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Is the transcriptional regulator BACH1 an activator or a repressor? | BACH1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. In the absence of elevated intracellular heme or oxidative stress, BACH1 functions as a repressor of the enhancers of heme oxygenase-1 (HO-1) gene (Hmox-1) by forming heterodimers with the small Maf proteins such as MafK. Bach1 is recruited to a subset of p53 target genes and contributes to impeding p53 action by promoting histone deacetylation. | Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative
stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have
been the focus of studies on the inducible expression of ho-1. Here we show that
a heme-binding factor, Bach1, is a critical physiological repressor of ho-1.
Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers
with MafK in vitro and repressed their activity in vivo, while heme abrogated
this repressor function of Bach1 by inhibiting its binding to the ho-1
enhancers. Gene targeting experiments in mice revealed that, in the absence of
Bach1, ho-1 became expressed constitutively at high levels in various tissues
under normal physiological conditions. By analyzing bach1/nrf2
compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2
in several tissues. Chromatin immunoprecipitation revealed that small Maf
proteins participate in both repression and activation of ho-1. Thus, regulation
of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac
repressor sensitivity to lactose), generating a simple feedback loop whereby the
substrate effects repressor-activator antagonism. Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron,
carbon monoxide, and bilirubin IXalpha. HO-1 is induced by its substrate heme
and various environmental factors, which represents a protective response
against oxidative stresses. Here we show that hypoxia represses HO-1 expression
in three human cell types but induces it in rat, bovine, and monkey cells,
indicating the inter-species difference in the hypoxic regulation of HO-1
expression. The hypoxia-mediated repression of HO-1 expression is consistently
associated with the induction of Bach1, a heme-regulated transcriptional
repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a
heterodimer with a small Maf protein. Expression of HO-1 was also reduced in
human cells when exposed to interferon-gamma or an iron chelator
desferrioxamine, each of which induced Bach1 expression. In contrast, induction
of HO-1 expression by CoCl(2) is associated with reduced expression of Bach1
mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have
identified a Maf recognition element in the human HO-1 gene that is required for
repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1
functions as a hypoxia-inducible repressor for the HO-1 gene, thereby
contributing to fine-tuning of oxygen homeostasis in human cells. The export of certain nuclear proteins is involved in the regulation of various
nuclear functions, including transcription. In some cases, the export of target
proteins is induced upon environmental or cellular cues, resulting in
conditional gene expression. The small Maf proteins appear to be critical
regulators of heme oxygenase (HO)-1, an anti-oxidant defense enzyme that
degrades heme into iron, carbon monoxide, and biliverdin. Although ho-1 is
repressed by Bach1/small Maf heterodimers, it is activated by Nrf2/small Maf
heterodimers, indicating that Bach1 and Nrf2 compete with each other. We
anticipated that the nuclear concentration of Bach1 might be regulated to ensure
that the entire system effectively responds to various stimuli. We carried out
detailed domain analysis of Bach1 in an effort to understand how various
inducers of HO-1 inactivate Bach1. We show here that cadmium, a strong inducer
of HO-1, activates the nuclear export of Bach1. This cadmium-induced export of
Bach1 was mediated in trans by its C-terminal region that is conserved between
Bach1 and Bach2. The nuclear export of Bach2 was also induced by cadmium,
indicating that the cadmium responsibility is shared between Bach1 and Bach2.
The nuclear export of Bach1 was dependent on Crm1/Exportin-1 as well as the
extracellular signal-regulated kinase-1/2 (ERK1/2) activity. These results
indicate that the nuclear export of Bach1 constitutes an important regulatory
mechanism to relieve the Bach1-mediated repression of genes such as ho-1. Heme must be synthesized and degraded within an individual nucleated cell. Heme
degradation is catalyzed by the two isozymes of heme oxygenase, heme oxygenase-1
(HO-1) and HO-2, eventually yielding biliverdin/bilirubin, CO, and iron. These
products possess important physiological roles but are potentially toxic to
cells. Characteristically, human HO-1 contains no Cys residues, whereas HO-2
contains the potential heme-binding motifs of the Cys-Pro dipeptide. Expression
of HO-1 is inducible or repressible, depending on cell types or cellular
microenvironments, but expression levels of HO-2 are fairly constant. Thus, the
main regulation of heme catabolism is a problem of the balance between induction
and repression of HO-1. Notably, HO-1 expression is induced by heme in all
mammalian cells examined, but is repressed by hypoxia in certain types of
cultured human cells. The recent discovery of Bach1 as a heme-regulated and
hypoxia-inducible repressor for transcription of the HO-1 gene has provided a
missing link in the feedback control of heme catabolism. On the other hand, the
human HO-1 gene promoter contains the (GT)n repeat polymorphism and a single
nucleotide polymorphism (-427A --> T), both of which may contribute to
fine-tuning of the transcription. Importantly, long (GT)n alleles are associated
with susceptibility to smoking-induced emphysema or coronary artery disease, but
may provide with resistance to cerebral malaria. The latter finding suggests a
novel therapeutic strategy with inhibitors of HO-1 for the treatment of cerebral
malaria. We discuss the potential regulatory role of Bach1 and HO-2 in heme
catabolism and update the understanding of the regulation of HO-1 expression. Both nuclear factor erythroid 2 45 kDa subunit (p45) and BTB and CNC homolog 1
(Bach) transcription factors can form dimers with one of the small Maf proteins,
and these heterodimers bind to the musculoaponeurotic fibrosarcoma oncogene
(Maf) recognition element (MARE). MARE is known to act as a critical
cis-regulatory element of erythroid and megakaryocytic genes. Although detailed
analyses of p45-null mutant mice and small maf compound mutant mice revealed
that these factors are both critical for platelet production, the functional
contributions of Bach1 and the relationship or redundancy between Bach1 and p45
in megakaryocytes remain to be clarified. To address these issues, we generated
transgenic lines of mice bearing human BACH1 cDNA under the control of the
GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed
significant thrombocytopenia associated with impaired maturation of the
megakaryocytes, and they developed myelofibrosis. The megakaryocytes in the
transgenic mice exhibited reduced proplatelet formation, and the modal ploidy
class of megakaryocytes was 2N, indicating the impairment of endomitosis.
Transcription of the p45 target genes was down-regulated and we indeed found
that BACH1 binds to the thromboxane synthase gene, one of the target genes for
p45 in megakaryocytes. These findings thus provide evidence that BACH1 acts as a
transcriptional repressor in the regulation of MARE-dependent genes in
megakaryocytes. The antioxidant response element (ARE) and Nrf2 are known to regulate the
expression and coordinated induction of genes encoding detoxifying enzymes
including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In
this report, we demonstrate that overexpression of the transcription factor
Bach1 in Hep-G2 cells negatively regulated NQO1 gene expression and induction in
response to antioxidant t-BHQ. Bandshift and supershift assays revealed that
Bach1 binds to the ARE as a heterodimer with small Maf proteins but not as a
homodimer or heterodimer with Nrf2. The transfection and ChIP assays revealed
that Bach1 and Nrf2 competed with each other to regulate ARE-mediated gene
expression. Heme, a negative regulator of Bach1 relieved the Bach1 repression of
NQO1 gene expression in transfected cells. The transcription of Bach1 and Nrf2
did not change in response to t-BHQ. Immunofluorescence assays and Western blot
analysis revealed that both Bach1 and Nrf2 localized in the cytoplasm and
nucleus of the untreated cells. The treatment of cells with t-BHQ resulted in
the nuclear accumulation of both Bach1 and Nrf2. Interestingly, the
t-BHQ-induced nuclear accumulation of Bach1 was significantly delayed over that
of Nrf2. These results led to the conclusion that a balance of Nrf2 versus Bach1
inside the nucleus influences up- or down-regulation of ARE-mediated gene
expression. The results further suggest that antioxidant-induced delayed
accumulation of Bach1 contributes to the down-regulation of ARE-regulated genes,
presumably to reduce the antioxidant enzymes to normal levels. Bach1 is a transcriptional repressor of the cytoprotective enzyme heme
oxygenase-1 (HO-1). Although HO-1 protects against atherosclerosis, the function
of Bach1 in this process is poorly understood. We isolated peritoneal
macrophages and aortic smooth muscle cells (SMC) from wild-type and
bach1-deficient mice. bach1-Deficient macrophages expressed increased levels of
HO-1 and showed elevated phagocytic activity when incubated with 0.75 microm
microspheres. In SMC, bach1-ablation resulted in increased expression of HO-1
and decreased proliferation in bromodeoxyuridine incorporation assay as compared
with wild-type cells. The up-regulated phagocytic activity and reduced SMC
proliferation of bach1-deficient cells were not restored by Zinc (II)
protoporphyrin IX, an inhibitor of HO, suggesting that HO-independent mechanisms
are also involved in the regulation of phagocytosis of macrophages and
proliferation of SMC by Bach1. In wild-type mice, cuff placement around femoral
artery caused pronounced intimal proliferation without affecting the media, thus
resulting in intimal to medial (I/M) volume ratio of 65.6%. bach1-deficient mice
had less degree of intimal growth (I/M ratio of 45.6%). These results indicate
that Bach1 plays a critical role in the regulation of HO-1 expression,
macrophage function, SMC proliferation and neointimal formation. Bach1 may
regulate gene expression in these cells during inflammation and atherogenesis. Bach1 functions as a transcriptional repressor of heme oxygenase-1 (HO-1) and
the beta-globin genes. The enhancer regions of these genes contain multiple Maf
recognition elements (MAREs) to which Bach1 can bind. Previous studies have
shown that increased levels of heme and cadmium induce the nuclear export of
Bach1, resulting in cytoplasmic accumulation. By means of a yeast two hybrid
screening using Bach1 as bait, we identified the intracellular hyaluronic acid
binding protein (IHABP) as a potential regulator of Bach1. IHABP is a
microtubule-associated protein that may regulate the organization of the
cytoskeletal network. A series of domain analyses revealed that a region of
Bach1 previously implicated in cytoplasmic accumulation was necessary for
IHABP-binding. A C-terminal region of IHABP was necessary for Bach1-binding.
Overexpressed Bach1 colocalized with IHABP in the cytoplasm, forming fiber-like
structures on microtubules. Fluorescence recovery after photobleaching (FRAP)
analysis revealed a dynamic nature of the Bach1-IHABP interaction in living
cells. The repression of HO-1 reporter activity by Bach1 was attenuated by
co-transfecting IHABP in a dose-dependent manner. Moreover, the overexpression
of IHABP induced the endogenous HO-1 gene in NIH3T3 cells. The overall results
suggest that IHABP regulates the subcelluar localization of Bach1 in order to
fine-tune transactivation of Bach1 target genes such as HO-1. BACKGROUND/AIMS: Hepatitis C infection induces hepatic oxidative stress. Heme
oxygenase (HO), the rate-controlling enzyme of heme catabolism, plays a key role
as a protector against oxidative, and other stresses. Other recent work has
implicated Bach1, a heme binding protein that represses gene expression, in the
regulation of HO-1 gene expression.
METHODS: We investigated the effects of HCV polyprotein expression on expression
of HO-1 and Bach1 genes in human hepatoma cells (Huh-7 cells).
RESULTS: HO-1 was up-regulated in the cell line expressing HCV proteins from
core up to the aminoterminal domain of NS3. Addition of increasing
concentrations of N-acetylcysteine (NAC) led to down-regulation of HO-1 in cells
expressing HCV proteins. In contrast, Bach1 was significantly down-regulated in
these cells. Sodium arsenite, a strong inducer of oxidative stress and HO-1,
reduced Bach1 expression in wild type Huh-7 cells, and NAC partially abrogated
this decrease.
CONCLUSIONS: Huh-7 cells expressing HCV proteins show significant up-regulation
of the HO-1 gene, and reciprocal down-regulation of the Bach1 gene. Exogenous
oxidative stressors and anti-oxidants can modulate expression of these genes.
These and other results suggest a key role of down-regulation of Bach1 and
up-regulation of HO-1 in diminishing cytotoxic effects of HCV proteins in human
hepatocytes. Oxidative stress is involved in the mechanism of atherosclerotic lesion
formation and in the mechanisms underlying the development of other pathogenic
conditions of the cardiovascular system, including endothelial dysfunction,
hypertension, and heart failure. Reducing oxidative stress may be a reasonable
therapeutic approach to treat cardiovascular diseases. HO-1 is a cytoprotective
enzyme that is induced in response to oxidative stress and degrades heme into
carbon monoxide (CO) and bilirubin, both of which have cytoprotective effects. A
substantial body of evidence suggests that introduction of HO-1, either
pharmacologically or by a gene delivery technique, confers cytoprotection in
ischemic heart disease and atherosclerosis in animals. Recent studies have
revealed that CO has anti-inflammatory properties and that administration of CO
provides protection against atherosclerosis and ischemic heart disease.
Discovery of Bach1, a transcriptional repressor of HO-1, has greatly contributed
to the understanding of the regulation of HO-11 expression, providing a clue to
a development of alternative method to enhance HO activity. Bach1 normally
represses HO-1 expression. However, upon exposure to oxidative stress, Bach1
loses its repressive activity and is exported out of the nucleus, which in turn
results in the upregulation of HO-1. Bach1 knockout mice, expressing an
increased amount of HO-1, are resistant to pro-atherosclerotic and ischemic
stresses. These findings indicate that inhibition of Bach1 may be a novel
approach to enhance protection against stress. In summary, the Bach1-HO-1 system
is an important defense mechanism against oxidative stress. Development of a
safe and effective method to enhance this pathway, such as Bach1 inhibitor, may
be of great clinical relevance. Bach1 is a transcriptional repressor of heme oxygenase-1 gene (Hmox-1) and
beta-globin gene. Heme oxygenase (HO)-1 is an inducible cytoprotective enzyme
that degrades pro-oxidant heme to carbon monoxide (CO) and biliverdin/bilirubin,
which are thought to mediate anti-inflammatory and anti-oxidant actions of HO-1.
In the present study, we investigated the role of Bach1 in tissue protection
against myocardial ischemia/reperfusion (I/R) injury in vivo using mice lacking
the Bach1 gene (Bach1(-/-)) and wild-type (Bach1(+/+)) mice. In Bach1(-/-) mice,
myocardial expression of HO-1 protein was constitutively up-regulated by
3.4-fold compared to that in Bach1(+/+) mice. While myocardial I/R induced HO-1
protein in ischemic myocytes in both strains of mice, the extent of induction
was significantly greater in Bach1(-/-) mice than in Bach1(+/+) mice. Myocardial
infarction was markedly reduced in size by 48.4% in Bach1(-/-) mice.
Pretreatment of Bach1(-/-) mice with zinc-protoporphyrin, an inhibitor of HO
activity, abolished the infarction-reducing effect of Bach1 disruption,
indicating that reduction in the infarct size was mediated, at least in part, by
HO-1 activity. Thus, Bach1 plays a pivotal role in setting the levels of both
constitutive and inducible expression of HO-1 in the myocardium. Bach1
inactivation during I/R appears to be a key mechanism controlling the activation
level of cytoprotective program involving HO-1. Tin mesoporphyrin (SnMP), a competitive heme oxygenase (HO) inhibitor, also
induces HO-1 mRNA and protein expression by a mechanism that is not fully
understood. We examined whether the induction by SnMP is mediated by a
de-repression of Bach1, a transcription factor that suppresses the HO-1 gene.
Incubation of NIH3T3-HO-1-luc cells with SnMP attenuated HO activity with a
concomitant increase in HO-1 mRNA and protein and a decrease in Bach1 and HO-2
proteins, which was not due to transcriptional down-regulation, but accelerated
protein decay. Similarly, HO-1 protein degradation was increased by SnMP,
despite of an elevation in HO-1 transcription. Transfection of Bach1 shRNA in
Hepa cells raised basal HO-1 expression significantly, and SnMP treatment
further increased HO-1 mRNA. In conclusion, SnMP induces HO-1 expression not
only by de-repressing the HO-1 promoter by binding Bach1, but also by
accelerating Bach1 degradation. The mammalian transcription factor Bach1 functions as a repressor of the
enhancers of heme oxygenase-1 (HO-1) gene (Hmox-1) by forming heterodimers with
the small Maf proteins such as MafK. The transcription of Hmox-1 is regulated by
the substrate of HO-1, heme. Heme induces expression of Hmox-1 in part by
inhibiting the binding of Bach1 to the enhancers and inducing the nuclear export
of Bach1. A dipeptide motif of cysteine and proline (CP motif) in Bach1 is
essential for the heme-mediated regulation. In this study, we show that five
molecules of heme bind to Bach1 by the heme-titration assay. The Bach1-heme
complex exhibits an absorption spectrum with a major Soret peak at 371 nm and
Raman band at 343 cm(-1) in high amounts of heme and a spectrum containing the
major Soret peak at 423 nm at low heme concentrations. The spectroscopic
characterization indicates that Bach1 has two kinds of heme-binding sites with
different coordination structures. Mutagenesis studies have established that
four molecules of heme bind to the cysteine residues of four CP motifs in the C
terminus of Bach1. These results raise the possibility that two separated
activities of Bach1, DNA-binding and nuclear export, are regulated by heme
binding at the different CP motifs of Bach1 respectively, but not by cooperative
heme-binding. Ferritin gene transcription is regulated by heme as is ferritin mRNA
translation, which is mediated by the well studied mRNA.IRE/IRP protein complex.
The heme-sensitive DNA sequence in ferritin genes is the maf
recognition/antioxidant response element present in several other genes that are
induced by heme and repressed by Bach1. We now report that chromatin
immunoprecipitated with Bach1 antiserum contains ferritin DNA sequences. In
addition, overexpression of Bach1 protein in the transfected cells decreased
ferritin expression, indicating insufficient endogenous Bach1 for full
repression; decreasing Bach1 with antisense RNA increased ferritin expression.
Thioredoxin reductase1, a gene that also contains a maf recognition/antioxidant
response element but is less studied, responded similarly to ferritin, as did
the positive controls heme oxygenase1 and NADP(H) quinone (oxido) reductase1.
Bach1-DNA promoter interactions in cells were confirmed in vitro with soluble,
recombit Bach1 protein and revealed a quantitative range of Bach1/DNA
stabilities: ferritin L approximately ferritin H approximately beta-globin,
beta-globin approximately 2-fold >heme oxygenase1 = quinone reductase
beta-globin approximately 4-fold >thioredoxin reductase1. Such results indicate
the possibility that modulation of cellular Bach1 concentrations will have
variable effects among the genes coordinately regulated by maf
recognition/antioxidant response elements in iron/oxygen/antioxidant metabolism. Bach1 is a stress-responsive transcriptional factor that is thought to control
the expression levels of cytoprotective factors, including heme-oxygenase
(HO)-1. In the present study, we investigated the roles of Bach1 in the
development of left ventricular (LV) hypertrophy and remodeling induced by
transverse aortic constriction (TAC) in vivo using Bach1 gene-deficient
(Bach1(-/-)) mice. TAC for 3 weeks in wild-type control (Bach1(+/+)) mice
produced LV hypertrophy and remodeling manifested by increased heart weight,
histological findings showing increased myocyte cross-sectional area (CSA) and
interstitial fibrosis (picro Sirius red staining), reexpressions of ANP, BNP,
and betaMHC genes, and echocardiographic findings showing wall thickening, LV
dilatation, and reduced LV contraction. Deletion of Bach1 caused significant
reductions in heart weight (by 16%), CSA (by 36%), tissue collagen content (by
38%), and gene expression levels of ANP (by 75%), BNP (by 45%), and betaMHC (by
74%). Echocardiography revealed reduced LV dimension and ameliorated LV
contractile function. Deletion of Bach1 in the LV caused marked upregulation of
HO-1 protein accompanied by elevated HO activity in both basal or TAC-stimulated
conditions. Treatment of Bach1(-/-) mice with tin-protoporphyrin, an inhibitor
of HO, abolished the antihypertrophic and antiremodeling effects of Bach1 gene
ablation. These results suggest that deletion of Bach1 caused upregulation of
cytoprotective HO-1, thereby inhibiting TAC-induced LV hypertrophy and
remodeling, at least in part, through activation of HO. Bach1 repressively
controls myocardial HO-1 expression both in basal and stressed conditions,
inhibition of Bach1 may be a novel therapeutic strategy to protect the
myocardium from pressure overload. Intracellular heme is a redox active molecule that can be detrimental to cells
at high concentrations or under oxidizing conditions. To prevent accumulation,
the inducible enzyme heme oxygenase-1 (HMOX1) catalyzes degradation of heme. In
the absence of elevated intracellular heme or oxidative stress, the basic region
leucine zipper transcriptional regulator BACH1 binds HMOX1 antioxidant response
elements and represses transcription. Conversely, increased intracellular heme
or sulfhydryl oxidation inactivate BACH1, permitting transcriptional induction
of HMOX1. Here, we investigate the effect of BACH1 inactivation on the induction
of HMOX1 and as a mechanism for broader gene induction. We show that BACH1 is
inactivated at low micromolar arsenite concentrations and that BACH1
inactivation is necessary and sufficient for transcriptional induction of HMOX1.
