, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), and
FLOWERING LOCUS T (FT), and also super-induced cold-responsive reporter gene
expression. The chromatin immunoprecipitation experiments revealed the
amplification of specific DNA regions of FLC and COLD-REGULATED 15A (COR15A),
indicating that FVE may bind to the FLC and COR15A chromatin. Gel-filtration
chromatography and the immunoprecipitation of putative FVE complexes showed that
FVE forms a protein complex of approximately 1.0 MDa. These results demonstrate
that FVE may exist as a multiprotein complex, similar to the mammalian HDAC
complex harboring RbAp, to regulate flowering time and cold response by
associating with the FLC and COR chromatin. The histone chaperones play an important role in chromatin assembly and
disassembly during replication and transcription. We have assessed the global
roles of histone chaperones in Saccharomyces cerevisiae. Microarray
transcriptional analyzes indicate that histone chaperones have their own
specific target genes, and various histone chaperones have partially overlapping
functions during transcriptional regulation. The histone deacetylase inhibitor
TSA and histone chaperones Asf1, Vps75 and Rtt106 can function in parallel
pathways to regulate transcription. Moreover, TSA can specifically antagonize
histone chaperone Chz1-mediated telomere anti-silencing. This study demonstrates
that a mutual cross-talk mechanism exists between histone chaperones and histone
deacetylation in transcriptional regulation. Post-translational modifications of histone proteins play a crucial role in
responding to environmental stresses. Histone deacetylases (HDACs) catalyze the
removal of an acetyl group from histones and are generally believed to be a
transcriptional repressor. In this paper, we report that cold treatment highly
induces the up-regulation of HDACs, leading to global deacetylation of histones
H3 and H4. Treatment of maize with the HDAC inhibitor trichostatin A (TSA) under
cold stress conditions strongly inhibits induction of the maize cold-responsive
genes ZmDREB1 and ZmCOR413. However, up-regulation of the ZmICE1 gene in
response to cold stress is less affected. The expression of drought and salt
induced genes, ZmDBF1 and rab17, is almost unaffected by TSA treatment. Thus,
these observations show that HDACs may selectively activate transcription. The
time course of TSA effects on the expression of ZmDREB1 and ZmCOR413 genes
indicates that HDACs appear to directly activate the ZmDREB1 gene, which in turn
modulates ZmCOR413 expression. After cold treatment, histone hyperacetylation
and DNA demethylation occurs in the ICE1 binding region, accompanied by an
increase in accessibility to micrococcal nuclease (MNase). The two regions
adjacent to the ICE1 binding site remain hypoacetylated and methylated. However,
during cold acclimation, TSA treatment increases the acetylation status and
accessibility of MNase and decreases DNA methylation at these two regions.
However, TSA treatment does not affect histone hyperacetylation and DNA
methylation levels at the ICE1 binding regions of the ZmDREB1 gene. Altogether,
our findings indicate that HDACs positively regulate the expression of the
cold-induced ZmDREB1 gene through histone modification and chromatin
conformational changes and that this activation is both gene and site selective. Lysine acetylation of histones is one of the major epigenetic regulators of
chromatin conformation and gene expression. The dynamic nature of histone
acetylation is determined by the counterbalancing activity of histone
acetyltransferase and histone deacetylase (HDAC) enzymes. Acetylation of
histones is generally associated with open and transcriptionally active
chromatin, whereas the activity of HDACs leads to histone deacetylation,
condensation of chromatin, and inhibition of transcription. Aberrant silencing
of tumor suppressors and other genes has been found in different types of
cancer. Abnormal activity of HDACs has been implicated in tumorigenesis and
therefore considerable effort has been put into the development of HDAC
inhibitors as a means of modifying histone acetylation status and reexpressing
aberrantly silenced tumor suppressor genes. This has led to the generation of a
number of structurally diverse compounds that can effectively inhibit HDAC
activity, thus altering chromatin structure in cancer cells. This unit discusses
the methods and recent technological developments with respect to the studies of
HDAC inhibition in cancer. Histone deacetylation plays an important role in epigenetic control of gene
expression. HD2 is a plant-specific histone deacetylase that is able to mediate
transcriptional repression in many biological processes. To investigate the
epigenetic and transcriptional mechanisms of longan fruit senescence, one
histone deacetylase 2-like gene, DlHD2, and two ethylene-responsive factor-like
genes, DlERF1 and DlERF2, were cloned and characterized from longan fruit.
Expression of these genes was examined during fruit senescence under different
storage conditions. The accumulation of DlHD2 reached a peak at 2 d and 30 d in
the fruit stored at 25 °C (room temperature) and 4 °C (low temperature),
respectively, or 6 h after the fruit was transferred from 4 °C to 25 °C, when
fruit senescence was initiated. However, the DlERF1 transcript accumulated
mostly at the later stage of fruit senescence, reaching a peak at 5 d and 35 d
in the fruit stored at 25 °C and 4 °C, respectively, or 36 h after the fruit was
transferred from low temperature to room temperature. Moreover, application of
nitric oxide (NO) delayed fruit senescence, enhanced the expression of DlHD2,
but suppressed the expression of DlERF1 and DlERF2. These results indicated a
possible interaction between DlHD2 and DlERFs in regulating longan fruit
senescence, and the direct interaction between DlHD2 and DlERF1 was confirmed by
yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays.
Taken together, the results suggested that DlHD2 may act with DlERF1 to regulate
gene expression involved in longan fruit senescence. DNA methylation and histone acetylation/deacetylation are distinct biochemical
processes that control gene expression. While DNA methylation is a common
epigenetic signal that inhibits gene transcription, histone deacetylation
similarly represses transcription but can be both an epigenetic and
nonepigenetic phenomenon. Here we report that the histone deacetylase SIRT1
regulates the activities of DNMT1, a key enzyme responsible for DNA methylation.
In mass spectrometry analysis, 12 new acetylated lysine sites were identified in
DNMT1. SIRT1 physically associates with DNMT1 and can deacetylate acetylated
DNMT1 in vitro and in vivo. Interestingly, deacetylation of different lysines on
DNMT1 has different effects on the functions of DNMT1. For example,
deacetylation of Lys1349 and Lys1415 in the catalytic domain of DNMT1 enhances
DNMT1's methyltransferase activity, while deacetylation of lysine residues in
the GK linker decreases DNMT1's methyltransferase-independent transcriptional
repression function. Furthermore, deacetylation of all identified acetylated
lysine sites in DNMT1 abrogates its binding to SIRT1 and impairs its capability
to regulate cell cycle G(2)/M transition. Finally, inhibition of SIRT1
strengthens the silencing effects of DNMT1 on the expression of tumor suppressor
genes ER-α and CDH1 in MDA-MB-231 breast cancer cells. Together, these results
suggest that SIRT1-mediated deacetylation of DNMT1 is crucial for DNMT1's
multiple effects in gene silencing. CD43 (leukosialin, sialophorin), a cell surface protein on most hematopoietic
cells, is an important regulator of immune cell function and is involved in
regulation of cell adhesion and proliferation. Aberrant expression of CD43 is a
common event observed in human tumors of non-hematopoietic origin suggesting a
role in tumor development. We have previously shown that overexpression of CD43
causes activation of the ARF-p53 tumor-suppressor pathway and results in cell
death. In a non-functional ARF-p53 background, the cells overexpressing CD43
display an increased cell growth rate due to higher survival. Here we show that
p53 specifically downregulates the expression of CD43 at the protein and mRNA
level. Transactivating properties of p53 are necessary to affect the expression
of exogenous CD43. The downregulation of CD43 mRNA is caused by p53-dependent
transrepression, at least in part, via a histone deacetylation mechanism. These
studies establish that under certain conditions there exists a negative feedback
loop between p53 and CD43: CD43-dependent signaling activates p53, which in turn
downregulates the expression of CD43. OBJECTIVE: Class IIa histone deacetylases (HDACs) belong to a large family of
enzymes involved in protein deacetylation and play a role in regulating gene
expression and cell differentiation. Previously, we showed that HDAC inhibitors
modify the timing and determination of pancreatic cell fate. The aim of this
study was to determine the role of class IIa HDACs in pancreas development.
RESEARCH DESIGN AND METHODS: We took a genetic approach and analyzed the
pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a
novel method of lentiviral infection of pancreatic explants and performed
gain-of-function experiments.
RESULTS: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted
expression in the endocrine β- and δ-cells of the pancreas. Analyses of the
pancreas of class IIa HDAC mutant mice revealed an increased pool of
insulin-producing β-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased
pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice.
Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of
insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment
of pancreatic explants with the selective class IIa HDAC inhibitor MC1568
enhances expression of Pax4, a key factor required for proper β-and δ-cell
differentiation and amplifies endocrine β- and δ-cells.
CONCLUSIONS: We conclude that HDAC4, -5, and -9 are key regulators to control
the pancreatic β/δ-cell lineage. These results highlight the epigenetic
mechanisms underlying the regulation of endocrine cell development and suggest
new strategies for β-cell differentiation-based therapies. IL-2 is a key cytokine during proliferation and activation of T lymphocytes and
functions as an auto- and paracrine growth factor. Regardless of activating
effects on T lymphocytes, the absence of IL-2 has been linked to the development
of autoimmune pathology in mice and humans. Systemic lupus erythematosus (SLE)
is a multifactorial autoimmune disease and characterized by dysregulation of
lymphocyte function, transcription factor and cytokine expression, and antigen
presentation. Reduced IL-2 expression is a hallmark of SLE T lymphocytes and
results in decreased numbers of regulatory T lymphocytes which play an important
role in preventing autoimmunity. Reduced IL-2 expression was linked to
overproduction of the transcription regulatory factor cAMP-responsive element
modulator (CREM)α in SLE T lymphocytes and subsequent CREMα binding to a CRE
site within the IL2 promoter (-180 CRE). In this study, we demonstrate the
involvement of CREMα-mediated IL2 silencing in T lymphocytes from SLE patients
through a gene-wide histone deacetylase 1-directed deacetylation of histone
H3K18 and DNA methyltransferase 3a-directed cytosine phosphate guanosine
(CpG)-DNA hypermethylation. For the first time, we provide direct evidence that
CREMα mediates silencing of the IL2 gene in SLE T cells though histone
deacetylation and CpG-DNA methylation. Klotho is a single pass transmembrane protein, associated with premature aging.
We identified tumor suppressor activities for klotho, associated with reduced
expression in breast cancer. We now aimed to analyze klotho expression in early
stages of breast tumorigenesis and elucidate mechanisms leading to klotho
silencing in breast tumors. We studied klotho expression, using
immunohistochemistry, and found high klotho expression in all normal and mild
hyperplasia samples, whereas reduced expression was associated with moderate and
atypical ductal hyperplasia. Promoter methylation and histone deacetylation were
studied as possible mechanisms for klotho silencing. Using bisulfite sequencing,
and methylation-specific PCR, we identified KLOTHO promoter methylation in five
breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the
non-tumorous mammary cell line HB2. Importantly, methylation status inversely
correlated with klotho mRNA levels, and treatment of breast caner cells with
5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO
promoter methylation was detected in 8/23 of breast cancer samples but not in
normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO
promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was
deacetylated, and treatment with the histone deacetylase inhibitor
suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together,
these data indicate loss of klotho expression as an early event in breast cancer
development, and suggest a role for DNA methylation and histone deacetylation in
klotho silencing. Klotho expression and methylation may, therefore, serve as
early markers for breast tumorigenesis. Chronic inflammation impairs metabolic homeostasis and is intimately correlated
with the pathogenesis of type 2 diabetes. The pro-inflammatory cytokine IFN-γ is
an integral part of the metabolic inflammation circuit and contributes
significantly to metabolic dysfunction. The underlying mechanism, however,
remains largely unknown. In the present study, we report that IFN-γ disrupts the
expression of genes key to cellular metabolism and energy expenditure by
repressing the expression and activity of SIRT1 at the transcription level.
Further analysis reveals that IFN-γ requires class II transactivator (CIITA) to
repress SIRT1 transcription. CIITA, once induced by IFN-γ, is recruited to the
SIRT1 promoter by hypermethylated in cancer 1 (HIC1) and promotes
down-regulation of SIRT1 transcription via active deacetylation of core histones
surrounding the SIRT1 proximal promoter. Silencing CIITA or HIC1 restores SIRT1
activity and expression of metabolic genes in skeletal muscle cells challenged
with IFN-γ. Therefore, our data delineate an IFN-γ/HIC1/CIITA axis that
contributes to metabolic dysfunction by suppressing SIRT1 transcription in
skeletal muscle cells and as such shed new light on the development of novel
therapeutic strategies against type 2 diabetes. Cell fate depends on the interplay between chromatin regulators and
transcription factors. Here we show that activity of the Mi-2β
nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by
the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral
component of the NuRD complex in lymphocytes, tethered this complex to active
genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros
DNA-binding activity caused a local increase in chromatin remodeling and histone
deacetylation and suppression of lymphoid cell-specific gene expression. Without
Ikaros, the NuRD complex also redistributed to transcriptionally poised genes
that were not targets of Ikaros (encoding molecules involved in proliferation
and metabolism), which induced their reactivation. Thus, release of NuRD from
Ikaros regulation blocks lymphocyte maturation and mediates progression to a
leukemic state by engaging functionally opposing epigenetic and genetic
networks. RNA molecules such as small-interfering RNAs (siRNAs) and antisense RNAs
(asRNAs) trigger chromatin silencing of target loci. In the model plant
Arabidopsis, RNA-triggered chromatin silencing involves repressive histone
modifications such as histone deacetylation, histone H3 lysine-9 methylation,
and H3 lysine-27 monomethylation. Here, we report that two Arabidopsis homologs
of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48
(RbAp46/48), known as MSI4 (or FVE) and MSI5, function in partial redundancy in
chromatin silencing of various loci targeted by siRNAs or asRNAs. We show that
MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C (FLC),
which is a crucial floral repressor subject to asRNA-mediated silencing, FLC
homologs, and other loci including transposable and repetitive elements which
are targets of siRNA-directed DNA Methylation (RdDM). Both FVE and MSI5
associate with HISTONE DEACETYLASE 6 (HDA6) to form complexes and directly
interact with the target loci, leading to histone deacetylation and
transcriptional silencing. In addition, these two genes function in de novo CHH
(H = A, T, or C) methylation and maintece of symmetric cytosine methylation
(mainly CHG methylation) at endogenous RdDM target loci, and they are also
required for establishment of cytosine methylation in the previously
unmethylated sequences directed by the RdDM pathway. This reveals an important
functional divergence of the plant RbAp46/48 relatives from animal counterparts. Epigenetic regulation of gene expression is important in maintaining
self-renewal of embryonic stem (ES) and trophoblast stem (TS) cells. Histone
deacetylases (HDACs) negatively control histone acetylation by removing covalent
acetylation marks from histone tails. Because histone acetylation is a known
mark for active transcription, HDACs presumably associate with inactive genes.
Here, we used genome-wide chromatin immunoprecipitation to investigate targets
of HDAC1 in ES and TS cells. Through evaluation of genes associated with
acetylated histone H3 marks, and global expression analysis of Hdac1 knockout ES
and trichostatin A-treated ES and TS cells, we found that HDAC1 occupies mainly
active genes, including important regulators of ES and TS cells self-renewal. We
also observed occupancy of methyl-CpG binding domain protein 3 (MBD3), a subunit
of the nucleosome remodeling and histone deacetylation (NuRD) complex, at a
subset of HDAC1-occupied sequences in ES cells, including the pluripotency
regulators Oct4, Nanog and Kfl4. By mapping HDAC1 targets on a global scale, our
results describe further insight into epigenetic mechanisms of ES and TS cells
self-renewal. HIV-1 Vpr-binding protein (VprBP) has been implicated in the regulation of both
DNA replication and cell cycle progression, but its precise role remains
unclear. Here we report that VprBP regulates the p53-induced transcription and
apoptotic pathway. VprBP is recruited to p53-responsive promoters and suppresses
p53 transactivation in the absence of stress stimuli. To maintain target
promoters in an inactive state, VprBP stably binds to nucleosomes by recognizing
unacetylated H3 tails. Promoter-localized deacetylation of H3 tails is a
prerequisite for VprBP to tether and act as a bona fide inhibitor at p53 target
genes. VprBP knockdown leads to activation of p53 target genes and causes an
increase in DNA damage-induced apoptosis. Moreover, phosphorylation of VprBP at
serine 895 impairs the ability of VprBP to bind H3 tails and to repress p53
transactivation. Our results thus reveal a new role for VprBP in regulation of
the p53 signaling pathway, as well as molecular mechanisms of cancer development
related to VprBP misregulation. Relatively little is known about the regulatory mechanisms of the Drosha/DGCR8
complex, which processes miRNAs at the initial step of biogenesis. We found that
histone deacetylase 1 (HDAC1) increases the expression levels of mature miRNAs
despite repressing the transcription of host genes. HDAC1 is an integral
component of the Drosha/DGCR8 complex and enhances miRNA processing by
increasing the affinity of DGCR8 to primary miRNA transcripts via deacetylation
of critical lysine residues in the RNA-binding domains of DGCR8. This finding
suggests that HDACs have two arms for gene silencing: transcriptional repression
by promoter histone deacetylation and post-transcriptional inhibition by
increasing miRNA abundance. Huntington's disease (HD) is an autosomal domit progressive neurodegenerative
disorder caused by an expansion of a CAG/polyglutamine repeat for which there
are no disease modifying treatments. In recent years, transcriptional
dysregulation has emerged as a pathogenic process that appears early in disease
progression and has been recapitulated across multiple HD models. Altered
histone acetylation has been proposed to underlie this transcriptional
dysregulation and histone deacetylase (HDAC) inhibitors, such as suberoylanilide
hydroxamic acid (SAHA), have been shown to improve polyglutamine-dependent
phenotypes in numerous HD models. However potent pan-HDAC inhibitors such as
SAHA display toxic side-effects. To better understand the mechanism underlying
this potential therapeutic benefit and to dissociate the beneficial and toxic
effects of SAHA, we set out to identify the specific HDAC(s) involved in this
process. For this purpose, we are exploring the effect of the genetic reduction
of specific HDACs on HD-related phenotypes in the R6/2 mouse model of HD. The
study presented here focuses on HDAC3, which, as a class I HDAC, is one of the
preferred targets of SAHA and is directly involved in histone deacetylation. To
evaluate a potential benefit of Hdac3 genetic reduction in R6/2, we generated a
mouse carrying a critical deletion in the Hdac3 gene. We confirmed that the
complete knock-out of Hdac3 is embryonic lethal. To test the effects of HDAC3
inhibition, we used Hdac3(+/-) heterozygotes to reduce nuclear HDAC3 levels in
R6/2 mice. We found that Hdac3 knock-down does not ameliorate physiological or
behavioural phenotypes and has no effect on molecular changes including
dysregulated transcripts. We conclude that HDAC3 should not be considered as the
major mediator of the beneficial effect induced by SAHA and other HDAC
inhibitors in HD. The Epstein-Barr virus (EBV) predomitly establishes latent infection in B
cells, and the reactivation of the virus from latency is dependent on the
expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits
only low basal activity but is activated in response to chemical or biological
inducers, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium
ionophores, or histone deacetylase (HDAC) inhibitors. In some cell lines
latently infected with EBV, an HDAC inhibitor alone can induce BZLF1
transcription, while the treatment does not enhance expression in other cell
lines, such as B95-8 or Raji cells, suggesting unknown suppressive mechanisms
besides histone deacetylation in those cells. Here, we found the epigenetic
modification of the BZLF1 promoter in latent Raji cells by histone H3 lysine 27
trimethylation (H3K27me3), H3K9me2/me3, and H4K20me3. Levels of active markers
such as histone acetylation and H3K4me3 were low in latent cells but increased
upon reactivation. Treatment with 3-deazaneplanocin A (DZNep), an inhibitor of
H3K27me3 and H4K20me3, significantly enhanced the BZLF1 transcription in Raji
cells when in combination with an HDAC inhibitor, trichostatin A (TSA). The
knockdown of Ezh2 or Suv420h1, histone methyltransferases for H3K27me3 or
H4K20me3, respectively, further proved the suppression of Zp by the
methylations. Taken together, the results indicate that H3K27 methylation and
H4K20 methylation are involved, at least partly, in the maintece of latency,
and histone acetylation and H3K4 methylation correlate with the reactivation of
the virus in Raji cells. Runx2, a runt-related transcriptional factor family member, is involved in the
regulation of osteoblast differentiation. Interestingly, it is abundant in
growth-arrested 3T3-L1 preadipocytes and was dramatically down-regulated during
adipocyte differentiation. Knockdown of Runx2 expression promoted 3T3-L1
adipocyte differentiation, whereas overexpression inhibited adipocyte
differentiation and promoted the trans-differentiation of 3T3-L1 preadipocytes
to bone cells. Runx2 was down-regulated specifically by dexamethasone (DEX).
Only type I Runx2 was expressed in 3T3-L1 preadipocytes. Using luciferase assay
and chromatin immunoprecipitation-quantitative PCR analysis, it was found that
DEX repressed this type of Runx2 at the transcriptional level through direct
binding of the glucocorticoid receptor (GR) to a GR-binding element in the Runx2
P2 promoter. Further studies indicated that GR recruited histone deacetylase 1
to the Runx2 P2 promoter which then mediated the deacetylation of histone H4 and
down-regulated Runx2 expression. Runx2 might play its repressive role through
the induction of p27 expression, which blocked 3T3-L1 adipocyte differentiation
by inhibiting mitotic clonal expansion. Taken together, we identified Runx2 as a
new downstream target of DEX and explored a new pathway between DEX, Runx2, and
p27 which contributed to the mechanism of the 3T3-L1 adipocyte differentiation. Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by
recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins.
Ume6p repression is relieved in a two-step destruction process mediated by the
anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step
induces partial Ume6p degradation when vegetative cells shift from glucose- to
acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here
we demonstrate that the first step in Ume6p destruction is controlled by its
acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p,
respectively. Ume6p acetylation occurs in medium lacking dextrose and results in
a partial destruction of the repressor. Preventing acetylation delays Ume6p
meiotic destruction and retards both the transient transcription program and
execution of the meiotic nuclear divisions. Conversely, mimicking acetylation
induces partial destruction of Ume6p in dextrose medium and accelerates meiotic
degradation by the APC/C. These studies reveal a new mechanism by which
acetyltransferase activity induces gene expression through targeted destruction
of a transcriptional repressor. These findings also demonstrate an important
role for nonhistone acetylation in the transition between mitotic and meiotic
cell division. AFP-producing adenocarcinoma is a variant of adenocarcinoma with high
maligcy. Production of AFP suggests enteroblastic or hepatoid differentiation
of cancer cells. GATA4 is a key molecule involved in the prenatal development of
the stomach and liver. GATA4 is epigenetically silenced by hypermethylation of
primer region in many types of cancers including gastric cancer. The aim of this
study is to investigate the expression and epigenetic regulation of GATA4 in
AFP-producing adenocarcinoma. Immunohistochemical analysis revealed that GATA4
was positive in 3/8 cases of AFP-producing gastric adenocarcinomas and in 28/30
cases of common type adenocarcinomas. Epigenetic modification of GATA4 promoter
region was investigated with 3 AFP-producing and 4 common-type gastric cancer
cell lines. GATA4 mRNA was detected in 1/3 of AFP-producing and 2/4 of
common-type gastric cancer cell lines by RT-PCR. Methylation-specific PCR
revealed no GATA4 methylation in any of the AFP-producing gastric cancers,
whereas methylation was consistent with GATA4 expression in the common-type
gastric cancers. Chromatin immunoprecipitation assay for AFP-producing gastric
cancers revealed that histones H3 and H4 were hypoacetylated in the
GATA4-negative cells, while they were hyperacetylated in the GATA4-positive
cells. Treatment with trichostain A, an inhibitor for histone deacetylase,
induced acetylation of histones H3 and H4, and tri-methylation of lysine 4 of
histone H3, which was associated with the active transcription of GATA4 in
GATA4-negative AFP-producing cells. These results indicated that histone
deacetylation is a silencing mechanism for GATA4 expression in AFP-producing
gastric cancer cells. Differences between AFP-producing gastric cancer and
common-type gastric cancer in terms of the mechanism of GATA4 regulation may be
reflected in the phenotypic deviation of AFP-producing gastric cancer from
common-type gastric cancer. DNA methylation, methylation of histone H3 at Lys9 (H3K9me3) and hypoacetylated
histones are common molecular features of heterochromatin. Important details of
their functions and inter-relationships remain unclear, however. In Neurospora
crassa, H3K9me3 directs DNA methylation through a complex containing
heterochromatin protein 1 (HP1) and the DNA methyltransferase DIM-2. We
identified a distinct HP1 complex, HP1, CDP-2, HDA-1 and CHAP (HCHC), and found
that it is responsible for silencing independently of DNA methylation. HCHC
defects cause hyperacetylation of centromeric histones, greater accessibility of
DIM-2 and hypermethylation of centromeric DNA. Loss of HCHC also causes
mislocalization of the DIM-5 H3K9 methyltransferase at a subset of interstitial
methylated regions, leading to selective DNA hypomethylation. We demonstrate
that HP1 forms distinct DNA methylation and histone deacetylation complexes that
work in parallel to assemble silent chromatin in N. crassa. Higher order chromatin structure in eukaryotes can lead to differential gene
expression in response to the same transcription factor; however, how
transcription factor inputs integrate with quantitative features of the
chromatin environment to regulate gene expression is not clear. In vitro models
of HIV gene regulation, in which repressive mechanisms acting locally at an
integration site keep proviruses transcriptionally silent until appropriately
stimulated, provide a powerful system to study gene expression regulation in
different chromatin environments. Here we quantified HIV expression as a
function of activating transcription factor nuclear factor-κB RelA/p65 (RelA)
levels and chromatin features at a panel of viral integration sites. Variable
RelA overexpression demonstrated that the viral genomic location sets a
threshold RelA level necessary to induce gene expression. However, once the
induction threshold is reached, gene expression increases similarly for all
integration sites. Furthermore, we found that higher induction thresholds are
associated with repressive histone marks and a decreased sensitivity to nuclease
digestion at the LTR promoter. Increasing chromatin accessibility via inhibition
of histone deacetylation or DNA methylation lowered the induction threshold,
demonstrating that chromatin accessibility sets the level of RelA required to
activate gene expression. Finally, a functional relationship between gene
expression, RelA level, and chromatin accessibility accurately predicted
synergistic HIV activation in response to combinatorial pharmacological
perturbations. Different genomic environments thus set a threshold for
transcription factor activation of a key viral promoter, which may point toward
biological principles that underlie selective gene expression and inform
strategies for combinatorial therapies to combat latent HIV. BACKGROUND: Liposarcomas are the most common class of soft tissue sarcomas, and
myxoid liposarcoma is the second most common liposarcoma. EWSR1-DDIT3 is a
chimeric fusion protein generated by the myxoid liposarcoma-specific chromosomal
translocation t(12;22)(q13;q12). Current studies indicate that multipotent
mesenchymal cells are the origin of sarcomas. The mechanism whereby EWSR1-DDIT3
contributes to the phenotypic selection of target cells during oncogenic
transformation remains to be elucidated.
METHODOLOGY/PRINCIPAL FINDINGS: Reporter assays showed that the EWSR1-DDIT3
myxoid liposarcoma fusion protein, but not its wild-type counterparts EWSR1 and
DDIT3, selectively repressed the transcriptional activity of cell
lineage-specific marker genes in multipotent mesenchymal C3H10T1/2 cells.
Specifically, the osteoblastic marker Opn promoter and chondrocytic marker
Col11a2 promoter were repressed, while the adipocytic marker Ppar-γ2 promoter
was not affected. Mutation analyses, transient ChIP assays, and treatment of
cells with trichostatin A (a potent inhibitor of histone deacetylases) or
5-Aza-2'-deoxycytidine (a methylation-resistant cytosine homolog) revealed the
possible molecular mechanisms underlying the above-mentioned selective
transcriptional repression. The first is a genetic action of the EWSR1-DDIT3
fusion protein, which results in binding to the functional C/EBP site within Opn
and Col11a2 promoters through interaction of its DNA-binding domain and
subsequent interference with endogenous C/EBPβ function. Another possible
mechanism is an epigenetic action of EWSR1-DDIT3, which enhances histone
deacetylation, DNA methylation, and histone H3K9 trimethylation at the
transcriptional repression site. We hypothesize that EWSR1-DDIT3-mediated
transcriptional regulation may modulate the target cell lineage through target
gene-specific genetic and epigenetic conversions.
CONCLUSIONS/SIGNIFICANCE: This study elucidates the molecular mechanisms
underlying EWSR1-DDIT3 fusion protein-mediated phenotypic selection of putative
target multipotent mesenchymal cells during myxoid liposarcoma development. A
better understanding of this process is fundamental to the elucidation of
possible direct lineage reprogramming in oncogenic sarcoma transformation
mediated by fusion proteins. The epigenetic remodeling of chromatin histone proteins by acetylation has been
the subject of recent investigations searching for biomarkers indicative of late
onset cognitive loss. Histone acetylations affect the regulation of gene
transcription, and the loss of learning induced deacetylation at specific
histone sites may represent biomarkers for memory loss and Alzheimer's disease
(AD). Selected-reaction-monitoring (SRM) has recently been advanced to
quantitate peptides and proteins in complex biological systems. In this paper,
we provide evidence that SRM-based targeted proteomics can reliably quantify
specific histone acetylations in both AD and control brain by identifying the
patterns of H3 K18/K23 acetylations Results of targeted proteomics assays have
been validated by Western blot (WB) analysis. As compared with LC-MS/MS-TMT
(tandem-mass-tagging) and WB methods, the targeted proteomics method has shown
higher throughput, and therefore promised to be more suitable for clinical
applications. With this methodology, we find that histone acetylation is
significantly lower in AD temporal lobe than found in aged controls. Targeted
proteomics warrants increased application for studying epigenetics of
neurodegenerative diseases. BACKGROUND: Neurons containing proopiomelanocortin (POMC)-derived peptides,
known to control stress axis, metabolic, and immune functions, have a lower
function in patients with a family history of alcoholism, raising the
possibility that alcohol effects on the POMC system may transmit through
generations. Here we describe epigenetic modifications of Pomc gene that
transmit through generation via male germline and may be critically involved in
alcoholism-inherited diseases.
METHODS: Whether an epigenetic mechanism is involved in causing a Pomc
expression deficit in fetal alcohol-exposed rats is studied by determining Pomc
gene methylation, expression, and functional abnormalities and their
normalization following suppression of DNA methylation or histone acetylation.
Additionally, transgenerational studies were conducted to evaluate the
germline-transmitted effect of alcohol.
RESULTS: Fetal alcohol-exposed male and female rat offspring showed a
significant deficit in POMC neuronal functions. Associated with this was an
increased methylation status of several CpG dinucleotides in the proximal part
of the Pomc promoter region and altered level of histone-modifying proteins and
DNA methyltransferases levels in POMC neurons. Suppression of histone
deacetylation and DNA methylation normalized Pomc expression and functional
abnormalities. Fetal alcohol-induced Pomc gene methylation, expression, and
functional defects persisted in the F2 and F3 male but not in female germline.
Additionally, the hypermethylated Pomc gene was detected in sperm of fetal
alcohol-exposed F1 offspring that was transmitted through F3 generation via male
germline.
CONCLUSIONS: Trangenerational epigenetic studies should spur new insight into
the biological mechanisms that influence the sex-dependent difference in genetic
risk of alcoholism-inherited diseases. Anergy is induced in T cells as a consequence of a partial or suboptimal
stimulation. Anergic T cells become unresponsive and fail to proliferate and
produce cytokines. We had previously shown that in anergic CD4(+) T cells,
Ikaros participates in the transcriptional repression of the Il2 gene by
recruiting histone deacetylases that cause core histone deacetylation at the Il2
promoter. Here we show that deacetylation at the Il2 promoter is the initial
step in a process that leads to the stable silencing of the Il2 gene
transcription in anergic T cells. We have found that anergy-induced
deacetylation of the Il2 promoter permits binding of the histone
methyl-transferase Suv39H1, which trimethylates lysine-9 of histone H3
(Me3H3-K9). Furthermore, the establishment of the Me3H3-K9 mark allows the
recruitment of the heterochromatin protein HP1, allowing the silenced Il2 loci
to reposition close to heterochromatin-rich regions. Our results indicate that
silencing of Il2 transcription in anergic T cells is attained through a series
of epigenetic changes that involve the establishment of repressive marks and the
subsequent nuclear repositioning of the Il2 loci, which become juxtaposed to
transcriptionally silent regions. This mechanism may account for the stable
nature of the inhibition of IL-2 production in anergic cells. Tendon overuse injuries and tendinitis are accompanied by catabolic processes
and apoptosis of tenocytes. However, the precise molecular mechanisms of the
destructive processes in tendon are not fully understood. Sirt-1, a nicotinamide
adenine dinucleotide (NAD(+))-dependent deacetylase, has been linked to
transcriptional silencing and appears to play a key role in inflammation. The
purpose of this study was to examine whether down-regulation of Sirt-1 using
antisense oligonucleotides (ASO) affects inflammatory and apoptotic signaling in
tenocytes. Transient transfection of tenocytes with ASO against Sirt-1 induced
expression of Bax and other proteins involved in apoptosis (cleaved caspase-3
and poly(ADP-ribose)polymerase), acetylation of tumor suppressor p53, and
mitochondrial degradation. Interestingly, Sirt-1 was found to interact directly
with p53. In contrast, Sirt-1 activator resveratrol inhibited interleukin-1β
(IL-1β)- and nicotinamide-induced NF-κB activation and p65 acetylation and
suppressed the activation of IκB-α kinase. Resveratrol also reversed the IL-1β-
or nicotinamide-induced up-regulation of various gene products that mediate
inflammation (cyclooxygenase-2) and matrix degradation (matrix
metalloproteinase-9) that are known to be regulated by NF-κB. Knockdown of
Sirt-1 by using ASO abolished the inhibitory effects of resveratrol on
inflammatory and apoptotic signaling including Akt activation and SCAX
suppression. Down-regulation of histone deacetylase Sirt-1 by mRNA interference
abrogated the effect of resveratrol on NF-κB suppression, thus highlighting the
crucial homeostatic role of this enzyme. Overall, these results suggest for the
first time that Sirt-1 can regulate p53 and NF-κB signaling via deacetylation,
demonstrating a novel role for resveratrol in the treatment of
tendinitis/tendinopathy. Epigenetic silencing is one of the mechanisms leading to inactivation of a tumor
suppressor gene, either by DNA methylation or histone modification in a promoter
regulatory region. Mitogen inducible gene 6 (MIG-6), mainly known as a negative
feedback inhibitor of the epidermal growth factor receptor (EGFR) family, is a
tumor suppressor gene that is associated with many human cancers. To determine
if MIG-6 is inactivated by epigenetic alteration, we identified a group of human
lung cancer and melanoma cell lines in which its expression is either low or
undetectable and studied the effects of methylation and of histone deacetylation
on its expression. The DNA methyltransferase (DNMT) inhibitor
5-aza-2'-deoxycytidine (5-aza-dC) induced MIG-6 expression in melanoma cell
lines but little in lung cancer lines. By contrast, the histone deacetylase
(HDAC) inhibitor trichostatin A (TSA) induced MIG-6 expression in lung cancer
lines but had little effect in melanoma lines. However, the MIG-6 promoter
itself did not appear to be directly affected by either methylation or histone
deacetylation, indicating an indirect regulatory mechanism. Luciferase reporter
assays revealed that a short segment of exon 1 in the MIG-6 gene is responsible
for TSA response in the lung cancer cells; thus, the MIG-6 gene can be
epigenetically silenced through an indirect mechanism without having a physical
alteration in its promoter. Furthermore, our data also suggest that MIG-6 gene
expression is differentially regulated in lung cancer and melanoma. Sirtuin proteins regulate diverse cellular pathways that influence genomic
stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical
activity, molecular targets and physiological functions have been unclear. Here
we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of
histone H3) deacetylase that stabilizes the transformed state of cancer cells.
Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific
set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional
repression. The spectrum of SIRT7 target genes is defined in part by its
interaction with the cancer-associated E26 transformed specific (ETS)
transcription factor ELK4, and comprises numerous genes with links to tumour
suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to
oncogenic transformation, and in patients is associated with aggressive tumour
phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is
necessary for maintaining essential features of human cancer cells, including
anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7
is necessary for a global hypoacetylation of H3K18Ac associated with cellular
transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly
reduces the tumorigenicity of human cancer cell xenografts in mice. Together,
our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and
demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular
transformation programs and tumour formation in vivo. Intrathymic CD4/CD8 differentiation is a process that establishes the mutually
exclusive expression profiles of the CD4 and CD8 T cell lineage. The
RUNX3-mediated silencing of CD4 in CD8 lineage cells has been well documented;
however, it is unclear how CD8 is silenced during CD4 lineage differentiation.
In this study, we report that, by directly binding the CD8 locus, ThPOK works as
a negative regulator that mediates the deacetylation of Cd8 genes and
repositions the CD8 alleles close to heterochromatin during the development of
the CD4 lineage. The ectopic expression of ThPOK resulted in increased
recruitment of histone deacetylases at Cd8 loci; the enhanced deacetylation of
Cd8 genes eventually led to impaired Cd8 transcription. In the absence of ThPOK,
the enhanced acetylation and transcription of Cd8 genes were observed. The
results of these studies showed that Cd8 loci are the direct targets of ThPOK,
and, more importantly, they provide new insights into CD8 silencing during CD4
lineage commitment. During cancer development, tumor suppressor genes were silenced by promoter
methylation or histone deacetylation. Histone deacetylases (HDACs) are important
to maintain histone deacetylation. HDAC inhibitors (HDACis) were thus proposed
as a new therapeutic approach to cancer. The current study aims to understand
the effect and molecular mechanisms of HDACis on gastric cancer cells.
Trichostatin A (TSA) significantly inhibited the growth of gastric cancer cells
by inducing apoptosis. Gene profiling results showed PUMA (p53 upregulated
modulator of apoptosis) as one of 122 genes upregulated in TSA-treated gastric
cancer cells. PUMA was downregulated in gastric cancer cell lines and primary
gastric carcinoma tissues. Patients with low PUMA expression had significant
decreases in overall survival (HR, 2.04; p = 0.047). Ectopic PUMA expression
inhibited the growth of gastric cancer cells while PUMA depletion promoted
cellular growth. The knockdown of HDAC3 but not other HDACs upregulated PUMA
expression. HDAC3 could bind to PUMA promoter, which was abrogated after TSA
treatment. In contrast to TSA and SB, HDAC3 siRNA failed to upregulate p53
expression but promoted the interaction of p53 with PUMA promoter. In summary,
proapoptotic PUMA was downregulated in gastric cancer and its mRNA expression
level is a valuable prognosis factor for gastric cancer. HDAC3 is important to
downregulate PUMA expression in gastric cancer and HDACis, like TSA, promoted
PUMA expression through stabilizing p53 in addition to HDAC3 inhibition. In
combination with chemotherapy, targeting HDAC3 might be a promising strategy to
induce apoptosis of gastric cancer cells. Metastasis is a complex process facilitated by the action of several genes.
Metastasis associated 1 (MTA1) gene is one such gene which assists the process
of metastasis by regulating several molecular targets. MTA1 acts as part of a
nucleosome remodelling and histone deacetylation complex, which is involved in
transcriptional regulation. Expression of MTA1 has been shown to be closely
correlated with aggressiveness in several types of cancers, including breast
cancer. In the present study we show that MTA1 regulates SMAD7, a component of
Transforming growth factor beta (TGFbeta) signalling. TGFbeta signals are
transduced to the nucleus by the Smad family of proteins, which includes Smad7,
an inhibitory SMAD, which acts as a negative regulator of TGFbeta. On knockdown
of MTA1, SMAD7 expression increases. Treating cells with a histone deacetylase
inhibitor also increases SMAD7 expression. MTA1 is recruited to SMAD7 promoter
region. SMAD7 inhibits activation of SMAD2 and SMAD3 and we show that the levels
of these active SMAD proteins are decreased in cells expressing shRNA against
MTA1. We further show that on MTA1 knockdown, the expression of downstream
targets of SMAD7 is decreased. MTA1 thus appears to regulate a key inhibitor of
TGFbeta signalling, SMAD7. By regulating molecules like SMAD7 MTA1 might assist
the process of tumourigenesis and metastasis. The amount of arginine available at inflammatory loci is a limiting factor for
the growth of several cells of the immune system. IL-4-induced activation of
macrophages produced arginase-1, which converts arginine into ornithine, a
precursor of polyamines and proline. Trichostatin A (TSA), a pan-inhibitor of
histone deacetylases (HDACs), inhibited IL-4-induced arginase-1 expression. TSA
showed promoter-specific effects on the IL-4-responsive genes. While TSA
inhibited the expression of arginase-1, fizz1, and mrc1, other genes, such as
ym,1 mgl1, and mgl2, were not affected. The inhibition of arginase-1 occurred at
the transcriptional level with the inhibition of polymerase II binding to the
promoter. IL-4 induced STAT6 phosphorylation and binding to DNA. These
activities were not affected by TSA treatment. However, TSA inhibited C/EBPβ DNA
binding. This inhibitor induced acetylation on lysine residues 215-216, which
are critical for DNA binding. Finally, using macrophages from STAT6 KO mice we
showed that STAT6 is required for the DNA binding of C/EBPβ. These results
demonstrate that the acetylation/deacetylation balance strongly influences the
expression of arginase-1, a gene of alternative activation of macrophages. These
findings also provide a molecular mechanism to explain the control of gene
expression through deacetylase activity. Amphibians such as Xenopus laevis and Ambystoma mexicanum are capable of whole
structure regeneration. However, transcriptional control over these events is
not well understood. Here, we investigate the role of histone deacetylase (HDAC)
enzymes in regeneration using HDAC inhibitors. The class I/II HDAC inhibitor
valproic acid (VPA) inhibits tail regeneration in embryos of the anuran
amphibian Xenopus laevis, confirming a recent report by others (Tseng et al.,
2011). This inhibition correlates with a sixfold reduction in endogenous HDAC
activity. VPA also inhibited tail regeneration in post-refractory stage Xenopus
larvae and larvae of the urodele A. mexicanum (axolotl). Furthermore, Xenopus
limb regeneration was also significantly impaired by post-amputation treatment
with VPA, suggesting a general requirement for HDAC activity in the process of
appendage regeneration in amphibians. The most potent inhibition of tail
regeneration was observed following treatment with VPA during the wound healing,
pre-blastema phase. A second HDAC inhibitor, sodium butyrate, was also shown to
inhibit tail regeneration. While both VPA and sodium butyrate are reported to
block sodium channel function as well as HDACs, regeneration was not inhibited
by valpromide, an analogue of VPA that lacks HDAC inhibition but retains sodium
channel blocking activity. Finally, although VPA is a known teratogen, we show
that neither tailbud nor limb bud development are affected by exposure to this
compound. We conclude that histone deacetylation is specifically required for
the earliest events in appendage regeneration in amphibians, and suggest that
this may act as a switch to trigger re-expression of developmental genes. Following amino acid deprivation, the amino acid response (AAR) induces
transcription from specific genes through a collection of signaling mechanisms,
including the GCN2-eIF2-ATF4 pathway. The present report documents that the
histone demethylase JMJD3 is an activating transcription factor 4
(ATF4)-dependent target gene. The JMJD3 gene contains two AAR-induced promoter
activities and chromatin immunoprecipitation (ChIP) analysis showed that the AAR
leads to enhanced ATF4 recruitment to the C/EBP-ATF response element (CARE)
upstream of Promoter-1. AAR-induced histone modifications across the JMJD3 gene
locus occur upon ATF4 binding. Jmjd3 transcription is not induced in
Atf4-knock-out cells, but the AAR-dependent activation was rescued by inhibition
of histone deacetylation with trichostatin A (TSA). The TSA rescue of AAR
activation in the absence of Atf4 also occurred for the Atf3 and C/EBP homology
protein (Chop) genes, but not for the asparagine synthetase gene. ChIP analysis
of the Jmjd3, Atf3, and Chop genes in Atf4 knock-out cells documented that
activation of the AAR in the presence of TSA led to specific changes in
acetylation of histone H4. The results suggest that a primary function of ATF4
is to recruit histone acetyltransferase activity to a sub-set of AAR target
genes. Thus, absolute binding of ATF4 to these particular genes is not required
and no ATF4 interaction with the general transcription machinery is necessary.
The data are consistent with the hypothesis that ATF4 functions as a pioneer
factor to alter chromatin structure and thus, enhance transcription in a
gene-specific manner. Core histone modifications play an important role in chromatin remodeling and
transcriptional regulation. Histone acetylation is one of the best-studied gene
modifications and has been shown to be involved in numerous important biological
processes. Herein, we demonstrated that the depletion of histone deacetylase 3
(Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further
genetic studies showed that Hdac3 counteracted the organ overgrowth induced by
overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6
kinase (S6K), and the growth regulation by Hdac3 was mediated through the
deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations
of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of
males-absent-on-the-first (MOF), a histone acetyltransferase that specifically
targets H4K16, resulted in changes in body size. Furthermore, we found that
H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K
pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K
pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the
depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and
PI3K-mediated alterations in the transcription profile. Overall, our studies
indicated that Hdac3 served as an important regulator of the PI3K pathway and
revealed a novel link between histone acetylation and growth control. The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at
lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed
regions. To discern how Set3C affects gene expression, genome-wide transcription
was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3
primarily caused changes during transition periods, as genes were induced or
repressed. Surprisingly, a majority of Set3-affected genes are overlapped by
noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2
instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA
transcription from distal or antisense promoters. Set3C also represses internal
cryptic promoters, but in different regions of genes than the Set2/Rpd3S
pathway. Finally, Set3C stimulates some genes by repressing an overlapping
antagonistic antisense transcript. These results show that overlapping noncoding
transcription can fine-tune gene expression, not via the ncRNA but by depositing
H3K4me2 to recruit the Set3C deacetylase. Gene silencing technology, such as RNA interference (RNAi), is commonly used to
reduce gene expression in plant cells, and exogenous double-stranded RNA (dsRNA)
can induce gene silencing in higher plants. Previously, we showed that the
delivery of double-stranded DNA (dsDNA) fragments, such as PCR products of an
endogenous gene sequence, into fern (Adiantum capillus-veneris) gametophytic
cells induces a sequence-specific gene silencing that we termed DNAi. In this
study, we used a neochrome 1 gene (NEO1) that mediates both red light-induced
chloroplast movement and phototropism as a model of DNAi and confirmed that the
NEO1 function was suppressed by the repression of the NEO1 gene. Interestingly,
the gene silencing effect by DNAi was found in the progeny. Cytosine methylation
was detected in the NEO1-silenced lines. The DNA modifications was present in
the transcriptional region of NEO1, but no differences between wild type and the
silenced lines were found in the downstream region of NEO1. Our data suggest
that the DNAi gene silencing effect that was inherited throughout the next
generation is regulated by epigenetic modification. Furthermore, the histone
deacetylase inhibitor, trichostatin A (TSA), recovered the expression and
function of NEO1 in the silenced lines, suggesting that histone deacetylation is
essential for the direct suppression of target genes by DNAi. The class IIa histone deacetylases (HDACs) act as transcriptional repressors by
altering chromatin structure through histone deacetylation. This family of
enzymes regulates muscle development and phenotype, through regulation of
muscle-specific genes including myogenin and MyoD (MYOD1). More recently, class
IIa HDACs have been implicated in regulation of genes involved in glucose
metabolism. However, the effects of HDAC5 on glucose metabolism and insulin
action have not been directly assessed. Knockdown of HDAC5 in human primary
muscle cells increased glucose uptake and was associated with increased GLUT4
(SLC2A4) expression and promoter activity but was associated with reduced GLUT1
(SLC2A1) expression. There was no change in PGC-1α (PPARGC1A) expression. The
effects of HDAC5 knockdown on glucose metabolism were not due to alterations in
the initiation of differentiation, as knockdown of HDAC5 after the onset of
differentiation also resulted in increased glucose uptake and insulin-stimulated
glycogen synthesis. These data show that inhibition of HDAC5 enhances metabolism
and insulin action in muscle cells. As these processes in muscle are
dysregulated in metabolic disease, HDAC inhibition could be an effective
therapeutic strategy to improve muscle metabolism in these diseases. Therefore,
we also examined the effects of the pan HDAC inhibitor, Scriptaid, on muscle
cell metabolism. In myotubes, Scriptaid increased histone 3 acetylation, GLUT4
expression, glucose uptake and both oxidative and non-oxidative metabolic flux.
Together, these data suggest that HDAC5 regulates muscle glucose metabolism and
insulin action and that HDAC inhibitors can be used to modulate these parameters
in muscle cells. During initial development, both X chromosomes are active in females, and one of
them must be silenced at the appropriate time in order to dosage compensate
their gene expression levels to male counterparts. Silencing involves epigenetic
mechanisms, including histone deacetylation. Major X chromosome inactivation
(XCI) in bovine occurs between hatching and implantation, although in vitro
culture conditions might disrupt the silencing process, increasing or decreasing
X-linked gene expression. In this study, we aimed to address the roles of
histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation
development. We tested the hypothesis that by enhancing histone acetylation, TSA
would increase the percentage of embryos achieving 16-cell stage, reducing
percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF
embryos. We noticed that after TSA treatment, acetylation levels in individual
blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and
the same was detected for blastocysts. Changes among blastomere levels within
the same embryo were diminished on TSA group, as low-acetylated blastomeres were
no longer detected. The percentage of embryos that reached the 5th cleavage
cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after
TSA treatment. Then, we assessed XIST and G6PD expression in individual female
bovine blastocysts by quantitative real-time PCR. Even though G6PD expression
remained unaltered after TSA exposure, XIST expression was eightfold decreased,
and we also detected a major decrease in the percentage of blastocysts
expressing detectable XIST levels after TSA treatment. Based on these results,
we conclude that HDAC is involved on XCI process in bovine embryos, and its
inhibition might delay X chromosome silencing and attenuate aberrant XIST
expression described for IVF embryos. Brain tumors encompass a heterogeneous group of maligt tumors with variable
histopathology, aggressiveness, clinical outcome and prognosis. Current gene
expression profiling studies indicate interplay of genetic and epigenetic
alterations in their pathobiology. A central molecular event underlying
epigenetics is the alteration of chromatin structure by post-translational
modifications of DNA and histones as well as nucleosome repositioning. Dynamic
remodeling of the fundamental nucleosomal structure of chromatin or covalent
histone marks located in core histones regulate main cellular processes
including DNA methylation, replication, DNA-damage repair as well as gene
expression. Deregulation of these processes has been linked to tumor suppressor
gene silencing, cancer initiation and progression. The reversible nature of
deregulated chromatin structure by DNA methylation and histone deacetylation
inhibitors, leading to re-expression of tumor suppressor genes, makes
chromatin-remodeling pathways as promising therapeutic targets. In fact, a
considerable number of these inhibitors are being tested today either alone or
in combination with other agents or conventional treatments in the management of
brain tumors with considerable success. In this review, we focus on the
mechanisms underpinning deregulated chromatin remodeling in brain tumors,
discuss their potential clinical implications and highlight the advances toward
new therapeutic strategies. Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed
in both basic and preclinical gene transfer settings. Often, however, the IDLV
transgene expression profile is muted when compared to that of their
integration-proficient counterparts. We hypothesized that the episomal nature of
IDLVs turns them into preferential targets for epigenetic silencing involving
chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array
of cis-acting elements and transcriptional unit components were assembled with
the aid of packaging constructs encoding either the wild-type or the class I
mutant D116N integrase moieties. The transduction levels and transgene-product
yields provided by each vector class were assessed in the presence and absence
of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A.
To investigate the role of the target cell replication status, we performed
experiments in growth-arrested human mesenchymal stem cells and in post-mitotic
syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless
of their genetic makeup or target cell replication rate. Interestingly, the
magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a
vector backbone- and cell type-dependent manner. Finally, investigation of
histone modifications by chromatin immunoprecipitation followed by quantitative
PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV
genomes when compared to those measured on isogenic integration-competent vector
templates. These findings support the view that IDLVs constitute preferential
targets for epigenetic silencing involving histone deacetylation, which
contributes to dampening their full transcriptional potential. Our data provide
leads on how to most optimally titrate and deploy these promising episomal gene
delivery vehicles. Theoretical models suggest that gene silencing at the nuclear periphery may
involve "closing" of chromatin by transcriptional repressors, such as histone
deacetylases (HDACs). Here we provide experimental evidence confirming these
predictions. Histone acetylation, chromatin compactness, and gene repression in
lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2
cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were
downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the
repressed multigenic domain associated with its detachment from the lamina and
enhanced histone acetylation. Our data reveal the major role for HDAC1 in
mediating deacetylation, chromatin compaction, and gene silencing in the
multigenic domain, and an auxiliary role for HDAC3 that is required for
retention of the domain at the lamina. These findings demonstrate the manifold
and central involvement of class I HDACs in regulation of lamina-associated
genes, illuminating a mechanism by which these enzymes can orchestrate normal
and pathological development. ASYMMETRIC LEAVES 1 (AS1) is a MYB-type transcription repressor that controls
leaf development by regulating KNOX gene expression, but the underlying
molecular mechanism is still unclear. In this study, we demonstrated that AS1
can interact with the histone deacetylase HDA6 in vitro and in vivo. The KNOX
genes were up-regulated and hyperacetylated in the hda6 mutant, axe1-5,
indicating that HDA6 may regulate KNOX expression through histone deacetylation.
Compared with the single mutants, the as1-1/axe1-5 and as2-1/axe1-5 double
mutants displayed more severe serrated leaf and short petiole phenotypes. In
addition, the frequencies of leaf lobes and leaflet-like structures were also
increased in as1-1/axe1-5 and as2-1/axe1-5 double mutants, suggesting that HDA6
acts together with AS1 and AS2 in regulating leaf development. Chromatin
immunoprecipitation assays revealed that HDA6 and AS1 bound directly to KNAT1,
KNAT2, and KNATM chromatin. Taken together, these data indicate that HDA6 is a
part of the AS1 repressor complex to regulate the KNOX expression in leaf
development. DNA methylation and histone acetylation are epigenetic modifications that act as
regulators of gene expression. DNA methylation is considered an important
mechanism for silencing of retroelements in the mammalian genome. However, the
methylation of human endogenous retroviruses (HERVs) is not well investigated.
The aim of this study was to investigate the transcriptional potential of
HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of
CpG methylation on this LTR in number of cell lines. Specifically, the role of
demethylating chemicals e.g. 5-aza-2' deoxycytidine and Trichostatin-A, in
inducing or reactivating expression of HERV-Fc1 specific sequences and the
mechanisms were investigated. In our present study, 5-aza-dC is shown to be a
powerful inducer of HERV-Fc1, and at the same time it strongly inhibits
methylation of DNA. Treatment with this demethylating agent 5-aza-dC, results in
significantly increased levels of HERV-Fc1 expression in cells previously not
expressing HERV-Fc1, or with a very low expression level. The extent of
expression of HERV-Fc1 RNAs precisely correlates with the apparent extent of
demethylation of the related DNA sequences. In conclusion, the results suggest
that inhibition of DNA methylation/histone deacetylase can interfere with gene
silencing mechanisms affecting HERV-Fc1 expression in human cells. Histone deacetylases (HDACs) mediate histone deacetylation and act in concert
with histone acetyltransferases to regulate dynamic and reversible histone
acetylation which modifies chromatin structure and function, affects gene
transcription, thus, controlling multiple cellular processes. HDACs are widely
distributed in almost all eukaryotes, and there have been many researches
focusing on plant HDACs recently. An increasing number of HDAC genes have been
identified and characterized in a variety of plant species and the functions of
certain HDACs have been studied. The present studies indicate that HDACs play a
key role in regulating plant growth, development and stress responses. This
paper reviews recent findings on HDACs and their functions in plants, especially
their roles in development and stress responses. Contagious cancers that pass between individuals as an infectious cell line are
highly unusual pathogens. Devil facial tumor disease (DFTD) is one such
contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to
extinction. As both a pathogen and an allograft, DFTD cells should be rejected
by the host-immune response, yet DFTD causes 100% mortality among infected
devils with no apparent rejection of tumor cells. Why DFTD cells are not
rejected has been a question of considerable confusion. Here, we show that DFTD
cells do not express cell surface MHC molecules in vitro or in vivo, due to
down-regulation of genes essential to the antigen-processing pathway, such as
β2-microglobulin and transporters associated with antigen processing. Loss of
gene expression is not due to structural mutations, but to regulatory changes
including epigenetic deacetylation of histones. Consequently, MHC class I
molecules can be restored to the surface of DFTD cells in vitro by using
recombit devil IFN-γ, which is associated with up-regulation of the MHC class
II transactivator, a key transcription factor with deacetylase activity.
Further, expression of MHC class I molecules by DFTD cells can occur in vivo
during lymphocyte infiltration. These results explain why T cells do not target
DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells
may provide a vaccine to DFTD. In addition, we suggest that down-regulation of
MHC molecules using regulatory mechanisms allows evolvability of transmissible
cancers and could affect the evolutionary trajectory of DFTD. |
Which is the subcellular localization of the protein angiogenin? | Under growth conditions, ANG is located in nucleolus where it promotes ribosomal RNA (rRNA) transcription thereby stimulating cell growth. In adverse conditions, ANG is relocated to cytoplasm to promote damage repairs and cell survival. | Angiogenin is endocytosed by subconfluent endothelial cells, translocated to the
nucleus and accumulates in the nucleolus. It also localizes into the nucleolus
of digitonin-permeabilized endothelial cells. The peptide RRRGL corresponding to
residues 31-35 of human angiogenin specifically targets non-nuclear carrier
proteins such as albumin, an anti-human nucleolus monoclonal antibody and R33A
angiogenin to the nucleolus of permeabilized endothelial cells. Proteins
conjugated with a "mutant" peptide, RRAGL, are not imported. Fluorescein
isothiocyanate-conjugated RRRGL is also rapidly imported into the nucleus and
localized to the nucleolus, whereas the "mutant" peptide is not. Residue R33 is
essential for nuclear translocation and R31 and R32 appear to modulate this
process. Thus, 31RRRGL35 is a nuclear localization signal responsible for the
nucleolar targeting of human angiogenin. The review is devoted to angiogenin, one of the factors that induce formation of
blood vessels, which is unique among them in that it is a ribonuclease.
Consideration is given to the tertiary structure of human angiogenin; the
catalytic and cell-receptor binding sites, their significance for angiogenic
activity; the human angiogenin gene structure, chromosomal localization, and
expression; the specificity of angiogenin as a ribonuclease and abolishment of
protein synthesis; the nuclear localization of angiogenin in proliferating
endothelial cells and its significance for angiogenic activity; angiogenin
binding to a cell-surface actin as a plausible mechanism of inducing
neovascularization (enhancement of plasminogen activation by actin with
angiogenin, stimulation of the cell-associated proteolytic activity by
angiogenin; promotion of the cultured cells invasiveness); modulation of
mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by
angiogenin. The importance of angiogenin as an adhesive molecule for endothelial
and tumor cells is discussed too, as well as the modulation of tubular
morphogenesis by bovine angiogenin, prevention of tumor growth in vivo by
angiogenin antagonists, prospects of the use of angiogenin and
angiogenin-encoding recombit plasmids and vaccinia virus in therapeutic
practice. Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia
coli and then purified with gel filtration chromatography. Approximately 90%
pure angiogenin was obtained to generate a monoclonal antibody. Using western
immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted
from hybridoma cells stably and specifically binds to angiogenin. The fused
protein angiogenin-EGF was then expressed in HUVECs, and the subcellular
localization of the recombit protein was determined by confocal microscopy
and TEM assay. Recombit angiogenin was found to mainly concentrate in the
pars granulosa of the nucleus, where the protein accumulates to form
ribonucleoprotein particles. AIMS AND BACKGROUND: Human maligt melanoma is a very aggressive and highly
angiogenesis-dependent tumor. Basic fibroblast growth factor and angiogenin are
the potentially important angiogenic factors for melanoma progression and
metastasis. Many studies have mainly focused on how they induce angiogenesis. In
the present study, we investigated the effects of basic fibroblast growth factor
on the expression of angiogenin and melanoma cell growth.
METHODS AND STUDY DESIGN: Angiogenin mRNA and protein expression were
investigated by means of semi-quantitative reverse transcriptase polymerase
chain reaction assay and western blotting. We analyzed cell proliferation using
MTT, flow cytometry and soft agar assay. Immunofluorescence staining was applied
to investigate co-localization and nuclear translocation.
RESULTS: We found that basic fibroblast growth factor negatively affected the
expression of angiogenin in A375 cells. The result showed that down-regulation
of basic fibroblast growth factor induced decreased cell proliferation ofA375
cells, and in basic fibroblast growth factor up-regulated cells, cell
proliferation was increased. We demonstrated that basic fibroblast growth factor
protein was co-localized with angiogenin and that it underwent nuclear
translocation in A375 cells.
CONCLUSION: These findings suggest that there is a cooperation mechanism between
basic fibroblast growth factor and angiogenin in A375 cells, and the cooperation
mechanism affects the progress of tumor cell proliferation and angiogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with
the angioproliferative Kaposi's sarcoma (KS). KSHV infection and the expression
of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic
multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is
detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL)
cells. ANG knockdown or the inhibition of ANG's nuclear translocation resulted
in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and
PEL cell survival (Sadagopan et al., J. Virol. 83:3342-3364, 2009). Further
studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate
in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and
BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV
genome and other viral proteins. In gel filtration chromatography analyses of
BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in
high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also
coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected
cells, and colocalization between ANG and p53 was also observed in
LANA-1-negative cells. The deletion constructs of ANG suggested that the
C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53
interactions. Silencing ANG or inhibiting its nuclear translocation resulted in
decreased nuclear LANA-1 and ANG levels, decreased interactions between
ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax
proteins, the increased cytoplasmic localization of p53, the downregulation of
Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such
effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at
serine 15, which is essential for p53 stabilization and for p53's apoptotic and
cell cycle regulation functions, was increased in BCBL-1 cells transduced with
short hairpin RNA targeting ANG. Together, these studies suggest that the
antiapoptosis observed in KSHV-infected cells and the suppression of p53
functions are mediated in part by ANG, and KSHV has probably evolved to utilize
angiogenin's multiple functions for the maintece of its latency and cell
survival. Thus, targeting ANG to induce the apoptosis of cells latently infected
with KSHV is an attractive therapeutic strategy against KSHV infection and
associated maligcies. |
List FDA approved treatments for androgenetic allopecia | Recently, in addition to the two currently approved U.S. Food and Drug Administration (FDA) medications (minoxidil and finasteride), a novel device was FDA-approved for the treatment of hair loss, the laser hair comb. | BACKGROUND: Over the last decade surgical management of hair loss has become an
increasingly popular and satisfying procedure for both men and women, as
innovations in donor harvesting, graft size, and hairline design have resulted
in consistently natural-appearing hair restoration.
OBJECTIVE: In addition, a better understanding of the regulation of the
hair-growth cycle has led to advances in the pharmacologic treatment of
androgenetic alopecia.
METHODS: Currently there are two U.S. Food and Drug Administration
(FDA)-approved agents that promote hair regrowth: over-the-counter topical
minoxidil solution for men and women and prescription oral finasteride tablets
for men. In October 2001, a group of 11 international experts on hair loss and
hair transplantation convened to review the physiology and effects of
pharmacologic treatments of hair loss and to discuss the value of administering
topical minoxidil therapy as an adjunct to hair transplantation.
RESULTS: This article presents the key findings and consensus points among the
participants, including their current use of pharmacologic treatments,
strategies for optimal results both pre- and postsurgery, and the importance of
realistic patient expectations and compliance.
CONCLUSIONS: Based on the surgeons' clinical experience, the use of approved
hair regrowth agents in hair transplant patients with viable but suboptimally
functioning follicles in the region to be transplanted can increase hair
density, speed regrowth in transplanted follicles, and complement the surgical
result by slowing down or stopping further hair loss. BACKGROUND: Finasteride (Propecia) was approved by the FDA in 1998 for treating
men with androgenetic alopecia. The published clinical trials demonstrated
statistical differences between drug and placebo. Rarely do new drugs undergo
further non-drug-company-sponsored studies of efficacy. Concerns about clinical
studies and marketing of drugs prompted this evaluation of a large group of
patients taking this medication.
OBJECTIVE: Finasteride usage offered an opportunity not only to understand the
acceptance of a cosmetically oriented medication, but also to evaluate
subjective comments and compliance after a long period of time.
METHODS: A total of 1261 patients were monitored with phone calls every 3 months
after finasteride was initially prescribed. After 12 months, a detailed
questionnaire was sent to all patients with an additional letter and two
telephone calls if no response was received. Statistical analysis of the
patients' data was made.
RESULTS: Thirty-two percent or 414 men continued to take finasteride daily for 1
to 3 years. Twenty-four percent or 297 men discontinued the drug between 3 and
15 months owing to poor results. The remaining 44% or 549 men were lost to
follow-up despite numerous attempts to contact them.
CONCLUSION: A total of 414 men continued to take the medication, but only 211
returned detailed questionnaires. A small percentage of this group felt that
they grew hair. The remaining patients noted poor results. Androgenic alopecia (AGA), or pattern hair loss, is a common condition that
affects both men and women has been gradually increasing. The discovery of the
androgen receptor (AR) gene and related genes has expanded the knowledge on the
genetics of hair loss. These basic science studies, combined with more recent
clinical studies, have led to a better understanding of the pathogenesis of AGA
in both men and women. These genetic advances have also led to the development
of a new screening test for AGA. Recently, in addition to the two currently
approved U.S. Food and Drug Administration (FDA) medications (minoxidil and
finasteride), a novel device was FDA-approved for the treatment of hair loss,
the laser hair comb. Further studies are needed to verify the accuracy and
validity of the genetic screening test and the efficacy of the laser hair comb. PURPOSE: Androgenetic alopecia (AGA), or male pattern hair loss, affects up to
96% of Caucasian men. Characterized by gradual thinning and eventual loss of
hair along frontotemporal, parietal, and vertex areas of the scalp, AGA is
associated with low self-esteem, depression, and dissatisfaction with body
appearance.
DATA SOURCES: In this systematic review of the literature, six primary research
studies conducted in the United States are evaluated for their clinical
application to primary care provider practice.
CONCLUSIONS: Topical minoxidil 2%-5% 1 mL twice daily or finasteride 1 mg daily
are recommended as first line treatments, followed by the use of Food and Drug
Administration-cleared HairMax LaserComb® in patients who do not respond to
first line modalities.
IMPLICATIONS FOR PRACTICE: Further research in novel and established treatments
is recommended, along with an evidence-based clinical practice guideline for
practitioners in the United States. |
Which bone protein is used in archaelogy for dating and species identification? | Collagen is the main protein extracted from bones and analyzed by mass spectrometry. It is traditionally used for radiocarbon dating but sophisticated new technologies are using collagen for species identification as well. | Mass spectrometric analysis of the stable carbon isotope composition (13C/12C or
delta 13C) of bone collagen from human remains recovered at archaeological sites
provides a direct chemical method for investigating dietary patterns of
prehistoric human populations. This methodology is based on the facts that (1)
different food items within the human diet have distinct delta 13C values, and
(2) the delta 13C value of human bone collagen is determined by the delta 13C
value of the diet. Studies of the development of subsistence patterns based on
corn agriculture, one of the most significant developments in North American
prehistory, can benefit from the use of stable carbon isotope techniques because
corn has a high delta 13C value relative to other components of the human diet.
Measurements of delta 13C of bone collagen from prehistoric human skeletal
remains from southeastern Missouri and northeastern Arkansas indicate that
intensive corn agriculture began in this region around A.D. 1000, that the
incorporation of corn into the human diet was a rapid phenomenon, and that 35 to
77% of the human diet from A.D. 1000 to A.D. 1600 consisted of corn. Results
from an isochronous population in southeastern South Dakota (A.D. 1400) suggest
that 78 to 90% of the diet of this group consisted of corn, with no difference
between males and females. Coupled with more traditional archaeological methods,
stable carbon isotope analysis of bone collagen can significantly enhance
reconstruction of dietary patterns of prehistoric humans. Matjes River Rock Shelter is a large shell midden on the southern coast of South
Africa. Stable nitrogen (delta(15)N) and carbon (delta(13)C) isotope ratios were
measured in bone collagen and dentine from human skeletons excavated from this
site in order to establish a weaning curve in mid-Holocene hunter-gatherers.
delta(15)N results show a progressive increase in individuals from birth to 1.5
years old. delta(13)C results are more tightly clustered and mirror the steady
progressive change seen for delta(15)N. We deduce that children at Matjes River
Rock Shelter were breastfed for at least the first 1.5 years after birth, and
were weaned sometime between 2-4 years of age. A similar pattern was documented
for historic-era Kalahari foraging people, where the interbirth spacing was
approximately 3 years. This study provides the first direct evidence for an
extended period of breastfeeding, and thus long interbirth intervals, among
prehistoric foragers, even when those foragers lived in an environment with
abundant food resources. The scope of compound-specific stable isotope analysis has recently been
increased with the development of the LC IsoLink which interfaces
high-performance liquid chromatography (HPLC) and isotope ratio mass
spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement
of non-volatile compounds previously not amenable to compound-specific analysis
or requiring substantial modification for gas chromatography/combustion/isotope
ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino
acids are an example of such compounds. We present a new chromatographic method
for the HPLC separation of underivatized amino acids using an acidic, aqueous
mobile phase in conjunction with a mixed-mode stationary phase that can be
interfaced with the LC IsoLink for compound-specific delta13C analysis. The
method utilizes a reversed-phase Primesep-A column with embedded, ionizable,
functional groups providing the capability for ion-exchange and hydrophobic
interactions. Baseline separation of 15 amino acids and their carbon isotope
values are reported with an average standard deviation of 0.18 per thousand (n =
6). In addition delta13C values of 18 amino acids are determined from modern
protein and archaeological bone collagen hydrolysates, demonstrating the
potential of this method for compound-specific applications in a number of
fields including metabolic, ecological and palaeodietary studies. We used authentication tests developed for ancient DNA to evaluate claims by
Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences
recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon
samples pass these tests, absence of amino acid composition data, lack of
evidence for peptide deamidation, and association of alpha1(I) collagen
sequences with amphibians rather than birds suggest that T. rex does not. We report a novel method for the chromatographic separation and measurement of
stable carbon isotope ratios (delta(13)C) of individual amino acids in hair
proteins and bone collagen using the LC-IsoLink system, which interfaces liquid
chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper
provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen
and hair proteins, respectively. We also describe an approach to analysing small
hair samples for compound-specific analysis of segmental hair sections. The
LC/IRMS method is applied in a historical context by the delta(13)C analysis of
hair proteins and bone collagen recovered from six individuals from Uummannaq in
Greenland. The analysis of hair and bone amino acids from the same individual,
compared for the first time in this study, is of importance in palaeodietary
reconstruction. If hair proteins can be used as a proxy for bone collagen at the
amino acid level, this validates compound-specific isotope studies using hair as
a model for palaeodietary reconstruction. Our results suggest that a small
offset observed in the bulk delta(13)C values of the hair and bone samples may
be attributed to two factors: (i) amino acid compositional differences between
hair and bone proteins, and (ii) differential turnover rates of the tissues and
the amino acid pools contributing to their synthesis. This application proposes
that hair may be a useful complementary or alternative source of
compound-specific paleodietary information. Bones are potentially the best age indicators in a stratigraphic study, because
they are closely related to the layer in which they are found. Collagen is the
most suitable fraction and is the material normally used in radiocarbon dating.