Because BACH1 is thought to interact with antioxidant response element motifs,
we further examined the role of BACH1 as a regulator of inducible antioxidant
gene expression by assessing the global profile of gene expression following
BACH1 knockdown using small interfering RNA. The loss of BACH1 function in human
keratinocytes results almost exclusively in HMOX1 induction, suggesting that
BACH1 may function as a rheostat regulating levels of intracellular free heme. Oxidative stress has been implicated in tissue damage from traumatic brain
injury. Heme oxygenase-1 (HO-1) is an inducible enzyme that degrades prooxidant
heme to radical-scavenging biliverdin/bilirubin in order to protect cells from
oxidative stress. Although HO-1 is induced after induction of brain damage, the
regulatory mechanism of HO-1 in the brain is still unclear. Bach1 is a
transcriptional repressor of the HO-1 gene, and plays a critical role in tissue
protection from oxidative stress by reperfusion injury of the myocardium. In
this study, we examined the role of Bach1 in HO-1 regulation of the various
brain sites by investigating the expression of Bach1 and HO-1 in brain tissues
of mice bearing Bach1-deficient (Bach1(-/-)) or wild-type (Bach1(+/+)) genes.
While the expression levels of Bach1 mRNA in the olfactory bulb were
significantly higher than other brain areas, those at the cortex showed the
lowest activity. Bach1(-/-) mice showed significantly higher HO-1 mRNA
expression levels than Bach1(+/+) mice in all brain sites studied. Moreover,
higher induction of HO-1 was observed around damaged tissues after cold injury
in Bach1(-/-) than Bach1(+/+) mice. Thus, Bach1 plays an important role in
regulating the constitutive and inducible expression levels of HO-1 in the
brain. Although a significantly higher level of HO-1 was observed in Bach1(-/-)
than Bach1(+/+) mice, genetic ablation of the Bach1 gene failed to show any
tissue protective effect after cold injury was inflicted on the cortex. Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation and
antioxidant defense protein, is induced in the lungs of animals exposed to
hyperoxia. However, high levels of HO-1 expression may be deleterious, thus
necessitating tight regulation. Previous reports show maturational differences
in rat HO-1 regulation in hyperoxia, as newborns do not up-regulate HO-1mRNA
compared with adults. To better understand the differential response of lung
HO-1 to hyperoxia, we exposed newborn and adult mice to >95% oxygen. The newborn
lungs had reduced HO-1 mRNA induction compared with adults and newborn
transgenic mice over-expressing luciferase driven by the 15 kb HO-1 promoter
(HO-1/Luc Tg) had less increased light emission in hyperoxia compared with
adults. Compared with adults, levels of the repressor of HO-1 transcription,
Bach1, were higher in the neonatal lung as was nuclear protein-DNA binding to
the antioxidant response element (ARE) from HO-1. Furthermore, at baseline and
in hyperoxia, chromatin immunoprecipitation (ChIP) revealed increased Bach1
binding to the HO-1 distal enhancers (DEs) in the neonates compared with adults.
These data suggest that elevated levels of Bach1 may help to limit HO-1
induction in the newborn at baseline and in response to oxidative stress. Cellular senescence is one of the key strategies to suppress expansion of cells
with mutations. Senescence is induced in response to genotoxic and oxidative
stress. Here we show that the transcription factor Bach1 (BTB and CNC homology
1, basic leucine zipper transcription factor 1), which inhibits oxidative
stress-inducible genes, is a crucial negative regulator of oxidative
stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts
showed a propensity to undergo more rapid and profound p53-dependent premature
senescence than control wild-type cells in response to oxidative stress. Bach1
formed a complex that contained p53, histone deacetylase 1 and nuclear
co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and
contributed to impeding p53 action by promoting histone deacetylation. Because
Bach1 is regulated by oxidative stress and heme, our data show that Bach1
connects oxygen metabolism and cellular senescence as a negative regulator of
p53. OBJECT: Oxidative stress contributes to secondary injury after spinal cord
injury (SCI). The expression of heme oxygenase-1 (HO-1), which protects cells
from various insults including oxidative stress, is upregulated in injured
spinal cords. Mice deficient in Bach1 (Bach1-/-), a transcriptional repressor of
the HO-1 and beta-globin genes, express high levels of HO-1 mRNA and protein in
various organs. The authors hypothesized that HO-1 modulates the secondary
injury process after SCI in Bach1(-/-) mice.
METHODS: Male C57BL/6 (wild-type) and homozygous Bach1(-/-) C57BL/6 mice were
subjected to moderate SCI, and differences in hindlimb motor function, and
electrophysiological, molecular biological, and histopathological changes were
assessed for 2 weeks.
RESULTS: Functional recovery was greater, and motor evoked potentials were
significantly larger in Bach1(-/-) mice than in wild-type mice throughout the
observation period. The expression of HO-1 mRNA in the spinal cord was
significantly increased in both mice until 3 days after injury, and it was
significantly higher in Bach1(-/-) mice than in wild-type mice at every
assessment point. Histological examination using Luxol fast blue staining at 1
day after injury showed that the injured areas were smaller in Bach1(-/-) mice
than in wild-type mice. The HO-1 immunoreactivity was not detected in uninjured
spinal cord, but 3 days postinjury the number of HO-1-immunoreactive cells was
obviously higher in the injured area in both mice, particularly in Bach1(-/-)
mice. The HO-1 was primarily induced in microglia/macrophage in both mice.
CONCLUSIONS: These results suggest that HO-1 modulates the secondary injury
process, and high HO-1 expression may preserve spinal cord function in the early
stages after SCI in Bach1(-/-) mice. Treatment that induces HO-1 expression at
these early stages may preserve the functional outcome after SCI. Bach1 is a basic region-leucine zipper (bZip) protein that forms heterodimers
with the small Maf proteins and functions as a repressor of gene expression. One
of the target genes of Bach1 is Hmox-1 that encodes heme oxygenase-1 (HO-1).
HO-1 degrades heme into carbon monoxide (CO), biliverdin, and iron. HO-1 is
strongly induced by various stresses as well as its substrate heme, and protects
cells and tissues against insults through diverse cytoprotective functions of
the reaction products CO and biliverdin. Bach1-deficiency in mice leads to
higher expression of Hmox-1 in various tissues. Here we investigated the effects
of Bach1-deficiency in mice on tissue injuries: hepatic injury induced by
D-galactosamine (GalN) and lipopolysaccharide (LPS), and mouse paw edema induced
by carrageenin, polysaccharide derived from various seaweeds. Bach1-deficiency
suppressed induction of plasma alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) activities in response to the GalN/LPS-treatment.
However, production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide
(NO), both being cytotoxic mediators in LPS-induced hepatic injury, in
Bach1-deficient mice and their peritoneal macrophages was similar to wild type
controls. In contrast, Bach1-deficiency did not affect extent of mouse paw edema
induced by carrageenin, which enhances vascular permeability by activating kinin
release. These results indicate that Bach1 plays an inhibitory role in the
cytoprotection of LPS-induced liver injury but not in the kinin-mediated
inflammatory edema. The inhibitory role for Bach1 may stem from its activity to
repress gene expression including HO-1. Bach1 is a transcriptional repressor of the heme oxygenase (HO)-1 gene.
Bach1-null (Bach1(-/-)) mice are reported to be protected from myocardial
ischemia/reperfusion injury; however, the effect of Bach1 disruption on another
oxidative stress model of hyperoxic lung injury has yet to be determined. To
investigate the role of Bach1 in hyperoxic lung injury, Bach1(-/-) mice and
wild-type (WT) mice were exposed to 90% O(2). During hyperoxic exposure, the
survival of Bach1(-/-) mice was significantly longer than that of WT mice.
However, the administration of zinc protoporphyrin, an inhibitor of HO-1
activity, did not change the mortality in either of the mice, thus suggesting
that this protective effect was not mediated by an HO-1 overexpression in
Bach1(-/-) mice. The indices of lung injury in the lungs of Bach1(-/-) mice were
lower than those of WT mice; unexpectedly, however, the levels of IL-6 in
bronchoalveolar lavage (BAL) fluid from Bach1(-/-) mice were significantly
higher than those of WT mice. Interestingly, the intrapulmonary administration
of small interfering RNA against IL-6 was shown to reduce the IL-6 levels in BAL
fluids and shorten the survival in Bach1(-/-) mice during hyperoxic exposure. In
addition, a chromatin immunoprecipitation analysis revealed the binding of Bach1
to the IL-6 promoter and its detachment after oxidative stress. Considering the
previous observation that the transgenic mice overexpressing IL-6 are protected
from hyperoxic lung injury, these results therefore indicate that IL-6 mediates
an increased survival in Bach1(-/-) mice during hyperoxic exposure. The tumor suppressor p53 induces cellular senescence, an irreversible form of
proliferation arrest, to inhibit carcinogenesis as well as aging of organs and a
body. While the major cause of cellular senescence and aging is oxidative
stress, little is known about how the p53 activity is regulated under such
conditions. Bach1 inhibits expression of oxidative stress responsive genes by
competing with Nrf2, the key activator of oxidative stress response. Bach1
inhibits p53-dependent cellular senescence induced by oxidative stress. Bach1
forms a protein complex with p53, is recruited to p53 target genes, and inhibits
their expression. These findings provide completely novel insights into how the
activity of p53 is regulated under oxidative stress. Since p53 is the critical
tumor suppressor with huge clinical implications, the newly identified mechanism
should open new research fields. Hepatitis C virus (HCV) directly induces oxidative stress and liver injury.
Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively
regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has
antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small
noncoding RNAs ( approximately 22 nt) that are important regulators of gene
expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown.
The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1,
and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1
isolate and J6/JFH1-based HCV cell culture system were used in this study. The
effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were
measured by way of quantitative real-time polymerase chain reaction (qRT-PCR)
and Western blotting, respectively. The Dual Glo Luciferase Assay System was
used to determine reporter activities. miR-196 mimic significantly
down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV
expression. Dual luciferase reporter assays demonstrated that transfection of
miR-196 mimic resulted in a significant decrease in Bach1 3'-untranslated region
(UTR)-dependent luciferase activity but not in mutant Bach1 3'-UTR-dependent
luciferase activity. Moreover, there was no detectable effect of mutant miR-196
on Bach1 3'-UTR-dependent luciferase activity.
CONCLUSION: miR-196 directly acts on the 3'-UTR of Bach1 messenger RNA and
translationally represses the expression of this protein, and up-regulates
HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype
1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a
role in both HMOX1/Bach1 expression and the regulation of HCV expression in
human hepatocytes. Overexpression of miR-196 holds promise as a potential novel
strategy to prevent or ameliorate hepatitis C infection, and to protect against
liver injury in chronic HCV infection. Heme oxygenase (HO)-1 has anti-oxidative, anti-inflammatory, and anti-apoptotic
activities. However, little is known about the regulation of HO-1 in human
primary acute myeloid leukemia (AML) cells. Here we investigated the expression
of HO-1 in primary and established AML cells as well as other types of leukemic
cells and normal monocytes, and its regulatory mechanism by the transcriptional
repressor, BTB and CNC homology 1 (Bach1), and the activator, nuclear factor
erythroid-derived 2 related factor 2 (Nrf2). Leukemic cell lines such as U937
expressed little HO-1, whereas most freshly isolated AML cells and monocytes
expressed substantial amounts of HO-1, along with Bach1 and Nrf2. When U937
cells were treated with phorbol myristate acetate (PHA) or gamma-interferon,
they significantly expressed both HO-1 and Bach1, like primary AML cells.
Treatment with lipopolysaccharide (LPS) enhanced HO-1 expression in U937 cells
but suppressed it in primary monocytes and PMA-treated U937 cells. In
HO-1-expressing cells, Bach1 was localized in the cytoplasm, but Nrf2 was
localized in the nuclei. Chromatin immunoprecipitation assay of these cells
revealed the preferential binding of Nrf2 over Bach1 to Maf-recognition
elements, the enhancer regions of the HO-1 gene. The downregulation of the HO-1
gene with siRNA increased a cytotoxic effect of an anticancer drug on primary
AML cells, whereas the downregulation of Bach1 increased HO-1 expression,
leading to enhanced survival. These and other results show that Bach1 plays a
critical role in regulating HO-1 gene expression in AML cells and its expression
suppresses their survival by downregulating HO-1 expression. Thus, functional
upregulation of Bach1 is a potential strategy for antileukemic therapy. BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1
(HO-1). It plays an important role in the feedback regulation of HO-1
expression, which protects cells from various insults including oxidative stress
and inflammatory cytokines. However, the role of Bach1 in intestinal
inflammation remains unclear. In this study, the role of Bach1 in intestinal
mucosal injury was elucidated using 8-week-old female C57BL/6 (wild-type) and
homozygous Bach1-deficient C57BL/6 mice. Intestinal mucosal injuries induced by
a single subcutaneous administration of indomethacin were evaluated
macroscopically, histologically, and biochemically. Mucosal protein content and
chemokine mRNA levels were determined by real-time PCR. Our results showed that
the indomethacin-induced intestinal injury was remarkably improved in
Bach1-deficient mice. Histological examination showed that the area of injured
lesion was decreased in Bach1-deficient mice compared to wild-type mice.
Administration of indomethacin induced expression of inflammatory chemokines
such as KC, MIP1alpha and MCP1, which was suppressed in Bach1-deficient mice.
Myeloperoxidase activity in the intestinal mucosa was also significantly
decreased in Bach1-deficient mice. Additionally, Bach1 deficiency enhanced
immunopositivity of HO-1 in the intestinal mucosa after indomethacin
administration. Disruption of the Bach1 gene thus caused inhibition of mucosal
injury, indicating that inhibition of Bach1 may be a novel therapeutic strategy
for treating indomethacin-induced intestinal injury. Bach1 is a member of the basic leucine zipper transcription factor family, and
the Bach1/small Maf heterodimer specifically represses transcriptional activity
directed by the Maf recognition element (MARE). Because Bach1 is a repressor of
the oxidative stress response, we examined the function(s) of Bach1 in
keratinocytes subjected to oxidative stress. Oxidative stress induced by
H(2)O(2) led to an increase in MARE activity and expression of heme oxygenase-1
(HO-1), an inducible antioxidant defense enzyme. Bach1 depletion by small
interfering RNAs or by deletion of Bach1 enhanced HO-1 expression in the absence
of H(2)O(2), indicating that Bach1 is a critical repressor of HO-1 in
keratinocytes. Although Bach1-deficient or -reduced keratinocytes expressed
higher levels of HO-1 than control cells in response to H(2)O(2), Bach1
down-regulation did not attenuate the production of reactive oxygen species by
H(2)O(2). In contrast, Bach1 overexpression abolished HO-1 induction by
H(2)O(2), which led to increased reactive oxygen species accumulation. HO-1 was
induced during keratinocyte differentiation, but MARE activity did not change
during differentiation. Furthermore, Bach1 overexpression did not inhibit
differentiation-associated induction of HO-1 expression, suggesting that HO-1
induction in differentiation is independent of Bach1. Thus, in response to
oxidative stress, Bach1 regulates the oxidation state through the negative
control of HO-1 expression prior to terminal keratinocyte differentiation.
However, Bach1-mediated repression is negated during keratinocyte
differentiation. Bach1 is a transcriptional repressor of heme oxygenase-1 (HO-1, a.k.a. HSP-32),
which is an inducible enzyme and has anti-oxidation/anti-inflammatory properties
shown in various models of organ injuries. Since oxidative stress plays a
pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH), HO-1
induction would be expected to prevent the development of NASH. In this study,
we investigated the influence of Bach1 ablation in mice on the progression of
NASH in methionine-choline deficient (MCD) diet model. Bach1 ablation resulted
in significant induction of HO-1 mRNA and its activity in the liver. When fed
MCD diet, Bach1(-/-) mice exhibited negligible hepatic steatosis compared to
pronounced steatohepatitis in wild type mice with 6-fold increase in hepatic
triglyceride content. Whereas feeding of MCD diet decreased mRNA expressions of
peroxisome proliferator-activated receptor (PPAR) α and microsomal triglyceride
transfer protein (MTP) in wild type mice, there were no change in Bach1(-/-)
mice. In addition, hepatic concentration of malondialdehyde (MDA), a biomarker
for oxidative stress as well as plasma alanine aminotransferase (ALT) was
significantly lower in Bach1(-/-) mice. These findings suggest that Bach1
ablation exerts hepatoprotective effect against steatohepatitis presumably via
HO-1 induction and may be a potential therapeutic target. The regulation of gene expression in response to environmental signals and
metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and
CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the
transcription from a subset of MAF recognition elements at low intracellular
heme levels. Upon heme binding, BACH1 is released from the MAF recognition
elements, resulting in increased expression of antioxidant response genes. To
systematically address the gene regulatory networks involving BACH1, we combined
chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK
293 cells with knockdown of BACH1 using three independent types of small
interfering RNAs followed by transcriptome profiling using microarrays. The 59
BACH1 target genes identified by chromatin immunoprecipitation sequencing were
found highly enriched in genes showing expression changes after BACH1 knockdown,
demonstrating the impact of BACH1 repression on transcription. In addition to
known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1,
and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered
BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1,
SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport
processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of
BACH1 on genes involved in neurodegenerative processes and proliferation
provides an interesting basis for future dissection of BACH1-mediated gene
repression in neurodegeneration and virus-induced cancerogenesis. A central mechanism in cellular defence against oxidative or electrophilic
stress is mediated by transcriptional induction of genes via the ARE
(antioxidant-response element), a cis-acting sequence present in the regulatory
regions of genes involved in the detoxification and elimination of reactive
oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine
zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid
2-related factor 2) that functions as a transcriptional activator via
heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is
relatively well understood, the mechanisms by which ARE-mediated signalling is
down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex,
tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to
ARE-like sequences, functioning as a transcriptional repressor in a subset of
ARE-regulated genes, thus antagonizing the activator function of Nrf2. In the
present study, we have demonstrated that BACH1 itself is regulated by Nrf2 as it
is induced by Nrf2 overexpression and by Nrf2-activating agents in an
Nrf2-dependent manner. Furthermore, a functional ARE site was identified at
+1411 from the transcription start site of transcript variant 2 of BACH1. We
conclude that BACH1 is a bona fide Nrf2 target gene and that induction of BACH1
by Nrf2 may serve as a feedback-inhibitory mechanism for ARE-mediated gene
regulation. Up-regulation of heme oxygenase 1 (HO-1) by ultraviolet A (UVA; 320-380 nm)
irradiation of human skin cells protects them against oxidative stress. The role
of Nrf2 in up-regulation of HO-1 and other phase II genes is well established.
The mechanism underlying Bach1-mediated HO-1 repression is less well understood
although cellular localization seems to be crucial. Because prolonged HO-1
overexpression is likely to be detrimental, it is crucial that activation of the
gene is transient. We now show that UVA irradiation of cultured human skin
fibroblasts enhances accumulation of Bach1 mRNA and protein severalfold.
Endogenous Bach1 protein accumulates in the nucleus after 8h and may occupy MARE
sites after HO-1 activation thus providing a compensatory mechanism to control
HO-1 overexpression. Overexpression of Bach1, together with MafK, represses
basal and UVA-mediated HO-1 protein expression, whereas silencing of the Bach1
gene by Bach1-specific siRNAs causes robust enhancement of constitutive HO-1
levels. UVA treatment of cells in which Bach1 has been silenced leads to higher
levels of induction of the HO-1 protein. Although Bach1 protein is exported from
the nucleus 12h after UVA irradiation, the release of free cellular heme from
microsomal heme-containing proteins is immediate rather than delayed. Although
heme does promote the export of Bach1 via the Crm1/exportin 1 pathway and is
involved in the delayed UVA-mediated export of the protein, it is not clear how
this occurs. The transcriptional repressor Bach1 mediates various stress responses. Despite
its role in transcription, Bach1 is predomitly exported to the cytoplasm in a
Crm1-dependent manner, but the functional role of its cytoplasmic retention is
still unclear. We found that Bach1 was also excluded from mitotic chromatin by a
C-terminal cytoplasmic localization sequence dependent and leptomycin B
sensitive process. Bach1 depletion resulted in disordered mitotic chromosome
alignment, which was rescued by Bach1 mutants lacking the BTB or DNA binding
domains, suggesting its transcription-independent mechanism. We thus revealed a
novel role of Bach1 in the regulation of mitotic chromosome dynamics. The let-7 microRNA (miRNA) plays important roles in human liver development and
diseases such as hepatocellular carcinoma, liver fibrosis and hepatitis wherein
oxidative stress accelerates the progression of these diseases. To date, the
role of the let-7 miRNA family in modulation of heme oxygenase 1 (HMOX1), a key
cytoprotective enzyme, remains unknown. Our aims were to determine whether let-7
miRNA directly regulates Bach1, a transcriptional repressor of the HMOX1 gene,
and whether indirect up-regulation of HMOX1 by let-7 miRNA attenuates oxidant
injury in human hepatocytes. The effects of let-7 miRNA on Bach1 and HMOX1 gene
expression in Huh-7 and HepG2 cells were determined by real-time qRT-PCR,
Western blot, and luciferase reporter assays. Dual luciferase reporter assays
revealed that let-7b, let-7c, or miR-98 significantly decreased Bach1
3'-untranslated region (3'-UTR)-dependent luciferase activity but not mutant
Bach1 3'-UTR-dependent luciferase activity, whereas mutant let-7 miRNA
containing base complementarity with mutant Bach1 3'-UTR restored its effect on
mutant reporter activity. let-7b, let-7c, or miR-98 down-regulated Bach1 protein
levels by 50-70%, and subsequently up-regulated HMOX1 gene expression by 3-4
fold, compared with non-specific controls. Furthermore, Huh-7 cells transfected
with let-7b, let-7c or miR-98 mimic showed increased resistance against oxidant
injury induced by tert-butyl-hydroperoxide (tBuOOH), whereas the protection was
abrogated by over-expression of Bach1. In conclusion, let-7 miRNA directly acts
on the 3'-UTR of Bach1 and negatively regulates expression of this protein, and
thereby up-regulates HMOX1 gene expression. Over-expression of the let-7 miRNA
family members may represent a novel approach to protecting human hepatocytes
from oxidant injury. Phenylpropanoids have several highly significant biological properties in both
plants and animals. Four phenylpropanoid glycosides (PPGs), verbascoside (VB),
forsythoside B (FB), echinacoside (EC) and campneoside I (CP), were purified and
tested for their capability to activate NRF2 and induce phase II cytoprotective
enzymes in a human keratinocyte cell line (HaCaT). All four substances showed
similar strong antioxidant and radical-scavenging activities as determined by
diphenylpicrylhydrazyl assay. Furthermore, in HaCaT cells, FB and EC are strong
activators of NRF2, the nuclear transcription factor regulating many phase II
detoxifying and cytoprotective enzymes, such as heme oxygenase 1 (HMOX1). In
HaCaT cells, FB and EC (200 μM) induced nuclear translocation of NRF2 protein
after 24 h and reduced nuclear protein levels of BACH1, a repressor of the
antioxidant response element. FB and EC greatly HMOX1 mRNA levels by more than
40-fold in 72 h. Cytoplasmic HMOX1 protein levels were also increased at 48 h
after treatment. VB was less active compared to FB and EC, and CP was slightly
active only at later times of treatment. We suggest that hydroxytyrosol (HYD)
could be a potential bioactive metabolite of PPGs since HYD, in equimolar
amounts to PGGs, is able to both activate HO-1 transcription and modify
Nrf2/Bach1 nuclear protein levels. This is in agreement with the poor activity
of CP, which contains a HYD moiety modified by an O-methyl group. In conclusion,
FB and EC from plant cell cultures may provide long-lasting skin protection by
induction of phase II cytoprotective capabilities. Reactive oxygen species (ROS), by-products of aerobic respiration, promote
genetic instability and contribute to the maligt transformation of cells.
Among the genes related to ROS metabolism, Bach1 is a repressor of the oxidative
stress response, and a negative regulator of ROS-induced cellular senescence
directed by p53 in higher eukaryotes. While ROS are intimately involved in
carcinogenesis, it is not clear whether Bach1 is involved in this process. We
found that senescent Bach1-deficient mouse embryonic fibroblasts (MEFs)
underwent spontaneous immortalization the same as did the wild-type cells. When
transduced with constitutively active Ras (H-Ras(V12)), the proliferation and
colony formation of these cells in vitro were markedly reduced. When
transplanted into athymic nude mice, the growth and vascularization of tumors
derived from Bach1-deficient cells were also decreased. Gene expression
profiling of the MEFs revealed a new H-Ras(V12) signature, which was distinct
from the previously reported signatures in epithelial tumors, and was partly
dependent on Bach1. The Bach1-deficient cells showed diminished phosphorylation
of MEK and ERK1/2 in response to H-Ras(V12), which was consistent with the
alterations in the gene expression profile, including phosphatase genes.
Finally, Bach1-deficient mice were less susceptible to
4-nitroquinoline-1-oxidide (4-NQO)-induced tongue carcinoma than wild-type mice.
Our data provide evidence for a critical role of Bach1 in cell transformation
and tumor growth induced by activated H-Ras(V12). Previous studies have proved that the environmental toxicant, inorganic arsenic,
activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many
different cell types. This study tried to explore the hepatic Nrf2 pathway upon
arsenic treatment comprehensively, since liver is one of the major target organs
of arsenical toxicity. Our results showed that inorganic arsenic significantly
induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also
observed a dose-dependent increase of antioxidant response element- (ARE-)
luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone
oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated
dramatically. On the other hand, entry and accumulation of Nrf2 protein in the
nucleus, while exportting the transcriptional repressor BTB and CNC homology 1
(Bach1) from nucleus to cytoplasm, were also confirmed by western blot and
immunofluorescence assay. Our results therefore confirmed the arsenic-induced
Nrf2 pathway activation in hepatocytes and also suggested that the translocation
of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic
Nrf2 pathway plays indispensable roles for cellular defenses against arsenic
hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand
the self-defensive responses and the diverse biological effects of arsenicals. BTB and CNC homology 1 (Bach1) is a transcriptional repressor of antioxidative
enzymes, such as heme oxygenase-1 (HO-1). Oxidative stress is reportedly
involved in insulin secretion impairment and obesity-associated insulin
resistance. However, the role of Bach1 in the development of diabetes is
unclear. HO-1 expression in the liver, white adipose tissue, and pancreatic
islets was markedly upregulated in Bach1-deficient mice. Unexpectedly, glucose
and insulin tolerance tests showed no differences in obese wild-type (WT) and
obese Bach1-deficient mice after high-fat diet loading for 6 wk, suggesting
minimal roles of Bach1 in the development of insulin resistance. In contrast,
Bach1 deficiency significantly suppressed alloxan-induced pancreatic insulin
content reduction and the resultant glucose elevation. Furthermore,
TUNEL-positive cells in pancreatic islets of Bach1-deficient mice were markedly
decreased, by 60%, compared with those in WT mice. HO-1 expression in islets was
significantly upregulated in alloxan-injected Bach1-deficient mice, whereas
expression of other antioxidative enzymes, e.g., catalase, superoxide dismutase,
and glutathione peroxidase, was not changed by either alloxan administration or
Bach1 deficiency. Our results suggest that Bach1 deficiency protects pancreatic
β-cells from oxidative stress-induced apoptosis and that the enhancement of HO-1
expression plays an important role in this protection. Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor
proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor
proteins remain poorly studied due to a shortage of efficient methods to
identify biologically relevant substrates. Here, we report the development of
parallel adaptor capture (PAC) proteomics and its use to systematically identify
candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs)
that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments,
we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2
oxidative stress pathway. We demonstrate that FBXL17 controls the transcription
of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in
the absence or presence of extrinsic oxidative stress. This work identifies a
role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene
activation and provides a framework for elucidating the functions of CRL adaptor
proteins. |
Is Kanzaki disease associated with deficiency in alpha-N-acetylgalactosaminidase? | Yes, Kanzaki disease is attributable to a deficiency in alpha-N-acetylgalactosaminidase, which hydrolyzes GalNAcalpha1-O-Ser/Thr. | Schindler disease and Kanzaki disease are caused by a deficient lysosomal
enzyme, alpha-N-acetylgalactosaminidase (E.C.3.2.1.49). Two German children were
first reported in 1987 and other two Dutch children were recently reported in
1993. These children were very similar clinically and characterized by maked
neuroaxonal dystrophy of an infantile onset. This disease (type 1) was named
Schindler disease. On the other hand, an adult patient with profuse
angiokeratoma corporis diffusum but minimum involvement in nervous system was
reported in 1987 from Japan. This disease (type 2) was named Kanzaki disease
(Mckusick catalog No. 104170). Molecular analyses of these diseases revealed one
each point mutation in the encoding gene. Clinical, ultrastructural and
molecular studies of these disease were described. Alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency (Schindler/Kanzaki
disease) is a clinically and pathologically heterogeneous genetic disease with a
wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease)
and late onset angiokeratoma corporis diffusum (Kanzaki disease). In alpha-NAGA
deficiency, there are discrepancies between the genotype and phenotype, and also
between urinary excretion products (sialyl glycoconjugates) and a theoretical
accumulated material (Tn-antigen; Gal NAcalpha1-O-Ser/Thr) resulting from a
defect in alpha-NAGA. As for the former issue, previously reported genetic,
biochemical and pathological data raise the question whether or not E325K
mutation found in Schindler disease patients really leads to the severe
phenotype of alpha-NAGA deficiency. The latter issue leads to the question of
whether alpha-NAGA deficiency is associated with the basic pathogenesis of this
disease. To clarify the pathogenesis of this disease, we performed structural
and immunocytochemical studies. The structure of human alpha-NAGA deduced on
homology modeling is composed of two domains, domain I, including the active
site, and domain II. R329W/Q, identified in patients with Kanzaki disease have
been deduced to cause drastic changes at the interface between domains I and II.
The structural change caused by E325K found in patients with Schindler disease
is localized on the N-terminal side of the tenth beta-strand in domain II and is
smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that
the main lysosomal accumulated material in cultured fibroblasts from patients
with Kanzaki disease is Tn-antigen. These data suggest that a prototype of
alpha-NAGA deficiency in Kanzaki disease and factors other than the defect of
alpha-NAGA may contribute to severe neurological disorders, and Kanzaki disease
is thought to be caused by a single enzyme deficiency. We describe the neurologic findings in a patient with
alpha-N-acetylgalactosaminidase deficiency (Kanzaki disease). Clinical and
electrophysiologic studies revealed sensory-motor polyneuropathy, and sural
nerve pathology showed decreased density of myelinated fibers with axonal
degeneration. The patient had mildly impaired intellectual function with
abnormal brain MRI and sensory-neuronal hearing impairment with repeated
episodes of vertigo attacks. These findings suggest that Kanzaki disease may
develop neurologic complications in the CNS and peripheral nervous system. BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in
alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes
GalNAcalpha1-O-Ser/Thr. Missense mutations, R329W or R329Q were identified in
two Japanese Kanzaki patients. Although they are on the same codon, the clinical
manifestation was more severe in R329W because an amino acid substitution led to
protein instability resulting in structural change, which is greater in R329W
than in R329Q.
OBJECTIVE: To examine whether the different clinical phenotypes are attributable
to the two mutations.
METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were
measured and three-dimensional models of human alpha-NAGA and its complexes with
GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling.
RESULTS: Residual enzyme activity was significantly higher in the R329Q- than
the R329W mutant (0.022+/-0.005 versus 0.005+/-0.001 nmol/h/ml: p<0.05); the
urinary ratios of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10,
respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active
site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to
associate with alpha-NAGA because of the side chain (CH3) of the threonine
residue.
CONCLUSION: Our findings suggest that the association of alpha-NAGA with its
substrates is strongly affected by the amino acid substitution at R329 and that
the association with GalNAcalpha1-O-Thr is more highly susceptible to structural
changes. The residual mutant enzyme in R329W could not associate with
GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme
in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary
ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr was lower and the clinical
phenotype was milder in the R329Q mutation. Structural analysis revealed
biochemical and phenotypic differences in these Kanzaki patients with the R329Q
and R329W mutation. alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal
exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from
glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity
results in the lysosomal storage disorders Schindler disease and Kanzaki
disease. To better understand the molecular defects in the diseases, we
determined the crystal structure of human alpha-NAGAL after expressing wild-type
and glycosylation-deficient glycoproteins in recombit insect cell expression
systems. We measured the enzymatic parameters of our purified wild-type and
mutant enzymes, establishing their enzymatic equivalence. To investigate the
binding specificity and catalytic mechanism of the human alpha-NAGAL enzyme, we
determined three crystallographic complexes with different catalytic products
bound in the active site of the enzyme. To better understand how individual
defects in the alpha-NAGAL glycoprotein lead to Schindler disease, we analyzed
the effect of disease-causing mutations on the three-dimensional structure. |
Is Mycobacterium avium less susceptible to antibiotics than Mycobacterium tuberculosis? | Mycobacterium avium causes disseminated infection in patients with acquired immune deficiency syndrome. M tuberculosis disease is preventable and curable and yet communicable, physicians should maintain a high degree of suspicion for tuberculosis in HIV-infected adults. In comparison, the goal of treating M avium complex in patients with advanced HIV disease is to reduce constitutional symptoms and improve survival. Patients who were suspected to have disseminated mycobacterial infection, presenting fever and (preferably) a CD4 T cell count<100.0 cell/mL were investigated. Twelve (15%) of the 80 blood cultures were positive for mycobacteria, with Mycobacterium avium being identified in 7 (8.8%) samples and M. tuberculosis in 5 (6.2%). The antimycobacterial activities of RS-112997, RS-124922 and RS-118641, three capuramycin analogues that inhibit phospho-N-acetylmuramyl-pentapeptide translocase, were tested against clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare. The MIC50/90 (mg/L) results for RS-118641 were: M. tuberculosis, 1/2; multidrug-resistant (MDR) M. tuberculosis, 0.5/2; M. avium, 4/8; and M. intracellulare, 0.06/0.5 | Mycobacterium avium causes disseminated infection in patients with acquired
immune deficiency syndrome. Mycobacterium tuberculosis is a pathogen associated
with the deaths of millions of people worldwide annually. Effective therapeutic
regimens exist that are limited by the emergence of drug resistance and the
inability of antibiotics to kill dormant organisms. The present study describes
a system using Mycobacterium smegmatis, an avirulent mycobacterium, to deliver
the lytic phage TM4 where both M. avium and M. tuberculosis reside within
macrophages. These results showed that treatment of M. avium-infected, as well
as M. tuberculosis-infected, RAW 264.7 macrophages, with M. smegmatis
transiently infected with TM4, resulted in a significant time- and
titer-dependent reduction in the number of viable intracellular bacilli. In
addition, the M. smegmatis vacuole harboring TM4 fuses with the M. avium vacuole
in macrophages. These results suggest a potentially novel concept to kill
intracellular pathogenic bacteria and warrant future development. OBJECTIVES: The antimycobacterial activities of RS-112997, RS-124922 and
RS-118641, three capuramycin analogues that inhibit
phospho-N-acetylmuramyl-pentapeptide translocase, were tested against clinical
isolates of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium
intracellulare.
METHODS AND RESULTS: MICs were determined by the broth microdilution method
using a modified Middlebrook 7H9 broth. RS-118641 was the most potent compound
overall. The MIC50/90 (mg/L) results for RS-118641 were: M. tuberculosis, 1/2;
multidrug-resistant (MDR) M. tuberculosis, 0.5/2; M. avium, 4/8; and M.
intracellulare, 0.06/0.5. No statistically significant differences in MIC
distributions were observed between non-MDR and MDR M. tuberculosis for any of
the capuramycin analogues tested. In order to evaluate the therapeutic efficacy
of RS-112997 and RS-124922 in a murine lung model of tuberculosis, both
compounds were administered intranasally at 0.1 or 1 mg/mouse/day for 12 days.
The mycobacterial load in the lungs was significantly lower in all treatment
groups than in the untreated controls. Additional experiments were performed to
evaluate the therapeutic efficacy of the three compounds against the M.
intracellulare infection in mice. All compounds were administered intranasally
at 0.1 mg/mouse/day for 21 days. The mycobacterial load in the lungs was
significantly lower in all treatment groups than in the untreated controls.
CONCLUSIONS: These results suggest that capuramycin analogues exhibit strong
antimycobacterial potential and should be considered for further evaluation in
the treatment of M. tuberculosis and M. avium-M. intracellulare complex
infections in humans. The use of highly active antiretroviral therapy (HAART) for the treatment of HIV
infection has been associated with a marked reduction in the incidence of most
opportunistic infections. From April 2001 to February 2002, 80 blood samples
from patients who were suspected to have disseminated mycobacterial infection,
presenting fever and (preferably) a CD4 T cell count < 100.0 cell/mL were
investigated. Twelve (15%) of the 80 blood cultures were positive for
mycobacteria, with Mycobacterium avium being identified in 7 (8.8%) samples and
M. tuberculosis in 5 (6.2%). The TCD4+ count at the time of M. avium bacteremia
ranged from 7 cells/microL (average of 48.5 cell/microL), while in M.
tuberculosis bacteremia it ranged from 50.0 cells/microL (average of 80.0
cell/microL). The prevalence of M. avium bacteremia in our study follows the
expected decline in opportunistic infections observed after the introduction of
HAART; however, mycobacteremia by M. tuberculosis still indicates a high
prevalence of tuberculosis infection in AIDS patients. Pulmonary tuberculosis (TB) has again become a global problem: it infects 2.2
billion people world-wide, caused the deaths of over 3 million last year and
will produce over 8 million new cases of TB this coming year. Although effective
therapy is widely available for antibiotic susceptible strains of Mycobacterium
tuberculosis, current drugs are relatively useless against multi-drug resistant
infections (MDRTB). Mortality is almost complete within two years regardless of
therapy, and in the case of co-infection with HIV/AIDS, mortality is 100% within
a few months of diagnosis especially the M. tuberculosis strain in XDRTB. As of
the time of this writing no new effective anti-TB drugs have been made available
by the pharmaceutical industry and XDRTB. Because TB is an intracellular
infection of the non-killing macrophage of the lung, any agent that is to prove
effective must have activity against MDRTB and XDRTB strains that have been
phagocytosed by the human macrophage. This review intents to provide cogent in
vitro, ex vivo and in vivo evidence that supports the use of a variety of
commonly available phenothiazines for the therapy of MDRTB and XDRTB, especially
when the prognosis of the infection is poor and the use of the recommend agents
can take place along lines of "compassionate therapy". In addition, we will
describe the macrophage assay as indispensable when an agent is to be further
studied for its effectiveness as an anti-TB drug. In vitro studies if not
complemented by ex vivo studies will for the most part be dead-ended since few
agents that have activity in vitro have any activity against phagocytosed M.
tuberculosis. AIMS: Polymerase chain reaction (PCR) is the most rapid and sensitive method for
diagnosing mycobacterial infections and identifying the aetiological
Mycobacterial species in order to administer the appropriate therapy and for
better patient management.
METHODS AND RESULTS: Two hundred and thirty-five samples from 145 clinically
suspected cases of tuberculosis were processed for the detection of
Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L-J & BACTEC
MGIT-960 culture and multiplex PCR tests. The multiplex PCR comprised of
genus-specific primers targeting hsp65 gene, Mycobacterium tuberculosis
complex-specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium
avium complex-specific primer pairs targeting 16S-23S Internal Transcribed
Spacer sequences. The multiplex PCR developed had an analytical sensitivity of
10 fg (3-4 cells) of mycobacterial DNA. The multiplex PCR test showed the
highest (77.24%) detection rate, while ZN smear examination had the lowest (20%)
detection rate, which was bettered by L-J culture (34.4%) and BACTEC MGIT-960
culture (50.34%) methods. The mean isolation time for M. tuberculosis was 19.03
days in L-J culture and 8.7 days in BACTEC MGIT-960 culture. Using the multiplex
PCR, we could establish M. tuberculosis + M. avium co-infection in 1.3%
HIV-negative and 2.9% HIV-positive patients. The multiplex PCR was also highly
useful in diagnosing mycobacteraemia in 38.09% HIV-positive and 15.38%
HIV-negative cases.
CONCLUSIONS: The developed in-house multiplex PCR could identify and
differentiate the M. tuberculosis and M. avium complexes from other
Mycobacterial species directly from clinical specimens.
SIGNIFICANCE AND IMPACT OF THE STUDY: The triplex PCR developed by us could be
used to detect and differentiate M. tuberculosis, M. avium and other
mycobacteria in a single reaction tube. Few studies have compared the clinical and radiographic findings of tuberculomas
to those of solitary pulmonary nodules (SPNs) caused by Mycobacterium avium
complex (MAC). We retrospectively analyzed clinical and radiographic findings
from 26 patients with tuberculomas and 15 patients with SPNs caused by MAC.
Median SPN size was 22 mm. In 26 patients (63%), the SPN was detected during a
routine health checkup or evaluation of organs other than lungs. Patients with
SPNs due to MAC were slightly older (median = 59 years) compared with those with
tuberculomas (median = 50 years; P = 0.044). When we compared computed
tomography (CT) features between patients with tuberculomas and patients with
MAC, no significant differences were found in SPN location or the presence of
calcification, cavitation, central low attenuation, and the satellite lesions.
Although the maximum standardized uptake values were slightly higher in patients
with SPNs due to MAC (median = 8.5) compared with those with tuberculomas
(median = 2.2), this difference was not significant (P = 0.053). Of the 15
patients with SPNs due to MAC, 10 were initially diagnosed with "tuberculoma"
and administered antituberculosis medication. MAC pulmonary disease should be
considered in the differential diagnosis of SPNs, even when encountered in
geographic regions with a high prevalence of pulmonary tuberculosis. INTRODUCTION: The purpose of this study was to separate MAC lung disease from
colonization and to define indications for treatment.