Bone contamits can strongly alter the carbon isotopic fraction values of the
samples, so chemical pretreatment for (14)C dating by accelerator mass
spectrometry (AMS) is essential. The most widespread method for collagen
extraction is based on the Longin procedure, which consists in HCl
demineralization to dissolve the inorganic phase of the samples, followed by
dissolution of collagen in a weak acid solution. In this work the possible side
effects of this procedure on a modern bone are presented; the extracted collagen
was analyzed by ATR-IR spectroscopy. An alternative procedure, based on use of
HF instead of HCl, to minimize unwanted degradation of the organic fraction, is
also given. A study by ATR-IR spectroscopic analysis of collagen collected after
different demineralization times and with different acid volumes, and a study of
an archaeological sample, are also presented. In archaeological studies, the isotopic enrichment values of carbon and nitrogen
in bone collagen give a degree of information on dietary composition. The
isotopic enrichments of individual amino acids from bone collagen and dietary
protein have the potential to provide more precise information about the
components of diet. A limited amount of work has been done on this, although the
reliability of these studies is potentially limited by fractionation arising
through hydrolysis of whole plant tissue (where reaction between amino acids and
carbohydrates may occur) and, for certain amino acids, the use of derivatives
(particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio
mass spectrometry (GC/IRMS) analysis. The present study takes the approach of
extracting the protein components of plant tissues before hydrolysis and using
liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not
require derivatisation, for measurement of the isotopic enrichment of the amino
acids. The protocol developed offers a methodology for consistent measurement of
the δ(13)C values of amino acids, allowing isotopic differences between the
individual amino acids from different plant tissues to be identified. In
particular, there are highly significant differences between leaf and seed
protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰),
aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended
application in archaeology, the technique will be of value in the fields of
plant sciences, nutrition and environmental food-web studies. Results are presented of a comparison of the amino acid (AA) δ(13)C values
obtained by gas chromatography-combustion-isotope ratio mass spectrometry
(GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS).
Although the primary focus was the compound-specific stable carbon isotope
analysis of bone collagen AAs, because of its growing application for
palaeodietary and palaeoecological reconstruction, the results are relevant to
any field where AA δ(13)C values are required. We compare LC/IRMS with the most
up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives.
This comparison involves the analysis of standard AAs and hydrolysates of
archaeological human bone collagen, which have been previously investigated as
N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although
GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a
wider range of AAs, particularly those not amenable to GC analysis (e.g.
arginine). Accordingly, reconstructed bulk δ(13)C values based on
LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values
than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA
δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the
δ(13)C values of the TFA/IP derivatives compared with the NACME- and
LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP
derivatives, resulting from: (i) inefficient sample combustion, and/or (ii)
differences in the intra-molecular distribution of δ(13)C values between AAs,
which are manifested by incomplete combustion. Close similarities between the
NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the
TFA/IP derivatives should be abandoned for the natural abundance determinations
of AA δ(13)C values. As part of the road widening scheme between London and Dover, Oxford Archaeology
South uncovered a large boundary ditch of Iron Age origin that contained Iron
Age and Roman inhumations, adjacent to which was a small mid-late Roman
cemetery, interpreted as a rural cemetery for Romano-British farmers. Grave
goods in the cemetery were restricted to a few individuals with hobnailed boots.
Bulk bone collagen isotopic analysis of 11 skeletons of Iron Age and Roman date
gave a typical C(3) terrestrial signal (average δ(13) C = -19.8‰, δ(15) N =
9.3‰), but also revealed one (SK12671) with a diet which included a substantial
C(4) component (δ(13) C = -15.2‰, δ(15) N = 11.2‰). This is only the second such
diet reported in Roman Britain. Subsequent δ(18) O(c) and (87) Sr/(86) Sr
measurements on the dental enamel in this individual were, however, consistent
with a "local" origin, indicating that either C(4) protein was consumed in Late
Roman Britain, or that he came from somewhere else, but where conditions gave
rise to similar isotopic values. If we accept the latter, then it indicates that
using oxygen and strontium isotopes alone to identify "incomers" may be
problematic. The provision of hobnailed boots for the dead appears to have had a
strong symbolic element in Late Roman Britain. We suggest that in this case the
boots may be significant, in that he was being equipped for the long march home. RATIONALE: Non-enzymatic deamidation accumulates in aging tissues in vivo and
has been proposed to be potentially useful as a molecular clock. The process
continues post mortem, and here we explore the increase in levels of deamidation
in archaeological collagen, as measured during Zooarchaeology by Mass
Spectrometry (ZooMS) analysis.
METHODS: With the high sensitivity of current generation mass spectrometers,
ZooMS provides a non-destructive and highly cost-effective method to
characterise collagen peptides. Deamidation can be detected by mass spectrometry
as a +0.984 Da mass shift; therefore, aside from its original purpose, peptide
mass-fingerprinting for bone identification, ZooMS concurrently yields a
'thermal indicator' of the samples.
RESULTS: By analysis of conventional ZooMS spectra, we determined the
deamidation rate for glutamine residues in 911 bone collagen samples from 50
sites, with ages varying from medieval to Palaeolithic. The degree of
deamidation was compared to diagenetic parameters and nearby sequence
properties.
CONCLUSIONS: The extent of deamidation was found to be influenced more by burial
conditions and thermal age than, for example, chronological age, the extent of
bioerosion or crystallinity. The method lends itself mostly to screening
heterogenic deposits of bone to identify outliers. RATIONALE: This study presents the first sulphur isotope data of modern and
archaeological cod (Gadus morhua) bone collagen, undertaken to identify
large-scale spatial variability of significance as both baseline values for
studies of human diet and a potential variable in isotope-based studies of fish
trading.
METHODS: Collagen was extracted from modern and archaeological cod bones using a
weak HCl solution and analysed for its sulphur isotopic composition by isotope
ratio mass spectrometry (IRMS).
RESULTS: The archaeological cod have sulphur isotope values ranging from +9.1‰
to +18.2‰, whereas values for modern specimens range from +14.8‰ to +18.3‰. The
modern data show values implying less freshwater influence, consistent with
their offshore catch locations, but also corroborate some of the regional
variability evident from the archaeological evidence.
CONCLUSIONS: The archaeological data have a large range of sulphur isotope
values compared with the modern populations, probably indicating they were taken
from a wide range of geographic locations, including both coastal and offshore
locales. They show broad trends of regional difference that may relate to both
the fish populations targeted (e.g. 'inshore' versus 'offshore') and the
baseline values of the local ecosystem (e.g. degree of freshwater input from
river systems). Bones are one of the few organs in which uranyl (UO2(2+)) accumulates. This
large dioxo-cation displays affinity for carboxylates, phenolates and
phosphorylated functional groups in proteins. The noncollagenous protein
osteopontin (OPN) plays an important role in bone homeostasis. It is mainly
found in the extracellular matrix of mineralized tissues but also in body fluids
such as milk, blood and urine. Furthermore, OPN is an intrinsically disordered
protein, which, like other proteins of the SIBLING family, contains a
polyaspartic acid sequence and numerous patterns of alternating acidic and
phosphorylated residues. All these properties led to the hypothesis that this
protein could be prone to UO2(2+) binding. In this work, a simple purification
procedure enabling highly purified bovine (bOPN) and human OPN (hOPN) to be
obtained was developed. Various biophysical approaches were set up to study the
impact of phosphorylations on the affinity of OPN for UO2(2+) as well as the
formation of stable complexes originating from structural changes induced by the
binding of this metal cation. The results obtained suggest a new mechanism of
the interaction of UO2(2+) with bone metabolism and a new role for OPN as a
metal transporter. |
Does thyroid hormone affect cardiac remodeling ? | Cardiac function and mechanics are significantly affected by low thyroid function. l-T4 therapy improves but does not completely recover cardiac function in patients with mild hypothyroidism.
Long-term T4 treatment after myocardial infarction has beneficial effects on myocyte, arteriolar, and collagen matrix remodeling in the non-infarcted area. Most importantly, results suggest improved survival of myocytes in the peri-infarct area. | Thyroid hormone directly affects the heart and peripheral vascular system. The
hormone can increase myocardial inotropy and heart rate and dilate peripheral
arteries to increase cardiac output. An excessive deficiency of thyroid hormone
can cause cardiovascular disease and aggravate many preexisting conditions. In
severe systemic illness and after major surgical procedures changes in thyroid
function can occur, leading to the "euthyroid sick syndrome." Patients will have
normal or decreased levels of T4, decreased free and total T3, and usually
normal levels of thyroid stimulating hormone. This syndrome may be an adaptive
response to systemic illness that usually will revert to normal without hormone
supplementation as the illness subsides. Recently, however, many investigators
have explored the benefits of thyroid hormone supplementation in those diseases
associated with euthyroid sick syndrome. Thyroid hormone's effects on the
cardiovascular system make it an attractive therapy for those patients with
impaired hemodynamics and low T3. Thyroid hormone has also been considered a
treatment for patients with congestive heart failure, for patients undergoing
cardiopulmonary bypass and heart transplantation, and for patients with
hyperlipidemia. At present there is no evidence suggesting a favorable treatment
outcome using thyroid hormone supplementation for any systemic condition except
in those patients with documented hypothyroidism. Exercise training improves the aging-induced downregulation of myosin heavy
chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in
the regulation of cardiac contraction and relaxation. Thyroid hormone receptor
(TR), a transcriptional activator, affected the regulation of gene expression of
MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling
contributes to a molecular mechanism of exercise training-induced improvement of
MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated
whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged
heart are affected by exercise training, using the hearts of sedentary young
rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo
old, swimming training for 8 wk). Trained aged rats showed improvement in
cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart
were significantly lower in sedentary aged rats than in sedentary young rats and
were significantly higher in trained aged rats than in sedentary aged rats. The
activity of TR DNA binding to the transcriptional regulatory region in the
alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of
alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and
thyroxine levels were altered in association with changes in the myocardial TR
protein levels. These findings suggest that exercise training improves the
aging-induced downregulation of myocardial TR signaling-mediated transcription
of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of
cardiac function in trained aged hearts. Cardiac surgery using cardiopulmonary bypass produces a generalized systemic
inflammatory response, resulting in increased postoperative morbidity and
mortality. Under these circumstances, a typical pattern of thyroid abnormalities
is seen in the absence of primary disease, defined as sick euthyroid syndrome
(SES). The presence of postoperative SES mainly in small children and neonates
exposed to long bypass times and the pharmacological profile of thyroid hormones
and their effects on the cardiovascular physiology make supplementation therapy
an attractive treatment option to improve postoperative morbidity and mortality.
Many studies have been performed with conflicting results. In this article, we
review the important literature on the development of SES in paediatric
postoperative cardiac patients, analyse the existing information on thyroid
hormone replacement therapy in this patient group and try to summarize the
findings for a recommendation. Thyroid (dys)function in heart failure: is it a potential target for medical
treatment? This review evaluates the hypothesis that the cardiac effects of amiodarone can
be explained-at least partly-by the induction of a local 'hypothyroid-like
condition' in the heart. Evidence supporting the hypothesis comprises the
observation that amiodarone exerts an inhibitory effect on the binding of T3 to
thyroid hormone receptors (TR) alpha-1 and beta-1 in vitro, and on the
expression of particular T3-dependent genes in vivo. In the heart, amiodarone
decreases heart rate and alpha myosin heavy chain expression (mediated via TR
alpha-1), and increases sarcoplasmic reticulum calcium-activated ATPase and beta
myosin heavy chain expression (mediated via TR beta-1). Recent data show a
significant similarity in expression profiles of 8,435 genes in the heart of
hypothyroid and amiodarone-treated animals, although similarities do not always
exist in transcripts of ion channel genes. Induction of a hypothyroid cardiac
phenotype by amiodarone may be advantageous by decreasing energy demands and
increasing energy availability. In the post-reperfusion era, molecular and genetic mechanisms of
cardioprotection and regeneration represent new therapeutic challenges to limit
infarct size and minimize post-ischemic remodeling after acute myocardial
infarction (AMI). Activation of cell survival mechanisms can be promoted by the
administration of external drugs, stimulation of internal mechanisms, and
genetic manipulation to delete or replace pathological genes or enhance gene
expression. Among internal cardiovascular regulatory mechanisms, thyroid
hormones (THs) may play a fundamental role. TH has a critical role in
cardiovascular development and homeostasis in both physiological and
pathological conditions. In experimental AMI, TH has been shown to affect
cardiac contractility, left ventricular (LV) function, and remodeling. Several
experimental studies have clearly shown that THs participate in the regulation
of molecular mechanisms of angiogenesis, cardioprotection, cardiac metabolism,
and ultimately myocyte regeneration, changes that can reverse left ventricular
remodeling by favorably improving myocyte shape and geometry of LV cavity, thus
improving systolic and diastolic performance. This review is focused on the role
of thyroid on AMI evolution and on the potential novel option of thyroid-related
treatment of AMI. BACKGROUND: Left ventricular (LV) remodeling following large transmural
myocardial infarction (MI) remains a pivotal clinical issue despite the advance
of medical treatment over the past few decades. Identification of new
medications to improve the remodeling process and prevent progression to heart
failure after MI is critical. Thyroid hormones (THs) have been shown to improve
LV function and remodeling in animals post-MI and in the human setting. However,
changes in underlying cellular remodeling resulting from TH treatment are not
clear.
METHODS: MI was produced in adult female Sprague-Dawley rats by ligation of the
left descending coronary artery. L-thyroxine (T4) pellet (3.3 mg, 60 days
sustained release) was used to treat MI rats for 8 weeks. Isolated myocyte
shape, arterioles, and collagen deposition in the non-infarcted area were
measured at terminal study.
RESULTS: T4 treatment improved LV ±dp/dt, normalized TAU, and increased myocyte
cross-sectional area without further increasing myocyte length in MI rats. T4
treatment increased the total LV tissue area by 34%, increased the non-infarcted
tissue area by 41%, and increased the thickness of non-infarcted area by 36% in
MI rats. However, myocyte volume accounted for only ~1/3 of the increase in
myocyte mass in the non-infarct area, indicating the presence of more myocytes
with treatment. T4 treatment tended to increase the total length of smaller
arterioles (5 to 15 μm) proportional to LV weight increase and also decreased
collagen deposition in the LV non-infarcted area. A tendency for increased
metalloproteinase-2 (MMP-2) expression and tissue inhibitor of
metalloproteinases (TIMPs) -1 to -4 expression was also observed in T4 treated
MI rats.
CONCLUSIONS: These results suggest that long-term T4 treatment after MI has
beneficial effects on myocyte, arteriolar, and collagen matrix remodeling in the
non-infarcted area. Most importantly, results suggest improved survival of
myocytes in the peri-infarct area. Thyroid hormone receptor α1 (TRα1) is shown to be critical for the maturation of
cardiomyocytes and for the cellular response to stress. TRα1 is altered during
post ischemic cardiac remodeling but the physiological significance of this
response is not fully understood. Thus, the present study explored the potential
consequences of selective pharmacological inhibition of TRα1 on the mechanical
performance of the post-infarcted heart. Acute myocardial infarction was induced
in mice (AMI), while sham operated animals served as controls (SHAM). A group of
mice was treated with debutyl-dronedarone (DBD), a selective TRα1 inhibitor
(AMI-DBD). AMI resulted in low T3 levels in plasma and in down-regulation of
TRα1 and TRβ1 expression. Left ventricular ejection fraction (LVEF%) was
significantly reduced in AMI [33 (SEM 2.1) vs 79(2.5) in SHAM, p < 0.05] and was
further declined in AMI-DBD [22(1.1) vs 33(2.1), respectively, p < 0.05].
Cardiac mass was increased in AMI but not in AMI-DBD hearts, resulting in
significant increase in wall tension index. This increase in wall stress was
accompanied by marked activation of p38 MAPK, a kinase that is sensitive to
mechanical stretch and exerts negative inotropic effect. Furthermore, AMI
resulted in β-myosin heavy chain overexpression and reduction in the ratio of
SR(Ca)ATPase to phospholamban (PLB). The latter further declined in AMI-DBD
mainly due to increased expression of PLB. AMI induces downregulation of thyroid
hormone signaling and pharmacological inhibition of TRα1 further depresses
post-ischemic cardiac function. p38 MAPK and PLB may, at least in part, be
involved in this response. BACKGROUND: Subclinical hypothyroidism occurs during aging in humans and mice
and may contribute to the development of heart failure. Aging also impairs
myocardial fatty acid oxidation, causing increased reliance on flux through
pyruvate dehydrogenase (PDH) to maintain function. We hypothesize that the
metabolic changes in aged hearts make them less tolerant to acutely increased
work and that thyroid hormone supplementation reverses these defects.
METHODS: Studies were performed on young (Young, 4-6 months) and aged (Old,
22-24 months) C57/BL6 mice at standard (50 mmHg) and high afterload (80 mmHg).
Another aged group received thyroid hormone for 3 weeks (Old-TH, high afterload
only). Function was measured in isolated working hearts along with substrate
fractional contributions (Fc) to the citric acid cycle (CAC) using perfusate
with (13)C labeled lactate, pyruvate, glucose and unlabeled palmitate and
insulin.
RESULTS: Old mice maintained cardiac function under standard workload
conditions, despite a marked decrease in unlabeled (presumably palmitate) Fc and
relatively similar individual carbohydrate contributions. However, old mice
exhibited reduced palmitate oxidation with diastolic dysfunction exemplified by
lower -dP/dT. Thyroid hormone abrogated the functional and substrate flux
abnormalities in aged mice.
CONCLUSION: The aged heart shows diminished ability to increase cardiac work due
to substrate limitations, primarily impaired fatty acid oxidation. The heart
accommodates slightly by increasing efficiency through oxidation of carbohydrate
substrates. Thyroid hormone supplementation in aged mice significantly improves
cardiac function potentially through restoration of fatty acid oxidation. BACKGROUND: Concomitant thyroid and heart disease are frequently encountered in
clinical practice. There are many studies evaluating thyroid function in acute
and critical conditions. Information on thyroid dysfunction in ST-segment
elevation myocardial infarction (STEMI) is limited; its correlation with short
and long-term outcome is not fully known.
METHODS: Four hundred and fifty seven patients diagnosed with STEMI in our
emergency department were included in the study. Patients were divided into two
groups: patients with normal thyroid function (euthyroid) and patients with
thyroid dysfunction. STEMI was diagnosed with 12 derivation surface
electrocardiogram. Thyroid hormone levels (TSH, free T3 and free T4) were
measured. Patients with other acute coronary syndromes and endocrine pathologies
except diabetes mellitus were excluded. Two patient groups were compared in
terms of in-hospital and long-term outcome.
RESULTS: Out of 457, 72 (15%) patients with thyroid dysfunction were detected.
The other patients were euthyroid and constituted the control group. In-hospital
cardiogenic shock (15% vs. 3% in the control group; p < 0.01) and death (7% vs.
1% in the control group; p < 0.01) were more frequently observed in the thyroid
dysfunction group. In the subgroup analysis, it was observed that patients with
sick euthyroid syndrome have the poorest outcome. Other markers for poor outcome
were anemia and renal failure.
CONCLUSIONS: Thyroid dysfunction, particularly sick euthyroid syndrome, was
found to be related to in-hospital and long term mortality in patients with
STEMI undergoing primary percutaneous intervention. BACKGROUND: High blood pressure (BP) is a major health problem in Nigeria and
the involvement of thyroid hormones in this condition has not been evaluated in
this center.
OBJECTIVE: The objective of this study was to evaluate the proportion of
patients with an essential hypertension who had abnormal thyroid hormone levels
and the type of thyroid disorders commonly observed in this group of patients.
MATERIALS AND METHODS: A retrospective study of 94 patients (30 males, aged 30.4
± 2 years and 64 females, aged 43.4 ± 1.3 years) with essential hypertension was
carried out between January 2005 and December 2007. Demographic and other
medical information were obtained from the health records. Analysis of
laboratory results of triiodothyronine (T3), Thyroxine (T4), and thyroid
stimulating hormone (TSH) were made.
RESULTS: Of the 94 patients evaluated, 26 (27.7%) had abnormal thyroid hormone
levels, with 23.4% having hyperthyroidism, 4.3% had sub-clinical hypothyroidism
and none had overt hypothyroidism. Of the 26 subjects with abnormal thyroid
hormone levels, 18 (69.2%) were females while 8 (30.8%) were males. Out of the
18 female subjects with abnormal thyroid hormone levels, 16 had hyperthyroid
levels while 2 had sub-clinical hypothyroid levels. Out of the 8 male patients,
6 had hyperthyroid hormone levels while 2 had sub-clinical hypothyroid levels.
CONCLUSION: Thyroid hormone abnormalities are common in patients with essential
hypertension. Hyperthyroidism was the most common thyroid disorder observed.
Young patients presenting with essential hypertension should be screened for
thyroid hormone abnormalities since they can best be managed by treating the
underlying causes. Reduced function of the thyroid gland causes Hypothyroidism which is further
attributed to defects in the secretion of thyroid hormones triiodothyronine (T3)
and tetra-iodothyronine or thyroxine (T4). T3 and T4 hormones are not only known
to regulate the rate of metabolism but also affect the growth and rate of
function of many other systems in the body such as neuromuscular,
gastrointestinal and cardiovascular system. Hypothyroidism patient usually show
higher levels of total cholesterol, low-density lipoproteins (LDL),
triglycerides, and other lipid molecules associated with heart disease. The
question still remained to be addressed though is whether hypothyroidism affects
heart and result in cardiovascular disease. The current review updates us with
the recent progress in the hypothyroidism area especially in relation to its
connecting link with the heart disease. The present study will further enhance
our understanding of the intricacies involved in the secretion of thyroid
hormones (T3 & T4) and thyroid stimulating hormone (TSH) subsequently affecting
serum lipid levels. The study may help to dice-out cardiovascular risk factors
associated with hypothyroidism so that effective measures could be taken prior
to occurrence of coronary heart disease. BACKGROUND: Previous studies have suggested that hypothyroidism correlated with
coronary heart diseases (CHD) mortality in long-term cohort, but whether the
thyroid function status is associated with myocardial injury in acute
ST-elevation myocardial infarction (STEMI) has not been investigated
sufficiently.
METHODS: Five hundred and eighty-two hospitalized patients from January 2010 to
December 2011, with the diagnosis of STEMI, were enrolled in this study. All
patients underwent testing for thyroid function status, cardiac troponin I
(cTnI), cardiac enzymes, C-reactive protein (CRP). We investigated the
association between thyroid hormone levels and cardiac markers (creatine
kinase-MB and cTnI), and thus evaluated the potential role of thyroid function
status in predicting the myocardial injury.
RESULTS: There were 76 patients (13.06%) who had hypothyroidism including
low-T3-syndrome (34 patients, 5.84%), subclinical hypothyroidism (28 patients,
4.81%) and clinical hypothyroidism (14 patients, 2.41%). After adjusting for
conventional risk factors (age, gender, smoking, diabetes mellitus,
dyslipidemia, hypertension), free triiodothyronine (FT3) was significantly and
negatively correlated with log-CKMB (r = -0.244, P < 0.001) and log-cTnI (r =
-0.290, P < 0.001), indicating that the lower thyroid hormone level correlates
with the severer cardiac injury in STEMI patients. FT3 also had a moderate
negative correlation with CRP (r = -0.475, P < 0.001), which might indicate that
hypothyroidism may activate the inflammation response. No significant
correlation was found between other thyroid parameters (TSH, FT4) and cardiac
markers.
CONCLUSIONS: As the lower FT3 level correlates with higher level of cardiac
markers and lower left ventricular ejection fraction (LVEF), the hypothyroidism
may be a predictor for myocardial injury in STEMI. And these results may warrant
further study to investigate whether reversing the hypothyroidism could benefit
the STEMI patients. OBJECTIVES: The aim of this study was to investigate whether patients with
systolic heart failure (HF) and abnormal thyroid function are at increased risk
for death.
BACKGROUND: Thyroid hormone homeostasis is vital to the optimal functioning of
the cardiovascular system, but an independent prognostic effect of thyroid
abnormalities in patients with HF has not been established.
METHODS: In SCD-HeFT (Sudden Cardiac Death in Heart Failure Trial), which
randomized patients with ischemic or nonischemic HF to placebo or amiodarone or
implantable cardioverter-defibrillator therapy, thyroid-stimulating hormone
(TSH) was measured at baseline and at 6-month intervals throughout the 5-year
study.
RESULTS: Of 2,225 patients, the majority (87%) had normal TSH levels (0.3 to 5.0
μU/ml) at baseline, 12% had values suggestive of hypothyroidism, and 1% had
values consistent with hyperthyroidism. Compared with euthyroid patients, those
hypothyroid at baseline were older and included more women and Caucasians (all p
values <0.05). Over the median follow-up period of 45.5 months, among patients
euthyroid at baseline, 89 developed abnormally low TSH levels, and 341 developed
abnormally high values. Patients randomized to amiodarone (median dose 300 mg)
had an elevated risk for developing abnormal TSH levels compared with
implantable cardioverter-defibrillator therapy or placebo (p < 0.0001). Patients
with baseline or new-onset abnormal thyroid function had a higher mortality than
those with normal thyroid function, even after controlling for other known
mortality predictors (hazard ratio: 1.58; 95% confidence interval: 1.29 to 1.94;
p < 0.0001 for hypothyroid; hazard ratio: 1.85; 95% confidence interval: 1.21 to
2.83; p = 0.0048 for hyperthyroid). Implantable cardioverter-defibrillator
benefit did not vary with thyroid function.
CONCLUSIONS: Abnormal thyroid function in patients with symptomatic HF and
ejection fractions ≤35% is associated with significantly increased risk for
death, even after controlling for known mortality predictors. CONTEXT: Subclinical hypothyroidism (SHT) is associated with left ventricular
(LV) remodeling. The LV mechanics has not been previously assessed by two- and
three-dimensional (2DE and 3DE) speckle tracking imaging in the SHT patients.
OBJECTIVES: The objective of the study was to investigate LV mechanics by 2DE
and 3DE speckle tracking in the SHT patients and evaluate the influence of
levothyroxine therapy on LV remodeling.
DESIGN: We conducted a prospective study. All SHT patients received
levothyroxine therapy and were followed up for 1 year after the euthyroid state
had been achieved.
SETTING: The study was performed at a university hospital.
PATIENTS: We included 54 untreated women with SHT and 40 healthy control women
who were of similar age.
MAIN OUTCOME MEASURES: The 2DE strain and strain rates, 3DE volumes, 3DE strain,
and thyroid hormones levels were assessed.
RESULTS: The 2DE LV longitudinal and circumferential strain and systolic and
early diastolic strain rates were significantly decreased in the SHT patients
before therapy in comparison with the controls or the SHT patients after
therapy. The 3DE LV cardiac output and ejection fraction were significantly
reduced in the SHT patients at baseline compared with the controls or patients
after 1 year of treatment. The 3DE LV longitudinal and radial strains were
significantly lower in the SHT group before treatment in comparison with the
controls or patients after therapy, whereas the 3DE LV circumferential and area
strains gradually increased from untreated SHT patients, among the treated SHT
patients, to the controls.
CONCLUSION: SHT significantly affects LV deformation assessed by 2DE and 3DE
speckle tracking. The improvement of LV mechanics after 1 year of levothyroxine
treatment is significant but incomplete. BACKGROUND: Thyroid stimulating hormone (TSH) abnormalities have been associated
with various cardiac arrhythmias. Effect of TSH abnormalities on mortality and
implantable cardioverter defibrillators (ICD) behavior in patients with
cardiomyopathy has not been investigated.
METHODS: Our ICD database includes 1,445 patients between December 1997 and
January 2008. TSH levels using the ultra sensitive assay were available in 371
patients. Patients were classified based on TSH levels as: High TSH (N = 102,
TSH > 5) and Low TSH (N = 26, TSH < 0.4). TSH groups were compared for
mortality, appropriate and inappropriate ICD therapies.
RESULTS: A high or low TSH was an independent predictor of all-cause mortality:
Hazard ratio (HR) 1.46 (95% CI 1.03-2.08), P = 0.033 and HR 1.76 (95% confidence
interval 1.02-3.03), P = 0.043 respectively. TSH abnormalities were not
associated with an increased incidence in appropriate or inappropriate ICD
therapy.
CONCLUSION: A low or high TSH was associated with a higher mortality; however,
neither were associated with appropriate or inappropriate ICD therapy after
multivariate analysis. Univariate analysis demonstrated an association of high
TSH and appropriate ICD therapy. However, multivariate analysis did not show
this association. Therefore, we are concluding that TSH levels are predictors of
all-cause mortality but not ICD therapy. BACKGROUND: In dialysis patients, the prevalence of thyroid disorders and their
impact on specific cardiovascular (CV) events and mortality are largely unknown.
The aim of the present study was to analyze whether subclinical thyroid
disorders were associated with CV events and mortality.
STUDY DESIGN: Prospective multicenter cohort study.
SETTING & PARTICIPANTS: Thyroid status and clinical outcomes were explored in
1,000 diabetic hemodialysis patients from 178 centers in Germany.
PREDICTOR: Thyroid status, defined by the following cutoff values: euthyroidism
(thyrotropin [TSH], 0.30-4.0 mIU/L; free triiodothyronine [T3], 2.7-7.6 pmol/L;
and free thyroxine [T4], 11.0-24.0 pmol/L), subclinical hyperthyroidism (TSH<0.3
mIU/L and free T3/free T4 within reference ranges), subclinical hypothyroidism
(TSH, 4.1-15.0 mIU/L and free T3/free T4 within reference ranges), euthyroid
sick syndrome (free T3<2.7 pmol/L and TSH/free T4 low or within reference
ranges).
OUTCOMES: During 4 years' follow-up, prespecified adjudicated end points were
determined: sudden cardiac death, myocardial infarction, stroke, combined CV
events, and overall mortality. Short-term effects within the first 12 months
were contrasted to long-term effects (years 2-4).
MEASUREMENTS: TSH, free T3, and free T4 levels at baseline.
RESULTS: Euthyroidism was present in 78.1% of patients; subclinical
hyperthyroidism, in 13.7%; and subclinical hypothyroidism, in 1.6%. Euthyroid
sick syndrome was exhibited by 5.4% of patients. The adjusted short-term risk of
sudden cardiac death was more than doubled (HR, 2.03; 95% CI, 0.94-4.36) in
patients with subclinical hyperthyroidism, and similarly for patients with
euthyroid sick syndrome (HR, 2.74; 95% CI, 0.94-7.98) compared with patients
with euthyroidism. Short-term mortality was increased almost 3-fold for patients
with euthyroid sick syndrome (HR, 2.97; 95% CI, 1.66-5.29), but this effect was
not seen in the long term. Subclinical hypothyroidism was not associated with CV
events or all-cause mortality. Risks of stroke and myocardial infarction were
not affected meaningfully by thyroid disorders.
LIMITATIONS: Observational study design.
CONCLUSIONS: Sudden cardiac death may be influenced by subclinical
hyperthyroidism and euthyroid sick syndrome in the short term. Furthermore,
euthyroid sick syndrome is associated strongly with mortality in hemodialysis
patients. Regular assessment of thyroid status may help estimate the cardiac
risk of dialysis patients. Cardiomyocytes continuously generate the contractile force to circulate blood
through the body. Imbalances in contractile performance or energy supply cause
adaptive responses of the heart resulting in adverse rearrangement of regular
structures, which in turn might lead to heart failure. At the cellular level,
cardiomyocyte remodeling includes (1) restructuring of the contractile
apparatus; (2) rearrangement of the cytoskeleton; and (3) changes in energy
metabolism. Dedifferentiation represents a key feature of cardiomyocyte
remodeling. It is characterized by reciprocal changes in the expression pattern
of "mature" and "immature" cardiomyocyte-specific genes. Dedifferentiation may
enable cardiomyocytes to cope with hypoxic stress by disassembly of the energy
demanding contractile machinery and by reduction of the cellular energy demand.
Dedifferentiation during myocardial repair might provide cardiomyocytes with
additional plasticity, enabling survival under hypoxic conditions and increasing
the propensity to enter the cell cycle. Although dedifferentiation of
cardiomyocytes has been described during tissue regeneration in zebrafish and
newts, little is known about corresponding mechanisms and regulatory circuits in
mammals. The recent finding that the cytokine oncostatin M (OSM) is pivotal for
cardiomyocyte dedifferentiation and exerts strong protective effects during
myocardial infarction highlights the role of cytokines as potent stimulators of
cardiac remodeling. Here, we summarize the current knowledge about transient
dedifferentiation of cardiomyocytes in the context of myocardial remodeling, and
propose a model for the role of OSM in this process. |
What is the effect of SAHA treatment in Huntington's disease? | Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, protects dopaminergic neurons from neurotoxin-induced damage. SAHA is predominantly an inhibitor of class I HDACs. However, it can also bind to class IIa HDACs and has been shown to degrade class IIa HDACs at the protein level in vitro. The neuroprotective effects of SAHA were mediated in part by promoting release of neurotrophic factors from astroglia through inhibition of histone deacetylation. SAHA can lead to the degradation of class IIa HDACs 4 and 5 via RANBP2 mediated proteasome degradation in vitro. | Huntington's disease (HD) is an inherited, progressive neurological disorder
caused by a CAG/polyglutamine repeat expansion, for which there is no effective
disease modifying therapy. In recent years, transcriptional dysregulation has
emerged as a pathogenic process that appears early in disease progression.
Administration of histone deacetylase (HDAC) inhibitors such as suberoylanilide
hydroxamic acid (SAHA) have consistently shown therapeutic potential in models
of HD, at least partly through increasing the association of acetylated histones
with down-regulated genes and by correcting mRNA abnormalities. The HDAC enzyme
through which SAHA mediates its beneficial effects in the R6/2 mouse model of HD
is not known. Therefore, we have embarked on a series of genetic studies to
uncover the HDAC target that is relevant to therapeutic development for HD.
HDAC7 is of interest in this context because SAHA has been shown to decrease
HDAC7 expression in cell culture systems in addition to inhibiting enzyme
activity. After confirming that expression levels of Hdac7 are decreased in the
brains of wild type and R6/2 mice after SAHA administration, we performed a
genetic cross to determine whether genetic reduction of Hdac7 would alleviate
phenotypes in the R6/2 mice. We found no improvement in a number of
physiological or behavioral phenotypes. Similarly, the dysregulated expression
levels of a number of genes of interest were not improved suggesting that
reduction in Hdac7 does not alleviate the R6/2 HD-related transcriptional
dysregulation. Therefore, we conclude that the beneficial effects of HDAC
inhibitors are not predomitly mediated through the inhibition of HDAC7. |
What is known about the role of mHealth in the prevention of disease? | Most reported uses of mHealth are for treatment, we identified report on approaches to child obesity prevention. | Mobile phones have been shown effective in several public health domains.
However, there are few evaluations of the effectiveness of mobile health in
health promotion. Also, although many studies have referenced behavioral theory,
none appears to have explicitly tested theoretical assumptions or demonstrated
mechanisms of change. More robust evaluation models that incorporate theory and
measurement of behavioral mediators are needed. As in all public health
programs, mobile health operates within a social ecological context. For
example, organizational- and individual-level programs seek to influence health
and health care practices and individual health behaviors. New programs such as
Text4baby demonstrate how theory and explicit testing of mediators can be
incorporated in evaluations. There are challenges and opportunities facing
mHealth evaluations given the nature of the mobile channel. Mobile communication
is ubiquitous, available at all times and places, and thus experimental control
is often difficult. Natural experiments using variation in dosage of mHealth and
place- or location-based designs may increase experimental control. Text4baby is
a text messaging program that provides prenatal care messages to pregt women
and new mothers. It uses a partnership model with health care facilities often
serving as local implementation partners. The authors review a case example of
the evaluation of Text4baby at Madigan Army Medical Center. Participants were
randomized to usual prenatal care plus text messaging or usual care alone. The
evaluation has a theoretical model of behavior change and measures mediators as
well as behavioral outcomes. Results will inform how behavioral theory works
within mobile health programs. There is a growing interest in the effect of mobile phones in health care
(mHealth) service delivery, but more research is needed to determine whether
short message service (SMS)-based campaigns are appropriate for developing
countries. This pilot study explored the efficacy of an mHealth campaign using
SMS as a platform to disseminate and measure HIV/AIDS knowledge, and to promote
HIV/AIDS testing at clinics in rural Uganda. Over a 1-month period, 13 HIV/AIDS
quiz questions were sent to 10,000 mobile subscribers. Despite participation
incentives, only one-fifth of the mobile subscribers responded to any of the
questions. The campaign had proportionately limited success in increasing
knowledge levels on a mass scale. Furthermore, the program design may be
reinforcing entrenched knowledge gaps. The results suggest that it is important
to be conservative when considering the potential overall effect of SMS-based
programs. However, the authors recognize the potential of mHealth tools when
extended to millions of mobile phone users as part of an integrated health
campaign approach. The authors propose several steps to improve the program
design to reach a larger portion of the intended audience and increase campaign
effectiveness. HIV prevalence in Uganda has leveled off, however trends indicate that incidence
is on the rise and disproportionately affects certain vulnerable groups, such as
women. There is growing support for using mobile health (mHealth) programs to
reach vulnerable populations. Using the Extended Technology-Community-Management
model for mHealth, we examined a text message campaign in Arua, Uganda, that
disseminated and measured HIV/AIDS knowledge and promoted HIV/AIDS testing at
clinics. Empirical data from this campaign illustrated that mHealth campaigns
need to address socio-cultural, informational, economic and individual
vulnerabilities. We found, for example, that the interactive SMS quiz design
motivated recipients with the correct HIV/AIDS knowledge to respond (and thus
become eligible for free HIV screening). In doing so, however, the more
vulnerable sections of the population, such as those with incorrect knowledge,
were further put at risk. We discuss the programmatic implications and provide
strategic recommendations for campaign focus, particularly to reach young women. BACKGROUND: Mobile Health (mHealth) proposes health care delivering anytime and
anywhere. It aims to answer several emerging problems in health services,
including the increasing number of chronic diseases, high costs on national
health services, and the need to provide direct access to health services,
regardless of time and place. mHealth systems include the use of mobile devices
and apps that interact with patients and caretakers. However, mobile devices
present several constraints, such as processor, energy, and storage resource
limitations. The constant mobility and often-required Internet connectivity also
exposes and compromises the privacy and confidentiality of health information.
OBJECTIVE: This paper presents a proposal, construction, performance evaluation,
and validation of a data encryption solution for mobile health apps (DE4MHA),
considering a novel and early-proposed cooperation strategy. The goal was to
present a robust solution based on encryption algorithms that guarantee the best
confidentiality, integrity, and authenticity of users health information. In
this paper, we presented, explained, evaluated the performance, and discussed
the cooperation mechanisms and the proposed encryption solution for mHealth
apps.
METHODS: First, we designed and deployed the DE4MHA. Then two studies were
performed: (1) study and comparison of symmetric and asymmetric
encryption/decryption algorithms in an mHealth app under a cooperation
environment, and (2) performance evaluation of the DE4MHA. Its performance was
evaluated through a prototype using an mHealth app for obesity prevention and
cares, called SapoFit. We then conducted an evaluation study of the mHealth app
with cooperation mechanisms and the DE4MHA using real users and a real
cooperation scenario. In 5 days, 5 different groups of 7 students selected
randomly agreed to use and experiment the SapoFit app using the 7 devices
available for trials.
RESULTS: There were 35 users of SapoFit that participated in this study. The
performance evaluation of the app was done using 7 real mobile devices in 5
different days. The results showed that confidentiality and protection of the
users' health information was guaranteed and SapoFit users were able to use the
mHealth app with satisfactory quality. Results also showed that the app with the
DE4MHA presented nearly the same results as the app without the DE4MHA. The
performance evaluation results considered the probability that a request was
successfully answered as a function of the number of uncooperative nodes in the
network. The service delivery probability decreased with the increase of
uncooperative mobile nodes. Using DE4MHA, it was observed that performance
presented a slightly worse result. The service average was also slightly worse
but practically insignificantly different than with DE4MHA, being considered
negligible.
CONCLUSIONS: This paper proposed a data encryption solution for mobile health
apps, called DE4MHA. The data encryption algorithm DE4MHA with cooperation
mechanisms in mobile health allow users to safely obtain health information with
the data being carried securely. These security mechanisms did not deteriorate
the overall network performance and the app, maintaining similar performance
levels as without the encryption. More importantly, it offers a robust and
reliable increase of privacy, confidentiality, integrity, and authenticity of
their health information. Although it was experimented on a specific mHealth
app, SapoFit, both DE4MHA and the cooperation strategy can be deployed in other
mHealth apps. BACKGROUND: Today's generation of young adults are gaining weight faster than
their parents; however, there remains insufficient evidence to inform
interventions to prevent this weight gain. Mobile phones are a popular means of
communication that may provide a convenient, inexpensive means to deliver health
intervention programmes. This pilot study aimed to measure the effect of a
12-week mobile health (mHealth) intervention on body weight, body mass index and
specific lifestyle behaviours addressed by the programme.
METHODS: University students and staff aged 18-35 years (n = 51) were randomised
(ratio 1 : 1, intervention : control). Both groups received a printed diet
booklet with instructions prepared by a dietitian. The intervention group also
received Short Message Service (SMS) text messages (four per week), e-mails
(four per week), and had access to smartphone applications and Internet forums.
RESULTS: Pre- to post-intervention, participants in the intervention group
decreased their body weight [mean (SD)] [-1.6 (2.6) kg], increased their light
intensity activity [34 (35) min day(-1)] and reported an increased vegetable
(1.0 median serving day(-1)) and decreased sugar-sweetened beverage intake [-355
(836) mL week(-1)]. Despite this, post-intervention changes in outcomes were not
significantly different from controls.
CONCLUSIONS: The piloted mHealth programme provided some short-term positive
changes in weight, nutrition and physical activity using a low cost, convenient
delivery method for this population. However, changes were no different from
those observed among controls. This might partly be explained by intervention
participants' low engagement with the programme, which is likely to require
further modification to provide more regular, personalised, monitored support. BACKGROUND: Using regulatory focus theory, an intervention of daily weight
loss-sustaining messages was developed and tested for acceptability,
feasibility, and efficacy on helping people sustain weight loss.
METHODS: Participants (n = 120) were randomized to a promotion, prevention, or
an attention-control text message group after completion of a weight loss
program. Participants completed baseline assessments, and reported their weight
at 1 and 3 months postbaseline.
RESULTS: Participants found the message content and intervention acceptable and
valuable. A minimum of one message per day delivered at approximately 8:00 am
was deemed the optimal delivery time and frequency. The sustained weight loss
rate at month 3 for the control, promotion, and prevention groups was 90%, 95%,
and 100%, respectively. Medium-to-large effects were observed for the promotion
and prevention groups at month 1 and for prevention at month 3 relative to
controls. The mean weight loss for promotion and prevention was 15 pounds,
compared with 10 in the controls at month 3.
CONCLUSION: A clinically significant decrease in mean weight, higher rate of
sustained weight loss, and medium-to-large effects on sustained weight loss
occurred in the promotion and prevention interventions. Tools such as this text
message-based intervention that are constructed and guided by evidence-based
content and theoretical constructs show promise in helping people sustain
healthy behaviors that can lead to improved health outcomes. This systematic review assesses the published literature to describe the
landscape of mobile health technology (mHealth) for HIV/AIDS and the evidence
supporting the use of these tools to address the HIV prevention, care, and
treatment cascade. The speed of innovation, broad range of initiatives and
tools, and heterogeneity in reporting have made it difficult to uncover and
synthesize knowledge on how mHealth tools might be effective in addressing the
HIV pandemic. To do address this gap, a team of reviewers collected literature
on the use of mobile technology for HIV/AIDS among health, engineering, and
social science literature databases and analyzed a final set of 62 articles.
Articles were systematically coded, assessed for scientific rigor, and sorted
for HIV programmatic relevance. The review revealed evidence that mHealth tools
support HIV programmatic priorities, including: linkage to care, retention in
care, and adherence to antiretroviral treatment. In terms of technical features,
mHealth tools facilitate alerts and reminders, data collection, direct voice
communication, educational messaging, information on demand, and more. Studies
were mostly descriptive with a growing number of quasi-experimental and
experimental designs. There was a lack of evidence around the use of mHealth
tools to address the needs of key populations, including pregt mothers, sex
workers, users of injection drugs, and men who have sex with men. The science
and practice of mHealth for HIV are evolving rapidly, but still in their early
stages. Small-scale efforts, pilot projects, and preliminary descriptive studies
are advancing and there is a promising trend toward implementing mHealth
innovation that is feasible and acceptable within low-resource settings,
positive program outcomes, operational improvements, and rigorous study design. Childhood obesity continues to be a significant public health issue. mHealth
systems offer state-of-the-art approaches to intervention design, delivery, and
diffusion of treatment and prevention efforts. Benefits include cost
effectiveness, potential for real-time data collection, feedback capability,
minimized participant burden, relevance to multiple types of populations, and
increased dissemination capability. However, these advantages are coupled with
unique challenges. This commentary discusses challenges with using mHealth
strategies for child obesity prevention, such as lack of scientific evidence
base describing effectiveness of commercially available applications; relatively
slower speed of technology development in academic research settings as compared
with industry; data security, and patient privacy; potentially adverse
consequences of increased sedentary screen time, and decreased focused attention
due to technology use. Implications for researchers include development of more
nuanced measures of screen time and other technology-related activities, and
partnering with industry for developing healthier technologies. Implications for
health practitioners include monitoring, assessing, and providing feedback to
child obesity program designers about users' data transfer issues, perceived
security and privacy, sedentary behavior, focused attention, and maintece of
behavior change. Implications for policy makers include regulation of claims and
quality of apps (especially those aimed at children), supporting standardized
data encryption and secure open architecture, and resources for
research-industry partnerships that improve the look and feel of technology.
Partnerships between academia and industry may promote solutions, as discussed
in this commentary. |
Which receptors are targeted by suvorexant? | Suvorexant is a potent, selective, and orally bioavailable antagonist of orexin 1 receptor and orexin 2 receptor currently under clinical investigation as a novel therapy for insomnia. | The first example of an intramolecular asymmetric reductive amination of a
dialkyl ketone with an aliphatic amine has been developed for the synthesis of
Suvorexant (MK-4305), a potent dual Orexin antagonist under development for the
treatment of sleep disorders. This challenging transformation is mediated by a
novel Ru-based transfer hydrogenation catalyst that provides the desired
diazepane ring in 97% yield and 94.5% ee. Mechanistic studies have revealed that
CO(2), produced as a necessary byproduct of this transfer hydrogenation
reaction, has pronounced effects on the efficiency of the Ru catalyst, the form
of the amine product, and the kinetics of the transformation. A simple kinetic
model explains how product inhibition by CO(2) leads to overall first-order
kinetics, but yields an apparent zero-order dependence on initial substrate
concentration. The deleterious effects of CO(2) on reaction rates and product
isolation can be overcome by purging CO(2) from the system. Moreover, the rate
of ketone hydrogenation can be greatly accelerated by purging of CO(2) or
trapping with nucleophilic secondary amines. A concise, etioselective synthesis of the potent dual orexin inhibitor
suvorexant (1) is reported. Key features of the synthesis include a mild
copper-catalyzed amination, a highly chemoselective conjugate addition, and a
tandem etioselective transamination/seven-membered ring annulation. The
synthesis requires inexpensive starting materials and only four linear steps for
completion. Suvorexant is a dual orexin antagonist currently in Phase III clinical trials
for the modulation of sleep and is being developed by Merck. Recent Phase III
results showed that patients taking the drug fell asleep faster and slept longer
than those on placebo. Application WO2012081692 from Taisho Pharmaceutical Co. Ltd. claims
pyrazole-based antagonists of the orexin-1 and orexin-2 receptors. Utility in a
number of therapeutic areas is claimed, including the treatment of sleep
disorders; the most likely use of the claimed compounds. Data from in vitro
functional assays are presented, with the claimed compounds typically being dual
orexin receptor antagonists (DORAs) or having moderate selectivity for orexin-1.
Structurally, the claimed compounds represent a variation on established DORA
SAR themes and translate features of clinical compounds into a pyrazole-based
scaffold. Example 52, the most potent molecule in the application, has similar
molecular weight and lipophilicity to suvorexant, the most advanced DORA, with
broadly comparable potency in functional assays. Orexin A and orexin B are hypothalamic neuropeptides initially identified as
endogenous ligands for two orphan G-protein coupled receptors (GPCRs). They play
critical roles in the maintece of wakefulness by regulating function of
monoaminergic and cholinergic neurons that are implicated in the regulation of
wakefulness. Loss of orexin neurons in humans is associated with narcolepsy, a
sleep disorder characterized by excessive daytime sleepiness and cataplexy,
further suggesting the particular importance of orexin in the maintece of the
wakefulness state. These findings have encouraged pharmaceutical companies to
develop drugs targeting orexin receptors as novel medications of sleep
disorders, such as narcolepsy and insomnia. Indeed, phase III clinical trials
were completed last year of suvorexant, a non-selective (dual) antagonist for
orexin receptors, for the treatment of primary insomnia, and demonstrate
promising results. The New Drug Application (NDA) for suvorexant has been
submitted to the US FDA. Thus, the discovery of a critical role played by the
orexin system in the regulation of sleep/wakefulness has opened the door of a
new era for sleep medicine. STUDY OBJECTIVES: Suvorexant (MK-4305) is an orexin receptor antagonist being
developed for the treatment of insomnia. This report describes the effects of
nighttime administration of suvorexant on polysomnography (PSG) sleep parameters
in healthy young men.
DESIGN: Randomized, double-blind, placebo-controlled, 4-period crossover PSG
study, followed by an additional 5(th) period to assess pharmacokinetics.
SETTING: Sleep laboratory.
PARTICIPANTS: Healthy young men between 18 and 45 years of age (22 enrolled, 19
completed).
INTERVENTIONS: Periods 1-4: suvorexant (10 mg, 50 mg, or 100 mg) or placebo 1 h
before nighttime PSG recording. Period 5: suvorexant 10 mg, 50 mg, or 100 mg.
MEASUREMENTS AND RESULTS: In Periods 1-4, overnight sleep parameters were
recorded by PSG and next-morning residual effects were assessed by psychomotor
performance tests and subjective assessments. Statistically significant
sleep-promoting effects were observed with all doses of suvorexant compared to
placebo. Suvorexant 50 mg and 100 mg significantly decreased latency to
persistent sleep and wake after sleep onset time, and increased sleep
efficiency. Suvorexant 10 mg significantly decreased wake after sleep onset
time. There were no statistically significant effects of suvorexant on EEG
frequency bands including delta (slow wave) activity based on power spectral
analysis. Suvorexant was well tolerated. There was no evidence of next-day
residual effects for suvorexant 10 mg. Suvorexant 50 mg statistically
significantly reduced subjective alertness, and suvorexant 100 mg significantly
increased reaction time and reduced subjective alertness. There were no
statistically significant effects of any suvorexant dose on digit symbol
substitution test performance. In Period 5, plasma samples of suvorexant were
collected for pharmacokinetic evaluation. The median T(max) was 3 hours and
apparent terminal t(½) was 9-13 hours.
CONCLUSIONS: In healthy young men without sleep disorders, suvorexant promoted
sleep with some evidence of residual effects at the highest doses. Insomnia persistently affects the quality and quantity of sleep. Currently
approved treatments for insomnia primarily target γ-aminobutyric acid-A (GABA-A)
receptor signalling and include benzodiazepines and GABA-A receptor modulators.
These drugs are used to address this sleep disorder, but have the potential for
side effects such as tolerance and dependence, making them less attractive as
maintece therapy. Forward and reverse genetic approaches in animals have
implicated orexin signalling (also referred to as hypocretin signalling) in the
control of vigilance and sleep/wake states. Screening for orexin receptor
antagonists using in vitro and in vivo methods in animals has identified
compounds that block one or other of the orexin receptors (single or dual orexin
receptor antagonists [SORAs and DORAs], respectively) in animals and humans.
SORAs have primarily been used as probes to further elucidate the roles of the
individual orexin receptors, while a number of DORAs have progressed to clinical
development as pharmaceutical candidates for insomnia. The DORA almorexant
demonstrated significant improvements in a number of clinically relevant sleep
parameters in animal models and in patients with insomnia but its development
was halted. SB-649868 and suvorexant have demonstrated efficacy and tolerability
in Phase II and III trials respectively. Furthermore, suvorexant is currently
under review by the Food and Drug Administration for the treatment of insomnia.
Based on the publication of recent non-clinical and clinical data, orexin
receptor antagonists potentially represent a targeted, effective and
well-tolerated new class of medications for insomnia. Orexins (also known as hypocretins) play critical roles in the regulation of
sleep/wakefulness states by activating two G-protein coupled receptors (GPCRs),
orexin 1 (OX1R) and orexin 2 receptors (OX2R). In order to understand the
differential contribution of both receptors in regulating sleep/wakefulness
states we compared the pharmacological effects of a newly developed OX2R
antagonist (2-SORA), Compound 1 m (C1 m), with those of a dual orexin receptor
antagonist (DORA), suvorexant, in C57BL/6J mice. After oral administration in
the dark period, both C1m and suvorexant decreased wakefulness time with similar
efficacy in a dose-dependent manner. While C1m primarily increased total
non-rapid eye movement (NREM) sleep time without affecting episode durations and
with minimal effects on REM sleep, suvorexant increased both total NREM and REM
sleep time and episode durations with predomit effects on REM sleep.
Fos-immunostaining showed that both compounds affected the activities of
arousal-related neurons with different patterns. The number of Fos-IR
noradrenergic neurons in the locus coeruleus was lower in the suvorexant group
as compared with the control and C1m-treated groups. In contrast, the numbers of
Fos-IR neurons in histaminergic neurons in the tuberomamillary nucleus and
serotonergic neurons in the dorsal raphe were reduced to a similar extent in the
suvorexant and C1m groups as compared with the vehicle-treated group. Together,
these results suggest that an orexin-mediated suppression of REM sleep via
potential activation of OX1Rs in the locus coeruleus may possibly contribute to
the differential effects on sleep/wakefulness exerted by a DORA as compared to a
2-SORA. BACKGROUND: Suvorexant (MK-4305) is an orexin receptor antagonist shown to be
efficacious for insomnia over 3 months. We aimed to assess its clinical profile
during and after 1 year of treatment.
METHODS: We did a randomised, placebo-controlled, parallel-group trial at 106
investigational centres in the Americas, Australia, Europe, and South Africa
from December, 2009, to August, 2011. Patients aged 18 years or older with
primary insomnia by DSM-IV-TR criteria were assigned using a computer-generated
randomised allocation schedule to receive nightly suvorexant (40 mg for patients
younger than 65 years, 30 mg for patients aged 65 years or older) or placebo at
a 2:1 ratio for 1 year with a subsequent 2-month randomised discontinuation
phase in which patients on suvorexant either continued suvorexant or were
abruptly switched to placebo while patients on placebo remained on placebo.
Treatment assignment was masked from patients and investigators. The primary
objective was to assess the safety and tolerability of suvorexant for up to 1
year. Secondary objectives were to assess the efficacy of suvorexant for
improving patient-reported subjective total sleep time (sTST) and time to sleep
onset (sTSO) over the first month of treatment. Efficacy endpoints over the
first month were assessed with a mixed model with terms for baseline value of
the response variable, age, sex, region, treatment, time, and treatment by time
interaction. This trial is registered with ClinicalTrials.gov, number
NCT01021813.
FINDINGS: 322 (62%) of 522 patients randomly assigned to receive suvorexant and
162 (63%) of 259 assigned to receive placebo completed the 1-year phase. Over 1
year, 362 (69%) of 521 patients treated with suvorexant experienced any adverse
events compared with 164 (64%) of 258 treated with placebo. Serious adverse
events were recorded in 27 patients (5%) who received suvorexant and 17 (7%) who
received placebo. The most common adverse event, somnolence, was reported for 69
patients (13%) who received suvorexant and seven (3%) who received placebo. At
month 1, suvorexant (517 patients in the efficacy population) showed greater
efficacy than placebo (254 in the efficacy population) in improving sTST (38·7
min vs 16·0 min; difference 22·7, 95% CI 16·4 to 29·0; p<0·0001) and sTSO (-18·0
min vs -8·4 min, difference -9·5, -14·6 to -4·5; p=0·0002).
INTERPRETATION: Our findings show that suvorexant was generally safe and well
tolerated over 1 year of nightly treatment in patients with insomnia, with
efficacy noted for subjective measures of sleep onset and maintece.
FUNDING: Merck & Co Inc. Addiction is a chronic relapsing disorder which presents a significant global
health burden and unmet medical need. The orexin/hypocretin system is an
attractive potential therapeutic target as demonstrated by the successful
clinical trials of antagonist medications like Suvorexant for insomnia. It is
composed of two neuropeptides, orexin-A and orexin-B and two excitatory and
promiscuous G-protein coupled receptors, OX1 and OX2. Orexins are known to have
a variety of functions, most notably in regulating arousal, appetite and reward.
The orexins have been shown to have a role in mediating the effects of several
drugs of abuse, such as cocaine, morphine and alcohol via projections to key
brain regions such as the ventral tegmental area, nucleus accumbens and
prefrontal cortex. However, it has not yet been demonstrated whether the dual
orexin receptor antagonists (DORAs) under development for insomnia are ideal
drugs for the treatment of addiction. The question of whether to use a DORA or
single orexin receptor antagonist (SORA) for the treatment of addiction is a key
question that will need to be answered in order to maximize the clinical utility
of orexin receptor antagonists. This review will examine the role of the
orexin/hypocretin system in addiction, orexin-based pharmacotherapies under
development and factors affecting the selection of one or both orexin receptors
as drug targets for the treatment of addiction. STUDY OBJECTIVES: Suvorexant, an orexin receptor antagonist, improves sleep in
healthy subjects (HS) and patients with insomnia. We compared the
electroencephalographic (EEG) power spectral density (PSD) profile of suvorexant
with placebo using data from a phase 2 trial in patients with insomnia. We also
compared suvorexant's PSD profile with the profiles of other insomnia treatments
using data from 3 HS studies.
DESIGN: Phase 2 trial--randomized, double-blind, two-period (4 w per period)
crossover. HS studies--randomized, double-blind, crossover.
SETTING: Sleep laboratories.
PARTICIPANTS: Insomnia patients (n = 229) or HS (n = 124).
INTERVENTIONS: Phase 2 trial--suvorexant 10 mg, 20 mg, 40 mg, 80 mg, placebo; HS
study 1--suvorexant 10 mg, 50 mg, placebo; HS study 2--gaboxadol 15 mg, zolpidem
10 mg, placebo; HS study 3--trazodone 150 mg, placebo.
MEASUREMENTS AND RESULTS: The PSD of the EEG signal at 1-32 Hz of each PSG
recording during nonrapid eye movement (NREM) and rapid eye movement (REM) sleep
were calculated. The day 1 and day 28 PSD profiles of suvorexant at all four
doses during NREM and REM sleep in patients with insomnia were generally flat
and close to 1.0 (placebo) at all frequencies. The day 1 PSD profile of
suvorexant in HS was similar to that in insomnia patients. In contrast, the
other three drugs had distinct PSD profiles in HS that differed from each other.
CONCLUSIONS: Suvorexant at clinically effective doses had limited effects on
power spectral density compared with placebo in healthy subjects and in patients
with insomnia, in contrast to the three comparison insomnia treatments. These
findings suggest the possibility that antagonism of the orexin pathway might
lead to improvements in sleep without major changes in the patient's
neurophysiology as assessed by electroencephalographic. Suvorexant (Belsomra(®)), a first-in-class, orally active dual orexin-1 receptor
and orexin-2 receptor antagonist, has been developed by Merck for the treatment
of insomnia. Variations in the levels of the neuropeptides orexin A and orexin B
have been linked to circadian rhythms and wakefulness. Orexin-producing neurons
in the lateral hypothalamus regulate wakefulness by signalling through orexin
receptors. Blockade of orexin receptors is known to promote sleep. Suvorexant
was approved in the US in August 2014 for the treatment of adults with sleep
onset and/or sleep maintece insomnia. The drug is also preregistration in
Japan, with approval submissions planned for other countries worldwide for this
indication. This article summarizes the milestones in the development of
suvorexant leading to this first approval for insomnia. The orexin-1 and orexin-2 receptors are two G protein-coupled receptors that
bind the neuropeptides orexin-A and orexin-B. Dual antagonism of the receptors
by small molecules is clinically efficacious in the treatment of insomnia, where
the most advanced molecule suvorexant has recently been approved. The scope of
this article is to review the small molecule orexin receptor antagonist patent
literature between January 2012 and January 2014. |
Is armodafinil used for treatment of insomnia? | No, armodafinil is not used for treatment of insomnia. Armodafinil is a wakefulness-promoting medication. Its efficacy and tolerability have been established in patients with excessive sleepiness (ES) associated with treated obstructive sleep apnea (OSA), shift work disorder (SWD), or narcolepsy. The wakefulness-promoting agents armodafinil and modafinil are FDA approved for the treatment of ES in patients with SWD. | OBJECTIVE: This study assessed the efficacy and safety of armodafinil, the
longer half-life etiomer of modafinil, for the treatment of excessive
sleepiness in patients with narcolepsy.
RESEARCH DESIGN AND METHODS: This was a multicenter double-blind study with 196
patients (aged 18-65 years) randomized to receive armodafinil 150 mg (n = 65),
armodafinil 250 mg (n = 67), or placebo (n = 64) once daily for 12 weeks.
MAIN OUTCOME MEASURES: Efficacy was assessed using the Maintece of
Wakefulness Test (MWT) (six 20-min subtests across the day), the Clinical Global
Impression of Change (CGI-C), subjective measures of sleepiness (Epworth
Sleepiness Scale), patient diaries, and evaluations of cognitive performance
(Cognitive Drug Research) and fatigue (Brief Fatigue Inventory).
RESULTS: Armodafinil significantly increased MWT mean sleep latency (at
0900-1500) compared with placebo. The mean change from baseline at final visit
for armodafinil was an increase of 1.3, 2.6, and 1.9 min in the 150-mg, 250-mg,
and combined groups, respectively, compared with a decrease of 1.9 min for
placebo (p < 0.01 for all three comparisons). Mean late-day MWT latency
(1500-1900) was also significantly improved (difference of armodafinil combined
group relative to placebo at final visit: 2.8 min, p = 0.0358). The proportions
of patients who showed at least minimal improvement in the CGIC rating from
baseline to final visit in the armodafinil 150-mg, 250-mg, and combined groups
were 69%, 73%, and 71%, respectively, compared with 33% for placebo (p <
0.0001). Both doses were associated with statistically significant improvements
in memory, attention, and fatigue (p < 0.05). The most common adverse events in
patients receiving armodafinil were headache, nausea, and dizziness.
CONCLUSIONS: Armodafinil significantly improved ability to sustain wakefulness
throughout the day in patients with narcolepsy. Armodafinil also significantly
improved overall clinical condition, memory, attention, and fatigue when
compared with placebo. BACKGROUND: Some patients with obstructive sleep apnea/hypopnea syndrome
(OSA/HS) experience excessive sleepiness (ES) that might not resolve with nasal
continuous positive airway pressure (nCPAP) treatment.
OBJECTIVE: The aim of the present study was to assess the efficacy and
tolerability of armodafinil 150 or 250 mg QD when used as adjunctive treatment
for residual ES associated with OSA/HS in patients who are adherent to nCPAP
therapy.
METHODS: This 12-week, multicenter, double-blind, randomized, placebo-controlled
study was conducted at 37 centers in the United States and Canada. Male and
female patients aged 18 to 65 years with residual ES associated with OSA/HS were
enrolled. Patients were randomly assigned to receive armodafinil 150 or 250 mg
or placebo PO QD for 12 weeks. Assessments were conducted at baseline and study
weeks 4, 8, and 12 and included the Maintece of Wakefulness Test (MWT) to
determine wakefulness, the Clinical Global Impression of Change (CGI-C) to
determine improvement in clinical condition, the Epworth Sleepiness Scale (ESS)
to determine patient-estimated wakefulness, the Brief Fatigue Inventory (BFI) to
determine global fatigue, and the Cognitive Drug Research computerized
assessment battery. To distinguish between earlier and later effects, sleep
latencies, assessed using the MWT, were averaged across the first 4 (9 and 11
AM, and 1 and 3 PM) and last 3 (3, 5, and 7 PM) tests. Tolerability assessments
included monitoring of adverse events (AEs), clinical laboratory tests, vital
sign measurements, and electrocardiography.
RESULTS: A total of 395 patients were enrolled in the study (armodafinil 150
mg/d, 133; armodafinil 250 mg/d, 131; placebo, 131); 392 received >or=1 dose of
study drug (armodafinil 150 mg/d, 131; armodafinil 250 mg/d, 131; placebo, 130).
The armodafinil and placebo groups were well matched with regard to age (mean
[SD], 49.2 [8.9] vs 50.1 [9.4] years), sex (71 vs 69% men), race (84% vs 87%
white), and body weight (mean [SD], 110.3 [24.9] vs 111.9 [24.0] kg). At the
final visit, the mean (SD) change from baseline in MWT sleep latency across the
morning and afternoon was significantly greater in the armodafinil combined
group compared with the placebo group (+1.9 [7.3] vs 1.7 [8.6] minutes; P <
0.001). Also at the final visit, the proportions of patients who showed at least
minimal improvement on the CGI-C, and the mean (SD) changes from baseline in ESS
and BFI scores, were significantly greater in the armodafinil group compared
with those in the placebo group (72% vs 37%, -5.5 [5.0] vs -3.3 [4.7], and -1.2
[2.2] vs -0.6 [2.0], respectively; P < 0.001, P < 0.001, and P < 0.01,
respectively). No significant effects on nighttime sleep, as assessed using
polysomnography, were found with armodafinil. AEs reported in the armodafinil
combined and placebo groups were headache, nausea, insomnia, anxiety, and
dizziness. Serious AEs (ulcerative colitis, migraine, worsening of Axis II and
mood disorder, and duodenal ulcer) were reported in 4 (1.5%) patients receiving
armodafinil and were considered by the investigator not or unlikely to be drug
related.
CONCLUSIONS: In this selected population of patients with OSA/HS and residual ES
despite effective treatment with nCPAP, armodafinil QD used as an adjunct to
nCPAP treatment was associated with improved wakefulness and overall clinical
condition. Clinical benefit was shown at the first assessment and maintained for
the 12-week duration of the study. Armodafinil was also associated with
significantly reduced interference of ES with daily activities and global
fatigue. Armodafinil was well tolerated, with no adverse effect on nighttime
sleep or nCPAP use. More than 6 million Americans work night shifts on a regular or rotating basis.
The negative consequences of shift work have been established, and recent
evidence suggests that patients with shift work sleep disorder (SWSD) are at
increased risk of these consequences and co-morbidities. SWSD is a relatively
common but under-recognised, and hence undertreated, condition with potentially
serious medical, social, economic and quality-of-life consequences. In addition
to increased risk of gastrointestinal and cardiovascular disease, patients with
SWSD experience clinically significant excessive sleepiness or insomnia
associated with work during normal sleep times, which has important safety
implications. A number of studies have evaluated countermeasures or
interventions in shift workers; proposed treatments include chronobiotic
interventions, such as light exposure, melatonin, hypnotic agents, caffeine and
CNS stimulants (amphetamine), and the wake-promoting agents modafinil and
armodafinil. However, most studies evaluating pharmacological therapies and
nonpharmacological interventions simulate night-shift work under conditions that
may not accurately reflect real-world activities. Pharmacological and
nonpharmacological countermeasures evaluated mostly in simulated laboratory
conditions have been shown to improve alertness or sleep in shift workers but
have not yet been evaluated in patients with SWSD. To date, three randomised,
double-blind clinical studies have evaluated pharmacological therapies in
patients with SWSD. These studies showed that modafinil and armodafinil
significantly improve the ability to sustain wakefulness during waking
activities (e.g. working, driving), overall clinical condition, and sustained
attention or memory in patients with SWSD. In conclusion, SWSD is a common
condition that remains under-recognised and undertreated. Further research is
needed to evaluate different treatment approaches for this condition, to clarify
the substantial health and economic consequences of SWSD, and to determine the
potential for interventions or treatments to reduce the negative consequences of
this condition. Residual excessive sleepiness (ES) and impaired cognition can occur despite
effective and regular nasal continuous positive airway pressure (nCPAP) therapy
in some patients with obstructive sleep apnea (OSA). A pooled analysis of two
12-week, randomized, double-blind studies in nCPAP-adherent patients with ES
associated with OSA evaluated the effect of armodafinil on wakefulness and
cognition. Three hundred and ninety-one patients received armodafinil (150 or
250 mg) and 260 patients received placebo once daily for 12 weeks. Efficacy
assessments included the Maintece of Wakefulness Test (MWT), Cognitive Drug
Research cognitive performance battery, Epworth Sleepiness Scale, and Brief
Fatigue Inventory. Adverse events were monitored. Armodafinil increased mean MWT
sleep latency from baseline to final visit by 2.0 min vs a decrease of 1.5 min
with placebo (P < 0.0001). Compared with placebo, armodafinil significantly
improved quality of episodic secondary memory (P < 0.05) and patients' ability
to engage in activities of daily living (P < 0.0001) and reduced fatigue (P <
0.01). The most common adverse events were headache, nausea, and insomnia.
Armodafinil did not adversely affect desired nighttime sleep, and nCPAP use
remained high (approximately 7 h/night). Adjunct treatment with armodafinil
significantly improved wakefulness, long-term memory, and patients' ability to
engage in activities of daily living in nCPAP-adherent individuals with ES
associated with OSA. Armodafinil also reduced patient-reported fatigue and was
well tolerated. OBJECTIVE: To assess the effect of armodafinil, 150 mg, on the physiologic
propensity for sleep and cognitive performance during usual night shift hours in
patients with excessive sleepiness associated with chronic (> or =3 months)
shift work disorder (SWD) of moderate or greater severity.
PATIENTS AND METHODS: This 12-week, randomized controlled study was conducted at
42 sleep research facilities in North America from April 2 through December 23,
2004, and enrolled 254 permanent or rotating night shift workers with SWD. Entry
criteria included excessive sleepiness during usual night shifts for 3 months or
longer (corroborated by mean sleep latency of < or =6 minutes on a Multiple
Sleep Latency Test), insomnia (sleep efficiency < or =87.5% during daytime
sleep), and SWD that was judged clinically to be of moderate or greater
severity. Patients received armodafinil, 150 mg, or placebo 30 to 60 minutes
before each night shift. Physiologic sleep propensity during night shift hours,
clinical impression of severity, patient-reported sleepiness, and cognitive
function were assessed during laboratory night shifts at weeks 4, 8, and 12.
RESULTS: Armodafinil significantly improved mean (SD) sleep latency from 2.3
(1.6) minutes at baseline to 5.3 (5.0) minutes at final visit, compared with a
change from 2.4 (1.6) minutes to 2.8 (2.9) minutes in the placebo group
(P<.001). Clinical condition ratings improved in more patients receiving
armodafinil (79%) vs placebo (59%) (P=.001). As reported by patients' diaries,
armodafinil significantly reduced sleepiness during laboratory nights (P<.001),
night shifts at work (P<.001), and the commute home (P=.003). Armodafinil
improved performance on standardized memory (P<.001) and attention (power,
P=.001; continuity, P<.001) tests compared with placebo. Armodafinil was well
tolerated and did not affect daytime sleep, as measured by polysomnography.
CONCLUSION: In patients with excessive sleepiness associated with chronic SWD of
moderate or greater severity, armodafinil significantly improved wakefulness
during scheduled night work, raising mean nighttime sleep latency above the
level considered to indicate severe sleepiness during the daytime. Armodafinil
also significantly improved measures of overall clinical condition, long-term
memory, and attention.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00080288. Behavioral measures, eg, exercise and improved sleep hygiene, can enhance sleep
quality and combat insomnia and excessive sleepiness (ES) in shift workers and
individuals with shift-work disorder (SWD). Napping before a shift followed by
consumption of a caffeinated drink and, if appropriate, scheduled naps at work,
may improve ES in patients with SWD. Use of bright light therapy to partially
re-entrain the circadian clock should be explored for all night-shift
workers-particularly those with SWD. The wakefulness-promoting agents
armodafinil and modafinil are FDA approved for the treatment of ES in patients
with SWD. Alongside nonpharmacologic interventions, they can be included in a
comprehensive management plan for SWD. Melatonin or other sleep-promoting agents
may help shift workers achieve sleep during required rest periods and when
adjusting to night-shift work; studies are needed in patients with SWD to better
evaluate the utility of these agents in this population. BACKGROUND: Armodafinil (Nuvigil(®), Cephalon, Inc., Frazer, PA, USA), the
longer-lasting isomer of racemic modafinil, is a nonamphetamine,
wakefulness-promoting medication. In patients with excessive sleepiness
associated with shift work disorder, treated obstructive sleep apnoea, or
narcolepsy, armodafinil has been found to improve wakefulness throughout the
shift or day. In addition, while not approved for this indication, armodafinil
has been found to improve excessive sleepiness associated with jet-lag disorder.