MATERIALS AND METHODOLOGY: Over 4 years, we evaluated patients who had positive
MAC cultures, MAC infection and coinfection with MTB. In the first study, 42
immunocompetent patients with sputum or BAL culture positive only for MAC during
a single year (2004) were reviewed. On clinical and radiographic review, they
were classified as disease related to MAC, likely related to MAC or unrelated to
MAC. In the second study, we reviewed all immunocompetent patients, during two
years (2004-2005), whose respiratory secretions cultured both MTB and
nontuberculous mycobacteria (NTM). In the last study, we evaluated pulmonary
function (PF) in patients with MAC infection before and after therapy (2006-
2007). PF was evaluated in patients following ATS guidelines.
RESULTS: Lung disease was related/likely related to MAC in 21 patients (50%) and
not related in 21 (50%). In patients with MAC-related lung disease, the primary
physician did not consider the diagnosis except when that physician was a
pulmonologist. Half of those with MAC-related lung disease were smokers, white
and US-born. There were 12 immunocompetent patients with MTB and NTM cultures.
Eleven were non-white and all were foreign-born. Presentation and clinical
course were consistent with MTB. All 8 patients with abnormal PF improved.
CONCLUSIONS: The prevalence of MAC lung infection in two inner city hospitals
was four times higher than that of TB. The indication for treatment of MAC
infection should also rely heavily on clinical and radiological evidence when
there is only one positive sputum culture. The diagnosis was considered only
when the admitting physician was a pulmonologist. Most patients with combined
infection were clinically consistent with MTB and responded to anti MTB
treatment alone. Treatment with anti-MAC therapy improved PF in those patients
whose PF was abnormal to begin with. |
What molecule is targeted by suvorexant? | Suvorexant is a dual orexin receptor antagonist for the treatment of sleep onset and sleep maintenance 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. Insomnia is a highly prevalent disorder that can occur in conjunction with other
medical or psychiatric conditions or can occur in the absence of a coexisting
disorder. Regardless, treatment of insomnia is beneficial to the patient and may
benefit comorbidities if they exist. Nonpharmacologic modalities such as sleep
hygiene and stimulus controls are important mainstays of insomnia therapy, but
may not be sufficient to treat the disorder. Dual orexin receptor antagonists
(DORAs) are a new class of insomnia medication that target wakefulness-promoting
neuropeptides to regulate the sleep-wake cycle. Suvorexant is the first DORA to
be approved and has demonstrated efficacy at decreasing both time to sleep onset
and increasing total sleep time compared with placebo. Suvorexant has a novel
mechanism of action and may represent an alternative for patients who cannot
tolerate or do not receive benefit from traditional sleep agents. Suvorexant is
generally effective and well tolerated, but has not been compared head to head
with traditional sleep agents and being only newly available, lacks a
longer-term 'real-world' experience base. |
For which type of diabetes can empagliflozin be used? | The oral antidiabetes agent, empagliflozin, can be used as monotherapy or alongside other glucose-lowering treatments, including insulin, to treat T2DM. | AIM: This Phase IIb, randomized, double-blind, placebo-controlled trial
evaluated the efficacy, safety, tolerability and pharmacokinetics of
empagliflozin in patients with type 2 diabetes.
METHODS: Four hundred and eight patients (treatment-naïve or after a 4-week
wash-out period) were randomized to receive empagliflozin 5, 10 or 25 mg once
daily, placebo or open-label metformin for 12 weeks. The primary endpoint was
change in haemoglobin A1c (HbA1c) after 12 weeks.
RESULTS: After 12 weeks' treatment, empagliflozin showed dose-dependent
reductions in HbA1c from baseline [5 mg: -0.4%, 10 mg: -0.5%, 25 mg: -0.6%; all
doses p < 0.0001 vs. placebo (+0.09%)]. Fasting plasma glucose (FPG) decreased
with empagliflozin [5 mg: -1.29 mmol/l, 10 mg: -1.61 mmol/l, 25 mg:
-1.72 mmol/l; all doses p < 0.0001 vs. placebo (+0.04 mmol/l)]. Body weight
decreased in all empagliflozin groups (all doses p < 0.001 vs. placebo). The
incidence of adverse events (AEs) was similar in the placebo (32.9%) and
empagliflozin (29.1%) groups. The most frequently reported AEs on empagliflozin
were pollakiuria (3.3% vs. 0% for placebo), thirst (3.3% vs. 0% for placebo) and
nasopharyngitis (2.0% vs. 1.2% for placebo). AEs consistent with urinary tract
infections (UTIs) were reported in four (1.6%) patients on empagliflozin vs. one
(1.2%) on placebo. Genital infections were reported in five (2%) patients on
empagliflozin vs. 0% on placebo. No UTIs or genital infections led to premature
discontinuation.
CONCLUSIONS: In patients with type 2 diabetes, empagliflozin resulted in
dose-dependent, clinically meaningful reductions in HbA1c and FPG, and
reductions in body weight compared with placebo. Empagliflozin was
well-tolerated with a favourable safety profile. AIMS: To evaluate the effects of the sodium glucose cotransporter 2 (SGLT2)
inhibitor empagliflozin added to metformin for 12 weeks in patients with type 2
diabetes.
METHODS: This dose-ranging, double-blind, placebo-controlled trial randomized
495 participants with type 2 diabetes inadequately controlled on metformin
[haemoglobin A1c (HbA1c) >7 to ≤10%] to receive 1, 5, 10, 25, or 50 mg
empagliflozin once daily (QD), or placebo, or open-label sitagliptin (100 mg
QD), added to metformin for 12 weeks. The primary endpoint was change in HbA1c
from baseline to week 12 (empagliflozin groups versus placebo).
RESULTS: Reductions in HbA1c of -0.09 to -0.56% were observed with empagliflozin
after 12 weeks, versus an increase of 0.15% with placebo (baseline: 7.8-8.1%).
Compared with placebo, empagliflozin doses from 5 to 50 mg resulted in
reductions in fasting plasma glucose (-2 to -28 mg/dl vs. 5 mg/dl with placebo;
p < 0.0001) and body weight (-2.3 to -2.9 kg vs. -1.2 kg; p < 0.01). Frequency
of adverse events was generally similar with empagliflozin (29.6-48.6%), placebo
(36.6%) and sitagliptin (35.2%). Hypoglycaemia rates were very low and balanced
among groups. Most frequent adverse events with empagliflozin were urinary tract
infections (4.0% vs. 2.8% with placebo) and pollakiuria (2.5% vs. 1.4% with
placebo). Genital infections were reported only with empagliflozin (4.0%).
CONCLUSIONS: Once daily empagliflozin as add-on therapy to metformin was well
tolerated except for increased genital infections and resulted in reductions in
HbA1c, fasting plasma glucose and body weight in patients with type 2 diabetes
inadequately controlled on metformin monotherapy. Data from five randomized, placebo-controlled, multiple oral dose studies of
empagliflozin in patients with type 2 diabetes mellitus (T2DM; N = 974; 1-100 mg
q.d.; ≤12 weeks) were used to develop a population pharmacokinetic (PK) model
for empagliflozin. The model consisted of two-compartmental disposition, lagged
first-order absorption and first-order elimination, and incorporated appropriate
covariates. Population estimates (interindividual variance, CV%) of oral
apparent clearance, central and peripheral volumes of distribution, and
inter-compartmental clearance were 9.87 L/h (26.9%), 3.02 L, 60.4 L (30.8%), and
5.16 L/h, respectively. An imposed allometric weight effect was the most
influential PK covariate effect, with a maximum effect on exposure of ±30%,
using 2.5th and 97.5th percentiles of observed weights, relative to the median
observed weight. Sex and race did not lend additional description to PK
variability beyond allometric weight effects, other than ∼25% greater oral
absorption rate constant for Asian patients. Age, total protein, and
smoking/alcohol history did not affect PK parameters. Predictive check plots
were consistent with observed data, implying an adequate description of
empagliflozin PKs following multiple dosing in patients with T2DM. The lack of
marked covariate effects, including weight, suggests that no exposure-based dose
adjustments were required within the study population and dose range. BACKGROUND: Sulfonylureas (SUs) are commonly used in the treatment of type 2
diabetes (T2DM), usually as second-line treatment after the failure of
metformin. However, SUs are associated with poor durability, hypoglycemia and
weight gain. Empagliflozin is a sodium glucose cotransporter 2 (SGLT2) inhibitor
in development for the treatment of T2DM. In Phase II/III trials, empagliflozin
reduced hyperglycemia, body weight and blood pressure, with a low incidence of
hypoglycemia. The aim of this Phase III study is to compare the effects of
empagliflozin and the SU glimepiride as second-line therapy in patients with
T2DM inadequately controlled with metformin immediate release (IR) and
diet/exercise.
METHOD: After a 2-week placebo run-in, patients were randomized to receive
empagliflozin 25 mg once daily (qd) or glimepiride 1-4 mg qd double-blind for 2
years, in addition to metformin IR. Patients who participate in the initial
2-year randomization period will be eligible for a 2-year double-blind
extension. The primary endpoint is change from baseline in HbA1c. Secondary
endpoints are change from baseline in body weight, the incidence of confirmed
hypoglycemia and changes in systolic and diastolic blood pressure. Exploratory
endpoints include markers of insulin secretion, body composition and responder
analyses. Safety endpoints include the incidence of adverse events (AEs)
(including macro- and microvascular adverse events) and changes from baseline in
clinical laboratory parameters.
RESULTS: Between August 2010 and June 2011, 1549 patients were randomized and
1545 patients were treated. At baseline, mean (SD) age was 55.9 (10.4) years,
HbA1c was 7.92 (0.84)%, body mass index was 30.11 (5.59) kg/m², systolic blood
pressure was 133.5 (15.9) mmHg and diastolic blood pressure was 79.5 (9.4) mmHg.
DISCUSSION: This is the largest study to compare the efficacy and safety of an
SGLT2 inhibitor with an SU in patients with T2DM inadequately controlled on
metformin to date. In addition to determining the effects of these treatments on
glycemic control over the long term, this study will investigate effects on
beta-cell function, cardiovascular risk factors and markers of renal
function/damage. The results will help to inform the choice of second-line
treatment in patients with T2DM who have failed on metformin.
TRIAL REGISTRATION: Clinicaltrials.gov NCT01167881. OBJECTIVE: To investigate the long-term safety and efficacy of empagliflozin, a
sodium glucose cotransporter 2 inhibitor; sitagliptin; and metformin in patients
with type 2 diabetes.
RESEARCH DESIGN AND METHODS: In this randomized, open-label, 78-week extension
study of two 12-week, blinded, dose-finding studies of empagliflozin
(monotherapy and add-on to metformin) with open-label comparators, 272 patients
received 10 mg empagliflozin (166 as add-on to metformin), 275 received 25 mg
empagliflozin (166 as add-on to metformin), 56 patients received metformin, and
56 patients received sitagliptin as add-on to metformin.
RESULTS: Changes from baseline in HbA1c at week 90 were -0.34 to -0.63% (-3.7 to
-6.9 mmol/mol) with empagliflozin, -0.56% (-6.1 mmol/mol) with metformin, and
-0.40% (-4.4 mmol/mol) with sitagliptin. Changes from baseline in weight at week
90 were -2.2 to -4.0 kg with empagliflozin, -1.3 kg with metformin, and -0.4 kg
with sitagliptin. Adverse events (AEs) were reported in 63.2-74.1% of patients
on empagliflozin and 69.6% on metformin or sitagliptin; most AEs were mild or
moderate in intensity. Hypoglycemic events were rare in all treatment groups,
and none required assistance. AEs consistent with genital infections were
reported in 3.0-5.5% of patients on empagliflozin, 1.8% on metformin, and none
on sitagliptin. AEs consistent with urinary tract infections were reported in
3.8-12.7% of patients on empagliflozin, 3.6% on metformin, and 12.5% on
sitagliptin.
CONCLUSIONS: Long-term empagliflozin treatment provided sustained glycemic and
weight control and was well tolerated with a low risk of hypoglycemia in
patients with type 2 diabetes. BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors lower glycemia by
enhancing urinary glucose excretion. The physiologic response to
pharmacologically induced acute or chronic glycosuria has not been investigated
in human diabetes.
METHODS: We evaluated 66 patients with type 2 diabetes (62 ± 7 years, BMI = 31.6
± 4.6 kg/m(2), HbA1c = 55 ± 8 mmol/mol, mean ± SD) at baseline, after a single
dose, and following 4-week treatment with empagliflozin (25 mg). At each time
point, patients received a mixed meal coupled with dual-tracer glucose
administration and indirect calorimetry.
RESULTS: Both single-dose and chronic empagliflozin treatment caused glycosuria
during fasting (median, 7.8 [interquartile range {IQR}, 4.4] g/3 hours and 9.2
[IQR, 5.2] g/3 hours) and after meal ingestion (median, 29.0 [IQR, 12.5] g/5
hours and 28.2 [IQR, 15.4] g/5 hours). After 3 hours of fasting, endogenous
glucose production (EGP) was increased 25%, while glycemia was 0.9 ± 0.7 mmol/l
lower (P < 0.0001 vs. baseline). After meal ingestion, glucose and insulin AUC
decreased, whereas the glucagon response increased (all P < 0.001). While oral
glucose appearance was unchanged, EGP was increased (median, 40 [IQR, 14] g and
37 [IQR, 11] g vs. 34 [IQR, 11] g, both P < 0.01). Tissue glucose disposal was
reduced (median, 75 [IQR, 16] g and 70 [IQR, 21] g vs. 93 [IQR, 18] g, P <
0.0001), due to a decrease in both glucose oxidation and nonoxidative glucose
disposal, with a concomitant rise in lipid oxidation after chronic
administration (all P < 0.01). β Cell glucose sensitivity increased (median, 55
[IQR, 35] pmol • min(-1) • m(-2) • mM(-1) and 55 [IQR, 39] pmol • min(-1) •
m(-2) • mM(-1) vs. 44 [IQR, 32] pmol • min(-1) • m(-2) • mM(-1), P < 0.0001),
and insulin sensitivity was improved. Resting energy expenditure rates and those
after meal ingestion were unchanged.
CONCLUSIONS: In patients with type 2 diabetes, empagliflozin-induced glycosuria
improved β cell function and insulin sensitivity, despite the fall in insulin
secretion and tissue glucose disposal and the rise in EGP after one dose,
thereby lowering fasting and postprandial glycemia. Chronic dosing shifted
substrate utilization from carbohydrate to lipid. Trial registration.
ClinicalTrials.Gov NCT01248364 (EudraCT no. 2010-018708-99). Funding. This study
was funded by Boehringer Ingelheim. BACKGROUND: Diabetes is a leading cause of chronic kidney disease (CKD)
worldwide. Optimum glycaemic control in patients with type 2 diabetes is
important to minimise the risk of microvascular and macrovascular complications
and to slow the progression of CKD. We assessed the efficacy and safety of
empagliflozin as an add-on treatment in patients with type 2 diabetes and CKD.
METHODS: We did a phase 3, randomised, double-blind, parallel-group,
placebo-controlled trial at 127 centres in 15 countries. Patients with HbA1c of
7% or greater to 10% or less were eligible for inclusion. Patients with stage 2
CKD (estimated glomerular filtration rate [eGFR] ≥60 to <90 mL/min per 1·73
m(2); n=290) were randomly assigned (1:1:1) to receive empagliflozin 10 mg or 25
mg or placebo once daily for 52 weeks. Patients with stage 3 CKD (eGFR ≥30 to
<60 mL/min per 1·73 m(2); n=374) were randomly assigned (1:1) to receive
empagliflozin 25 mg or placebo for 52 weeks. Randomisation was done with a
computer-generated random sequence and stratified by renal impairment, HbA1c,
and background antidiabetes medication. Treatment assignment was masked from
patients and investigators. The primary endpoint was change from baseline in
HbA1c at week 24 by ANCOVA in the full analysis set. This study is registered
with ClinicalTrials.gov, number NCT01164501.
FINDINGS: In patients with stage 2 CKD, adjusted mean treatment differences
versus placebo in changes from baseline in HbA1c at week 24 were -0·52% (95% CI
-0·72 to -0·32) for empagliflozin 10 mg and -0·68% (-0·88 to -0·49) for
empagliflozin 25 mg (both p<0·0001). In patients with stage 3 CKD, adjusted mean
treatment difference versus placebo in change from baseline in HbA1c at week 24
was -0·42% (-0·56 to -0·28) for empagliflozin 25 mg (p<0·0001). In patients with
stage 2 CKD, adverse events were reported over 52 weeks by 83 patients (87%) on
placebo (15 severe [16%] and 11 serious [12%]), 86 (88%) on empagliflozin 10 mg
(six severe [6%] and six serious [6%]) and 78 (80%) on empagliflozin 25 mg
(eight severe [8%] and seven serious [7%]). In patients with stage 3 CKD,
adverse events were reported over 52 weeks by 156 patients (83%) on placebo (15
severe [8%] and 23 serious [12%]) and 156 (83%) on empagliflozin 25 mg (18
severe [10%] and 22 serious [12%]).
INTERPRETATION: In patients with type 2 diabetes and stage 2 or 3 CKD,
empagliflozin reduced HbA1c and was well tolerated. However, our findings might
not be applicable to the general population of patients with type 2 diabetes and
renal impairment.
FUNDING: Boehringer Ingelheim, Eli Lilly. INTRODUCTION: To evaluate the pharmacodynamics, pharmacokinetics, safety and
tolerability of empagliflozin in Japanese patients with type 2 diabetes
mellitus.
MATERIALS AND METHODS: In this 4-week, multiple dose, randomized,
parallel-group, double-blind, placebo-controlled trial, patients (n = 100) were
randomized to receive 1, 5, 10 or 25 mg of empagliflozin, or placebo once daily.
Key end-points were urinary glucose excretion (UGE), fasting plasma glucose
(FPG) and eight-point glucose profile.
RESULTS: Data are presented for 1, 5, 10, 25 mg of empagliflozin and placebo
groups, respectively. Adjusted mean changes from baseline to day 27 in UGE were
40.8, 77.1, 80.9, 93.0 and -2.1 g (P < 0.0001 for all empagliflozin groups vs
placebo). Adjusted mean changes from baseline to day 28 in FPG were -1.56,
-1.96, -2.31, -2.37 and -0.86 mmol/L (P < 0.01 for all empagliflozin groups vs
placebo). Adjusted mean changes from baseline to day 27 in eight-point glucose
profile were -1.96, -2.21, -2.42, -2.54 and -0.97 mmol/L (P < 0.01 for all
empagliflozin groups vs placebo). Empagliflozin reached peak plasma
concentration 1.5-2 h after dosing. Mean steady state terminal elimination
half-lives ranged from 13.2 to 18.0 h. Of 100 patients, 25 experienced an
adverse event, occurring more frequently for empagliflozin (29.1%) than placebo
(9.5%); frequency was not dose related.
CONCLUSIONS: In Japanese patients with type 2 diabetes mellitus, empagliflozin
at doses up to 25 mg once daily for 4 weeks was well tolerated and resulted in
significant improvements in glycemic control compared with placebo. This trial
was registered with ClinicalTrials.gov (no. NCT00885118). BACKGROUND: Evidence concerning the importance of glucose lowering in the
prevention of cardiovascular (CV) outcomes remains controversial. Given the
multi-faceted pathogenesis of atherosclerosis in diabetes, it is likely that any
intervention to mitigate this risk must address CV risk factors beyond glycemia
alone. The SGLT-2 inhibitor empagliflozin improves glucose control, body weight
and blood pressure when used as monotherapy or add-on to other antihyperglycemic
agents in patients with type 2 diabetes. The aim of the ongoing EMPA-REG
OUTCOME™ trial is to determine the long-term CV safety of empagliflozin, as well
as investigating potential benefits on macro-/microvascular outcomes.
METHODS: Patients who were drug-naïve (HbA1c ≥7.0% and ≤9.0%), or on background
glucose-lowering therapy (HbA1c ≥7.0% and ≤10.0%), and were at high risk of CV
events, were randomized (1:1:1) and treated with empagliflozin 10 mg,
empagliflozin 25 mg, or placebo (double blind, double dummy) superimposed upon
the standard of care. The primary outcome is time to first occurrence of CV
death, non-fatal myocardial infarction, or non-fatal stroke. CV events will be
prospectively adjudicated by an independent Clinical Events Committee. The trial
will continue until ≥691 confirmed primary outcome events have occurred,
providing a power of 90% to yield an upper limit of the adjusted 95% CI for a
hazard ratio of <1.3 with a one-sided α of 0.025, assuming equal risks between
placebo and empagliflozin (both doses pooled). Hierarchical testing for
superiority will follow for the primary outcome and key secondary outcomes (time
to first occurrence of CV death, non-fatal myocardial infarction, non-fatal
stroke or hospitalization for unstable angina pectoris) where non-inferiority is
achieved.
RESULTS: Between Sept 2010 and April 2013, 592 clinical sites randomized and
treated 7034 patients (41% from Europe, 20% from North America, and 19% from
Asia). At baseline, the mean age was 63 ± 9 years, BMI 30.6 ± 5.3 kg/m2, HbA1c
8.1 ± 0.8%, and eGFR 74 ± 21 ml/min/1.73 m2. The study is expected to report in
2015.
DISCUSSION: EMPA-REG OUTCOME™ will determine the CV safety of empagliflozin in a
cohort of patients with type 2 diabetes and high CV risk, with the potential to
show cardioprotection.
TRIAL REGISTRATION: Clinicaltrials.gov NCT01131676. Diabetic nephropathy is the leading cause of end-stage renal disease in humans
in the Western world. The recent development of Na+-glucose cotransporter 2
(SGLT2) inhibitors offers a new antidiabetic therapy via enhanced glucose
excretion. Whether this strategy exerts beneficial effects on the development of
type 2 diabetic nephropathy is still largely unclear. We investigated the
effects of the specific SGLT2 inhibitor empagliflozin in BTBR.Cg-Lep<ob>/WiscJ
(BTBR ob/ob) mice, which spontaneously develop type 2 diabetic nephropathy. In
the first experiment, BTBR ob/ob mice received either a diet containing 300 ppm
empagliflozin or equicaloric placebo chow for 12 wk. In the second experiment,
BTBR ob/ob mice received 1 μg·kg body wt(-1)·day(-1) ANG II to induce arterial
hypertension and were separated into the same two diet groups for 6 wk. In both
experiments, empagliflozin treatment enhanced glucosuria, thereby lowering blood
glucose. Independently of hypertension, empagliflozin reduced albuminuria in
diabetic mice. However, empagliflozin treatment affected diabetes-related
glomerular hypertrophy, markers of renal inflammation, and mesangial matrix
expansion only in BTBR ob/ob mice without hypertension. In summary,
empagliflozin demonstrated significant antihyperglycemic effects, differentially
ameliorating early features of diabetic nephropathy in BTBR ob/ob mice with and
without hypertension. BACKGROUND: Metformin is the recommended first-line pharmacotherapy for patients
with type 2 diabetes. There is no consensus on the optimum second-line
pharmacotherapy. We compared the efficacy and safety of the sodium glucose
cotransporter 2 inhibitor empagliflozin and the sulfonylurea glimepiride as
add-on to metformin in patients with type 2 diabetes.
METHODS: In this double-blind phase 3 trial, patients (aged ≥18 years) with type
2 diabetes and HbA1c concentrations of 7-10%, despite metformin treatment and
diet and exercise counselling, were randomly assigned in a 1:1 ratio with a
computer-generated random sequence, stratified by HbA1c, estimated glomerular
filtration rate (eGFR), and region, to empagliflozin (25 mg once daily, orally)
or glimepiride (1-4 mg once daily, orally) as add-on to metformin for 104 weeks.
Patients and investigators were masked to treatment assignment. The primary
endpoint was change from baseline in HbA1c levels at weeks 52 and 104.
Differences in the primary endpoint were first tested for non-inferiority (based
on a margin of 0·3%). If non-inferiority was shown, differences in the primary
endpoint at week 104 were then tested for superiority. Analysis was done on the
full-analysis set-ie, patients who were treated with at least one dose of study
drug and had a baseline HbA1c value. This study is registered with
ClinicalTrials.gov, number NCT01167881. A 104-week extension is ongoing.
FINDINGS: Between August, 2010, and June, 2011, 1549 patients were randomly
assigned to receive empagliflozin (n=769) or glimepiride (n=780); four patients
in the empagliflozin group did not receive the assigned treatment. Empagliflozin
was non-inferior to glimepiride at both timepoints. At week 104, adjusted mean
difference in change from baseline in HbA1c with empagliflozin versus
glimepiride was -0·11% (95% CI -0·19 to -0·02; p=0·0153 for superiority).
Adverse events were reported in 661 (86%) patients treated with empagliflozin
and 673 (86%) patients treated with glimepiride. Severe adverse events were
reported in 72 (9%) patients in the empagliflozin group and 68 (9%) in the
glimepiride group. Serious adverse events were reported in 119 (16%) patients in
the empagliflozin group and 89 (11%) in the glimepiride group. Confirmed
hypoglycaemic adverse events (plasma glucose ≤3·9 mmol/L or requiring
assistance) at week 104 were reported in 19 (2%) patients treated with
empagliflozin and 189 (24%) patients treated with glimepiride.
INTERPRETATION: Empagliflozin might be an effective and a well tolerated
second-line treatment option for patients with type 2 diabetes who have not
achieved good glycaemic control on metformin.
FUNDING: Boehringer Ingelheim and Eli Lilly. AIMS: To provide model-based clinical development decision support including
dose selection guidance for empagliflozin, an orally administered sodium glucose
cotransporter 2 inhibitor, through developed exposure-response (E-R) models for
efficacy and tolerability in patients with type 2 diabetes mellitus (T2DM).
METHODS: Five randomized, placebo-controlled, multiple oral dose studies of
empagliflozin in patients with T2DM (n = 974; 1-100 mg once daily, duration ≤12
weeks) were used to develop E-R models for efficacy (glycosylated haemoglobin
[HbA1c ], fasting plasma glucose [FPG] and urinary glucose excretion). Two
studies (n = 748, 12 weeks) were used to evaluate tolerability E-R.
RESULTS: The efficacy model predicted maximal decreases in FPG and HbA1c of 16%
and 0.6%, respectively, assuming a baseline FPG concentration of 8 mm
(144 mg dl(-1) ) and 10-25 mg every day empagliflozin targeted 80-90% of these
maximums. Increases in exposure had no effect on incidence rates of
hypoglycaemia (n = 4), urinary tract infection (n = 17) or
genital/vulvovaginal-related (n = 16) events, although low prevalence rates may
have precluded more accurate evaluation.
CONCLUSIONS: E-R analyses indicated that 10 and 25 mg once daily empagliflozin
doses achieved near maximal glucose lowering efficacy. Type 2 diabetes is increasing in prevalence worldwide, and hyperglycemia is
often poorly controlled despite a number of therapeutic options. Unlike
previously available agents, sodium-glucose co-transporter 2 (SGLT2) inhibitors
offer an insulin-independent mechanism for improving blood glucose levels, since
they promote urinary glucose excretion (UGE) by inhibiting glucose reabsorption
in the kidney. In addition to glucose control, SGLT2 inhibitors are associated
with weight loss and blood pressure reductions, and do not increase the risk of
hypoglycemia. Empagliflozin is a selective inhibitor of SGLT2, providing
dose-dependent UGE increases in healthy volunteers, with up to 90 g of glucose
excreted per day. It can be administered orally, and studies of people with
renal or hepatic impairment indicated empagliflozin needed no dose adjustment
based on pharmacokinetics. In Phase II trials in patients with type 2 diabetes,
empagliflozin provided improvements in glycosylated hemoglobin (HbA1c) and other
measures of glycemic control when given as monotherapy or add-on to metformin,
as well as reductions in weight and systolic blood pressure. As add-on to basal
insulin, empagliflozin not only improved HbA1c levels but also reduced insulin
doses. Across studies, empagliflozin was generally well tolerated with a similar
rate of hypoglycemia to placebo; however, patients had a slightly increased
frequency of genital infections, but not urinary tract infections, versus
placebo. Phase III studies have also reported a good safety profile along with
significant improvements in HbA1c, weight and blood pressure, with no increased
risk of hypoglycemia versus placebo. Based on available data, it appears that
empagliflozin may be a useful option in a range of patients; however, clinical
decisions will be better informed by the results of ongoing studies, in
particular, a large cardiovascular outcome study (EMPA-REG OUTCOME™). INTRODUCTION: Despite the availability of numerous anti-diabetes drugs and
treatment guidelines, many patients with type 2 diabetes mellitus (T2DM) do not
reach recommended targets for glycemic control. There remains an unmet need for
effective and well-tolerated anti-diabetes agents that can be used as
monotherapy or in combination with other therapies to improve glycemic control
in patients with T2DM. Sodium glucose cotransporter 2 (SGLT2) inhibitors are a
new class of treatment for T2DM that reduce hyperglycemia by reducing renal
glucose reabsorption and thereby increasing urinary glucose excretion.
AREAS COVERED: This paper reviews the pharmacokinetic and pharmacodynamic
properties of the SGLT2 inhibitor empagliflozin , the results of clinical trials
investigating the efficacy of empagliflozin given as monotherapy or as add-on
therapy on glycemic control, body weight, and blood pressure in patients with
T2DM, and the safety and tolerability profile of empagliflozin.
EXPERT OPINION: Empagliflozin offers good glycemic efficacy, weight loss, blood
pressure reduction, and a low risk of hypoglycemia. These attributes, coupled
with the ability to be used in virtually any combination with other
anti-diabetes agents and at any stage in the disease process, provide a welcome
new agent to our armamentarium of drugs to help manage T2DM. Empagliflozin,
(2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol
was recently approved by the FDA for the treatment of chronic type 2 diabetes
mellitus. Herein, we report the synthesis of carbon-13 and carbon-14 labeled
empagliflozin. Carbon-13 labeled empagliflozin was prepared in five steps and in
34% overall chemical yield starting from the commercially available
α-D-glucose-[(13)C6]. For the radiosynthesis, the carbon-14 atom was introduced
in three different positions of the molecule. In the first synthesis, Carbon-14
D-(+)-gluconic acid δ-lactone was used to prepare specifically labeled
empagliflozin in carbon-1 of the sugar moiety in four steps and in 19% overall
radiochemical yield. Carbon-14 labeled empagliflozin with the radioactive atom
in the benzylic position was obtained in eight steps and in 7% overall
radiochemical yield. In the last synthesis carbon-14 uniformly labeled phenol
was used to give [(14)C]empagliflozin in eight steps and in 18% overall
radiochemical yield. In all these radiosyntheses, the specific activities of the
final compounds were higher than 53 mCi/mmol, and the radiochemical purities
were above 98.5%. Although several treatment options are available to reduce hyperglycemia, only
about half of individuals with diagnosed diabetes mellitus (DM) achieve
recommended glycemic targets. New agents that reduce blood glucose
concentrations by novel mechanisms and have acceptable safety profiles are
needed to improve glycemic control and reduce the complications associated with
type 2 diabetes mellitus (T2DM). The renal sodium-glucose co-transporter 2
(SGLT2) is responsible for reabsorption of most of the glucose filtered by the
kidney. Inhibitors of SGLT2 lower blood glucose independent of the secretion and
action of insulin by inhibiting renal reabsorption of glucose, thereby promoting
the increased urinary excretion of excess glucose. Canagliflozin, dapagliflozin,
and empagliflozin are SGLT2 inhibitors approved as treatments for T2DM in the
United States, Europe, and other countries. Canagliflozin, dapagliflozin, and
empagliflozin increase renal excretion of glucose and improve glycemic
parameters in patients with T2DM when used as monotherapy or in combination with
other antihyperglycemic agents. Treatment with SGLT2 inhibitors is associated
with weight reduction, lowered blood pressure, and a low intrinsic propensity to
cause hypoglycemia. Overall, canagliflozin, dapagliflozin, and empagliflozin are
well tolerated. Cases of genital infections and, in some studies, urinary tract
infections have been more frequent in canagliflozin-, dapagliflozin-, and
empagliflozin-treated patients compared with those receiving placebo. Evidence
from clinical trials suggests that SGLT2 inhibitors are a promising new
treatment option for T2DM. The combination of metformin and a sulfonylurea is commonly used in type 2
diabetes mellitus. Many patients on this combination therapy do not achieve or
maintain glycemic targets and require the addition of a third antihyperglycemic
agent. Among the options are the sodium glucose cotransporter 2 (SGLT2)
inhibitors, a recently developed class of medications that effectively improve
glycemic control and are associated with reduction in body weight and blood
pressure. This article evaluates a 24-week, randomized, placebo-controlled study
of the SGLT2 inhibitor empagliflozin, added to metformin plus sulfonylurea
regimens. Empagliflozin led to significant reductions in glycated hemoglobin and
fasting plasma glucose, as well as body weight and systolic blood pressure.
Adverse events typically recorded with SGLT2 inhibitors were observed; notably,
genital infections occurred in more patients on empagliflozin than placebo.
Overall, empagliflozin was well tolerated. These results indicate that SGLT2
inhibitors can be successfully added to metformin plus sulfonylurea regimens.
SGLT2 inhibitors are not the only therapeutic option in this clinical situation;
however, based on the secondary effects observed in this and other studies, they
appear to be of particular value for patients who are obese or overweight. The treatment of patients with type 2 diabetes is typically accompanied by
hypoglycemia, if insulin or derivatives of sulfonylurea are used within the
treatment. Apart from the fact that hypoglycemias are the major obstacle to
achieving the desirable compensation of diabetes, hypoglycemia also has a number
of serious clinical consequences. A long term serious hypoglycemia may lead to a
sudden death, heart attack or irreversible brain damage. Clinically significant
are also the light or asymptomatic hypoglycemias which in a considerably
negative way affect the patient's quality of life. The use of modern
technologies in continuous monitoring of glycemias has shown that the occurrence
of asymptomatic hypoglycemias is much higher than we anticipated and that they
largely involve nocturnal hypoglycemia. Hypoglycemia is associated with an
increased level of depression, anxiety, dissatisfaction with the treatment and
with a greater number of physician office visits. Nocturnal hypoglycemia has a
negative impact on the quality of sleep, it may impair cognitive functions and
performance efficiency next day. The prevention of hypoglycemia is therefore one
of the basic goals of diabetes treatment and the low risk of hypoglycemia is
among the main requirements that we place on the newly developed antidiabetic
drugs. The negligible risk of hypoglycemia, which is comparable to placebo both
in monotherapy and in most combinations with the antidiabetic drugs available
today, is evidenced by the data from the studies undertaken with empagliflozin.
It shows that the low risk of hypoglycemia is one of the benefits of gliflozins,
the new group of medications with a unique mechanism of effect which has quite
recently appeared on our market. OBJECTIVE: To review available studies of empagliflozin, a sodium glucose
co-transporter-2 (SGLT2) inhibitor approved in 2014 by the European Commission
and the United States Food and Drug Administration for the treatment of type 2
diabetes mellitus (T2DM).
DATA SOURCES: PubMed was searched using the search terms empagliflozin, BI
10773, and BI10773, for entries between January 1, 2000, and December 1, 2014.
Reference lists from retrieved articles were searched manually for additional
peer-reviewed publications.
STUDY SELECTION AND DATA EXTRACTION: All publications reporting clinical trials
of empagliflozin were eligible for inclusion.
DATA SYNTHESIS: Empagliflozin is a new once-daily oral SGLT2 inhibitor with a
mechanism of action that is independent of β-cell function and the insulin
pathway. Data from a comprehensive phase III clinical trial program have
demonstrated its efficacy as monotherapy, as add-on to other glucose-lowering
agents, and in different patient populations. In these studies, empagliflozin
resulted in improvements in blood glucose levels as well as reductions in body
weight and blood pressure. Empagliflozin was well tolerated and was not
associated with an increased risk of hypoglycemia versus placebo.
CONCLUSION: The oral antidiabetes agent, empagliflozin, can be used as
monotherapy or alongside other glucose-lowering treatments, including insulin,
to treat T2DM. BACKGROUND: Treatment of type 2 diabetes mellitus invariably requires the use of
multiple daily medications which can impact negatively on patient adherence. As
a result, there is growing interest in the use of single-pill combinations that
can reduce the pill burden. Many such formulations incorporate metformin,
although this agent is not suitable for all patients. The single-pill
combination of the dipeptidyl peptidase-4 inhibitor linagliptin with the sodium
glucose co-transporter 2 inhibitor empagliflozin offers a new and attractive
option, given their complementary mechanisms of action.
SCOPE: Publications with titles containing the keywords 'linagliptin' or
'empagliflozin' were identified from a non-systematic search of PubMed without
date restrictions, together with abstracts presented at the annual meetings of
the American Diabetes Association and the European Association for the Study of
Diabetes 2012-2014. ClinicalTrials.gov was searched for entries containing these
two keywords. Additional references known to the author were included.
FINDINGS: The efficacy and safety of linagliptin and empagliflozin as
monotherapy or in combination with other oral antidiabetic drugs has been
established through extensive clinical trial programs. Studies specifically
evaluating the efficacy/safety of a dipeptidyl peptidase-4 inhibitor/sodium
glucose co-transporter 2 inhibitor in combination are limited, but do include
two studies of linagliptin/empagliflozin of up to 52 weeks in duration. These
studies show that the single-pill combination of linagliptin and empagliflozin
produced clinical improvements in glycemic control that were generally superior
to the improvements seen with linagliptin and empagliflozin alone, but with a
safety profile comparable to that of the individual constituents.
CONCLUSIONS: The single-pill combination of linagliptin and empagliflozin, with
their complementary mechanisms of action, is a promising treatment option for
patients with type 2 diabetes mellitus. It would reduce the daily pill burden in
this population, potentially improving adherence to, and optimizing the benefits
of, treatment of diabetes mellitus. BACKGROUND: The sodium-glucose cotransporter 2 (SGLT2) inhibitors, which include
canagliflozin, dapagliflozin, and empagliflozin, represent a new class of
antihyperglycemic agents. Few studies have assessed their cost per response,
with "cost per response" being the total cost of a select drug, divided by the
resulting change in glycated hemoglobin (HbA1c) levels.
OBJECTIVE: To examine the drug cost of SGLT2 inhibitors per a reduction in
placebo-adjusted 1% HbA1c in patients with type 2 diabetes mellitus who received
treatment during 26 weeks with canagliflozin, dapagliflozin, or empagliflozin.
METHODS: The drug cost per response for each of the 3 agents individually was
assessed based on data from a subset of clinical trials discussed in the
prescribing information for each drug that were all placebo-controlled studies
evaluating each drug as monotherapy, dual therapy (combined with metformin), and
triple therapy (combined with metformin and a sulfonylurea) in patients with
uncontrolled, type 2 diabetes mellitus. The US 2015 wholesale acquisition cost
for each drug was used to calculate each drug's treatment costs over 26 weeks.
The average cost per response for each drug was defined as the prescription drug
cost of each SGLT2 inhibitor, divided by the average, placebo-adjusted HbA1c
reduction at 26 weeks.
RESULTS: The drug cost per unit dose was the same for canagliflozin (100 mg or
300 mg), dapagliflozin (5 mg or 10 mg), and empagliflozin (10 mg or 25 mg), at
$11.43. The drug cost per placebo-adjusted 1% HbA1c reduction varied by agent
and by dose, as a result of the differences in the treatment responses for each
of the 3 drugs. The costs per response for canagliflozin 100 mg as monotherapy,
dual therapy, and triple therapy regimens ranged from $2286 to $3355, and for
canagliflozin 300 mg, from $1793 to $2702. The costs per response for
dapagliflozin 5 mg as monotherapy and dual therapy (triple therapy was not
available at the time of the study) ranged from $4161 to $5201; the cost for
dapagliflozin 10 mg ranged from $2972 to $4161. The costs per response for
empagliflozin 10 mg ranged from $2972 to $3467 across the monotherapy, dual
therapy, and triple therapy regimens; the cost for empagliflozin 25 mg ranged
from $2311 to $3467.
CONCLUSION: Simple analyses, such as the drug cost per placebo-adjusted 1%
reduction in HbA1c, may be useful when considering the addition of
antihyperglycemic agents to the health plan's formulary. |
Which are the main methods for pharmacophore modelling? | A pharmacophore describes the arrangement of molecular features a ligand must contain to efficaciously bind a receptor. Pharmacophore models are developed to improve molecular understanding of ligand–protein interactions, and can be used as a tool to identify novel compounds that fulfil the pharmacophore requirements and have a high probability of being biologically active. Protein structure-based pharmacophores (SBPs) derive these molecular features by conversion of protein properties to reciprocal ligand space. Unlike ligand-based pharmacophore models, which require templates of ligands in their bioactive conformation, SBPs do not depend on ligand information. | Generation of reliable pharmacophore models is a key strategy in drug design.
The quality of a pharmacophore model is known to depend on several factors, with
the quality of the conformer sets used perhaps being one of the most important.
The goal of this study was to compare different conformational analysis methods
to determine if one was superior to the others for pharmacophore generation
using Catalyst/HypoGen. The five methods selected were Catalyst/Fast,
Catalyst/Best, Omega, Chem-X and MacroModel. Data sets for which Catalysts
models had previously been published were selected using defined quality
measures. Hypotheses were generated for each of the data sets and the
performance of the different conformational analysis methods was compared using
both quantitative (cost and correlation coefficients) and qualitative measures
(by comparing the hypotheses in terms of the features present and their spatial
relationships). Two main conclusions emerged from the study. First, it was not
always possible to replicate the literature results. The reasons for these
failures are explored in detail, and a template for use in publications that
apply the Catalyst methodology is proposed. Second, the faster rule-based
methods for conformational analysis give pharmacophore models that are just as
good as, and in some cases better than, the models generated using the slower,
more rigorous approaches. Whereas computational methods for molecular design are well established in
medicinal chemistry research, their application in the field of natural products
is still not exhaustively explored. This article gives a short introduction into
both the potential for the application of computer-assisted approaches, such as
pharmacophore modelling, virtual screening, docking, and neural networking to
efficiently access the bioactive metabolites, and the requirements and
limitations related to this specific field. The challenge is which selection
criteria and/or multiple filtering tools to apply for a target-oriented
isolation of potentially bioactive secondary metabolites. Application examples
are provided where in silico tools and classical methods used by natural product
scientists are used in an effort to maximize their efficacy in drug discovery.