OBJECTIVE: This study evaluated systemic exposure to armodafinil and its two
major circulating metabolites, R-modafinil acid and modafinil sulfone, and
assessed the tolerability profile of armodafinil in elderly and young subjects.
METHODS: The pharmacokinetics and tolerability of armodafinil were assessed in
an open-label, multiple-dose, parallel-group study in two groups (n = 25 in each
group) of healthy men (elderly group aged ≥65 years and young group aged 18-45
years) who received armodafinil 50 mg on day 1, 100 mg on day 2 and 150 mg once
daily on days 3 through 7. Plasma concentrations of armodafinil and its
metabolites were quantified over 72 hours following the last dose on day 7.
Pharmacokinetic parameters, including area under the plasma drug
concentration-versus-time curve during a dosing interval (AUC(τ)) and maximum
observed plasma drug concentration (C(max)), and tolerability were assessed.
RESULTS: All 50 subjects enrolled in the study were evaluable for tolerability
and 49 were included in the pharmacokinetic analysis. One elderly subject was
excluded from the pharmacokinetic analyses because of apparent noncompliance
with armodafinil dosing. Systemic exposure following administration of
armodafinil, as measured by steady-state AUC(τ) and C(max) values, was
approximately 15% greater in elderly subjects compared with young subjects.
Geometric mean ratios for AUC(τ) and C(max) in the two groups were 1.14 (95% CI
1.03, 1.25; p = 0.0086) and 1.15 (95% CI 1.08, 1.24; p = 0.0002), respectively.
When data were analysed for elderly subgroups, systemic exposure in the
old-elderly group (age ≥75 years; n = 7) was 27% greater than in young subjects,
as compared with 10% greater in the young-elderly group (age 65-74 years;
n = 17). Although steady-state exposure to the metabolite R-modafinil acid was
also higher in elderly than in young subjects (geometric mean ratios for AUC(τ)
and C(max) were 1.73 and 1.61, respectively; p < 0.0001), there were no
significant differences in systemic exposure to modafinil sulfone. Armodafinil
was generally well tolerated by both groups. Headache (four subjects in each
group), nausea (one in the elderly group and four in the young group), insomnia
(two in the elderly group and one in the young group), and dizziness (two in the
young group) were the most common adverse events.
CONCLUSIONS: Systemic exposure following administration of armodafinil is
increased in the elderly in comparison with younger subjects, particularly in
those aged ≥75 years. Although the increase in plasma armodafinil concentration
in elderly subjects does not appear to result in more adverse events compared
with young subjects, consideration should be given to the use of lower dosages
of armodafinil for the management of excessive sleepiness in older patients,
particularly the very elderly. "Shift work" is a term that applies to a wide array of nontraditional work
schedules. Shift work disorder (SWD) is a circadian rhythm sleep disorder
experienced by a subset of shift workers that is characterized by excessive
sleepiness during work and/or insomnia during scheduled sleep times. It is
estimated to affect up to 2 million Americans, and is associated with increased
morbidity and mortality from metabolic risk factors, cardiovascular and
gastrointestinal diseases, depression, accidents, and some kinds of cancers.
Patient history is all that is needed to make a diagnosis with the International
Classification of Sleep Disorders-Second Edition criteria as described herein.
Circadian rhythm disorders, in which an underlying misalignment of circadian
rhythm with the sleep-wake cycle occurs, may be treated by behavioral and
pharmacologic approaches, including the use of hypnotics to improve the duration
of sleep. However, evidence is limited with these approaches in patients
diagnosed with SWD. Other treatment options may include pharmacologic
interventions such as modafinil and armodafinil, which have shown efficacy in
this population. Combined therapy can reduce insomnia and excessive sleepiness,
and improve attention and alertness during work shifts and the subsequent
commute home. BACKGROUND: Shift work disorder (SWD) is characterized by symptoms of excessive
sleepiness during work hours or insomnia during allotted daytime sleep hours, as
well as by a disruption of the circadian rhythm. Many shift workers with SWD
experience significant social, behavioral, and health problems as a result of
this disorder. SWD is associated with a higher risk of occupational and motor
vehicle accidents, and thus poses a public health risk.
METHODS: Currently there are both pharmacologic and non-pharmacologic treatments
for this disorder that can be used to normalize the disruption of the circadian
cycle or alleviate the symptoms of excessive sleepiness or insomnia. The
American Academy of Sleep Medicine and the British Society of Psychopharmacology
have developed guidelines for the diagnosis and treatment of patients with SWD.
RESULTS: Recommended therapies for altering the circadian cycle include
chronobiotics such as melatonin or melatonin agonists and non-pharmacologic
interventions such as timed light exposure. Other therapies, such as sedative
hypnotics, target daytime insomnia, while pharmacologic agents such as
modafinil, armodafinil, and caffeine and non-pharmacologic approaches such as
napping promote nighttime alertness.
CONCLUSIONS: While no therapies (pharmacological or nonpharmacological) can
restore altered circadian cycles to baseline levels, proper identification and
management of SWD will likely reduce its co-morbidities and improve the quality
of life for individuals with this disorder. |
A common problem in proteomics is the contamination of samples with exogenous proteins (often from other species). These proteins can be found in specific databases. List some contaminants. | Some common contaminants in proteomics are proteases (used for the digestion of the proteins), keratins (usually from the skin), proteins originated from the serum of the culture media and antibodies if used in the experiment. | Unmatched masses are often observed in the experimental peptide mass spectra
when database searching is performed with the ProFound program. Comparison
between theoretical and experimental mass spectra of standard proteins shows
that contamination accounts for most of the unmatched masses. In this
retrospective analysis, the top 100 most probable contaminating masses, as
listed in order of their probability, are statistically filtered out from 118
different experimental peptide mass fingerprinting (PMF) maps. Most of the
interfering masses originate from trypsin autolysis and human keratins.
Subtraction of known contamits from raw data and using cleaner masses for
searching can enhance protein identification by PMF. Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques
for studying protein-protein interactions. These methods utilize immobilized
Protein A or Protein G to isolate antibody-bound target antigens. The main
disadvantage of traditional IP and co-IP is that the conditions used to elute
the precipitated antigen also release the antibody thus contaminating the
antigen and destroying the antibody support. To overcome these problems, we
describe two methods to generate a reusable antibody support by cross-linking
the antibody to immobilized Protein A or Protein G, or by coupling it directly
to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while
antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the
antibody resin with the protein sample. Washes and elutions are carried out in a
spin column to reduce resin loss and decrease assay time. Target proteins are
eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is
re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have
demonstrated that the immobilization efficiency for the antibody coupling method
was similar for several species of antibody. Furthermore, we illustrate that
using both methods of antibody immobilization yield IP and co-IP results similar
to traditional protocols but eliminate the antibody heavy and light chain
contamination. Cell culture is a fundamental tool in proteomics where mammalian cells are
cultured in vitro using a growth medium often supplemented with 5-15% FBS.
Contamination by bovine proteins is difficult to avoid because of adherence to
the plastic vessel and the cultured cells. We have generated peptides from
bovine serum using four sample preparation methods and analyzed the peptides by
high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is
difficult because of a considerable overlap of identical tryptic peptide
sequences. Pitfalls in interpretation, different database search strategies to
minimize erroneous identifications and an augmented contamit database are
presented. Mass spectrometry is widely used in bioanalysis, including the fields of
metabolomics and proteomics, to simultaneously measure large numbers of
molecules in complex biological samples. Contamits routinely occur within
these samples, for example, originating from the solvents or plasticware.
Identification of these contamits is crucial to enable their removal before
data analysis, in particular to maintain the validity of conclusions drawn from
uni- and multivariate statistical analyses. Although efforts have been made to
report contamits within mass spectra, this information is fragmented and its
accessibility is relatively limited. In response to the needs of the
bioanalytical community, here we report the creation of an extensive manually
well-annotated database of currently known small molecule contamits.
AVAILABILITY: The Mass spectrometry Contamits Database (MaConDa) is freely
available and accessible through all major browsers or by using the MaConDa web
service http://www.maconda.bham.ac.uk. |
Which gene has been implicated in Majeed Syndrome? | Homozygous mutations in LPIN2 are responsible for the syndrome of chronic recurrent multifocal osteomyelitis and congenital dyserythropoietic anaemia (Majeed syndrome). | BACKGROUND: Majeed syndrome is an autosomal recessive, autoinflammatory disorder
characterised by chronic recurrent multifocal osteomyelitis and congenital
dyserythropoietic anaemia. The objectives of this study were to map, identify,
and characterise the Majeed syndrome causal gene and to speculate on its
function and role in skin and bone inflammation.
METHODS: Six individuals with Majeed syndrome from two unrelated families were
identified for this study. Homozygosity mapping and parametric linkage analysis
were employed for the localisation of the gene responsible for Majeed syndrome.
Direct sequencing was utilised for the identification of mutations within the
genes contained in the region of linkage. Expression studies and in silico
characterisation of the identified causal gene and its protein were carried out.
RESULTS: The phenotype of Majeed syndrome includes inflammation of the bone and
skin, recurrent fevers, and dyserythropoietic anaemia. The clinical picture of
the six affected individuals is briefly reviewed. The gene was mapped to a 5.5
cM interval (1.8 Mb) on chromosome 18p. Examination of genes in this interval
led to the identification of homozygous mutations in LPIN2 in affected
individuals from the two families. LPIN2 was found to be expressed in almost all
tissues. The function of LPIN2 and its role in inflammation remains unknown.
CONCLUSIONS: We conclude that homozygous mutations in LPIN2 result in Majeed
syndrome. Understanding the aberrant immune response in this condition will shed
light on the aetiology of other inflammatory disorders of multifactorial
aetiology including isolated chronic recurrent multifocal osteomyelitis, Sweet
syndrome, and psoriasis. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory
disorder that primarily affects bone but is often accompanied by inflammation of
the skin and/or gastrointestinal tract. The etiology is unknown but evidence
suggests a genetic component to disease susceptibility. Although most cases of
CRMO are sporadic, there is an autosomal recessive syndromic form of the
disease, called Majeed syndrome, which is due to homozygous mutations in LPIN2.
In addition, there is a phenotypically similar mouse, called cmo (chronic
multifocal osteomyelitis) in which the disease is inherited as an autosomal
recessive disorder. The cmo locus has been mapped to murine chromosome 18. In
this report, we describe phenotypic abnormalities in the cmo mouse that include
bone, cartilage and skin inflammation. Utilizing a backcross breeding strategy,
we refined the cmo locus to a 1.3 Mb region on murine chromosome 18. Within the
refined region was the gene pstpip2, which shares significant sequence homology
to the PSTPIP1. Mutations in PSTPIP1 have been shown to cause the
autoinflammatory disorder PAPA syndrome (pyogenic arthritis, pyoderma
gangrenosum and acne). Mutation analysis, utilizing direct sequencing, revealed
a single base pair change c.293T --> C in the pstpip2 gene resulting in a highly
conserved leucine at amino acid 98 being replaced by a proline (L98P). No other
mutations were found in the coding sequence of the remaining genes in the
refined interval, although a 50 kb gap remains unexplored. These data suggest
that mutations in pstpip2 may be the genetic explanation for the
autoinflammatory phenotype seen in the cmo mouse. Majeed syndrome is an autoinflammatory disorder consisting of chronic recurrent
multifocal osteomyelitis, congenital dyserythropoietic anemia, and neutrophilic
dermatosis. To date, 2 unrelated families with Majeed syndrome have been
reported. Mutations in LPIN2 have been found in both families. Here we report a
third consanguineous family with Majeed syndrome with a novel mutation. The
patient, a 3-year-old Arabic girl, had hepatosplenomegaly and anemia as a
neonate. At age 15 months, she developed recurrent episodes of fever and
multifocal osteomyelitis. In addition, bone marrow aspiration demonstrated
significant dyserythropoiesis, suggesting Majeed syndrome. Coding sequences and
splice sites of LPIN2 were sequenced in the patient and her mother. A homozygous
single-basepair change was detected in the donor splice site of exon 17
(c.2327+1G>C) in the patient; her mother was heterozygous at this site. These
data confirm the role of LPIN2 mutations in the etiology of Majeed syndrome. Mammalian lipins (lipin-1, lipin-2, and lipin-3) are Mg2+-dependent
phosphatidate phosphatase (PAP) enzymes, which catalyze a key reaction in
glycerolipid biosynthesis. Lipin-1 also functions as a transcriptional
coactivator in conjunction with members of the peroxisome proliferator-activated
receptor family. An S734L mutation in LPIN2 causes Majeed syndrome, a human
inflammatory disorder characterized by recurrent osteomyelitis, fever,
dyserythropoietic anemia, and cutaneous inflammation. Here we demonstrate that
mutation of the equivalent serine in mouse lipin-1 and lipin-2 to leucine or
aspartate abolishes PAP activity but does not impair lipin association with
microsomal membranes, the major site of glycerolipid synthesis. We also
determined that lipin-2 has transcriptional coactivator activity for peroxisome
proliferator-activated receptor-response elements similar to lipin-1 and that
this activity is not affected by mutating the conserved serine. Therefore, our
results indicate that the symptoms of the Majeed syndrome result from a loss of
lipin-2 PAP activity. To characterize sites of lipin-2 action, we detected
lipin-2 expression by in situ hybridization on whole mouse sections and by
quantitative PCR of tissues relevant to Majeed syndrome. Lipin-2 was most
prominently expressed in liver, where levels were much higher than lipin-1, and
also in kidney, lung, gastrointestinal tract, and specific regions of the brain.
Lipin-2 was also expressed in circulating red blood cells and sites of
lymphopoiesis (bone marrow, thymus, and spleen). These results raise the
possibility that the loss of lipin-2 PAP activity in erythrocytes and
lymphocytes may contribute to the anemia and inflammation phenotypes observed in
Majeed syndrome patients. BACKGROUND AND OBJECTIVE: Majeed syndrome is an autosomal recessive disorder
characterised by the triad of chronic recurrent multifocal osteomyelitis,
congenital dyserythropoietic anaemia and a neutrophilic dermatosis that is
caused by mutations in LPIN2. Long-term outcome is poor. This is the first
report detailing the treatment of Majeed syndrome with biological agents and
demonstrates clinical improvement with IL-1blockade.
METHODS: We describe the clinical presentation, genetic analysis, cytokine
profiles and response to biological therapy in two brothers with Majeed
syndrome.
RESULTS: Both boys were homozygous for a novel 2-base pair deletion in LPIN2
(c.1312_1313delCT; p.Leu438fs+16X), confirming the diagnosis. Their bone disease
and anaemia were refractory to treatment with corticosteroids. Both siblings had
elevated proinflammatory cytokines in their serum, including tumour necrosis
factor α (TNF-α), however a trial of the TNF inhibitor etanercept resulted in no
improvement. IL-1 inhibition with either a recombit IL-1 receptor antagonist
(anakinra) or an anti-IL-1β antibody (canakinumab) resulted in dramatic clinical
and laboratory improvement.
CONCLUSIONS: The differential response to treatment with TNF-α or IL-1 blocking
agents sheds light into disease pathogenesis; it supports the hypothesis that
Majeed syndrome is an IL-1β dependent autoinflammatory disorder, and further
underscores the importance of IL-1 in sterile bone inflammation. Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate
step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA)
to generate diacylglycerol. The lipin family of PA phosphatases is composed of
lipins 1-3, which are members of the conserved haloacid dehalogenase
superfamily. Although genetic alteration of LPIN2 in humans is known to cause
Majeed syndrome, little is known about the biochemical regulation of its PAP
activity. Here, in an attempt to gain a better general understanding of the
biochemical nature of lipin 2, we have performed kinetic and phosphorylation
analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the
electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis.
Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15
phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not
induced by insulin signaling nor is it sensitive to inhibition of the mammalian
target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively
regulate either membrane binding or PAP activity. This suggests that lipin 2
functions as a constitutively active PA phosphatase in stark contrast to the
high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of
lipin 2 regulation is important for a deeper understanding of how the lipin
family functions with respect to lipid synthesis and, more generally, as an
example of how the membrane environment around PA can influence its effector
proteins. Triglyceride (TG) synthesis, storage, and degradation together constitute
cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where
starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are
synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds
sequentially, producing FAs and glycerol. Several reactions of CTGM can be
catalyzed by more than one enzyme, creating great potential for complex
tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic
energy source during fasting and is related to obesity. Inborn errors and mouse
models have demonstrated the importance of CTGM for non-adipose tissues,
including skeletal muscle, myocardium and liver, because steatosis and
dysfunction can occur. We discuss known inborn errors of CTGM, including
deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood
rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described
elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1
(partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease
(NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear
leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy,
cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE,
hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol
metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia)
and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis).
Mouse models often resemble human phenotypes but may diverge markedly. Inborn
errors have been described for less than one-third of CTGM enzymes, and new
phenotypes may yet be identified. |
What is Trypan blue used for? | Trypan blue is used in the "trypan blue exclusion assay" for assessing cell viability/cell death. | Baicalin is one of the major compounds in the traditional Chinese medicinal herb
from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of
cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin
inhibited cell survival as shown by MTT assay and increased cell death by trypan
blue exclusion assay in a concentration-dependent manner. Baicalin did not
induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay.
Baicalin induced the acidic vesicular organelle cell autophagy marker,
manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and
cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein
expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were
upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by
3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA)
reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore,
protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT
were downregulated in T24 cells after baicalin treatment. In conclusion,
baicalin triggered cell autophagy through the AKT signaling pathway in T24
cells. BACKGROUND: In this study, responses to fatty acid treatments in commonly used
prostate cancer cell culture models and variability of gene expression between
them were determined.
MATERIALS AND METHODS: PC3, DU145, LNCaP, VCaP and PNT2 cells were treated with
100 μM of either oleate, stearate or conjugated linoleate. Cell proliferation
and viability were assessed using trypan blue and 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
respectively. Gene expression was measured using real-time polymerase chain
reaction (PCR).
RESULTS: Conjugated linoleic acid reduced cell proliferation and viability in
all prostate cancer cell lines, whilst the effects of oleic and stearic acid on
proliferation were found to be cell line-dependent. A reduction in gene
expression of fatty acid desaturases was observed in prostate cancer cell lines
compared to normal prostate cells.
CONCLUSION: Differential responses of the cell lines investigated here to fatty
acid treatment suggest that multiple prostate cancer cell line models should be
used when designing experiments aimed at examining lipid metabolism in prostate
cancer. AIM: To immobilize dental pulp stem cells (DPSC) in alginate microspheres and to
determine cell viability, proliferation, stem cell characteristics and
osteogenic potential of the immobilized DPSCs.
METHODOLOGY: Human DPSCs isolated from the dental pulp were immobilized in 1%
w/v alginate microspheres. Viability and proliferation of immobilized DPSCs were
determined by trypan blue and MTT assay, respectively. Stem cell characteristics
of DPSCs post immobilization were verified by labelling the cells with CD73 and
CD90. Osteogenic potential of immobilized DPSCs was assessed by the presence of
osteocalcin. Alizarin red staining and O-cresolphthalein complexone method
confirmed and quantified calcium deposition. A final reverse transcriptase PCR
evaluated the expression of osteogenic markers - ALP, Runx-2 and OCN.
RESULTS: More than 80% of immobilized DPSCs were viable throughout the 3-week
study. Proliferation appeared controlled and consistent unlike DPSCs in the
control group. Presence of CD73 and CD90 markers confirmed the stem cell nature
of immobilized DPSCs. The presence of osteocalcin, an osteoblastic marker, was
confirmed in the microspheres on day 21. Mineralization assays showed high
calcium deposition indicating elevated osteogenic potential of immobilized
DPSCs. Osteogenic genes- ALP, Runx-2 and OCN were also upregulated in
immobilized DPSCs. Surprisingly, immobilized DPSCs in the control group cultured
in conventional stem cell media showed upregulation of osteogenic genes and
expressed osteocalcin.
CONCLUSION: Dental pulp stem cells immobilized in alginate hydrogels exhibit
enhanced osteogenic potential while maintaining high cell viability both of
which are fundamental for bone tissue regeneration. The development of resistance to imatinib mesylate may partly depend on high
bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has
bcr-abl suppressing activity in vitro, without cross-resistance to imatinib.
Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor
(VEGF), which is associated with tumor-related angiogenesis and is involved in
chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to
improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary
CML cells were transfected with a VEGF antisense sequence. Cell viability and
survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow
cytometry following annexin V and propidium iodide staining. The results showed
that VEGF depletion effectively promotes enhanced ATO antileukemic activity by
repressing bcr-abl protein levels. These data provide a rationale for the
clinical development of optimized ATO-based regimens that incorporate VEGF
modulator for CML treatment. BACKGROUND: Dipyrithione (PTS2) is widely used as a bactericide and fungicide.
Here, we investigated whether PTS2 has broad-spectrum antitumor activity by
studying its cytotoxicity and proapoptotic effects in four cancer cell lines.
METHODS: We used MTT assays and trypan blue staining to test the viability of
cancer cell lines. Hoechst 33258 and DAPI staining were used to observe cell
apoptosis. Cell-cycle percentages were analyzed by flow cytometry. Apoptosis was
assayed using caspase-3 and poly (ADP-ribose) polymerase (PARP) combined with
Western blotting. Student's t-test was used for statistical analysis.
RESULTS: PTS2 inhibited proliferation in four cancer cell lines in a
dose-dependent manner. Treated cells showed shrinkage, irregular fragments,
condensed and dispersed blue fluorescent particles compared with control cells.
PTS2 induced cycle-arrest and death. Cleavage of caspase-9, caspase-3, and PARP
were detected in PTS2-treated cells. Antitumor activity of PTS2 was more
effective against widely used cancer drugs and its precursor.
CONCLUSIONS: PTS2 appears to have novel cytotoxicity and potent broad-spectrum
antitumor activity, which suggests its potential as the basis of an anticancer
drug. BACKGROUND: The main objective of the present study was to investigate chemical
composition and possible cyto/genotoxic potential of several medical implant
materials commonly used in total hip joint replacement.
METHODS: Medical implant metal alloy (Ti6Al4V and CoCrMo) and high density
polyethylene particles were analyzed by energy dispersive X-ray spectrometry
while toxicological characterization was done on human lymphocytes using
multi-biomarker approach.
RESULTS: Energy dispersive X-ray spectrometry showed that none of the elements
identified deviate from the chemical composition defined by appropriate ISO
standard. Toxicological characterization showed that the tested materials were
non-cyto/genotoxic as determined by the comet and cytokinesis-block micronucleus
(CBMN) assay. Particle morphology was found (by using scanning electron and
optical microscope) as flat, sharp-edged, irregularly shaped fiber-like grains
with the mean particle size less than 10µm; this corresponds to the so-called
"submicron wear". The very large surface area per wear volume enables high
reactivity with surrounding media and cellular elements.
CONCLUSIONS: Although orthopedic implants proved to be non-cyto/genotoxic, in
tested concentration (10μg/ml) there is a constant need for monitoring of
patients that have implanted artificial hips or other joints, to minimize the
risks of any unwanted health effects.
GENERAL SIGNIFICANCE: The fractal and multifractal analyses, performed in order
to evaluate the degree of particle shape effect, showed that the fractal and
multifractal terms are related to the "remt" level of the particles' toxicity
especially with the cell viability (trypan blue method) and total number of
nucleoplasmic bridges and nuclear buds as CBMN assay parameters. BACKGROUND: Autophagy induction can increase or decrease anticancer drug
efficacy. Anticancer drug-induced autophagy induction is poorly characterized in
osteosarcoma (OS). In this study, we investigated the impact of autophagy
inhibition on camptothecin (CPT)-induced cytotoxicity in OS.
METHODS: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were
generated by infection with lentiviral shRNA directed against the essential
autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of
autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively.
Metabolic activity was determined by MTT assay and cell viability was determined
by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II
protein expression were used to determine autophagy induction. Oxidative stress
was assessed by staining cells with HE and DCFH-DA followed by flow cytometry
analysis. Mitochondrial membrane potential was determined by staining cells with
TMRE followed by flow cytometry analysis. Immunoblotting was used to detect
caspase activation, Parp cleavage and p53 phosphorylation.
RESULTS: Autophagy inhibition caused a greater deficit in metabolic activity and
cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher
basal autophagy levels than DLM8 cells and non-transformed murine MCT3
osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage.
Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while
increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced
CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8
cells. Buthionine sulfoximine (BSO)-induced cell death was greater in
autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment.
Camptothecin-induced and BSO-induced autophagy induction was also reversed by
antioxidant pretreatment. Significantly, autophagy inhibition not only reduced
CPT-induced oxidative stress but also reduced basal oxidative stress.
CONCLUSIONS: The results of this study indicate that autophagy inhibition can
have an opposing effect on CPT-induced cytotoxicity within OS. The
cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves
reduced CPT-induced oxidative stress and not reduced DNA damage. Our results
also reveal the novel finding that knockdown of ATG5 protein reduces both basal
oxidative stress and drug-induced oxidative stress. PURPOSE: The proteasome inhibitor bortezomib (PS-341) has displayed significant
efficiency against pancreatic cancer cells. However, the underlying mechanisms
are not fully understood. Here, we tested if ceramide production was involved in
the bortezomib's effect.
METHODS: Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the
primary pancreatic cancer cells were used. Cell death was analyzed by MTT
viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone
DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal
protein changes. The cellular ceramide level after bortezomib treatment was also
determined.
RESULTS: In cultured pancreatic cancer cells, bortezomib increased cellular
ceramide production to promote cell apoptosis. The ceramide de novo synthase
inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production
and apoptosis, while exogenously added C6-ceramide facilitated
bortezomib-induced pancreatic cancer cell death. Meanwhile,
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of
glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors
(SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and
subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun
N-terminal kinase (JNK) activation was also associated with ceramide production.
JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II
and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP
dramatically enhanced bortezomib-induced cytotoxicity in primary cultured
pancreatic cancer cells.
CONCLUSIONS: We found that bortezomib-induced apoptosis was associated with
ceramide production in primary and transformed pancreatic cancer cells. Janus kinase (JAK) is one of the main upstream activators of signal transducers
and activators of transcription (STAT) that are constitutively activated in
various maligcies and are associated with cell growth, survival, and
carcinogenesis. Here, we investigated the role of JAKs in colorectal cancer in
order to develop effective therapeutic targets for INCB018424, which is the
first JAK1/2 inhibitor to be approved by FDA. After examining the basal
expression levels of phospho-JAK1 and phospho-JAK2, we measured the effects of
INCB018424 on the phosphorylation of JAK1/2 using western blot analysis. Cell
viability was determined using the trypan blue exclusion assay. The cell death
mechanism was identified by the activation of caspase 3 using western blot and
annexin V staining. The basal levels of phospho-JAK1 and phospho-JAK2 were
cancer cell type dependent. Colorectal cancer cell lines that phosphorylate both
JAK1 and JAK2 include DLD-1 and RKO. INCB018424 inactivates both JAK1 and JAK2
in DLD-1 cells but inactivates only JAK1 in RKO cells. Cell death was
proportional to the inactivation of JAK1 but not JAK2. INCB018424 causes
caspase-dependent cell death, which is prevented by treatment with z-VAD. The
inhibition of JAK1 phosphorylation seemed sufficient to allow
INCB018424-mediated apoptosis. JAK1 is a key molecule that is involved in colon
cancer cell survival and the inhibition of JAK1 by INCB01424 results in
caspase-dependent apoptosis in colorectal cancer cells. The use of selective
JAK1 inhibitors could be an attractive therapy against colorectal cancer, but
further clinical investigations are needed to test this possibility. BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer
deaths reported worldwide. The incidence is higher in Asia and Africa, where
there is greater endemic prevalence of hepatitis B and C. The devastating
outcome of cancer can be minimized only by the use of potent therapeutic agents.
Tridham (TD) has been acknowledged since olden days for its wide spectrum of
biological properties and was used by traditional practitioners of Siddha and
other indigenous systems of medicine. The present study aims at investigating
the mechanistic action of TD by assessing the antiproliferative and
pro-apoptotic effects on human hepatocellular carcinoma cell line (Huh7).
METHODS: Cell viability and apoptosis assay using MTT analysis and trypan blue
staining, DAPI staining, DNA fragmentation, cell cycle analysis, mitochondrial
membrane potential, real-time reverse transcription-polymerase chain reaction,
western blotting and immunofluorescence staining were determined in Huh7 cells.
RESULTS: Viability studies of TD treated Huh7 cells showed an inhibition in cell
growth in time and dose dependent manner. Chromatin condensation, DNA
fragmentation and apoptotic bodies, which are structural changes characteristic
of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and
agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell
cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of
cells in the sub-G1 phase of the cell cycle in a dose dependent manner.
Immunofluorescent staining for Ki-67 showed a higher level of expression in
untreated cells as compared to TD treated cells. We observed a significant loss
in the mitochondrial membrane potential and the release of cytochrome c into the
cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and
Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene
expression was found to be unaltered in TD treated cells.
CONCLUSION: These results suggest that TD induces apoptosis of Huh7 cells
through activation of Bax and triggered caspase cascade, independent of p53
function. This study throws light on the mechanistic action of TD in triggering
apoptosis in Huh 7 cells. AIMS: Muscarinic acetylcholine receptor agonist pilocarpine reduces intraocular
pressure (IOP) of glaucoma mainly by stimulating ciliary muscle contraction and
then increasing aqueous outflow. It is of our great interest to know whether
pilocarpine has the additional properties of retinal neuroprotection independent
of IOP lowering in vitro and in vivo models.
METHODS: In rat primary retinal cultures, cell viability was measured using an
MTT assay and the trypan blue exclusion method, respectively. Retinal ganglion
cells (RGCs) were identified by immunofluorescence and quantified by flow
cytometry. For the in vivo study, the retinal damage after retinal
ischemia/reperfusion injury in rats was evaluated by histopathological study
using hematoxylin and eosin staining, transmission electron microscopy, and
immunohistochemical study on cleaved caspase-3, caspase-3, and ChAT.
RESULTS: Pretreatment of pilocarpine attenuated glutamate-induced neurotoxicity
of primary retinal neurons in a dose-dependent manner. Protection of pilocarpine
in both retinal neurons and RGCs was largely abolished by the nonselective
muscarinic receptor antagonist atropine and the M1-selective muscarinic receptor
antagonist pirenzepine. After ischemia/reperfusion injury in retina, the inner
retinal degeneration occurred including ganglion cell layer thinning and neuron
lost, and the optic nerve underwent vacuolar changes. These degenerative changes
were significantly lessened by topical application of 2% pilocarpine. In
addition, the protective effect of pilocarpine on the ischemic rat retina was
favorably reflected by downregulating the expression of activated apoptosis
marker cleaved caspase-3 and caspase-3 and upregulating the expression of
cholinergic cell marker ChAT.
CONCLUSIONS: Taken together, this highlights pilocarpine through the activation
of muscarinic receptors appear to afford significant protection against retinal
neurons damage and optic nerve degeneration at clinically relevant
concentrations. These data also further support muscarinic receptors as
potential therapeutic neuroprotective targets in glaucoma. Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to
treat disorders like diabetes, high cholesterol, wounds, inflammation,
gastrointestinal ailments, and it is believed to have anti-tumor properties,
although the mechanisms for the activity remain to be elucidated. In this study,
we prepared a methanol extract from Fenugreek whole plants and investigated the
mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer
cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue
exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR
assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48
hours. FME induced apoptosis was mediated by the death receptor pathway as
demonstrated by the increased level of Fas receptor expression after FME
treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53,
FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent
manner. In summary, these data demonstrate that at least 90% of FME induced
apoptosis in breast cell is mediated by Fas receptor-independently of either
FADD, Caspase 8 or 3, as well as p53 interdependently. Dietary habit affects the composition of human feces thus determining intestinal
environment and exposure of colon mucosa to risk factors. Fecal water (FW)
citotoxicity and genotoxicity were investigated in 33 healthy young Italian
people, as well as the relationship between genotoxicity and nutrient intake or
microflora composition. Two fecal samples were collected at 2 weeks apart and
3-d dietary diary was recorded for each volunteer. Cytotoxicity was measured
using the Trypan Blue Dye Exclusion assay and genotoxicity using the Comet Assay
(alkaline single-cell electrophoresis). Fecal bifidobacteria, total microbial
count and nutrient intakes were also assessed. High intra- and inter-variability
in genotoxicity data and in bacteria counts were found. None of the FW samples
were citotoxic, but 90% of FW samples were genotoxic. Seventy five percent
indicated intermediate and 15% were highly genotoxic. There was a different
sex-related distribution. Genotoxicity was positively correlated to the total
lipid intake in females and to the bifidobacteria/total bacteria count ratio in
male volunteers. These results demonstrate that the majority of FW samples
isolated from free-living Italian people show intermediate level of genotoxicity
and sustain a relation between this possible non-invasive marker of colorectal
cancer risk with both dietary habits and colonic ecosystem. Contrast-induced nephropathy represents the third cause of hospital-acquired
acute renal failure. This study investigated the effects of low- vs iso-osmolar
contrast medium (CM) exposure on NADPH-dependent reactive oxygen species (ROS)
generation by tubular cells. X-ray attenuation of iohexol, iopamidol, and
iodixanol was assessed at equimolar iodine concentrations and their effects on
human renal proximal tubular cells (PTCs) were evaluated with equally
attenuating solutions of each CM. Cytotoxicity, apoptosis, and necrosis were
investigated by trypan blue exclusion, MTT assay, and annexin V/propidium iodide
assay, respectively. ROS production was assessed by DCF assay, NADPH oxidase
activity by the lucigenin-enhanced chemiluminescence method, and Nox4 expression
by immunoblot. Yielding the same X-ray attenuation, CM cytotoxicity was assessed
in PTCs at equimolar iodine concentrations. More necrosis was present after
incubation with iohexol and iopamidol than after incubation with equal
concentrations of iodixanol. Iohexol and iodixanol at low iodine concentrations
induced less cytotoxicity than iopamidol. Moreover, both iohexol and iopamidol
induced more apoptosis than iodixanol, with a dose-dependent effect. ROS
generation was significantly higher with iopamidol and iohexol compared to
iodixanol. NADPH oxidase activity and Nox4 protein expression significantly
increased after exposure to iopamidol and iohexol, with a dose-dependent effect,
compared with iodixanol. CM-induced Nox4 expression and activity depended upon
Src activation. In conclusion, at angiographic concentrations, iodixanol induces
fewer cytotoxic effects on cultured tubular cells than iohexol and iopamidol
along with a lower induction of Nox4-dependent ROS generation. This enzyme may,
thus, represent a potential therapeutic target to prevent iodinated CM-related
oxidative stress. Pterostilbene, a naturally occurring structural analog of resveratrol, has been
reported to exert antiproliferative and proapoptotic effects in various cancer
types. Recently, it has been demonstrated to induce both autophagy and apoptosis
in human bladder and breast cancer cell lines. The aim of this study was to
evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell
morphology was examined using confocal and electron microscopy. Cell viability
was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3
processing was studied based on Western blotting and immunofluorescence
analyses. Flow cytometry was used to study cell cycle distribution,
phosphatidylserine externalization, caspase activation, disruption of
mitochondrial membrane potential and intracellular production of reactive oxygen
species. DNA degradation was examined by gel electrophoresis. We found that
treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in
the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I
to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar
structures. Pterostilbene also led to phosphatidylserine externalization,
internucleosomal DNA fragmentation, caspase activation and disruption of
mitochondrial membrane potential. Moreover, it did not induce oxidative stress.
Our results suggest that pterostilbene induces accumulation of autophagic
vacuoles followed by cell death in HL60 cells. BACKGROUND: F1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating
mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on
hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to
investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase
modulates the H/RO-induced mitochondrial dysfunction. In addition, the potential
for melatonin, a potent antioxidant with multiple mitochondrial protective
properties, to protect NARP cells exposed to H/RO was assessed.
METHODS AND FINDINGS: NARP cybrids harboring 98% of mtDNA T8993G genes were
established as an in vitro model for cells with F1F0-ATPase defect; their
parental osteosarcoma 143B cells were studied for comparison. Treating the cells
with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP
significantly enhanced apoptotic death upon H/RO detected by MTT assay and the
trypan blue exclusion test of cell viability. Based on fluorescence
probe-coupled laser scanning imaging microscopy, NARP significantly enhanced
mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca(2+)
(mCa(2+)) accumulation in response to H/RO, which augmented the depletion of
cardiolipin, resulting in the retardation of mitochondrial movement. With
stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia
duration, or administrating secondary oxidative stress following H/RO), NARP
augmented H/RO-induced mROS formation to significantly depolarize mitochondrial
membrane potential (ΔΨm), and enhance mCa(2+) accumulation and nitric oxide
formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted
cardiolipin, thereby promoting permanent mitochondrial permeability transition,
retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly
reduced NARP-augmented H/RO-induced mROS formation and therefore significantly
reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa(2+),
stabilized cardiolipin, and then improved mitochondrial movement and cell
survival.
CONCLUSION: NARP-induced inhibition of F1F0-ATPase enhances mROS formation upon
H/RO, which augments the depletion of cardiolipin and retardation of
mitochondrial movement. Melatonin may have the potential to rescue patients with
ischemia/reperfusion insults, even those associated with NARP symptoms. PURPOSE: To assess the viability of cultured epithelium and preserved by
freezing for periods varying from one month to one year.
METHODS: Samples of cultured epithelium were incubated in cryoprotectant medium
(Group A), packed in aluminum envelopes and packed in polystyrene boxes. The
boxes were subjected to a temperature of-70 ºC. After freezing for a period of
time ranging from one to 12 months, cultured epithelial samples were assessed
for their viability by vital staining (Trypan blue) and metabolic analysis based
on glucose consumption and lactate production. Samples of not frozen cultured
epithelium (Group B) were also tested for viability and the results obtained
were used as comparison parameter for the variation of viability.
RESULTS: Statistical analysis between the group A and B indicate that the mean
age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical
difference. In 30 days we obtained 37% of the original viability of cultured
epithelium, 25% at six months and one year, less than 15%. This trend was
confirmed statistically with a reduction of approximately 1.8% of the original
viability epithelium cultured every 30 days of storage. In the analysis by
lactate production, similar results were observed. In the analysis by the
glucose consumption results were not significant. The viability indices show
statistically significant difference between the group A and B (p<0.0001).
CONCLUSIONS: Although cryopreserved cultured epithelium showed significant
reduction of viability, all samples remained viable. It was also found that the
viability of cryopreserved cultured epithelial decreased as a function of
storage time. Uncoupling protein 2 (UCP2) is a mitochondrial membrane protein that regulates
energy metabolism and reactive oxygen species (ROS) production. We generated
mouse carboxy- and amino-terminal green fluorescent protein (GFP)-tagged UCP2
constructs to investigate the effect of UCP2 expression on cell proliferation
and viability. UCP2-transfected Hepa 1-6 cells did not show reduced cellular
adenosine triphosphate (ATP) but showed increased levels of glutathione. Flow
cytometry analysis indicated that transfected cells were less proliferative than
nontransfected controls, with most cells blocked at the G1 phase. The effect of
UCP2 on cell cycle arrest could not be reversed by providing exogenous ATP or
oxidant supply, and was not affected by the chemical uncoupler carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). However, this effect of UCP2
was augmented by treatment with genistein, a tyrosine kinase inhibitor, which by
itself did not affect cell proliferation on control hepatocytes. Western
blotting analysis revealed decreased expression levels of CDK6 but not CDK2 and
D-type cyclins. Examination of cell viability in UCP2-transfected cells with
Trypan Blue and Annexin-V staining revealed that UCP2 transfection led to
significantly increased cell death. However, characteristics of apoptosis were
absent in UCP2-transfected Hepa 1-6 cells, including lack of oligonucleosomal
fragmentation (laddering) of chromosomal DNA, release of cytochrome c from
mitochondria, and cleavage of caspase-3. In conclusion, our results indicate
that UCP2 induces cell cycle arrest at G1 phase and causes nonapoptotic cell
death, suggesting that UCP2 may act as a powerful influence on hepatic
regeneration and cell death in the steatotic liver. |
Selexipag is used for which disease? | Selexipag is a novel, oral, selective prostacyclin (PGI2) receptor agonist in clinical development for the treatment of pulmonary arterial hypertension. | In this phase 2 proof-of-concept study we examined the safety and efficacy of
selexipag, an orally available, selective prostacyclin receptor (IP receptor)
agonist, as a treatment for pulmonary arterial hypertension (PAH). 43 adult
patients with symptomatic PAH (receiving stable endothelin receptor antagonist
and/or a phosphodiesterase type-5 inhibitor therapy) were randomised three to
one to receive either selexipag or placebo. Dosage was up-titrated in 200-μg
increments from 200 μg twice daily on day 1 to the maximum tolerated dose by day
35 (maximum allowed dose of 800 μg twice daily). Change in pulmonary vascular
resistance at week 17 expressed as a percentage of the baseline value was the
primary efficacy end-point, and was analysed in the per protocol set first and
then in the all-treated set to assess robustness of results. A statistically
significant 30.3% reduction in geometric mean pulmonary vascular resistance was
observed after 17 weeks' treatment with selexipag compared with placebo (95%
confidence limits -44.7- -12.2; p=0.0045, Wilcoxon rank sum test). This was
supported by a similar result from the all-treated set. Selexipag was well
tolerated with a safety profile in line with the expected pharmacological
effect. Our results encourage the further investigation of selexipag for the
treatment of PAH. INTRODUCTION: Selexipag is a first-in-class orally available selective
non-prostanoid IP receptor agonist. This review was based on a PubMed search and
focuses on the potential role of selexipag in the treatment of pulmonary
arterial hypertension (PAH).
AREAS COVERED: Selexipag is rapidly hydrolyzed to an active metabolite,
ACT-333679. Both selexipag and its metabolite are highly selective for the IP
receptor compared with other prostanoid receptors. This selectivity for the IP
receptor offers the potential for improved tolerability with selexipag, as side
effects (e.g., nausea and vomiting) that might result from activation of the
other prostanoid receptors may be minimized. In addition, the selexipag
metabolite has a half-life of 7.9 h, thus permitting oral dosing twice daily.
Selexipag showed effects on pharmacodynamic end points obtained with right heart
catheterization in a Phase II trial in patients with PAH, and is being evaluated
in the ongoing Phase III trial (GRIPHON trial, Clinicaltrials.gov NCT01106014).
EXPERT OPINION: The signal of a beneficial effect of selexipag on disease
progression may become more robust for long term under prolonged exposure.
Pending the GRIPHON trial results, selexipag could provide a convenient
first-line prostacyclin treatment option for patients with PAH. OBJECTIVE: Selexipag is a novel, oral, selective prostacyclin (PGI2) receptor
agonist in clinical development for the treatment of pulmonary arterial
hypertension. Film-coated tablets with strength between 200 and 1,600 μg were
used. Bioequivalence between 8 x 200 μg and a new 1,600 μg tablet was evaluated
at steady state in healthy male subjects.
MATERIALS AND METHODS: This was an open-label, 2-treatment, 2-period, crossover,
up-titration, phase 1 study. The treatments were selexipag at 1,600 μg b.i.d.
for 4.5 days either as 8 x 200 μg tablets (reference: A) or 1 x 1,600 μg tablet
(test: B), both preceded by an up-titration phase starting from 400 μg b.i.d.
doses, in 200-μg steps every 4th day. Subjects were randomized 1 : 1 to the A-B
or B-A sequence. The pharmacokinetics and tolerability of selexipag and its
active metabolite, ACT-333679, were investigated.
RESULTS: 80 subjects were enrolled in the study: 65 subjects completed the study
according to protocol, and 15 subjects withdrew from the study. The most
frequent adverse events (AEs) were headache (86%), myalgia (73%), and jaw pain
(73%). There was no difference in nature and overall frequency of AEs between
the two treatments. Steady state was attained within 3 days of the selexipag
1,600 μg b.i.d.
TREATMENTS: The 90% confidence intervals (CIs) of the geometric mean ratio (B/A)
at steady state for AUCÏ and Cmax,ss were within (0.80, 1.25) bioequivalence
interval: (0.92, 1.06) and (0.95, 1.14), respectively, for selexipag and (0.95,
1.06) and (0.94, 1.07), respectively, for the active metabolite, ACT-333679.
CONCLUSIONS: Bioequivalence was demonstrated between 8 x 200 μg and 1 x 1,600 μg
selexipag at steady state. |
How does ranolazine affect kinase signaling activation in the heart? | Ranolazine inhibits Ca(2+)/calmodulin kinase II (CaMKII) activity | Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II
(CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to
phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL).
Conversely, an increase of I(NaL) would be expected to cause elevation of
intracellular Ca(2+) and activation of CaMKII. However, a relationship between
enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We
investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and
explored the functional significance of this pathway. In neonatal rat
ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin
II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased
phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2.
Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced
CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in
CaMKII activation. In support of this view, CaMKII activity was also increased
in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant
(N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed
by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII
inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore,
ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same
association between CaMKII and Na(v)1.5 was also found in N(1325)S mice,
suggesting a direct protein-protein interaction. Pharmacological inhibitions of
either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and
reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II
in rat perfused hearts. Taken together, these results suggest that a
Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which
in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological
inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction
caused by excessive Na(+) influx. BACKGROUND: Hypertrophic cardiomyopathy (HCM), the most common mendelian heart
disorder, remains an orphan of disease-specific pharmacological treatment
because of the limited understanding of cellular mechanisms underlying
arrhythmogenicity and diastolic dysfunction.
METHODS AND RESULTS: We assessed the electromechanical profile of cardiomyocytes
from 26 HCM patients undergoing myectomy compared with those from nonfailing
nonhypertrophic surgical patients by performing patch-clamp and intracellular
Ca(2+) (Ca(2+)(i)) studies. Compared with controls, HCM cardiomyocytes showed
prolonged action potential related to increased late Na(+) (I(NaL)) and Ca(2+)
(I(CaL)) currents and decreased repolarizing K(+) currents, increased occurrence
of cellular arrhythmias, prolonged Ca(2+)(i) transients, and higher diastolic
Ca(2+)(i). Such changes were related to enhanced Ca(2+)/calmodulin kinase II
(CaMKII) activity and increased phosphorylation of its targets. Ranolazine at
therapeutic concentrations partially reversed the HCM-related cellular
abnormalities via I(NaL) inhibition, with negligible effects in controls. By
shortening the action potential duration in HCM cardiomyocytes, ranolazine
reduced the occurrence of early and delayed afterdepolarizations. Finally, as a
result of the faster kinetics of Ca(2+)(i) transients and the lower diastolic
Ca(2+)(i), ranolazine accelerated the contraction-relaxation cycle of HCM
trabeculae, ameliorating diastolic function.
CONCLUSIONS: We highlighted a specific set of functional changes in human HCM
myocardium that stem from a complex remodeling process involving alterations of
CaMKII-dependent signaling, rather than being a direct consequence of the causal
sarcomeric mutations. Among the several ion channel and Ca(2+)(i) handling
proteins changes identified, an enhanced I(NaL) seems to be a major contributor
to the electrophysiological and Ca(2+)(i) dynamic abnormalities of ventricular
myocytes and trabeculae from patients with HCM, suggesting potential therapeutic
implications of I(NaL) inhibition. |
List features of the DEND syndrome. | Clinical features of the DEND syndrome include developmental delay, epilepsy and neonatal diabetes. | Permanent neonatal diabetes (PNDM) is diagnosed in the first three months of
life and is a major management problem as patients require lifelong insulin
injections. Recently, activating mutations in the KCNJ11 gene which encodes the
Kir6.2 subunit of the KATP channels in the pancreatic beta-cells were found to
be an important cause of PNDM. The mutated KATP channels do not close in the
presence of adenosine triphosphate (ATP) so the beta-cell membrane is
hyperpolarized and insulin secretion does not occur. Some patients have DEND
syndrome (developmental delay, epilepsy and neonatal diabetes) with the
neurological features arising from mutated KATP channels in muscle, nerve and
brain. Defining a genetic aetiology has not only given insights into clinical
classification and disease mechanism, but has also influenced treatment.
Sulphonylureas, by binding the sulphonylurea receptor, can close the KATP
channel. This has led to patients who were insulin-dependent being able to
discontinue insulin injections and achieve excellent control with sulphonylurea
tablets. In this article we discuss the work that established Kir6.2 mutations
as a common cause of neonatal diabetes, the clinical features, the underlying
mechanism and the impact on patient treatment. Closure of ATP-sensitive K(+) channels (K(ATP) channels) in response to
metabolically generated ATP or binding of sulfonylurea drugs stimulates insulin
release from pancreatic beta-cells. Heterozygous gain-of-function mutations in
the KCJN11 gene encoding the Kir6.2 subunit of this channel are found in
approximately 47% of patients diagnosed with permanent diabetes at <6 months of
age. There is a striking genotype-phenotype relationship with specific Kir6.2
mutations being associated with transient neonatal diabetes, permanent neonatal
diabetes alone, and a novel syndrome characterized by developmental delay,
epilepsy, and neonatal diabetes (DEND) syndrome. All mutations appear to cause
neonatal diabetes by reducing K(ATP) channel ATP sensitivity and increasing the
K(ATP) current, which inhibits beta-cell electrical activity and insulin
secretion. The severity of the clinical symptoms is reflected in the ATP
sensitivity of heterozygous channels in vitro with wild type > transient
neonatal diabetes > permanent neonatal diabetes > DEND syndrome channels.
Sulfonylureas still close mutated K(ATP) channels, and many patients can
discontinue insulin injections and show improved glycemic control when treated
with high-dose sulfonylurea tablets. In conclusion, the finding that Kir6.2
mutations can cause neonatal diabetes has enabled a new therapeutic approach and
shed new light on the structure and function of the Kir6.2 subunit of the K(ATP)
channel. Heterozygous activating mutations in the gene encoding for the ATP-sensitive
potassium channel subunit Kir6.2 (KCNJ11) have recently been shown to be a
common cause of permanent neonatal diabetes. Kir6.2 is expressed in muscle,
neuron and brain as well as the pancreatic beta-cell, so patients with KCNJ11
mutations could have a neurological phenotype in addition to their diabetes. It
is proposed that some patients with KCNJ11 mutations have neurological features
that are part of a discrete neurological syndrome termed developmental Delay,
Epilepsy and Neonatal Diabetes (DEND), but there are also neurological
consequences of chronic or acute diabetes. We identified KCNJ11 mutations in
four of 10 probands with permanent neonatal diabetes and one affected parent;
this included the novel C166F mutation and the previously described V59M and
R201H. Four of the five patients with mutations had neurological features: the
patient with the C166F mutation had marked developmental delay, severe
generalised epilepsy, hypotonia and muscle weakness; mild developmental delay
was present in the patient with the V59M mutation; one patient with the R201H
mutation had acute and chronic neurological consequences of cerebral oedema and
another had diabetic neuropathy from chronic hyperglycaemia. In conclusion, the
clinical features in these patients support the existence of a discrete
neurological syndrome with KCNJ11 mutations. The severe DEND syndrome was seen
with the novel C166F mutation and mild developmental delay with the V59M
mutation. These features differ markedly from the neurological consequences of
acute or chronic diabetes. OBJECTIVES: Activating mutations in the human KCNJ11 gene, encoding the
pore-forming subunit (Kir6.2) of the ATP-sensitive potassium (K(ATP)) channel,
are one cause of neonatal diabetes mellitus. In a few patients, KCNJ11 mutations
cause a triad of developmental delay, epilepsy, and neonatal diabetes (DEND
syndrome). The aim of this study was to determine the clinical effects,
functional cause, and sensitivity to sulfonylurea treatment of a novel KCNJ11
mutation producing DEND syndrome.
METHODS: We screened the DNA of a 3-year-old patient with neonatal diabetes,
severe developmental delay, and therapy-resistant epilepsy for mutations in
KCNJ11. We carried out electrophysiologic analysis of wild-type and mutant
K(ATP) channels heterologously expressed in Xenopus oocytes.
RESULTS: We identified a novel Kir6.2 mutation (I167L) causing DEND syndrome.
Functional analysis showed both homomeric and heterozygous mutant channels were
less inhibited by MgATP leading to an increase in whole-cell K(ATP) currents.
This effect was due to an increase in the intrinsic open probability.
Heterozygous channels were strongly inhibited by the sulfonylurea tolbutamide.
Treatment of the patient with the sulfonylurea glibenclamide not only enabled
insulin therapy to be stopped, but also resulted in improvement in epilepsy and
psychomotor abilities.
CONCLUSIONS: We report a case of developmental delay, epilepsy, and neonatal
diabetes (DEND) syndrome that shows neurologic improvement with sulfonylurea
therapy. Early recognition of patients with DEND syndrome may have considerable
therapeutic benefit for the patient. Heterozygous activating mutations of KCNJ11 (Kir6.2) are the most common cause
of permanent neonatal diabetes mellitus (PNDM) and several cases have been
successfully treated with oral sulfonylureas. We report on the attempted
transfer of insulin therapy to glibenclamide in a 4-year old child with PNDM and
DEND syndrome, bearing a C166Y mutation in KCNJ11. An inpatient transition from
subcutaneous NPH insulin (0.2 units/kg/d) to oral glibenclamide (1 mg/kg/d and
1.5 mg/kg/d) was performed. Glucose and C-peptide responses stimulated by oral
glucose tolerance test (OGTT), hemoglobin A1c levels, the 8-point self-measured
blood glucose (SMBG) profile and the frequency of hypoglycemia episodes were
analyzed, before and during treatment with glibenclamide. Neither diabetes
control nor neurological improvements were observed. We concluded that C166Y
mutation was associated with a form of PNDM insensitive to glibenclamide. Mutations in the KCNJ11 gene, which encodes the Kir6.2 subunit of the
ATP-sensitive potassium channel, often result in neonatal diabetes. Patients
with this mutation have been successfully transitioned from insulin to
sulfonylurea (SU) therapy without compromise in their glycemic control. Among
patients with neonatal diabetes due to KCNJ11 mutations, approximately 25% have
neurological findings including developmental delay, motor dysfunction, and
epilepsy, known as DEND syndrome. There have been rare cases of juvenile
patients with intermediate DEND syndrome (iDEND) reporting variable improvement
in neurological function following transition from insulin to SU treatment. We
describe the response to glyburide in a 15-yr-old boy with severe global
developmental delays resulting from the KCNJ11 mutation V59M. The patient was
discovered to have diabetes mellitus at 11.5 months of age, making this the
oldest age at diagnosis of a KCNJ11 mutation-related case of neonatal diabetes.
Because consensus has been to screen patients for this mutation only if younger
than 6 months at the time of diagnosis, we suggest that all patients under the
age of 12 months at diagnosis should receive genetic testing for monogenic
causes of diabetes. BACKGROUND: A 17-year-old female was referred for the reassessment of her type 1
diabetes mellitus, with which she had been diagnosed at the age of 15 weeks
owing to symptoms of ketoacidosis. The patient had mild learning difficulties,
which resulted in her requiring additional support at school. There was no
family history of diabetes.
INVESTIGATIONS: Measurements of plasma C-peptide and glutamate decarboxylase
autoantibodies. Molecular genetic testing was performed.
DIAGNOSIS: Intermediate developmental delay, epilepsy and neonatal diabetes
mellitus (DEND) syndrome as a result of a 59V>M Kir6.2 mutation.
MANAGEMENT: Treatment with high-dose oral glibenclamide replaced insulin
treatment. Good glycemic control was achieved with levels of HbA(1c)
consistently below 6.5% and no hypoglycemia. AIM: ATP-sensitive potassium channels are important regulators of insulin
secretion. They consist of four sulphonylurea receptor (encoded by ABCC8) and
four inwardly rectifying protein (encoded by KCNJ11) subunits. Activating ABCC8
mutations lead to decreased insulin secretion and to diabetes. Wide phenotype
variability is associated with single ABCC8 mutations, ranging from transient or
permanent neonatal diabetes (ND) with or without developmental delay (DEND
syndrome) to very mild phenotypes. This report describes the case of a Caucasian
infant diagnosed with ND at the age of 2 months due to a novel ABCC8 missense
mutation.
METHODS: ABCC8 was analyzed by sequence analysis. The mutation was present in
the patient and her family and was found to be associated with phenotypes
ranging from ND to asymptomatic impaired fasting glucose (IFG).
RESULTS: A novel His863Tyr ABCC8 mutation was identified in a 2-month-old girl
diagnosed with ND. After an initial insulin treatment, treatment with
glibenclamide was initiated and the treatment with insulin discontinued. The
same mutation was found in her father, who had been fortuitously diagnosed with
diabetes and had an HbA(1c) level of 9% (74.8 mmol/mol). The patient's brother
and mother both had normal fasting glucose, and were not found to be carriers of
the mutation. However, the same mutation was found in her grandmother, who had
been asymptomatic and discovered IFG (6.9 mmol/L) with an HbA(1c) of 6.8% (50.8
mmol/mol).
CONCLUSION: This case describes a novel ABCC8 mutation and offers a further
illustration of the highly variable phenotypes associated with an identical
mutation present across three generations. Gain-of-function mutations of KCNJ11 can cause permanent neonatal diabetes
mellitus, but only rarely after 6 months of age. Specific uncommon mutations
KCNJ11give rise to a syndrome defined as developmental delay, epilepsy, and
neonatal diabetes (DEND), or - more frequently - to a milder sub-type lacking
epilepsy, denoted as intermediate-DEND (iDEND). Our aim was to consider a
possible monogenic etiology in a 12-yr-old boy with early onset diabetes and
mild neurological features. We studied a subject diagnosed with diabetes at
21 months of age, and negative to type 1 diabetes autoantibodies testing. He had
learning difficulties during primary school, and a single episode of seizures at
the age of 10 yr. We performed direct DNA sequencing of the KCNJ11 gene with
subsequent functional study of mutated channels in COSm6 cells. The patient's
clinical response to oral glyburide (Glyb) was assessed. Motor coordination was
evaluated before and after 6 and 12 months of Glyb therapy. Sequencing of the
KCNJ11 gene detected the novel, spontaneous mutation S225T, combined with
deletion of amino acids 226-232. In vitro studies revealed that the mutation
results in a K(ATP) channel with reduced sensitivity to the inhibitory action of
ATP. Glyb improved diabetes control (hemoglobin A1c on insulin:
52 mmol/mol/6.9%; on Glyb: 36 mmol/mol/5.4%) and also performance on motor
coordination tests that were impaired before the switch of therapy. We conclude
that KCNJ11/S225T, del226-232 mutation caused a mild iDEND form in our patient.
KCNJ11 should be considered as the etiology of diabetes even beyond the neonatal
period if present in combination with negative autoantibody testing and even
mild neurological symptoms. Heterozygous activating mutations of KCNJ11 (Kir6.2) are the most common cause
of permanent neonatal diabetes mellitus (NDM), and successful glycemic control
has been obtained in several cases with oral sulfonylureas (SU). We have
verified a lack of clinical response for both glycemic control and neurological
features in an infant with permanent neonatal diabetes mellitus and DEND
syndrome due to a V59A mutation in the KCNJ11 gene. Thus, our case reinforces
that most cases with DEND syndrome are insensitive to SU. OBJECTIVE: Activating mutations in the KCNJ11 gene, encoding the Kir6.2 subunit
of the KATP channel, result in permanent neonatal diabetes mellitus. They also
may cause neurologic symptoms such as mental retardation and motor problems
(iDEND syndrome) and epilepsy (DEND syndrome). Sulphonylurea (SU) treatment is
reported to alleviate both the neurologic symptoms and diabetes in such cases.
The study aimed to establish the magnitude and functional basis of the effect of
SUs on the neurologic phenotype in children with iDEND using neuroimaging before
and after insulin replacement with glibenclamide.
RESEARCH DESIGN AND METHODS: To localize and quantify the effect of
glibenclamide administration, we performed single-photon emission computed
tomography in seven patients with different mutations in KCNJ11. In five
patients, measurements before and after initiation of SU treatment were
performed. RESULTS Significant changes in single-photon emission computed
tomography signal intensity after transfer to SU therapy were restricted to the
cerebellum, consistent with previous data showing high Kir6.2 expression in this
brain region. Cerebellar perfusion improved for both left (P = 0.006) and right
(P = 0.01) hemispheres, with the mean improvement being 26.7 ± 7.1% (n = 5). No
patients showed deterioration of cerebellar perfusion on SU therapy.
Electrophysiological studies revealed a good correlation between the magnitude
of KATP channel dysfunction and the clinical phenotype; mutant channels with the
greatest reduction in adenosine 5'-triphosphate inhibition were associated with
the most severe neurologic symptoms.
CONCLUSIONS: We conclude it is likely that at least some of the beneficial
effects of SU treatment on neurodevelopment in iDEND patients result from
improved cerebellar perfusion. Neonatal diabetes mellitus and organic acidemias, may present with similar
features like hyperglycemia, ketoacidosis and failure to thrive. A four-mo-old
girl presented with diabetic ketoacidosis following a febrile respiratory
illness during which high anion gap metabolic acidosis and hyperglycemia were
detected. She also had hyperammonemia, which led to diagnostic uncertainty.
Euglycemia was achieved with insulin injections. Genotyping revealed a
homozygous novel mutation of the ABCC8 gene coding for the SUR1 subunit of the
pancreatic beta cell potassium channel. Subsequently, the child was successfully
transitioned to oral glibenclamide therapy. Developmental delay was noted on
follow-up which raised the possibility of intermediate DEND syndrome. A possible
cause for hyperammonemia in neonatal diabetes mellitus has been postulated in
the discussion. Mutations in the KCNJ11 gene are responsible for the majority of permanent
neonatal diabetes mellitus (PNDM) cases. Some mutations in this gene, including
p.Q52R, are associated with the developmental delay, epilepsy, neonatal diabetes
(DEND) syndrome. We describe a patient with PNDM who had no neurological finding
although she was determined to have a novel mutation (p.Q52L) in the same
residue of the KCNJ11 as in the previously reported cases with DEND syndrome.
This case suggests that not all Q52 mutations in the KCNJ11 gene are necessarily
related to DEND syndrome. |
What is the RESID database? | The RESID Database of Protein Modifications is a comprehensive collection of annotations and structures for protein modifications and cross-links including pre-, co-, and post-translational modifications | Because the number of post-translational modifications requiring standardized
annotation in the PIR-International Protein Sequence Database was large and
steadily increasing, a database of protein structure modifications was
constructed in 1993 to assist in producing appropriate feature annotations for
covalent binding sites, modified sites and cross-links. In 1995 RESID was
publicly released as a PIR-International text database distributed on CD-ROM and
accessible through the ATLAS program. In 1998 it was made available on the PIR
Web site at http://www-nbrf.georgetown.edu/pir/searchdb++ +.html . The RESID
Database includes such information as: systematic and frequently observed
alternate names; Chemical s Service registry numbers; atomic formulas and
weights; enzyme activities; indicators forN-terminal, C-terminal or peptide
chain cross-link modifications; keywords; and literature citations with database
cross-references. The RESID Database can be used to predict atomic masses for
peptides, and is being enhanced to provide molecular structures for graphical
presentation on the PIR Web site using widely available molecular viewing
programs. The RESID Database is a comprehensive collection of annotations and structures
for protein pre-, co- and post-translational modifications including
amino-terminal, carboxyl-terminal and peptide chain cross-link modifications.
The RESID Database includes: systematic and alternate names, atomic formulas and
masses, enzyme activities generating the modifications, keywords, literature
citations, Gene Ontology cross-references, Protein Information Resource (PIR)
and SWISS-PROT protein sequence database feature table annotations, structure
diagrams and molecular models. This database is freely accessible on the
Internet through the European Bioinformatics Institute at
http://srs.ebi.ac.uk/srs6bin/cgi-bin/wgetz?-page+LibInfo+-lib+RESID, through the
National Cancer Institute - Frederick Advanced Biomedical Computing Center at
http://www.ncifcrf.gov/RESID, or through the Protein Information Resource at
http://pir.georgetown.edu/pirwww/dbinfo/resid.html. |
Pridopidine has been tested for treatment of which disorder? | Pridopidine is a dopaminergic stabilizer that has shown promising results for treatment of Huntington disease patients. | The CHDI Fifth Annual HD Therapeutics Conference, held in Palm Springs, CA,
included topics covering new therapeutic developments in the field of
Huntington's disease (HD). This conference report highlights presentations on
biomarkers in HD; emerging topics in drug targeting, such as the lysosomal
degradation pathway and target prediction by network-based modeling;
understanding phenotype and neuronal circuit dysfunction in animal models;
regulation of huntingtin protein expression and function; RNAi and antisense
technology to deplete the mutant huntingtin protein; and small-molecule drugs
that are progressing quickly through the clinic. Investigational drugs discussed
include ALN-HTT (Alnylam Pharmaceuticals Inc/Medtronic Inc), EPI-743 (Edison
Pharmaceuticals Inc), LNK-754 (Link Medicine Corp) and pridopidine (NeuroSearch
A/S). OBJECTIVES: To evaluate the efficacy and safety of the dopaminergic stabilizer
pridopidine (ACR16) in patients with Huntington's disease (HD).
METHODS: In a randomized, double-blind, placebo-controlled, 4-week trial,
patients with HD received pridopidine (50 mg/d, n = 28) or placebo (n = 30). The
primary outcome measure was the change from baseline in weighted cognitive
score, assessed by cognitive tests (Symbol Digit Modalities, verbal fluency, and
Stroop tests). Secondary outcome measures included changes in the Unified
Huntington's Disease Rating Scale, Hospital Anxiety and Depression Scale, Leeds
Sleep Evaluation Questionnaire, Reitan Trail-Making Test A, and Clinical Global
Impression of Change. Safety assessments were also performed.
RESULTS: There was no significant difference between pridopidine and placebo in
the change from baseline of the weighted cognitive score. However, secondary
measures such as affective symptoms showed trends toward improvement, and there
was significant improvement in voluntary motor symptoms compared with placebo (P
< 0.05). Pridopidine was well tolerated, with a safety profile similar to
placebo.
CONCLUSIONS: Pridopidine shows promise as a treatment for some of the symptoms
of HD. In this small-scale study, the most notable effect was improvement in
voluntary motor symptoms. Larger, longer-term trials are warranted. Pridopidine (ACR16) belongs to a new pharmacological class of agents affecting
the central nervous system called dopaminergic stabilizers. Dopaminergic
stabilizers act primarily at dopamine type 2 (D(2)) receptors and display
state-dependent behavioural effects. This article aims to give an overview of
the preclinical neurochemical and behavioural in vivo pharmacological properties
of pridopidine. Pridopidine was given s.c. to male Sprague-Dawley rats
(locomotor, microdialysis and tissue neurochemistry) and i.p. to Swiss male mice
(tail suspension test). Pridopidine dose-dependently increased striatal tissue
levels of the dopamine metabolite 3,4-dihydroxyphenylalanin (ED(50)=81
micromol/kg), and prefrontal cortex dialysate levels of dopamine and
noradrenaline as measured by high performance liquid chromatography. The agent
reduced hyperlocomotion (d-amphetamine: ED(50)=54 micromol/kg; MK-801: ED(50)=40
micromol/kg), but preserved spontaneous locomotor activity, confirming
state-dependent behavioural effects. In addition, pridopidine significantly
reduced immobility time in the tail suspension test. We conclude that
pridopidine state-dependently stabilizes psychomotor activity by the dual
actions of functional dopamine D(2) receptor antagonism and strengthening of
cortical glutamate functions in various settings of perturbed neurotransmission.
The putative restoration of function in cortico-subcortical circuitry by
pridopidine is likely to make it useful for ameliorating several neurological
and psychiatric disorders, including Huntington's disease. OBJECTIVES: Huntington disease is a hereditary neurodegenerative disorder
resulting in loss of motor, cognitive, and behavioral functions and is
characterized by a distinctive pattern of cerebral metabolic abnormalities.
Pridopidine (ACR16) belongs to a novel class of central nervous system compounds
in development for the treatment of Huntington disease. The objective of the
study was to investigate the metabolic changes in patients with Huntington
disease before and after pridopidine treatment.
METHODS: [(18)F]Fluorodeoxyglucose positron emission tomographic imaging was
used to measure the regional cerebral metabolic rate of glucose at baseline and
after 14 days of open-label pridopidine treatment in 8 patients with Huntington
disease. Clinical assessments were performed using the Unified Huntington's
Disease Rating Scale.
RESULTS: Statistical parametric mapping analysis showed increased metabolic
activity in several brain regions such as the precuneus and the mediodorsal
thalamic nucleus after treatment. In addition, after pridopidine treatment, the
correlation between the clinical status and the cerebral metabolic activity was
strengthened.
CONCLUSIONS: Our findings suggest that pridopidine induces metabolic changes in
brain regions implicated as important for mediating compensatory mechanisms in
Huntington disease. In addition, the finding of a strong relationship between
clinical severity and metabolic activity after treatment also suggests that
pridopidine treatment targets a Huntington disease-related metabolic activity
pattern. BACKGROUND: Huntington's disease is a progressive neurodegenerative disorder,
characterised by motor, cognitive, and behavioural deficits. Pridopidine belongs
to a new class of compounds known as dopaminergic stabilisers, and results from
a small phase 2 study in patients with Huntington's disease suggested that this
drug might improve voluntary motor function. We aimed to assess further the
effects of pridopidine in patients with Huntington's disease.
METHODS: We undertook a 6 month, randomised, double-blind, placebo-controlled
trial to assess the efficacy of pridopidine in the treatment of motor deficits
in patients with Huntington's disease. Our primary endpoint was change in the
modified motor score (mMS; derived from the unified Huntington's disease rating
scale) at 26 weeks. We recruited patients with Huntington's disease from 32
European centres; patients were aged 30 years or older and had an mMS of 10
points or greater at baseline. Patients were randomly assigned (1:1:1) to
receive placebo, 45 mg per day pridopidine, or 90 mg per day pridopidine by use
of centralised computer-generated codes. Patients and investigators were masked
to treatment assignment. We also assessed the safety and tolerability profile of
pridopidine. For our primary analysis, all patients were eligible for inclusion
in our full analysis set, in which we used the last observation carried forward
method for missing values. We used an analysis of covariance model and the
Bonferroni method to adjust for multiple comparisons. We used a prespecified
per-protocol population as our sensitivity analysis. The α level was 0·025 for
our primary analysis and 0·05 overall. This trial is registered with
ClinicalTrials.gov, number NCT00665223.
FINDINGS: At 26 weeks, in our full analysis set the difference in mean mMS was
-0·99 points (97·5% CI -2·08 to 0·10, p=0·042) in patients who received 90 mg
per day pridopidine (n=145) versus those who received placebo (n=144), and -0·36
points (-1·44 to 0·72, p=0·456) in those who received 45 mg per day pridopidine
(n=148) versus those who received placebo. At the 90 mg per day dose, in our
per-protocol population (n=114), the reduction in the mMS was of -1·29 points
(-2·47 to -0·12; p=0·014) compared with placebo (n=120). We did not identify any
changes in non-motor endpoints at either dose. Pridopidine was well tolerated
and had an adverse event profile similar to that of placebo.
INTERPRETATION: This study did not provide evidence of efficacy as measured by
the mMS, but a potential effect of pridopidine on the motor phenotype of
Huntington's disease merits further investigation. Pridopidine up to 90 mg per
day was well tolerated in patients with Huntington's disease.
FUNDING: NeuroSearch A/S. Huntington's disease (HD) is a neurodegenerative disease characterized by
progressive motor, cognitive and psychiatric deficits, associated with
predomit loss of striatal neurons and caused by a polyglutamine expansion in
the huntingtin protein. There is so far neither cure nor approved
disease-slowing therapy for HD, though recent clinical studies have shown a
beneficial long-term effect of pridopidine in patients with HD. The nature of
this effect, purely symptomatic or, in addition, neuroprotective, is difficult
to elucidate in clinical trials. Pridopidine and (-)-OSU6162 are members of a
new family of compounds referred to as dopaminergic stabilizers, which normalize
abnormal dopamine neurotransmission. We investigated the effects of (-)-OSU6162
on huntingtin knocked-in striatal neurons in culture. Control neurons had normal
full-length huntingtin with 7 glutamines while "mutant" neurons had large
expansions (Q=111). We studied the dose-effect curves of (-)-OSU6162 on
mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and
Q111 cells. In addition, we investigated the effects of (-)-OSU6162 on Q7 and
Q111 neurons challenged with different neurotoxins such as sodium glutamate,
H(2)O(2), rotenone and 3-nitropropionic acid (3NP). As we found prevention of
toxicity of some of these neurotoxins, we investigated the putative
neuroprotective mechanisms of action of (-)-OSU6162 measuring the effects of
this dopaminergic stabilizer on expression and release of BDNF, the ratios of
Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the
effects of (-)-OSU6162 on dopamine uptake and release. We found that
(-)-OSU6162, 3-150 μM, produces a dose dependent increase of mitochondrial
activity and a reduction of cell death. (-)-OSU6162 does not change glutamate
toxicity, but it partially prevents that of H(2)O(2), rotenone and
3-nitropropionic acid. (-)-OSU6162 increases the intracellular levels of BDNF
and Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells.
(-)-OSU6162 increased (3)H-dopamine uptake and amphetamine-induced (3)H-dopamine
release in E13 mouse mid brain neurons. Our studies demonstrate that (-)-OSU6162
improves survival and mitochondrial function in striatal Q111 neurons and the
resistance of these cells to several striatal neurotoxins, suggesting that
(-)-OSU6162 and related compounds should be tested for neuroprotection in animal
models and, eventually, in patients with HD. Pridopidine is being developed for the treatment of impaired motor function
associated with Huntington's disease and belongs to a new class of compounds
known as dopidines, which act as dopaminergic stabilizers. In vitro studies have
shown that pridopidine is a substrate for the P450 cytochrome 2D6 enzyme
(CYP2D6), and clinical data show that the half-life of pridopidine is different
following single dosing versus at steady state. To further investigate the
pharmacokinetic profile of pridopidine and to establish whether dose adjustment
is needed in poor CYP2D6 metabolizers, a single-centre, open-label,
multiple-dose study in healthy volunteers was performed. In total, 24 extensive
CYP2D6 metabolizers (EMs) and 12 poor CYP2D6 metabolizers (PMs) were enrolled.
Both groups received 45 mg pridopidine twice daily (b.i.d.). Plasma samples were
taken during the first day of b.i.d. dosing (Day 1) and at steady state,
following 14 days of b.i.d. dosing. At Day 1, total exposure in PMs was almost
three times higher than those in EMs (AUC0-∞ = 11,192 and 3,782 h·ng/mL,
respectively; PM/EM ratio = 2.96; p < 0.001). However, at steady state, PMs and
EMs had comparable exposure due to a reduction in pridopidine elimination in EMs
over time. Thus, at steady-state peak (C max) and total (AUC0-24) exposures were
only 1.24 and 1.29 times higher, respectively, in PMs than EMs. These results
support that pridopidine is a CYP2D6 auto-inhibitor. Pridopidine was well
tolerated in both EMs and PMs. The slightly higher exposure level in PMs at
steady state does not indicate a need for dose adjustment or genotyping for
CYP2D6 metabolizer status. The dopaminergic stabilizer pridopidine demonstrates state-dependent effects on
locomotor activity, counteracting both hypo- and hyperactivity in rats.
Pridopidine has been shown to display both functional dopamine D2 receptor
antagonist properties and increase in biomarkers associated with NMDA-mediated
glutamate transmission in the frontal cortex. To further characterise the
effects of pridopidine on prefrontal cortex (PFC) neurons, a series of in vivo
electrophysiological studies were performed in urethane-anaesthetised rats.
Pridopidine, administered at doses from 10 to 60 mg/kg (i.v.), dose dependently
increased pyramidal cell firing in the majority of the neurons tested.
Pridopidine induced a significant increase of 162 % in mean firing activity of
PFC neurons, versus initial basal firing activity as the cumulative dose of 30
mg/kg, i.v., was administered. This enhancement of activity was due to increased
firing frequency of already spontaneously active neurons, rather than an
increase in population activity. The increase was partially reversed or
prevented by a sub-threshold dose of the dopamine D1 receptor antagonist
SCH23390 (0.5 mg/kg, i.v.). Microiontophoretic application of pridopidine had
only moderate activating effects. The selective dopamine D1 receptor agonist
A-68930 also had limited effects when administered by microiontophoretic
application, but exerted a dose dependent (0.2-3 mg/kg, i.v.) activation of
firing in the majority of neurons tested (10/16). However, inhibition of firing
by systemic administration of A-68930 was also observed in a subgroup of neurons
(6/16). Both activation and inhibition of firing induced by systemic
administration of A-68930 were reversed by the systemic administration of
SCH23390. The present data suggests that pridopidine enhances pyramidal cell
firing via an indirect dopamine D1 receptor-mediated mechanism. These effects of
pridopidine may serve to strengthen the cortico-striatal communication and to
improve motor control in Huntington's disease for which pridopidine is currently
in development. |
Are there currently applications of deep learning in genomics? | Yes. Deep learning has been used so far in genomics for predicting splicing patterns in individual tissues and differences in splicing patterns across tissues. The deep architecture surpasses the performance of the previous Bayesian method for predicting alternative splicing (AS) patterns. | Author information:
(1)Department of Electrical and Computer Engineering, University of Toronto,
Toronto, Ontario M5S 3G4, Banting and Best Department of Medical Research,
University of Toronto, Toronto, Ontario M5S 3E1, Canada and Canadian Institute
for Advanced Research, Toronto, Ontario M5G 1Z8, CanadaDepartment of Electrical
and Computer Engineering, University of Toronto, Toronto, Ontario M5S 3G4,
Banting and Best Department of Medical Research, University of Toronto, Toronto,
Ontario M5S 3E1, Canada and Canadian Institute for Advanced Research, Toronto,
Ontario M5G 1Z8, Canada.
(2)Department of Electrical and Computer Engineering, University of Toronto,
Toronto, Ontario M5S 3G4, Banting and Best Department of Medical Research,
University of Toronto, Toronto, Ontario M5S 3E1, Canada and Canadian Institute
for Advanced Research, Toronto, Ontario M5G 1Z8, CanadaDepartment of Electrical
and Computer Engineering, University of Toronto, Toronto, Ontario M5S 3G4,
Banting and Best Department of Medical Research, University of Toronto, Toronto,
Ontario M5S 3E1, Canada and Canadian Institute for Advanced Research, Toronto,
Ontario M5G 1Z8, CanadaDepartment of Electrical and Computer Engineering,
University of Toronto, Toronto, Ontario M5S 3G4, Banting and Best Department of
Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada and
Canadian Institute for Advanced Research, Toronto, Ontario M5G 1Z8, Canada. |
Are CpG islands located close to housekeeping genes? | Our analysis indicates that the association of CGIs with housekeeping genes is not as strong as previously estimated. These regions represent about 1% of genomic DNA and are generally found in the promoter region of housekeeping genes. In housekeeping and many tissue-specific genes, the promoter is embedded in a so-called CpG island. Methylation-free CpG clusters, so-called HTF islands, are most often associated with the promoter regions of housekeeping genes, whereas genes expressed in a single-cell type are usually deficient in these sequences. All housekeeping and widely expressed genes have a CpG island covering the transcription start, whereas 40% of the genes with a tissue-specific or limited expression are associated with islands. It has been envisaged that CpG islands are often observed near the transcriptional start sites (TSS) of housekeeping genes. CpG islands, which are found almost exclusively at the 5'-end of housekeeping genes. CpG islands were associated with the 5' ends of all housekeeping genes and many tissue-specific genes, and with the 3' ends of some tissue-specific genes. | CpG islands are short, dispersed regions of unmethylated DNA with a high
frequency of CpG dinucleotides relative to the bulk genome. We have analyzed 375
genes and 58 pseudogenes from the human entries in the EMBL Database for the
presence of CpG islands. All 240 islands identified are associated with genes,
and almost all cover at least a part of one exon; i.e., they are useful
landmarks in the genome for identifying genes. More than half of the genes
analyzed were associated with islands. All housekeeping and widely expressed
genes have a CpG island covering the transcription start, whereas 40% of the
genes with a tissue-specific or limited expression are associated with islands.
In this latter group of genes, the position of the islands was not biased toward
the 5' end of the transcription unit. Patterns of DNA methylation at CpG dinucleotides and their relations with gene
expression are complex. Methylation-free CpG clusters, so-called HTF islands,
are most often associated with the promoter regions of housekeeping genes,
whereas genes expressed in a single-cell type are usually deficient in these
sequences. However, in the human carbonic anhydrase (CA) gene family, both the
ubiquitously expressed CAII and the muscle specific CAIII appear to have such
CpG islands although erythrocyte-specific CAI does not. The CAII island is
quantitatively more CpG rich than that of CAIII, with a CpG:GpC ratio of 0.94
compared with 0.82 for CAIII. Estimation of CpG:GpC ratios in the
proximal-promoter regions of 44 vertebrate genes suggest that 40% of genes with
tissue-specific or limited tissue distribution may show methylation-free CpG
clusters in their promoter regions. In many cases the CpG:GpC ratio is less than
that found in housekeeping genes and this may reflect variation in the
interaction of CpG clusters with regulatory factors that define different
patterns of tissue expression. Unmethylated CpG rich islands are a feature of vertebrate DNA: they are
associated with housekeeping and many tissue specific genes. CpG islands on the
active X chromosome of mammals are also unmethylated. However, islands on the
inactive X chromosome are heavily methylated. We have identified a CpG island in
the 5' region of the G6PD gene, and two islands forty Kb 3' from the G6PD gene,
on the human X chromosome. Expression of the G6PD gene is associated with
concordant demethylation of all three CpG islands. We have shown that one of the
two islands is in the promoter region of a housekeeping gene, GdX. In this paper
we show that the second CpG island is also associated with a gene, P3. The P3
gene has no homology to previously described genes. It is a single copy, 4 kb
gene, conserved in evolution, and it has the features of a housekeeping two
genes is within the CpG island and that sequences in the islands have promoter
function. In housekeeping and many tissue-specific genes, the promoter is embedded in a
so-called CpG island. We have compared the available human and mouse DNA
sequences with respect to their CpG island properties. While mouse sequences
showed a simple gradient distribution of G + C content and CpG densities, man
had a distinct peak of sequences with typical CpG island characteristics.
Pairwise comparison of 23 orthologous genes revealed that mouse almost always
had a less pronounced CpG island than man, or none at all. In both species the
requirements for a functional CpG island may be similar in that most DNA regions
with a density of six or more CpG per 100 bp remain unmethylated. However, the
mouse has apparently experienced more accidental CpG island methylation,
suggested by local TpG and CpA excess. We propose that: (1) in mouse the CpG
islands do not represent the ancestral state but have been eroded during
evolution, and (2) this erosion may be related to the mouse's small body mass
and short life-span, allowing for a more relaxed control of gene activity. In vivo and in vitro experiments carried out on L929 mouse fibroblasts suggested
that the poly(ADP-ribosyl) ation process acts somehow as a protecting agent
against full methylation of CpG dinucleotides in genomic DNA. Since CpG islands,
which are found almost exclusively at the 5'-end of housekeeping genes, are rich
in CpG dinucleotides, which are the target of mammalian DNA methyltransferase,
we examined the possibility that the poly(ADP-ribosyl)ation reaction is involved
in maintaining the unmethylated state of these DNA sequences. Experiments were
conducted by two different strategies, using either methylation-dependent
restriction enzymes on purified genomic DNA or a sequence-dependent restriction
enzyme on an aliquot of the same DNA, previously modified by a bisulfite
reaction. With the methylation-dependent restriction enzymes, it was observed
that the "HpaII tiny fragments" greatly decreased when the cells were
preincubated with 3-aminobenzamide, a well known inhibitor of poly(ADP-ribose)
polymerase. The other experimental approach allowed us to prove that, as a
consequence of the inhibition of the poly(ADP-ribosyl)ation process, an
anomalous methylation pattern could be evidenced in the CpG island of the
promoter fragment of the Htf9 gene, amplified from DNA obtained from fibroblasts
preincubated with 3-aminobenzamide. These data confirm the hypothesis that, at
least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects
the unmethylated state of the CpG island. CpG islands are stretches of DNA sequence that are enriched in the (CpG)n repeat
and are present in close association with all housekeeping genes as well as some
tissue-specific genes in the mammalian genome. Methylation of CpG islands
strongly influences both structural organization and function of chromatin. The
presence of a CpG island in a given chromosomal domain can, by itself, give rise
to relatively open and active chromatin. Recently, several histone
acetyltransferases, histone deacetylases, and chromatin remodeling factors have
been found to be part of the transcription machinery. It is becoming
increasingly clear that CpG islands and their methylation status may influence
the function or recruitment of these newly discovered chromatin remodeling
factors, especially the histone deacetylases. In addition, CpG islands may also
play a significant role in the reorganization of chromatin during mammalian
spermiogenesis. DNA methylation is the epigenetic modification, which introduces 5mC as fifth
base onto DNA. As for the distribution of 5mCs, it is well known that they
distribute themselves in a non-random fashion in genomic DNA so that methylation
pattern is characterized by the presence of methylated cytosines on the bulk of
DNA while the unmethylated ones are mainly located within particular regions
termed CpG islands. These regions represent about 1% of genomic DNA and are
generally found in the promoter region of housekeeping genes. Their unmethylated
state, which is an essential condition for the correct expression of correlated
genes, is paradoxical if one considers that these regions are termed CpG islands
because they are particularly rich in this dinucleotide, which is the best
substrate for enzymes involved in DNA methylation. Anomalous insertion of methyl
groups in these regions generally leads to the lack of transcription of
correlated genes. An interesting scientific problem is to clarify the
mechanism(s) whereby CpG islands, which remain protected from methylation in
normal cells, are susceptible to methylation in tumor cells. How the CpG
moieties in CpG islands become vulnerable or resistant to the action of DNA
methyltransferases and can thus lose or maintain their characteristic pattern of
methylation is still an open question. Our aim is to gather some mechanisms
regarding this intriguing enigma, which, despite all energy spent, still remains
an unresolved puzzle. It has been envisaged that CpG islands are often observed near the
transcriptional start sites (TSS) of housekeeping genes. However, neither the
precise positions of CpG islands relative to TSS of genes nor the correlation
between the presence of the CpG islands and the expression specificity of these
genes is well-understood. Using thousands of sequences with known TSS in human
and mouse, we found that there is a clear peak in the distribution of CpG
islands around TSS in the genes of these two species. Thus, we classified human
(mouse) genes into 6600 (2948) CpG+ genes and 2619 (1830) CpG- ones, based on
the presence of a CpG island within the -100: +100 region. We estimated the
degree of each gene being a housekeeper by the number of cDNA libraries where
its ESTs were detected. Then, the tendency that a gene lacking CpG islands
around its TSS is expressed with a higher degree of tissue specificity turned
out to be evolutionarily conserved. We also confirmed this tendency by analyzing
the gene ontology annotation of classified genes. Since no such clear
correlation was found in the control data (mRNAs, pre-mRNAs, and chromosome
banding pattern), we concluded that the effect of a CpG island near the TSS
should be more important than the global GC content of the region where the gene
resides. Abnormal development and fetal loss during postimplantation period are concerns
for production of nuclear transferred animals. Aberrant DNA methylation is one
of the reasons for poor survival of cloned animals. In mammalian genome DNA, CpG
islands are preferentially located at the start of transcription of housekeeping
genes and are associated with tissue-specific genes. The correct and consecutive
mechanisms of DNA methylation in the CpG islands are necessary for selective
gene expressions that determine the properties of individual cells, tissues, and
organs. In this study, we investigated the methylation status of the CpG islands
of the bovine Leptin and POU5F1 genes in fetal and placental tissues from
fetuses produced by artificial insemination (AI) and nuclear transfer (NT) at
days 48 and 59 of pregcy. Altered DNA methylation was observed in the normal
and cloned fetal, placental, and endometrial tissues using bisulfite sequencing
and pyrosequencing. Different tissue-specific methylated regions in the bovine
Leptin and POU5F1 genes show a variable methylation status in NT fetuses
compared to AI control. CpG islands (CGIs) are often considered as gene markers, but the number of CGIs
varies among mammalian genomes that have similar numbers of genes. In this
study, we investigated the distribution of CGIs in the promoter regions of 3,197
human-mouse orthologous gene pairs and found that the mouse genome has notably
fewer CGIs in the promoter regions and less pronounced CGI characteristics than
does the human genome. We further inferred CGI's ancestral state using the dog
genome as a reference and examined the nucleotide substitution pattern and the
mutational direction in the conserved regions of human and mouse CGIs. The
results reveal many losses of CGIs in both genomes but the loss rate in the
mouse lineage is two to four times the rate in the human lineage. We found an
intriguing feature of CGI loss, namely that the loss of a CGI usually starts
from erosion at the both edges and gradually moves towards the center. We found
functional bias in the genes that have lost promoter-associated CGIs in the
human or mouse lineage. Finally, our analysis indicates that the association of
CGIs with housekeeping genes is not as strong as previously estimated. Our study
provides a detailed view of the evolution of promoter-associated CGIs in the
human and mouse genomes and our findings are helpful for understanding the
evolution of mammalian genomes and the role of CGIs in gene function. |
Is COL5A2 gene associated to ischemic heart disease? | Analysis of a gene co-expression network establishes robust association between Col5a2 and ischemic heart disease | |
Which is the target of bortezomib used in cancer therapy? | Bortezomib is a potent and specific reversible ubiquitin/proteasome pathway inhibitor, which has shown strong in vitro antitumor activity as single agent and in combination with other cytotoxic drugs in a broad spectrum of hematological and solid malignancies. | E3 ubiquitin ligases are a large family of proteins that can be classified into
three major structurally distinct types: N-end rule E3s, E3s containing the HECT
(Homology to E6AP C-Terminus) domain, and E3s with the RING (Really Interesting
New Gene) finger, including its derivatives, the U- Box and the PHD (Plant
Homeo-Domain). E3 ubiquitin ligases exist as single polypeptide or multimeric
complexes. Together with ubiquitin activating enzyme E1 and ubiquitin
conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a
variety of protein substrates for targeted degradation via the 26S proteasome.
E3 ubiqutin ligases, therefore, play an essential role in regulation of many
biological processes. Furthermore, E3s are enzymes that determine the
specificity of protein substrates; they represent a class of "drugable" targets
for pharmaceutical intervention. In this review, I will mainly focus on E3
ubiquitin ligases as potential cancer targets and discuss three of the most
promising E3s, Mdm2/Hdm2, IAPs, and SCF, for their target rationales, target
validation, and critical issues associated with them. These E3 ligases or their
components are overexpressed in many human cancers and their inhibition leads to
growth suppression or apoptosis. In addition, I will evaluate two current
methodologies available for the high throughput screening for small molecular
weight chemical inhibitors of the E3 ubiquitin ligases. Although targeting E3
ubiquitin ligases is still in its infancy, speedy approval of the general
proteasome inhibitor, Velcade (bortezomib) by the FDA for the treatment of
relapsed and refractory multiple myeloma suggests the promise of specific E3
inhibitors in anti-cancer therapy. Emerging technologies, such as siRNA, will
provide a better validation of many E3s. It is anticipated that E3 ubiquitin
ligases will represent an important new target platform for future
mechanism-driven drug discovery. The proteasome is a multicatalytic proteinase complex responsible for the
degradation of most intracellular proteins, including proteins crucial to cell
cycle regulation and programmed cell death, or apoptosis. In preclinical cancer
models, proteasome inhibitors induce apoptosis, have in vivo antitumor efficacy,
and sensitize maligt cells and tumors to the proapoptotic effects of
conventional chemotherapeutics and radiation therapy. Interestingly, transformed
cells display greater susceptibility to proteasome inhibition than nonmaligt
cells. Therefore, proteasome inhibition holds promise as a novel approach to the
treatment of cancer. Inhibitors of the proteasome impact on cells in part
through down-regulation of nuclear factor kappaB, but also through modulation of
cell cycle proteins and other pro- and antiapoptotic pathways. Bortezomib
(VELCADE; formerly PS-341), the first such inhibitor to undergo clinical
testing, has demonstrated impressive antitumor activity and manageable
toxicities in Phase I and II trials both as a single agent, and in combination
with other drugs. It has been approved recently by the Food and Drug
Administration for therapy of patients with multiple myeloma who have received
at least two prior regimens and progressed on the last of these. Ongoing
preclinical evaluations of the mechanisms that underlie the antitumor effects of
proteasome inhibitors, and clinical trials in a variety of tumor types, will
allow additional refinement of the role these agents will play in cancer
therapy. Below we discuss the rationale behind targeting the proteasome for
cancer therapy, and review the preclinical and clinical data on proteasome
inhibitors alone, and in combination with conventional chemotherapeutics. The 26S proteasome is a promising new target for breast cancer therapy. The
degradation of proteins by the proteasome is an essential metabolic process, and
inhibition of the proteasome results in cell-cycle arrest and apoptosis.
However, cancer cells and proliferating blood vessels appear to be particularly
sensitive to the effects of proteasome inhibition. Studies carried out in breast
cancer cells and murine xenograft models of breast cancer have demonstrated the
potent antitumor effects of proteasome inhibition in this disease. Proteasome
inhibition interferes with many cell signaling pathways, including those
involved in the development and progression of breast cancer. The potent and
selective proteasome inhibitor bortezomib (VELCADE; formerly known as PS-341) is
particularly promising as a potential anticancer agent. PS-341 is the first
proteasome inhibitor to be extensively studied in murine models of cancer and to
progress to clinical trials in cancer patients. Preliminary clinical data from
patients with a range of maligcies indicate that the drug effectively
inhibits proteasome activity at doses associated with manageable toxicity. Early
clinical trials are currently recruiting participants for the analysis of PS-341
activity in breast cancer. Critical cellular processes are regulated, in part, by maintaining the
appropriate intracellular levels of proteins. Whereas de novo protein synthesis
is a comparatively slow process, proteins are rapidly degraded at a rate
compatible with the control of cell cycle transitions and cell death induction.
A major pathway for protein degradation is initiated by the addition of multiple
76-amino acid ubiquitin monomers via a three-step process of ubiquitin
activation and substrate recognition. Polyubiquitination targets proteins for
recognition and processing by the 26S proteasome, a cylindrical organelle that
recognizes ubiquitinated proteins, degrades the proteins, and recycles
ubiquitin. The critical roles played by ubiquitin-mediated protein turnover in
cell cycle regulation makes this process a target for oncogenic mutations.
Oncogenes of several common maligcies, for example colon and renal cell
cancer, code for ubiquitin ligase components. Cervical oncogenesis by human
papillomavirus is also mediated by alteration of ubiquitin ligase pathways.
Protein degradation pathways are also targets for cancer therapy, as shown by
the successful introduction of bortezomib, an inhibitor of the 26S proteasome.
Further work in this area holds great promise toward our understanding and
treatment of a wide range of cancers. Cellular homeostasis requires routine degradation of key regulatory proteins,
including tumor suppressor gene products, transcription factors, cell-cycle
proteins and their inhibitors, as well as damaged and misfolded proteins. A
critical part of this process is mediated by the 26S proteasome, a multi-subunit
enzyme found in the nucleus and cytoplasm of all eukaryotic cells. Because of
its essential role in many cellular processes controlling growth and survival,
the proteasome has been identified as a potential target for cancer therapy.
Drugs known to inhibit proteasome activity have been shown to induce cell-cycle
arrest and programmed cell death (apoptosis). The impact of this finding is
heightened by research showing that cancer cells are more sensitive to the
proapoptotic effects of proteasome inhibition than normal cells. Preclinical
evidence using bortezomib, the only proteasome inhibitor to enter clinical
trials, suggests that proteasome inhibition may be effective in the treatment of
hematologic and solid maligcies by promoting apoptosis, retarding
angiogenesis, and inhibiting tumor cell adhesion and production of growth
factors by acting on molecules such as nuclear factor-kappaB. Further
preclinical evidence suggests that the antitumor effects of cytotoxic
chemotherapy or radiotherapy may be enhanced by the addition of a proteasome
inhibitor. Bortezomib was recently approved for the treatment of multiple
myeloma. It is currently being investigated, both as a single agent and in
combination, in phase I and II trials in a variety of tumor types. A major challenge in cancer therapy is tumor drug resistance. To overcome it, it
is essential to understand the mechanisms and identify the molecules involved,
so that they can be specifically targeted in combination therapies. The
proteasome is such a validated target: it plays a key role in cancer cell
proliferation, inhibition of chemotherapy-induced apoptosis and drug resistance
development. Bortezomib (Velcade, PS-341) was the first proteasome inhibitor to
receive regulatory approval from the US Food and Drug Administration for the
treatment of multiple myeloma. Clinical combination trials have demonstrated a
chemo-sensitizing effect of bortezomib on conventional agents in hematological
maligcies and some solid tumors such as androgen-independent prostate and
ovarian cancer. Although generally well-tolerated, bortezomib still generates
toxicity which underscores the need for less toxic proteasome inhibitors.
Several naturally occurring products, such as green tea polyphenols and the
antibiotic lactacystin, have been shown to be potent proteasome inhibitors.
Significantly, green tea polyphenols, as well as several flavonoids such as
genistein, curcumin and resveratrol, have also been shown to have
chemo-sensitizing properties in prostate, breast, hepatic, and lung tumors.
Further studies on natural proteasome inhibitors as chemo-sensitizers could lead
to identification of more potent and less toxic compounds that could be used in
combination therapies for drug-resistant tumors. The ubiquitin-proteasome pathway plays a central role in the degradation of
proteins involved in several pathways including the cell cycle, cellular
proliferation and apoptosis. Bortezomib is the first proteasome inhibitor to
enter clinical use, and received approval by the Food and Drug Administration
(FDA) for the treatment of patients with multiple myeloma, therefore validating
inhibition of the proteasome as an anticancer target. The approval of Bortezomib
was based on a large, international, multicenter phase III trial showing its
efficacy and safety compared with conventional therapy. Preclinical data also
demonstrates the synergistic effect of bortezomib with other chemotherapeutic
agents and its ability to overcome drug resistance. Since then several other
proteasome inhibitors have been developed. The anti-tumor activities of
bortezomib have been attributed to its effect on pro-apoptotic pathways
including the inhibition of NF-kappaB and induction of endoplasmic reticulum
stress. However, the molecular mechanisms are not fully understood. In this
review, we will summarize the molecular mechanism of apoptosis by bortezomib. The proteasome has emerged as an important target for cancer therapy with the
approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for
relapsed/refractory multiple myeloma (MM). However, many patients have disease
that does not respond to bortezomib, whereas others develop resistance,
suggesting the need for other inhibitors with enhanced activity. We therefore
evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor,
carfilzomib. In models of MM, this agent potently bound and specifically
inhibited the chymotrypsin-like proteasome and immunoproteasome activities,
resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a
dose- and time-dependent inhibition of proliferation, ultimately leading to
apoptosis. Programmed cell death was associated with activation of
c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of
cytochrome c, and activation of both intrinsic and extrinsic caspase pathways.
This agent also inhibited proliferation and activated apoptosis in
patient-derived MM cells and neoplastic cells from patients with other
hematologic maligcies. Importantly, carfilzomib showed increased efficacy
compared with bortezomib and was active against bortezomib-resistant MM cell
lines and samples from patients with clinical bortezomib resistance. Carfilzomib
also overcame resistance to other conventional agents and acted synergistically
with dexamethasone to enhance cell death. Taken together, these data provide a
rationale for the clinical evaluation of carfilzomib in MM. BACKGROUND: The ubiquitin-proteasome system has become a promising novel
molecular target in cancer due to its critical role in cellular protein
degradation, its interaction with cell cycle and apoptosis regulation and its
unique mechanism of action.
OBJECTIVE: This review focuses both on preclinical results and on data from
clinical trials with proteasome inhibitors in cancer.
METHODS: Results in hematological maligcies and solid tumors were included,
and important data presented in abstract form were considered in this review.
RESULTS/CONCLUSION: Bortezomib as first-in-class proteasome inhibitor has proven
to be highly effective in some hematological maligcies, overcomes
conventional chemoresistance, directly induces cell cycle arrest and apoptosis,
and also targets the tumor microenvironment. It has been granted approval by the
FDA for relapsed multiple myeloma, and recently for relapsed mantle cell
lymphoma. Combination chemotherapy regimens have been developed providing high
remission rates and remission quality in frontline treatment or in the relapsed
setting in multiple myeloma. The combination of proteasome inhibition with novel
targeted therapies is an emerging field in oncology. Moreover, novel proteasome
inhibitors, such as NPI-0052 and carfilzomib, have been developed. This review
summarizes our knowledge of the ubiquitin-proteasome system and recent data from
cancer clinical trials. The ubiquitin-proteasome system plays a critical role in controlling the level,
activity and location of various cellular proteins. Significant progress has
been made in investigating the molecular mechanisms of ubiquitination,
particularly in understanding the structure of the ubiquitination machinery and
identifying ubiquitin protein ligases, the primary specificity-determining
enzymes. Therefore, it is now possible to target specific molecules involved in
ubiquitination and proteasomal degradation to regulate many cellular processes
such as signal transduction, proliferation and apoptosis. In particular,
alterations in ubiquitination are observed in most, if not all, cancer cells.
This is manifested by destabilization of tumor suppressors, such as p53, and
overexpression of oncogenes such as c-Myc and c-Jun. In addition to the
development and clinical validation of proteasome inhibitor, bortezomib, in
myeloma therapy, recent studies have demonstrated that it is possible to develop
inhibitors for specific ubiquitination and deubiquitination enzymes. With the
help of structural studies, rational design and chemical synthesis, it is
conceivable that we will be able to use 'druggable' inhibitors of the ubiquitin
system to evaluate their effects in animal tumor models in the not-so-distant
future. The ubiquitin-proteasome system has recently emerged as a major target for drug
development in cancer therapy. The proteasome inhibitor bortezomib has clinical
activity in multiple myeloma and mantle cell lymphoma. Here we report that
Eeyarestatin I (EerI), a chemical inhibitor that blocks endoplasmic reticulum
(ER)-associated protein degradation, has antitumor and biologic activities
similar to bortezomib and can synergize with bortezomib. Like bortezomib,
EerI-induced cytotoxicity requires the up-regulation of the Bcl-2 homology3
(BH3)-only pro-apoptotic protein NOXA. We further demonstrate that both EerI and
bortezomib activate NOXA via an uticipated mechanism that requires
cooperation between two processes. First, these agents elicit an integrated
stress response program at the ER to activate the CREB/ATF transcription factors
ATF3 and ATF4. We show that ATF3 and ATF4 form a complex capable of binding to
the NOXA promoter, which is required for NOXA activation. Second, EerI and
bortezomib also block ubiquitination of histone H2A to relieve its inhibition on
NOXA transcription. Our results identify a class of anticancer agents that
integrate ER stress response with an epigenetic mechanism to induce cell death. The ubiquitin-proteasome system has become a promising molecular target in
cancer therapy due to its critical role in cellular protein degradation,
interaction with cell cycle and apoptosis regulation, and unique mechanism of
action. Bortezomib (PS-341) is a potent and specific reversible proteasome
inhibitor, which has shown strong in vitro antitumor activity as single agent
and in combination with other cytotoxic drugs in a broad spectrum of
hematological and solid maligcies. In preclinical studies, bortezomib induced
apoptosis of maligt cells through the inhibition of NF-|B and stabilization
of pro-apoptotic proteins. Bortezomib also promotes chemo- and
radiosensitization of maligt cells in vitro and inhibits tumor growth in
murine xenograft models. The proteasome has been established as a relevant
target in hematologic maligcies and bortezomib has been approved for the
treatment of multiple myeloma. This review summarizes recent data from clinical
trials in solid tumors. The increasing appreciation of the importance of autophagy as consequence of
cancer therapy or underlying disease biology is illustrated by the large number
of papers that are evaluating autophagy as a cancer target. While autophagy is
often linked to the generation of metabolic precursors, it is also important in
diseases where protein production is a hallmark of the disease itself, such as
pancreatic cancer and multiple myeloma. Multiple myeloma is characterized by
ongoing autophagy as a consequence of constitutive immunoglobulin production,
which creates the need for efficient transfer and disposal of misfolded or
unfolded proteins. In order to survive this cellular stress, plasma cells depend
on proteasomal degradation of the large volume of misfolded proteins as well as
the autophagy pathway. It has previously been suggested that the excess proteins
not targeted to the proteasome, or that accumulate when the proteasome is
inhibited through the use of chemically active agents such as bortezomib, are
linked to impaired cell survival, and that their packaging in the form of an
aggresome somehow minimizes their 'proteotoxicity' allowing these toxic proteins
to be sequestered away from normal cellular machinery. Selective degradation of proteins by the ubiquitin-proteasome pathway is a
critical determit for maintaining cellular homeostasis. Most intracellular
proteins are degraded by the proteasome, a multicatalytic enzyme complex
containing a 20S catalytic core and two 19S regulatory complexes. Many
proteasome target proteins are involved in the regulation of important processes
of carcinogenesis and cancer cell survival, such as cell cycle progression, cell
proliferation, differentiation and apoptosis. Indeed, the
ubiquitin-proteasome-dependent degradation pathway plays an essential role in
both the up-regulation of cell proliferation and down-regulation of cell death
in human cancer cells. Both in vitro and in vivo experimental and clinical
results have demonstrated the potential use of proteasome inhibitors as novel
anticancer drugs. Proteasome inhibition in cancer cells leads to accumulation of
pro-apoptotic target proteins followed by induction of cell death. The clinical
efficacy of the proteasome inhibitor bortezomib toward multiple myeloma and
other hematologic maligcies provides the "proof of concept" that targeting
the proteasome is a promising strategy for cancer treatment. Several other
proteasome inhibitors have also been identified from natural resources, such as
marine microbial metabolites, green tea polyphenols, flavonoids, and medicinal
compounds. Additionally, the use of metal complexes as proteasome inhibitors has
also been investigated as a potential anticancer strategy. The clinical
significance of targeting the tumor survival-associated proteasome pathway for
cancer treatment, intervention and prevention will be discussed. The 26S proteasome complex engages in an ATP-dependent proteolytic degradation
of a variety of oncoproteins, transcription factors, cell cycle specific
cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other
key regulatory cellular proteins. Thus, the proteasome regulates either directly
or indirectly many important cellular processes. Altered regulation of these
cellular events is linked to the development of cancer. Therefore, the
proteasome has become an attractive target for the treatment of numerous
cancers. Several proteasome inhibitors that target the proteolytic active sites
of the 26S proteasome complex have been developed and tested for anti-tumor
activities. These proteasome inhibitors have displayed impressive anti-tumor
functions by inducing apoptosis in different tumor types. Further, the
proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit
angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory
responses, and DNA repair response. A number of proteasome inhibitors are now in
clinical trials to treat multiple myeloma and solid tumors. Many other
proteasome inhibitors with different efficiencies are being developed and tested
for anti-tumor activities. Several proteasome inhibitors currently in clinical
trials have shown significantly improved anti-tumor activities when combined
with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein
kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90)
inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the
clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the
26S proteasome complex in carcinogenesis and different proteasome inhibitors
with their potential therapeutic applications in treatment of numerous cancers. Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a
multisubunit complex comprising a proteolytic 20S core particle capped by 19S
regulatory particles. The approval of bortezomib for the treatment of multiple
myeloma validated the 20S core particle as an anticancer drug target. Here we
describe the small molecule b-AP15 as a previously unidentified class of
proteasome inhibitor that abrogates the deubiquitinating activity of the 19S
regulatory particle. b-AP15 inhibited the activity of two 19S
regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5
(UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation
of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to
TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that
treatment with b-AP15 inhibited tumor progression in four different in vivo
solid tumor models and inhibited organ infiltration in an acute myeloid leukemia
model. Our results show that the deubiquitinating activity of the 19S regulatory
particle is a new anticancer drug target. The ubiquitin-proteasome pathway regulates many basic cellular processes and has
been proven to be a promising target for cancer therapy. Bortezomib is the first
U.S. Food and Drug Administration (FDA) approved proteasome inhibitor used in
the treatment of newly diagnosed multiple myeloma, relapsed/refractory multiple
myeloma, and mantle cell lymphoma. The anti-cancer mechanisms of bortezomib
elucidated by preclinical studies include: upregulation of proapoptotic proteins
(e.g., Noxa, IκB), inhibition of NFκB and its anti-apoptotic target genes,
suppression of several anti-apoptotic proteins (e.g., Bcl-XL, Bcl-2, and
STAT-3), down-regulation of expression of several proteins involved in DNA
repair pathways, and induction of endoplasmic reticulum (ER) stress and
pro-apoptotic Unfolded Protein Response (UPR). Bortezomib has potent
chemo-/radio-sensitizing effects and can overcome traditional drug resistance in
tumors when used in combination with potential chemotherapies. Although
bortezomib has been successful in improving clinical outcomes when used in
hematological maligcies, relapse may occur in those patients who responded
initially. Furthermore, some cytotoxicities (such as peripheral neuropathy) were
found to be associated with bortezomib treatment. These observations have
encouraged researchers to search for the next generation proteasome inhibitors
(including carfilzomib and marizomib) that could overcome bortezomib resistance
and have improved properties, reduced toxicities, and broader anticancer
activities, based on the lessons learned from the mechanisms and use of
bortezomib. This review summarizes the current status of bortezomib as well as
several other proteasome inhibitors that are currently under clinical and
preclinical investigation. The ubiquitin-proteasome system (UPS) is a conserved pathway regulating numerous
biological processes including protein turnover, DNA repair, and intracellular
trafficking. Tumor cells are dependent on a functioning UPS, making it an ideal
target for the development of novel anti-cancer therapies. The development of
bortezomib (Velcade(®)) as a treatment for multiple myeloma and mantle cell
lymphoma has verified this and suggests that targeting other components of the
UPS may be a viable strategy for the treatment for cancer. We recently described
a novel class of proteasome inhibitors that function by an alternative mechanism
of action (D'Arcy et al., 2011). The small molecule b-AP15 blocks the
deubiquitinase (DUB) activity of the 19S regulatory particle (19S RP) without
inhibiting the proteolytic activities of the 20S core particle (20S CP). b-AP15
inhibits two proteasome-associated DUBs, USP14 and UCHL5, resulting in a rapid
accumulation of high molecular weight ubiquitin conjugates and a functional
proteasome shutdown. Interestingly, b-AP15 displays several differences to
bortezomib including insensitivity to over-expression of the anti-apoptotic
mediator Bcl-2 and anti-tumor activity in solid tumor models. In this review we
will discuss the potential of proteasome deubiquitinase inhibitors as additions
to the therapeutic arsenal against cancer. |
Have microRNAs been implicated in pharmacogenomics? | Yes. MicroRNAs have been implicated in pharmacogenomics. | MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles
by repressing translation or cleaving RNA transcripts. Although the number of
verified human miRNA is still expanding, only few have been functionally
described. However, emerging evidences suggest the potential involvement of
altered regulation of miRNA in pathogenesis of cancers and these genes are
thought to function as both tumours suppressor and oncogenes. In our study, we
examined by Real-Time PCR the expression of 156 mature miRNA in colorectal
cancer. The analysis by several bioinformatics algorithms of colorectal tumours
and adjacent non-neoplastic tissues from patients and colorectal cancer cell
lines allowed identifying a group of 13 miRNA whose expression is significantly
altered in this tumor. The most significantly deregulated miRNA being miR-31,
miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression
level of miR-31 was correlated with the stage of CRC tumor. Our results suggest
that miRNA expression profile could have relevance to the biological and
clinical behavior of colorectal neoplasia. Advances in the understanding of cancer cell biology and response to drug
treatment have benefited from new molecular technologies and methods for
integrating information from multiple sources. The NCI-60, a panel of 60 diverse
human cancer cell lines, has been used by the National Cancer Institute to
screen >100,000 chemical compounds and natural product extracts for anticancer
activity. The NCI-60 has also been profiled for mRNA and protein expression,
mutational status, chromosomal aberrations, and DNA copy number, generating an
unparalleled public resource for integrated chemogenomic studies. Recently,
microRNAs have been shown to target particular sets of mRNAs, thereby preventing
translation or accelerating mRNA turnover. To complement the existing NCI-60
data sets, we have measured expression levels of microRNAs in the NCI-60 and
incorporated the resulting data into the CellMiner program package for
integrative analysis. Cell line groupings based on microRNA expression were
generally consistent with tissue type and with cell line clustering based on
mRNA expression. However, mRNA expression seemed to be somewhat more informative
for discriminating among tissue types than was microRNA expression. In addition,
we found that there does not seem to be a significant correlation between
microRNA expression patterns and those of known target transcripts. Comparison
of microRNA expression patterns and compound potency patterns showed significant
correlations, suggesting that microRNAs may play a role in chemoresistance.
Combined with gene expression and other biological data using multivariate
analysis, microRNA expression profiles may provide a critical link for
understanding mechanisms involved in chemosensitivity and chemoresistance. MicroRNAs are non-coding RNAs that can block mRNA translation and influence mRNA
stability. Recent evidence indicates that miRNA variations can affect drug
resistance, efficacy, and metabolism, opening new avenues of pharmacogenomics
research. We investigated associations between polymorphisms in both
miRNA-containing genomic regions (primary and precursor miRNA) and in genes
related to miRNA biogenesis with clinical outcome in metastatic colorectal
cancer (mCRC) patients treated with 5-fluorouracil and irinotecan (CPT-11).
Eighteen single-nucleotide polymorphisms (SNPs) were analyzed in 61 patients. A
significant association with tumor response and time to progression (TTP) was
found for SNP rs7372209 in pri-miR26a-1 (P=0.041 and P=0.017, respectively). The
genotypes CC and CT were favorable when compared with the TT variant genotype.
In addition, SNP rs1834306, located in the pri-miR-100 gene, significantly
correlated with a longer TTP (P=0.04). In the miRNA-biogenesis pathway, a trend
was identified between SNP rs11077 in the exportin-5 gene and disease control
rate (P=0.076). This study is the first to suggest a relationship between
treatment outcome and SNPs in the miRNA-biogenesis machinery, in both primary
and precursor miRNAs. Our results suggest that miRNA polymorphic variants might
be useful predictors of clinical outcome in mCRC patients treated with
5-fluorouracil and CPT-11 combination. AIMS: Polymorphisms of genes involved in the pharmacokinetic and pharmacodynamic
processes underlie the divergent drug responses among individuals. Despite some
successes in identifying these polymorphisms, the candidate gene approach
suffers from insufficient gene coverage whereas the genome-wide association
approach is limited by less than ideal coverage of SNPs in some important genes.
To expand the potential of the candidate approach, we aim to delineate a
comprehensive network of drug-response genes for in-depth genetic studies.
MATERIALS & METHODS: Pharmacologically important genes were extracted from
various sources including literatures and web resources. These genes, along with
their homologs and regulatory miRNAs, were organized based on their
pharmacological functions and weighted by literature evidence and confidence
levels. Their coverage was evaluated by analyzing three commercial SNP chips
commonly used for genome-wide association studies: Affymetrix SNP array 6.0,
Illumina HumanHap1M and Illumina Omni.
RESULTS: A panel of drug-response genes was constructed, which contains 923
pharmacokinetic genes, 703 pharmacodynamic genes and 720 miRNAs. There are only
16.7% of these genes whose all known SNPs can be directly or indirectly (r(2) >
0.8) captured by the SNP chips with coverage of more than 80%. This is possibly
because these SNPs chips have notably poor performance over rare SNPs and miRNA
genes.
CONCLUSION: We have compiled a panel of candidate genes that may be
pharmacologically important. Using this knowledgebase, we are able to
systematically evaluate genes and their variants that govern an individual's
response to a given pharmaceutical therapy. This approach can serve as a
necessary complement to genome-wide associations. Pharmacogenomics, toxicogenomics, and small RNA expression analysis are three of
the most active research topics in the biological, biomedical, pharmaceutical,
and toxicological fields. All of these studies are based on gene expression
analysis, which requires reference genes to reduce the variations derived from
different amounts of starting materials and different efficiencies of RNA
extraction and cDNA synthesis. Thus, accurate normalization to one or several
constitutively expressed reference genes is a prerequisite to valid gene
expression studies. Although selection of reliable reference genes has been
conducted in previous studies in several animals and plants, no research has
been focused on pharmacological targets, and very few studies have had a
toxicological context. More interestingly, no studies have been performed to
identify reference genes for small RNA analysis although small RNA, particularly
microRNA (miRNA)-related research is currently one of the fastest-moving topics.
In this study, using MCF-7 breast cancer cells as a model, we employed
quantitative real-time PCR (qRT-PCR), one of the most reliable methods for gene
expression analysis in many research fields, to evaluate and to determine the
most reliable reference genes for pharmacogenomics and toxicogenomics studies as
well as for small RNA expression analysis. We tested the transcriptional
expression of five protein-coding genes as well as five non-coding genes in
MCF-7 cells treated with five different pharmaceuticals or toxicants [paclitaxel
(PTX), gossypol (GOS), methyl jasmonate (JAS), L-nicotine (NIC), and melamine
(mela)] and analyzed the stability of the selected reference genes by four
different methods: geNorm, NormFinder, BestKeeper, and the comparative ΔCt
method. According to our analysis, a protein-coding gene, hTBCA and four
non-coding genes, hRNU44, hRNU48, hU6, and hRNU47, appear to be the most
reliable reference genes for the five chemical treatments. Similar results were
also obtained in dose-response and time-course assays with gossypol (GOS)
treatment. Our results demonstrated that traditionally used reference genes,
such as 18s RNA, β-actin, and GAPDH, are not reliable reference genes for
pharmacogenomics and toxicogenomics studies. In contrast, hTBCA and small RNAs
are more stable during drug treatment, and they are better reference genes for
pharmacogenomics and toxicogenomics studies. To widely use these genes as
reference genes, these results should be corroborated by studies with other
human cell lines and additional drugs classes and hormonal treatments. OBJECTIVE: The aim of pharmacogenomics is to identify individual differences in
genome and transcriptome composition and their effect on drug efficacy.
MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression
of the majority of animal genes, including many genes involved in drug efficacy.
Consequently, differences in the miRNA expression among individuals could be an
important factor contributing to differential drug response. Pharmacogenomics
genes can be divided into drug target genes termed as pharmacodynamics genes
(PD) and genes involved in drug transport and metabolism termed as
pharmacokinetics genes (PK). To clarify the regulatory potential of miRNAs in
pharmacogenomics, we have examined the potential regulation by miRNAs of PK and
PD genes.
METHODS: We identified PK and PD genes as annotated by the Pharmacogenomics
Knowledge Base and examined miRNA targeting of genes in the two groups according
to several miRNA target prediction databases. We furthermore studied how
differences between the two groups are reflected in the gene structure and
across gene families. Lastly, we studied changes in expression levels of PK
versus PD genes in cells depleted for miRNAs by shRNA-mediated knockdown of the
miRNA-processing enzyme Dicer.
RESULTS: Our analysis identify a striking difference in the level of miRNA
regulation between PK and PD genes, with the former having less than half
predicted conserved miRNA binding sites compared with the latter. Importantly,
this finding is reflected in a highly significant difference in the shift in
expression levels of PD versus PK genes after depletion of miRNAs.
CONCLUSION: Our study emphasizes an intrinsic difference between PK and PD genes
and helps clarify the role of miRNAs in pharmacogenomics. BACKGROUND: The function of microRNAs (miRNAs) depends on the binding of miRNAs
to their target sequences in the 3'UTR of messenger RNAs (mRNAs), which enhances
the degradation of mRNAs and consequently, represses their expression. Single
nucleotide polymorphisms (SNPs) in the miRNA target sequences may affect or
impair the binding of miRNAs. Studies have shown that SNPs in miRNA target sites
(miR-TS-SNPs) have a great influence on diverse biological functions, including
pharmacogenomics and disease susceptibilities in human.
METHODS: High-resolution melting (HRM) analysis was applied for investigating
the allele frequencies of 3 miR-TS-SNPs (PLA2G2A, IL-16, and NOD2) in acute
leukemia. We also compared the genotypes of acute lymphoblastic leukemia
patients at initial diagnosis and complete remission.
RESULTS: HRM analysis revealed 3 genotypes (both homozygous and heterozygous) in
the 3 miR-TS-SNPs. The allele frequencies of all 3 miR-TS-SNPs were similar in
normal individuals and patients with acute myelogenous leukemia. Most patients
with acute lymphoblastic leukemia had the same genotypes at initial diagnosis
and complete remission.
CONCLUSIONS: Large scale scanning of case-control studies for miR-TS-SNPs may
contribute to the investigation of their roles and pathogenesis mechanisms in
human diseases. Our study showed that HRM analysis can be an efficient tool for
studies of miR-TS-SNPs. Regulatory elements play an important role in the variability of individual
responses to drug treatment. This has been established through studies on three
classes of elements that regulate RNA and protein abundance: promoters,
enhancers and microRNAs. Each of these elements, and genetic variants within
them, are being characterized at an exponential pace by next-generation
sequencing (NGS) technologies. In this review, we outline examples of how each
class of element affects drug response via regulation of drug targets,
transporters and enzymes. We also discuss the impact of NGS technologies such as
chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing
(RNA-Seq), and the ramifications of new techniques such as high-throughput
chromosome capture (Hi-C), chromatin interaction analysis by paired-end tag
sequencing (ChIA-PET) and massively parallel reporter assays (MPRA). NGS
approaches are generating data faster than they can be analyzed, and new methods
will be required to prioritize laboratory results before they are ready for the
clinic. However, there is no doubt that these approaches will bring about a
systems-level understanding of the interplay between genetic variants and drug
response. An understanding of the importance of regulatory variants in
pharmacogenomics will facilitate the identification of responders versus
non-responders, the prevention of adverse effects and the optimization of
therapies for individual patients. MicroRNAs (miRNAs) are short regulatory RNAs that down-regulate gene expression.
They are essential for cell homeostasis and active in many disease states. A
major discovery is the ability of miRNAs to determine the efficacy of drugs,
which has given rise to the field of 'miRNA pharmacogenomics' through
'Pharmaco-miRs'. miRNAs play a significant role in pharmacogenomics by
down-regulating genes that are important for drug function. These interactions
can be described as triplet sets consisting of a miRNA, a target gene and a drug
associated with the gene. We have developed a web server which links miRNA
expression and drug function by combining data on miRNA targeting and
protein-drug interactions. miRNA targeting information derive from both
experimental data and computational predictions, and protein-drug interactions
are annotated by the Pharmacogenomics Knowledge base (PharmGKB). Pharmaco-miR's
input consists of miRNAs, genes and/or drug names and the output consists of
miRNA pharmacogenomic sets or a list of unique associated miRNAs, genes and
drugs. We have furthermore built a database, named Pharmaco-miR Verified Sets
(VerSe), which contains miRNA pharmacogenomic data manually curated from the
literature, can be searched and downloaded via Pharmaco-miR and informs on
trends and generalities published in the field. Overall, we present examples of
how Pharmaco-miR provides possible explanations for previously published
observations, including how the cisplatin and 5-fluorouracil resistance induced
by miR-148a may be caused by miR-148a targeting of the gene KIT. The information
is available at www.Pharmaco-miR.org. |
Which genes are known to be involved in Diamond-Blackfan anemia? | Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26, as well as in GATA1, in about 60-65 % of patients. | BACKGROUND: Diamond-Blackfan anemia is a rare, clinically heterogeneous,
congenital red cell aplasia: 40% of patients have congenital abnormalities.
Recent studies have shown that in western countries, the disease is associated
with heterozygous mutations in the ribosomal protein (RP) genes in about 50% of
patients. There have been no studies to determine the incidence of these
mutations in Asian patients with Diamond-Blackfan anemia.
DESIGN AND METHODS: We screened 49 Japanese patients with Diamond-Blackfan
anemia (45 probands) for mutations in the six known genes associated with
Diamond-Blackfan anemia: RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35A. RPS14 was
also examined due to its implied involvement in 5q- syndrome.
RESULTS: Mutations in RPS19, RPL5, RPL11 and RPS17 were identified in five,
four, two and one of the probands, respectively. In total, 12 (27%) of the
Japanese Diamond-Blackfan anemia patients had mutations in ribosomal protein
genes. No mutations were detected in RPS14, RPS24 or RPL35A. All patients with
RPS19 and RPL5 mutations had physical abnormalities. Remarkably, cleft palate
was seen in two patients with RPL5 mutations, and thumb anomalies were seen in
six patients with an RPS19 or RPL5 mutation. In contrast, a small-for-date
phenotype was seen in five patients without an RPL5 mutation.
CONCLUSIONS: We observed a slightly lower frequency of mutations in the
ribosomal protein genes in patients with Diamond-Blackfan anemia compared to the
frequency reported in western countries. Genotype-phenotype data suggest an
association between anomalies and RPS19 mutations, and a negative association
between small-for-date phenotype and RPL5 mutations. Diamond-Blackfan anemia (DBA) is a rare, pure red-cell aplasia that presents
during infancy. Approximately 40% of cases are associated with other congenital
defects, particularly malformations of the upper limb or craniofacial region.
Mutations in the gene coding for the ribosomal protein RPS19 have been
identified in 25% of patients with DBA, with resulting impairment of 18S rRNA
processing and 40S ribosomal subunit formation. Moreover, mutations in other
ribosomal protein coding genes account for about 25% of other DBA cases.
Recently, the analysis of mice from which the gene coding for the heme exporter
Feline Leukemia Virus subgroup C Receptor (FLVCR1) is deleted suggested that
this gene may be involved in the pathogenesis of DBA. FLVCR1-null mice show a
phenotype resembling that of DBA patients, including erythroid failure and
malformations. Interestingly, some DBA patients have disease linkage to
chromosome 1q31, where FLVCR1 is mapped. Moreover, it has been reported that
cells from DBA patients express alternatively spliced isoforms of FLVCR1 which
encode non-functional proteins. Herein, we review the known roles of RPS19 and
FLVCR1 in ribosome function and heme metabolism respectively, and discuss how
the deficiency of a ribosomal protein or of a heme exporter may result in the
same phenotype. Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome (five
to seven cases per million live births) characterized by an aregenerative,
usually macrocytic anemia with an absence or less than 5% of erythroid
precursors (erythroblastopenia) in an otherwise normal bone marrow. The platelet
and the white cell counts are usually normal but neutropenia, thrombopenia or
thrombocytosis have been noted at diagnosis. In 40 to 50% of DBA patients,
congenital abnormalities mostly in the cephalic area and in thumbs and upper
limbs have been described. Recent analysis did show a phenotype/genotype
correlation. Congenital erythroblastopenia of DBA is the first human disease
identified to result from defects in ribosomal biogenesis. The first ribosomal
gene involved in DBA, ribosomal protein (RP) gene S19 (RPS19 gene), was
identified in 1999. Subsequently, mutations in 12 other RP genes out of a total
of 78 RP genes have been identified in DBA. All RP gene mutations described to
date are heterozygous and domit inheritance has been documented in 40 to 45%
of affected individuals. As RP mutations are yet to be identified in
approximately 50% of DBA cases, it is likely that other yet to be identified
genes involved in ribosomal biogenesis or other pathways may be responsible for
DBA phenotype. Diamond-Blackfan anemia is an autosomal domit disease due to mutations in
nine ribosomal protein encoding genes. Because most mutations are loss of
function and detected by direct sequencing of coding exons, we reasoned that
part of the approximately 50% mutation negative patients may have carried a copy
number variant of ribosomal protein genes. As a proof of concept, we designed a
multiplex ligation-dependent probe amplification assay targeted to screen the
six genes that are most frequently mutated in Diamond-Blackfan anemia patients:
RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that
deletions represent approximately 20% of all mutations. The combination of
sequencing and multiplex ligation-dependent probe amplification analysis of
these six genes allows the genetic characterization of approximately 65% of
patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy. Diamond-Blackfan anemia (DBA) is a hypoplastic anemia characterized by impaired
production of red blood cells, with approximately half of all cases attributed
to ribosomal protein gene mutations. We performed exome sequencing on two
siblings who had no known pathogenic mutations for DBA and identified a mutation
in the gene encoding the hematopoietic transcription factor GATA1. This
mutation, which occurred at a splice site of the GATA1 gene, impaired production
of the full-length form of the protein. We further identified an additional
patient carrying a distinct mutation at the same splice site of the GATA1 gene.
These findings provide insight into the pathogenesis of DBA, showing that the
reduction in erythropoiesis associated with the disease can arise from causes
other than defects in ribosomal protein genes. These results also illustrate the
multifactorial role of GATA1 in human hematopoiesis. This study was aimed to explore the mutations of ribosomal protein (RP) genes in
patients with Diamond Blackfan anemia (DBA). Twenty-one cases of DBA admitted in
our hospital from Dec 2008 to Aug 2012 were screened by PCR for mutations in the
nine known genes associated with DBA: RPS19, RPS24, RPS17, RPL5, RPL11, RPS7,
RPL35a, RPS10 and RPS26. The results found that 8 patients (38.1%) with DBA had
mutations in the genes coding for ribosomal protein, in which RPS19 mutation was
identified in 3 patients, RPS24, RPS7, RPL5, RPL11 and RPL35A mutations were
identified respectively in 1 of the patient. No mutations were detected in
RPS17, RPS10 or RPS26 genes. Thumb anomalies were found in 2 patients with RPL11
or RPL5 mutation, and hypospadias was found in 1 patient with RPS19 mutation. It
is concluded that the mutation frequency of the genes coding for ribosomal
protein in the patients with DBA here is lower than that in western countries.
The hypospadias can be observed in some patients with RPS19 mutation and some
dactyl anomalies are associated with RPL11 and RPL5 mutations. Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that
usually presents during the first year of life. The main features of the disease
are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent
erythroid progenitors in the bone marrow. The patients also present with growth
retardation and craniofacial, upper limb, heart and urinary system congenital
malformations in ~30-50 % of cases. The disease has been associated with point
mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24,
RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about
60-65 % of patients. Here, we report a novel large deletion in RPL15, a gene not
previously implicated to be causative in DBA. Like RPL26, RPL15 presents the
distinctive feature of being required both for 60S subunit formation and for
efficient cleavage of the internal transcribed spacer 1. In addition, we
detected five deletions in RP genes in which mutations have been previously
shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17.
Pre-ribosomal RNA processing was affected in cells established from the patients
bearing these deletions, suggesting a possible molecular basis for their
pathological effect. These data identify RPL15 as a new gene involved in DBA and
further support the presence of large deletions in RP genes in DBA patients. |
Is nicotinamide effective for skin cancer prevention? | Yes, oral nicotinamide is safe and effective in reducing the rates of new nonmelanoma skin cancers and actinic keratoses in high-risk patients. | BACKGROUND: Ultraviolet (UV) radiation can profoundly suppress the cutaneous
immune system, thus enhancing carcinogenesis. Agents that prevent UV-induced
immunosuppression may thus reduce skin cancer. Nicotinamide (vitamin B3)
prevents UV-induced immunosuppression and carcinogenesis in mice, and
solar-simulated (ss) UV-induced immunosuppression in humans. Its effectiveness
against different UV wavebands and mechanism of action is as yet unknown.
OBJECTIVES: To determine the effects and mechanisms of topical nicotinamide on
UV-induced suppression of delayed type hypersensitivity (DTH) responses in
humans.
METHODS: Healthy Mantoux-positive volunteers in four randomised, double-blinded
studies were irradiated with solar-simulated (ss)UV (UVB + UVA) or narrowband
UVB (300 nm) or UVA (385 nm). Topical nicotinamide (0.2% or 5%) or its vehicle
were applied immediately after each irradiation. Mantoux testing was performed
at irradiated sites and adjacent unirradiated control sites 48 h after the first
irradiation and measured 72 h later. Immunosuppression was calculated as the
difference in Mantoux-induced erythema and induration at test sites compared to
control sites. Human keratinocyte cell cultures, with and without ssUV and
nicotinamide, were used for quantitative real-time reverse
transcriptase-polymerase chain reaction assessment of TP53 and enzymes that
regulate oxidative phosphorylation.
RESULTS: Nicotinamide cooperated with ssUV to increase enzymes involved in
cellular energy metabolism and p53, and significantly protected against
immunosuppression caused by UVB, longwave UVA and single and repeated ssUV
exposures.
CONCLUSIONS: Longwave UVA, which is poorly filtered by most sunscreens, was
highly immune suppressive even at doses equivalent to 20 min of sun exposure.
Nicotinamide, which protected against both UVB and UVA, is a promising agent for
skin cancer prevention. Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced
carcinogenesis in mice and from UV-induced immunosuppression in mice and humans.
Recent double-blinded randomized controlled Phase 2 studies in heavily
sun-damaged individuals have shown that oral nicotinamide significantly reduces
premaligt actinic keratoses, and may reduce new non-melanoma skin cancers.
Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an
essential coenzyme in adenosine triphosphate (ATP) production. Previously, we
showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes.
Energy-dependent DNA repair is a key determit of cellular survival after
exposure to DNA-damaging agents such as UV radiation. Hence, in this study we
investigated whether nicotinamide protection from cellular energy loss
influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and
exposed them to low-dose solar-simulated UV (ssUV). Excision repair was
quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased
both the proportion of cells undergoing excision repair and the repair rate in
each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers
(CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by
comet assay in keratinocytes and with immunohistochemistry in human skin.
Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to
be due to enhancement of DNA repair. These results show that nicotinamide
enhances two different pathways for repair of UV-induced photolesions,
supporting nicotinamide's potential as an inexpensive, convenient and non-toxic
agent for skin cancer chemoprevention. Cutaneous melanoma is a significant cause of morbidity and mortality.
Nicotinamide is a safe, widely available vitamin that reduces the immune
suppressive effects of UV, enhances DNA repair in keratinocytes and has shown
promise in the chemoprevention of non-melanoma skin cancer. Here, we report the
effect of nicotinamide on DNA damage and repair in primary human melanocytes.
Nicotinamide significantly enhanced the repair of oxidative DNA damage
(8-oxo-7,8-dihydro-2'-deoxyguanosine) and cyclobutane pyrimidine dimers induced
by UV exposure. It also enhanced the repair of
8-oxo-7,8-dihydro-2'-deoxyguanosine induced by the culture conditions in
unirradiated melanocytes. A significant increase in the percentage of
melanocytes undergoing unscheduled but not scheduled DNA synthesis was observed,
confirming that nicotinamide enhances DNA repair in human melanocytes. In
summary, nicotinamide, by enhancing DNA repair in melanocytes, is a potential
agent for the chemoprevention of cutaneous melanoma. Nicotinamide has shown potential as a safe and effective intervention for the
prevention of maligt and premaligt skin lesions. Recent studies have shown
that nicotinamide, in both oral and topical forms, is able to prevent
ultraviolet-induced immunosuppression in humans [1,2,3] and mice [4,5].
Immunosuppression is a known factor for the progression of premaligt lesions,
such as actinic keratosis [6]. Murine studies have shown that nicotinamide is
also able to protect against photocarcinogenesis [4,5]. Preliminary human
studies suggest that nicotinamide may help prevent skin cancers and enhance the
regression of actinic keratoses. BACKGROUND: Nonmelanoma skin cancers, such as basal-cell carcinoma and
squamous-cell carcinoma, are common cancers that are caused principally by
ultraviolet (UV) radiation. Nicotinamide (vitamin B3) has been shown to have
protective effects against damage caused by UV radiation and to reduce the rate
of new premaligt actinic keratoses.
METHODS: In this phase 3, double-blind, randomized, controlled trial, we
randomly assigned, in a 1:1 ratio, 386 participants who had had at least two
nonmelanoma skin cancers in the previous 5 years to receive 500 mg of
nicotinamide twice daily or placebo for 12 months. Participants were evaluated
by dermatologists at 3-month intervals for 18 months. The primary end point was
the number of new nonmelanoma skin cancers (i.e., basal-cell carcinomas plus
squamous-cell carcinomas) during the 12-month intervention period. Secondary end
points included the number of new squamous-cell carcinomas and basal-cell
carcinomas and the number of actinic keratoses during the 12-month intervention
period, the number of nonmelanoma skin cancers in the 6-month postintervention
period, and the safety of nicotinamide.
RESULTS: At 12 months, the rate of new nonmelanoma skin cancers was lower by 23%
(95% confidence interval [CI], 4 to 38) in the nicotinamide group than in the
placebo group (P=0.02). Similar differences were found between the nicotinamide
group and the placebo group with respect to new basal-cell carcinomas (20% [95%
CI, -6 to 39] lower rate with nicotinamide, P=0.12) and new squamous-cell
carcinomas (30% [95% CI, 0 to 51] lower rate, P=0.05). The number of actinic
keratoses was 11% lower in the nicotinamide group than in the placebo group at 3
months (P=0.01), 14% lower at 6 months (P<0.001), 20% lower at 9 months
(P<0.001), and 13% lower at 12 months (P=0.001). No noteworthy between-group
differences were found with respect to the number or types of adverse events
during the 12-month intervention period, and there was no evidence of benefit
after nicotinamide was discontinued.
CONCLUSIONS: Oral nicotinamide was safe and effective in reducing the rates of
new nonmelanoma skin cancers and actinic keratoses in high-risk patients.
(Funded by the National Health and Medical Research Council; ONTRAC Australian
New Zealand Clinical Trials Registry number, ACTRN12612000625875.). |
Which is the chromosomal location of the gene MAOA? | The MAOA gene is locatad on chromosome X (Xp21-p11). | An essentially full-length cDNA clone for the human enzyme monoamine oxidase
type A (MAO-A) has been used to determine the chromosomal location of a gene
encoding it. This enzyme is important in the degradative metabolism of biogenic
amines throughout the body and is located in the outer mitochondrial membrane of
many cell types. Southern blot analysis of PstI-digested human DNA revealed
multiple fragments that hybridized to this probe. Using rodent-human somatic
cell hybrids containing all or part of the human X chromosome, we have mapped
these fragments to the region Xp21-p11. A restriction fragment length
polymorphism (RFLP) for this MAOA gene was identified and used to evaluate
linkage distances between this locus and several other loci on Xp. The MAOA
locus lies between DXS14 and OTC, about 29 cM from the former. |
Do Parkinson's disease patients experience stridor? | Yes. Stridor has been described in Parkinon's disease patients. Stridor has been linked to deep brain stimulation and L-Dopa medication use.
However, stridor was shown to be more common in multiple system atrophy patients. | A patient is described with idiopathic Parkinson's disease and severe laryngeal
stridor. Other than urinary frequency and urgency, not uncommon in this
condition, and postoperative levodopa-sensitive postural hypotension, there were
no features of generalized autonomic failure. The laryngeal stridor responded to
levodopa therapy, and we are not aware that this has been reported previously. Striatonigral degeneration (SND) is difficult to diagnose in vivo. The purpose
of this study was to detect the best indicators for an early and reliable
diagnosis of the disease. Eighteen patients clinically diagnosed as having SND
were selected with rigorous inclusion criteria and compared to 18 patients with
Parkinson's disease (PD) matched for age and disease duration. Apart from
dysautonomia, the principal discrimit clinical features that distinguished
SND from PD were the early appearance of the following symptoms and signs: (a)
severe and atypical progressive parkinsonism characterized by bilateral
bradykinesia and rigidity, slowness of gait, postural instability, and falls,
and poor or absent response to adequate levodopa treatment; (b) increased tendon
reflexes associated or not with frank pyramidal signs, severe dysarthria, and
less consistently, dysphagia, stridor, antecollis, and stimulus-sensitive
myoclonus, which, when present, are highly suggestive of the disease. INTRODUCTION: Autonomic dysfunction (AD) can be a feature of both multiple
system atrophy (MSA) and idiopathic Parkinson's disease (IPD), conditions that
are frequently misdiagnosed in life. Most studies on AD in MSA and IPD are based
on clinical cases without pathological verification.
MATERIAL AND METHODS: We retrospectively analysed AD in 135 pathologically
confirmed cases of IPD and in 33 of MSA from the UK PD Society Brain Bank.
RESULTS: MSA started at a younger age than IPD (54.4 +/- 10.7 yrs versus 60.6
+/- 10.8 yrs), and AD began earlier in the course of the illness All MSA
patients had some degree of AD in life whereas AD was absent in 24% of IPD
patients. Although each of five autonomic domains was affected in variable
numbers of IPD patients, AD in MSA generally involved more autonomic domains
than in IPD, and to a more severe degree, in particular with regard to
inspiratory stridor.
CONCLUSIONS: These results indicate that the presence of autonomic disturbance
alone does not distinguish between MSA and IPD in individual cases. However, the
presence of severe AD, of AD preceding parkinsonism, or of inspiratory stridor,
are all individually suggestive of MSA. We report a 60-year-old woman with juvenile Parkinson disease (PD) with vocal
cord abductor paralysis (VCAP). She had suffered from juvenile PD for 30 years.
She was admitted in February 1998 to our clinical unit, because of maligt
syndrome induced by dehydration. Neurological examination revealed disturbance
of consciousness, hand tremor, dyskinesia of the trunk and all extremities, and
rigidity. Laboratory examinations disclosed leukocytosis, renal dysfunction,
hypermyoglobinemia, and elevation of the serum creatine kinase. Six days after
admission, dyspnea and inspiratory stridor were noted, and the respiratory
distress worsened. Endoscopy of the upper airways revealed that the vocal cord
was in the midline or paramedian position. There are some cases of PD with VCAP,
but such a case is very rare in Japan. We discussed the pathogenic mechanisms of
these conditions, and speculated that VCAP was associated with maligt
syndrome in our case. A 69-year-old woman suffering from Parkinson's disease for 22 years was admitted
because of frequent occurrence of paroxysmal dyspnea for 3 months. Her dyspneic
attacks consisting of inspiratory stridor and cyanosis occurred mainly during
the wearing-off time and continued for less than 30 min. During nonictal period
her respiration and phonation were normal and endoscopic investigation of the
vocal cord and upper respiratory tract revealed no abnormality. Based on these
findings, she was diagnosed to have focal laryngeal dystonia. The 24-hr
monitoring with pulseoxymeter recorded frequent occurrence of paroxysmal
asymptomatic hopoxemia during both daytime and sleep. With the treatment of
tracheostomy and the reduction and alteration of anti-Parkinsonian drugs,
dyspneic attacks disappeared gradually. We also confirmed the complete
disappearance of paroxysmal asymptomatic hopoxemia with the 24-hr monitoring by
pulseoxymeter, which is considered to be a useful method for early detection of
asymptomatic focal laryngeal dystonia. We report a case of involuntary phonation caused by abnormal vocal cord
movements during expiration in a patient with Parkinson's disease. A 60-year-old
woman had been treated for parkinsonism at the outpatient clinic of the
Department of Neurology since August 1999. She began to groan involuntarily in
the daytime in September 2001. She could not eat well while groaning. Stridor
was not noted during sleep at night. Endoscopic examination of the larynx
revealed insufficient abduction of the bilateral vocal cords, although the
glottis was not so small as to cause stridor during inspiration. During
expiration, however, the vocal cords adducted, resulting in the involuntary
production of voice. Electromyography showed an increase in the activity of the
thyroarytenoid and lateral cricoarytenoid muscles. This muscle activity was
further enhanced during inspiration. The involuntary phonation disappeared when
the patient's dose of L-dopa was decreased, although she had a decrease in her
systemic mobility as well. When the dose of L-dopa was increased to the
therapeutic level, involuntary phonation recurred, and her voluntary systemic
activity improved. In the present case, it was considered that excessive
dopaminergic denervation occurred in the nerve innervating the laryngeal
adductors. Involuntary voice appeared to be produced by hypertonus of the
laryngeal adductors because of a lowering in the threshold level for L-dopa,
even though the drug was administered at the usual dose. INTRODUCTION: The aim of this study is to show if the exploration of the
autonomic nervous system is useful to improve the specificity of clinical
criteria of Parkinson's Disease (PD) and Multiple System Atrophy (MSA).
PATIENTS AND METHODS: 20 patients with PD and 13 patients with MSA were studied.
After 12 hours in off medication, NE and GH were measured in supine position and
NE after 5 minutes standing. Later, GH levels were recorded at 15, 30, 45 and 60
minutes after a dose of 0.005 mg/kg of apomorphine. Finally, analysis of the
symptoms of autonomic dysfunction and levodopa test were carried out.