Thus, integrated computer-assisted strategies may help to process the huge
amount of available structural and biological information in a reasonably short
time for a straightforward search of bioactive natural products. Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes involved in protein
biosynthesis in all living organisms and are an unexploited antibacterial
targets, as many strains of bacteria have become resistant to all established
classes of antibiotics. Therefore, the main aim of this study is to discover new
lead molecules which would be useful as anti-bacterial compounds. Pharmacophore
models were developed by using CATALYST HypoGen with a training set of 29
diverse methionyl-tRNA synthetase (MetRS) inhibitors. The best quantitative
pharmacophore hypothesis (Hypo1) obtained a correlation coefficient of 0.975,
root mean square deviation (RMSD) of 0.55 and cost difference (null cost-total
cost) of 70.32. This Hypo1 was validated by two methods, first by using 104 test
set molecules which resulted a correlation of 0.926 between HypoGen estimated
activities versus experimental activities and secondly by Cat-Scramble
validation method. This validated pharmacophore model was further used for
screening databases for discovery of new MetRS inhibitors. The new lead
compounds were further analyzed for drug-like properties. Homology modeled
structure of Staphylococcus aureus MetRS was built and molecular docking studies
were performed with many inhibitors using the newly built protein structure.
Finally, it was found that the new leads exhibited good estimated inhibitory
activity, calculated binding properties similar to experimentally proven
compounds and also favorable drug-like properties. Aurora-A has been identified as one of the most attractive targets for cancer
therapy and a considerable number of Aurora-A inhibitors have been reported
recently. In order to clarify the essential structure-activity relationship for
the known Aurora-A inhibitors as well as identify new lead compounds against
Aurora-A, 3D pharmacophore models were developed based on the known inhibitors.
The best hypothesis, Hypo1, was used to screen molecular structural databases,
including Specs and China Natural Products Database for potential lead
compounds. The hit compounds were subsequently subjected to filtering by
Lipinski's rules and docking study to refine the retrieved hits and as a result
to reduce the rate of false positive. Finally, 39 compounds were purchased for
further in vitro assay against several human tumour cell lines including A549,
MCF-7, HepG2 and PC-3, in which Aurora-A is overexpressed. Two compounds show
very low micromolar inhibition potency against some of these tumour cells. And
they have been selected for further investigation. Pharmacophore approaches have become one of the major tools in drug discovery
after the past century's development. Various ligand-based and structure-based
methods have been developed for improved pharmacophore modeling and have been
successfully and extensively applied in virtual screening, de novo design and
lead optimization. Despite these successes, pharmacophore approaches have not
reached their expected full capacity, particularly in facing the demand for
reducing the current expensive overall cost associated with drug discovery and
development. Here, the challenges of pharmacophore modeling and applications in
drug discovery are discussed and recent advances and latest developments are
described, which provide useful clues to the further development and application
of pharmacophore approaches. A pharmacophore model has been developed using diverse classes of epidermal
growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors useful in the
treatment of human tumours. Among the top 10 generated hypotheses, the second
hypothesis, with one hydrogen bond acceptor, one ring aromatic and three
hydrophobic features, was found to be the best on the basis of Cat Scramble
validation as well as test set prediction (r(training) = 0.89, r(test) = 0.82).
The model also maps well to the external test set molecules as well as
clinically active molecules and corroborates the docking studies. Finally, 10
hits were identified as potential leads after virtual screening of ZINC database
for EGFR TK inhibition. The study may facilitate the designing and discovery of
novel EGFR TK inhibitors. A pharmacophore is a model which represents the key physico-chemical
interactions that mediate biological activity. There is a long history of using
pharmacophore modeling methods to select subsets of compounds, focused towards a
specific target of interest. This paper will review existing computational
methods for deriving and comparing pharmacophore models. We outline a new
classification of pharmacophore methods based on the abstraction of the
underlying chemical interactions which embody a pharmacophore, and the methods
available to quantitatively compare them. Within the context of this
classification, example studies, using specific pharmacophore modeling methods
for focused library selection, will be discussed. Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme that plays a vital
role in obesity-induced type 2 diabetes and fatty acid metabolism. To identify
dual inhibitors of Acetyl-CoA carboxylase1 and Acetyl-CoA carboxylase2, a
pharmacophore modelling approach has been employed. The best HypoGen
pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consists of one hydrogen
bond acceptor, one hydrophobic aliphatic and one hydrophobic aromatic feature,
whereas the best pharmacophore (Hypo1_ACC1) for ACC1 consists of one additional
hydrogen-bond donor (HBD) features. The best pharmacophore hypotheses were
validated by various methods such as test set, decoy set and Cat-Scramble
methodology. The validated pharmacophore models were used to screen several
small-molecule databases, including Specs, NCI, ChemDiv and Natural product
databases to identify the potential dual ACC inhibitors. The virtual hits were
then subjected to several filters such as estimated [Formula: see text] value,
quantitative estimation of drug-likeness and molecular docking analysis.
Finally, three novel compounds with diverse scaffolds were selected as potential
starting points for the design of novel dual ACC inhibitors. Protein-based pharmacophore models derived from protein binding site atoms
without the inclusion of any ligand information have become more popular in
virtual screening studies. However, the accuracy of protein-based pharmacophore
models for reproducing the critical protein-ligand interactions has never been
explicitly assessed. In this study, we used known protein-ligand contacts from a
large set of experimentally determined protein-ligand complexes to assess the
quality of the protein-based pharmacophores in reproducing these critical
contacts. We demonstrate how these contacts can be used to optimize the
pharmacophore generation procedure to produce pharmacophore models that
optimally cover the known protein-ligand interactions. Finally, we explored the
potential of the optimized protein-based pharmacophore models for pose
prediction and pose rankings. Our results demonstrate that there are significant
variations in the success of protein-based pharmacophore models to reproduce
native contacts and consequently native ligand poses dependent on the details of
the pharmacophore generation process. We show that the generation of optimized
protein-based pharmacophore models is a promising approach for ligand pose
prediction and pose rankings. Protein:protein interactions are becoming increasingly significant as potential
drug targets; however, the rational identification of small molecule inhibitors
of such interactions remains a challenge. Pharmacophore modelling is a popular
tool for virtual screening of compound libraries, and has previously been
successfully applied to the discovery of enzymatic inhibitors. However, the
application of pharmacophore modelling in the field of protein:protein
interaction inhibitors has historically been considered more of a challenge and
remains limited. In this review, we explore the interaction mimicry by known
inhibitors that originate from in vitro screening, demonstrating the validity of
pharmacophore mapping in the generation of queries for virtual screening. We
discuss the pharmacophore mapping methods that have been successfully employed
in the discovery of first-in-class inhibitors. These successful cases
demonstrate the usefulness of a "tool kit" of diverse strategies for application
across a range of situations depending on the available structural information. A pharmacophore model does not describe a real molecule or a real association of
functional groups but illustrates a molecular recognition of a biological target
shared by a group of compounds. Pharmacophores also represent the spatial
arrangement of essential interactions in a receptor-binding pocket. Structure
based pharmacophores (SBPs) can work both with a free (apo) structure or a
macromolecule-ligand complex (holo) structure. The SBP methods that derive
pharmacophore from protein-ligand complexes use the potential interactions
observed between ligand and protein, whereas, the SBP method that aims to derive
pharmacophore from ligand free protein, uses only protein active site
information. Therefore SBPs do not encounter to challenging problems such as
ligand flexibility, molecular alignment as well as proper selection of training
set compounds in ligand based pharmacophore modeling. The current review deals
with Hot Spot' analysis of binding site to feature generation, several
approaches to feature reduction, and considers shape and excluded volumes to SBP
model building. This review continues to represent several applications of SBPs
in virtual screening especially in parallel screening approach and multi-target
drug design. Also it reports the applications of SBPs in QSAR. This review
emphasizes that SBPs are valuable tools for hit to lead optimization, virtual
screening, scaffold hopping, and multi-target drug design. Pharmacophore approaches have evolved to be one of the most successful tools in
drug discovery, especially since the past two decades. 3D pharmacophore methods
are now commonly used as part of more complex workflows in drug discovery
campaigns, and have been successfully and extensively applied in virtual
screening (VS) approaches. This review provides a perspective of how to assess
the performance of 3D pharmacophore models to be used in VS. Since 3D VS
protocols are in general assessed by their ability to discriminate between
active and inactive compounds, we summarize the impact of the composition and
preparation of modeling and external sets on the outcome of evaluations.
Moreover, we highlight the significance of both classic enrichment parameters
and advanced descriptors for the performance of 3D pharmacophore-based virtual
screening methods. One of the major reasons for late-stage failure of drug candidates is due to
problems uncovered in pharmacokinetics during clinical trials. There is now a
general consensus for earlier consideration of these effects in the drug
discovery process. Computer-aided design technology provides us with tools to
develop predictive models for such pharmacokinetic properties. Among these
tools, we focus on pharmacophore modeling techniques in this article.
Pharmacophore models that are reported for various cytochrome P450 (CYP) enzymes
are reviewed for the isoenzymes CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4. In
addition pharmacophore models for related metabolic processes through CYP19
(aromatase), CYP51 (14.α-lanosterol demethylase), PXR (prege X-receptor), and
finally for human intrinsic clearance are also reviewed. The models reported by
various scientists are schematically represented in the figures in order to
visually demonstrate their similarities and differences. The models developed by
different researchers or sometimes even by the same research group for different
sets of ligands, provide a clear picture of the challenges in coming up with a
single model with good predictive values. One of the main reasons for this
challenge is related to relatively large size of the active sites and
flexibility of the CYP isoenzymes, which results in multiple binding sites. We
propose development of multiple- diverse pharmacophore models for each binding
mode (as opposed to a single predictive model for each CYP isoenzyme). After
scoring and prioritization of the models, we propose the use of a battery of
pharmacophore models for each CYP isoenzyme binding mode to computationally
obtain a P450 interaction profile for drug candidates early in the drug
development cycle, when decisions on their fate can be made before incurring the
costs of synthesis and testing. Accurate prediction of ligand-binding poses is crucial for understanding
molecular interactions and is very important for drug discovery, structural
design, and optimization. In this study, we developed a novel scoring program,
HotLig, which applies the Connolly surface of a protein to calculate hydrophobic
interaction and paired pharmacophore interactions with ligands. In addition to
molecular surface distance, ligand-contacting areas and hydrogen-bond angles
were also introduced to the scoring functions in HotLig. Four individual energy
scoring functions for H-bonds, ionic pairs, metal coordination, and hydrophobic
effects were derived from 600 protein-ligand complexes, and then, their
weighting factors were optimized through an interaction-characterized training
set. Success rates of ligand-binding-pose predictions (with a root mean squared
deviation of ≤2 Å) for the Wang, GOLD, and Cheng data sets were respectively
validated to be 91.0%, 87.0%, and 85.6%. HotLig was found to possess equally
good predictive powers for the hydrophilic (88.6%) and hydrophobic subsets
(87.5%), and the success rate for the mixed subset was as high as 96.9%. The
Spearman correlation coefficients were as good as 0.609 to 0.668, which
indicates HotLig also has satisfactory predictive power for binding affinities.
These results suggested that the HotLig can analyze diverse ligands, including
peptides, and is expected to be a powerful tool for drug design and discovery. Herein, a combined molecular docking-based and pharmacophore-based target
prediction strategy is presented, in which a probabilistic fusion method is
suggested for target ranking. Establishment and validation of the combined
strategy are described. A target database, termed TargetDB, was firstly
constructed, which contains 1105 drug targets. Based on TargetDB, the molecular
docking-based target prediction and pharmacophore-based target prediction
protocols were established. A probabilistic fusion method was then developed by
constructing probability assignment curves (PACs) against a set of selected
targets. Finally the workflow for the combined molecular docking-based and
pharmacophore-based target prediction strategy was established. Evaluations of
the performance of the combined strategy were carried out against a set of
structurally different single-target compounds and a well-known multi-target
drug, 4H-tamoxifen, which results showed that the combined strategy consistently
outperformed the sole use of docking-based and pharmacophore-based methods.
Overall, this investigation provides a possible way for improving the accuracy
of in silico target prediction and a method for target ranking. Recent technological breakthroughs in medicinal chemistry arena had ameliorated
the perspectives of quantitative structure-activity relationship (QSAR) methods.
In this direction, we developed a group-based QSAR method based on
pharmacophore-similarity concept which takes into account the 2D topological
pharmacophoric descriptors and predicts the group-specific biological
activities. This activity prediction may assist the contribution of certain
pharmacophore features encoded by respective fragments toward activity
improvement and/or detrimental effects. We termed this method as
pharmacophore-similarity-based QSAR (PS-QSAR) and studied the activity
contribution of fragments from 3-hydroxypyridinones derivatives possessing
antimalarial activities. |
Are there conserved noncoding elements identified between genomes of human and teleosts? | Vertebrate genomes contain thousands of conserved noncoding elements (CNEs) that often function as tissue-specific enhancers. In this study, we have identified CNEs in human, dog, chicken, Xenopus, and four teleost fishes (zebrafish, stickleback, medaka, and fugu) using elephant shark, a cartilaginous vertebrate, as the base genome and investigated the evolution of these ancient vertebrate CNEs (aCNEs) in bony vertebrate lineages | Evolutionary sequence conservation is an accepted criterion to identify
noncoding regulatory sequences. We have used a transposon-based transgenic assay
in zebrafish to evaluate noncoding sequences at the zebrafish ret locus,
conserved among teleosts, and at the human RET locus, conserved among mammals.
Most teleost sequences directed ret-specific reporter gene expression, with many
displaying overlapping regulatory control. The majority of human RET noncoding
sequences also directed ret-specific expression in zebrafish. Thus, vast amounts
of functional sequence information may exist that would not be detected by
sequence similarity approaches. We report evidence for a mechanism for the maintece of long-range conserved
synteny across vertebrate genomes. We found the largest mammal-teleost conserved
chromosomal segments to be spanned by highly conserved noncoding elements
(HCNEs), their developmental regulatory target genes, and phylogenetically and
functionally unrelated "bystander" genes. Bystander genes are not specifically
under the control of the regulatory elements that drive the target genes and are
expressed in patterns that are different from those of the target genes.
Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2,
rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the
expression patterns of these genes even if located inside or beyond bystander
genes, suggesting that the regulatory domain of a developmental regulatory gene
can extend into and beyond adjacent transcriptional units. We termed these
chromosomal segments genomic regulatory blocks (GRBs). After whole genome
duplication in teleosts, GRBs, including HCNEs and target genes, were often
maintained in both copies, while bystander genes were typically lost from one
GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy
GRBs of higher vertebrates intact. We show that loss of bystander genes and
other mutational events suffered by duplicated GRBs in teleost genomes permits
target gene identification and HCNE/target gene assignment. These findings
explain the absence of evolutionary breakpoints from large vertebrate
chromosomal segments and will aid in the recognition of position effect
mutations within human GRBs. BACKGROUND: Embryonic development is coordinated by sets of cis-regulatory
elements that are collectively responsible for the precise spatio-temporal
organization of regulatory gene networks. There is little information on how
these elements, which are often associated with highly conserved noncoding
sequences, are combined to generate precise gene expression patterns in
vertebrates. To address this issue, we have focused on Six3, an important
regulator of vertebrate forebrain development.
RESULTS: Using computational analysis and exploiting the diversity of teleost
genomes, we identified a cluster of highly conserved noncoding sequences
surrounding the Six3 gene. Transgenesis in medaka fish demonstrates that these
sequences have enhancer, silencer, and silencer blocker activities that are
differentially combined to control the entire distribution of Six3.
CONCLUSION: This report provides the first example of the precise regulatory
code necessary for the expression of a vertebrate gene, and offers a unique
framework for defining the interplay of trans-acting factors that control the
evolutionary conserved use of Six3. A large-scale enhancer detection screen was performed in the zebrafish using a
retroviral vector carrying a basal promoter and a fluorescent protein reporter
cassette. Analysis of insertional hotspots uncovered areas around developmental
regulatory genes in which an insertion results in the same global expression
pattern, irrespective of exact position. These areas coincide with vertebrate
chromosomal segments containing identical gene order; a phenomenon known as
conserved synteny and thought to be a vestige of evolution. Genomic comparative
studies have found large numbers of highly conserved noncoding elements (HCNEs)
spanning these and other loci. HCNEs are thought to act as transcriptional
enhancers based on the finding that many of those that have been tested direct
tissue specific expression in transient or transgenic assays. Although gene
order in hox and other gene clusters has long been known to be conserved because
of shared regulatory sequences or overlapping transcriptional units, the
chromosomal areas found through insertional hotspots contain only one or a few
developmental regulatory genes as well as phylogenetically unrelated genes. We
have termed these regions genomic regulatory blocks (GRBs), and show that they
underlie the phenomenon of conserved synteny through all sequenced vertebrate
genomes. After teleost whole genome duplication, a subset of GRBs were retained
in two copies, underwent degenerative changes compared with tetrapod loci that
exist as single copy, and that therefore can be viewed as representing the
ancestral form. We discuss these findings in light of evolution of vertebrate
chromosomal architecture and the identification of human disease mutations. Teleost fishes are the largest and most diverse group of vertebrates. The
diversity of teleosts has been attributed to a whole-genome duplication (WGD)
event in the ray-finned fish lineage. Recent comparative genomic studies have
revealed that teleost genomes have experienced frequent gene-linkage disruptions
compared to other vertebrates, and that protein-coding sequences in teleosts are
evolving faster than in mammals, irrespective of their duplication status. A
significant number of conserved noncoding elements (CNEs) shared between
cartilaginous fishes and tetrapods have diverged beyond recognition in teleost
fishes. The divergence of CNEs seems to have been initiated in basal ray-finned
fishes before the WGD. The fast evolving singleton and duplicated genes as well
as the divergent CNEs might have contributed to the diversity of teleost fishes. Fibroblast growth factors (Fgfs) encode small signaling proteins that help
regulate embryo patterning. Fgfs fall into seven families, including FgfD.
Nonvertebrate chordates have a single FgfD gene; mammals have three (Fgf8,
Fgf17, and Fgf18); and teleosts have six (fgf8a, fgf8b, fgf17, fgf18a, fgf18b,
and fgf24). What are the evolutionary processes that led to the structural
duplication and functional diversification of FgfD genes during vertebrate
phylogeny? To study this question, we investigated conserved syntenies, patterns
of gene expression, and the distribution of conserved noncoding elements (CNEs)
in FgfD genes of stickleback and zebrafish, and compared them with data from
cephalochordates, urochordates, and mammals. Genomic analysis suggests that
Fgf8, Fgf17, Fgf18, and Fgf24 arose in two rounds of whole genome duplication at
the base of the vertebrate radiation; that fgf8 and fgf18 duplications occurred
at the base of the teleost radiation; and that Fgf24 is an ohnolog that was lost
in the mammalian lineage. Expression analysis suggests that ancestral
subfunctions partitioned between gene duplicates and points to the evolution of
novel expression domains. Analysis of CNEs, at least some of which are candidate
regulatory elements, suggests that ancestral CNEs partitioned between gene
duplicates. These results help explain the evolutionary pathways by which the
developmentally important family of FgfD molecules arose and the deduced
principles that guided FgfD evolution are likely applicable to the evolution of
developmental regulation in many vertebrate multigene families. |
How early during pregnancy does non-invasive cffDNA testing allow sex determination of the fetus? | Using cffDNA from maternal blood, the fetal gender can be determined as early as 6 to 10 weeks of gestation (during the first trimester of pregnancy). | Fetal sex prediction can be achieved using PCR targeted at the SRY gene by
analysing cell-free fetal DNA in maternal serum. Unfortunately, the results
reported to date show a lack of sensitivity, especially during the first
trimester of pregcy. Therefore, determination of fetal sex by maternal serum
analysis could not replace karyotype analysis following chorionic villus
sampling. A new highly sensitive real-time PCR was developed to detect an SRY
gene sequence in maternal serum. Analysis was performed on 121 pregt women
during the first trimester of pregcy (mean gestational age: 11.8 weeks).
Among them, 51 had at least one previous male-bearing pregcy. Results were
compared with fetal sex. SRY PCR analysis of maternal serum was in complete
concordance with fetal sex. Among the 121 pregt women, 61 were bearing a male
fetus and 60 a female fetus. No false-negative results were observed.
Furthermore, no false-positive results occurred, even though 27 women carrying a
female fetus during the current pregcy had at least one previous male-bearing
pregcy. This study demonstrates that a reliable, non-invasive sex
determination can be achieved by PCR analysis of maternal serum during the first
trimester of pregcy. This non-invasive approach for fetal sex prediction
should have great implications in the management of pregt women who are
carriers of an X-linked genetic disorder. Prenatal diagnosis might thus be
performed for male fetuses only, avoiding invasive procedures and the risk of
the loss of female fetuses. OBJECTIVE: Fetal sex prediction can be achieved using PCR targeted at the SRY
gene by analyzing cell-free fetal DNA in maternal serum. Unfortunately, the
results reported to date, show lack of sensitivity, especially in the first
trimester of pregcy. Therefore, determination of fetal sex by maternal serum
analysis can not replace caryotype analysis following chorionic villus sampling.
PATIENTS AND METHODS: A new highly sensitive real-time PCR was developed to
detect a SRY gene sequence in maternal serum. Analysis was performed on 121
pregt women during their first trimester of pregcy (mean gestational age:
11.8 weeks). Among them, 61 had at least one previous male-bearing pregcy.
Results were compared to fetal sex.
RESULTS: SRY PCR analysis of maternal serum was in complete concordance with
fetal sex. Among the 121 pregt women, 61 were bearing a male fetus and 60 a
female fetus No false negative results were observed. Furthermore, no false
positive results results occurred although 27 women carried female fetus during
the current pregcy, had at least one previous male-bearing pregcy.
DISCUSSION AND CONCLUSION: This study demonstrates that a reliable, non-invasive
sex determination can be achieved by PCR analysis of maternal serum during the
first trimester of pregcy. This non-invasive approach for fetal sex
prediction should have great implications in the management of pregt women
carriers of an X-linked genetic disorder. Prenatal diagnosis is thus performed
for male fetuses only, avoiding invasive procedures and the risk of fetal loss
for female fetuses. OBJECTIVE: Couples with a risk of transmitting X-linked diseases included in a
preimplantation genetic diagnosis (PGD) center need early and rapid fetal sex
determination during pregcy in two situations. The first situation
corresponds to control of embryo sexing after PGD, the second one being that of
couples in PGD program having a spontaneous pregcy. Determination of fetal
sex can be achieved by karyotyping using invasive procedures such as chorionic
villus sampling (CVS), amniocentesis or cordocentesis and by non-invasive
procedures such as ultrasound (US) examination. CVS is the earliest invasive
procedure for fetal sex determination and molecular analysis of X-linked genetic
disorders during the first trimester but it is associated with a risk of fetal
loss. US allows reliable fetal sex determination only during the second
trimester. Recently, reliable non-invasive fetal sex determination was realized
by using SRY gene amplification in maternal serum.
PATIENTS AND METHODS: We report the prospective use of fetal sex determination
in maternal serum in our PGD center. Management of pregcies was performed
using this non-invasive procedure in four cases of embryo sexing control and
nine cases of spontaneous pregcies in couples included in PGD program for
X-linked diseases.
RESULTS: Fetal sex results using SRY gene amplification on maternal serum were
in complete concordance with fetal sex observed by cytogenetic analysis or US
examination, as well as at birth.
DISCUSSION AND CONCLUSION: This new strategy allowed rapid sex determination
during the first trimester and permitted to avoid performing invasive procedures
in nine pregcies. BACKGROUND: Detection of cell-free fetal DNA (cffDNA) in maternal plasma has
given rise to the possibility of new non-invasive approaches for early prenatal
diagnoses. We evaluated the feasibility and accuracy of non-invasive fetal
gender determination using quantitative fluorescent-polymerase chain reaction
(QF-PCR) analysis of circulating cffDNA in the first-trimester maternal plasma.
METHODS: Plasma samples were prospectively collected from 202 singleton
pregcies at 4 to 13 weeks of gestation. Fetal gender was determined by QF-PCR
with the sex-determining region Y (SRY) and amelogenin X/Y (AMELX/Y) genes. The
result was confirmed by fetal karyotyping or phenotype at birth.
RESULTS: Of the 202 pregcies, 162 had pregcy outcomes available and could
be included in our evaluation. The accuracies of AMELX/Y, SRY, and combined
AMELX/Y+SRY analysis for fetal gender determination were 83.3%, 82.1%, and
97.5%, respectively, compared with those of the invasive approach and the fetal
gender outcome at birth (82 males and 80 females). Combined AMELX/Y+SRY analysis
had the highest sensitivity (98.8%) for fetal gender determination with a
specificity of 96.3%. Moreover, fetal gender detection by the combined
AMELX/Y+SRY analysis at 11 to 13 weeks of gestation was 100% correct.
CONCLUSION: Fetal gender determination could be accurately determined from
maternal cffDNA in the first-trimester using QF-PCR analysis of combined
AMELX/Y+SRY. In previous years, identification of fetal cells in maternal blood circulation
has caused a new revolution in non-invasive method of prenatal diagnosis. Low
number of fetal cells in maternal blood and long-term survival after pregcy
limited the use of fetal cells in diagnostic and clinical applications. With the
discovery of cell-free fetal DNA (cffDNA) in plasma of pregt women, access to
genetic material of the fetus had become possible to determine early gender of a
fetus in pregcies at the risk of X-linked genetic conditions instead of
applying invasive methods. Therefore in this study, the probability of detecting
sequences on the Y chromosome in pregt women has been evaluated to identify
the gender of fetuses. Peripheral blood samples were obtained from 80 pregt
women at 6(th) to 10(th) weeks of gestation and then the fetal DNA was extracted
from the plasma. Nested PCR was applied to detect the sequences of single copy
SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male
fetuses. At the end, all the obtained results were compared with the actual
gender of the newborns. In 40 out of 42 born baby boys, the relevant gene
sequences were identified and 95.2% sensitivity was obtained. Non-invasive early
determination of fetal gender using cffDNA could be employed as a pre-test in
the shortest possible time and with a high reliability to avoid applying
invasive methods in cases where a fetus is at the risk of genetic diseases. Chorionic villus sampling (CVS) or amniocentesis for fetal sex determination is
generally the first step in the prenatal diagnosis of X-linked genetic disorders
such as Duchenne muscular dystrophy (DMD). However, non-invasive prenatal
diagnostic (NIPD) techniques such as measurement of cell-free fetal DNA (cffDNA)
in maternal plasma are preferable given the procedure-related miscarriage rate
of CVS. We determined fetal sex during the first trimester using a quantitative
real-time polymerase chain reaction (PCR) assay of cffDNA in pregt carriers
of DMD. The fetal sex was confirmed by amniocentesis karyotype analysis and
multiplex ligation-dependent probe amplification (MLPA) at 16 weeks. This
procedure may avoid unnecessary CVS or amniocentesis of female fetuses. |
Does cortical spreading depression appear in ischemic penumbra following ischemic stroke? | Yes, cortical spreading depression appears in ischemic penumbra following ischemic stroke and is associated with expansion of ischemic injury. This has been shown in humans and in animal models. | The classic concept of the viability thresholds of ischemia differentiates
between two critical flow rates, the threshold of electrical failure and the
threshold of membrane failure. These thresholds mark the upper and lower flow
limits of the ischemic penumbra which is thought to suffer only functional but
not structural injury. Recent studies of the functional and metabolic
disturbances suggest a more complex pattern of thresholds. At declining flow
rates, protein synthesis is inhibited at first (at a threshold of about 0.55
ml/gm/min), followed by a stimulation of anaerobic glycolysis (at 0.35
ml/gm/min), the release of neurotransmitters and the beginning disturbance of
energy metabolism (at about 0.20 ml/min), and finally the anoxic depolarization
(< 0.15 ml/gm/min). The penumbra, as defined by the classic flow thresholds,
does not remain viable for extended periods. Since viability of the tissue
requires maintece of energy-dependent metabolic processes, penumbra is
redefined as a region of constrained blood supply in which the energy metabolism
is preserved. Imaging of the penumbra by combining autoradiographic cerebral
blood flow measurements with bioluminescent images of adenosine triphosphate
(ATP) demonstrates a gradual expansion of the infarct core (in which ATP is
depleted) into the penumbra until, after a few hours, the penumbra has
disappeared. It is suggested that the limited survival of the penumbra is due to
periinfarct depolarizations, which result in repeated episodes of tissue
hypoxia, because the increased metabolic workload is not coupled to an adequate
increase of collateral blood supply. This explains pharmacological suppression
of periinfarct depolarizations lowering the threshold of metabolic disturbances
and reducing the volume of the ischemic infarct. When a cerebral infarction occurs, surrounding the core of dying tissue there
usually is an ischemic penumbra of nonfunctional but still viable tissue. One
current but controversial hypothesis is that this penumbra tissue often
eventually dies because of the metabolic stress imposed by multiple cortical
spreading depression (CSD) waves, that is, by ischemic depolarizations. We
describe here a computational model of CSD developed to study the implications
of this hypothesis. After simulated infarction, the model displays the linear
relation between final infarct size and the number of CSD waves traversing the
penumbra that has been reported experimentally, although damage with each
individual wave progresses nonlinearly with time. It successfully reproduces the
experimental dependency of final infarct size on midpenumbra cerebral blood flow
and potassium reuptake rates, and predicts a critical penumbra blood flow rate
beyond which damage does not occur. The model reproduces the dependency of CSD
wave propagation on N-methyl-D-aspartate activation. It also makes testable
predictions about the number, velocity, and duration of ischemic CSD waves and
predicts a positive correlation between the duration of elevated potassium in
the infarct core and the number of CSD waves. These findings support the
hypothesis that CSD waves play an important causal role in the death of ischemic
penumbra tissue. BACKGROUND: Spreading depression (SD) is known to go along with temporary
breakdown of ion gradients and cell swelling which spontaneously normalizes.
Here, the effects of SD at reduced flow conditions as encountered in the
ischemic penumbra are examined.
METHODS: In rats the right carotid artery was permanently occluded. MABP was
lowered to 50 mmHg for 30 min. This is sufficient to reduce CBF to penumbra-like
conditions in the right hemisphere. The following parameters were assessed:
rCBF, DC potential, and tissue impedance. 5 or 15 min after onset of flow
reduction one SD wave was initiated by microinjection of KCl. Histology was
performed after 7 days.
RESULTS: In animals with hypotension there was depolarization resembling anoxic
depolarization after SD induction and an uncoupling of CBF and metabolism only
in the right hemisphere. Impedance increased with SD but did not recover
spontaneously as long as rCBF remained reduced. 15 min of SD-induced cell
swelling was tolerated without permanent damage, whereas 25 min were followed by
severe neuron loss in the affected cortex after 7 days.
CONCLUSIONS: The study demonstrates the induction of penumbra conditions in the
cortex of one hemisphere. SD is followed by cell swelling which persists as long
as flow is critically reduced. The experiments illustrate how peri-infarct
depolarizations may detrimentally affect the penumbra. The effect of the free radical spin-trap alpha-phenyl-butyl-tert-nitrone
(alpha-PBN) in permanent focal cerebral ischemia in rats was examined in two
series of experiments. In the first, rats were subjected to permanent occlusion
of the middle cerebral artery (MCAO) and treated 1 h after occlusion with a
single dose of alpha-PBN (100 mg/kg) or saline. Body temperature was measured
and controlled for the first 24 h to obtain identical temperature curves in the
two groups. Cortical infarct volumes were determined on histological sections 7
days later. alpha-PBN did not significantly reduce infarct volume (control:
28.3+/-16.3 mm3 vs. alpha-PBN 23.7+/-7.4 mm3). In the second series of
experiments, periinfarct depolarizations (PIDs) were recorded with an
extracellular DC electrode at two locations in the ischemic penumbra for the
initial 3 h following MCAO. alpha-PBN (100 mg/kg, single dose in conjunction
with occlusion) significantly reduced the total number (median value of 3 PIDs
in the control groups vs. 1 PID in alpha-PBN groups, p<0.001) and total duration
of the PIDs (median value 662 s in the control groups vs. 162 s in the alpha-PBN
groups, p<0.006). In spite of this, cortical infarct volumes determined 7 days
later in the same rats were not smaller in alpha-PBN-treated rats. The study
thus demonstrates that attenuation of PIDs does not always lead to smaller
infarcts if permanent arterial occlusion is followed by long survival time and
does not support the hypothesis that PIDs per se are critical determits of
infarct size in this situation. Spreading depression (SD) has been demonstrated following focal ischemia, and
the additional workload imposed by SD on a tissue already compromised by a
marked reduction in blood flow may contribute to the evolution of irreversible
damage in the ischemic penumbra. SD was elicited in one group of rats by
injecting KCl directly into a frontal craniectomy and the wave of depolarization
was recorded in two craniectomies 3 and 6 mm posterior to the first one. In a
second group, the middle cerebral artery was occluded using the monofilament
technique and a recording electrode was placed 5 mm lateral to the midline and
0.2 mm posterior to bregma. To determine the metabolic response in the penumbral
region of the cortex ipsilateral to the occlusion, brains from both groups were
frozen in situ when the deflection of the SD was maximal. The spatial metabolic
response of SD in the ischemic cortex was compared to that in the non-ischemic
cortex. Coronal sections of the brains were lyophilized, pieces of the
dorsolateral cortex were dissected and weighed, and analyzed for ATP,
P-creatine, inorganic phosphate (Pi), glucose, glycogen and lactate at varying
distances anterior and posterior to the recording electrode. ATP and P-creatine
levels were significantly decreased at the wavefront in both groups and the
levels recovered after passage of the wavefront in the normal brain, but not in
the ischemic brain. Glucose and glycogen levels were significantly decreased and
lactate levels significantly increased in the tissue after the passage of the
wavefront. While the changes in the glucose-related metabolites persisted during
recovery even in anterior portions of the cortex in both groups in the aftermath
of the SD, the magnitude of the changes was greater in the penumbra than in the
normal cortex. SD appears to impose an equivalent increase in energy demands in
control and ischemic brain, but the ability of the penumbra to recover from the
insult is compromised. Thus, increasing the energy imbalance in the penumbra
after multiple SDs may hasten the deterioration of the energy status of the
tissue and eventually contribute to terminal depolarization and cell death,
particularly in the penumbra. Focal brain infarcts are surrounded by extended perilesional zones that comprise
the partially ischemic penumbra but also completely non-ischemic cortex of the
remote ipsilateral hemisphere. To delineate the impact of lesion-associated vs.
remote processes on transcriptional programming after focal ischemia, we used
cDNA array analysis, quantitative real-time polymerase chain reaction and
immunohistochemistry in the photothrombosis model of circumscribed cortical
ischemia in rats. At an early stage of 4 h after ischemia, gene induction
occurred to a similar extent in the ischemic infarct and remote non-ischemic
cortex of the ipsilateral hemisphere. Among the genes induced in non-ischemic
cortex we found the NGF-inducible genes PC3, VGF and Arc, the transcriptional
regulators I kappa B-alpha and Stat3, and the beta-chemokine MIP-1 alpha (CCL3).
At 3 days, the spatial pattern of gene expression had changed dramatically with
brain fatty acid-binding protein as the only gene significantly induced in
non-ischemic ipsilateral cortex. In contrast, numerous genes were exclusively
regulated at the lesion site, comprising genes involved in cell cycle
regulation, proteolysis, apoptosis, lipid homeostasis and anti-inflammatory
counter-regulation. Cortical spreading depression was identified as the main
mechanism underlying gene induction in remote non-ischemic cortex. Our data
demonstrate a dynamic spatiotemporal pattern of gene induction, which may
contribute to delayed progression of damage or, alternatively, mediate
neuroprotection, tissue remodeling and functional compensation. BACKGROUND AND PURPOSE: Spreading depression (SD)-like depolarizations may
augment neuronal damage in neurovascular disorders such as stroke and traumatic
brain injury. Spreading ischemia (SI), a particularly maligt variant of
SD-like depolarization, is characterized by inverse coupling between the
spreading depolarization wave and cerebral blood flow. SI has been implicated in
particular in the pathophysiology of subarachnoid hemorrhage. Under
physiological conditions, SD is blocked by N-methyl-D-aspartate receptor (NMDAR)
antagonists. However, because both SD-like depolarizations and SI occur in
presence of an increased extracellular K+ concentration ([K+]o), we tested
whether this increase in baseline [K+]o would reduce the efficacy of NMDAR
antagonists.
METHODS: Cranial window preparations, laser Doppler flowmetry, and
K+-sensitive/reference microelectrodes were used to record SD, SD-like
depolarizations, and SI in rats in vivo; microelectrodes and intrinsic optical
signal measurements were used to record SD and SD-like depolarizations in human
and rat brain slices.
RESULTS: In vivo, the noncompetitive NMDAR antagonist dizocilpine (MK-801)
blocked SD propagation under physiological conditions, but did not block SD-like
depolarizations or SI under high baseline [K+]o. Similar results were found in
human and rat neocortical slices with both MK-801 and the competitive NMDAR
antagonist D-2-amino-5-phosphonovaleric acid.
CONCLUSIONS: Our data suggest that elevated baseline [K+]o reduces the efficacy
of NMDAR antagonists on SD-like depolarizations and SI. In conditions of
moderate energy depletion, as in the ischemic penumbra, or after subarachnoid
hemorrhage, NMDAR inhibition may not be sufficient to block these
depolarizations. Spreading depression (SD) is a slowly propagating wave of transient neuronal and
glial depolarization that develops after stroke, trauma and subarachnoid
hemorrhage. In compromised tissue, repetitive SD-like injury depolarizations
reduce tissue viability by worsening the mismatch between blood flow and
metabolism. Although the mechanism remains unknown, SDs show delayed
electrophysiological recovery within the ischemic penumbra. Here, we tested the
hypothesis that the recovery rate of SD can be varied by modulating tissue
perfusion pressure and oxygenation. Systemic blood pressure and arterial pO(2)
were simultaneously manipulated in anesthetized rats under full physiologic
monitoring. We found that arterial hypotension doubled the SD duration, whereas
hypertension reduced it by a third compared with normoxic normotensive rats.
Hyperoxia failed to shorten the prolonged SD durations in hypotensive rats,
despite restoring tissue pO(2). Indeed, varying arterial pO(2) (40 to 400 mm Hg)
alone did not significantly influence SD duration, whereas blood pressure (40 to
160 mm Hg) was inversely related to SD duration in compromised tissue. These
data suggest that cerebral perfusion pressure is a critical determit of SD
duration independent of tissue oxygenation over a wide range of arterial pO(2)
levels, and that hypotension may be detrimental in stroke and subarachnoid
hemorrhage, where SD-like injury depolarizations have been observed. Spontaneous spreading depolarizations (SDs) occur in the penumbra surrounding
ischemic core. These SDs, often referred to as peri-infarct depolarizations,
cause vasoconstriction and recruitment of the penumbra into the ischemic core in
the critical first hours after focal ischemic stroke; however, the real-time
spatiotemporal dynamics of SD-induced injury to synaptic circuitry in the
penumbra remain unknown. A modified cortical photothrombosis model was used to
produce a square-shaped lesion surrounding a penumbra-like "area at risk" in
middle cerebral artery territory of mouse somatosensory cortex. Lesioning
resulted in recurrent spontaneous SDs. In vivo two-photon microscopy of green
fluorescent protein-expressing neurons in this penumbra-like area at risk
revealed that SDs were temporally correlated with rapid (<6 s) dendritic
beading. Dendrites quickly (<3 min) recovered between SDs to near-control
morphology until the occurrence of SD-induced terminal dendritic injury,
signifying acute synaptic damage. SDs are characterized by a breakdown of ion
homeostasis that can be recovered by ion pumps if the energy supply is adequate.
Indeed, the likelihood of rapid dendritic recovery between SDs was correlated
with the presence of nearby flowing blood vessels, but the presence of such
vessels was not always sufficient for rapid dendritic recovery, suggesting that
energy needs for recovery exceeded energy supply of compromised blood flow. We
propose that metabolic stress resulting from recurring SDs facilitates acute
injury at the level of dendrites and dendritic spines in metabolically
compromised tissue, expediting penumbral recruitment into the ischemic core. Spreading depolarizations are a key event in the pathophysiology of stroke,
resulting in rapid dendritic beading, which represents acute damage to synaptic
circuitry. The impact of spreading depolarizations on the real-time injury of
astrocytes during ischemia is less clear. We used simultaneous in vivo 2-photon
imaging and electrophysiological recordings in adult mouse somatosensory cortex
to examine spreading depolarization-induced astroglial structural changes
concurrently with signs of neuronal injury in the early periods of focal and
global ischemia. Astrocytes in the metabolically compromised ischemic
penumbra-like area showed a long lasting swelling response to spontaneous
spreading depolarizations despite rapid dendritic recovery in a photothrombotic
occlusion model of focal stroke. Astroglial swelling was often facilitated by
recurrent depolarizations and the magnitude of swelling strongly correlated with
the total duration of depolarization. In contrast, spreading
depolarization-induced astroglial swelling was transient in normoxic healthy
tissue. In a model of transient global ischemia, the occurrence of a single
spreading depolarization elicited by a bilateral common carotid artery occlusion
coincided with astroglial swelling alongside dendritic beading. With immediate
reperfusion, dendritic beading subsides. Astroglial swelling was either
transient during short ischemic periods distinguished by a short-lasting
spreading depolarization, or persistent during severe ischemia characterized by
a long-lasting depolarization with the ultraslow negative voltage component. We
propose that persistent astroglial swelling is initiated and exacerbated during
spreading depolarization in brain tissue with moderate to severe energy
deficits, disrupting astroglial maintece of normal homeostatic function thus
contributing to the negative outcome of ischemic stroke as astrocytes fail to
provide neuronal support. An ischemic penumbra has the potential for functional recovery provided that
local blood flow can be reestablished, but irreversible damage will develop
without sufficient reperfusion, depending on the interaction of severity and
duration of ischemia. With acute flows below the threshold required for
maintece of basic housekeeping, injury in the core is established within a
few minutes. During the subacute phase, the irreversible damage expands into the
penumbra: multiple electrical and biological signals are triggered by
periinfarct, spreading depression-like depolarizations leading to hypoxia and
stepwise increase in lactate. Usually within 6 to 8 hours, all the penumbra are
converted into irreversible infarcts. In a delayed phase, secondary phenomena
may cause additional tissue damage: disruption of the tight junctions results in
vasogenic edema, leading to increase of water content and damage expansion.