RESULTS: Sympathetic response to postural changes was significantly higher in
patients with PD (NE increase in relation to basal: PD: 170.90 +/- 110.08 pg/ml;
MSA: 91.33 +/- 73.79 pg/ml; p = 0.029). No differences were found in the
response of GH to apomorphine (GH peak at 45 minutes: PD: 2.37 +/- 2.7 ng/ml;
MSA: 1.69 +/- 1.90 ng/ml; ns). The symptoms of autonomic dysfunction were more
frequently in patients with MSA. The stridor was specific to MSA. Improvement in
motor scores in the levodopa test was higher in patients with PD (PD: 39.7 %;
MSA: 17.89; p = 0.019).
DISCUSSION: Sympathetic response to postural changes, description of symptoms of
autonomic dysfunction, and motor response to levodopa test are useful tools in
order to improve specificity of the diagnostic criteria of PD and MSA. The GH
test with apomorphine was not useful for a differential diagnosis. Multiple system atrophy (MSA) is a neurodegenerative disorder that usually
presents clinically as a combination of parkinsonism, cerebellar syndrome and
autonomic failure. Patients with MSA can present other clinical features, such
as inspiratory stridor and rapid eye movement (REM) sleep behaviour disorder
(RBD). We report a patient with pathologically confirmed MSA who presented with
a longstanding history of stridor, RBD and autonomic disturbances but did not
develop overt parkinsonism or cerebellar signs. This case illustrates that MSA
may present clinically without its cardinal motor symptoms, and that stridor and
RBD may be clues to recognise the disease in a patient with autonomic failure. OBJECTIVES: (1) To present a rare case of stridor secondary to prolonged
laryngospasm in a patient with Parkinson's disease, and (2) to review the
literature on stridor in Parkinson's disease.
METHODS: We report a 73-year-old Parkinson's disease patient who developed acute
stridor due to prolonged laryngospasm triggered by overspill of excessive
secretions. The literature was reviewed, following a Medline search using the
keywords 'Parkinson's disease' and 'stridor' or 'airway obstruction' or
'laryngospasm' or 'laryngeal dystonia' or 'bilateral vocal cord palsy'.
RESULT: Only 12 previously reported cases of stridor in Parkinson's disease
patients were identified. Causes included bilateral vocal fold palsy (eight
cases), laryngospasm (five), and dystonia of the jaw and neck muscles (two). The
mechanism of laryngospasm in our patient was similar to 'dry drowning', and has
not previously been described.
CONCLUSION: Laryngospasm can be triggered in Parkinson's disease by excessive
secretions entering the larynx. The mechanism is similar to 'dry drowning'.
Treatment focuses on reducing secretions. The use of botulinum toxin to reduce
spasm is inappropriate in this situation. This case emphasises the importance of
recognising different causes of stridor in Parkinson's disease patients, as this
affects management. INTRODUCTION: In addition to the classic triad (tremor, rigidity and akinesia),
Parkinson's disease (PD) is also accompanied by several non-motor disorders.
AIM: To carry out an updated review of these non-motor symptoms in terms of
their pathophysiology, epidemiology, clinical features and treatment.
DEVELOPMENT: Autonomic disorders such as seborrhoeic dermatitis and disorders
involving sweating, fatigue, weight loss or respiratory problems (dyspnea,
inspiratory stridor) are highly prevalent and very disabling symptoms. In
addition, they may be the main problem in a particular phase of PD (fatigue,
stridor) and condition the quality of life of patients with Parkinson. They are
often neglected and remain undetected. Although they may respond to dopaminergic
agents, they usually require a different therapeutic approach. Studies are
needed to evaluate new therapeutic perspectives that act against the
pathophysiological mechanisms of these disorders.
CONCLUSIONS: PD affects systems other than the dopaminergic nigrostriatal. A
better understanding of this pathology will make it possible to approach and
optimise the treatment of the conditioning symptoms, thereby enhancing the
welfare of patients with PD. Refractory symptoms in Parkinson disease show good response to deep brain
stimulation (DBS). This procedure improves United Parkinson's Disease Rating
Scale scores and reduces dyskinesias, whereas speech and swallowing dysfunction
typically do not improve and may even worsen. Rarely, DBS can cause
idiosyncratic dystonias of muscle groups, including those of the neck and
throat. The authors describe a patient experiencing stridor and dysphagia with
confirmed pulmonary restriction and aspiration following subthalamic nucleus
deep brain stimulator adjustment, with a resolution of symptoms and signs when
the stimulator was switched off. |
Which depression rating scales were shown to have acceptable psychometric properties for screening of poststroke depression? | The Center of Epidemiological Studies-Depression Scale, Hamilton Depression Rating Scale, and the Patient Health Questionnaire, The Beck Depression Inventory, Hospital Anxiety and Depression Scale-depression subscale, Montgomery-Asberg Depression Rating Scale, Poststroke Depression Rating Scale and Clinical Global Impression scale appeared to have acceptable psychometric properties for identifying depression in patients after stroke. Therefore, these scales can be used for screening of post-stroke depression. Signs of Depression Scale and Visual Analogue Mood Scale showed inferior psychometric properties and therefore should not be used for screening for post-stroke depression. | OBJECTIVE: To determine which screening instrument is most suitable for
detecting depression in Chinese stroke patients.
METHOD: A random sample of 60 Chinese subjects recruited from consecutively
admitted patients with first-ever stroke to a rehabilitation facility were
assessed using the Visual Analogue Mood Scale (VAMS), the Hospital Anxiety and
Depression Scale (HADS), and the Geriatric Depression Scale (GDS). Psychiatric
diagnoses, which served as a benchmark for the comparison of screening
instruments, were made using the SCID-DSM-III-R. Rating instruments were
compared with respect to response rate, sensitivity, specificity, positive and
negative predictive values, and receiver operating characteristic (ROC) curves.
RESULTS: The VAMS was not useful in screening for depression in Chinese stroke
patients while both the HADS and the GDS demonstrated satisfactory accuracy in
detecting depression in Chinese stroke patients.
CONCLUSIONS: Screening instruments for poststroke depression are
culture-specific and even scales well established in other cultures should be
tested before being used in clinical practice and research. AIM: This paper is a report of a study to determine the accuracy and utility of
an observational screening tool (Signs of Depression Scale), when rated by
nurses and carers for detecting depression in patients who have recently had a
stroke.
BACKGROUND: Depression following a stroke is common and adversely affects
recovery and rehabilitation. Identifying depression when patients have
communication and cognitive difficulties is especially problematic. Screening
tools which rely solely on observation may be beneficial in this group.
METHOD: A cross-sectional study of people admitted with an acute stroke compared
a clinical diagnosis of depression by a psychiatrist (the gold standard) with
the Signs of Depression Scale completed by nurses and carers. The agreement
between nurses' and carers' ratings was also explored. Data were collected over
10 months (December 2004-October 2005).
FINDINGS: Seventy-one patients were included in the study, median age 70
[inter-quartile ranges (IQR) 59-76], including 40 (56.3%) males. The
psychiatrist classified 25/71 (35.2%) patients as depressed. Using the
recommended cut-point of 2 or more on the Signs of Depression Scale, the nurse
and carer respectively rated 27/71 (38.0%) and 18/30 (60.0%) patients as
potentially depressed. The proportion of patients correctly identified as
depressed by the test (sensitivity) when rated by nurses was 64%, and the
proportion of patients not depressed who were correctly identified by the test
(specificity) was 61%, whereas carers achieved sensitivity 90% and specificity
35%. The optimal cut-point for carers was higher at 4 or more. Inter-rater
agreement on the Signs of Depression Scale between nurses and carers was fair
(ICC = 0.43, 95% CI: 0.09-0.68).
CONCLUSION: The Signs of Depression Scale is easily completed by clinical staff,
although we found the sensitivity when completed by nurses to be low.
Information from carers shows potential to improve screening and it is important
for nurses to value the knowledge and skills of carers in detecting depression
following a stroke. Further refinement of the Signs of Depression Scale, with
accompanying research, is required. BACKGROUND: Poststroke depression (PSD) is a common and invalidating condition,
requiring a prompt diagnosis to provide appropriate treatment. The diagnosis of
PSD is based on clinical evaluation, supported by psychiatric scales,
predomitly constructed to assess 'functional' depressive disorders, which can
miss important clinical information about PSD. This study evaluated the
diagnostic accuracy of the Poststroke Depression Rating Scale (PSDRS), a
diagnostic tool specifically devised to assess depression after stroke, in
comparison to the Hamilton Depression Rating Scale (Ham-D).
METHODS: 143 patients were enrolled at their first-ever stroke; 46 subjects
received a diagnosis of major depression-like disorder (MDL) and 53 a diagnosis
of mood disorder with depressive manifestations (MDDM). Each patient underwent
the PSDRS, Ham-D and Mini Mental State Examination. Areas under
receiver-operating characteristic curves were calculated to evaluate diagnostic
accuracy.
RESULTS: At their optimum cut-off points, the Ham-D and PSDRS showed good
sensitivity and specificity for MDL or MDL + MDDM; the PSDRS had a higher
positive predictive value for MDL in respect of the Ham-D (78 vs. 59%).
Furthermore, the diagnostic accuracy of the PSDRS was higher in respect of the
Ham-D in aphasic patients. Regression analyses showed that a longer latency from
stroke onset predicted misdiagnosis on both PSDRS and Ham-D; the Ham-D was
significantly influenced by cognitive impairment while the PSDRS was affected by
age.
CONCLUSIONS: The Ham-D and PSDRS are both reliable diagnostic tools for the
diagnosis of PSD. The PSDRS showed a slightly superior positive predictive value
for MDL, particularly in aphasic patients, and was not influenced by cognitive
dysfunction, a common consequence of stroke affecting the diagnostic performance
of the Ham-D. We suggest that the PSDRS could be a useful tool in clinical
practice and in therapeutic trials. BACKGROUND AND PURPOSE: Assessing poststroke depression may be complicated by
aphasia, other cognitive deficits, and several somatic stroke-related symptoms.
We studied the possible differences in performance of some commonly used
instruments in screening depression after stroke.
METHODS: We compared the Beck Depression Inventory, Hamilton Rating Scale for
Depression, Visual Analogue Mood Scale, proxy assessment, and Clinical Global
Impression of the nursing and study personnel, together with Diagnostic and
Statistical Manual of Mental Disorders, 3rd Edition, Revised diagnosis, in
assessing depression after stroke in a follow-up study of 100 patients. The
patients were studied at 2 weeks and at 2, 6, 12, and 18 months after stroke.
RESULTS: The feasibility rates of all assessment instruments studied were fairly
similar, but the prevalence rates differed according to the assessment
instruments, varying from the lowest rates with a Diagnostic and Statistical
Manual of Mental Disorders, 3rd Edition, Revised-based diagnosis up to 3-fold
with caregiver ratings. The sensitivity and specificity against the Diagnostic
and Statistical Manual of Mental Disorders criteria were acceptable with the
Clinical Global Impression, Beck Depression Inventory, and Hamilton Rating Scale
for Depression, mostly in the range of 0.70 to 1.00. The caregiver ratings were
higher than the patient ratings (P<0.001) and correlated with the caregiver's
own Beck Depression Inventory (0.60 to 0.61, P<0.001). The Visual Analogue Mood
Scale was not a sensitive instrument (sensitivity, 0.20 to 0.60) and did not
correlate with the Beck Depression Inventory during the first year after stroke.
CONCLUSIONS: Beck Depression Inventory, Hamilton Rating Scale for Depression,
and Clinical Global Impression assessment by professionals, in addition to the
Diagnostic and Statistical Manual of Mental Disorders, 3rd Edition, Revised
diagnosis, are useful in assessing depression, but none of these instruments
clearly stood apart from the others. Proxy ratings should be used with caution,
and the use of the Visual Analogue Mood Scale among patients with aphasia and
other cognitive impairments cannot be recommended. BACKGROUND: Major depression is common in stroke patients and associated with
increased rates of disability and mortality. Identifying depression may improve
mental and physical health. The aim of this review was to determine the most
accurate tool for detecting poststroke depression.
METHODS: Seven databases were searched up to November 2012. Two authors selected
studies using International Classification of Disease or Diagnostic and
Statistical Manual diagnosis of depression as the reference standard. Two
authors extracted data and assessed methodological quality. Included studies
were synthesised using meta-analyses.
RESULTS: A total of 24 included studies provided data on 2907 participants. The
Center of Epidemiological Studies-Depression Scale (CESD) (sensitivity: 0.75;
95% CI 0.60 to 0.85; specificity: 0.88; 95% CI 0.71 to 0.95), the Hamilton
Depression Rating Scale (HDRS) (sensitivity: 0.84; 95% CI 0.75 to 0.90;
specificity:0.83; 95% CI 0.72 to 0.90) and the Patient Health Questionnaire
(PHQ)-9 (sensitivity: 0.86; 95% CI 0.70 to 0.94; specificity: 0.79; 95% CI 0.60
to 0.90) appeared to be the optimal measures for screening measures. However,
the clinical utility of all tools was modest for case-finding.
INTERPRETATION: There are a number of possible instruments that may help in
screening for poststroke depression but none are satisfactory for case-finding.
Preliminary data suggests the CESD, HDRS or the PHQ-9 as the most promising
options. Although it should be noted such scales should not be used in isolation
but followed up with a more detailed clinical assessment. While there is
promising data for the PHQ-2 in other populations, it performed less well than
other measures. |
What is the mechanism of action of trichostatin A (TSA) as an antitumoral agent? | Trichostatin A (TSA) exerts antitumoral activity as a histone deacetylase inhibitor | The potential anticancer activities of histone deacetylase (HDAC) inhibitors and
DNA methyltransferase (DNMT) inhibitors have been extensively studied in recent
years. HDAC inhibitors suppress the activities of multiple HDACs, leading to an
increase in histone acetylation. This histone acetylation induces an enhancement
of the expression of specific genes that elicit extensive cellular morphologic
and metabolic changes, such as growth arrest, differentiation and apoptosis.
DNMT inhibitors, such as 5-aza-cytidine (5-aza-CR) and 5-aza-2'-deoxycytidine
(5-aza-CdR) are also widely studied because DNA hypomethylation induces the
re-activation of tumor suppressor genes that are silenced by
methylation-mediated mechanisms. Recently, the combination of HDAC inhibitors or
demethylating agents with other chemo-therapeutics has gained increasing
interest as a possible molecularly targeted therapeutic strategy. In particular,
the combination of HDAC inhibitors with demethylating agents has become
attractive since histones are connected to DNA by both physical and functional
interactions. To date, the accumulating evidence has confirmed the hypothesis
that the combination of HDAC and DNMT inhibition is very effective (and
synergistic) in inducing apoptosis, differentiation and/or cell growth arrest in
human lung, breast, thoracic, leukemia and colon cancer cell lines. This review
will discuss the in vitro effects of HDAC inhibitors, such as trichostatin A
(TSA), sodium butyrate, depsipeptide (FR901228, FK228), valproic acid (VPA) and
suberoylanilide hydroxamic acid (SAHA), and the demethylating agent, 5-aza-CdR
used alone and in combination treatment of human cancer cells and the possible
mechanisms involved. Histone deacetylase inhibitors (HDACis) are emerging as a promising new class of
anticancer agents displaying growth-inhibitory activity and low toxicity in
vivo. In this study, we examined the effect of sodium butyrate (NaB) and
trichostatin A (TSA) on the growth of human bladder carcinoma cell lines in
culture and TSA on the growth of EJ and UM-UC-3 human bladder xenografts in nude
mice. NaB and TSA suppressed the growth of bladder cell lines at millimolar
(1.5-4.3 mM) and micromolar (0.03-0.33 microM) concentrations, respectively,
inducing concentration-dependent cell death. Bladder carcinoma cells within the
experimental panel displayed the phenotype of late-stage bladder lesions
expressing N-cadherin in the absence of E-cadherin accompanied by low levels of
plakoglobin expression. Exposure of these cells to HDACis resulted in
upregulation of plakoglobin with no change in E-cadherin expression. A 2-hr
exposure to TSA was the minimal time required to upregulate plakoglobin in cells
with downregulation to baseline levels occurring within 24 hr following drug
removal. In mice bearing EJ and UM-UC-3 bladder xenografts, TSA (500
microg/kg/day) caused suppression of tumor growth compared with mice receiving
vehicle alone. A > 70% reduction in mean final tumor volume was recorded in both
bladder xenograft models with no detectable toxicity. The results suggest that
TSA inhibits bladder carcinoma cell growth and may be a useful, relatively
nontoxic agent for consideration in the treatment of late-stage bladder tumors. Epigenetic alterations of the histone acetylation play an important role in the
regulation of gene expression associated with cell cycles and apoptosis that may
affect the chemosensitivity of gastric carcinomas. Recently, a histone
deacetylase inhibitor, trichostatin A (TSA), was proven to be a chemo-sensitizer
on human erythroleukemia cells. With the aim of improving the chemotherapeutic
efficacy of gastric carcinoma, the effect of TSA on the chemosensitivity of
several anticancer drugs in gastric carcinoma cells was investigated. Human
gastric cancer cell lines, OCUM-8 and MKN-74, and 5 anticancer drugs,
5-fluorouracil (5-FU), paclitaxel (PTX), oxaliplatin (OXA), irinotecan (SN38)
and gemcitabine (GEM) were used. In both gastric cancer cell lines, a
synergistic anti-proliferative effect by the combination of TSA (30 ng/ml) with
5-FU, PTX or SN38 showed a synergistic anti-proliferative effect in OCUM-8 and
MKN-74 cells. TSA increases the expression of p21, p53, DAPK-1 and the DAPK-2
gene in both OCUM-8 and MKN-74 cells. In conclusion, TSA is a promising
chemotherapeutical agent in combination with anticancer drugs of 5-FU, PTX and
SN38 in gastric cancer cell lines. The up-regulation of p53, p21, DAPK-1 and
DAPK-2 might be associated with the synergistic effect of TSA. The use of histone deacetylase (HDAC) inhibitors has shown promise for a variety
of maligcies. In this investigation, we define the activity of this class of
inhibitors in combination with traditional cytotoxic chemotherapy in endometrial
cancer cells. Significant reductions in growth were observed in Ark2 and KLE
endometrial cancer cells following treatment with paclitaxel, doxorubicin,
carboplatin, or the HDAC inhibitor trichostatin A (TSA). However, only combined
treatment with TSA/paclitaxel caused synergistic inhibition of cell growth. This
combination also resulted in significant changes in cell morphology. Using cell
cycle analysis, nuclear staining, and Western blot analysis for poly(ADP-ribose)
polymerase and caspase-9 degradation products, TSA/paclitaxel showed the most
dramatic activation of the apoptotic cascade. These effects were also observed
when the HDAC inhibitors HDAC inhibitor-1 or oxamflatin were substituted for
TSA. The anticancer properties of paclitaxel are known to result in part from
inhibition of microtubule depolymerization, which results in apoptosis. We show
that TSA administration also stabilizes microtubules via alpha-tubulin
acetylation. Furthermore, using Western blot and immunohistochemical analysis,
treatment with TSA/paclitaxel led to a significant increase in acetylated
tubulin and microtubule stabilization. These effects were confirmed in a mouse
xenograft model. Moreover, TSA/paclitaxel resulted in a 50% reduction in tumor
weight compared with either agent alone. This study provides in vivo evidence of
nonhistone protein acetylation as one possible mechanism by which HDAC
inhibitors reduce cancer growth. The TSA/paclitaxel combination seems to hold
promise for the treatment of serous endometrial carcinoma and other maligcies
with limited sensitivity to paclitaxel. Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with
trichostatin A (TSA) as the reference compound, are potential antitumoral drugs
and show promise in the creation of long-term primary cell cultures. However,
their metabolic properties have barely been investigated. TSA is rapidly
inactivated in rodents both in vitro and in vivo. We previously found that
5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH
(compound 1) is metabolically more stable upon incubation with rat hepatocyte
suspensions. In this study, we show that human hepatocytes also metabolize TSA
more rapidly than compound 1 and that similar pathways are involved.
Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to
have the same favorable metabolic properties. Removal of the dimethylamino
substituent of compound 1 creates a very stable but 50% less potent inhibitor.
Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and
metabolic stability, favoring HA reduction to hydrolysis. On the other hand,
Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but
also increase the rate of metabolic inactivation approximately 2-fold, mainly
through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1
is shown to be extensively metabolized by phase II conjugation. In conclusion,
this study suggests that simple structural modifications of amide-linked TSA
analogs can improve their phase I metabolic stability in both rat and human
hepatocyte suspensions. Phase II glucuronidation, however, can compensate for
their lower phase I metabolism in rat hepatocyte monolayers and could play a yet
unidentified role in the determination of their in vivo clearance. Oncolytic herpes simplex viruses (HSVs) possess direct oncolytic and
antiangiogenic activities and are promising anticancer agents, but their
efficacy, when used as single agents, leaves room for improvement. We
investigated whether combination therapy of HSV with histone deacetylase
inhibitor trichostatin A (TSA), an agent that also targets cancer cells and
tumor vasculature, would result in enhanced efficacy. In vitro, TSA and G47Delta
showed strong synergy of action against proliferating endothelial cells, varying
degrees of synergistic action against most cancer cell lines, but no effect in
quiescent, normal endothelial and prostate epithelial cells. Synergy is
dependent on viral replication; however, it is not dependent on the dosing
sequence of TSA and G47Delta, viral genetic alterations, infectivity, or
replication kinetics of G47Delta. Using an isogenic cell system, we found that a
high level of cellular cyclin D1 is also critically important for the
interaction. Normal cells with low cyclin D1 levels were not subjected to
toxicity by either agent. In tumor cells and proliferating endothelial cells,
the combination treatment enhanced the inhibition of cyclin D1 and vascular
endothelial growth factor (VEGF). Concurrent systemic TSA and intratumoral
G47Delta administration resulted in enhanced antiangiogenesis and enhanced
antitumoral efficacy in animal models. Therefore, combination treatment with TSA
and oncolytic HSV provides a novel approach to cancer therapy. OBJECTIVES: Valproic acid (VPA) and trichostatin A (TSA) exert antitumor
activity as histone deacetylase inhibitors, whereas ellipticine action is based
mainly on DNA intercalation, inhibition of topoisomerase II and formation of
cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the
first report on the molecular mechanism of combined treatment of human
neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds.
METHODS: HPLC with UV detection was employed for the separation and
characterization of ellipticine metabolites formed by microsomes and
peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells
and in incubations with microsomes and peroxidases were detected by
32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was
examined by Western blot.
RESULTS: The cytotoxicity of ellipticine to neuroblastomas was increased by
pre-treating these cells with VPA or TSA. A higher sensitivity of cells to
ellipticine correlated with an increase in formation of covalent
ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of
this finding, we investigated the modulation by VPA and TSA of CYP- and
peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The
effects of ellipticine in the presence of VPA and TSA on expression of CYPs and
peroxidases relevant for ellipticine activation and levels of cytochrome b5 and
P-glycoprotein in neuroblastoma cells were also investigated. Based on these
studies, we attribute most of the enhancing effects of VPA and TSA on
ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by
an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma
cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine
with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of
P-glycoprotein expressed in this cell line.
CONCLUSION: The results found here warrant further studies and may help in the
design of new protocols geared to the treatment of high risk neuroblastomas. Breast cancer patients with HER-2 positive or estrogen receptor negative tumors
have a poor prognosis because these tumors are aggressive and respond poorly to
standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to
decreased cell survival, which suggests that HDAC inhibitors may be developed
for preventing and treating breast cancer. Curcumin has anti-inflammatory and
proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor,
Tricostatin A (TSA) in combination with curcumin would produce greater
antiproliferative and apoptotic effects than either agent alone. Increasing the
concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory
effects of the combination in SkBr3 and 435eB breast cancer cells, which was
accompanied by decreased viability along with decreased phosphorylation of ERK
and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was
combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27,
and decreased Cyclin D1 protein expression. The combination induced cleavage of
caspase 3 and poly(ADP-ribose) polymerase-1, suggesting that cell death occurred
by apoptosis. There were no changes in protein expression of Bcl2, Bax, or
Bcl-xL and decreased expression of p53. The combination increased protein
expression of phosphorylated JNK and phosphorylated p38. Pharmacological
inhibition of JNK, but not p38, attenuated the decreased viability induced by
the curcumin and TSA combination. We conclude that p53 independent apoptosis
induced by combining curcumin and TSA involves JNK activation. These findings
provide a rationale for exploring the potential benefits of the combination of
curcumin with TSA for treatment of breast cancer. Antiestrogen is one type of the endocrine therapeutic agents for estrogen
receptor α (ERα)-positive breast cancer. Unfortunately, this treatment alone is
insufficient. Here we reported a novel potential anticancer strategy by using
histone deacetylase (HDAC) inhibitor to enhance the action of endocrine therapy
in ERα-positive breast cancer cell. The well-described HDAC inhibitor,
trichostatin A (TSA), and antiestrogen raloxifene were found to, respectively,
inhibit E2-induced proliferation of MCF-7 breast cancer cell in a
dose-responsive and time-dependent manner. TSA and raloxifene enhanced the
antiproliferative activity of each other by promoting cell death via apoptosis
and cell cycle arrest. Thus, they displayed better antiproliferative effects in
combined treatment than that with either agent alone. The expression level of
estrogen receptor β (ERβ) showed a marked increase after TSA or/and raloxifene
treatment. Treatments with TSA or/and raloxifene resulting in the up-regulation
of ERβ are in accordance with the antiproliferative effects of the two agents.
Furthermore, the over-expression of ERβ by adenovirus delivery could inhibit the
proliferation of MCF-7 tumor cells and drastically enhanced the
antiproliferative effects of TSA and raloxifene. These results demonstrated that
the interference of ERβ on the antiproliferative effects of HDAC inhibitor and
antiestrogen constitutes a promising approach for breast cancer treatment. Hypoxia is an essential feature of the microenvironment of solid tumors, which
regulates a variety of transcription factors including hypoxia-inducible
factor-1α (HIF-1α). HIF-1α overexpression enhances tumor angiogenesis via
upregulation of vascular endothelial growth factor (VEGF) and some other
hypoxia-inducible angiogenic factors, which lead to a more aggressive tumor
phenotype, tumor metastasis and resistance to radiation and chemotherapy. In
this study, we found that a histone deacetylase (HDAC) inhibitor, trichostatin A
(TSA), inhibited cell proliferation and invasion, blocked the cell cycle, and
induced cell apoptosis in a dose- and time-dependent manner in the human tongue
squamous cell carcinoma (TSCC) SCC-6 cell line in vitro. Furthermore, TSA
reduced both basal levels and hypoxia-induced HIF-1α protein accumulation but
not HIF-1α mRNA levels, and both protein and mRNA levels of VEGF expression.
These results showed that TSA had a potent anticancer activity on TSCC cells,
suggesting that TSA could be a promising drug targeting tumor angiogenesis via
inhibition of HIF-1α and VEGF expression in the development of an effective
chemopreventive and anticancer agent on human TSCCs. BACKGROUND AND AIMS: Trichostatin A (TSA) is a potent histone deacetylase
inhibitor and widely used as a promising anticancer agent. Recently, a novel
insight for TSA has been shown to protect the heart from ischemia/reperfusion
(I/R) injury in mice, but the underlying mechanism remains unclear. The purpose
of this study is to investigate whether TSA can influence endoplasmic reticulum
stress (ERS) and whether its cardioprotective effect is mediated by inhibiting
myocardial ERS-induced apoptosis in rats.
METHODS: Male Wistar rats were used and pretreated with saline or TSA (0.05, 0.1
and 0.2 mg·kg(-1)) once daily i.p. for 5 days. I/R model was established by
occlusion/release of the left anterior descending coronary artery.
RESULTS: TSA significantly reduced myocardial infarct size and plasma activities
of lactate dehydrogenase and creatine kinase in a dose-dependent manner in rats.
Accompanied by the reduced injury, TSA also markedly reduced I/R-induced
myocardial apoptosis (30 min/24 h) by the TUNEL assay. In addition, increased
expression of glucose-regulated protein 78 (an ERS marker) by Western blot
showed the effects of TSA on ERS. Induction of C/EBP homologous protein (CHOP),
a critical mediator for ERS-induced apoptosis, was attenuated by TSA after
reperfusion for 6 h and 24 h.
CONCLUSIONS: Our findings showed that inhibition of histone deacetylase
ameliorated I/R-induced myocardial injury in vivo and for the first time
provided the evidence that suppression of CHOP expression and attenuation of the
CHOP-induced apoptosis may contribute to the cardioprotection of TSA against
myocardial I/R injury. BACKGROUND AND OBJECTIVE: Histone deacetylase (HDAC) inhibitors represent a
promising class of potential anticancer agents for treatment of human
maligcies. In this study, we investigated the effect of trichostatin A (TSA),
one such HDAC inhibitor, in combination with docetaxel (TXT), a cytotoxic
chemotherapy agent or erlotinib, a novel molecular target therapy drug, on lung
cancer A549 cells.
METHODS: A549 cells were treated with TXT, erlotinib alone or in combination
with TSA, respectively. Cell viability, apoptosis, and cell cycle distribution
were evaluated using MTT (3- (4, 5-dimethylthiazol-2-yl) -2,
5-diphenyltetrazolium bromide) assay, Hochst33258 staining and flow cytometry.
Moreover, immunofluorescent staining and Western blot analysis were employed to
examine alterations of α-tubulin, heat shock protein 90 (hsp90), epidermal
growth factor receptor (EGFR), and caspase-3 in response to the different
exogenous stimuli.
RESULTS: Compared with single-agent treatment, co-treatment of A549 cells with
TSA/TXT or TSA/erlotinib synergistically inhibited cell proliferation, induced
apoptosis, and caused cell cycle delay at the G2/M transition. Treatment with
TSA/TXT or TSA/erlotinib led to a significant increase of cleaved caspase-3
expression, also resulting in elevated acetylation of α-tubulin or hsp90 and
decreased expression of EGFR, which was negatively associated with the level of
acetylated hsp90.
CONCLUSIONS: Synergistic anti-tumor effects are observed between TXT or
erlotinib and TSA on lung cancer cells. Such combinations may provide a more
effective strategy for treating human lung cancer. BACKGROUND: Histone deacetylase (HDAC) inhibitors are promising antitumoral
drugs. Currently there are no data regarding the comparison of different HDAC
inhibitors on hepatoma cells.
MATERIALS AND METHODS: Hepatoma cells were incubated with the HDAC inhibitors
MS-275, SAHA, FK901228 and trichostatin. Proliferation was assessed via BrdU
incorporation and apoptosis rate via flow cytometry. Trichostatin, SAHA and
MS-275 were applied in a rat hepatoma model.
RESULTS: The agents showed antiproliferative and pro-apoptotic effects time- and
dose-dependently. SAHA and MS-275 were moderately effective at 10 μM, while
trichostatin A and FK901228 showed higher potency. Caspases 3 and 8 were
activated upon treatment with the drugs. The agents increased the acetylation
rate. Hyperacetylation did not correlate with antitumoral efficacy. In vivo,
SAHA was superior to MS-275 and trichostatin A.
CONCLUSION: The HDAC inhibitors were effective both in vitro and in vivo. The
potency of SAHA and MS-275 was similar. In spite of differing affinity to the 11
known HDACs, the agents induced comparable effects. These findings suggest that
these agents have further antitumoral effects apart from HDAC inhibition. BACKGROUND: Multiple myeloma (MM) is still a fatal plasma cell cancer. Novel
compounds are currently clinically tested as a single agent in relapsing
patients, but in best cases with partial response of a fraction of patients,
emphasising the need to design tools predicting drug efficacy. Histone
deacetylase inhibitors (HDACi) are anticancer agents targeting epigenetic
regulation of gene expression and are in clinical development in MM.
METHODS: To create a score predicting HDACi efficacy, five MM cell lines were
treated with trichostatin A (TSA) and gene expression profiles were determined.
RESULTS: The expression of 95 genes was found to be upregulated by TSA, using
paired supervised analysis with Significance Analysis of Microarrays software.
Thirty-seven of these 95 genes had prognostic value for overall survival in a
cohort of 206 newly diagnosed MM patients and their prognostic information was
summed up in a histone acetylation score (HA Score); patients with the highest
HA Score had the shorter overall survival. It is worth noting that MM cell lines
or patients' primary MM cells with a high HA Score had a significant higher
sensitivity to TSA, valproic acid, panobinostat or vorinostat.
CONCLUSION: In conclusion, the HA Score allows identification of MM patients
with poor survival, who could benefit from HDACi treatment. Radiotherapy is one of the main treatments for clinical cancer therapy. However,
its application was limited due to lack of radiosensitivity in some cancers.
Trichostatin A (TSA) is a classic histone deacetylases inhibitor (HDACi) that
specifically inhibits the biochemical functions of HDAC and is demonstrated to
be an active anticancer drug. However, whether it could sensitize colon cancer
to radiation is not clear. Our results showed that TSA enhanced the
radiosensitivity of colon cancer cells as determined by CCK-8 and clonogenic
survival assay. Moreover, apoptotic cell death induced by radiation was enhanced
by TSA treatment. Additionally, TSA also induced autophagic response in colon
cancer cells, while autophagy inhibition led to cell apoptosis and enhanced the
radiosensitivity of colon cancer cells. Our data suggested that inhibition of
cytoprotective autophagy sensitizes cancer cell to radiation, which might be
further investigated for clinical cancer radiotherapy. |
Is there an association between serum interleukin-6 concentrations and outcomes of stroke patients? | Yes. Greater serum interleukin-6 concentrations are associated with worse outcomes in ischemic and hemmorhagic stroke patients | The onset of cerebral ischaemia triggers a cascade of proinflammatory molecular
and cellular events. Clinical studies suggest that the strength of this acute
response is important in early and late clinical outcomes, early clinical
worsening, and extent of brain damage. Variables that are predictors of adverse
stroke outcome include erythrocyte sedimentation rate, and levels of C-reactive
protein (CRP), interleukin-6, tumour necrosis factor-alpha and intercellular
adhesion molecule-1. Current data indicate that inflammation serves to fuel
atherosclerosis and can act as the link between atherosclerosis and
atherothrombosis. Growing evidence indicates that platelets act as prominent
players in the inflammatory component of these disease processes. Thus, upon
activation, platelets release a series of cytokines and growth factors and
express CD40 ligand, which interacts with the CD40 receptor on other major cell
types involved in atherosclerosis/atherothrombosis. In healthy volunteers, CD40L
expression in platelets is not significantly inhibited by acetylsalicylic acid
(ASA) alone, but is inhibited after treatment with the ADP-receptor antagonist
clopidogrel or with clopidogrel plus ASA. Of a range of potential inflammatory
biomarkers that have been reported in the literature, the best studied is CRP.
Such biomarkers may have clinical utility for refined identification of patients
at high risk for atherothrombosis in different arterial beds and for monitoring
of therapeutic agents in clinical trials. OBJECTIVE: The role of inflammation in the pathogenesis of acute ischemic stroke
is well known, but its association with the clinical picture is as yet unclear.
MATERIAL AND METHODS: In our study, we measured the serum levels of the
proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor
alpha (TNFalpha) and interleukin-6 (IL-6) within the first 50 h of stroke in 60
acute stroke patients, and examined the association with the natural
anticoagulants protein C and free protein S. We compared the results with a
control group that consisted of 30 volunteers. We also correlated their levels
with the clinical outcomes by using the Canadian Neurological Scale (CNS).
RESULTS: Neither stroke patients nor the control group had any elevations in
IL-1beta serum levels. However, the levels of serum IL-6 were significantly
higher in stroke patients (13.7 +/- 19.46 vs. 4.3 +/- 15.88, p = 0.002). In
addition, the protein S levels of patients were lower than those of the controls
(84.36 +/- 27.97 vs. 95.9 +/- 25.64, p = 0.007). Although IL-6 showed negative
correlation with protein S (r = -0.504, p = 0.000), the other studied cytokines
TNFalpha and IL-1beta did not correlate with these natural anticoagulants.
Another negative correlation was found between IL-6 and CNS scores (r = -0.451,
p = 0.000). In addition, both protein C and protein S positively correlated with
CNS (r = 0.263, p = 0.042; r = 0.381, p = 0.003). There was also a positive
correlation between protein C and protein S (r = 0.408, p = 0.001).
CONCLUSIONS: Our results suggest that TNFalpha and IL1beta serum levels are not
elevated in the acute phase of stroke and have no correlation with the natural
anticoagulants protein C and protein S. However, a decrease in free protein S
may be related to elevated IL-6 levels. In addition, increased levels of IL-6
and reduced levels of protein C and protein S may play a role in acute ischemic
stroke severity. INTRODUCTION: Several studies have highlighted the role of interleukin-6 (IL-6)
as an early signal of the inflammatory response following acute ischemic stroke.
This study examines the potential advantage of employing high-sensitivity
(hs)-IL-6 as a possible biomarker at the early stages of acute stroke for
identifying an acute phase response and its potential rheological and clinical
implications.
METHODS: Venous blood was obtained from 186 stroke patients within 24 h of
hospital admission and 3-5 days thereafter in order to characterize an
inflammatory and hemorheological profile (including erythrocyte aggregation).
Neurological state was assessed by the National Institutes of Health Stroke
Scale (NIHSS) and the modified Rankin scale (mRs).
RESULTS: While most biomarkers displayed elevated concentrations with time,
serum concentrations of hs-IL-6 declined 3-5 days following acute stroke.
Initially elevated levels of hs-IL-6 at presentation further correlated with
unfavorable clinical outcomes (by NIHSS and mRs) at both time points. Analysis
of variance in the different quartiles identified an hs-IL-6 gradient-dependent
correlation at both time points, such that the higher the initial hs-IL-6
concentration, the higher the elevation in inflammatory biomarkers and the
poorer the neurological state at both time points (p<0.001 for NIHSS and p=0.001
for mRs, for trend across quartiles).
CONCLUSIONS: This study demonstrates the potential of employing hs-IL-6 as an
early stage biomarker for the prognosis of acute ischemic stroke. Such an
advance would provide the means to identify at an early stage the patients who
would require closer clinical surveillance and/or administration of therapeutic
interventions. PURPOSE: Proinflammatory and anti-inflammatory cytokines may play a pivotal role
in cerebral inflammation, which is implicated in the development of brain
injury. Systemic cytokine release is mediated by the sympathetic nervous system
and catecholamines. The aim of this study was to investigate which parameters,
among plasma levels of interleukin-1beta (IL-1beta), interleukin-6 (IL-6),
interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) and the
levels of the catecholamines, epinephrine and norepinephrine, contribute to the
clinical outcome in acute stroke patients.
METHODS: Thirty-seven acute stroke patients (ischemic, n = 19; hemorrhagic, n =
18) were enrolled. All of them were admitted to our hospital within 8 h after
stroke onset. Neurological status was evaluated by a modified National Institute
of Health Stroke Scale (mNIHSS) on admission and by a modified Rankin Scale
(mRS) at 1 month. An mRS score of 3 or more at 1 month was considered to
indicate poor outcome. Serum samples for the cytokine and catecholamine
measurements were collected on admission. Plasma levels of IL-1beta, IL-6,
IL-10, and TNF-alpha were determined by an enzyme-linked immunosorbent assay
(ELISA) method and epinephrine and norepinephrine concentrations were determined
by high-performance liquid chromatography with electrochemical detection
(HPLC-EC).
RESULTS: In the ischemic stroke patients, poor outcome was noted in 9 (47%).
There were no significant differences in cytokine or catecholamine
concentrations between patients with poor and good outcomes, and there was no
association between clinical outcome and cytokine and catecholamine
concentrations. In the hemorrhagic stroke patients, poor outcome was noted in 10
(56%). IL-6 and IL-10 levels were higher in patients with poor outcome. On
logistic regression analysis, higher values of IL-6 were significantly
associated with clinical outcome at 1 month (odds ratio [OR], 1.25; 95%
confidence interval [CI], 1.02-1.54).
CONCLUSION: In ischemic stroke, plasma cytokines and catecholamines were not
predictors of neurological outcome at 1 month. In hemorrhagic stroke, high
levels of IL-6 in the early phase indicated a poor neurological outcome. |
Could bioprinting be used in regenerative medicine against bone disease? | The developments in bioprinting (3D cell printing) techniques will be used in regenerative medicine and may allow the fabrication of customized implants for patients suffering in bone diseases in the future, once several technological limitations are addressed. | Improvements have been made in regenerative medicine, due to the development of
tissue engineering and cellular therapy. Bone regeneration is an ambitious
project, leading to many applications involving skull, maxillofacial, and
orthopaedic surgery. Scaffolds, stem cells, and signals support bone tissue
engineering. The scaffold physical and chemical properties promote cell
invasion, guide their differentiation, and enable signal transmission. Scaffold
may be inorganic or organic. Their conception was improved by the use of new
techniques: self-assembled ofibres, electrospinning, solution-phase
separation, micropatterned hydrogels, bioprinting, and rapid prototyping.
Cellular biology processes allow us to choose between embryonic stem cells or
adult stem cells for regenerative medicine. Finally, communication between cells
and their environment is essential; they use various signals to do so. The study
of signals and their transmission led to the discovery and the use of Bone
Morphogenetic Protein (BMP). The development of cellular therapy led to the
emergence of a specific field: gene therapy. It relies on viral vectors, which
include: retroviruses, adenoviruses and adeno-associated vectors (AAV).
Non-viral vectors include plasmids and lipoplex. Some BMP genes have
successfully been transfected. The ability to control transfected cells and the
capacity to combine and transfect many genes involved in osseous healing will
improve gene therapy. Additive manufacturing, otherwise known as three-dimensional (3D) printing, is
driving major innovations in many areas, such as engineering, manufacturing,
art, education and medicine. Recent advances have enabled 3D printing of
biocompatible materials, cells and supporting components into complex 3D
functional living tissues. 3D bioprinting is being applied to regenerative
medicine to address the need for tissues and organs suitable for
transplantation. Compared with non-biological printing, 3D bioprinting involves
additional complexities, such as the choice of materials, cell types, growth and
differentiation factors, and technical challenges related to the sensitivities
of living cells and the construction of tissues. Addressing these complexities
requires the integration of technologies from the fields of engineering,
biomaterials science, cell biology, physics and medicine. 3D bioprinting has
already been used for the generation and transplantation of several tissues,
including multilayered skin, bone, vascular grafts, tracheal splints, heart
tissue and cartilaginous structures. Other applications include developing
high-throughput 3D-bioprinted tissue models for research, drug discovery and
toxicology. In recent years a paradigm shift in understanding of human bone formation has
occurred that starts to change current concepts in tissue engineering of bone
and cartilage. New discoveries revealed that fundamental steps in
biomineralization are enzyme driven, not only during hydroxyapatite deposition,
but also during initial bioseed formation, involving the transient deposition
and subsequent transformation of calcium carbonate to calcium phosphate mineral.
The principal enzymes mediating these reactions, carbonic anhydrase and alkaline
phosphatase, open novel targets for pharmacological intervention of bone
diseases like osteoporosis, by applying compounds acting as potential activators
of these enzymes. It is expected that these new findings will give an innovation
boost for the development of scaffolds for bone repair and reconstruction, which
began with the use of bioinert materials, followed by bioactive materials and
now leading to functional regenerative tissue units. These new developments have
become possible with the discovery of the morphogenic activity of bioinorganic
polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a
biogenic, enzymatic mechanism, a driving force along with the development of
novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting
(3D cell printing) techniques that may allow a fabrication of customized
implants for patients suffering in bone diseases in the future. 3D Printing promises to produce complex biomedical devices according to computer
design using patient-specific anatomical data. Since its initial use as
pre-surgical visualization models and tooling molds, 3D Printing has slowly
evolved to create one-of-a-kind devices, implants, scaffolds for tissue
engineering, diagnostic platforms, and drug delivery systems. Fueled by the
recent explosion in public interest and access to affordable printers, there is
renewed interest to combine stem cells with custom 3D scaffolds for personalized
regenerative medicine. Before 3D Printing can be used routinely for the
regeneration of complex tissues (e.g. bone, cartilage, muscles, vessels, nerves
in the craniomaxillofacial complex), and complex organs with intricate 3D
microarchitecture (e.g. liver, lymphoid organs), several technological
limitations must be addressed. In this review, the major materials and
technology advances within the last five years for each of the common 3D
Printing technologies (Three Dimensional Printing, Fused Deposition Modeling,
Selective Laser Sintering, Stereolithography, and 3D
Plotting/Direct-Write/Bioprinting) are described. Examples are highlighted to
illustrate progress of each technology in tissue engineering, and key
limitations are identified to motivate future research and advance this
fascinating field of advanced manufacturing. |
Idarucizumab is an antidote of which drug? | Idarucizumab is an antidote of Dabigatran. It is used for Dabigatran Reversal. | Lack of specific antidotes is a major concern in intracerebral hemorrhage (ICH)
related to direct anticoagulants including dabigatran (OAC-ICH). We examined the
efficacy of idarucizumab, an antibody fragment binding to dabigatran, in a mouse
model of OAC-ICH. Dabigatran etexilate (DE) dose-dependently prolonged diluted
thrombin time and tail-vein bleeding time, which were reversed by idarucizumab.
Pretreatment with DE increased intracerebral hematoma volume and cerebral
hemoglobin content. Idarucizumab in equimolar dose prevented excess hematoma
expansion for both DE doses. In more extensive ICH, idarucizumab significantly
reduced mortality. Thus, idarucizumab prevents excess intracerebral hematoma
formation in mice anticoagulated with dabigatran and reduces mortality. BACKGROUND: Idarucizumab is a monoclonal antibody fragment that binds dabigatran
with high affinity in a 1:1 molar ratio. We investigated the safety,
tolerability, and efficacy of increasing doses of idarucizumab for the reversal
of anticoagulant effects of dabigatran in a two-part phase 1 study (rising-dose
assessment and dose-finding, proof-of-concept investigation). Here we present
the results of the proof-of-concept part of the study.
METHODS: In this randomised, placebo-controlled, double-blind, proof-of-concept
phase 1 study, we enrolled healthy volunteers (aged 18-45 years) with a
body-mass index of 18·5-29·9 kg/m(2) into one of four dose groups at SGS Life
Sciences Clinical Research Services, Belgium. Participants were randomly
assigned within groups in a 3:1 ratio to idarucizumab or placebo using a
pseudorandom number generator and a supplied seed number. Participants and care
providers were masked to treatment assignment. All participants received oral
dabigatran etexilate 220 mg twice daily for 3 days and a final dose on day 4.
Idarucizumab (1 g, 2 g, or 4 g 5-min infusion, or 5 g plus 2·5 g in two 5-min
infusions given 1 h apart) was administered about 2 h after the final dabigatran
etexilate dose. The primary endpoint was incidence of drug-related adverse
events, analysed in all randomly assigned participants who received at least one
dose of dabigatran etexilate. Reversal of diluted thrombin time (dTT), ecarin
clotting time (ECT), activated partial thromboplastin time (aPTT), and thrombin
time (TT) were secondary endpoints assessed by measuring the area under the
effect curve from 2 h to 12 h (AUEC2-12) after dabigatran etexilate ingestion on
days 3 and 4. This trial is registered with ClinicalTrials.gov, number
NCT01688830.
FINDINGS: Between Feb 23, and Nov 29, 2013, 47 men completed this part of the
study. 12 were enrolled into each of the 1 g, 2 g, or 5 g plus 2·5 g
idarucizumab groups (nine to idarucizumab and three to placebo in each group),
and 11 were enrolled into the 4 g idarucizumab group (eight to idarucizumab and
three to placebo). Drug-related adverse events were all of mild intensity and
reported in seven participants: one in the 1 g idarucizumab group (infusion site
erythema and hot flushes), one in the 5 g plus 2·5 g idarucizumab group
(epistaxis); one receiving placebo (infusion site haematoma), and four during
dabigatran etexilate pretreatment (three haematuria and one epistaxis).
Idarucizumab immediately and completely reversed dabigatran-induced
anticoagulation in a dose-dependent manner; the mean ratio of day 4 AUEC2-12 to
day 3 AUEC2-12 for dTT was 1·01 with placebo, 0·26 with 1 g idarucizumab (74%
reduction), 0·06 with 2 g idarucizumab (94% reduction), 0·02 with 4 g
idarucizumab (98% reduction), and 0·01 with 5 g plus 2·5 g idarucizumab (99%
reduction). No serious or severe adverse events were reported, no adverse event
led to discontinuation of treatment, and no clinically relevant difference in
incidence of adverse events was noted between treatment groups.
INTERPRETATION: These phase 1 results show that idarucizumab was associated with
immediate, complete, and sustained reversal of dabigatran-induced
anticoagulation in healthy men, and was well tolerated with no unexpected or
clinically relevant safety concerns, supporting further testing. Further
clinical studies are in progress.
FUNDING: Boehringer Ingelheim Pharma GmbH & Co KG. The direct thrombin inhibitor dabigatran and the anti-Xa agents rivaroxaban,
edoxaban, and apixaban are a new generation of oral anticoagulants. Their
advantage over the vitamin K antagonists is the lack of the need for monitoring
and dose adjustment. Their main disadvantage is currently the absence of a
specific reversal agent. Dabigatran's, unlike the anti-Xa agents, absorption can
be reduced by activated charcoal if administered shortly after ingestion and it
can be removed from the blood with hemodialysis. Prothrombin complex
concentrate, activated prothrombin complex concentrate, and recombit factor
VIIa all show some activity in reversing the anticoagulant effect of these drugs
but this is based on ex vivo, animal, and volunteer studies. It is unclear,
which, if any, of these drugs is the most suitable for emergency reversal. Three
novel molecules (idarucizumab, andexanet, and PER977) may provide the most
effective and safest way of reversal. These agents are currently in premarketing
studies. BACKGROUND: Specific reversal agents for non-vitamin K antagonist oral
anticoagulants are lacking. Idarucizumab, an antibody fragment, was developed to
reverse the anticoagulant effects of dabigatran.
METHODS: We undertook this prospective cohort study to determine the safety of 5
g of intravenous idarucizumab and its capacity to reverse the anticoagulant
effects of dabigatran in patients who had serious bleeding (group A) or required
an urgent procedure (group B). The primary end point was the maximum percentage
reversal of the anticoagulant effect of dabigatran within 4 hours after the
administration of idarucizumab, on the basis of the determination at a central
laboratory of the dilute thrombin time or ecarin clotting time. A key secondary
end point was the restoration of hemostasis.
RESULTS: This interim analysis included 90 patients who received idarucizumab
(51 patients in group A and 39 in group B). Among 68 patients with an elevated
dilute thrombin time and 81 with an elevated ecarin clotting time at baseline,
the median maximum percentage reversal was 100% (95% confidence interval, 100 to
100). Idarucizumab normalized the test results in 88 to 98% of the patients, an
effect that was evident within minutes. Concentrations of unbound dabigatran
remained below 20 ng per milliliter at 24 hours in 79% of the patients. Among 35
patients in group A who could be assessed, hemostasis, as determined by local
investigators, was restored at a median of 11.4 hours. Among 36 patients in
group B who underwent a procedure, normal intraoperative hemostasis was reported
in 33, and mildly or moderately abnormal hemostasis was reported in 2 patients
and 1 patient, respectively. One thrombotic event occurred within 72 hours after
idarucizumab administration in a patient in whom anticoagulants had not been
reinitiated.
CONCLUSIONS: Idarucizumab completely reversed the anticoagulant effect of
dabigatran within minutes. (Funded by Boehringer Ingelheim; RE-VERSE AD
ClinicalTrials.gov number, NCT02104947.). |
What are the main clinical characteristics of Pendred syndrome? | Pendred syndrome is an autosomal recessive disorder characterized by congenital sensorineural deafness, goiter, and impaired iodide organification. | OBJECTIVE: Pendred's syndrome is an association between congenital neurosensory
deafness and goitre with abnormal discharge of iodide following perchlorate
challenge, indicating a defect of iodide organification. Although Pendred's
syndrome may cause up to 7.5% of all cases of congenital deafness, the molecular
basis of the association between the hearing loss and the thyroid organification
defect remains unknown. We chose to investigate the role of the thyroid
peroxidase (TPO) gene as the genetic defect in Pendred's syndrome.
DESIGN: A highly informative variable number tandem repeat (VNTR), located 1.5
kb downstream of exon 10 of the TPO gene, was used to search for genetic linkage
in multiple sibships affected by Pendred's syndrome.
PATIENTS: Seven kindreds were recruited from the UK, each with at least two
affected members. We have also examined a large inbred Israeli family with two
affected offspring and five unaffected children.
MEASUREMENTS: Individuals were assigned affected status based on the
characteristic clinical features of Pendred's syndrome, namely the presence of
congenital sensorineural hearing loss and the appearance in early life of a
goitre. Additionally, at least one affected member from each sibship had a
characteristic positive perchlorate discharge test (Morgans & Trotter, 1958).
PCR amplification of genomic DNA at the TPO VNTR allowed assignment of genotypes
to each individual and the calculation of a two-point LOD score.
RESULTS: In six of the nine sibships analysed we found obligatory recombination
between TPO and Pendred's syndrome. Non-complementation observed in affected
parents with an affected offspring excluded TPO in an affected sibship with
genotype sharing and supports a hypothesis of genetic homogeneity for Pendred's
syndrome. In two sibships, mutation of the TPO gene as the cause of Pendred's
syndrome could not be excluded.
CONCLUSIONS: These data suggest that defects at the thyroid peroxidase locus on
chromosome 2 are not the major cause of Pendred's syndrome. Pendred syndrome is an autosomal recessive inherited disorder. Obligatory
features are profound deafness in childhood and defective organic binding of
iodine in the thyroid gland. Therefore, goiter is a common symptom. Hypoplasia
of the cochlea is another feature. Recently, the gene for Pendred syndrome was
identified. We describe a boy whose sensorineural hearing loss in both ears
progressed rapidly from about 50 to 60 dB at the age of 3 years and 3 months to
more than 100 dB at the age of 4 years and 4 months. This loss was preceded by a
medical history of a progressive hearing loss. The progressive nature of the
hearing loss motivated a search for the cause. Dysplasia of the cochlea and a
widened vestibular aqueduct were found. The results of thyroid function tests
were normal, but he had an elevated level of thyroglobulin. The diagnosis of
Pendred syndrome was confirmed by the positive results of a potassium
perchlorate test, indicating defective organic binding of iodine in the thyroid
gland. It is possible that the widened vestibular aqueduct was responsible for
the increase in the hearing impairment. Aside from the branchio-otorenal
syndrome, Pendred syndrome is the only other known genetic disorder with a
widened vestibular aqueduct. If a child has progressive sensorineural deafness
and a widened vestibular aqueduct, it is important to consider a diagnosis of
Pendred syndrome. A widened vestibular aqueduct may help to elucidate the
pathophysiologic characteristics of hearing loss in these genetic types of
deafness in childhood. Long-term hearing threshold-on-age follow-up data, including non-linear
regression analysis, are given for 12 consecutive Pendred patients. The clinical
diagnosis of Pendred's syndrome was confirmed by a mutation analysis of the PDS
gene in 11 out of the 11 cases tested. Recent imaging of the temporal bones in
seven out of these 12 patients showed widened vestibular aqueducts in each case.
The diagnostic perchlorate test was negative in one patient, but this test was
positive in her affected sister. Mutation analysis of the PDS gene in these
patients confirmed that Pendred's syndrome is a monogenetic disorder.
Progressive sensorineural hearing loss and widened vestibular aqueducts are
characteristic features of Pendred's syndrome, which provides the opportunity to
diagnose Pendred's syndrome clinically in the first few years of life, as has
recently been suggested in a case report (Cremers et al., Progressive
sensorineural hearing loss and a widend vestibular aqueduct in Pendred syndrome,
Arch. Otolaryngol. 124 (1998) 501-505). Mutation analysis of the involved gene
can be used to confirm the clinical diagnosis. Although the textbook view of Pendred syndrome is that of an autosomal recessive
condition characterized by deafness and goitre, it is increasingly clear that
not all such patients present this classical clinical picture. Malformations of
the inner ear, specifically enlargement of the vestibular aqueduct, are common
in Pendred syndrome and mutations in the PDS (Pendred Syndrome) gene have been
recorded in patients presenting with deafness and vestibular aqueduct dilatation
only, without other features of Pendred syndrome. Since this is the most common
radiological malformation of the cochlea in deaf patients, we investigated what
proportion of such cases were due to mutation of the PDS gene. We assessed 57
patients referred with radiological evidence of vestibular aqueduct enlargement,
by history, clinical examination, perchlorate discharge test and molecular
analysis of the PDS locus. Forty-one patients (72%) had unequivocal evidence of
Pendred syndrome. The finding of a single heterozygous mutation at the PDS gene
in a further eight was strongly suggestive of a critical role for pendrin, the
protein product of the PDS gene, in the generation of enlarged vestibular
aqueducts in at least 86% (49/57 cases) of patients with this radiological
malformation. Securing the diagnosis of Pendred syndrome may be difficult,
especially in the single case. Goitre is an inconstant finding, and the
perchlorate discharge test, although helpful, is of diagnostic value only if
abnormal. Enlargement of the vestibular aqueduct should be considered as the
most likely presentation of Pendred syndrome and should prompt specific
investigation of that diagnostic possibility. Pendred syndrome might henceforth
be recharacterized as deafness with enlargement of the vestibular aqueduct,
which is sometimes associated with goitre. Pendred's syndrome is a combination of congenital sensorineural hearing loss and
iodine organification defect leading to a positive perchlorate test and goiter.
Although it is the commonest form of syndromic hearing loss, the variable
clinical presentation contributes to the difficulty in securing a diagnosis. The
identification of the disease gene (PDS) prompts the need to reevaluate the
syndrome to identify possible clues for the diagnosis. To this purpose, in three
Italian families presenting with the clinical features of Pendred's syndrome,
the molecular analysis was accompanied by full clinical, biochemical, and
radiological examination. A correlation between genotype and phenotype was found
in the only patient with enlargement of vestibular aqueduct and endolymphatic
duct and sac at magnetic resoce imaging. This subject was a compound
heterozygote for a deletion in PDS exon 10 (1197delT, FS400) and a novel
insertion in exon 19 (2182-2183insG, Y728X). The present study demonstrates for
the first time the value of the combination of clinical/radiological and genetic
studies in the diagnosis of Pendred's syndrome. The positivity of a perchlorate
discharge test and the malformations of membranous labyrinth fit well with the
recent achievements on the role of pendrin in thyroid hormonogenesis and the
maintece of endolymph homeostasis. Sensorineural hearing defect and goiter are common features of Pendred's
syndrome. The clinical diagnosis of Pendred's syndrome remains difficult because
of the lack of sensitivity and specificity of the thyroid signs. The
identification of PDS as the causative gene allowed molecular screening and
enabled a re-evaluation of the syndrome to identify potential diagnostic
characteristics. This report presents the clinical and genotypic findings of 30
French families, for whom a diagnosis of Pendred's syndrome had been made.
Twenty-seven families had at least one mutated allele. Twenty-eight different
mutations were identified, 11 of which had never been previously reported. The
main clinical characteristics were: early hearing loss, fluctuation in terms of
during deafness evolution, and the presence of an enlarged vestibular aqueduct. BACKGROUND: Pendred-syndrome is an autosomal recessive disease that is
classically characterised by sensorineural hearing loss and enlargement of the
thyroid gland. The gene SLC26A4/PDS for the pendred-syndrome has been localised
by linkage analysis on chromosome 7q31. This protein is expressed in the inner
ear, thyroid gland, kidney and placenta. Functional analysis in Xenopus laevis
oocytes revealed that it acts as an iodide/chloride and chloride/formate
exchanger.
METHOD: Each of the exons and flanking splice regions of the SLC26A4/PDS gene
was analysed by direct sequencing.
RESULTS: In the involved family two heterozygous mutations could be detected
which results by combination in hearing loss and deafness.
CONCLUSION: By evidences of familial background in hearing loss and thyroid
disorder it is reasonable to analyse the PDS gene for mutation to have early the
possibility for medical care of linguistic development through hearing aid or
CI-implantation. Inherited as an autosomal recessive trait, Pendred syndrome is a disease that
shows congenital sensorineural hearing loss and goiter, with a positive finding
in the perchlorate discharge test. Pendred syndrome results from various
mutations in the PDS/SLC26A4 gene that cause production of an abnormal pendrin
protein. More than 90 mutations in the PDS/SLC26A4 gene have been reported
throughout the world. A recent study of 26 Korean patients with a relatively
high frequency (65%) of a mutated PDS/SLC26A4 gene exhibited nonsyndromic
deafness and an enlarged vestibular aqueduct. We report two patients with
characteristics of typical Pendred syndrome, a 26-yr-old female and a 61-yr-old
male, who were both homozygous for a previously reported missense mutation,
H723R (Histidine 723Arginine) in the PDS/SLC26A4 gene. OBJECTIVE: Pendred syndrome (PS) is characterized by the association of
sensorineural hearing loss (SNHL) and a partial iodide organification defect at
the thyroid level. It is caused by mutations in the SLC26A4 gene. The encoded
transmembrane protein, called pendrin, has been found to be able to transport
chloride and other anions.
DESIGN: The aim of the present study was to characterize a family with PS, which
shows a strong intrafamilial phenotypic variability, including kidney atrophy in
one member. The age of disease-onset was significantly different in all three
affected siblings, ranging from 2 to 21 years for thyroid alterations and from
1.5 to 11 years for SNHL.
METHODS: Clinical and genetic studies were carried out in affected siblings. The
functional activity of the novel duplication found was studied by a fluorimetric
method in a human renal cell line (HEK293 Phoenix) in which the protein was
overexpressed.
RESULTS: All three siblings were found to be compound heterozygotes for the
missense mutation (1226G>A, R409H) and for a novel 11 bp duplication
(1561_1571CTTGGAATGGC, S523fsX548). The latter mutation creates a frame shift
leading to the loss of the entire carboxy-terminus domain. Functional studies of
this mutant demonstrated impaired transport of chloride and iodide when
expressed in HEK 293 Phoenix cells, when compared with wild type pendrin.
CONCLUSIONS: A novel 11 bp duplication was found in a family with Pendred
syndrome, showing a high intrafamilial phenotypic variability. An impaired
transmembrane anionic transport of the mutated SLC26A4 protein was demonstrated
in functional studies using a heterologous cell system. Pendred syndrome is an autosomal recessive disorder characterised by
sensorineural hearing loss and thyroid dyshormonogenesis. It is caused by
mutations in the PDS/SLC26A4 gene (OMIM 605646) encoding for pendrin.
Hypothyroidism in Pendred syndrome can be--although rarely--present from birth
and therefore diagnosed by neonatal screening. The aim of our study was to
identify patients with Pendred syndrome among a historical cohort of patients
with congenital hypothyroidism (CH) identified by neonatal screening, and to
find their mutations in the PDS/SLC26A4 gene. We investigated 197 Czech
Caucasian children with CH detected by the neonatal screening between the years
1985 and 2005. The clinical diagnosis of Pendred syndrome was based on the
laboratory and sonographic signs of thyroid dyshormonogenesis in association
with sensorineural hearing loss. In subjects clinically diagnosed with Pendred
syndrome, we sequenced all exons and exon-intron boundaries of the PDS/SLC26A4
gene. Hearing loss was present in 10/197 children with screening-detected CH. Of
these, three fulfilled the diagnostic criteria of Pendred syndrome. Two patients
were compound heterozygotes for PDS/SLC26A4 mutations: patient 1 carried
c.2089+1G>A / c.3G>C and patient 2 carried p.Tyr530His / p.Val422Asp. Two of the
four identified mutations were novel (c.3G>C in patient 1 and p.Val422Asp in
patient 2). The third patient was free of mutations in the PDS/SLC26A4 gene,
representing a phenocopy. In conclusion, our results indicate the rarity of
Pendred syndrome as a cause of CH. The identification of two novel mutations
expands the spectrum of mutations in the PDS/SLC26A4 gene and emphasizes their
marked allelic heterogeneity. Pendred syndrome is an autosomal recessive disorder characterized by congenital
sensorineural deafness, goiter, and impaired iodide organification. It is caused
by mutations in the PDS gene. Most published mutation studies of Pendred
syndrome have dealt with Western populations. In this study, we examined
clinical and molecular characteristics of 16 affected individuals in 6 unrelated
Thai families. Of all the affected, 100% (16/16) had bilateral deafness, 68.8%
(11/16) goiters, and 25% (4/16) hypothyroidism. Follicular thyroid carcinoma and
Hürthle cell adenoma were found in affected members of a family, raising the
possibility of an increased risk of thyroid carcinoma in Pendred syndrome
patients. Sequence analysis of the entire coding region of the PDS gene
successfully identified all 12 mutant alleles in these 6 families. The 12
identified mutant alleles constituted 6 distinct mutations including 3 splice
site mutations (IVS4-1G>A, IVS7-2A>G, IVS9- 1G>A), one frame shift mutation
(1548insC) and 2 missense mutations (T67S, H723R). Eight mutations out of 12
were constituted by IVS7- 2A>G and 1548insC, each one being present in 4
distinct alleles in our studied group. The identification of these two frequent
PDS mutations will facilitate the molecular diagnosis of Pendred syndrome in
Thai populations. In addition, three newly identified mutations, T67S,
IVS4-1G>A, and IVS9-1G>A, were not observed in 50 unrelated healthy Thai
controls. INTRODUCTION: The Pendred syndrome (PS) is an autosomally recessively inherited
disease. Its diagnosis requires identification of the classical triad of
symptoms, including hypoacusis, thyroid goitre and iodine organification defect
in the thyroid, which may lead to thyroid functional disorders of
hypothyroidism. SP is accompanied by anatomical anomalies. The objective is the
hearing and balance system evaluation and the analysis of the inner ear
structure and also the assessment of the function and structure of thyroid
gland.
MATERIAL AND METHODS: For the research four families were qualified, 7 persons
with PS, 12 persons altogether. In all the patients the anamnesis in the form of
a questionnaire and laryngological examination were performed. It was followed
by pure tone, speech and impedance audiometry and brainstem response testing as
well. ENG was also conducted. Patients with hearing loss were subjected to
magnetic resoce of temporal bone. For the whole group thyroid hormones levels
and iodine organification in the thyroid identified in a test with potassium
perchlorate were measured and also USG and scyntography were conducted.
RESULTS: In audiological examination in 3 cases deafness, in 2 cases profound
hypoacusis and in 2 mild hypoacusis were recognised. In the group in 2 patients
the hypoacusis was of a mixed type. In radiological assessment the labirynth
showed anatomical anomalies in the form of enlargement of the vestibular
aqueduct and the endolyphatic sac, yet in 3 patients the anomalies also
concerned the structure of cochlear and semicircular canals. Endocrine
examination showed hypothyroidism in 5, its subclinical form in 1, diffuse
thyroid goitre in 4 and nodular thyroid goiter in 2 cases.
CONCLUSIONS: A complex clinical evaluation: endocrine and audiological, together
with radiological diagnostic imaging, supported by molecular studies of SLC26A4
gene, are the procedures, necessary for complete and accurate diagnosis of PS
and EVAS. Pendred syndrome is an autosomal recessive disorder defined by sensorineural
deafness, goiter and a partial defect in the organification of iodide. It is
caused by biallelic mutations in the SLC26A4 gene, which encodes pendrin, a
multifunctional anion exchanger. At the level of the inner ear, pendrin is
important for the creation of a normal endolymph composition and the maintece
of the endocochlear potential. In the thyroid, pendrin is expressed at the
apical membrane of thyroid follicular cells and it appears to be involved in
mediating iodide efflux into the lumen and/or maintece of the follicular pH.
Goiter development and hypothyroidism vary among affected individuals and seem
to be partially dependent on nutritional iodide intake. In the kidney, pendrin
functions as a chloride/bicarbonate exchanger. Elucidation of the molecular
basis of Pendred syndrome and the function of pendrin has provided unexpected
novel insights into the pathophysiology of the inner ear, thyroid hormone
synthesis, and chloride/bicarbonate exchange in the kidney. |
What is the Pfam database? | The Pfam database provides a collection of curated protein families. | Pseudofam (http://pseudofam.pseudogene.org) is a database of pseudogene families
based on the protein families from the Pfam database. It provides resources for
analyzing the family structure of pseudogenes including query tools, statistical
summaries and sequence alignments. The current version of Pseudofam contains
more than 125,000 pseudogenes identified from 10 eukaryotic genomes and aligned
within nearly 3000 families (approximately one-third of the total families in
PfamA). Pseudofam uses a large-scale parallelized homology search algorithm
(implemented as an extension of the PseudoPipe pipeline) to identify
pseudogenes. Each identified pseudogene is assigned to its parent protein family
and subsequently aligned to each other by transferring the parent domain
alignments from the Pfam family. Pseudogenes are also given additional
annotation based on an ontology, reflecting their mode of creation and
subsequent history. In particular, our annotation highlights the association of
pseudogene families with genomic features, such as segmental duplications. In
addition, pseudogene families are associated with key statistics, which identify
outlier families with an unusual degree of pseudogenization. The statistics also
show how the number of genes and pseudogenes in families correlates across
different species. Overall, they highlight the fact that housekeeping families
tend to be enriched with a large number of pseudogenes. Protein domains are the common currency of protein structure and function. Over
10,000 such protein families have now been collected in the Pfam database. Using
these data along with animal gene phylogenies from TreeFam allowed us to
investigate the gain and loss of protein domains. Most gains and losses of
domains occur at protein termini. We show that the nature of changes is similar
after speciation or duplication events. However, changes in domain architecture
happen at a higher frequency after gene duplication. We suggest that the bias
towards protein termini is largely because insertion and deletion of domains at
most positions in a protein are likely to disrupt the structure of existing
domains. We can also use Pfam to trace the evolution of specific families. For
example, the immunoglobulin superfamily can be traced over 500 million years
during its expansion into one of the largest families in the human genome. It
can be shown that this protein family has its origins in basic animals such as
the poriferan sponges where it is found in cell-surface-receptor proteins. We
can trace how the structure and sequence of this family diverged during
vertebrate evolution into constant and variable domains that are found in the
antibodies of our immune system as well as in neural and muscle proteins. The Pfam database is an important tool in genome annotation, since it provides a
collection of curated protein families. However, a subset of these families,
known as domains of unknown function (DUFs), remains poorly characterized. We
have related sequences from DUF404, DUF407, DUF482, DUF608, DUF810, DUF853,
DUF976 and DUF1111 to homologs in PDB, within the midnight zone (9-20%) of
sequence identity. These relationships were extended to provide functional
annotation by sequence analysis and model building. Also described are examples
of residue plasticity within enzyme active sites, and change of function within
homologous sequences of a DUF. It is a worthy goal to completely characterize all human proteins in terms of
their domains. Here, using the Pfam database, we asked how far we have
progressed in this endeavour. Ninety per cent of proteins in the human proteome
matched at least one of 5494 manually curated Pfam-A families. In contrast,
human residue coverage by Pfam-A families was <45%, with 9418 automatically
generated Pfam-B families adding a further 10%. Even after excluding predicted
signal peptide regions and short regions (<50 consecutive residues) unlikely to
harbour new families, for ∼38% of the human protein residues, there was no
information in Pfam about conservation and evolutionary relationship with other
protein regions. This uncovered portion of the human proteome was found to be
distributed over almost 25 000 distinct protein regions. Comparison with
proteins in the UniProtKB database suggested that the human regions that
exhibited similarity to thousands of other sequences were often either divergent
elements or N- or C-terminal extensions of existing families. Thirty-four per
cent of regions, on the other hand, matched fewer than 100 sequences in
UniProtKB. Most of these did not appear to share any relationship with existing
Pfam-A families, suggesting that thousands of new families would need to be
generated to cover them. Also, these latter regions were particularly rich in
amino acid compositional bias such as the one associated with intrinsic
disorder. This could represent a significant obstacle toward their inclusion
into new Pfam families. Based on these observations, a major focus for
increasing Pfam coverage of the human proteome will be to improve the definition
of existing families. New families will also be built, prioritizing those that
have been experimentally functionally characterized. Database URL:
http://pfam.sanger.ac.uk/ The database iPfam, available at http://ipfam.org, catalogues Pfam domain
interactions based on known 3D structures that are found in the Protein Data
Bank, providing interaction data at the molecular level. Previously, the iPfam
domain-domain interaction data was integrated within the Pfam database and
website, but it has now been migrated to a separate database. This allows for
independent development, improving data access and giving clearer separation
between the protein family and interactions datasets. In addition to
domain-domain interactions, iPfam has been expanded to include interaction data
for domain bound small molecule ligands. Functional annotations are provided
from source databases, supplemented by the incorporation of Wikipedia articles
where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500
domain-ligand interactions. The new website provides access to this data in a
variety of ways, including interactive visualizations of the interaction data. |
What is a popular mesaure of gene expression in RNA-seq experiments? | A commonly used measure for gene expression in RNA-seq experiments is Reads Per Kilobase per Million mapped reads (RPKM). In ocasions, and to account for partially mapped read, read Fragments per Kilobase per Million mapped reads (FPKM) is alternatively used. | Targeted RNA-Seq combines next-generation sequencing with capture of sequences
from a relevant subset of a transcriptome. When testing by capturing sequences
from a tumor cDNA library by hybridization to oligonucleotide probes specific
for 467 cancer-related genes, this method showed high selectivity, improved
mutation detection enabling discovery of novel chimeric transcripts, and
provided RNA expression data. Thus, targeted RNA-Seq produces an enhanced view
of the molecular state of a set of "high interest" genes. An essential question in human biology is how cells and tissues differ in gene
and protein expression and how these differences delineate specific biological
function. Here, we have performed a global analysis of both mRNA and protein
levels based on sequence-based transcriptome analysis (RNA-seq), SILAC-based
mass spectrometry analysis and antibody-based confocal microscopy. The study was
performed in three functionally different human cell lines and based on the
global analysis, we estimated the fractions of mRNA and protein that are cell
specific or expressed at similar/different levels in the cell lines. A highly
ubiquitous RNA expression was found with >60% of the gene products detected in
all cells. The changes of mRNA and protein levels in the cell lines using SILAC
and RNA ratios show high correlations, even though the genome-wide dynamic range
is substantially higher for the proteins as compared with the transcripts. Large
general differences in abundance for proteins from various functional classes
are observed and, in general, the cell-type specific proteins are low abundant
and highly enriched for cell-surface proteins. Thus, this study shows a path to
characterize the transcriptome and proteome in human cells from different
origins. Abstract Next generation high-throughput sequencing (NGS) is poised to replace
array-based technologies as the experiment of choice for measuring RNA
expression levels. Several groups have demonstrated the power of this new
approach (RNA-seq), making significant and novel contributions and
simultaneously proposing methodologies for the analysis of RNA-seq data. In a
typical experiment, millions of short sequences (reads) are sampled from RNA
extracts and mapped back to a reference genome. The number of reads mapping to
each gene is used as proxy for its corresponding RNA concentration. A
significant challenge in analyzing RNA expression of homologous genes is the
large fraction of the reads that map to multiple locations in the reference
genome. Currently, these reads are either dropped from the analysis, or a naive
algorithm is used to estimate their underlying distribution. In this work, we
present a rigorous alternative for handling the reads generated in an RNA-seq
experiment within a probabilistic model for RNA-seq data; we develop maximum
likelihood-based methods for estimating the model parameters. In contrast to
previous methods, our model takes into account the fact that the DNA of the
sequenced individual is not a perfect copy of the reference sequence. We show
with both simulated and real RNA-seq data that our new method improves the
accuracy and power of RNA-seq experiments. RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is
hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic
to exponential PCR amplification. We developed a simple strategy for mitigating
these complications, allowing truly digital RNA-Seq. Following reverse
transcription, a large set of barcode sequences is added in excess, and nearly
every cDNA molecule is uniquely labeled by random attachment of barcode
sequences to both ends. After PCR, we applied paired-end deep sequencing to read
the two barcodes and cDNA sequences. Rather than counting the number of reads,
RNA abundance is measured based on the number of unique barcode sequences
observed for a given cDNA sequence. We optimized the barcodes to be
unambiguously identifiable, even in the presence of multiple sequencing errors.
This method allows counting with single-copy resolution despite
sequence-dependent bias and PCR-amplification noise, and is analogous to digital
PCR but amendable to quantifying a whole transcriptome. We demonstrated
transcriptome profiling of Escherichia coli with more accurate and reproducible
quantification than conventional RNA-Seq. The rapid expansion in the quantity and quality of RNA-Seq data requires the
development of sophisticated high-performance bioinformatics tools capable of
rapidly transforming this data into meaningful information that is easily
interpretable by biologists. Currently available analysis tools are often not
easily installed by the general biologist and most of them lack inherent
parallel processing capabilities widely recognized as an essential feature of
next-generation bioinformatics tools. We present here a user-friendly and fully
automated RNA-Seq analysis pipeline (R-SAP) with built-in multi-threading
capability to analyze and quantitate high-throughput RNA-Seq datasets. R-SAP
follows a hierarchical decision making procedure to accurately characterize
various classes of transcripts and achieves a near linear decrease in data
processing time as a result of increased multi-threading. In addition, RNA
expression level estimates obtained using R-SAP display high concordance with
levels measured by microarrays. RNA expression profiles produced by next-generation sequencing (NGS) technology
(RNA-seq) allow comprehensive investigation of transcribed sequences within a
cell or tissue. RNA-seq is rapidly becoming more cost-effective for
transcriptome profiling. However, its usage will expand dramatically if one
starts with low amount of RNA and obtains transcript directionality during the
analysis. Here, we describe a detailed protocol for the creation of a
directional RNA-seq library from 100 ng of starting total RNA. Measures of RNA abundance are important for many areas of biology and often
obtained from high-throughput RNA sequencing methods such as Illumina sequence
data. These measures need to be normalized to remove technical biases inherent
in the sequencing approach, most notably the length of the RNA species and the
sequencing depth of a sample. These biases are corrected in the widely used
reads per kilobase per million reads (RPKM) measure. Here, we argue that the
intended meaning of RPKM is a measure of relative molar RNA concentration (rmc)
and show that for each set of transcripts the average rmc is a constant, namely
the inverse of the number of transcripts mapped. Further, we show that RPKM does
not respect this invariance property and thus cannot be an accurate measure of
rmc. We propose a slight modification of RPKM that eliminates this inconsistency
and call it TPM for transcripts per million. TPM respects the average invariance
and eliminates statistical biases inherent in the RPKM measure. BACKGROUND: Thoroughbred horses are the most expensive domestic animals, and
their running ability and knowledge about their muscle-related diseases are
important in animal genetics. While the horse reference genome is available,
there has been no large-scale functional annotation of the genome using
expressed genes derived from transcriptomes.
RESULTS: We present a large-scale analysis of whole transcriptome data. We
sequenced the whole mRNA from the blood and muscle tissues of six thoroughbred
horses before and after exercise. By comparing current genome annotations, we
identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933
(36.87%) annotated genes. More than 60% (20,428) of the unigene clusters did not
match any current equine gene model. We also identified 189,973 single
nucleotide variations (SNVs) from the sequences aligned against the horse
reference genome. Most SNVs (171,558 SNVs; 90.31%) were novel when compared with
over 1.1 million equine SNPs from two SNP databases. Using differential
expression analysis, we further identified a number of exercise-regulated genes:
62 up-regulated and 80 down-regulated genes in the blood, and 878 up-regulated
and 285 down-regulated genes in the muscle. Six of 28 previously-known
exercise-related genes were over-expressed in the muscle after exercise. Among
the differentially expressed genes, there were 91 transcription factor-encoding
genes, which included 56 functionally unknown transcription factor candidates
that are probably associated with an early regulatory exercise mechanism. In
addition, we found interesting RNA expression patterns where different
alternative splicing forms of the same gene showed reversed expressions before
and after exercising.
CONCLUSION: The first sequencing-based horse transcriptome data, extensive
analyses results, deferentially expressed genes before and after exercise, and
candidate genes that are related to the exercise are provided in this study. To construct a regulatory map of the genome of the human pathogen, Mycobacterium
tuberculosis, we applied two complementary high-resolution approaches:
strand-specific RNA-seq, to survey the global transcriptome, and ChIP-seq, to
monitor the genome-wide dynamics of RNA polymerase (RNAP) and the
anti-terminator NusA. Although NusA does not bind directly to DNA, but rather to
RNAP and/or to the nascent transcript, we demonstrate that NusA interacts with
RNAP ubiquitously throughout the chromosome, and that its profile mirrors RNAP
distribution in both the exponential and stationary phases of growth. Generally,
promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease
in signal strength reflecting transcriptional polarity. Differential binding of
RNAP and NusA in the two growth conditions correlated with transcriptional
activity as reflected by RNA abundance. Indeed, a significant association
between expression levels and the presence of NusA throughout the gene body was
detected, confirming the peculiar transcription-promoting role of NusA.
Integration of the data sets pinpointed transcriptional units, mapped promoters
and uncovered new anti-sense and non-coding transcripts. Highly expressed
transcriptional units were situated mainly on the leading strand, despite the
relatively unbiased distribution of genes throughout the genome, thus helping
the replicative and transcriptional complexes to align. |
Is macroautophagy a selective degradation process? | Yes. Macroautophagy (commonly referred to simply as autophagy) is a catabolic process conserved throughout the eukaryotes, and is distinct from other forms of autophagy by the formation of the autophagosome: this is a vesicle-like formation surrounded by a double membrane that sequesters the cytoplasmic material to be degraded. It was initially considered that macroautophagy was a bulk process; however, recent findings illustrate that specific cargos (ranging from misfolded or excess proteins, to organelles or even bacterial cells) can be selectively targeted to the pre-autophagosome membrane (i.e. the phagophore) and finally to the vacuoles/lysosomes for their degradation. | The rate of proteolysis is an important determit of the intracellular protein
content. Part of the degradation of intracellular proteins occurs in the
lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very
active and almost completely accounts for starvation-induced proteolysis.
Factors inhibiting this process include amino acids, cell swelling and insulin.
In the mechanisms controlling macroautophagy, protein phosphorylation plays an
important role. Activation of a signal transduction pathway, ultimately leading
to phosphorylation of ribosomal protein S6, accompanies inhibition of
macroautophagy. Components of this pathway may include a heterotrimeric
Gi3-protein, phosphatidylinositol 3-kinase and p70S6 kinase. Recent evidence
indicates that lysosomal protein degradation can be selective and occurs via
ubiquitin-dependent and -independent pathways. Three overlapping pathways mediate the transport of cytoplasmic material to the
vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt)
pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas
pexophagy mediates the delivery of excess peroxisomes for degradation. Both the
Cvt and pexophagy pathways are selective processes that specifically recognize
their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol
to the vacuole for recycling. Most of the import machinery characterized thus
far is required for all three modes of transport. However, unique features of
each pathway dictate the requirement for additional components that
differentiate these pathways from one another, including at the step of specific
cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart
Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor
API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of
peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced
pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for
starvation-induced nonselective transport of bulk cytoplasmic cargo by
macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the
membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct
role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally
associate with a novel, perivacuolar membrane compartment and interact with
Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P.
pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that
contact peroxisomes in the process of being engulfed by pexophagy. These
biochemical and morphological results demonstrate that Cvt9 and the P. pastoris
homologue Gsa9 may function at the step of selective cargo sequestration. Eukaryotic cells have the ability to degrade proteins and organelles by
selective and nonselective modes of micro- and macroautophagy. In addition,
there exist both constitutive and regulated forms of autophagy. For example,
pexophagy is a selective process for the regulated degradation of peroxisomes by
autophagy. Our studies have shown that the differing pathways of autophagy have
many molecular events in common. In this article, we have identified a new
member in the family of autophagy genes. GSA12 in Pichia pastoris and its
Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two
WD40 domains. We have shown that these proteins are required for pexophagy and
autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S.
cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in
pexophagy. That is, the involution of the vacuole or extension of vacuole arms
to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with
its role in vacuole engulfment, we have found that this cytosolic protein is
also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular
localization that distinguishes it from the characterized proteins required for
cytoplasm-to-vacuole delivery pathways. Macroautophagy, which is a lysosomal pathway for the turnover of organelles and
long-lived proteins, is a key determit of cell survival and longevity. In
this study, we show that neuronal macroautophagy is induced early in Alzheimer's
disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the
presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis.
Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate
markedly in dystrophic dendrites, implying an impaired maturation of AVs to
lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of
intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are
highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity.
Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by
modulating mammalian target of rapamycin kinase elicits parallel changes in AV
proliferation and Abeta production. Our results, therefore, link
beta-amyloidogenic and cell survival pathways through macroautophagy, which is
activated and is abnormal in AD. ATG genes encode proteins that are required for macroautophagy, the Cvt pathway
and/or pexophagy. Using the published Atg protein sequences, we have screened
protein and DNA databases to identify putative functional homologs (orthologs)
in 21 fungal species (yeast and filamentous fungi) of which the genome sequences
were available. For comparison with Atg proteins in higher eukaryotes, also an
analysis of Arabidopsis thaliana and Homo sapiens databases was included. This
analysis demonstrated that Atg proteins required for non-selective
macroautophagy are conserved from yeast to man, stressing the importance of this
process in cell survival and viability. The A. thaliana and human genomes encode
multiple proteins highly similar to specific fungal Atg proteins (paralogs),
possibly representing cell type-specific isoforms. The Atg proteins specifically
involved in the Cvt pathway and/or pexophagy showed poor conservation, and were
generally not present in A. thaliana and man. Furthermore, Atg19, the receptor
of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless,
Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex)
to the autophagic machinery was identified in all yeast species and filamentous
fungi under study. This suggests that in fungi an organism-specific form of
selective autophagy may occur, for which specialized Atg proteins have evolved. Throughout their life, cells must maintain homeostasis while facing constantly
fluctuating demands on their different organelles. A major mechanism for the
homeostatic control of organelle function is the unfolded protein response
(UPR), a signaling pathway that triggers a comprehensive remodeling of the
endoplasmic reticulum (ER) and the biosynthetic pathway according to need. We
discovered that activation of the UPR in yeast also induces a new branch of
macroautophagy that selectively targets the ER. We term this process "ER-phagy",
in analogy to pexophagy and mitophagy, the two other known forms of
organelle-specific marcoautophagy. ER-phagy involves the generation of
autophagosomes that selectively include ER membranes and whose delimiting double
membranes also derive, at least in part, from the ER. This finding provides
direct evidence that the ER can serve as a membrane source for autophagosome
formation and indicates that ER-phagy entails engulfment of the ER by itself.
ER-phagy could remove damaged or redundant parts of the ER and thus represent an
important degradative functionality of the UPR that helps to afford homeostatic
control. Growing evidence supports an active role for dysregulated macroautophagy
(autophagic stress) in neuronal cell death and neurodegeneration. Alterations in
mitochondrial function and dynamics are also strongly implicated in
neurodegenerative diseases. Interestingly, whereas the core autophagy machinery
is evolutionarily conserved and shared among constitutive and induced or
selective autophagy, recent studies implicate distinct mechanisms regulating
mitochondrial autophagy (mitophagy) in response to general autophagic stimuli.
Little is known about pathways regulating selective, damage-induced mitophagy.
We found that the parkinsonian neurotoxin MPP(+) induces autophagy and
mitochondrial degradation that is inhibited by siRNA knockdown of autophagy
proteins Atg5, Atg7 and Atg8, but occurs independently of Beclin 1, a component
of the class III (PIK3C3/Vps34) phosphoinositide 3-kinase (PI3K) complex.
Instead, MPP(+)-induced mitophagy is dependent upon MAPK signaling.
Interestingly, all treatments that inhibited autophagy also conferred protection
from MPP(+)-induced cell death. A prior human tissue study further supports a
role for ERK/MAPK-regulated autophagy in Parkinson's and Lewy body diseases. As
competition for limiting amounts of Beclin 1 may serve to prevent harmful
overactivation of autophagy, understanding mechanisms that bypass or complement
a requirement for PI3K-Beclin 1 activity could lead to strategies to modulate
autophagic stress in injured or degenerating neurons. Autophagy is a cellular process whose primary function is to degrade long-lived
proteins and recycle cellular components. Beside macroautophagy, there are
several forms of selective autophagy, including chaperone-mediated autophagy
(CMA), cytoplasm to vacuole targeting (Cvt), pexophagy and mitophagy. In this
review, we summarize what is currently known about selective autophagy, and
discuss its role in cell death and survival. We also discuss possible mechanisms
underlying the selectivity of macroautophagy. Production of a red blood cell's hemoglobin depends on mitochondrial heme
synthesis. However, mature red blood cells are devoid of mitochondria and rely
on glycolysis for ATP production. The molecular basis for the selective
elimination of mitochondria from mature red blood cells remains controversial.
Recent evidence suggests that clearance of both mitochondria and ribosomes,
which occurs in reticulocytes following nuclear extrusion, depends on autophagy.
Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast
atg1p, is a critical regulator of mitochondrial and ribosomal clearance during
the final stages of erythroid maturation. However, in contrast to the core
autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for
induction of macroautophagy in response to nutrient deprivation or for survival
of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is
a component of the selective autophagy machinery that leads to the elimination
of organelles in erythroid cells rather that an essential mechanistic component
of autophagy. Mitochondrial quality control is important in maintaining proper cellular
homeostasis. Although selective mitochondrial degradation by autophagy
(mitophagy) is suggested to have an important role in quality control, and
though there is evidence for a direct relation between mitophagy and
neurodegenerative diseases, the molecular mechanism of mitophagy is poorly
understood. Using a screen for mitophagy-deficient mutants, we found that
YIL146C/ECM37 is essential for mitophagy. This gene is not required for other
types of selective autophagy or for nonspecific macroautophagy. We designated
this autophagy-related (ATG) gene as ATG32. The Atg32 protein localizes on
mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an
adaptor protein for selective types of autophagy, and is then recruited to and
imported into the vacuole along with mitochondria. Therefore, Atg32 confers
selectivity for mitochondrial sequestration as a cargo and is necessary for
recruitment of this organelle by the autophagy machinery for mitophagy. To cause rice blast disease, the fungus Magnaporthe oryzae elaborates
specialized infection structures called appressoria, which use enormous turgor
to rupture the tough outer cuticle of a rice leaf. Here, we report the
generation of a set of 22 isogenic M. oryzae mutants each differing by a single
component of the predicted autophagic machinery of the fungus. Analysis of this
set of targeted deletion mutants demonstrated that loss of any of the 16 genes
necessary for nonselective macroautophagy renders the fungus unable to cause
rice blast disease, due to impairment of both conidial programmed cell death and
appressorium maturation. In contrast, genes necessary only for selective forms
of autophagy, such as pexophagy and mitophagy, are dispensable for
appressorium-mediated plant infection. A genome-wide analysis therefore
demonstrates the importance of infection-associated, nonselective autophagy for
the establishment of rice blast disease. Mitophagy is the process of selective mitochondrial degradation via autophagy,
which has an important role in mitochondrial quality control. Very little is
known, however, about the molecular mechanism of mitophagy. A genome-wide yeast
mutant screen for mitophagy-defective strains identified 32 mutants with a block
in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We
further characterized one of these mutants, ylr356wDelta that corresponds to a
gene whose function has not been identified. YLR356W is a mitophagy-specific
gene that was not required for other types of selective autophagy or
macroautophagy. The deletion of YLR356W partially inhibited mitophagy during
starvation, whereas there was an almost complete inhibition at post-log phase.
Accordingly, we have named this gene ATG33. The new mutants identified in this
analysis will provide a useful foundation for researchers interested in the
study of mitochondrial homeostasis and quality control. Macroautophagy was thought to be an unspecific bulk degradation process.
However, Ponpuak et al. (2010) show in this issue of Immunity that cytosolic
proteins are selectively recruited to autophagosomes to become metabolized to
bactericidal peptides. Macroautophagy (hereafter autophagy) is a cellular degradation process, which in
yeast is induced in response to nutrient deprivation. In this process, a
double-membrane vesicle, an autophagosome, surrounds part of the cytoplasm and
fuses with the vacuole to allow the breakdown and subsequent recycling of the
cargo. In yeast, many autophagy-related (ATG) genes have been identified that
are required for selective and/or nonselective autophagy. In all
autophagy-related pathways, core Atg proteins are required for the formation of
the autophagosome, which is one of the most unique aspects of autophagy and is
unlike other vesicle transport events. In contrast to nonselective autophagy,
the selective processes are induced in response to various specific
physiological conditions such as alterations in the carbon source. In this
review, we provide an overview of the common aspects concerning the mechanism of
autophagy-related pathways, and highlight recent advances in our understanding
of the machinery that controls autophagy induction in response to nutrient
starvation conditions. Mitochondria autophagy (mitophagy) is the process of selective degradation of
mitochondria that has an important role in mitochondrial quality control. To
gain insight into the molecular mechanism of mitophagy, we screened a yeast
knockout library for strains that are defective in mitophagy. We found 32
strains that showed a complete or partial block of mitophagy. One of the genes
identified, YLR356W, is required for mitophagy, but not for macroautophagy or
other types of selective autophagy. The deletion of YLR356W partially inhibits
mitophagy during starvation, whereas there is almost complete inhibition at
post-log phase. Accordingly, we hypothesize that Ylr356w is required to detect
or present aged or dysfunctional mitochondria when cells reach the post-log
phase. Dynamic regulation of cell shape underlies many developmental and immune
functions. Cortical remodeling is achieved under the central control of Rho
GTPase pathways that modulate an exquisite balance in the dynamic assembly and
disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy),
associated with bulk cytoplasmic remodeling through lysosomal degradation, has
clearly defined roles in cell survival and death. Moreover, it is becoming
apparent that proteins, organelles, and pathogens can be targeted for autophagic
clearance by selective mechanisms, although the extent and roles of such
degradation are unclear. Here we report a conserved role for autophagy
specifically in the cortical remodeling of Drosophila blood cells (hemocytes)
and mouse macrophages. Continuous autophagy was required for integrin-mediated
hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes
disrupted for autophagy were impaired in their recruitment to epidermal wounds.
Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating
selective autophagy as a novel mechanism for modulating cortical dynamics. These
results illuminate a specific and conserved role for autophagy as a regulatory
mechanism for cortical remodeling, with implications for immune cell function. BACKGROUND & AIMS: Alcohol abuse is a major cause of liver injury. The
pathologic features of alcoholic liver disease develop over prolonged periods,
yet the cellular defense mechanisms against the detrimental effects of alcohol
are not well understood. We investigated whether macroautophagy, an
evolutionarily conserved cellular mechanism that is commonly activated in
response to stress, could protect liver cells from ethanol toxicity.
METHODS: Mice were acutely given ethanol by gavage. The effects of ethanol on
primary hepatocytes and hepatic cell lines were also studied in vitro.
RESULTS: Ethanol-induced macroautophagy in the livers of mice and cultured cells
required ethanol metabolism, generation of reactive oxygen species, and
inhibition of mammalian target of rapamycin signaling. Suppression of
macroautophagy with pharmacologic agents or small interfering RNAs significantly
increased hepatocyte apoptosis and liver injury; macroautophagy therefore
protected cells from the toxic effects of ethanol. Macroautophagy induced by
ethanol seemed to be selective for damaged mitochondria and accumulated lipid
droplets, but not long-lived proteins, which could account for its protective
effects. Increasing macroautophagy pharmacologically reduced hepatotoxicity and
steatosis associated with acute ethanol exposure.
CONCLUSIONS: Macroautophagy protects against ethanol-induced toxicity in livers
of mice. Reagents that modify macroautophagy might be developed as therapeutics
for patients with alcoholic liver disease. Mitochondrial dysfunction is an early sign of many neurodegenerative diseases.
Very recently, two Parkinson disease (PD) associated genes, PINK1 and Parkin,
were shown to mediate the degradation of damaged mitochondria via selective
autophagy (mitophagy). PINK1 kinase activity is needed for prompt and efficient
Parkin recruitment to impaired mitochondria. PD-associated Parkin mutations
interfere with the process of mitophagy at distinct steps. Here we show that
whole mitochondria are turned over via macroautophagy. Moreover,
disease-associated PINK1 mutations also compromise the selective degradation of
depolarized mitochondria. This may be due to the decreased physical binding
activity of PD-linked PINK1 mutations to Parkin. Thus, PINK1 mutations abrogate
autophagy of impaired mitochondria upstream of Parkin. In addition to
compromised PINK1 kinase activity, reduced binding of PINK1 to Parkin leads to
failure in Parkin mitochondrial translocation, resulting in the accumulation of
damaged mitochondria, which may contribute to disease pathogenesis. Macroautophagy is a bulk degradation system conserved in all eukaryotic cells. A
ubiquitin-like protein, Atg8, and its homologues are essential for autophagosome
formation and act as a landmark for selective autophagy of aggregated proteins
and damaged organelles. In this study, we report evidence demonstrating that
OATL1, a putative Rab guanosine triphosphatase-activating protein (GAP), is a
novel binding partner of Atg8 homologues in mammalian cells. OATL1 is recruited
to isolation membranes and autophagosomes through direct interaction with Atg8
homologues and is involved in the fusion between autophagosomes and lysosomes
through its GAP activity. We further provide evidence that Rab33B, an
Atg16L1-binding protein, is a target substrate of OATL1 and is involved in the
fusion between autophagosomes and lysosomes, the same as OATL1. Because both its
GAP activity and its Atg8 homologue-binding activity are required for OATL1 to
function, we propose a model that OATL1 uses Atg8 homologues as a scaffold to
exert its GAP activity and to regulate autophagosomal maturation. Constitutive macroautophagy involved in the turnover of defective long-lived
proteins and organelles is crucial for neuronal homeostasis. We hypothesized
that macroautophagic dysregulation in selective brain regions was associated
with memory impairment in aged mice. We used the single-trial object recognition
test to measure short-term memory in 18 aged mice compared to 22 young mice and
employed immunohistochemistry to assess cellular distribution of proteins
involved in the selective degradation of ubiquitinated proteins via
macroautophagy. Values of the discrimination ratio (DR, a measure of short-term
recognition memory performance) in aged mice were significantly lower than those
in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in
aged mice, there were clusters of puncta immunoreactive for
microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding
protein p62, and ubiquitin in neuronal processes predomitly in the
hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The
hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited
inverse linear correlations with DR in aged mice (ρ = -0.48 and -0.55, p = 0.044
and 0.018, respectively, Spearman's rank correlation). These findings suggest
that increased accumulation of autophagosomes within neuronal processes in
selective brain regions is characteristic of aging. The dysregulation of
macroautophagy can adversely affect the turnover of aggregate-prone proteins and
defective organelles, which may contribute to memory impairment in aged mice. Macroautophagy (hereafter referred to simply as autophagy) is a catabolic
pathway that mediates the degradation of long-lived proteins and organelles in
eukaryotic cells. The regulation of mitochondrial degradation through autophagy
plays an essential role in the maintece and quality control of this
organelle. Compared with our understanding of the essential function of
mitochondria in many aspects of cellular metabolism such as energy production
and of the role of dysfunctional mitochondria in cell death, little is known
regarding their degradation and especially how upstream signaling pathways
control this process. Here, we report that two mitogen-activated protein kinases
(MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae.
Slt2 is required for the degradation of both mitochondria and peroxisomes (via
pexophagy), whereas Hog1 functions specifically in mitophagy. Slt2 also affects
the recruitment of mitochondria to the phagophore assembly site (PAS), a
critical step in the packaging of cargo for selective degradation. There is a reciprocal change in the expression of two members of the BAG
(Bcl-2-associated athanogen) family, BAG1 and BAG3, during cellular aging and
under acute stress ("BAG1-BAG3-switch"). BAG3 was recently described as a
mediator of a novel macroautophagy pathway that uses the specificity of heat
shock protein 70 (HSP70) to misfolded proteins and also involves other protein
partners, such as HSPB8. Also crucial for induction and execution of autophagy
are sequestosome-1/p62 (SQSTM1/p62) and LC3, an autophagosome-associated
protein. In this novel pathway, BAG3 mediates the targeting and transport of
degradation-prone substrates into aggresomes via the microtubule-motor dynein.
Interestingly, aggresome-targeting by BAG3 does not depend on substrate
ubiquitination and is, therefore, involved in the clearance of misfolded
proteins that are not ubiquitinated. Macroautophagy maintains cellular homeostasis through targeting cytoplasmic
contents and organelles into autophagosomes for degradation. This process begins
with the assembly of protein complexes on isolation membrane to initiate the
formation of autophagosome, followed by its nucleation, elongation and
maturation. Fusion of autophagosomes with lysosomes then leads to degradation of
the cargo. In the past decade, significant advances have been made on the
identification of molecular players that are implicated in various stages of
macroautophagy. Post-translational modifications of macroautophagy regulators
have also been demonstrated to be critical for the selective targeting of
cytoplasmic contents into autophagosomes. In addition, recent demonstration of
distinct macroautophagy regulators has led to the identification of different
subtypes of macroautophagy. Since deregulation of macroautophagy is implicated
in diseases including neurodegenerative disorders, cancers and inflammatory
disorders, understanding the molecular machinery of macroautophagy is crucial
for elucidating the mechanisms by which macroautophagy is deregulated in these
diseases, thereby revealing new potential therapeutic targets and strategies.
Here we summarize current knowledge on the regulation of mammalian
macroautophagy machineries and their disease-associated deregulation. Macroautophagy is a catabolic process by which the cell degrades cytoplasmic
components through the lysosomal machinery. While initially acknowledged as a
rather unspecific bulk degradation process, growing lines of evidence indicate
the selectivity of macroautophagy pathways in the removal of misfolded or
aggregated proteins. How such substrates are recognized and specifically
targeted to the macroautophagy machinery has become a hotspot of investigation,
and recent evidence suggests that here molecular chaperones and co-chaperones
play a central role. One emerging pathway is mediated by the co-chaperone
protein Bcl-2-associated athanogene 3 (BAG 3) which seems to utilize the
specificity of molecular chaperones (heat-shock proteins) towards non-native
proteins as basis for targeted macroautophagic degradation. In this short
review, we focus on the molecular interplay between the macroautophagy system
and molecular chaperones and highlight the relevance of the pathway mediated by
BAG3 to aging and age-associated protein-misfolding diseases. Macroautophagy (autophagy) is a bulk degradation system for cytoplasmic
components and is ubiquitously found in eukaryotic cells. Autophagy is induced
under starvation conditions and plays a cytoprotective role by degrading
unwanted cytoplasmic materials. The Ty1 transposon, a member of the Ty1/copia
superfamily, is the most abundant retrotransposon in the yeast Saccharomyces
cerevisiae and acts to introduce mutations in the host genome via Ty1 virus-like
particles (VLPs) localized in the cytoplasm. Here we show that selective
autophagy downregulates Ty1 transposition by eliminating Ty1 VLPs from the
cytoplasm under nutrient-limited conditions. Ty1 VLPs are targeted to
autophagosomes by an interaction with Atg19. We propose that selective autophagy
safeguards genome integrity against excessive insertional mutagenesis caused
during nutrient starvation by transposable elements in eukaryotic cells. Proper control of mitochondrial turnover is critical for maintece of cellular
energetics under basal and stressed conditions, and for prevention of endogenous
oxidative stress. Whole organelle turnover is mediated through macroautophagy, a
process by which autophagosomes deliver mitochondria to the lysosome for
hydrolytic degradation. While mitochondrial autophagy can occur as part of a
nonselective upregulation of autophagy, selective degradation of damaged or
unneeded mitochondria (mitophagy) is a rapidly growing area in development,
cancer, and neurodegeneration, particularly with regard to Parkinson's disease.
Due to its dynamic nature, and the potential for regulatory perturbation by
disease processes, no single technique is sufficient to evaluate mitophagy.
Here, we describe several complementary techniques that include electron
microscopy, single cell analysis of LC3 fluorescent puncta, and Western blot,
each used in conjunction with a flux inhibitor to trap newly formed
autophagosomes in order to monitor mitophagy in neuronal cells. During autophagy, cytosol, protein aggregates, and organelles are sequestered
into double-membrane vesicles called autophagosomes and delivered to the
lysosome/vacuole for breakdown and recycling of their basic components. In all
eukaryotes this pathway is important for adaptation to stress conditions such as
nutrient deprivation, as well as to regulate intracellular homeostasis by
adjusting organelle number and clearing damaged structures. For a long time,
starvation-induced autophagy has been viewed as a nonselective transport
pathway; however, recent studies have revealed that autophagy is able to
selectively engulf specific structures, ranging from proteins to entire
organelles. In this paper, we discuss recent findings on the mechanisms and
physiological implications of two selective types of autophagy: ribophagy, the
specific degradation of ribosomes, and reticulophagy, the selective elimination
of portions of the ER. Autophagy (macroautophagy), a highly conserved eukaryotic mechanism, is a
non-selective degradation process, helping to maintain a balance between the
synthesis, degradation and subsequent recycling of macromolecules to overcome
various stress conditions. The term autophagy denotes any cellular process which
involves the delivery of cytoplasmic material to the lysosome for degradation.
Autophagy, in filamentous fungi plays a critical role during cellular
development and pathogenicity. Autophagy, like the mitogen-activated protein
(MAP) kinase cascade and nutrient-sensing cyclic AMP (cAMP) pathway, is also an
important process for appressorium turgor accumulation in order to penetrate the
leaf surface of host plant and destroy the plant defense. Yeast, an autophagy
model, has been used to compare the multi-valued functions of ATG
(autophagy-related genes) in different filamentous fungi. The autophagy
machinery in both yeast and filamentous fungi is controlled by Tor kinase and
both contain two distinct phosphatidylinositol 3-kinase complexes. In this
review, we focus on the functions of ATG genes during pathogenic development in
filamentous fungi. The sorting nexins Atg20/Snx42 and Snx41 regulate membrane traffic and endosomal
protein sorting and are essential for Cvt and/or pexophagy in yeast. Previously,
we showed that macroautophagy is necessary for conidiation in the rice-blast
fungus Magnaporthe oryzae. Here, we analyzed the physiological function(s) of
selective autophagy in Magnaporthe through targeted deletion of MGG_12832, an
ortholog of yeast SNX41 and ATG20/SNX42. Loss of MGG_12832 (hereafter SNX41)
abolished conidia formation and pathogenesis in M. oryzae. Snx41-GFP localized
as dynamic puncta or short tubules that are partially associated with
autophagosomes and/or autophagic vacuoles. PX domain, but not macroautophagy per
se, was required for such localization of Snx41-GFP in Magnaporthe. Although not
required for nonselective autophagy, Snx41 was essential for pexophagy in
Magnaporthe. We identified Oxp1, an ATP-dependent oxoprolinase in the
gamma-glutamyl cycle, as a binding partner and potential retrieval target of
Snx41-dependent protein sorting. The substrate of Oxp1, 5-oxoproline, could
partially restore conidiation in the snx41Δ. Exogenous glutathione, a product of
the gamma-glutamyl cycle, significantly restored pathogenicity in the snx41Δ
mutant, likely through counteracting the oxidative stress imposed by the host.
We propose that the gamma-glutamyl cycle and glutathione biosynthesis are
subject to regulation by Snx41-dependent vesicular trafficking, and mediate
antioxidant defense crucial for in planta growth and pathogenic differentiation
of Magnaporthe at the onset of blast disease in rice. Selective macroautophagy uses double-membrane vesicles, termed autophagosomes,
to transport cytoplasmic pathogens, organelles and protein complexes to the
vacuole for degradation. Autophagosomes are formed de novo by membrane fusion
events at the phagophore assembly site (PAS). Therefore, precursor membrane
material must be targeted and transported to the PAS. While some
autophagy-related (Atg) proteins, such as Atg9 and Atg11, are known to be
involved in this process, most of the mechanistic details are not understood.
Previous work has also implicated the small Rab-family GTPase Ypt1 in the
process, identifying Trs85 as a unique subunit of the TRAPPIII targeting complex
and showing that it plays a macroautophagy-specific role; however, the
relationship between Ypt1, Atg9 and Atg11 was not clear. Now, a recent report
shows that Atg11 is a Trs85-specific effector of the Rab Ypt1, and may act as a
classic coiled-coil membrane tether that targets Atg9-containing membranes to
the PAS. Here, we review this finding in the context of what is known about
Atg11, other Rab-dependent coiled-coil tethers, and other tethering complexes
involved in autophagosome formation. Macroautophagy (hereafter autophagy) is a degradative cellular pathway that
protects eukaryotic cells from stress, starvation, and microbial infection. This
process must be tightly controlled because too little or too much autophagy can
be deleterious to cellular physiology. The phosphatidylinositol (PtdIns)
3-kinase Vps34 is a lipid kinase that regulates autophagy, but the role of other
PtdIns kinases has not been examined. Here we demonstrate a role for PtdIns
4-kinases and PtdIns4P 5-kinases in selective and nonselective types of
autophagy in yeast. The PtdIns 4-kinase Pik1 is involved in Atg9 trafficking
through the Golgi and is involved in both nonselective and selective types of
autophagy, whereas the PtdIns4P 5-kinase Mss4 is specifically involved in
mitophagy but not nonselective autophagy. Our data indicate that
phosphoinositide kinases have multiple roles in the regulation of autophagic
pathways. The current working definition of autophagy is the following: all processes in
which intracellular material is degraded within the lysosome/vacuole and where
the macromolecular constituents are recycled. There are several ways to classify
the different types of autophagy. For example, we can separate autophagy into
two primary types, based on the initial site of cargo sequestration. In
particular, during microautophagy and chaperone-mediated autophagy, uptake
occurs directly at the limiting membrane of the lysosome or vacuole. In
contrast, macroautophagy-whether selective or nonselective-and endosomal
microautophagy involve sequestration within an autophagosome or an omegasome, or
late endosomes/multivesicular bodies, respectively; the key point being that in
these types of autophagy the initial sequestration event does not occur at the
limiting membrane of the degradative organelle. In any case, the cargo is
ultimately delivered into the lysosome or vacuole lumen for subsequent
degradation. Thus, I think most autophagy researchers view the degradative
organelle as the ultimate destination of the pathway. Indeed, this fits with the
general concept that organelles allow reactions to be compartmentalized. With
regard to the lysosome or vacuole, this also confers a level of safety by
keeping the lytic contents away from the remainder of the cell. If we are
willing to slightly modify our definition of autophagy, with a focus on
"degradation of a cell's own components through the lysosomal/vacuolar
machinery," we can include a newly documented process, programmed nuclear
destruction (PND). Macroautophagy is a cellular catabolic process that involves the sequestration
of cytoplasmic constituents into double-membrane vesicles known as
autophagosomes, which subsequently fuse with lysosomes, where they deliver their
cargo for degradation. The main physiological role of autophagy is to recycle
intracellular components, under conditions of nutrient deprivation, so as to
supply cells with vital materials and energy. Selective autophagy also takes
place in nutrient-rich conditions to rid the cell of damaged organelles or
protein aggregates that would otherwise compromise cell viability. Mitophagy is
a selective type of autophagy, whereby damaged or superfluous mitochondria are
eliminated to maintain proper mitochondrial numbers and quality control. While
mitophagy shares key regulatory factors with the general macroautophagy pathway,
it also involves distinct steps, specific for mitochondrial elimination. Recent
findings indicate that parkin and the phosphatase and tensin homolog-induced
putative kinase protein 1 (PINK1), which have been implicated in the
pathogenesis of neurodegenerative diseases such as Parkinson's disease, also
regulate mitophagy and function to maintain mitochondrial homeostasis. Here, we
survey the molecular mechanisms that govern the process of mitophagy and discuss
its involvement in the onset and progression of neurodegenerative diseases
during aging. |
Inhibition of which enzyme is mechanism of action of alisertib? | Alisertib (MLN8237) is selective Aurora kinase inhibitor that acts by interfering with spindle organization and chromosome alignment during mitosis. It has been tested in patients with gastric cancer, breast cancer, relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas, epithelial ovarian, fallopian tube, and primary peritoneal carcinoma. | The Aurora kinases (serine/threonine kinases) were discovered in 1995 during
studies of mutant alleles associated with abnormal spindle pole formation in
Drosophila melanogaster. They soon became the focus of much attention because of
their importance in human biology and association with cancer. Aurora kinases
are essential for cell division and are primarily active during mitosis.
Following their identification as potential targets for cancer chemotherapy,
many Aurora kinase inhibitors have been discovered, and are currently under
development. The binding modes of Aurora kinase inhibitors to Aurora kinases
share specific hydrogen bonds between the inhibitor core and the back bone of
the kinase hinge region, while others parts of the molecules may point to
different parts of the active site via noncovalent interactions. Currently there
are about 30 Aurora kinase inhibitors in different stages of pre-clinical and
clinical development. This review summarizes the characteristics and status of
Aurora kinase inhibitors in preclinical, Phase I, and Phase II clinical studies,
with particular emphasis on the mechanisms of action and resistance to these
promising anticancer agents. We also discuss the validity of Aurora kinases as
oncology targets, on/off-target toxicities, and other important aspects of
overall clinical performance and future of Aurora kinase inhibitors. Novel therapies are urgently needed to improve clinical outcomes for patients
with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237)
is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II
studies. We investigated the preclinical efficacy and pharmacodynamics of
alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we
report that alisertib disrupted cell viability, diminished clonogenic survival,
induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A
link between Aurora A expression and sensitivity to ara-C was established,
suggesting that Aurora A inhibition may be a promising strategy to increase the
efficacy of ara-C. Accordingly, alisertib significantly potentiated the
antileukemic activity of ara-C in both AML cell lines and primary blasts.
Targeted FOXO3a knockdown significantly blunted the pro-apoptotic effects of the
alisertib/ara-C combination, indicating that it is an important regulator of
sensitivity to these agents. In vivo studies demonstrated that alisertib
significantly augmented the efficacy of ara-C without affecting its
pharmacokinetic profile and led to the induction of p27 and BIM. Our collective
data indicate that targeting Aurora A with alisertib represents a novel approach
to increase the efficacy of ara-C that warrants further investigation. PURPOSE: Aurora A kinase (AAK) is a key regulator of mitosis and a target for
anticancer drug development. This phase I study investigated the safety,
pharmacokinetics, and pharmacodynamics of MLN8237 (alisertib), an
investigational, oral, selective AAK inhibitor, in 59 adults with advanced solid
tumors.
EXPERIMENTAL DESIGN: Patients received MLN8237 once daily or twice daily for 7,
14, or 21 consecutive days, followed by 14 days recovery, in 21-, 28-, or 35-day
cycles. Dose-limiting toxicities (DLT) and the maximum-tolerated dose (MTD) for
the 7- and 21-day schedules were determined. Pharmacokinetic parameters were
derived from plasma concentration-time profiles. AAK inhibition in skin and
tumor biopsies was evaluated and antitumor activity assessed.
RESULTS: Neutropenia and stomatitis were the most common DLTs. The MTD for the
7- and 21-day schedules was 50 mg twice daily and 50 mg once daily,
respectively. MLN8237 absorption was fast (median time to maximum concentration,
2 hours). Mean terminal half-life was approximately 19 hours. At steady state,
pharmacodynamic effects were shown by accumulation of mitotic and apoptotic
cells in skin, and exposure-related increases in numbers of mitotic cells with
characteristic spindle and chromosomal abnormalities in tumor specimens,
supporting AAK inhibition by MLN8237. Stable disease was observed and was
durable with repeat treatment cycles, administered over 6 months, in 6 patients,
without notable cumulative toxicity.
CONCLUSIONS: The recommended phase II dose of MLN8237 is 50 mg twice daily on
the 7-day schedule, which is being evaluated further in a variety of
maligcies, including in a phase III trial in peripheral T-cell lymphoma. The mechanism by which cells decide to skip mitosis to become polyploid is
largely undefined. Here we used a high-content image-based screen to identify
small-molecule probes that induce polyploidization of megakaryocytic leukemia
cells and serve as perturbagens to help understand this process. Our study
implicates five networks of kinases that regulate the switch to polyploidy.
Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased
polyploidization, mature cell-surface marker expression, and apoptosis of
maligt megakaryocytes. An integrated target identification approach employing
proteomic and shRNA screening revealed that a major target of diMF is Aurora
kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective
inhibitor of AURKA, induced polyploidization and expression of mature
megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and
displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to
support clinical trials of MLN8237 and other inducers of polyploidization and
differentiation in AMKL. We investigated the effects of targeting the mitotic regulators aurora kinase A
and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia
(AML). Aurora protein expression levels in pediatric ALL and AML patient samples
were determined by western blot and reverse phase protein array. Both kinases
were overexpressed in ALL and AML patients (P<0.0002), especially in
E2A-PBX1-translocated ALL cases (P<0.002), compared with normal bone-marrow
mononuclear cells. Aurora kinase expression was silenced in leukemic cell lines
using short hairpin RNAs and locked nucleic acid-based mRNA antagonists. Aurora
B knockdown resulted in proliferation arrest and apoptosis, whereas aurora A
knockdown caused no or only minor growth delay. Most tested cell lines were
highly sensitive to the AURKB-selective inhibitor
barasertib-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA) in the omolar
range, as tested with an MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
assay. But most importantly, primary ALL cells with a high aurora B protein
expression, especially E2A-PBX1-positive cases, were sensitive as well. In adult
AML early clinical trials, clear responses are observed with barasertib. Here we
show that inhibition of aurora B, more than aurora A, has an antiproliferative
and pro-apoptotic effect on acute leukemia cells, indicating that particularly
targeting aurora B may offer a new strategy to treat pediatric ALL and AML. BACKGROUND: Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to
current chemotherapeutic regimes. The authors and others have previously
reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and
protein overexpression in UGCs. The objective of the current study was to
determine the therapeutic potential of alisertib (MLN8237) alone and in
combination with docetaxel in UGCs.
METHODS: After treatment with alisertib and/or docetaxel, clonogenic cell
survival, cell cycle analyses, Western blot analyses, and tumor xenograft growth
assays were carried out to measure cell survival, cell cycle progression,
apoptotic protein expression, and tumor xenograft volumes, respectively.
RESULTS: By using the AGS, FLO-1, and OE33 UGC cell lines, which have
constitutive AURKA overexpression and variable tumor protein 53 (p53) status,
significantly enhanced inhibition of cancer cell survival was observed with
alisertib and docetaxel treatment in combination (P < .001), compared with
single-agent treatments. Cell cycle analyses, after 48 hours of treatment with
alisertib, produced a significant increase in the percentage of polyploidy in
UGC cells (P < .01) that was further enhanced by docetaxel (P < .001). In
addition, an increase in the percentage of cells in sub-G1-phase observed with
alisertib (P < .01) was significantly enhanced with the combination treatment (P
< .001). Western blot analysis demonstrated higher induction of cleaved caspase
3 protein expression with the combined treatment compared with single-agent
treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated
enhanced antitumor activity for the alisertib and docetaxel combination compared
with single-agent treatments (P < .001).
CONCLUSIONS: The current study demonstrated that alisertib combined with
docetaxel can mediate a better therapeutic outcome in UGC cell lines. Peripheral T-cell lymphomas (PTCL) are a diverse group of rare non-Hodgkin
lymphomas (NHL) that carry a poor prognosis and are in need of effective
therapies. Alisertib (MLN8237) an investigational agent that inhibits Aurora A
Ser/Thr kinase has shown activity in PTCL patients. Here we demonstrate that
aurora A and B are highly expressed in T-cell lymphoma cell lines. In PTCL
patient samples aurora A was positive in 3 of 24 samples and co-expressed with
aurora B. Aurora B was positive in tumor cells in 22 of 32 samples. Of the
subtypes of PTCL, aurora B was over-expressed in PTCL (NOS) [73%], T-NHL [100%],
ALCL (Alk-Neg) [100%] and AITL [100%]. Treatment with MLN8237 inhibited PTCL
cell proliferation in CRL-2396 and TIB-48 cells with an IC50 of 80-100nM.
MLN8237 induced endo-reduplication in a dose and time dependent manner in PTCL
cell lines leading to apoptosis demonstrated by flow cytometry and PARP-cleavage
at concentrations achieved in early phase clinical trials. Moreover, inhibition
of HisH3 and aurora A phosphorylation was dose dependent and strongly correlated
with endo-reduplication. The data provide a sound rationale for aurora
inhibition in PTCL as a therapeutic modality and warrants clinical trial
evaluation. Osteosarcoma, the most common maligt bone tumor of childhood, is a high-grade
primary bone sarcoma that occurs mostly in adolescence. Standard treatment
consists of surgery in combination with multi-agent chemotherapy regimens. The
development and approval of imatinib for Philadelphia chromosome-positive acute
lymphoblastic leukemia in children and the fully human monoclonal antibody,
anti-GD2, as part of an immune therapy for high-risk neuroblastoma patients have
established the precedent for use of targeted inhibitors along with standard
chemotherapy backbones. However, few targeted agents tested have achieved
traditional clinical endpoints for osteosarcoma. Many biological agents
demonstrating anti-tumor responses in preclinical and early-phase clinical
testing have failed to reach response thresholds to justify randomized trials
with large numbers of patients. The development of targeted therapies for
pediatric cancer remains a significant challenge. To aid in the prioritization
of new agents for clinical testing, the Pediatric Preclinical Testing Program
(PPTP) has developed reliable and robust preclinical pediatric cancer models to
rapidly screen agents for activity in multiple childhood cancers and establish
pharmacological parameters and effective drug concentrations for clinical
trials. In this article, we examine a range of standard and novel agents that
have been evaluated by the PPTP, and we discuss the preclinical and clinical
development of these for the treatment of osteosarcoma. We further demonstrate
that committed resources for hypothesis-driven drug discovery and development
are needed to yield clinical successes in the search for new therapies for this
pediatric disease. Peripheral T-cell lymphomas (PTCL) are a diverse group of lymphoproliferative
disorders, which share a common denominator of overall poor prognosis, with few
exceptions. In this article, the authors review current standard of care
approaches for the treatment of PTCLs, the role of stem-cell/bone marrow
transplantation, and current developments in novel targeted therapies. PURPOSE: Aurora A kinase (AAK) is overexpressed in aggressive lymphomas and can
correlate with more histologically aggressive forms of disease. We therefore
designed a phase II study of alisertib, a selective AAK inhibitor, in patients
with relapsed and refractory aggressive non-Hodgkin lymphomas.
PATIENTS AND METHODS: Patients age ≥ 18 years were eligible if they had relapsed
or refractory diffuse large B-cell lymphoma (DLBCL), mantle-cell lymphoma (MCL),
transformed follicular lymphoma, Burkitt's lymphoma, or noncutaneous T-cell
lymphoma. Alisertib was administered orally at 50 mg twice daily for 7 days in
21-day cycles.
RESULTS: We enrolled 48 patients. Histologies included DLBCL (n = 21), MCL (n =
13), peripheral T-cell lymphoma (n = 8), transformed follicular lymphoma (n =
5), and Burkitt's (n = 1). Most common grade 3 to 4 adverse events were
neutropenia (63%), leukopenia (54%), anemia (35%), thrombocytopenia (33%),
stomatitis (15%), febrile neutropenia (13%), and fatigue (6%). Four deaths
during the study were attributed to progressive non-Hodgkin lymphoma (n = 2),
treatment-related sepsis (n = 1), and unknown cause (n = 1). The overall
response rate was 27%, including responses in three of 21 patients with DLBCL,
three of 13 with MCL, one of one with Burkitt's lymphoma, two of five with
transformed follicular lymphoma, and four of eight with noncutaneous T-cell
lymphoma. The alisertib steady-state trough concentration (n = 25) revealed the
expected pharmacokinetic variability, with a trend for higher incidence of
adverse event-related dose reductions at higher trough concentrations. Analysis
for AAK gene amplification and total AAK protein revealed no differences between
histologies or correlation with clinical response.
CONCLUSION: The novel AAK inhibitor alisertib seems clinically active in both B-
and T-cell aggressive lymphomas. On the basis of these results, confirmatory
single-agent and combination studies have been initiated. |
List the three most prevalent pathogenic species of Borrelia in Europe. | The most prevalent pathogenic species of Borrelia in Europe are: B. afzelii, B. garinii and B. burgdorferi ss. | Ixodes ricinus is the most important tick species in Europe as it is most widely
distributed and transmits the majority of tick-borne zoonotic pathogens. As
limited data are available for Hungary, the aim of the present study was to
investigate the seasonal timing of questing by I. ricinus and the infection rate
of this tick species with all major tick-borne zoonotic pathogens. Monthly
collections of I. ricinus were carried out over 3 consecutive years by dragging
a blanket in 6 biotopes representing different areas of Hungary. Altogether,
1800 nymphs (300 per collection point) were screened as pooled samples (each of
5 specimens) by PCR-based methods for tick-borne pathogens. I. ricinus larvae,
nymphs, and adults had bimodal activity patterns with a major peak in the
spring. As newly moulted ticks of all stages are thought to emerge in the autumn
of each year, it appears that most newly emerged ticks delayed their questing
until the following spring. The minimum prevalence of Borrelia burgdorferi sensu
lato was 2.5%. Borr. afzelii, Borr. burgdorferi sensu stricto, Borr. garinii,
Borr. lusitaniae, and Borr. valaisiana were identified by hybridization. The
minimum infection rate with spotted fever group rickettsiae was 1.9%. Rickettsia
helvetica was identified in all biotopes. The minimum prevalence of Anaplasma
phagocytophilum, Babesia divergens and Bab. microti was low (0.3-0.5%).
Bartonella spp.-, Francisella tularensis-, and TBE virus-specific amplification
products were not detected. Relative to the results of comparable studies
carried out in the Carpathian Basin, the prevalence of tick-borne pathogens was
low in Hungary. This might be attributed to the climatic difference between the
lowland areas of Hungary and submountain areas of the surrounding countries
involved in the studies. Between 2009-2010, a group of 259 patients suspected of contracting Lyme disease
were examined in the Provincial Sanitary-Epidemiological Station in Olsztyn for
the presence of IgM and IgG against specific Borrelia burgdorferi sensu lato
(s.l.) genospecies antigens by immunoblot. A total of 27.4% and 29.0% of the
blood serum samples showed positive and uncertain results for IgM and IgG
antibodies. The majority of positive and uncertain results were found in
patients aged 30-40 years (30%) for IgM, and people aged 50 and over (35.8%) for
IgG. Significantly more positive results for IgG were found in males (40.2%)
then females (19.7%). In both groups, similar proportions of positive results
for IgM anti-Borrelia were recorded (26.1% of females and 29% of males. In 71.5%
of patients, IgM against flagellin protein (p41) of B. burgdorferi sensu stricto
(s.s.) was found. For IgG, the most frequently detected antibodies were found
against the p41 protein of B. burgdorferi s.s. (64.8%) and the recombit
variable surface antigens (VlsE) (49%). Among all the analysed antigens those of
B. burgdorferi s.s. were the most frequent cause of immunological reaction,
followed by antigens of B. afzelii and B. garinii. Reaction to antigens of B.
spielmanii was rarely detected. The European red squirrel (Sciurus vulgaris) has long been suspected to be a
reservoir host of the agents of Lyme borreliosis, in particular B. burgdorferi
sensu stricto (s.s.). However, very few data support this hypothesis. Hereafter,
we investigated the infections with B. burgdorferi genospecies in road-killed
red squirrels collected across France. We also characterized the diversity of
hard tick species collected from a subsample of hosts. DNA of B. burgdorferi
genospecies were detected and identified from PCR products in ear biopsies using
reverse line blot hybridization. Variation in prevalence was investigated
accord-ing to biogeographic areas (Mediterranean, Atlantic, Continental, and
Alpine), season, sex, relative age, and body mass from 273 squirrels collected
2003–2010. Among the 746 identified tick specimens, no adult was observed, 63%
were nymphs, and 37% were larvae all belonging to the species Ixodes ricinus
except one nymph identified as I. trianguliceps. Overall, no squirrels of
Mediterranean origin and no unweaned juveniles were found infested by hard
ticks. Only season explained variation in I. ricinus abundance on squirrels,
with more ticks present in spring to summer than in autumn to winter. Squirrels
of Mediterranean origin (n = 20) were not found infected with B. burgdorferi
sensu-lato (s.l.), which is almost certainly related to the low occurrence of I.
ricinus in this region. Based on individuals analyzed in the other regions of
France, 11.5% (n = 26) unweaned young harboured B. burgdorferi s.l., which
indicates that infection occurred already in the nest. In adults (n = 227), the
prevalence of infection with B. burgdorferi s.l. was 27.3%, with 18.9% B.
burgdorferi s.s., 11.9% B. afzelii, and 3.5% B. garinii. The season and the body
mass, sex, and geographic origin of adults had no effect on the frequency of
infection. Infection prevalence of S. vulgaris is among the highest found in
rodents in Europe, particularly for B. burgdorferi s.s. supporting the
hypothesis that sciurids are particularly suitable hosts for this genospecies. Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi
sensu lato (sl) complex transmitted to humans by Ixodes ticks. B. burgdorferi sl
complex, currently comprising at least 19 genospecies, includes the main
pathogenic species responsible for human disease in Europe: B. burgdorferi sensu
stricto (ss), B. afzelii, and B. garinii. In this study, for the first time,
MALDI-TOF MS was applied to Borrelia spp., supplementing the existing database,
limited to the species B. burgdorferi ss, B . spielmanii and B. garinii, with
the species B. afzelii, in order to enable the identification of all the species
potentially implicated in LB in Europe. Moreover, we supplemented the database
also with B. hermsii, which is the primary cause of tick-borne relapsing fever
in western North America, B. japonica, circulating in Asia, and another
reference strain of B. burgdorferi ss (B31 strain). The dendrogram obtained by
analyzing the protein profiles of the different Borrelia species reflected
Borrelia taxonomy, showing that all the species included in the Borrelia sl
complex clustered in a unique branch, while Borrelia hermsii clustered
separately. In conclusion, in this study MALDI-TOF MS proved a useful tool
suitable for identification of Borrelia spp. both for diagnostic purpose and
epidemiological surveillance. Due to increased travel, climatic, and environmental changes, the incidence of
tick-borne disease in both humans and animals is increasing throughout Europe.
Therefore, extended surveillance tools are desirable. To accurately screen
tick-borne pathogens (TBPs), a large scale epidemiological study was conducted
on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the
Netherlands using a powerful new high-throughput approach. This advanced
methodology permitted the simultaneous detection of 25 bacterial, and 12
parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia,
Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and
Theileria genus) across 94 samples. We successfully determined the prevalence of
expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia
helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia
venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae)
pathogens in the three European countries. Moreover we detected Borrelia
spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the
first time in Danish ticks. This surveillance method represents a major
improvement in epidemiological studies, able to facilitate comprehensive testing
of TBPs, and which can also be customized to monitor emerging diseases. Borrelia miyamotoi is a newly described emerging pathogen transmitted to people
by Ixodes species ticks and found in temperate regions of North America, Europe,
and Asia. There is limited understanding of large scale entomological risk
patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the
agent of Lyme disease, in western North America. In this study, B. miyamotoi, a
relapsing fever spirochete, was detected in adult (n=70) and nymphal (n=36)
Ixodes pacificus ticks collected from 24 of 48 California counties that were
surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu
lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in
adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of
B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I.
pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to
B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in
California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi
from nymphal tick bites. While regional risk of exposure to these two
spirochetes varies, the highest risk for both species is found in north and
central coastal California and the Sierra Nevada foothill region, and the lowest
risk is in southern California; nevertheless, tick-bite avoidance measures
should be implemented in all regions of California. This is the first study to
comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi
for both adult and nymphal I. pacificus, an important human biting tick in
western North America. |
Which thyroid hormone transporter is implicated in thyroid hormone resistance syndrome? | thyroid hormone transporter MCT8 is implicated in thyroid hormone resistance syndrome | Generalized resistance to thyroid hormone (GRTH) encompasses a heterogeneous
group of conditions characterized by reduced responses of target tissues to
thyroid hormone due to defects at the site of hormone action. In the majority of
patients, GRTH is inherited as a domit trait associated with mutations in the
hormone-binding domain of the thyroid hormone receptor. GRTH serves as a
prototype of other resistance syndromes for hormones that act via nuclear
receptors. Known since 1967 and usually familial, the syndrome of tissue resistance to
thyroid hormones may take one of two different forms, depending on the receptors
involved. When resistance affects both peripheral and pituitary receptors,
plasma thyroid hormone levels are high despite the lack of thyrotoxicosis,
thyroxine and triiodothyronine are ineffective, even in high dosage, and plasma
TSH increases to explode under TRH. When resistance only affects pituitary
receptors, there is moderate thyrotoxicosis with paradoxical and inappropriate
TSH increase. Contrary to expectations, nuclear receptors to triiodothyronine
are perturbed in only a few cases. Reduction of thyrotropic hyperfunction, which
is the primary purpose of treatment, can be achieved with D-forms of thyroid
hormones or with somatostatin and its derivatives. Generalized resistance to thyroid hormone (GRTH), a syndrome of inherited tissue
hyposensitivity to thyroid hormone, is linked to thyroid hormone receptor (TR)
mutations. A typical feature of GRTH is variable severity of organ involvement
among families that, surprisingly, does not correlate with the degree of
T3-binding impairment of the corresponding in vitro synthesized mutant TRs.
Furthermore, variations in the clinical severity among family members harboring
identical TR beta mutations have been reported. We compared serum levels of
thyroid hormones that maintained a normal TSH in members of a large family with
GRTH divided in three groups: Group A, 8 affected subjects with a mutation
replacing arginine-320 with a histidine in the T3-binding domain of TR beta;
Group B, 11 first degree relatives (sibs and children of affected subjects) with
no TR beta mutation; Group C, 16 controls related by marriage. TSH values were
not different among the three groups. As expected, total and free T4 and T3, and
rT3 levels were significantly higher in Group A vs Groups B and C. However, with
the exception of T3, the same tests were also significantly higher in Group B vs
Group C. The latter differences are not due to thyroid hormone transport in
serum since TBG concentrations were not different. It is postulated that genetic
variability of factors that contribute to the action of thyroid hormone modulate
the phenotype of GRTH associated with TR beta mutations. The thyroid hormone resistance syndromes are disorders in which the body's
tissues are resistant to the effects of thyroid hormone. Generalized resistance
to thyroid hormone (GRTH) is characterized by resistance in the pituitary gland
and in most or all of the peripheral tissues. Affected individuals have elevated
serum thyroid hormone levels and inappropriately normal or elevated
thyroid-stimulating hormone (TSH) but are usually clinically euthyroid and
require no treatment. Selective pituitary resistance to thyroid hormone (PRTH)
is characterized by resistance in the pituitary gland but not in peripheral
tissues. Patients have elevated serum thyroid hormone levels and normal or
elevated TSH levels and are clinically thyrotoxic. Therapy is usually necessary,
but current choices are not completely satisfactory. Selective peripheral
resistance to thyroid hormone (PerRTH) is characterized by resistance in
peripheral tissues but not in the pituitary. The only patient thus far described
had normal serum thyroid hormone and TSH levels but was clinically hypothyroid
and improved with thyroid hormone administration. All of these disorders are
probably more common than is generally recognized and are often misdiagnosed and
inappropriately treated. GRTH, in most cases studied, results from a mutation in
the thyroid hormone receptor beta gene causing an amino acid substitution in or
a partial or complete deletion of the thyroid hormone-binding domain of the
receptor. The causes of PRTH and PerRTH remain to be determined. Thyroid hormones (T3, T4) exert multiple cellular effects through nuclear
thyroid hormone receptors (TR alpha, TR beta). Thyroid hormone receptors are
transcription factors that act by altering patterns of gene expression.
Resistance to thyroid hormone (RTH) is a rare disorder caused by mutations in
the TR beta gene. Biochemically, the syndrome is defined by elevated circulating
levels of free thyroid hormones due to reduced target tissue responsiveness and
normal, or elevated, levels of thyroid-stimulating hormone (TSH). This
"inappropriate" TSH elevation contrasts with the situation in hyperthyroidism,
where the pituitary secretion of TSH is suppressed. Patients with RTH usually
present with goiter and an euthyroid or mildly hypothyroid metabolic state.
Thus, pituitary resistance results in hypersecretion of TSH, which compensates,
at least in part, for hormone resistance in peripheral tissues. Despite this
compensation, clinical effects of RTH can include short stature, delayed bone
maturation, hyperactivity, learning disabilities, and hearing defects, as well
as variable features of hyper- and hypothyroidism. With the exception of a
single sibship, which harbored a deletion of the entire coding sequence of the
TR beta gene and a recessive pattern of inheritance, all other cases of RTH have
been inherited in an autosomal domit manner or have been de novo heterozygous
mutations of the TR beta gene. The domit pattern of inheritance is explained
by the functional properties of the mutant receptors which act in a domit
negative manner to block the activity of normal TR alpha and TR beta receptors.
Now that a large number of different RTH mutations have been identified, it is
striking that the mutations are clustered within restricted domains in the
carboxyterminal region of the receptor. Mutations in these regions have been
shown to preserve critical receptor functions such as dimerization and DNA
binding, while inactivating other activites such as T3 binding and
transcriptional activation. The examination of patients with RTH and their
mutated receptors has provided important insights into the mechanisms of thyroid
hormone action, the structure-function relationship of the receptors, and the
molecular mechanisms of domit negative activity. Nuclear hormone receptors are hormone-regulated transcription factors that play
critical roles in chordate development and homeostasis. Aberrant nuclear hormone
receptors have been implicated as causal agents in a number of endocrine and
neoplastic diseases. The syndrome of Resistance to Thyroid Hormone (RTH) is a
human genetic disease characterized by an impaired physiological response to
thyroid hormone. RTH is associated with diverse mutations in the thyroid hormone
receptor beta-gene. The resulting mutant receptors function as domit
negatives, interfering with the actions of normal thyroid hormone receptors
coexpressed in the same cells. We report here that RTH receptors interact
aberrantly with a newly recognized family of transcriptional corepressors
variously denoted as nuclear receptor corepressor (N-CoR), retinoid X receptor
interacting protein-13 (RIP-13), silencing mediator for retinoid and thyroid
hormone receptors (SMRT), and thyroid hormone receptor-associating cofactor
(TRAC). All RTH receptors tested exhibit an impaired ability to dissociate from
corepressors in the presence of thyroid hormone. Two of the RTH mutations
uncouple corepressor dissociation from hormone binding; two additional RTH
mutants exhibit an unusually strong interaction with corepressor under all
hormone conditions tested. Finally, artificial mutants that abolish corepressor
binding abrogate the domit negative activity of RTH mutants. We suggest that
an altered corepressor interaction is likely to play a critical role in the
domit negative potency of RTH mutants and may contribute to the variable
phenotype in this disorder. Resistance to thyroid hormone (RTH) is usually domitly inherited and is
characterized by elevated free thyroid hormones in the serum and failure to
suppress pituitary thyroid stimulating hormone (TSH) secretion with variable
refractoriness to hormone action in peripheral tissues. Two major forms of the
disorder are recognized: asymptomatic individuals with generalized resistance
(GRTH) and patients with thyrotoxic features, suggesting predomit pituitary
resistance (PRTH). Molecular genetic analyses indicate that both GRTH and PRTH
are associated with diverse mutations in the thyroid hormone receptor beta gene,
which localize to three regions in the hormone binding domain of the receptor.
In addition to being functionally impaired, the mutant receptors are also able
to inhibit their wild-type counterparts in a domit negative manner.
Recognized features of RTH include failure to thrive, growth retardation and
attention-deficit hyperactivity disorder in childhood, and goitre and thyrotoxic
cardiac symptoms in adults. The pathogenesis of variable tissue resistance is
not fully understood but may be related to the differing tissue distributions of
a and b thyroid hormone receptors and variable domit negative activity of
mutant receptors on different target genes. Thyroid hormone receptors (T3Rs) both repress and activate gene transcription by
interacting with auxiliary factors denoted corepressors and coactivators.
Resistance to thyroid hormone (RTH) syndrome in humans is manifested as a
failure to respond properly to elevated circulating thyroid hormone. RTH
syndrome has been mapped to T3Rbeta mutations that alter the transcriptional
properties of the receptor, resulting in a domit negative phenotype. We
report here a characterization of a series of RTH mutant T3Rs that exhibit
unusual interactions with corepressor. Two mutations in receptor helix 11
(delta430, delta432) greatly enhance the ability of the mutant receptors to bind
to corepressor. A distinct mutation, V264D, in an 'omega loop' region of the
receptor, impairs corepressor release but does not fully eliminate the ability
to recruit coactivator. These mutations reveal novel determits that regulate
the interaction of the T3R with important ancillary cofactors, and that are
disrupted in a human endocrine disease. The finding of increased thyroxine (T4) and tri-iodothyronine (T3) levels in a
patient with normal or increased thyroid-stimulating hormone is unexpected and
presents a differential diagnosis between a thyroid-stimulating
hormone-secreting pituitary adenoma, generalized resistance to thyroid hormone
(RTH) and laboratory artefact. Without careful clinical and biochemical
evaluation, errors may occur in patient diagnosis and treatment. In the case of
RTH, mutation of the thyroid hormone receptor beta gene results in generalized
tissue resistance to thyroid hormone. As the pituitary gland shares in this
tissue resistance, euthyroidism with a normal thyroid-stimulating hormone is
usually maintained by increased thyroid hormones. To date, we have identified
eight pedigrees in New Zealand with mutations in the thyroid hormone receptor
beta gene, including two novel mutations. Mutational analysis of the thyroid
hormone receptor beta gene allows definitive diagnosis of RTH, potentially
avoiding the need for protracted and expensive pituitary function testing and
imaging. Mutational analysis also enables family screening and may help to avoid
potential misdiagnosis and inappropriate treatment. At least six major steps are required for secreted thyroid hormone (TH) to exert
its action on target tissues. Mutations interfering with three of these steps
have been so far identified. The first recognized defect, which causes
resistance to TH, involves the TH receptor beta gene and has been given the
acronym RTH. Occurring in approximately 1 per 40,000 newborns, more than 1000
affected subjects, from 339 families, have been identified. The gene defect
remains unknown in 15% of subjects with RTH. Two novel syndromes causing reduced
sensitivity to TH were recently identified. One, producing severe psychomotor
defects in > 100 males from 26 families, is caused by mutations in the
cell-membrane transporter of TH, MCT8; the second, affecting the intracellular
metabolism of TH in four individuals from two families, is caused by mutations
in the SECISBP2 gene, which is required for the synthesis of selenoproteins,
including TH deiodinases. Thyroid hormone resistance (THR) is a rare syndrome of reduced end organ
sensitivity. Patients with THR have elevated serum free thyroxine (FT4), free
triiodothyronine (FT3), but normal or slightly elevated serum thyrotropin
values. The characteristic clinical feature is goitre without symptoms and
metabolic consequences of thyroid hormone excess. THR can be classified on the
basis of tissue resistance into pituitary, peripheral or generalised (both
pituitary and peripheral) types. Mutations in the TRbeta gene, cell membrane
transporter and genes controlling intracellular metabolism of thyroid hormone
have been implicated. THR is differentiated from thyroid stimulating hormone
(TSH) secreting pituitary adenoma by history of THR in the family. No specific
treatment is often required for THR; patients with features of hypo- or
hyperthyroidism are appropriately treated with levo-triiodothyronine (L-T3),
levo-thyroxine (L-T4), dextro-thyroxine(D-T4) or 3,3,5 triiodo-thyroacetic acid
(TRIAC). The diagnosis helps in appropriate genetic counselling of the family. Thyroid hormone is a pleiotropic hormone with widespread biological actions. For
instance, adequate levels of thyroid hormone are critical for the development of
different tissues such as the central nervous system, but are also essential for
the regulation of metabolic processes throughout life. The biological activity
of thyroid hormone depends not only on serum thyroid hormone levels, but is also
regulated at the tissue level by the expression and activity of deiodinases,
which activate thyroid hormone or mediate its degradation. In addition, thyroid
hormone transporters are necessary for the uptake of thyroid hormone into target
tissues. With the discovery of monocarboxylate transporter 8 (MCT8) as a
specific thyroid hormone transporter and the finding that mutations in this
transporter lead to a syndrome of severe psychomotor retardation and elevated
serum 3,3',5-tri-iodothyronine levels known as the Allan-Herndon-Dudley
syndrome, the interest in this area of research has greatly increased. In this
review, we will focus on the molecular aspects of thyroid hormone transporters,
including MCT8, MCT10, organic anion transporting polypeptides, and the effects
of genetic variation in these transporters. PURPOSE OF REVIEW: To discuss the recent advances on thyroid hormone transport
in the brain. A special attention is paid to the X-linked thyroid hormone cell
transport (THCT) defect (also known as the Allan-Herndon-Dudley syndrome),
caused by mutations of the specific thyroid hormone transporter MCT8 gene.
RECENT FINDINGS: MCT8 is involved in thyroid hormone transport in the brain. MRI
of patients with THCT defect showed myelination delays, probably related to
impaired thyroid hormone action on oligodendrocytes. MCT8 is also expressed in
the thyroid and has an important role in thyroid hormone secretion. The altered
circulating concentrations of thyroid hormone in the patients are partly because
of impaired secretion and altered peripheral metabolism. Increased deiodinase
activity is important in the pathophysiology of the syndrome. High D1 activity
in liver and kidney increases T4 and rT3 deiodination, and contributes to the
increased serum T3. High D2 activity in the brain contributes to compensate the
deficient T3 transport by increasing local T3 production.
SUMMARY: Patients with suspected X-linked leukoencephalopathy should be screened
for MCT8 gene mutations. Research on the brain pathophysiology of the THCT
defect should focus on the specific role of Mct8 on oligodendrocytes and
myelination. BACKGROUND: Six known steps are required for the circulating thyroid hormone
(TH) to exert its action on target tissues. For three of these steps, human
mutations and distinct phenotypes have been identified.
SCOPE OF REVIEW: The clinical, laboratory, genetic and molecular characteristics
of these three defects of TH action are the subject of this review. The first
defect, recognized 45years ago, produces resistance to TH and carries the
acronym, RTH. In the majority of cases it is caused by TH receptor β gene
mutations. It has been found in over 3000 individuals belonging to approximately
1000 families. Two relatively novel syndromes presenting reduced sensitivity to
TH involve membrane transport and metabolism of TH. One of them, caused by
mutations in the TH cell-membrane transporter MCT8, produces severe psychomotor
defects. It has been identified in more than 170 males from 90 families. A
defect of the intracellular metabolism of TH in 10 individuals from 8 families
is caused by mutations in the SECISBP2 gene required for the synthesis of
selenoproteins, including TH deiodinases.
MAJOR CONCLUSIONS: Defects at different steps along the pathway leading to TH
action at cellular level can manifest as reduced sensitivity to TH.
GENERAL SIGNIFICANCE: Knowledge of the molecular mechanisms involved in TH
action allows the recognition of the phenotypes caused by defects of TH action.
Once previously known defects have been ruled out, new molecular defects could
be sought, thus opening the avenue for novel insights in thyroid physiology.
This article is part of a Special Issue entitled Thyroid hormone signaling. Cellular entry is an important step preceding intracellular metabolism and
action of thyroid hormone (TH). Transport of TH across the plasma membrane does
not take place by simple diffusion but requires transporter proteins. One of the
most effective and specific TH transporters identified to date is
monocarboxylate transporter 8 (MCT8), the gene of which is located on the X
chromosome. Although MCT8 is expressed in many tissues, its function appears to
be most critical in the brain. Hemizygous MCT8 mutations in males cause severe
psychomotor retardation, known as the Allan-Herndon-Dudley syndrome (AHDS), and
abnormal serum TH levels. AHDS thus represents a type of TH resistance caused by
a defect in cellular TH transport. Allan-Herndon-Dudley Syndrome (AHDS), an X linked condition, is characterized by
congenital hypotonia that progresses to spasticity with severe psychomotor
delays, in combination with altered thyroid hormone levels, in particular, high
serum T3 levels. Recently, this disease was proved to be caused by mutations in
SLC16A2 coding for the monocarboxylate thyroid hormone transporter 8 (MCT8).
Here we describe a 26-year -old Japanese patient with AHDS who had deletion of
exon 3 of SLC16A2. |
Which are the subunits of the transcription factor NF-kappaB in the canonical pathway activation? | The NF-κB canonical pathway is mediated by the p65/relA and p50 subunits. | The inflammatory response is of major importance in host defence, but is
involved in all acute and chronic diseases. Multiple inflammatory cells and
molecules are involved. Among the latter, the Nuclear Factor -κB (NF-κB) has
been found to be most important and present in all cell types. NF-B regulates
the expression of a large number of genes involved in inflammation. NF-κB plays
a key role in the orchestration of the multifaceted inflammatory response, not
only in the first pro-inflammatory phase, but also later in the regulation of
the resolution of inflammation, when anti-inflammatory genes are expressed and
apoptosis is induced. The review describes NF-κB and its two pathways: the
canonical, mediated by the p65 and p50 subunits, and the non-canonical, mediated
by the subunits RelB, p52 and p50. The relevance of the kinases and interactions
leading to NF-κB activation is considered in different primary cells (i.e.
macrophages, dendritic cells, fibroblasts, cells from inflammatory tissues),
together with the response induced and the ligand involved. Then we overview the
different steps to NF-B activation that can be targeted (IKKs, IκBα or NF-κB
subunits themselves) with various technologies available i.e. small molecules
peptides or nucleic acids. A rationale is provided for possible targets to
consider, in the light that NF-κB signaling pathways regulates both
pro-inflammatory and anti-inflammatory responses. The possibility of using NF-κB
targeted dendritic cells in immunotherapy is considered. Herpes simplex virus 1 (HSV-1) is the archetypal member of the alphaherpesvirus
with a large genome encoding over 80 viral proteins, many of which are involved
in virus-host interactions and show immune modulatory capabilities. In this
study, we demonstrated that the HSV-1 UL42 protein, a DNA polymerase
processivity factor, was a novel antagonism of the canonical NF-κB signaling
pathway. UL42 was shown to significantly suppress TNF-α mediated NF-κB
activation. Co-immunoprecipitation experiment revealed that UL42 bound to the
NF-κB subunits p65 and p50. Fluorescence microscopy demonstrated that UL42
abolished nuclear translocation of p65 and p50 upon TNF-α-stimulation. But the
inhibiting capacity of UL42 2R/2A (R279A, R280A) and UL42 3R/3A (R113A, R279A
and R280A) mutants were less than wild type UL42. Also UL42 bound to the Rel
homology domain of the NF-κB subunit p65 and p50. Notably, the N-terminal of
UL42 was sufficient to interact with p65 and p50 and abolished NF-κB reporter
gene activity. Thus, it was first time we demonstrated that HSV-1 UL42 appeared
to prevent NF-κB-dependent gene expression by retaining p65 and p50 in the
cytoplasm, and UL42-dependent transcriptional activation were inherently coupled
to promote HSV-1 lytic replication, which also may contribute to immune evasion
and pathogenesis of HSV-1. |
Is there any link between conserved noncoding elements and alternative splicing in vertebrates? | Yes. Some of the most highly conserved sequences occur in genes encoding RNA binding proteins, particularly the RNA splicing-associated SR genes. | In vertebrates, conserved noncoding elements (CNEs) are functionally constrained
sequences that can show striking conservation over >400 million years of
evolutionary distance and frequently are located megabases away from target
developmental genes. Conserved noncoding sequences (CNSs) in plants are much
shorter, and it has been difficult to detect conservation among distantly
related genomes. In this article, we show not only that CNS sequences can be
detected throughout the eudicot clade of flowering plants, but also that a
subset of 37 CNSs can be found in all flowering plants (diverging ∼170 million
years ago). These CNSs are functionally similar to vertebrate CNEs, being highly
associated with transcription factor and development genes and enriched in
transcription factor binding sites. Some of the most highly conserved sequences
occur in genes encoding RNA binding proteins, particularly the RNA
splicing-associated SR genes. Differences in sequence conservation between
plants and animals are likely to reflect differences in the biology of the
organisms, with plants being much more able to tolerate genomic deletions and
whole-genome duplication events due, in part, to their far greater fecundity
compared with vertebrates. |
Which protein is affected by dusp8 activation? | dusp8 (M3/6) is a dual-specificity phosphatase selective for JNK. | Oxidative stress activates the c-Jun N-terminal kinase (JNK) pathway. However,
the exact mechanisms by which reactive oxygen species (ROS) activate JNK are
unclear. We found that the ability of hydrogen peroxide (H(2)O(2)) to induce JNK
activation varied in different cell types. Pyrrolidine dithiocarbamate (PDTC), a
presumed antioxidant, induced JNK activation on its own and enhanced JNK
activation by H(2)O(2) in many cell types, including Jurkat, HEK293, and LNCaP
and Tsu-Pr1 prostate cancer cells. The activation of JNK by PDTC, in the
presence or absence of exogenous H(2)O(2), was dependent on its chelating
ability to metal ions, most likely copper ions. Despite the strong
JNK-activating ability, H(2)O(2) plus PDTC did not induce significant activation
of the upstream kinases, SEK1/MKK4 and MKK7. However, the JNK inactivation rate
was slower in cells treated with H(2)O(2) plus PDTC compared with the rate in
cells treated with ultraviolet C (UV-C). Treatment of H(2)O(2) plus PDTC
significantly decreased the expression levels of a JNK phosphatase, M3/6 (also
named hVH-5), but not the levels of other phosphatases (PP2A and PP4). In
contrast, UV-C irradiation did not cause the down-regulation of M3/6. These
results suggest that JNK activation by H(2)O(2) plus PDTC resulted from the
down-regulation of JNK phosphatases. Our data also reveal a necessity to
carefully evaluate the pharmacological and biochemical properties of PDTC. Cells respond to stresses such as osmotic shock and heat shock by activating
stress-activated protein kinases (SAPKs), including c-Jun N-terminal kinase
(JNK) [1]. Activation of JNK requires phosphorylation of threonine and tyrosine
residues in the TPY activation loop motif [2, 3] and can be reversed by the
removal of either phosphate group. Numerous JNK phosphatases including
dual-specificity phosphatases [4, 5], have been identified. Many stimuli
activate JNK by increasing its rate of phosphorylation; however, JNK
dephosphorylation is inhibited in cells after heat shock [6], suggesting that a
JNK phosphatase(s) is inactivated. M3/6 is a dual-specificity phosphatase
selective for JNK [7, 8]. We have previously expressed M3/6 in the mouse bone
marrow cell line BAF3 in order to show that JNK activation by IL-3 is necessary
for cell survival and proliferation [9]. Here we report that M3/6 dissociates
from JNK and appears in an insoluble fraction after heat shock. These data
identify M3/6 as a JNK phosphatase that is inactivated by heat shock and provide
a molecular mechanism for the activation of JNK by heat shock. Stress signals elicit a wide variety of cellular responses, many of which
converge on the phosphorylation of JNK and p38 kinases, the activation of which
has been well-characterized. How these kinases are switched off by
dephosphorylation is not well understood. Here we describe how diverse cellular
stresses affect differently the stability and activity of a JNK-inactivating
dual-specificity threonine-tyrosine phosphatase M3/6. Both anisomycin and
arsenite activate the JNK pathway and, in addition, inactivate the M3/6
phosphatase. However, while anisomycin treatment of cells leads to M3/6 protein
degradation, arsenite appears to inactivate M3/6 directly. These results might
have implications for the mechanism of tumour promotion by arsenic. Polyglutamine diseases, including Huntington's disease, designate a group of
nine neurodegenerative disorders characterized by the presence of a toxic
polyglutamine expansion in specific target proteins. Using cell and mouse
models, we have shown that expanded polyglutamine led to activation of the
stress kinase JNK and the transcription factor AP-1, which are implicated in
neuronal death. Polyglutamine expansion-induced stress shared common features
with protein-damaging stress such as heat shock, because activation of JNK
involved inhibition of JNK phosphatase activities. Indeed, expanded
polyglutamine impaired the solubility of the dual-specificity JNK phosphatase
M3/6. Aggregation of M3/6 by polyglutamine expansion appeared to be indirect,
because M3/6 was not recruited into polyglutamine inclusions. The heat shock
protein HSP70, which is known to inhibit JNK during the heat shock response,
suppressed polyglutamine-mediated aggregation of M3/6 and activation of JNK.
Interestingly, levels of HSP70 were down-regulated by polyglutamine expansion.
We suggest that reduction of HSP70 by expanded polyglutamine is implicated in
aggregation and inhibition of M3/6 and in activation of JNK and AP-1. Specific outcomes upon activation of the c-Jun N-terminal kinase (JNK) pathway
critically depend on the intensity and duration of signal transmission.
Dual-specificity phosphatases (DUSPs) play a very important role in these events
by modulating the extent of JNK phosphorylation and activation and thus
regulating cellular responses to stress. M3/6 (DUSP8) is one of the
dual-specificity protein phosphatases with distinct specificity towards JNK. It
has been shown that M3/6 itself is phosphorylated by JNK upon stimulation with
arsenite, but the role of this phosphorylation has not been investigated. In
this study, we mapped JNK-induced phosphorylation sites on M3/6 using mass
spectrometry. Phosphorylated residues Ser 515, Thr 518 and Ser 520 were
identified and site-directed mutagenesis was employed to investigate their role.
Upon arsenite stimulation, M3/6 mutated at these sites exhibited decreased
phosphorylation compared to the wild-type protein. No difference was observed in
terms of the enzyme's in vitro phosphatase activity, its substrate specificity
towards JNK isoforms, its interactions with JNK and the scaffold family of
JNK-interacting proteins (JIPs), its stability or its subcellular localization.
Interestingly, expression of M3/6 phosphorylation mutants delayed the
time-course of JNK phosphorylation and activation by arsenite. We propose that
phosphorylation of the M3/6 phosphatase by JNK in response to stress stimuli
results in attenuation of phosphatase activity and acceleration of JNK
activation. Mitogen-activated protein kinase (MAPK) cascades are involved in the regulation
of cellular proliferation, differentiation, survival, apoptosis, as well as in
inflammatory responses. Signal intensity and duration have been recognized as
crucial parameters determining MAPK signaling output. Phosphatases play a
particularly important role in this respect, by tightly controlling MAPK
phosphorylation and activation. M3/6 (DUSP8) is a dual-specificity phosphatase
implicated in the dephosphorylation and inactivation of JNK and, to a lesser
extent, p38 MAPKs and is found in a complex with these kinases, along with other
pathway components, held together by scaffold proteins. The JNK family consists
of three genes, giving rise to at least ten different splice variants. Some
functional differences between these gene products have been demonstrated, but
the underlying molecular mechanisms and the roles of individual splice variants
are still incompletely understood. We have investigated the interaction of M3/6
with JNK isoforms, as well as scaffold proteins of the JNK interacting protein
(JIP) family, in order to elucidate the contribution of M3/6 to the regulation
of distinct JNK signaling modules. M3/6 exhibited stronger binding towards JNK1β
and JNK2α isoforms and this was reflected in higher enzymatic activity towards
JNK2α2 when compared to JNK1α1 in vitro. After activation of the pathway by
exposure of cells to arsenite, the interaction of M3/6 with JNK1α and JNK3 was
enhanced, whereas that with JNK1β or JNK2α decreased. The modulation of binding
affinities was found to be independent of JNK-mediated M3/6 phosphorylation.
Furthermore, arsenite treatment resulted in an inducible recruitment of M3/6 to
JNK-interacting protein 3 (JIP3) scaffold complexes, while its interaction with
JIP1 or JIP2 was constitutive. The presented data suggest an isoform-specific
role for the M3/6 phosphatase and the dynamic targeting of M3/6 towards distinct
JNK-containing signaling complexes. |
Which receptors are targeted by a drug Macitentan? | Endothelin receptor A and endothelin receptor B are targeted by a drug Macitentan. Macitentan is a potent, orally active, non-peptide dual antagonist of endothelin receptors A and B that is approved for the treatment of pulmonary arterial hypertension. | Macitentan, also called Actelion-1 or ACT-064992
[N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide],
is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for
tissue targeting. Selection of macitentan was based on inhibitory potency on
both ET receptors and optimization of physicochemical properties to achieve high
affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major
and pharmacologically active metabolite, ACT-132577. Macitentan and its
metabolite antagonized the specific binding of ET-1 on membranes of cells
overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium
mobilization in various natural cell lines, with inhibitory constants within the
omolar range. In functional assays, macitentan and ACT-132577 inhibited
ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A)
receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea
(ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented
both the increase of pulmonary pressure and the right ventricle hypertrophy, and
it markedly improved survival. In diabetic rats, chronic administration of
macitentan decreased blood pressure and proteinuria and prevented end-organ
damage (renal vascular hypertrophy and structural injury). In conclusion,
macitentan, by its tissue-targeting properties and dual antagonism of ET
receptors, protects against end-organ damage in diabetes and improves survival
in pulmonary hypertensive rats. This profile makes macitentan a new agent to
treat cardiovascular disorders associated with chronic tissue ET system
activation. Macitentan (ACT-064992), under development by Actelion Ltd in collaboration with
Japanese licensee Nippon Shinyaku Co Ltd, is an orally active, non-peptide dual
endothelin (ET)(A) and ET(B) receptor antagonist for the potential treatment of
idiopathic pulmonary fibrosis (IPF) and pulmonary arterial hypertension (PAH).
Scientific evidence suggests that the ET system may play an important role in
the pathobiology of several cardiovascular diseases. A major therapeutic advance
for the treatment of patients with PAH and IPF has been the pharmacological
control of the activated ET system with ET receptor antagonists. Macitentan,
because of its ability to target the tissues and to block both ET(A) and ET(B)
receptors, is emerging as a new agent to treat cardiovascular disorders
associated with chronic tissue ET system activation. The phase I and II clinical
trials conducted to date have demonstrated that macitentan increases plasma
levels of ET-1, displays dose-dependent pharmacokinetics, and was well tolerated
in healthy volunteers and patients. At the time of publication, a phase II trial
in patients with IPF and a phase III trial in patients with PAH was ongoing. It
is expected that the results of these trials will validate the safety and
efficacy of macitentan. Pulmonary hypertension (PH) is a debilitating disease with a poor prognosis.
Therapeutic options remain limited despite the introduction of prostacyclin
analogues, endothelin receptor antagonists and phosphodiesterase 5 inhibitors
within the last 15 years; these interventions address predomitly the
endothelial and vascular dysfunctionS associated with the condition, but simply
delay progression of the disease rather than offer a cure. In an attempt to
improve efficacy, emerging approaches have focused on targeting the
pro-proliferative phenotype that underpins the pulmonary vascular remodelling in
the lung and contributes to the impaired circulation and right heart failure.
Many novel targets have been investigated and validated in animal models of PH,
including modulation of guanylate cyclases, phosphodiesterases, tyrosine
kinases, Rho kinase, bone morphogenetic proteins signalling, 5-HT, peroxisome
proliferator activator receptors and ion channels. In addition, there is hope
that combinations of such treatments, harnessing and optimizing vasodilator and
anti-proliferative properties, will provide a further, possibly synergistic,
increase in efficacy; therapies directed at the right heart may also offer an
additional benefit. This overview highlights current therapeutic options,
promising new therapies, and provides the rationale for a combination approach
to treat the disease. Potential treatments for ovarian cancers that have become resistant to standard
chemotherapies include modulators of tumor cell survival, such as endothelin
receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual
ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1,
growing orthotopically in nude mice. Mice with established disease were treated
with vehicle (control), paclitaxel (weekly, intraperitoneal injections),
macitentan (daily oral administrations), or a combination of paclitaxel and
macitentan. Treatment with paclitaxel decreased tumor weight and volume of
ascites. Combination therapy with macitentan and paclitaxel reduced tumor
incidence and further reduced tumor weight and volume of ascites when compared
with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but
alone had no effect on tumor incidence or ascites. Immunohistochemical analyses
revealed that treatment with macitentan and macitentan plus paclitaxel inhibited
the phosphorylation of ETRs and suppressed the survival pathways of tumor cells
by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan
necessary for inhibition of phosphorylation correlated with the dose required to
increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced
the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis
in tumor cells and tumor-associated endothelial cells. Collectively, these
results show that administration of macitentan in combination with paclitaxel
prevents the progression of ovarian cancer in the peritoneal cavity of nude mice
in part by inhibiting survival pathways of both tumor cells and tumor-associated
endothelial cells. PURPOSE: To study the pharmacokinetics, pharmacodynamics, and tolerability of
rising single doses of macitentan, an endothelin receptor antagonist, in healthy
male subjects.
METHODS: This double-blind, placebo-controlled study was performed in seven
groups of eight healthy male subjects. Doses of 0.2, 1, 5, 25, 100, 300 and
600 mg or placebo (two subjects per group) were administered. Plasma macitentan
and endothelin-1 and serum total bile salt concentrations were measured and
analysed non-compartmentally. Plasma and urine were analysed qualitatively for
the presence of metabolites and one of these, ACT-132577, was also measured
quantitatively in plasma. Standard tolerability measurements were performed
throughout the study.
RESULTS: Macitentan was slowly absorbed and, at a dose of 300 mg, the t(1/2)
(95% confidence interval, CI) was 17.5 h (14.1, 21.8). The dose-proportionality
coefficient β for C(max) (95% CI) was 0.83 (0.79, 0.87) indicating less than
dose-proportional pharmacokinetics of macitentan. In plasma, a pharmacologically
active oxidative depropyl metabolite, ACT-132577, was found whereas in urine two
minor metabolites were detected. The t(1/2) of ACT-132577 (95% CI) was 65.6 h
(53.1, 80.9). Macitentan dose-dependently increased endothelin-1 concentrations
up to 2.2-fold (95% CI 1.4, 2.4) at a dose of 600 mg, but had no consistent
effect on total bile salts. Macitentan was well tolerated up to and including a
dose of 300 mg, the maximum tolerated dose. Headache, nausea and vomiting were
dose-limiting adverse events.
CONCLUSION: The pharmacokinetic and tolerability profile of macitentan is
consistent with a once-a-day dosing regimen and warrants further investigation
in clinical studies. Angiogenesis inhibition is an established treatment for several tumor types.
Unfortunately, this therapy is associated with adverse effects, including
hypertension and renal toxicity, referred to as "preeclampsia." Recently, we
demonstrated in patients and in rats that the multitarget tyrosine kinase
inhibitor sunitinib induces a rise in blood pressure (BP), renal dysfunction,
and proteinuria associated with activation of the endothelin system. In the
current study we investigated the effects of sunitinib on rat renal histology,
including the resemblance with preeclampsia, as well as the roles of endothelin
1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress
in the development of sunitinib-induced hypertension and renal toxicity. In rats
on sunitinib, light and electron microscopic examination revealed marked
glomerular endotheliosis, a characteristic histological feature of preeclampsia,
which was partly reversible after sunitinib discontinuation. The histological
abnormalities were accompanied by an increase in urinary excretion of endothelin
1 and diminished NO metabolite excretion. In rats on sunitinib alone, BP
increased (ΔBP: 31.6±0.9 mm Hg). This rise could largely be prevented with the
endothelin receptor antagonist macitentan (ΔBP: 12.3±1.5 mm Hg) and only mildly
with Tempol, a superoxide dismutase mimetic (ΔBP: 25.9±2.3 mm Hg). Both
compounds could not prevent the sunitinib-induced rise in serum creatinine or
renal histological abnormalities and had no effect on urine nitrates but
decreased proteinuria and urinary endothelin 1 excretion. Our findings indicate
that both the endothelin system and oxidative stress play important roles in the
development of sunitinib-induced proteinuria and that the endothelin system
rather than oxidative stress is important for the development of
sunitinib-induced hypertension. Macitentan is a dual endothelin receptor antagonist under phase 3 investigation
in pulmonary arterial hypertension. We investigated the effect of cyclosporine
(Cs) and rifampin on the pharmacokinetics of macitentan and its metabolites
ACT-132577 and ACT-373898 in healthy male subjects. In addition, in vitro
studies were performed to investigate interactions between macitentan and its
active metabolite ACT-132577 with human organic anion-transporting polypeptides
(OATPs). The clinical study (AC-055-111) was conducted as a two-part,
one-sequence, crossover study. Ten subjects in each part received multiple-dose
macitentan followed by multiple-dose co-administration of Cs (part A) or
rifampin (part B). In the presence of Cs, steady-state area under the plasma
concentration-time profiles during a dose interval (AUC(τ)) for macitentan and
ACT-373898 increased 10% and 7%, respectively, and decreased 3% for ACT-132577.
Steady-state AUC(τ) of macitentan and ACT-373898 in the presence of rifampin
decreased 79% and 64%, respectively. For ACT-132577, no relevant difference in
AUC(τ) between the two treatments was observed. Macitentan co-administered with
Cs or rifampin was well tolerated. The complementary in vitro studies
demonstrated no marked differences in uptake rates of macitentan and ACT-132577
between the wild-type and OATP over-expressing cells over the concentration
range tested. Concomitant treatment with Cs did not have any clinically relevant
effect on the exposure to macitentan or its metabolites, at steady-state.
Concomitant treatment with rifampin reduced significantly the exposure to
macitentan and its metabolite ACT-373898 at steady-state but did not affect the
exposure to the active metabolite ACT-132577 to a clinically relevant extent. Endothelin receptor antagonists and phosphodiesterase type 5 inhibitors are used
to treat pulmonary arterial hypertension. We tested the hypothesis that a
selective endothelin type A receptor antagonist (ambrisentan) and a
phosphodiesterase type 5 inhibitor (tadalafil) may act synergistically to relax
endothelin-constricted pulmonary arteries. Rat isolated intrapulmonary arterial
rings contracted with 8 nmol/L endothelin-1 were relaxed by 10 nmol/L
ambrisentan and 30 nmol/L tadalafil alone by 26±3% and 21±1%, respectively,
whereas both drugs in combination acted synergistically to relax arterial rings
by 83±6%. The nonselective endothelin type A and B receptor antagonists bosentan
(100 nmol/L) and macitentan (30 nmol/L) alone relaxed endothelin-contracted
rings by 30±5% and 24±3%, respectively. Combinations of 30 nmol/L tadalafil with
100 nmol/L bosentan or 30 nmol/L macitentan relaxed endothelin-contracted rings
by 53±5% or 46±7%, respectively; these values are similar to the calculated sums
of the individual effects of these compounds. Denudation of endothelium from
pulmonary arterial rings abolished the vasodilator response to 30 nmol/L
tadalafil and the synergistic vasorelaxant effect of tadalafil with ambrisentan.
In the presence of 1 μmol/L BQ-788, a selective endothelin type B receptor
antagonist, the vasorelaxant effects of 10 nmol/L ambrisentan and 30 nmol/L
tadalafil were additive but not synergistic. These data can be interpreted to
suggest that ambrisentan and tadalafil synergistically inhibit
endothelin-1-induced constriction of rat intrapulmonary arteries and that
endothelin type B receptors in endothelium are necessary to enable a synergistic
vasorelaxant effect of the drug combination. Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can
be resistant to conventional therapies. Thus, we investigated whether blockage
of the ETR pathways using the dual ETR antagonist macitentan combined with taxol
or cisplatinum can produce therapy for orthotopically growing
multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice
were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer
cells. Ten days later, mice were randomized to receive vehicle (saline),
macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum
(intraperitoneal, weekly), macitentan plus taxol, or macitentan plus
cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and
prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond
to taxol, cisplatinum, or macitentan administered as single agents. In contrast,
combination therapy with macitentan and taxol or macitentan and cisplatinum
significantly decreased the tumor incidence and weight and significantly
increased the survival of mice and their general condition. Multiple
immunohistochemical analyses revealed that treatment with macitentan and
macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs,
decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of
treatment and induced a first wave of apoptosis in tumor-associated endothelial
cells followed by apoptosis in surrounding tumor cells. Our study shows that
ovarian cancer cells, which express the endothelin axis and are multidrug
resistant, are exquisitely sensitive to treatment with a dual ET antagonist and
can be resensitized to both taxol and cisplatinum. This combined therapy led to
a significant reduction in tumor weight. Macitentan is a tissue-targeting, dual endothelin receptor antagonist, currently
under phase 3 investigation in pulmonary arterial hypertension. In this study
the disposition and metabolism of macitentan were investigated following
administration of a single oral 10 mg dose of (14)C-macitentan to six healthy
male subjects. The total radioactivity in matrices was determined using liquid
scintillation counting. The proposed structure of metabolites was based on mass
spectrometry characteristics and, when available, confirmed by comparison with
reference compounds. Mean (± SD) cumulative recovery of radioactivity from
faeces and urine was 73.6% (± 6.2%) of the administered radioactive dose, with
49.7% (± 3.9%) cumulative recovery from urine, and 23.9% (± 4.8%) from faeces.
In plasma, in addition to parent macitentan, ACT-132577, a pharmacologically
active metabolite elicited by oxidative depropylation and the carboxylic acid
metabolite ACT-373898 were identified. In urine, four entities were identified,
with the hydrolysis product of ACT-373898 as the most abundant one. In faeces,
five entities were identified, with the hydrolysis product of macitentan and
ACT-132577 as the most abundant one. Concentrations of total radioactivity in
whole blood were lower compared to plasma, which indicates that macitentan and
its metabolites poorly bind to or penetrate into erythrocytes. Starting from the structure of bosentan (1), we embarked on a medicinal
chemistry program aiming at the identification of novel potent dual endothelin
receptor antagonists with high oral efficacy. This led to the discovery of a
novel series of alkyl sulfamide substituted pyrimidines. Among these, compound
17 (macitentan, ACT-064992) emerged as particularly interesting as it is a
potent inhibitor of ET(A) with significant affinity for the ET(B) receptor and
shows excellent pharmacokinetic properties and high in vivo efficacy in
hypertensive Dahl salt-sensitive rats. Compound 17 successfully completed a
long-term phase III clinical trial for pulmonary arterial hypertension. Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are
currently approved for the treatment of pulmonary arterial hypertension (PAH), a
devastating disease involving an activated endothelin system and aberrant
contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC).
The novel ERA macitentan has recently concluded testing in a Phase III
morbidity/mortality clinical trial in PAH patients. Since the association and
dissociation rates of G protein-coupled receptor antagonists can influence their
pharmacological activity in vivo, we used human PASMC to characterize inhibitory
potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan
using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium
release assays macitentan, ambrisentan and bosentan were highly potent ERAs with
K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not
ambrisentan and bosentan, displayed slow apparent receptor association kinetics
as evidenced by increased antagonistic potency upon prolongation of antagonist
pre-incubation times. In compound washout experiments, macitentan displayed a
significantly lower receptor dissociation rate and longer receptor occupancy
half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes
versus 70 seconds and 40 seconds, respectively). Because of its lower
dissociation rate macitentan behaved as an insurmountable antagonist in calcium
release and IP(1) assays, and unlike bosentan and ambrisentan it blocked
endothelin receptor activation across a wide range of endothelin-1 (ET-1)
concentrations. However, prolongation of the ET-1 stimulation time beyond
ROt(1/2) rendered macitentan a surmountable antagonist, revealing its
competitive binding mode. Bosentan and ambrisentan behaved as surmountable
antagonists irrespective of the assay duration and they lacked inhibitory
activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with
significantly slower receptor dissociation kinetics than the currently approved
ERAs. Slow dissociation caused insurmountable antagonism in functional
PASMC-based assays and this could contribute to an enhanced pharmacological
activity of macitentan in ET-1-dependent pathologies. It is well established that the endothelin, nitric oxide and prostacyclin
pathways play an important role in the development of pulmonary arterial
hypertension (PAH). Indeed, the therapeutic options currently available for the
management of PAH all act on one of these mechanistic pathways. However, this is
an exciting time for both clinicians and scientists, as increased understanding
of the mechanisms involved in the pathogenesis and progression of PAH has
resulted in the development of a number of novel therapeutic options. This
article highlights how the introduction of new compounds such as macitentan,
riociguat and selexipag, which act on the endothelin, nitric oxide and
prostacyclin pathways, respectively, have the potential to further improve the
prognosis for patients with PAH. Macitentan is a new non-selective endothelin-1 receptor antagonist under
development for the treatment of pulmonary arterial hypertension. Information on
the potential for macitentan to influence the pharmacokinetics of concomitantly
administered drugs by inhibition or induction of drug metabolising enzymes or
drug transporters is sparse. We therefore studied the potential of macitentan to
inhibit and induce critical targets of drug metabolism and drug distribution
(transporters) in vitro. Induction was quantified at the mRNA level by real-time
RT-PCR in LS180 cells and revealed that macitentan significantly induced mRNA
expression of cytochrome P450 3A4 (CYP3A4), P-glycoprotein (P-gp, ABCB1), solute
carrier of organic anions 1B1 (SLCO1B1), and
uridinediphosphate-glucuronosyltransferase 1A3 (UGT1A9). By means of a reporter
gene assay our study establishes macitentan as a potent activator of prege X
receptor (PXR). Inhibition of drug transporters was evaluated by using
transporter over-expressing cell lines and fluorescent specific substrates of
the respective transporters and revealed that macitentan is an inhibitor of
P-gp, breast cancer resistance protein (BCRP), SLCO1B1, and SLCO1B3. Using
commercial kits macitentan was demonstrated to be a moderate inhibitor of CYP3A4
and CYP2C19. In conclusion our data provide a comprehensive analysis of the
interaction profile of macitentan with drug metabolising and transporting
enzymes in vitro. Although macitentan has a similar or higher potency for
induction and inhibition of drug metabolising enzymes and transporters than
bosentan, its low plasma concentrations and minimal accumulation in the liver
suggest that it will be markedly less prone to drug-drug interactions than
bosentan. Idiopathic pulmonary fibrosis is a progressive, fatal disease. This prospective,
randomised, double-blind, multicentre, parallel-group, placebo-controlled phase
II trial (NCT00903331) investigated the efficacy and safety of the endothelin
receptor antagonist macitentan in idiopathic pulmonary fibrosis. Eligible
subjects were adults with idiopathic pulmonary fibrosis of <3 years duration and
a histological pattern of usual interstitial pneumonia on surgical lung biopsy.
The primary objective was to demonstrate that macitentan (10 mg once daily)
positively affected forced vital capacity versus placebo. Using a centralised
system, 178 subjects were randomised (2:1) to macitentan (n=119) or placebo
(n=59). The median change from baseline up to month 12 in forced vital capacity
was -0.20 L in the macitentan arm and -0.20 L in the placebo arm. Overall, no
differences between treatments were observed in pulmonary function tests or time
to disease worsening or death. Median exposures to macitentan and placebo were
14.5 months and 15.0 months, respectively. Alanine and/or aspartate
aminotransferase elevations over three times upper limit of normal arose in 3.4%
of macitentan-treated subjects and 5.1% of placebo recipients. In conclusion,
the primary objective was not met. Long-term exposure to macitentan was well
tolerated with a similar, low incidence of elevated hepatic aminotransferases in
each treatment group. This multiple-ascending-dose study investigated safety, tolerability,
pharmacokinetics, and pharmacodynamics, of macitentan, a new endothelin receptor
antagonist (ERA) with sustained receptor binding and enhanced tissue penetration
properties compared to other ERAs. Healthy male subjects (n = 32) received once
daily oral doses of macitentan (1 - 30 mg) or placebo for 10 days.
Administration of macitentan was safe and well tolerated. Macitentan had no
effect on bile salts, suggesting an improved liver safety profile. The
multiple-dose pharmacokinetics of macitentan were dose-proportional and were
characterized by a median tmax and apparent elimination half-life varying from
6.0 to 8.5 and 14.3 to 18.5 hours, respectively, for the different doses and
minimal accumulation. ACT-132577, a metabolite with lower potency than
macitentan, had a half-life of about 48 hours and accumulated approximately
8.5-fold. Compared to placebo, administration of macitentan caused a
dose-dependent increase in plasma ET-1 with maximum effects attained at 10 mg. A
small dose-dependent increase in the 6β-hydroxycortisol/cortisol urinary
excretion ratio was observed, although there were no statistically significant
differences between treatments including placebo. Effects of macitentan on
cytochrome P450 enzyme 3A4 should be further evaluated in dedicated studies. The
present results support investigation of macitentan in the management of
pulmonary arterial hypertension and ET-1-dependent pathologies. Pulmonary arterial hypertension (PAH) is a progressive disease characterised by
remodelling of small pulmonary arteries leading to an increased pulmonary
vascular resistance, right ventricular failure and death. Available treatments
try to re-establish the equilibrium on three signalling pathways: the
prostacyclin, the endothelin (ET)-1 and the nitric oxide. Prostanoids, such as
epoprostenol or treprostinil have a vasodilator, antiproliferative and
immunomodulatory effect and, despite the administration inconveniences,
represent established therapies for severe cases of PAH. Recently oral
prostacyclin receptor agonists have shown encouraging results. Many clinical
studies targeting the vasoconstrictor ET-1 pathway with receptor antagonists
like bosentan and ambrisentan have shown strong results, even more optimism
coming from macitentan, the newest drug. Sildenafil and tadalafil, two
phosphodiesterase type-5 inhibitors, have shown improved exercise capacity by
increasing the nitric oxide level. Riociguat, acting on the same nitric oxide
pathway, as a guanylatecyclase activator, has shown promising results in
clinical trials and will be available soon. Long-awaited results for
tyrosin-kinase inhibitor, imatinib, as an antiproliferative therapy in PAH have
been disappointing, due to severe adverse events. In conclusion, although it
remains a disease with severe prognosis, the past 20 years have represented a
huge progress in terms of treatments for PAH with interesting opportunities for
the future. Over the past 20 years, great progress has been made in the treatment of
pulmonary arterial hypertension (PAH). Available therapies target one of three
principal pathways: the endothelin (ET), nitric oxide (NO) or the prostacyclin
(PGI2) pathway. Evidence shows that current drugs, used either as monotherapy or
in different combinations, can improve exercise capacity, clinical symptoms,
hemodynamics and even survival in PAH. Unfortunately, the disease remains
incurable and the prognosis of the disease is still poor. However, existing and
novel potent antiproliferative therapies are being explored, and new agents
targeting different and/or additional pathways are likely to become available to
clinicians in the near future. Promising candidates include tyrosine kinase
antagonists (e.g. imatinib); soluble guanylate cyclase stimulators (riociguat);
an oral analog of prostacyclin (selexipag); and a tissue targeting endothelin
receptor antagonist (macitentan). Phase II or III trials have either been
completed or are underway to evaluate the safety and efficacy of these various
therapies. Macitentan is under development for the treatment of pulmonary arterial
hypertension (PAH). Patients with PAH may suffer from comorbidities such as
renal or hepatic impairment. Two prospective, single-center, open-label studies
evaluated the pharmacokinetics of macitentan and its metabolites
(pharmacologically active ACT-132577 and inactive ACT-373898) in healthy
subjects and in subjects with mild, moderate, and severe hepatic impairment or
severe renal function impairment (SRFI). After administering a single oral dose
of 10 mg macitentan the pharmacokinetic parameters including area under the
curve from zero to infinity (AUC∞) were derived from plasma concentration-time
profiles. Exposure to macitentan and ACT-132577 was lower in hepatically
impaired versus healthy subjects, with no correlation with the degree of hepatic
impairment. Exposure to ACT-373898 was lower in subjects with moderate hepatic
impairment only. Plasma concentration-time profiles for macitentan and
ACT-132577 (active) were similar in healthy subjects and subjects with SRFI.
AUC∞ of ACT-373898 (inactive) was 7.3-fold higher in subjects with SRFI versus
healthy subjects. No safety concerns were raised in either study. Based on these
observations, pharmacokinetic alterations of macitentan due to hepatic or renal
function impairment are not considered clinically relevant and no dose
adjustment is necessary in these patients. INTRODUCTION: Macitentan is a novel dual endothelin receptor antagonist (ERA)
showing sustained receptor occupancy. In vitro and in vivo animal studies have
demonstrated its potency in antagonizing endothelin-induced disorders. A large
morbidity/mortality study in patients with pulmonary arterial hypertension (PAH)
taking macitentan has been completed recently.
AREAS COVERED: This drug evaluation reviews the efficacy, safety and clinical
pharmacology of macitentan in the treatment of PAH.
EXPERT OPINION: The large Phase III study (SERAPHIN) tested macitentan in more
than 700 PAH patients and has provided unique long-term outcome data for this
ERA, not available for other members of this class. The effect on a composite
clinically relevant morbidity/mortality end point was highly significant at a 10
mg/day dose. The safety profile of macitentan appears to be superior with
respect to hepatic safety and edema/fluid retention than bosentan and
ambrisentan, respectively, and is similar when considering decrease in
hemoglobin concentration. The drug has a low propensity for drug-drug
interactions and has one circulating pharmacologically active metabolite. The
pharmacokinetics of macitentan in patients with renal or hepatic impairment does
not require dose adjustments. Based on its characteristics, macitentan is an
important addition to the therapeutic armamentarium in the long-term treatment
of PAH. Its potential use in other disorders is under investigation. Macitentan (Opsumit®) is a novel dual endothelin receptor antagonist (ERA) with
sustained receptor binding properties developed by Actelion Pharmaceuticals Ltd.
In October 2013, oral macitentan 10 mg once daily received its first global
approval in the US, followed closely by Canada, for the treatment of pulmonary
arterial hypertension (PAH). The drug has also received a positive opinion in
the EU from the Committee for Medicinal Products for Human Use for the treatment
of PAH, and is under regulatory review in several other countries for the same
indication. Endothelin (ET)-1 influences pathological changes via two ET
receptor subtypes (ETA and ETB), to which it binds with high affinity. ET-1 is
implicated in several forms of vascular disease making it a valid target for the
treatment of pulmonary vascular diseases such as PAH. Clinical development is
underway for other indications, including Eisenmenger syndrome, ischaemic
digital ulcers secondary to systemic sclerosis, and glioblastoma. Macitentan was
also evaluated in idiopathic pulmonary fibrosis; however, a phase 2 trial did
not meet its primary endpoint and further investigation in this indication was
discontinued. Macitentan was developed by modifying the structure of bosentan in
the search for an optimal dual ERA with improved efficacy and tolerability
compared with other ERAs. This article summarizes the milestones in the
development of macitentan leading to this first approval for PAH. |
What is known as the cause of subacute thyroiditis? | Most cases of subacute thyroiditis are caused by a variety of viruses, for example, Coxsackie, cytomegalovirus, Epstein-Barr virus, and adenovirus | A 34-year-old woman was referred to our hospital complaining of sore throat and
arthralgia. She had low-grade fever, tachycardia, and goiter with tenderness.
Laboratory data revealed thyrotoxicosis and tests for acute inflammatory markers
were positive. Thyroidal radioactive iodine uptake was below normal.
Ultrasonography of thyroid revealed mild thyroid enlargement and hypoechogenic
areas consistent with tenderness. Subacute thyroiditis was diagnosed and
prednisone was administered. Two years later, her identical twin sister, who
lives separately, was referred to our hospital because of neck pain, low-grade
fever, and palpitation. She exhibited the same clinical picture as her twin
sister, and was also diagnosed as having subacute thyroiditis. Although the
cause of subacute thyroiditis remains unclear, viral infection has been
implicated in the onset of subacute thyroiditis in genetically predisposed
individuals. We could not identify the viruses, but heterozygotes for HLA-B35,
which has been reported to be linked with subacute thyroiditis, were found in
the twins. This supports the suspicion that genetic factors, including this HLA
haplotype, play a critical role in the onset of subacute thyroiditis. Subacute (de Quervain's) thyroiditis is a rare but important cause of fever of
unknown origin. Most cases of subacute thyroiditis are caused by a variety of
viruses, for example, Coxsackie, cytomegalovirus, Epstein-Barr virus, and
adenovirus. Influenza immunization or infection may cause subacute thyroiditis.
We present the first reported case of a fever of unknown origin due to seasonal
influenza A in a 67-year-old woman. |
Which drugs are utilized to treat eosinophilic esophagitis? | Therapeutic options of eosinophilic esophagitis include use of proton-pump inhibitors, immunosuppressive drugs, elimination diets, and esophageal dilatation.
Oral viscous budesonide (OVB) is an effective treatment of pan-esophageal disease in children with EoE. OVB improves symptoms and endoscopic and histologic features. | BACKGROUND: Both gastroesophageal reflux disease and allergy/atopy have been
implicated in the pathogenesis of eosinophilic esophagitis (EoE). There are no
prospective studies comparing treatment of EoE with acid suppression versus
topical corticosteroids.
OBJECTIVE: To determine the outcome of adult eosinophilic esophagitis patients
treated with esomeprazole versus topical fluticasone.
DESIGN: Prospective randomized controlled trial.
SETTING: Academic medical center.
PARTICIPANTS: Adults (18-80) diagnosed with EoE by symptoms of dysphagia and
esophageal biopsies with >or=15 eosinophils/hpf.
INTERVENTIONS: Subjects were randomized to esomeprazole (40 mg by mouth every
morning) or aerosolized, swallowed fluticasone (440 mcg by mouth twice a day)
for 8 weeks.
MAIN OUTCOME MEASUREMENTS: Improvement in dysphagia (8-point scale), esophageal
eosinophil infiltration before and after treatment, prevalence of GERD measured
by validated questionnaire and baseline pH study.
RESULTS: About 56% (14/25) had acid reflux by pH study. There was no difference
between treatment groups in improvement in dysphagia scores [3/12 (25%) of the
esomeprazole group versus 6/12 (50%) in the fluticasone group, P = 0.40].
Eosinophil infiltration decreased with treatment in both groups, and there was
no difference in the amount of decrease between groups (P = 0.70).
LIMITATIONS: Small sample size, unexpectedly high drop-out rate.
CONCLUSIONS: Gastroesophageal reflux disease is common in adult eosinophilic
esophagitis patients. Dysphagia improves and esophageal eosinophilic
infiltration decreases with either treatment. There was no difference in degree
of improvement in dysphagia or eosinophil infiltration in patients treated with
either topical fluticasone or oral esomeprazole. GERD may be important in the
pathogenesis of adult EoE. BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is caused by immunologic
reactions to ingested/inhaled allergens. The diagnosis is considered if >or=15
eosinophils per high-powered field (eos/hpf) are detected in mucosal biopsies.
Placebo-controlled studies have not been conducted to evaluate the safety and
efficacy of oral viscous budesonide (OVB).
METHODS: Children with EoE were randomly assigned to groups that were given OVB
(n=15) or placebo (n=9). Patients<5 feet and >or=5 feet tall received 1 mg and 2
mg OVB daily, respectively. All patients received lansoprazole. Duration of
treatment was 3 months, followed by repeat endoscopy and biopsies. Patients were
classified as responders if their peak eosinophil counts were <or=6 eos/hpf,
partial responders were 7-19 eos/hpf, and nonresponders were >or=20 eos/hpf.
Baseline and post-treatment symptoms and endoscopic and histologic features were
scored.
RESULTS: Thirteen (86.7%) children given OVB (P<.0001) and none who received
placebo (P=.3) were classified as responders. Mean pre-/post-treatment peak
eosinophil counts were 66.7 and 4.8 eos/hpf, respectively, in the group given
OVB (P<.0001); they were 83.9 and 65.6 eos/hpf, respectively, in the group given
placebo (P=.3). In the group given OVB, there were significant reductions from
baseline values in proximal (P=.002), mid (P=.0003), and distal (P=.001)
esophageal eosinophilia. After OVB therapy, compared with baseline, the mean
symptom (P=.0007), endoscopy (P=.0005), and histology scores improved (P=.0035)
significantly.
CONCLUSIONS: OVB is an effective treatment of pan-esophageal disease in children
with EoE. OVB improves symptoms and endoscopic and histologic features. Proton
pump inhibitor single therapy did not significantly improve esophageal
eosinophilia or symptoms of EoE. BACKGROUND & AIMS: Topical corticosteroids are effective in inducing clinical
and histologic remission in patients with eosinophilic esophagitis (EoE).
However, the best long-term management strategy for this chronic inflammatory
disease has not been determined.
METHODS: In a randomized, double-blind, placebo-controlled, 50-week trial, we
evaluated in 28 patients the efficacy of twice-daily swallowed budesonide (0.25
mg each) to maintain quiescent EoE in remission. Pretreatment and posttreatment
activity was assessed clinically, endoscopically, histologically,
immunohistologically, and by endosonography. The primary end point was the
therapy's ability to maintain EoE in histologic remission. Secondary end points
were efficacy in symptom control, prevention of tissue remodeling, and safety.
RESULTS: In patients given low-dose budesonide, the load of esophageal
eosinophils increased from 0.4 to 31.8 eosinophils/high-power field (P = .017).
In patients given placebo, the load increased from 0.7 to 65.0
eosinophils/high-power field (P = .0001); this increase was significantly
greater than in patients given budesonide (P = .024). The symptom scores
developed in a similar manner in the 2 groups. Budesonide, but not placebo,
reduced noneosinophilic markers of inflammation, epithelial cell apoptosis, and
remodeling events. Compared with control individuals, patients had significantly
thickened esophageal walls, based on endosonography (3.05 vs 2.18 mm; P <
.0001). Budesonide therapy was associated with a significant reduction in
mucosal thickness (0.75-0.45 mm; P = .025), but epithelial thickness remained
stable (261.22 vs 277.23 μm; P = .576). No serious adverse events occurred.
CONCLUSIONS: Low-dose budesonide is more effective than placebo in maintaining
EoE in histologic and clinical remission. Signs of esophageal remodeling showed
a trend toward normalization. Long-term administration of topical
corticosteroids was well tolerated without induction of epithelial atrophy. BACKGROUND & AIMS: We evaluated the effect of aerosolized fluticasone therapy on
symptomatic dysphagia and histologic eosinophilia in adults with eosinophilic
esophagitis (EoE).
METHODS: We performed a double-blind, randomized, placebo-controlled trial of
fluticasone in 42 adult patients with a new diagnosis of EoE (30 men; mean age,
37.5 y). Participants were assigned randomly to groups that swallowed 880 μg of
aerosolized fluticasone twice daily (n = 21), or took a placebo inhaler twice
daily (n = 15) for 6 weeks. End points of the study were symptomatic and
histologic response.
RESULTS: A complete histologic response (>90% decrease in mean eosinophil count)
was observed in 11 of 15 subjects who received 6 weeks of fluticasone (62%),
compared with none of the 15 subjects who received placebo (P < .001), based on
intention-to-treat analysis; histologic responses were observed in 68% of
subjects who received fluticasone (13 of 19) compared with none of those who
received placebo (0 of 15) by per-protocol analysis (P < .001). Intracellular
staining for eosinophil-derived neurotoxin was reduced in 81% of subjects who
received fluticasone (13 of 16) compared with 8% who received placebo (1 of 13)
(P < .001). Dysphagia was reduced in 57% of subjects who received fluticasone
(12 of 21) compared with 33% who received placebo (7 of 21) (P = .22) by
intention-to-treat analysis; dysphagia was reduced in 63% of patients who
received fluticasone (12 of 19) and 47% of those who received placebo (7 of 15)
(P = .49) based on per-protocol analysis. Esophageal candidiasis developed in
26% of subjects who received fluticasone (5 of 19), but in none of the subjects
in the placebo group (P = .05).
CONCLUSIONS: Aerosolized, swallowed fluticasone leads to a histologic but not a
symptomatic response in adults with EoE. We performed a randomized trial to compare nebulized and viscous topical
corticosteroid treatments for eosinophilic esophagitis (EoE). Subjects with
incident EoE (n = 25) received budesonide 1 mg twice daily, either nebulized and
then swallowed (NEB) or as an oral viscous slurry (OVB), for 8 weeks. Baseline
eosinophil counts for the NEB and OVB groups were 101 and 83 (P = .62).
Posttreatment counts were 89 and 11 (P = .02). The mucosal medication contact
time, measured by scintigraphy, was higher for the OVB group than the NEB group
(P < .005) and was inversely correlated with eosinophil count (R = -0.67; P =
.001). OVB was more effective than NEB in reducing numbers of esophageal
eosinophils in patients with EoE. OVB provided a significantly higher level of
esophageal exposure to the therapeutic agent, which correlated with lower
eosinophil counts. Eosinophilic esophagitis is characterized by symptoms of esophageal dysfunction
and eosinophil-predomit esophageal inflammation. Eosinophilic inflammation in
other parts of the gastrointestinal tract is absent and several differential
diagnoses for esophageal eosinophilia have to be excluded before diagnosing
eosinophilic esophagitis. Most patients are male and have concomitant atopic
disorders. Therapeutic options are based on drugs, diet and dilation. Eosinophilic esophagitis (EoE) is a relatively new disease that is increasingly
recognized as a chronic inflammatory condition with currently evolving
diagnostic and therapeutic aspects. There is data to support the rising
prevalence of EOE especially in western countries. EoE is an emerging cause of
dysphagia and food bolus impaction in adults as well as abdominal pain, feeding
disorders, and gastroesophageal reflux-like symptoms in younger patients. EoE is
ever more recognized as a separate disease process that is more complicated than
eosinophilic infiltration from gastroesophageal reflux disease. Food and
environmental antigens both play significant roles in stimulating T-helper
(Th-2) inflammatory response. Current therapeutic options include use of
proton-pump inhibitors, immunosuppressive drugs, elimination diets, and
esophageal dilatation. Simple elimination of food and environmental antigen
exposure can be challenging in adults due to the difficulty in accurately
identifying triggering antigens and adherence to restrictive diets from a wide
range of putative food allergens. Novel therapeutic options are being presented
as potential treatments that target chemokines and specific immunologic
mechanisms for EoE. This review will aim to summarize the latest and evolving
approaches to EoE diagnosis and management. In the future, biomarkers of
inflammatory response may help diagnose, treat, and stratify individual patients
for better treatment outcomes with this chronic disease. |
What is the incidence of cystic fibrosis in the caucasian population? | Estimates of the newborn frequency of cystic fibrosis in different Caucasian groups range from 4 times more to 40 times less common than the generally accepted figure of 1:2000. | Estimates of the newborn frequency of cystic fibrosis in different Caucasian
groups range from 4 times more to 40 times less common than the generally
accepted figure of 1:2000. Current meconium screening trials which may be
effective in populations with the incidence equal to or greater than 1:2000, may
be useful for populations with an incidence as low as 1:7000 only after maximum
improvement of the methods. Once the true incidence or the variable incidence is
proven for Caucasian populations, screening trails in Negro, Oriental and Indian
populations will be required. Cystic fibrosis is the most common, severe, inherited disease in the Caucasian
population. As a consequence, the demand for genetic counselling of patients
with cystic fibrosis and their families is large. In Denmark the incidence of
cystic fibrosis is 1:4700, which is quite low compared to other European
countries. We have investigated 268 Danish cystic fibrosis patients with respect
to DNA markers (haplotypes) and the most common mutation delta F508. The delta
F508 mutation is found on 88% of all cystic fibrosis chromosomes, the highest
frequency reported so far. This had had an important impact on genetic
counselling, prenatal diagnosis and eventually population screening. In the
Danish population 78% of all couples at risk will be informative for delta F508
and will be identifiable by simple screening methods. Cystic fibrosis (CF) is the most common autosomal recessive disorder in the
Caucasian population, affecting approximately 1 in 2,000 newborns but the actual
estimate varies with the geographic location. The incidence of CF in
non-Caucasian populations is low. Intensive efforts using genetic linkage
information ultimately led to the cloning of the CF gene prior to the
identification of the gene product or its function. The gene encodes what is
believed to be a transmembrane protein, which has been named the cystic fibrosis
transmembrane conductance regulator (CFTR). The CFTR contains two
nucleotide-binding folds (NBF) which show homology to numerous transport
proteins with the greatest homology to the P-glycoproteins that are encoded by
the multiple drug-resistance loci. A three- base deletion resulting in the loss
of phenylalanine residue (delta F508) in the tenth exon of the CFTR gene is the
mutation occurring on the majority of CF chromosomes. The overall frequency of
delta F508 in the present mutant CF gene pool is about 70%, but the study
populations are not equally represented: there is marked variation in the
population of delta F508 among different geographic populations. Recently,
numerous additional, less common mutations have been found. Some mutations occur
on 2-5% of the CF chromosomes. Many of these are rare 'private' mutations,
occurring in individual families of all racial and ethnic backgrounds. By
contrast over 80% of Western European CF mutations have been identified. The
highly heterogeneous nature of the remaining CF mutations provides important
insights into the structure and function of the protein, but further
improvements are needed in DNA-based genetic screening for CF carrier status. Cystic fibrosis (CF) is an autosomal recessive disorder with a prevalence at
birth estimated at 1/2000-1/2500 livebirths in Caucasian populations. Some 127
CF individuals are known in Saguenay-Lac-St-Jean (SLSJ), a geographically
isolated region of Quebec. The prevalence at birth was estimated at 1/902 live
borns, and the carrier rate was estimated at 1/15 inhabitants in the SLSJ
region. The mean inbreeding coefficient was only slightly elevated in the CF
group compared with three control groups, and was due to remote consanguinity.
The mean kinship coefficient was 2.4 times higher in the CF group than in the
control groups. In SLSJ region, the places of origin of the CF individuals and
their parents did not show a clustered nonuniform distribution. Endogamy was not
higher in the CF group than in control groups. The incidence of cystic fibrosis (CF) in Finland is one tenth that in other
Caucasian populations. To study the genetics of CF in Finland, we used a
combined molecular and genealogical approach. Out of the 20 Finnish families
with a living CF patient, 19 were typed for eight closely linked restriction
fragment length polymorphisms (RFLP) at the MET, D7S8, and D7S23 loci. The
birthplaces of the parents and grandparents were traced using population
registries. Allele and haplotype frequencies in Finland are similar to those of
other European and North American populations, but are modified by sampling: two
regional CF gene clusters, evidently the results of a founder effect, were
identified. Generally, the gene was evenly distributed over the population,
carrier frequency being estimated at approximately 1.3%. We conclude that CF in
Finland is caused by the common Caucasian mutation(s), and that the low
frequency of the gene can be explained by a negative sampling effect and genetic
drift. The incidence of cystic fibrosis (CF) in Finland, 1:25,000 newborn, is one of
the lowest in Caucasian populations. The delta F508 mutation accounts for 18/40
(45%) of CF chromosomes in Finland. Other mutations were therefore sought among
the remaining 55%. Twelve out of 40 chromosomes (30%) were found to carry
394delTT, whereas G542X and 3732delA were each detected in one chromosome. Eight
mutations remained unidentified using a testing panel for 26 mutations. Mutation
394delTT was associated exclusively with haplotype 23-36-13. Five unknown
mutations were associated with different haplotypes for microsatellite markers,
whereas three shared the same haplotype. Most delta F508 mutations and all
unidentified mutations originated from regions of old and dense settlement in
the coastal regions, whereas 394delTT was geographically clustered and enriched
in a rural location, consistent with a local founder effect. The remote location
of Finland and her population history give a plausible explanation for the
rarity of CF in Finland. Cyprus is an island in the eastern Mediterranean basin inhabited by people of
Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease
among Caucasians is cystic fibrosis (CF). Although no careful scientific study
had ever been done the impression was that CF was extremely rare among the
Greek-Cypriots, with an incidence estimated at around 1:30,000. About 2 years
ago, we introduced molecular diagnostic methodology in an effort to assist
clinicians in safer diagnosis of patients presenting with atypical CF
symptomatology, and also for testing the hypothesis that mutations that cause
milder phenotypes might be responsible for misdiagnosis or for missing entirely
some cases of CF. Initial screening for delta F508 revealed that it is indeed
rare in the general population. Further screening of suspected CF patients
revealed a novel mutation that converted leucine at position 346 to proline
(L346P) in two unrelated families. The second CF mutation was delta F508 and
1677delTA in the two families respectively, both reportedly associated with
severe phenotypes. Yet our patients did not present with typical CF pictures
possibly because of the domit nature of this novel mild mutation in exon 7.
Symptoms included failure to thrive, chest infections and electrolyte
disturbances. These findings raise the possibility that Cyprus might have been
spared very severe CF phenotypes but not cystic fibrosis transmembrane
conductance regulator (CFTR) mutations. The incidence of cystic fibrosis (CF) and the frequency of disease-causing
mutations varies among different ethnic and geographic populations. The Jewish
population around the world is comprised of two major ethnic groups; Ashkenazi
and non-Ashkenazi. The latter is further classified according to country of
origin. In this study, we analyzed the incidence of CF and the distribution of
CF mutations in the general Jewish population in Israel and in most of the
Jewish ethnic subgroups. The disease frequency varies considerably among the
latter. Among Ashkenazi Jews, the frequency of CF is 1:3300, which is similar to
the frequency in most Caucasian populations. Among non-Ashkenazi Jews, the
disease occurs at a similar frequency among Jews from Libya (1:2700), Georgia
(1:2700), Greece and Bulgaria (1:2400), but is rare in Jews from Yemen (1:8800),
Morocco (1:15000), Iraq (1:32000), and Iran (1:39000). So far, only 12 mutations
have been identified in Israeli Jews, and this enables the identification of 91%
of the CF chromosomes in the entire Jewish CF population. However, in each
Jewish ethnic group, the disease is caused by a different repertoire of
mutations. The frequency of identified mutations is high in Ashkenazi Jews
(95%), and in Jews originating from Tunisia (100%), Libya (91%), Turkey (90%),
and Georgia (88%). However, a lower frequency of mutations can be identified in
Moroccan (85%), Egyptian (50%), and Yemenite (0%) Jews. For genetic counseling
of a Jewish individual, it is necessary to calculate the residual risk according
to ethnic origin. Carrier screening of healthy Jewish individuals is currently
feasible for Ashkenazi Tunisian, Libyan, Turkish, and Georgian Jews. These
results provide the required information for genetic counseling of Jewish CF
families and screening programs of Jewish populations worldwide. Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasian
populations. The generally accepted incidence of CF in the United States is 1 in
3,200 in the Caucasian population. Intestinal obstructions and atresias have
been described among patients with CF. An association of CF with infantile
hypertrophic pyloric stenosis (IHPS) has not been previously documented. A
review in our clinic of 72 patients with CF revealed IHPS in two. The incidence
of 2.7% is greater than the 0.3% incidence expected in the general population.
This ninefold increase in IHPS in patients with CF suggests an association
between the two and warrants further investigation. Cystic Fibrosis (CF) is the most common and severe autosomal recessive disease
in Caucasian populations, with an incidence of 1 in 2500 live births. It is
characterized by a generalized disturbance in exocrine glands and it is caused
by over one thousand mutations at the cystic fibrosis conductance regulator gene
(CFTR) mapped at 7q31. AF508 is the most frequent mutation worldwide and it
consists in a deletion of the codon that encodes fenilalanine at the 508
protein's position. The aim of this study was to determine the frequency of the
delta F508 mutation in Venezuelan patients with CF using the Polymerase Chain
Reaction (PCR). We studied thirty patients of twenty eight families who were
diagnosed with CF based on their clinical features and sweat chloride level > 60
mEq/l in two determinations. Detection of the mutation was performed from the
amplification of a 98 pair of bases (pb) CF gene segment which contains the
codon that encodes fenilalanine in the 508 position by PCR. This PCR product is
absent in those who have the mutation. The delta F508 allelic frequency was
26.79%, distributed in six homozygous and seven compound heterozygote delta
F508/X. The reminder mutations (no delta F508) represent 73.21%. The delta F508
frequency in our sample is less than the reported in European countries. On the
other hand, a delta F508 frequency highly heterogeneous has been observed in
Latin-American countries. This variation results from mixed populations with a
different genetic background influenced by external migration and CF molecular
alterations, which exists in the analyzed populations. In this study, the delta
F508 mutation comes mainly from grandparents (79.41%) who were born in
Mediterranean countries and Colombia, while the no delta F508 mutations come
from grandparents who were born in Venezuela (79.27%) and Colombia (17.07%). Cystic fibrosis (CF) was considered to be non-existent in Indian subcontinent.
Reports in last one decade have suggested that cystic fibrosis occurs in India
but its precise magnitude is not known. Studies on migrant Indian population in
United States and United Kingdom estimate frequency of CF as 1:10,000 to
1:40,000. The clinical features are similar to that reported in Caucasian
population. CF in Indian children is usually diagnosed late and in advanced
stage. Children are more malnourished and may have clinically evident deficiency
of fat soluble vitamins. The frequency of clubbing, colonization with
Pseudomonas, and laboratory evidence of pseudo-Bartter syndrome is relatively
more at the time of diagnosis. Diagnostic facilities in form of sweat chloride
estimation and genetic studies are not available readily. Mutation profile is
different. The frequency of common mutation F508del in Indian children is
between 19% and 34%. Other mutations are heterogeneous. Management of CF in
India is difficult due to less number of trained manpower, limited availability,
and high cost of pharmacologic agents. The determits of early death include:
severe malnutrition and colonization with Pseudomonas at the time of diagnosis,
more than four episodes of lower respiratory infection per year and age of onset
of symptoms before 2 months of age. To conclude, CF does occur in India;
however, precise magnitude of problem is not known. There is need to create
awareness amongst pediatricians, developing diagnostic facilities, and
management protocols based on locally available resources. Cystic fibrosis is the most frequent autosomal recessive disease in the
Caucasian population, with an incidence of 1:2500 newborn and a frequency of
1:25. The associated gene is Cystic Fibrosis Transmembrane Conductance Regulator
(CFTR) and it encodes the CFTR protein that functions as a chloride (Cl(-))
channel. It is found in the apical membrane of exocrine epithelial cells,
responsible for the regulation of the movement of water and solutes through
biological membranes. To our knowledge, there are no studies on protein
localization in the different cell types of the seminiferous epithelium with
different pathologies. The aim of the present study was to analyze the
expression of the CFTR protein in the human seminiferous epithelium of infertile
males with different pathologies. CFTR protein expression was studied by
immunohistochemistry in paraffin sections of testicular biopsies of six
infertile men: Sertoli cell only syndrome, maturation arrest, secondary
obstructive azoospermia, primary obstructive azoospermia due to congenital
bilateral absence of the vas deferens (CBAVD), severe oligozoospermia, and
retrograde ejaculation. All cell types of the seminiferous epithelium were
studied: Sertoli cells, spermatogonia, primary spermatocytes at the
leptotene/zygotene and at the pachytene stages, secondary spermatocytes, round,
elongating and elongated spermatids, and spermatozoa. With the exception of
sperm, all cells were labeled in the cytoplasm and in the cytoplasmic membrane.
In the patient with CBAVD labeling was light at the cell membrane and absent in
the cytoplasm of Sertoli cells and diploid germ cells. Generally, labeling was
stronger after the diploid stage, which is probably related to cell volume
reduction during spermiogenesis. The results obtained also suggest that the CFTR
protein may impact CBAVD spermatogenesis and other pathologies. |
What is the effect of thapsigargin treatment? | Thapsigargin is an endoplasmic stress inducer. It is a sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor. | Autophagy is the process by which portions of cytoplasm are enclosed by
membranous organelles, phagophores, which deliver the sequestered cytoplasm to
degradative autophagic vacuoles. Genes and proteins involved in phagophore
manufacture have been extensively studied, but little is known about how mature
phagophores proceed through the subsequent steps of expansion, closure and
fusion. Here we have addressed these issues by combining our unique autophagic
cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a
cargo marker) with quantitative measurements of the lipidation-dependent
anchorage and turnover of the phagophore-associated protein LC3. In isolated rat
hepatocytes, amino acid starved to induce maximal autophagic activity, the two
unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin
(TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited
LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to
prevent phagophore closure. Intriguingly, the resumption of cargo sequestration
seen upon release from a reversible TG block was completely suppressed by 3MA,
revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks
phagophore closure at a post-lipidation step. 3MA did not, however, prevent the
resumption of lysosomal LC3 degradation, indicating that phagophores could fuse
directly with degradative autophagic vacuoles without carrying cytosolic cargo.
This fusion step was clearly blocked by TG. Furthermore, density gradient
centrifugation revealed that a fraction of the LC3-marked phagophores retained
by TG could be density-shifted by the acidotropic drug propylamine along with
the lysosomal marker cathepsin B, suggesting physical association of some
phagophores with lysosomes prior to cargo sequestration. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of
the tumor necrosis factor superfamily, whose members are capable of inducing
apoptosis and inflammation. Endoplasmic reticulum stress (ERS) plays a key role
in immune surveillance in macrophages. TRAIL mRNA and protein expression have
previously been detected in macrophages; however, whether ERS has any effects on
TRAIL expression in macrophages has not yet been determined. Here, we
demonstrate that thapsigargin (TG) and tunicamycin (TM), two ERS inducers
activated macrophages were able to increase TRAIL mRNA and protein expression in
RAW264.7 macrophages, the culture supernatant of THP-1 cells, and mouse
peritoneal macrophages, indicating that ERS as a potent inducer of TRAIL
transcription and expression in macrophages. This effect was blocked by the
specific JNK inhibitor SP600125 and transcription factor AP-1 inhibitor SR 1130.
Interestingly, at the molecular level, regulation of TRAIL expression by ERS was
accompanied by a significant decrease in cytokine signaling suppressor 3
(SOCS3). SOCS3 siRNA clearly increased the expression of TRAIL mRNA and protein
under ERS by activating the AP-1 components phosphorylated c-Jun and
phosphorylated c-Fos in RAW264.7 cells. In contrast, over-expression of SOCS3
reversed ERS-induced TRAIL expression. These findings provide in vitro evidence
that SOCS3 plays a critical negative role in the regulation of ERS-induced TRAIL
expression via the Jun N-terminal kinase/AP-1 signaling pathway in macrophages. Abnormal accumulation of undigested macromolecules, often disease-specific, is a
major feature of lysosomal and neurodegenerative disease and is frequently
attributed to defective autophagy. The mechanistic underpinnings of the
autophagy defects are the subject of intense research, which is aided by genetic
disease models. To gain an improved understanding of the pathways regulating
defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis
(JNCL or Batten disease), a neurodegenerative disease of childhood, we developed
and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3)
screening assay to identify, in an unbiased fashion, genotype-sensitive small
molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the
most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic
reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed
significantly more activity in the mouse JNCL cells, an effect that was also
observed in human-induced pluripotent stem cell-derived JNCL neural progenitor
cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and
autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation.
Interrogation of intracellular Ca(2+) handling highlighted alterations in
endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in
store-operated Ca(2+) uptake in JNCL cells. These results further support an
important role for the CLN3 protein in intracellular Ca(2+) handling and in
autophagic pathway flux and establish a powerful new platform for therapeutic
screening. AIMS: Adipocytes play a critical role in energy balance. Growth of fat tissue is
achieved via an increase in adipocyte mass and the formation of newly
differentiated adipocytes from precursor cells. Understanding the cellular and
molecular mechanisms of adipocyte differentiation is crucial for the study of
obesity- and fat-related diseases. The present study was designed to study
whether small heterodimer partner-interacting leucine zipper protein (SMILE), a
novel co-repressor, could regulate differentiation of adipocyte in 3T3-L1 cells.
MATERIALS AND METHODS: Treatment of endoplasmic stress inducers, thapsigargin
and tunicamycin, inhibited adipocyte differentiation, stimulated Smile mRNA
expression, and repressed the expression of adiponectin (Adipoq) in 3T3-L1
pre-adipocyte. Overexpression of SMILE in 3T3-L1 cells decreased the expression
of the mRNA encoding Adipoq, a major marker of adipocytes, significantly.
Furthermore, knockdown of SMILE recovered the thapsigargin-mediated repression
of Adipoq transcription. Co-immunoprecipitation experiments revealed that SMILE
interacted physically with PPARγ in 3T3-L1 cells. In addition, chromatin
immunoprecipitation experiments revealed that SMILE suppressed the binding
affinity of PPARγ for the Adipoq promoter.
KEY FINDINGS: We demonstrate that SMILE controls adipocyte differentiation by
regulating the transactivity of peroxisome proliferator-activated receptor γ
(PPARγ).
SIGNIFICANCE: These findings demonstrate that SMILE represses adipocyte
differentiation by regulating PPARγ transactivity; hence, SMILE is a potential
regulator of PPARγ-related diseases. Calcium-activated chloride channel (CaCC) plays an important role in modulating
epithelial secretion. It has been suggested that in salivary tissues, sustained
fluid secretion is dependent on Ca(2+) influx that activates ion channels such
as CaCC to initiate Cl(-) efflux. However direct evidence as well as the
molecular identity of the Ca(2+) channel responsible for activating CaCC in
salivary tissues is not yet identified. Here we provide evidence that in human
salivary cells, an outward rectifying Cl(-) current was activated by increasing
[Ca(2+)]i, which was inhibited by the addition of pharmacological agents
niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a
inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and
activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was
inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or
removal of external Ca(2+). Stimulation with Tg also increased plasma membrane
expression of TMEM16a protein, which was also dependent on Ca(2+) entry.
Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3,
inhibited CaCC especially upon store depletion. Moreover, primary acinar cells
isolated from submandibular gland also showed outward rectifying Cl(-) currents
upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the
addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar
cells isolated from the submandibular glands of TRPC1 knockout mice showed
significant inhibition of the outward Cl(-) currents without decreasing TMEM16a
expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels
is essential for the activation of CaCC. Genome-wide association studies in human type 2 diabetes (T2D) have renewed
interest in the pancreatic islet as a contributor to T2D risk. Chronic low-grade
inflammation resulting from obesity is a risk factor for T2D and a possible
trigger of β-cell failure. In this study, microarray data were collected from
mouse islets after overnight treatment with cytokines at concentrations
consistent with the chronic low-grade inflammation in T2D. Genes with a
cytokine-induced change of >2-fold were then examined for associations between
single nucleotide polymorphisms and the acute insulin response to glucose (AIRg)
using data from the Genetics Underlying Diabetes in Hispanics (GUARDIAN)
Consortium. Significant evidence of association was found between AIRg and
single nucleotide polymorphisms in Arap3 (5q31.3), F13a1 (6p25.3), Klhl6
(3q27.1), Nid1 (1q42.3), Pamr1 (11p13), Ripk2 (8q21.3), and Steap4 (7q21.12). To
assess the potential relevance to islet function, mouse islets were exposed to
conditions modeling low-grade inflammation, mitochondrial stress, endoplasmic
reticulum (ER) stress, glucotoxicity, and lipotoxicity. RT-PCR revealed that one
or more forms of stress significantly altered expression levels of all genes
except Arap3. Thapsigargin-induced ER stress up-regulated both Pamr1 and Klhl6.
Three genes confirmed microarray predictions of significant cytokine
sensitivity: F13a1 was down-regulated 3.3-fold by cytokines, Ripk2 was
up-regulated 1.5- to 3-fold by all stressors, and Steap4 was profoundly cytokine
sensitive (167-fold up-regulation). Three genes were thus closely associated
with low-grade inflammation in murine islets and also with a marker for islet
function (AIRg) in a diabetes-prone human population. This islet-targeted
genome-wide association scan identified several previously unrecognized
candidate genes related to islet dysfunction during the development of T2D. The protective effects of 5'-AMP-activated protein kinase (AMPK) on the
metabolic syndrome may include direct effects on resistance artery vasomotor
function. However, the precise actions of AMPK on microvessels and their
potential interaction are largely unknown. Thus, we set to determine the effects
of AMPK activation on vascular smooth muscle tone and the underlying mechanisms.
Resistance arteries isolated from hamster and mouse exhibited a pronounced
endothelium-independent dilation on direct pharmacological AMPK activation by 2
structurally unrelated compounds (PT1 and A769662). The dilation was associated
with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth
muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by
patch clamp studies in freshly isolated hamster vascular smooth muscle cell and
confirmed by direct proof of membrane hyperpolarization in intact arteries. The
BKCa channel blocker iberiotoxin abolished the hyperpolarization but only
partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By
contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor
thapsigargin largely reduced these effects, whereas combined inhibition of SERCA
and BKCa channels virtually abolished them. AMPK stimulation significantly
increased the phosphorylation of the SERCA modulator phospholamban at the
regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent
vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular
smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium
sequestration via SERCA activation, as well as activation of BKCa channels.
There is in part a mutual compensation of both calcium-lowering mechanisms.
However, SERCA activation which involves an AMPK-dependent phosphorylation of
phospholamban is the predomit mechanism in resistance vessels. Reactive oxygen species regulate cardiovascular and renal function in health and
disease. Superoxide participates in acute calcium signaling in afferent
arterioles and renal vasoconstriction produced by angiotensin II, endothelin,
thromboxane, and pressure-induced myogenic tone. Known mechanisms by which
superoxide acts include quenching of nitric oxide and increased ADP ribosyl
cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on
other calcium signaling pathways in the renal microcirculation is poorly
understood. The present experiments examined the acute effect of superoxide
generated by paraquat on calcium entry pathways in isolated rat afferent
arterioles. The peak increase in cytosolic calcium concentration caused by KCl
(40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was
mediated exclusively by L-type channels because it was abolished by nifedipine
but was unaffected by the T-type channel blocker mibefradil. Paraquat increased
superoxide production (dihydroethidium fluorescence), tripled the peak response
to KCl to 314±68 nmol/L (P<0.001) and doubled the plateau response. These
effects were abolished by tempol and nitroblue tetrazolium, but not by catalase,
confirming actions of superoxide and not of hydrogen peroxide. Unaffected by
paraquat and superoxide was calcium entry through store-operated calcium
channels activated by thapsigargin-induced calcium depletion of sarcoplasmic
reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated
calcium-induced calcium release from the sarcoplasmic reticulum. Our results
provide new evidence that superoxide enhances calcium entry through L-type
channels activated by membrane depolarization in rat cortical afferent
arterioles, without affecting calcium entry through store-operated entry or
ryanodine receptor-mediated calcium mobilization. Author information:
(1)Section of Biochemistry and Molecular Biology, Department of Medical
Sciences, Faculty of Medicine, University of Miyazaki, 5200, Kihara, Kiyotake,
Miyazaki 889-1692, Japan; Division of Bacteriology, Department of Infectious
Diseases Control, International Research Center for Infectious Diseases, The
Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo 108-8639, Japan. Electronic address:
[email protected].
(2)Division of Bacteriology, Department of Infectious Diseases Control,
International Research Center for Infectious Diseases, The Institute of Medical
Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan; Department of Biochemistry, Kawasaki Medical School, 577, Matsushima,
Kurashiki, Okayama 701-0192, Japan.
(3)Division of Bacteriology, Department of Infectious Diseases Control,
International Research Center for Infectious Diseases, The Institute of Medical
Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan.
(4)Laboratory Center for Proteomics Research, Graduate School of Frontier
Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
(5)Section of Biochemistry and Molecular Biology, Department of Medical
Sciences, Faculty of Medicine, University of Miyazaki, 5200, Kihara, Kiyotake,
Miyazaki 889-1692, Japan. RATIONALE: Hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1
(Hrd1) is an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase that
has been studied in yeast, where it contributes to ER protein quality control by
ER-associated degradation (ERAD) of misfolded proteins that accumulate during ER
stress. Neither Hrd1 nor ERAD has been studied in the heart, or in cardiac
myocytes, where protein quality control is critical for proper heart function.
OBJECTIVE: The objective of this study were to elucidate roles for Hrd1 in ER
stress, ERAD, and viability in cultured cardiac myocytes and in the mouse heart,
in vivo.
METHODS AND RESULTS: The effects of small interfering RNA-mediated Hrd1
knockdown were examined in cultured neonatal rat ventricular myocytes. The
effects of adeno-associated virus-mediated Hrd1 knockdown and overexpression
were examined in the hearts of mice subjected to pressure overload-induced
pathological cardiac hypertrophy, which challenges protein-folding capacity. In
cardiac myocytes, the ER stressors, thapsigargin and tunicamycin increased ERAD,
as well as adaptive ER stress proteins, and minimally affected cell death.
However, when Hrd1 was knocked down, thapsigargin and tunicamycin dramatically
decreased ERAD, while increasing maladaptive ER stress proteins and cell death.
In vivo, Hrd1 knockdown exacerbated cardiac dysfunction and increased apoptosis
and cardiac hypertrophy, whereas Hrd1 overexpression preserved cardiac function
and decreased apoptosis and attenuated cardiac hypertrophy in the hearts of mice
subjected to pressure overload.
CONCLUSIONS: Hrd1 and ERAD are essential components of the adaptive ER stress
response in cardiac myocytes. Hrd1 contributes to preserving heart structure and
function in a mouse model of pathological cardiac hypertrophy. Baicalein (5,6,7-trihydroxyflavone) (1) has been found to be active against a
wide variety of cancer cells. However, the molecular mechanism underlying the
effects of 1 on the induction of Ca(2+) movement and cytotoxicity in human
breast cancer cells is unknown. This study examined the relationship between
1-induced Ca(2+) signaling and cytotoxicity in ZR-75-1 human breast cancer
cells. The in vitro investigations reported herein produced the following
results: (i) Compound 1 increased intracellular Ca(2+) concentration ([Ca(2+)]i)
in a concentration-dependent manner. The signal was decreased by approximately
50% by removal of extracellular Ca(2+). (ii) Compound 1-triggered [Ca(2+)]i
increases were significantly suppressed by store-operated Ca(2+) channel
blockers 2-aminoethoxydiphenyl borate (2-APB) and the PKC inhibitor GF109203X.
(iii) In Ca(2+)-free medium, compound 1-induced [Ca(2+)]i increases were also
inhibited by GF109203X. Furthermore, pretreatment with the endoplasmic reticulum
Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-ditert-butylhydroquinone (BHQ)
abolished 1-induced [Ca(2+)]i increases. Inhibition of phospholipase C (PLC)
with U73122 abolished 1-induced [Ca(2+)]i increases. (iv) Compound 1 (20-40 μM)
caused cytotoxicity, increased reactive oxygen species (ROS) production, and
activated caspase-9/caspase-3. Furthermore, compound 1-induced apoptosis was
significantly inhibited by prechelating cytosolic Ca(2+) with BAPTA-AM
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester)
or by decreasing ROS with the antioxidant NAC (N-acetylcysteine). Together,
baicalein (1) induced a [Ca(2+)]i increase by inducing PLC-dependent Ca(2+)
release from the endoplasmic reticulum and Ca(2+) entry via PKC-dependent,
2-APB-sensitive store-operated Ca(2+) channels. Moreover, baicalein (1) induced
Ca(2+)-associated apoptosis involved ROS production in ZR-75-1 cells. |
What is the Her2 status in Li-Fraumeni syndrome? | In the background of a germline TP53 mutation of the Li-Fraumeni syndrome, the Her2 status was found to be positive in 63-83% of the cases. | BACKGROUND: Germline TP53 mutations predispose to early onset breast cancer in
women and are associated with Li-Fraumeni syndrome. Published data on the
pathological characteristics of breast cancer among women with TP53 mutations is
limited.
METHODS: We retrospectively reviewed the clinical records of women who underwent
genetic testing for suspected germline TP53 mutations and who were diagnosed
with breast cancer between 2000 and 2011. The pathological characteristics of
the breast tumors from patients testing positive for a mutation (cases) were
compared with those testing negative (controls).
RESULTS: Patients who tested positive for germlineTP53 mutations (n = 30) were
compared with controls (n = 79). Human epidermal growth factor receptor 2 (HER2)
amplification and/or overexpression was found in 67% of the tumors from the
cases, compared with 25% for the controls (P = .0001). Among patients with a
mutation, 70% had estrogen receptor- and/or progesterone receptor-positive
tumors, compared with 68% in the control group (P = .87). After adjusting for
age at breast cancer diagnosis, having a HER2-positive tumor increased the odds
of testing positive for a germline TP53 mutation (odds ratio, 6.9; 95%
confidence interval, 2.6-18.2). For each yearly increment in age at breast
cancer diagnosis, there was decreased likelihood of having a TP53 mutation of 5%
(odds ratio, 0.95; 95% confidence interval0.91-0.99).
CONCLUSIONS: This study suggests an association between germline TP53 mutations
and early onset HER2-positive breast cancer. If confirmed in a larger cohort,
these results could guide genetic testing strategies, lead to chemoprevention
trials incorporating HER2-targeted therapies, and elucidate some of the
molecular pathways involved in breast cancer. Li-Fraumeni syndrome (LFS) is one of the most penetrant forms of familial cancer
susceptibility syndromes, characterized by early age at tumor onset and a wide
spectrum of maligt tumors. Identifying LFS in patients with cancer is
clinically imperative because they have an increased sensitivity to ionizing
radiation and are more likely to develop radiation-induced secondary
maligcies. This case report describes a young woman whose initial
presentation of LFS was early-onset breast cancer and whose treatment of this
primary maligcy with breast conservation likely resulted in a secondary
maligcy arising in her radiation field. As seen in this case, most breast
cancers in patients with LFS exhibit a triple-positive phenotype (estrogen
receptor-positive/progesterone receptor-positive/HER2-positive). Although this
patient met classic LFS criteria based on age and personal and family history of
cancer, the NCCN Clinical Practice Guidelines in Oncology for Genetic/Familial
High-Risk Assessment: Breast and Ovarian Cancer endorse genetic screening for
TP53 mutations in a subset of patients with early-onset breast cancer, even in
the absence of a suggestive family history, because of the potential for de novo
TP53 mutations. |
Where is X-ray free electron laser used? | X-ray free electron laser (XFEL) technologies provide coherent and extremely intense photon pulses of short duration. XFELs are particularly useful in structural biology and imaging, in structural studies of single biological macromolecules (e.g. high resolution protein structure determination) and assemblies (e.g. viruses) or nanocrystals, which are not amenable to investigation with traditional crystallographic methods. Moreover, XFELs have the potential to be used for studying enzyme kinetics. | Membrane proteins constitute > 30% of the proteins in an average cell, and yet
the number of currently known structures of unique membrane proteins is < 300.
To develop new concepts for membrane protein structure determination, we have
explored the serial ocrystallography method, in which fully hydrated protein
ocrystals are delivered to an x-ray beam within a liquid jet at room
temperature. As a model system, we have collected x-ray powder diffraction data
from the integral membrane protein Photosystem I, which consists of 36 subunits
and 381 cofactors. Data were collected from crystals ranging in size from 100 nm
to 2 μm. The results demonstrate that there are membrane protein crystals that
contain < 100 unit cells (200 total molecules) and that 3D crystals of membrane
proteins, which contain < 200 molecules, may be suitable for structural
investigation. Serial ocrystallography overcomes the problem of x-ray damage,
which is currently one of the major limitations for x-ray structure
determination of small crystals. By combining serial ocrystallography with
x-ray free-electron laser sources in the future, it may be possible to produce
molecular-resolution electron-density maps using membrane protein crystals that
contain only a few hundred or thousand unit cells. X-ray crystallography provides the vast majority of macromolecular structures,
but the success of the method relies on growing crystals of sufficient size. In
conventional measurements, the necessary increase in X-ray dose to record data
from crystals that are too small leads to extensive damage before a diffraction
signal can be recorded. It is particularly challenging to obtain large,
well-diffracting crystals of membrane proteins, for which fewer than 300 unique
structures have been determined despite their importance in all living cells.
Here we present a method for structure determination where single-crystal X-ray
diffraction 'snapshots' are collected from a fully hydrated stream of
ocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the
Linac Coherent Light Source. We prove this concept with ocrystals of
photosystem I, one of the largest membrane protein complexes. More than
3,000,000 diffraction patterns were collected in this study, and a
three-dimensional data set was assembled from individual photosystem I
ocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation
damage in crystallography by using pulses briefer than the timescale of most
damage processes. This offers a new approach to structure determination of
macromolecules that do not yield crystals of sufficient size for studies using
conventional radiation sources or are particularly sensitive to radiation
damage. X-ray lasers offer new capabilities in understanding the structure of biological
systems, complex materials and matter under extreme conditions. Very short and
extremely bright, coherent X-ray pulses can be used to outrun key damage
processes and obtain a single diffraction pattern from a large macromolecule, a
virus or a cell before the sample explodes and turns into plasma. The continuous
diffraction pattern of non-crystalline objects permits oversampling and direct
phase retrieval. Here we show that high-quality diffraction data can be obtained
with a single X-ray pulse from a non-crystalline biological sample, a single
mimivirus particle, which was injected into the pulsed beam of a hard-X-ray
free-electron laser, the Linac Coherent Light Source. Calculations indicate that
the energy deposited into the virus by the pulse heated the particle to over
100,000 K after the pulse had left the sample. The reconstructed exit wavefront
(image) yielded 32-nm full-period resolution in a single exposure and showed no
measurable damage. The reconstruction indicates inhomogeneous arrangement of
dense material inside the virion. We expect that significantly higher
resolutions will be achieved in such experiments with shorter and brighter
photon pulses focused to a smaller area. The resolution in such experiments can
be further extended for samples available in multiple identical copies. 1960 Theodore Maiman built the first Ruby-LASER, starting-point for half a
century of R&D on Biomedical LASER continuous improvement. The purpose of this
paper is to contribute a review of the often disregarded, however, extremely
important Industrial Property documents of LASER-based in vitro Diagnostics
devices. It is an attempt to sketch-out the patent-trail leading towards the
modern Biomedical Laboratory and to offer an introduction to the employment of
"exotic" systems, such as the Free Electron LASER (FEL), that are expected to
focus on the fundamental processes of life, following chemical reactions and
biological processes as they happen, on unprecedented time and size scales.
There are various in vitro LASER applications, however, the most important ones
include: Hybrid Coulter Principle-LASER Hematology Analyzers. Flow Cytometry
systems. Fluorescent in situ Hybridization (FISH Techniques). Confocal LASER
Scanning Microscopy and Cytometry. From the first fluorescence-based flow
Cytometry device developed in 1968 by Wolfgang Göhde until nowadays, numerous
improvements and new features related to these devices appeared. The relevant
industrial property milestone-documents and their overall numeral trends are
presented. In 1971, J. Madey invented and developed the Free Electron LASER
(FEL), a vacuum-tube that uses a beam of relativistic electrons passing through
a periodic, transverse magnetic field (wiggler) to produce coherent radiation,
contained in an optical cavity defined by mirrors. A resoce condition that
involves the energy of the electron beam, the strength of the magnetic field,
and the periodicity of the magnet determines the wavelength of the radiation.
The FEL Coherent Light Sources like the Linac Coherent Light Source (LCLS) at
Stanford, CA, USA or the Xray Free Electron LASER (XFEL) at Hamburg, Germany,
will work much like a high-speed (< 100 femtoseconds) camera, enabling
scientists to take stop-motion pictures, on the oscale, of atoms and
molecules in motion. The curve of FEL-related patents of the last 20 years is
much smoother than the corresponding one for in vitro Diagnostics conventional
LASERS. If the diodes brought a LASER into almost everyone's pocket, the
above-mentioned super-imaging systems are huge facilities of enormous cost--the
price to steal a look at the fundamental processes of life. Coherent diffractive imaging using x-ray free-electron lasers (XFELs) may
provide a unique opportunity for high-resolution structural analysis of single
particles sprayed from an aqueous solution into the laser beam. As a result,
diffraction images are measured from randomly oriented objects covered by a
water layer. We analyze theoretically how the thickness of the covering water
layer influences the structural and orientational information contained in the
recorded diffraction images. This study has implications for planned experiments
on single-particle imaging with XFELs. The multiwavelength anomalous diffraction (MAD) method is used to determine
phase information in x-ray crystallography by employing anomalous scattering
from heavy atoms. X-ray free-electron lasers (FELs) show promise for revealing
the structure of single molecules or ocrystals, but the phase problem remains
largely unsolved. Because of the ultrabrightness of x-ray FEL, samples
experience severe electronic radiation damage, especially to heavy atoms, which
hinders direct implementation of MAD with x-ray FELs. Here, we propose a
generalized version of MAD phasing at high x-ray intensity. We demonstrate the
existence of a Karle-Hendrickson-type equation in the high-intensity regime and
calculate relevant coefficients with detailed electronic damage dynamics of
heavy atoms. The present method offers a potential for ab initio structural
determination in femtosecond x-ray ocrystallography. Johansson LC(1), Arnlund D, White TA, Katona G, Deponte DP, Weierstall U, Doak
RB, Shoeman RL, Lomb L, Malmerberg E, Davidsson J, Nass K, Liang M, Andreasson
J, Aquila A, Bajt S, Barthelmess M, Barty A, Bogan MJ, Bostedt C, Bozek JD,
Caleman C, Coffee R, Coppola N, Ekeberg T, Epp SW, Erk B, Fleckenstein H, Foucar
L, Graafsma H, Gumprecht L, Hajdu J, Hampton CY, Hartmann R, Hartmann A, Hauser
G, Hirsemann H, Holl P, Hunter MS, Kassemeyer S, Kimmel N, Kirian RA, Maia FR,
Marchesini S, Martin AV, Reich C, Rolles D, Rudek B, Rudenko A, Schlichting I,
Schulz J, Seibert MM, Sierra RG, Soltau H, Starodub D, Stellato F, Stern S,
Strüder L, Timneanu N, Ullrich J, Wahlgren WY, Wang X, Weidenspointner G,
Wunderer C, Fromme P, Chapman HN, Spence JC, Neutze R. We demonstrate the use of an X-ray free electron laser synchronized with an
optical pump laser to obtain X-ray diffraction snapshots from the photoactivated
states of large membrane protein complexes in the form of ocrystals flowing
in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals
were observed at time delays of 5 to 10 µs after excitation. The result
correlates with the microsecond kinetics of electron transfer from Photosystem I
to ferredoxin. The undocking process that follows the electron transfer leads to
large rearrangements in the crystals that will terminally lead to the
disintegration of the crystals. We describe the experimental setup and obtain
the first time-resolved femtosecond serial X-ray crystallography results from an
irreversible photo-chemical reaction at the Linac Coherent Light Source. This
technique opens the door to time-resolved structural studies of reaction
dynamics in biological systems. A growing number of X-ray sources based on the free-electron laser (XFEL)
principle are presently under construction or have recently started operation.
The intense, ultrashort pulses of these sources will enable new insights in many
different fields of science. A key problem is to provide x-ray optical elements
capable of collecting the largest possible fraction of the radiation and to
focus into the smallest possible focus. As a key step towards this goal, we
demonstrate here the first ofocusing of hard XFEL pulses. We developed
diamond based Fresnel zone plates capable of withstanding the full beam of the
world's most powerful x-ray laser. Using an imprint technique, we measured the
focal spot size, which was limited to 320 nm FWHM by the spectral band width of
the source. A peak power density in the focal spot of 4×10(17)W/cm(2) was
obtained at 70 fs pulse length. The recent development of x-ray free electron lasers providing coherent,
femtosecond-long pulses of high brilliance and variable energy opens new areas
of scientific research in a variety of disciplines such as physics, chemistry,
and biology. Pump-probe experimental techniques which observe the temporal
evolution of systems after optical or x-ray pulse excitation are one of the main
experimental schemes currently in use for ultrafast studies. The key challenge
in these experiments is to reliably achieve temporal and spatial overlap of the
x-ray and optical pulses. Here we present measurements of the x-ray pulse
induced transient change of optical reflectivity from a variety of materials
covering the soft x-ray photon energy range from 500eV to 2000eV and outline the
use of this technique to establish and characterize temporal synchronization of
the optical-laser and FEL x-ray pulses. The short and intense pulses of the new X-ray free electron lasers, now
operational or under construction, may make possible diffraction experiments on
single molecule-sized objects with high resolution, before radiation damage
destroys the sample. In a single molecule imaging (SMI) experiment thousands of
diffraction patterns of single molecules with random orientations are recorded.
One of the most challenging problems of SMI is how to assemble these noisy
patterns of unknown orientations into a consistent single set of diffraction
data. Here we present a new method which can solve the orientation problem of
SMI efficiently even for large biological molecules and in the presence of
noise. We show on simulated diffraction patterns of a large protein molecule,
how the orientations of the patterns can be found and the structure to atomic
resolution can be solved. The concept of our algorithm could be also applied to
experiments where images of an object are recorded in unknown orientations
and/or positions like in cryoEM or tomography. In this work, the Coulomb explosion of the octamer water cluster has been
studied employing a theoretical approach. Instead of the usual methodology that
makes use of classical molecular dynamics, time-dependent density functional
theory has been applied to tackle the problem. This method explicitly accounts
for the laser field and thus does not impose any constraint on the interaction
between the laser pulse and the cluster. We focus on the effects of energetic
changes in the system under high-intensity soft X-ray laser pulses. The motions
of the ions and their velocities during this process show significant
differences for the three applied laser intensities (10(14), 10(15) and 10(16) W
cm(-2)). Very strong soft X-ray free electron laser (FEL) pulses must be short
to allow for investigations of ultra-fast wet chemistry, according to the
principle of collect and destroy. The emergence of femtosecond diffractive imaging with X-ray lasers has enabled
pioneering structural studies of isolated particles, such as viruses, at
ometer length scales. However, the issue of missing low frequency data
significantly limits the potential of X-ray lasers to reveal sub-ometer
details of micrometer-sized samples. We have developed a new technique of
dark-field coherent diffractive imaging to simultaneously overcome the missing
data issue and enable us to harness the unique contrast mechanisms available in
dark-field microscopy. Images of airborne particulate matter (soot) up to two
microns in length were obtained using single-shot diffraction patterns obtained
at the Linac Coherent Light Source, four times the size of objects previously
imaged in similar experiments. This technique opens the door to femtosecond
diffractive imaging of a wide range of micrometer-sized materials that exhibit
irreproducible complexity down to the oscale, including airborne particulate
matter, small cells, bacteria and gold-labeled biological samples. A multi-crystal wavelength dispersive hard x-ray spectrometer with high-energy
resolution and large solid angle collection is described. The instrument is
specifically designed for time-resolved applications of x-ray emission
spectroscopy (XES) and x-ray Raman scattering (XRS) at X-ray Free Electron
Lasers (XFEL) and synchrotron radiation facilities. It also simplifies resot
inelastic x-ray scattering (RIXS) studies of the whole 2d RIXS plane. The
spectrometer is based on the Von Hamos geometry. This dispersive setup enables
an XES or XRS spectrum to be measured in a single-shot mode, overcoming the
scanning needs of the Rowland circle spectrometers. In conjunction with the XFEL
temporal profile and high-flux, it is a powerful tool for studying the dynamics
of time-dependent systems. Photo-induced processes and fast catalytic reaction
kinetics, ranging from femtoseconds to milliseconds, will be resolvable in a
wide array of systems circumventing radiation damage. Ultrashort X-ray pulses from free-electron laser X-ray sources make it feasible
to conduct small- and wide-angle scattering experiments on biomolecular samples
in solution at sub-picosecond timescales. During these so-called fluctuation
scattering experiments, the absence of rotational averaging, typically induced
by Brownian motion in classic solution-scattering experiments, increases the
information content of the data. In order to perform shape reconstruction or
structure refinement from such data, it is essential to compute the theoretical
profiles from three-dimensional models. Based on the three-dimensional Zernike
polynomial expansion models, a fast method to compute the theoretical
fluctuation scattering profiles has been derived. The theoretical profiles have
been validated against simulated results obtained from 300 000 scattering
patterns for several representative biomolecular species. Research opportunities and techniques are reviewed for the application of hard
x-ray pulsed free-electron lasers (XFEL) to structural biology. These include
the imaging of protein ocrystals, single particles such as viruses,
pump--probe experiments for time-resolved ocrystallography, and snapshot
wide-angle x-ray scattering (WAXS) from molecules in solution. The use of
femtosecond exposure times, rather than freezing of samples, as a means of
minimizing radiation damage is shown to open up new opportunities for the
molecular imaging of biochemical reactions at room temperature in solution. This
is possible using a 'diffract-and-destroy' mode in which the incident pulse
terminates before radiation damage begins. Methods for delivering hundreds of
hydrated bioparticles per second (in random orientations) to a pulsed x-ray beam
are described. New data analysis approaches are outlined for the correlated
fluctuations in fast WAXS, for protein ocrystals just a few molecules on a
side, and for the continuous x-ray scattering from a single virus. Methods for
determining the orientation of a molecule from its diffraction pattern are
reviewed. Methods for the preparation of protein ocrystals are also reviewed.
New opportunities for solving the phase problem for XFEL data are outlined. A
summary of the latest results is given, which now extend to atomic resolution
for ocrystals. Possibilities for time-resolved chemistry using fast WAXS
(solution scattering) from mixtures is reviewed, toward the general goal of
making molecular movies of biochemical processes. X-ray free electron lasers hold the promise of enabling atomic-resolution
diffractive imaging of single biological molecules. We develop a hybrid
continuum-particle model to describe the x-ray induced damage and find that the
photoelectron dynamics and electrostatic confinement strongly affect the time
scale of the damage processes. These phenomena are not fully captured in
hydrodynamic modeling approaches. We determined the pulse duration of x-ray free electron laser light at 10 keV
using highly resolved single-shot spectra, combined with an x-ray free electron
laser simulation. Spectral profiles, which were measured with a spectrometer
composed of an ultraprecisely figured elliptical mirror and an analyzer flat
crystal of silicon (555), changed markedly when we varied the compression
strength of the electron bunch. The analysis showed that the pulse durations
were reduced from 31 to 4.5 fs for the strongest compression condition. The
method, which is readily applicable to evaluate shorter pulse durations,
provides a firm basis for the development of femtosecond to attosecond sciences
in the x-ray region. In single-particle coherent x-ray diffraction imaging experiments, performed at
x-ray free-electron lasers (XFELs), samples are exposed to intense x-ray pulses
to obtain single-shot diffraction patterns. The high intensity induces
electronic dynamics on the femtosecond time scale in the system, which can
reduce the contrast of the obtained diffraction patterns and adds an isotropic
background. We quantify the degradation of the diffraction pattern from
ultrafast electronic damage by performing simulations on a biological sample
exposed to x-ray pulses with different parameters. We find that the contrast is
substantially reduced and the background is considerably strong only if almost
all electrons are removed from their parent atoms. This happens at fluences of
at least one order of magnitude larger than provided at currently available XFEL
sources. The recent development of X-ray free-electron laser sources has created new
opportunities for the structural analysis of protein ocrystals. The extremely
small sizes of the crystals, as well as imperfections of the crystal structure,
result in an interference phenomenon in the diffraction pattern. With decreasing
crystallite size the structural imperfections play a role in the formation of
the diffraction pattern that is comparable in importance to the size effects and
should be taken into account during the data analysis and structure
reconstruction processes. There now exists a need to develop new methods of
protein structure determination that do not depend on the availability of
good-quality crystals and that can treat proteins under conditions close to the
active form. This paper demonstrates an approach that is specifically tailored
to ocrystalline samples and offers a unique crystallographic solution. The phase problem is inherent to crystallographic, astronomical and optical
imaging where only the intensity of the scattered signal is detected and the
phase information is lost and must somehow be recovered to reconstruct the
object's structure. Modern imaging techniques at the molecular scale rely on
utilizing novel coherent light sources like X-ray free electron lasers for the
ultimate goal of visualizing such objects as individual biomolecules rather than
crystals. Here, unlike in the case of crystals where structures can be solved by
model building and phase refinement, the phase distribution of the wave
scattered by an individual molecule must directly be recovered. There are two
well-known solutions to the phase problem: holography and coherent diffraction
imaging (CDI). Both techniques have their pros and cons. In holography, the
reconstruction of the scattered complex-valued object wave is directly provided
by a well-defined reference wave that must cover the entire detector area which
often is an experimental challenge. CDI provides the highest possible, only
wavelength limited, resolution, but the phase recovery is an iterative process
which requires some pre-defined information about the object and whose outcome
is not always uniquely-defined. Moreover, the diffraction patterns must be
recorded under oversampling conditions, a pre-requisite to be able to solve the
phase problem. Here, we report how holography and CDI can be merged into one
superior technique: holographic coherent diffraction imaging (HCDI). An inline
hologram can be recorded by employing a modified CDI experimental scheme. We
demonstrate that the amplitude of the Fourier transform of an inline hologram is
related to the complex-valued visibility, thus providing information on both,
the amplitude and the phase of the scattered wave in the plane of the
diffraction pattern. With the phase information available, the condition of
oversampling the diffraction patterns can be relaxed, and the phase problem can
be solved in a fast and unambiguous manner. We demonstrate the reconstruction of
various diffraction patterns of objects recorded with visible light as well as
with low-energy electrons. Although we have demonstrated our HCDI method using
laser light and low-energy electrons, it can also be applied to any other
coherent radiation such as X-rays or high-energy electrons. X-ray free electron lasers (XFELs) deliver short (<100 fs) and intense (∼10(12)
photons) pulses of hard X-rays, making them excellent sources for time-resolved
studies. Here we show that, despite the inherent instabilities of current (SASE
based) XFELs, they can be used for measuring high-quality X-ray absorption data
and we report femtosecond time-resolved X-ray absorption near-edge spectroscopy
(XANES) measurements of a spin-crossover system, iron(II) tris(2,2'-bipyridine)
in water. The data indicate that the low-spin to high-spin transition can be
modeled by single-exponential kinetics convoluted with the overall time
resolution. The resulting time constant is ∼160 fs. The opening of hard X-ray free-electron laser facilities, such as the Linac
Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory in the
United States, has ushered in a new era in structural determination. With X-ray
pulse durations down to 10 fs or shorter, and up to 10(13) transversely coherent
photons per pulse in a narrow spectral bandwidth, focused irradiances of 10(18)
to 10(21) W cm(-2) or higher can be produced at X-ray energies ranging from 500
eV to 10 keV. New techniques for determining the structure of systems that
cannot be crystallized and for studying the time-resolved behavior of
irreversible reactions at femtosecond timescales are now available. Diffractive imaging using the intense and coherent beam of X-ray free-electron
lasers opens new perspectives for structural studies of single oparticles and
biomolecules. Simulations were carried out to generate 3D oversampled
diffraction patterns of non-crystalline biological samples, ranging from
peptides and proteins to megadalton complex assemblies, and to recover their
molecular structure from ometer to near-atomic resolutions. Using these
simulated data, we show here that iterative reconstruction methods based on
standard and variant forms of the charge flipping algorithm, can efficiently
solve the phase retrieval problem and extract a unique and reliable molecular
structure. Contrary to the case of conventional algorithms, where the estimation
and the use of a compact support is imposed, our approach does not require any
prior information about the molecular assembly, and is amenable to a wide range
of biological assemblies. Importantly, the robustness of this ab initio approach
is illustrated by the fact that it tolerates experimental noise and
incompleteness of the intensity data at the center of the speckle pattern. X-ray free-electron lasers (XFELs) generate sequences of ultra-short spatially
coherent pulses of X-ray radiation. A diffraction focusing spectrometer (DFS),
which is able to measure the whole energy spectrum of the radiation of a single
XFEL pulse with an energy resolution of ΔE/E 2 × 10(-6), is proposed. This is
much better than for most modern X-ray spectrometers. Such resolution allows one
to resolve the fine spectral structure of the XFEL pulse. The effect of
diffraction focusing occurs in a single-crystal plate due to dynamical
scattering, and is similar to focusing in a Pendry lens made from a metamaterial
with a negative refraction index. Such a spectrometer is easier to operate than
those based on bent crystals. It is shown that the DFS can be used in a wide
energy range from 5 keV to 20 keV. |
Which protein kinases have been found to phosphorylate Phospholamban and affect its biological activity? | Phosphorylation of phospholamban at Ser16 is mainly mediated by PKA and at Thr17 by Ca(2+) /calmodulin-dependent protein kinase (CaMKII). Phospholamban is also a reporter for PKG activity and akt kinase interacts with and phosphorylates PLN at Thr(17). | Phospholamban is the major membrane protein of the heart phosphorylated in
response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent
protein kinase catalyzes exclusive phosphorylation of serine 16 of
phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive
phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert,
J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341).
In this work we have localized the sites of phospholamban phosphorylation in
intact ventricles treated with the beta-adrenergic agonist isoproterenol.
Isolation of phosphorylated phospholamban from 32P-perfused guinea pig
ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis,
revealed phosphorylation of both serine and threonine residues. At steady state
after isoproterenol exposure, phospholamban contained approximately equimolar
amounts of these two phosphoamino acids. Two major tryptic phosphopeptides
containing greater than 90% of the incorporated radioactivity were obtained from
phospholamban labeled in intact ventricles. The amino acid sequences of these
two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine
cardiac phospholamban, thus localizing the sites of in situ phosphorylation to
serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in
heart perfused with isoproterenol was supported by detection of 11 distinct
mobility forms of the pentameric protein by use of the Western blotting method,
consistent with each phospholamban monomer containing two phosphorylation sites,
and with each pentamer containing from 0 to 10 incorporated phosphates. Our
results localize the sites of in situ phospholamban phosphorylation to serine 16
and threonine 17 and, furthermore, are consistent with the phosphorylations of
these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein
kinases, respectively. The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for
sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and
regulating cardiac muscle relaxation. Protein-protein interactions indicated
that it exists in complex with Ca(2+)/calmodulin-dependent protein kinase II
(CaMKII) and its anchoring protein alphaKAP. Confocal imaging of isolated
cardiomyocytes revealed the colocalization of CAMKII and alphaKAP with SERCA2a
at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different
regions in the association domain of alphaKAP but not with each other. Although
deletion of the putative N-terminal hydrophobic amino acid stretch in alphaKAP
prevented its membrane targeting, it did not influence binding to SERCA2a or
CaMKII. Both CaMKIIdelta(C) and the novel CaMKIIbeta(4) isoforms were found to
exist in complex with alphaKAP and SERCA2a at the SR and were able to
phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known
regulator of SERCA2a activity. Interestingly, the presence of alphaKAP was also
found to significantly modulate the Ca(2+)/calmodulin-dependent phosphorylation
of Thr-17 on PLN. These data demonstrate that alphaKAP exhibits a novel
interaction with SERCA2a and may serve to spatially position CaMKII isoforms at
the SR and to uniquely modulate the phosphorylation of PLN. Cardiomyocytes adapt to physical stress by increasing their size while
maintaining cell function. The serine/threonine kinase Akt plays a critical role
in this process of adaptation. We previously reported that transgenic
overexpression of an active form of Akt (Akt-E40K) in mice results in increased
cardiac contractility and cell size, as well as improved sarcoplasmic reticulum
(SR) Ca(2+) handling. Because it is not fully elucidated, we decided to study
the molecular mechanism by which Akt-E40K overexpression improves SR Ca(2+)
handling. To this end, SR Ca(2+) uptake and the phosphorylation status of
phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K
mice and mice harboring inducible and cardiac specific knock-out of
phosphatidylinositol-dependent kinase-1, the upstream activator of Akt.
Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt
targeted preferentially to the SR, and by biochemical assays to evaluate
potential interaction with PLN. We found that when activated, Akt interacts with
and phosphorylates PLN at Thr(17), the Ca(2+)-calmodulin-dependent kinase
IIdelta site, whereas silencing Akt signaling, through the knock-out of
phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of
PLN at Thr(17). Furthermore, overexpression of SR-targeted Akt in cardiomyocytes
improved Ca(2+) handling without affecting cell size. Thus, we describe here a
new mechanism whereby the preferential translocation of Akt to the SR is
responsible for enhancement of contractility without stimulation of hypertrophy. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role
mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the
mechanisms of this deleterious effect. Langendorff perfused rat and transgenic
mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP)
were subjected to global IR. The onset of reperfusion increased the
phosphorylation of Thr(17) site of phospholamban, without changes in total
protein, consistent with an increase in CaMKII activity. Instead, there was a
proportional decrease in the phosphorylation of Ser2815 site of ryanodine
receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the
ratio Ser2815/RyR2 did not change. Inhibition of the reverse
Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban
phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery.
CaMKII-inhibition (KN-93), significantly decreased phospholamban
phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis),
TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced
mitochondrial swelling (apoptosis), and increased contractile recovery when
compared with non-treated IR hearts or IR hearts pretreated with the inactive
analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene),
mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial
permeability transition pore (cyclosporine A), significantly decreased infarct
size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the
phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited
a significant decrease in infarct size, apoptosis and necrosis respect to
controls. The results reveal an apoptotic-necrotic pathway mediated by
CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode
and the mitochondria as trigger and end effectors, respectively, of the cascade. Nitric oxide (NO) and B-type natriuretic peptide (BNP) are protective against
ischemia-reperfusion injury as they increase intracellular cGMP level via
activation of soluble (sGC) or particulate guanylate cyclases (pGC),
respectively. The aim of the present study was to examine if the cGMP-elevating
mediators, NO and BNP, share a common downstream signaling pathway via
cGMP-dependent protein kinase (PKG) in cardiac cytoprotection. Neonatal rat
cardiac myocytes in vitro were subjected to 2.5 h simulated ischemia (SI)
followed by 2 h reoxygenation. Cell viability was tested by trypan blue
exclusion assay. PKG activity of cardiac myocytes was assessed by phospholamban
(PLB) phosphorylation determined by western blot. Cell death was 34 +/- 2% after
SI/reoxygenation injury in the control group. cGMP-inducing agents significantly
decreased irreversible cell injury: the cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10
nM) decreased it to 13 +/- 1% (p < 0.001), the direct NO-donor
S-nitroso-N-acetylpenicillamine (SNAP, 1 microM) to 18 +/- 6% (p < 0.05) and BNP
(10 nM) to 12 +/- 2% (p < 0.001), respectively. This protective effect was
abolished by the selective PKG inhibitor KT-5823 (600 nM) in each case. As PLB
is not a unique reporter for PKG activity since it is also phosphorylated by
protein kinase A (PKA), we examined PLB phosphorylation in the presence of the
PKA inhibitor KT-5720 (1 microM). The ratio of pPLB/PLB significantly increased
after administration of both BNP and 8-Br-cGMP under ischemic conditions, which
was abolished by the PKG inhibitor. This is the first demonstration that
elevated cGMP produced either by the sGC activator SNAP or the pGC activator BNP
exerts cytoprotective effects via a common downstream signaling pathway
involving PKG activation. Phospholamban (PLN) is a small phosphoprotein closely associated with the
cardiac sarcoplasmic reticulum (SR). Dephosphorylated PLN tonically inhibits the
SR Ca-ATPase (SERCA2a), while phosphorylation at Ser16 by PKA and Thr17 by
Ca(2+) /calmodulin-dependent protein kinase (CaMKII) relieves the inhibition,
and this increases SR Ca(2+) uptake. For this reason, PLN is one of the major
determits of cardiac contractility and relaxation. In this review, we
attempted to highlight the functional significance of PLN in vertebrate cardiac
physiology. We will refer to the huge literature on mammals in order to describe
the molecular characteristics of this protein, its interaction with SERCA2a and
its role in the regulation of the mechanic and the electric performance of the
heart under basal conditions, in the presence of chemical and physical stresses,
such as β-adrenergic stimulation, response to stretch, force-frequency
relationship and intracellular acidosis. Our aim is to provide the basis to
discuss the role of PLN also on the cardiac function of nonmammalian
vertebrates, because so far this aspect has been almost neglected. Accordingly,
when possible, the literature on PLN will be analysed taking into account the
nonuniform cardiac structural and functional characteristics encountered in
ectothermic vertebrates, such as the peculiar and variable organization of the
SR, the large spectrum of response to stresses and the disaptive absence of
crucial proteins (i.e. haemoglobinless and myoglobinless species). The sarcoplasmic reticulum calcium pump (SERCA) and its regulator,
phospholamban, are essential components of cardiac contractility. Phospholamban
modulates contractility by inhibiting SERCA, and this process is dynamically
regulated by β-adrenergic stimulation and phosphorylation of phospholamban.
Herein we reveal mechanistic insight into how four hereditary mutants of
phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14)
deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein
kinase A recognition motif, which abrogates phospholamban phosphorylation and
results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex
changes in function, where hydrophobic substitutions such as cysteine and
leucine eliminate both SERCA inhibition and phospholamban phosphorylation,
whereas an aromatic substitution such as histidine selectively disrupts
phosphorylation. We demonstrate that the role of Arg(9) in phospholamban
function is multifaceted: it is important for inhibition of SERCA, it increases
the efficiency of phosphorylation, and it is critical for protein kinase A
recognition in the context of the phospholamban pentamer. Given the synergistic
consequences on contractility, it is not surprising that the mutants cause
lethal, hereditary dilated cardiomyopathy. Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR
Ca-transport and Ca-release, is a key and universal characteristic of human and
experimental heart failure. These SR processes are regulated by multimeric
protein complexes, including protein kinases and phosphatases as well as their
anchoring and regulatory subunits that fine-tune Ca-handling in specific SR
sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and
its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an
inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or
calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory
effects. Recent studies identified additional regulatory proteins, associated
with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of
protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the
HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal
histidine-rich calcium binding protein (HRC) has been shown to interact with
both SERCA2a and triadin. Notably, there is physical and direct interaction
between these protein players, mediating a fine-cross talk between SR Ca-uptake,
storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA
interactome does not only impact cardiomyocyte contractility, but also survival
and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes
modulate their activity and interactions with other protein partners, resulting
in depressed contractility and accelerated remodeling. These genetic variants
may serve as potential prognostic or diagnostic markers in cardiac
pathophysiology. |
Why can't humans synthesize Neu5Gc (N-Glycolylneuraminic acid)? | N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid synthesized by animals, but not by humans or birds. Humans lack a functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) protein and cannot synthesize the sugar Neu5Gc, an innate mammalian signal of self. Losing this sugar changed how humans interact with some of our deadliest pathogens: malaria, influenza, and streptococcus among others. | A concern recently has been raised that human embryonic stem cell (HESC) lines
cultured with currently available methods may have limited clinical usefulness
due to the immunogenicity of the nonhuman sialic acid Neu5Gc incorporated into
their membranes during culturing. We find this concern has little relevance to
neural differentiation protocols with B27/N2/noggin because of the gradual
decline of Neu5Gc to less than 1% in differentiating cells upon switching to
B27/N2 medium. Human heterophile antibodies that agglutinate animal erythrocytes are known to
detect the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). This
monosaccharide cannot by itself fill the binding site (paratope) of an antibody
and can also be modified and presented in various linkages, on diverse
underlying glycans. Thus, we hypothesized that the human anti-Neu5Gc antibody
response is diverse and polyclonal. Here, we use a novel set of natural and
chemoenzymatically synthesized glycans to show that normal humans have an
abundant and diverse spectrum of such anti-Neu5Gc antibodies, directed against a
variety of Neu5Gc-containing epitopes. High sensitivity and specificity assays
were achieved by using N-acetylneuraminic acid (Neu5Ac)-containing probes
(differing from Neu5Gc by one less oxygen atom) as optimal background controls.
The commonest anti-Neu5Gc antibodies are of the IgG class. Moreover, the range
of reactivity and Ig classes of antibodies vary greatly amongst normal humans,
with some individuals having remarkably large amounts, even surpassing levels of
some well-known natural blood group and xenoreactive antibodies. We purified
these anti-Neu5Gc antibodies from individual human sera using a newly developed
affinity method and showed that they bind to wild-type but not Neu5Gc-deficient
mouse tissues. Moreover, they bind back to human carcinomas that have
accumulated Neu5Gc in vivo. As dietary Neu5Gc is primarily found in red meat and
milk products, we suggest that this ongoing antigen-antibody reaction may
generate chronic inflammation, possibly contributing to the high frequency of
diet-related carcinomas and other diseases in humans. BACKGROUND: Humans are genetically defective in synthesizing the common
mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically
incorporate it from dietary sources (particularly red meat and milk) into
glycoproteins and glycolipids of human tumors, fetuses and some normal tissues.
Metabolic incorporation of Neu5Gc from animal-derived cells and medium
components also results in variable contamination of molecules and cells
intended for human therapies. These Neu5Gc-incorporation phenomena are
practically significant, because normal humans can have high levels of
circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and
specific detection of Neu5Gc in human tissues and biotherapeutic products.
Unlike monoclonal antibodies that recognize Neu5Gc only in the context of
underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can
recognize Neu5Gc in broader contexts. However, prior preparations of such
antibodies (including our own) suffered from some non-specificity, as well as
some cross-reactivity with the human sialic acid N-acetylneuraminic acid
(Neu5Ac).
METHODOLOGY/PRINCIPAL FINDINGS: We have developed a novel affinity method
utilizing sequential columns of immobilized human and chimpanzee serum
sialoglycoproteins, followed by specific elution from the latter column by free
Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or
cells from mice with a human-like defect in Neu5Gc production. It allows
sensitive and specific detection of Neu5Gc in all underlying glycan structural
contexts studied, and is applicable to immunohistochemical, enzyme-linked
immunosorbent assay (ELISA), Western blot and flow cytometry analyses.
Non-immune chicken IgY is used as a reliable negative control. We show that
these approaches allow sensitive detection of Neu5Gc in human tissue samples and
in some biotherapeutic products, and finally show an example of how Neu5Gc might
be eliminated from such products, by using a human cell line grown under defined
conditions.
CONCLUSIONS: We report a reliable antibody-based method for highly sensitive and
specific detection of the non-human sialic acid Neu5Gc in human tissues and
biotherapeutic products that has not been previously described. N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a
significant role in human pathologies, such as cancer and vascular disease.
Further studies into the role of Neu5Gc in human disease are hindered by limited
sources of this carbohydrate. Using a chemo-enzymatic approach, Neu5Gc was
accessed in six steps from glucose. The synthesis allows access to gram-scale
quantities quickly and economically and produces Neu5Gc in superior quality to
commercial sources. Finally, we demonstrate that the synthesized Neu5Gc can be
incorporated into the cell glycocalyx of human cells, which do not naturally
synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or in
vivo use. N-glycolylneuraminic acid (Neu5Gc) is an immunogenic sugar of dietary origin
that metabolically incorporates into diverse native glycoconjugates in humans.
Anti-Neu5Gc antibodies are detected in all human sera, though with variable
levels and epitope-recognition profiles. These antibodies likely play a role in
several inflammation-mediated pathologies including cardiovascular diseases and
cancer. In cancer, they have dualistic and opposing roles, either stimulating or
repressing disease, as a function of their dose, and some of these antibodies
serve as carcinoma biomarkers. Thus, anti-Neu5Gc antibodies may signify risk of
inflammation-mediated diseases, and changes in their levels could potentially be
used to monitor disease progression and/or response to therapy. Currently, it is
difficult to determine levels of anti-Neu5Gc antibodies in individual human
samples because these antibodies recognize multiple Neu5Gc-epitopes. Here we
describe a simple and specific method for detection and overall estimation of
human anti-Neu5Gc antibodies. We exploit the difference between two mouse models
that differ only by Neu5Gc-presence (wild-type) or Neu5Gc-absence (Cmah(-/-)
knockout). We characterize mouse serum from both strains by HPLC, lectin and
mass-spectrometry analysis and show the target Neu5Gc-epitopes. We then use
Cmah(-/-) knockout sera to inhibit all non-Neu5Gc-reactivity followed by binding
to wild-type sera to detect overall anti-Neu5Gc response in a single assay. We
applied this methodology to characterize and quantify anti-Neu5Gc IgG and IgA in
sera of patients with Kawasaki disease (KD) at various stages compared to
controls. KD is an acute childhood febrile disease characterized by inflammation
of coronary arteries that untreated may lead to coronary artery aneurysms with
risk of thrombosis and myocardial infarction. This estimated response is
comparable to the average of detailed anti-Neu5Gc IgG profile analyzed by a
sialoglycan microarray. Both assays revealed an elevated response in acute KD
patients with normal coronaries compared to patients with aneurysm or dilated
coronaries. Implications of these findings are discussed. Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid
(Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been
discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to
dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac
to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is
able to be metabolically incorporated into surfaces of tissue cells and may be
related to enhancement of the infectivity and severity of IAV. Here, we
investigated the receptor function of Neu5Gc on IAV infection in
Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells
or by incubation with human cells in the presence of N-glycolylmannosamine.
Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that
possess Neu5Gc binding ability. Furthermore, there was no difference in
infectivity of a transfectant virus that included the wild-type HA gene from
A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7
cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the
other hand, cell entry of the transfectant virus that included the
Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was
arrested at the stage of internalization from the plasma membrane of the
CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface
of human epithelial cells suppresses infection of IAVs that possess Neu5Gc
binding ability. Neu5Gc is suggested to work as a decoy receptor of
Neu5Gc-binding IAVs but not a functional receptor for IAV infection.
IMPORTANCE: Influenza A viruses (IAVs) bind to the host cell surfaces through
sialic acids at the terminal of glycoconjugates. For IAV binding to sialic
acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor
but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a
receptor of human and animal IAVs. Our results showed that Neu5Gc expression on
human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding
ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but
not a functional receptor for IAV infection. Human cells cannot synthesize
Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase
gene but can exogenously and metabolically incorporate Neu5Gc from dietary
sources. The expression of Neu5Gc on human epithelial cells by taking in
exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction
of the infection of IAVs that have acquired Neu5Gc binding ability. Human sialic acid biology is unusual and thought to be unique among mammals.
Humans lack a functional cytidine monophosphate-N-acetylneuraminic acid
hydroxylase (CMAH) protein and cannot synthesize the sugar Neu5Gc, an innate
mammalian signal of self. Losing this sugar changed how humans interact with
some of our deadliest pathogens: malaria, influenza, and streptococcus among
others. We show that the New World monkeys, comprising the third of all primate
species, have human-like sialic acid biology. They have lost Neu5Gc because of
an independent CMAH inactivation ~30 million years ago (mya) (compared to ~3 mya
in hominids). This parallel loss of Neu5Gc opens sialic acid biology to
comparative phylogenetic analysis and reveals an unexpected conservation
priority. New World monkeys risk infection by human pathogens that can recognize
cells in the absence of Neu5Gc. This striking molecular convergence provides a
mechanism that could explain the long-standing observation that New World
monkeys are susceptible to some human diseases that cannot be transmitted to
other primates. Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and
N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors
for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from
Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase
(CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets
are susceptible to human-adapted IAV strains and have been the domit animal
model for IAV studies. Here we show that ferrets, like humans, do not synthesize
Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret
CMAH gene that is shared by the Pinnipedia and Musteloidia members of the
Carnivora. Interactions between two human strains of IAV with the sialyllactose
receptor (sialic acid--α2,6Gal) confirm that the type of terminal sialic acid
contributes significantly to IAV receptor specificity. Our results indicate that
exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to
human-adapted IAV strains. |
Which enzyme is inhibited by Orteronel? | Orteronel inhibits the 17,20 lyase activity of the enzyme CYP17A1, which is important for androgen synthesis in the testes, adrenal glands and prostate cancer cells. Orteronel is used for treatment for castration-resistant prostate cancer. | A novel naphthylmethylimidazole derivative 1 and its related compounds were
identified as 17,20-lyase inhibitors. Based on the structure-activity
relationship around the naphthalene scaffold and the results of a docking study
of 1a in the homology model of 17,20-lyase, the
6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and
identified as a potent and highly selective 17,20-lyase inhibitor. Biological
evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked
reductions in both serum testosterone and dehydroepiandrosterone concentrations.
Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for
clinical evaluation and is currently in phase III clinical trials for the
treatment of castration-resistant prostate cancer. Endogenous androgens play a role in the development and progression of prostate
cancer (PC), thus androgen suppression may offer an effective therapeutic
strategy for this disease. Orteronel (TAK-700),
6-[(7S)-7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl]-N-methyl-2-naphthamide,
is a novel, non-steroidal, selective inhibitor of the 17,20-lyase activity of
CYP17A--a key enzyme in the production of steroidal hormones--and is being
developed as a therapy for PC. The purpose of this study was to elucidate the
inhibitory activity of orteronel, in particular its specificity for androgen
synthesis enzymes, in male rats--an androgen-synthesis model that largely
reflects this pathway in humans. Orteronel inhibited 17,20-lyase activity in
rats with an IC(50) of 1200 nM but did not inhibit 17α-hydroxylase or
11β-hydroxylase (CYP11B1) activity in rats at concentrations up to 10 μM. In
cellular steroidogenesis assays using rat testicular cells, orteronel suppressed
testosterone and androstenedione production with an IC(50) of 640 nM and 210 nM,
respectively, but did not suppress either corticosterone or aldosterone
production in rat adrenal cells at concentrations up to 30 μM. In addition,
serum testosterone and androstenedione levels in human chorionic
gonadotropin-injected hypophysectomized rats were significantly reduced by
single oral administration of orteronel at a dose of 30 mg/kg (both p ≤ 0.01);
serum corticosterone and aldosterone levels in ACTH-injected hypophysectomized
rats did not result in significant differences compared with controls, following
orteronel administration at doses up to 300 mg/kg. Serum testosterone levels in
intact male rats were significantly reduced by orteronel 4h after dosing at
100mg/kg (p ≤ 0.01); testosterone levels showed a tendency to recover afterward.
In intact male rats, the weight of the prostate glands and seminal vesicles was
decreased in a dose-dependent manner following multiple doses of orteronel at
37.5, 150, and 600 mg/kg, TID for 4 days. The reversibility of orteronel was
further confirmed using a human adrenocortical tumor cell line. In summary,
orteronel is a selective and reversible 17,20-lyase inhibitor, and decreases the
weight of androgen-dependent organs in male rats. Our data suggests that
orteronel would therefore be effective for androgen-dependent disorders such as
PC. Androgen and the androgen receptor (AR) pathway remain the key targets for
emerging new therapies against castration-resistant prostate cancer (CRPC).
Adrenal androgens and intratumoral testosterone production appear to be
sufficient to activate AR in the castration-resistant setting. This process
re-engages AR and allows it to continue to be the primary target responsible for
prostate cancer progression. Adrenal androgen production can be blocked by
inhibiting cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17), a key enzyme for
androgen synthesis in adrenal glands and peripheral tissues. Therapeutic CYP17
inhibition by ketoconazole or by the recently approved adrenal inhibitor
abiraterone acetate is the only available choice to target this pathway in CRPC.
A new CYP17 inhibitor, with more selective inhibition of 17,20-lyase over
17α-hydroxylase, orteronel (TAK-700), is currently undergoing phase III clinical
trials in pre- and postchemotherapy CRPC. In a completed phase II trial in CRPC
patients, orteronel demonstrated its efficacy by lowering the levels of
circulating androgens, reducing prostate-specific antigen (PSA) levels, and
decreasing the levels of circulating tumor cells. Ongoing studies evaluating
orteronel in CRPC will further define its safety and role in the management of
this disease. A novel, simple, specific, sensitive and reproducible high-performance liquid
chromatography assay method has been developed and validated for the estimation
of Orteronel in rat plasma. The bioanalytical procedure involves extraction of
Orteronel and phenacetin (internal standard) from rat plasma with a simple
liquid-liquid extraction process. The chromatographic analysis was performed on
a Waters Alliance system using a gradient mobile phase conditions at a flow rate
of 1 mL/min and a C18 column maintained at ambient room temperature. The eluate
was monitored using a photodiode array detector set at 242. Orteronel and
internal standard eluted at 4.8 and 6.2 min, respectively and the total run time
was 9 min. Method validation was performed as per US Food and Drug
Administration guidelines and the results met the acceptance criteria. The
calibration curve was linear over a concentration range of 100-3149 ng/mL (r(2)
= 0.995). The intra- and inter-day precisions were in the ranges of 0.31-7.87
and 3.97-6.35, respectively, in rat plasma. The validated HPLC method was
successfully applied to a pharmacokinetic study of Orteronel in rats. Orteronel (TAK-700) is an investigational, non-steroidal inhibitor of CYP17A1
with preferential inhibition of 17,20-lyase in NCI-H295 cells. Estrogen is
synthesized from androgen by aromatase activity, and the effect of orteronel on
estrogen synthesis was therefore evaluated. First, it was confirmed that
orteronel does not directly inhibit aromatase activity. Second, the specific
decline of serum estradiol and androgen levels in hypophysectomized female rats
by orteronel in comparison with aromatase inhibitor anastrozole was evaluated;
orteronel at doses ≥3mg/kg significantly suppressed serum estradiol,
testosterone, androstenedione and 17-hydroxyprogesterone levels, and increased
progesterone levels in the estrogen-synthesis pathway. Orteronel, at a dose of
300mg/kg, suppressed serum estradiol concentrations to a similar degree as
0.1mg/kg anastrozole. In contrast, in the corticoid-synthesis pathway, serum
aldosterone, corticosterone, and progesterone levels did not change
significantly following administration of 300mg/kg of orteronel. Third, the
effect of multiple oral administration of orteronel on serum estradiol levels in
regularly cycling female cynomolgus monkeys was evaluated. Orteronel at
15mg/kg/day (7.5mg/kg/treatment, twice daily [bid]) continued to suppress the
estradiol surge prior to the start of luteal phase for 1.5-times the average
duration of three consecutive, pre-treatment menstrual cycles, while serum
progesterone was maintained at levels almost equal to those in the luteal phase
although a certain portion of this increased level of progesterone could be of
adrenal-origin. This suppressive effect on estradiol surge was thought to be
reversible since serum estradiol levels started to rise immediately after the
discontinuation of orteronel. Estradiol surge was not abrogated by treatment
with anastrozole 0.2mg/kg/day (0.1mg/kg/treatment, bid). In summary, orteronel
can suppress serum estradiol concentrations in hypophysectomized female rats and
monkeys through selective inhibition of CYP17A1 activity, suggesting that
orteronel might be effective for hormone-dependent breast cancers and
estrogen-dependent diseases. Since 2010, six drugs have been approved for the treatment of
castration-resistant prostate cancer, i.e., CYP17 inhibitor Abiraterone,
androgen receptor antagonist Enzalutamide, cytotoxic agent Cabazitaxel, vaccine
Sipuleucel-T, antibody Denosumab against receptor activator of nuclear factor
kappa B ligand and radiopharmaceutical Alpharadin. All these drugs demonstrate
improvement on overall survival, expect for Denosumab, which increases the bone
mineral density of patients under androgen deprivation therapy and prolongs
bone-metastasis-free survival. Besides further CYP17 inhibitors (Orteronel,
Galeterone, VT-464 and CFG920), androgen receptor antagonists (ARN-509, ODM-201,
AZD-3514 and EZN-4176) and vaccine Prostvac, more drug candidates with various
mechanisms or new indications of launched drugs are currently under evaluation
in different stages of clinical trials, including various kinase inhibitors and
platinum complexes. Some novel strategies have also been proposed aimed at
further potentiation of antitumor effects or reduction of side effects and
complications related to treatments. Under these flourishing circumstances, more
investigations should be performed on the optimal combination or the sequence of
treatments needed to delay or reverse possible resistance and thus maximize the
clinical benefits for the patients. As the first in class steroid 17α-hydroxylase/C17,20-lyase (CYP17) inhibitor,
abiraterone acetate (of which the active metabolite is abiraterone) has been
shown to improve overall survival in patients with castration-resistant prostate
cancer (CRPC)--in those who are chemotherapy-naive and those previously treated
with docetaxel. Furthermore, the clinical success of abiraterone demonstrated
that CRPC, which has previously been regarded as an androgen-independent
disease, is still driven, at least in part, by androgens. More importantly,
abiraterone is a 'promiscuous' drug that interacts with a number of targets,
which dictate its clinical benefits and adverse effects profile. Besides CYP17
inhibition, abiraterone acts as an antagonist to the androgen receptor and
inhibits 3β-hydroxysteroid dehydrogenase--two effects that potentially
contribute to its antitumour effects. However, the inhibition of the
17α-hydroxylase activity of CYP17, CYP11B1 and a panel of hepatic CYP enzymes
leads to adverse effects and toxicities that include secondary mineralocorticoid
excess. Abiraterone is also associated with increased incidence of cardiac
disorders. Under such circumstances, development of new CYP17 inhibitors as an
additional line of defence is urgently needed. To achieve enhanced clinical
benefits, new strategies are being explored that include selective inhibition of
the C17,20-lyase activity of CYP17 and multi-targeting strategies that affect
androgen synthesis and signalling at different points. Some of these
strategies-including the drugs orteronel, VT-464 and galeterone--are supported
by preclinical data and are being explored in the clinic. INTRODUCTION: New drugs have recently been developed, through a better
understanding of the mechanisms involved in the progression of prostate cancer,
including castration-resistant ones (CRPC). This article aims to describe the
mechanisms of action of these new hormonal treatments and their major clinical
outcomes and development programs.
MATERIALS AND METHODS: A bibliographic research in French and English using
Medline(®) and Embase(®) using the keywords "castration-resistant prostate
cancer", "abiraterone acetate", "orteronel", "enzalutamide", and "clinical
trials" was performed.
RESULTS: the androgen signaling pathway remains the cornerstone of advanced
cancers management. Hence, some molecules target the androgen biosynthesis, as
abiraterone acetate and orteronel, which are selective inhibitors of the enzyme
CYP17. Others act as antagonists of the androgen receptor: the enzalutamide,
RNA-509 and ODM201. Finally, galeterone combines the two effects.
CONCLUSION: Progress conferred by these molecules in terms of overall survival
and quality of life in patients with metastatic CRPC, suggest that their use at
earlier stages of the disease could reduce morbidity and mortality from prostate
cancer. Determining the best strategy for sequence or combination therapy to
optimize the use of these new molecules should be investigated. PURPOSE: The androgen receptor pathway remains active in men with prostate
cancer whose disease has progressed following surgical or medical castration.
Orteronel (TAK-700) is an investigational, oral, nonsteroidal, selective,
reversible inhibitor of 17,20-lyase, a key enzyme in the production of
androgenic hormones.
EXPERIMENTAL DESIGN: We conducted a phase I/II study in men with progressive,
chemotherapy-naïve, metastatic castration-resistant prostate cancer, and serum
testosterone <50 ng/dL. In the phase I part, patients received orteronel 100 to
600 mg twice daily or 400 mg twice a day plus prednisone 5 mg twice a day. In
phase II, patients received orteronel 300 mg twice a day, 400 mg twice a day
plus prednisone, 600 mg twice a day plus prednisone, or 600 mg once a day
without prednisone.
RESULTS: In phase I (n = 26), no dose-limiting toxicities were observed and 13
of 20 evaluable patients (65%) achieved ≥50% prostate-specific antigen (PSA)
decline from baseline at 12 weeks. In phase II (n = 97), 45 of 84 evaluable
patients (54%) achieved a ≥50% decline in PSA and at 12 weeks, substantial mean
reductions from baseline in testosterone (-7.5 ng/dL) and
dehydroepiandrosterone-sulfate (-45.3 μg/dL) were observed. Unconfirmed partial
responses were reported in 10 of 51 evaluable phase II patients (20%). Decreases
in circulating tumor cells were documented. Fifty-three percent of phase II
patients experienced grade ≥3 adverse events irrespective of causality; most
common were fatigue, hypokalemia, hyperglycemia, and diarrhea.
CONCLUSIONS: 17,20-Lyase inhibition by orteronel was tolerable and results in
declines in PSA and testosterone, with evidence of radiographic responses. Orteronel (also known as TAK-700) is a novel hormonal therapy that is currently
in testing for the treatment of prostate cancer. Orteronel inhibits the 17,20
lyase activity of the enzyme CYP17A1, which is important for androgen synthesis
in the testes, adrenal glands and prostate cancer cells. Preclinical studies
demonstrate that orteronel treatment suppresses androgen levels and causes
shrinkage of androgen-dependent organs, such as the prostate gland. Early
reports of clinical studies demonstrate that orteronel treatment leads to
reduced prostate-specific antigen levels, a marker of prostate cancer tumor
burden, and more complete suppression of androgen synthesis than conventional
androgen deprivation therapies that act in the testes alone. Treatment with
single-agent orteronel has been well tolerated with fatigue as the most common
adverse event, while febrile neutropenia was the dose-limiting toxicity in a
combination study of orteronel with docetaxel. Recently, the ELM-PC5 Phase III
clinical trial in patients with advanced-stage prostate cancer who had received
prior docetaxel was unblinded as the overall survival primary end point was not
achieved. However, additional Phase III orteronel trials are ongoing in men with
earlier stages of prostate cancer. It is now well established that hormonal pathways are involved in the
development of prostate cancer towards the castration resistant (CRPC) stage and
can be effective molecular targets for novel treatment strategies. Most CRPC are
sensitive to androgens and this can be due to the intratumoral production of
androgens, androgen receptor (AR) amplification/ mutations and epigenetic
modifications of AR expression/signaling. Based on these observations, potent
agents targeting the AR axis were developed: 1) inhibitors of CYP17 (a key
enzyme in the production of androgens), such as abiraterone and orteronel; 2) AR
antagonists that bind to AR and impair AR activation, such as enzalutamide and
ARN-509. Moreover, gonadotropin-releasing hormone receptors (GnRH-R), associated
with a strong antitumor activity, are expressed in CRPC cells, indicating that
they might represent an important target for GnRH analog-based therapeutic
strategies. In addition to GnRH agonists and antagonists (i.e., degarelix),
cytotoxic GnRH-based bioconjugates, delivering chemotherapeutic drugs to cancer
cells expressing the GnRH-R, were developed and reported to exert antitumor
effects on CRPC cells; some of them (i.e., AN-152) have already entered clinical
trials. This review discusses the most relevant patents and recent observations
on the anti-cancer efficacy of novel drugs targeting the AR and the GnRH-R
pathways in CRPC. PURPOSE: Orteronel (TAK-700) is an investigational, nonsteroidal, oral,
inhibitor of androgen synthesis with greater specificity for 17,20-lyase than
for 17α-hydroxylase. We investigated orteronel without steroids in patients with
nonmetastatic castration-resistant prostate cancer (nmCRPC; M0).
EXPERIMENTAL DESIGN: Patients with nmCRPC and rising prostate-specific antigen
(PSA) received orteronel 300 mg twice daily until PSA progression, metastases,
or unacceptable toxicity. The primary endpoint was percentage of patients
achieving PSA ≤0.2 ng/mL (undetectable levels) at 3 months. Secondary endpoints
included safety, PSA response, time to metastases, and correlated endpoints.
RESULTS: Thirty-nine patients with a median baseline PSA doubling time of 2.4
months (range, 0.9-9.2) received a median of fourteen 28-day treatment cycles.
PSA decreased >30% in 35 patients and 6 (16%) achieved PSA ≤ 0.2 ng/mL at 3
months. Median times to PSA progression and metastasis were 13.8 and 25.4
months, respectively. Kaplan-Meier estimates of freedom from PSA progression
were 57% and 42% at 12 and 24 months, and of freedom from metastasis were 94%
and 62% at 12 and 24 months, respectively. At 3 months, median testosterone
declined by 89% from baseline. Adverse events led to therapy discontinuation in
12 patients and grade ≥3/4 adverse events occurred in 22 patients. Most frequent
all-cause adverse events included fatigue (64%), hypertension (44%), diarrhea
(38%), and nausea (33%), which were primarily grade 1/2.
CONCLUSIONS: Single-agent orteronel produced marked and durable declines in PSA
in patients with nmCRPC. Orteronel has moderate but manageable toxicities and
its chronic administration without steroids appears feasible. Orteronel (TAK-700) is a novel and selective inhibitor of CYP17A1, which is
expressed in testicular, adrenal and prostate tumor tissues. Orteronel is
currently in Phase-III clinical development for metastatic castration-resistant
prostate patients. The objective of the study is to assess the permeability,
metabolic stability (in various preclinical and human liver microsomes),
identify the major CYPs involved in the metabolism of Orteronel. We have also
studied the pharmacokinetics and excretion of Orteronel in Sprague-Dawley rats.
Orteronel was found to be stable in various liver microsomes tested. The
half-life (t ½) of Orteronel with intravenous (i.v.) route was found to be 1.65
± 0.22 h. The clearance and volume of distribution by i.v. route for Orteronel
were found to be 27.5 ± 3.09 mL/min/kg and 3.94 ± 0.85 L/kg, respectively. The
absorption of Orteronel was rapid, with maximum concentrations of drug in plasma
of 614 ± 76.4, 1,764 ± 166, 4,652 ± 300 and 17,518 ± 3,178 ng/mL attained at
0.38, 0.75, 0.50 and 0.83 h, respectively, after oral administration of
Orteronel at 5, 10, 30 and 100 mg/kg as a suspension. In the dose proportional
oral pharmacokinetic study, the mean t ½ by oral route was found to be ~3.5 h
and bioavailability ranged between 69 and 89 %. The primary route of elimination
for Orteronel is urine. |
What is the disorder in which mutations in U4atac snRNA are detected? | Mutations in U4atac snRNA are thought to be the cause of Microcephalic Osteodysplastic Primordial Dwarfism type I (MOPDI), a recessive form of developmental disorder. | Microcephalic osteodysplastic primordial dwarfism type I (MOPD I) is a rare
autosomal recessive developmental disorder characterized by extreme intrauterine
growth retardation, severe microcephaly, central nervous system abnormalities,
dysmorphic facial features, skin abnormalities, skeletal changes, limb
deformations, and early death. Recently, mutations in the RNU4ATAC gene, which
encodes U4atac, a small nuclear RNA that is a crucial component of the minor
spliceosome, were found to cause MOPD I. MOPD I is the first disease known to be
associated with a defect in small nuclear RNAs. We describe here the clinical
and molecular data for 17 cases of MOPD I, including 15 previously unreported
cases, all carrying biallelic mutations in the RNU4ATAC gene. |
Subsets and Splits