Neutrophils and cytokinins cause secondary inflammation, inducing further damage
in periinfarct regions and connecting fiber tracts. Multimodal imaging might be
able to differentiate among the tissue compartments affected by acute, subacute,
or delayed ischemic damage, and thereby might provide the basis for
phase-specific treatment strategies. |
Is phospholamban a regulatory/inhibitory protein of the Ca ATPase SERCA? | Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca2+]i and contractility in these muscles.The membrane protein complex between the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) controls Ca(2+) transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. | Regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA 2a) depends on
the phosphorylation state of phospholamban (PLB). When PLB is phosphorylated,
its inhibitory effect towards SERCA 2a is relieved, leading to an enhanced
myocardial performance. This process is reversed by a sarcoplasmic reticulum
(SR)-associated type 1 protein phosphatase (PP1) composed of a catalytic subunit
PP1C and a regulatory subunit GM. Human GM and PLB have been produced in an in
vitro transcription/translation system and used for co-immunoprecipitation and
biosensor experiments. The detected interaction between the two partners
suggests that cardiac PPI is targeted to PLB via GM and we believe that this
process occurs with the identified transmembrane domains of the two proteins.
Thus, the interaction between PLB and GM may represent a specific way to
modulate the SR function in human cardiac muscle. Vascular endothelial cells regulate vascular smooth muscle tone through
Ca2+-dependent production and release of vasoactive molecules. Phospholamban
(PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the
sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is
reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which
PLB is an important regulator of [Ca2+]i and contractility in these muscles. In
the present study, we report the existence of PLB in the vascular endothelium, a
nonmuscle tissue, and provide functional data on PLB regulation of vascular
contractility through its actions in the endothelium. Endothelium-dependent
relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO)
mice compared with wild-type (WT) controls. This effect was not due to actions
of nitric oxide on the smooth muscle, because sodium nitroprusside-mediated
relaxation in either denuded or endothelium-intact aortas was unaffected by PLB
ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in
WT endothelium-intact aortas. The endothelium-dependent component of this
relaxation was attenuated in PLB-KO aortas. To investigate whether these changes
were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse
transcriptase-polymerase chain reaction and Western blot analyses revealed the
presence of PLB in endothelial cells, which were shown to be >98% pure by
diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that
PLB is present and modulates vascular function as a result of its actions in
endothelial cells. The presence of PLB in endothelial cells opens new fields for
investigation of Ca2+ regulatory pathways in nonmuscle cells and for modulation
of endothelial-vascular interactions. Transient elevations of intracellular Ca2+ play a signalling role in such
complex cellular functions as contraction, secretion, fertilization,
proliferation, metabolism, heartbeat and memory. However, prolonged elevation of
Ca2+ above about 10 microM is deleterious to a cell and can activate apoptosis.
In muscle, there is a narrow window of Ca2+ dysregulation in which abnormalities
in Ca2+ regulatory proteins can lead to disease, rather than apoptosis. Key
proteins in the regulation of muscle Ca2+ are the voltage-dependent,
dihydropyridine-sensitive, L-type Ca2+ channels located in the transverse tubule
and Ca2+ release channels in the junctional terminal cisternae of the
sarcoplasmic reticulum. Abnormalities in these proteins play a key role in
maligt hyperthermia (MH), a toxic response to anesthetics, and in central
core disease (CCD), a muscle myopathy. Sarco(endo)plasmic reticulum Ca2+ ATPases
(SERCAs) return sarcoplasmic Ca2+ to the lumen of the sarcoplasmic reticulum.
Loss of SERCA1a Ca2+ pump function is one cause of exercise-induced impairment
of the relaxation of skeletal muscle, in Brody disease. Phospholamban expressed
in cardiac muscle and sarcolipin expressed in skeletal muscle regulate SERCA
activity. Studies with knockout and transgenic mice show that gain of inhibitory
function of phospholamban alters cardiac contractility and could be a causal
feature in some cardiomyopathies. Calsequestrin, calreticulin, and a series of
other acidic, lumenal, Ca2+ binding proteins provide a buffer for Ca2+ stored in
the sarcoplasmic reticulum. Overexpression of cardiac calsequestrin leads to
cardiomyopathy and ablation of calreticulin alters cardiac development. 1. Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR)
Ca2+-ATPase (SERCA). Its presence and/or functional significance in
contractility of bladder, a smooth muscle tissue particularly dependent on SR
function, is unknown. We investigated this by measuring the effects of carbachol
(CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was
ablated (PLB-KO mice). In the PLB-KO bladder, the maximum increases in [Ca2+]i
and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50
values increased. 2. Inhibition of SERCA with cyclopiazonic acid (CPA) abolished
these differences between WT and PLB-KO bladder, localizing the effects to the
SR. 3. To determine whether these effects were specific to PLB, we generated
mice with smooth-muscle-specific expression of PLB (PLB-SMOE mice), using the
SMP8 alpha-actin promoter. Western blot analysis of PLB-SMOE mice showed
approximately an eightfold overexpression of PLB while SERCA was downregulated
12-fold. 4. In PLB-SMOE bladders, in contrast, the response of [Ca2+]i and force
to CCh was significantly increased and the EC50 values were decreased. CPA had
little affect on the CCh-induced increases in [Ca2+]i and force in PLB-SMOE
bladder. 5. These results show that alteration of the PLB:SERCA ratio can
significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that
PLB can play a major role in modulation of bladder contractility. We used EPR spectroscopy to probe directly the interaction between phospholamban
(PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA).
Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at
residue 11, which was then spin labeled. PLB was reconstituted into membranes in
the presence or absence of SERCA, and spin label mobility and accessibility were
measured. The spin label was quite rotationally mobile in the absence of SERCA,
but became more restricted in the presence of SERCA. SERCA also decreased the
dependence of spin label mobility on PLB concentration in the membrane,
indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL,
attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the
absence of SERCA. In the presence of SERCA, the spin label was released and a
covalent bond was formed between PLB and SERCA, indicating direct interaction of
one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the
PLB-bound spin label IPSL to paramagnetic agents, localized in different phases
of the membrane, indicates that SERCA greatly reduces the level of interaction
of the spin label with the membrane surface. We propose that the cytoplasmic
domain of PLB associates with the lipid surface, and that association with SERCA
induces a major conformational change in PLB in which the cytoplasmic domain is
drawn away from the lipid surface by SERCA. Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase
1 modulate the inhibitory activity of phospholamban (PLN), the endogenous
regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA).
This cyclic mechanism constitutes the driving force for calcium reuptake from
the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN
undergoes a conformational transition between a relaxed (R) and tense (T) state,
an equilibrium perturbed by the addition of SERCA. Here, we show that the single
phosphoryl transfer at Ser16 induces a more pronounced conformational switch to
the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA
is not affected (K(d) values for the transmembrane domains of pPLN and PLN are
approximately 60 microM), supporting the hypothesis that phosphorylation at
Ser16 does not dissociate PLN from SERCA. However, the binding surface and
dynamics in domain Ib (residues 22-31) change substantially upon
phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and
Thr17, we propose that these sites remotely control the conformation of domain
Ib. These findings constitute a paradigm for how post-translational
modifications such as phosphorylation in the cytoplasmic portion of membrane
proteins control intramembrane protein-protein interactions. Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase
(SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein
kinase II (CaM kinase II) phosphorylation. We previously reported significant
differences in contractility, SR Ca(2+) release, and CaM kinase II activity in
gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase
II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the
effect of PLB absence on contractility, CaM kinase II activity, and
intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and
amplitudes of spontaneous phasic contractions were elevated in antrum smooth
muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic
contractions in antrum smooth muscles from both control and PLB-KO mice.
Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of
antrum smooth muscle strips from PLB-KO mice, but the effects were less
pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was
less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle
strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not
further increased by caffeine, in antrum smooth muscles from PLB-KO mice.
Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further
increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings
suggest that PLB is an important modulator of gastric antrum smooth muscle
contractility by modulation of SR Ca(2+) release and CaM kinase II activity. Calcium transport across the membrane of the sarcoplasmic reticulum (SR) plays
an important role in the regulation of heart muscle contraction and relaxation.
The sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2a is responsible for
Ca(2+) up-take by this organelle and is inhibited in a reversible manner by
phospholamban, another SR membrane protein. Thus, alleviation of
phospholamban-mediated inhibition of SERCA2a is a potential therapeutic option
for heart failure and cardiomyopathy. We have now applied the systematic
evolution of ligands by exponential enrichment protocol to a library of
single-stranded DNA molecules containing a randomized 40-nucleotide sequence to
isolate aptamers that bind phospholamban. One of the obtained aptamers,
designated Apt-9, was found to specifically bind to the cytoplasmic region of
phospholamban in vitro with high affinity (dissociation constant, approximately
20 nM). Apt-9 increased the Ca(2+)-dependent ATPase activity of cardiac SR
vesicles but not that of SR vesicles from skeletal muscle in a
concentration-dependent manner. It also shifted the Ca(2+)
concentration-response curve for this ATPase activity to the left. These effects
of Apt-9 were not mimicked by an oligonucleotide with a scrambled version of the
Apt-9 sequence. Thus, our results indicate that Apt-9 activates SERCA2a by
alleviating the inhibitory effect of phospholamban on this ATPase, and they
suggest that phospholamban-specific aptamers warrant further investigation as
potential therapeutic agents for heart failure and cardiomyopathy. Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to
interactions between their respective transmembrane domains. In spite of
numerous mutagenesis and kinetic studies, we still do not have a clear
mechanistic picture of how PLB influences the calcium transport cycle of SERCA.
Herein, we have created alanine mutants for each residue in the transmembrane
domain of PLB, we have co-reconstituted these mutants with SERCA into
proteoliposomes, and we have performed kinetic simulations of the
calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a
variable effect on the calcium affinity, maximal activity, and cooperativity of
SERCA, such that a range of values was observed. Kinetic simulations using a
well-established reaction scheme for SERCA then allowed us to correlate the
effects on SERCA activity with changes in the reaction scheme rate constants.
Only three steps in the reaction scheme were affected by the presence of PLB,
namely, binding of the first calcium ion, a subsequent conformational change in
SERCA, and binding of the second calcium ion. The ability of wild-type and
mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated
with a decreased rate of binding of the second calcium ion. In addition, the
ability of wild-type and mutant forms of PLB to alter the maximal activity of
SERCA correlated with a change in the forward rate constant for the slow
conformational change in SERCA following binding of the first calcium ion. Phospholamban (PLB) is an integral membrane protein regulating Ca(2+) transport
through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase
(SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a
superinhibitor of the affinity of SERCA for Ca(2+) and of cardiac contractility
in vivo. The effects of this N27A mutation on the side-chain and backbone
dynamics of PLB were investigated with (2)H and (15)N solid-state NMR
spectroscopy in phospholipid multilamellar vesicles (MLVs). (2)H and (15)N NMR
spectra indicate that the N27A mutation does not significantly change the
side-chain or backbone dynamics of the transmembrane and cytoplasmic domains
when compared to wild-type PLB. However, dynamic changes are observed for the
hinge region, in which greater mobility is observed for the CD(3)-labeled Ala24
N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation
may allow the cytoplasmic helix to more easily interact with the Ca(2+)-ATPase;
thus, showing increased inhibition of Ca(2+)-ATPase. Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic
reticulum Ca(2+)-ATPase (SERCA), thereby regulating calcium homeostasis in
cardiac muscle. In membranes, PLN forms pentamers that have been proposed to
function either as a storage for active monomers or as ion channels. Here, we
report the T-state structure of pentameric PLN solved by a hybrid solution and
solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a
narrow hydrophobic pore, which excludes ion transport. In the T state, the
cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer
with the transmembrane domains arranged in a left-handed coiled-coil
configuration, crossing the bilayer with a tilt angle of approximately 11° with
respect to the membrane normal. The tilt angle difference between the monomer
and pentamer is approximately 13°, showing that intramembrane helix-helix
association forces dominate over the hydrophobic mismatch, driving the overall
topology of the transmembrane assembly. Our data reveal that both topology and
function of PLN are shaped by the interactions with lipids, which fine-tune the
regulation of SERCA. Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic
reticulum Ca(2+)-ATPase (SERCA), the integral membrane enzyme responsible for
70 % of the removal of Ca(2+) from the cytosol, inducing cardiac muscle
relaxation in humans. Dysfunctions in SERCA:PLN interactions have been
implicated as having a critical role in cardiac disease, and targeting Ca(2+)
transport has been demonstrated to be a promising avenue in treating conditions
of heart failure. Here, we designed a series of new mutants able to tune SERCA
function, targeting the loop sequence that connects the transmembrane and
cytoplasmic helices of PLN. We found that a variable degree of loss of
inhibition mutants is attainable by engineering glycine mutations along PLN's
loop domain. Remarkably, a double glycine mutation results in a complete
loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using
nuclear magnetic resoce spectroscopy, we rationalized the effects of these
mutations in terms of entropic control on PLN function, whose inhibitory
function can be modulated by increasing its conformational dynamics. However, if
PLN mutations go past a threshold set by the phosphorylated state, they break
the structural coupling between the transmembrane and cytoplasmic domains,
resulting in a species that behaves as the inhibitory transmembrane domain
alone. These studies provide new potential candidates for gene therapy to
reverse the effects of heart failure. |
What is TFBSshape? | To utilize DNA shape information when analysing the DNA binding specificities of TFs, the TFBSshape database was developed for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone. | |
Is Alpers disease inherited in an autosomal recessive mode? | Alpers disease is a fatal neurogenetic disorder first described more than 70 years ago. It is an autosomal recessive, developmental mitochondrial DNA depletion disorder characterized by deficiency in mitochondrial DNA polymerase gamma (POLG) catalytic activity, refractory seizures, neurodegeneration, and liver disease. | Alpers disease consists of diffuse cerebral degeneration manifested as
developmental delay, seizures, vomiting, and progressive neuromuscular
deterioration, with liver disease and death. We report the clinical course of
the liver disease, histologic progression of the hepatic lesions, and etiologic
investigations in five patients (four girls, three kinships). All had grown and
developed normally until seen at 6 to 36 months of age (mean 20 months), with
vomiting (n = 5), progressive hypotonia (n = 3), or seizures (n = 2). All had
been given anticonvulsants, including valproic acid in three. Liver disease was
noted at a mean age of 35 months (range 9 to 67 months), with hepatomegaly (two
patients), abnormal hepatic synthetic function (three) or transaminase values
(three), and cirrhosis in one. Patients survived for a mean of 4.6 weeks (range
1 to 8 weeks) after the identification of liver disease; all died of hepatic
failure. Results of evaluation for infectious and metabolic causes of liver
disease and causes of degenerative neuromuscular disease were negative in all
patients. Premortem liver biopsy specimens (n = 3) demonstrated an early lesion
consisting of lobular disarray, microvesicular steatosis, periportal acute and
chronic inflammation, and individual hepatocyte necrosis. Autopsy findings (n =
5) consisted of macrovesicular steatosis, massive hepatocyte dropout, and
proliferation of bile ductular elements, with almost complete replacement of
hepatocytes by proliferating bile ductular elements in two patients. Brain
showed characteristic neuronal degeneration. We conclude that Alpers disease can
be a cause of rapidly progressive liver failure in early childhood. Although the
cause of this autosomal recessive disease is not known, it does not appear to be
related to peroxisomal dysfunction. Two unrelated and previously healthy girls, aged 17 and 18, presented with a
subacute encephalopathy, visual and sensory symptoms and signs, and prominent
seizures that were difficult to control. Brain MRI showed lesions (high signal
on T2 weighted images) in the occipital lobes and thalamus; EEG showed slow wave
activity with superimposed polyspikes. Inexorable downhill progression led to
death in hepatic failure within eight months of onset. Histopathological
findings in both patients ((a) chronic hepatitis with prominent bile duct
proliferation, fatty change, and fibrosis; (b) in the brain a patchy destruction
of the cerebral cortex, predomitly involving striate cortex) were
characteristic of progressive neuronal degeneration of childhood with liver
disease--Alpers-Huttenlocher syndrome--a rare autosomal recessive disorder
usually seen in infants and young children. Alpers' syndrome is a fatal neurogenetic disorder first described more than 70
years ago. It is an autosomal recessive, developmental mitochondrial DNA
depletion disorder characterized by deficiency in mitochondrial DNA polymerase
gamma (POLG) catalytic activity, refractory seizures, neurodegeneration, and
liver disease. In two unrelated pedigrees of Alpers' syndrome, each affected
child was found to carry a homozygous mutation in exon 17 of the POLG locus that
led to a Glu873Stop mutation just upstream of the polymerase domain of the
protein. In addition, each affected child was heterozygous for the G1681A
mutation in exon 7 that led to an Ala467Thr substitution in POLG, within the
linker region of the protein. Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder
that affects children and young adults. It is characterized by a progressive,
fatal brain and liver disease. This syndrome has been associated with mutations
in POLG, the gene encoding the mitochondrial DNA polymerase (pol gamma). Most
patients with Alpers syndrome have been found to be compound heterozygotes,
carrying two pathogenic mutations in trans at the POLG locus. POLG is a
nuclear-encoded gene whose protein product is imported into mitochondria, where
it is essential for mtDNA replication and repair. We studied the skin
fibroblasts of a patient with Alpers syndrome having the genotype
E873stop/A467T. The E873stop mutation produces a premature termination codon
(TAG) in exon 17. The A467T mutation produces a threonine to alanine
substitution at a highly conserved site in exon 7. The allele bearing the stop
codon (E873-TAG) is predicted to produce a truncated, catalytically inactive
polymerase. However, only full-length pol gamma protein was detected by Western
blot analysis. Here, we show that transcripts containing this stop codon undergo
nonsense-associated alternative splicing and nonsense-mediated decay. More than
95% of the functional POLG mRNA was derived from the allele bearing the A467T
mutation and less than 5% contained the E873stop mutation. These events ensured
that virtually all POLG protein in the cell was expressed from the A467T allele.
Therefore, the Alpers phenotype in this patient was a consequence of a
single-copy gene dose of the A467T allele, and selective elimination of
transcripts bearing the E873stop mutation. Alpers disease is a recessive mitochondrial disorder caused by mutations in
POLG1 and characterized primarily by progressive neurological and hepatic
degeneration. Intestinal dysmotility is a frequent symptom, but it is often
overshadowed by other clinical manifestations. The onset and progression of
Alpers disease vary; however, most patients die during childhood, often before a
specific diagnosis has been established. The gastrointestinal neuromuscular
pathology of 4 patients, obtained largely from postmortem specimens, showed
distinctive eosinophilic cytoplasmic granules in a subset of enteric ganglia and
patchy atrophy of small intestinal muscularis externa. The cytoplasmic
inclusions corresponded to abnormal mitochondria, which have been reported
previously in another mitochondrial disorder (mitochondrial
neurogastrointestinal encephalomyopathy) but not in Alpers disease. Recognition
of these distinctive light microscopic findings, in an appropriate clinical
setting, should prompt the evaluation of an underlying primary
mitochondriopathy. |
Is vemurafenib effective for hairy-cell leukemia? | Yes, vemurafenib is highly effective in patients with relapsed or refractory hairy-cell leukemia. | The paper describes a case of a patient with refractory hairy cell leukemia. In
spite of the absence of CD25 expression, the disease was classified as a
classical form according to the WHO classification (2008), as also confirmed by
the detection of BRAFV600E mutation. The disease was characterized by resistance
to all lines of therapy (interferon-a, splenectomy, cladribin). Clinical and
hematological remission was achieved within 2 months of administration of the
BRAF kinase inhibitor vemurafenib. Hairy cell leukemia (HCL) shows unique clinicopathological and biological
features. HCL responds well to purine analogs but relapses are frequent and
novel therapies are required. BRAF-V600E is the key driver mutation in HCL and
distinguishes it from other B-cell lymphomas, including HCL-like
leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The
kinase-activating BRAF-V600E mutation also represents an ideal therapeutic
target in HCL. Here, we investigated the biological and therapeutic importance
of the activated BRAF-mitogen-activated protein kinase kinase
(MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in
vitro primary leukemic cells purified from 26 patients to clinically available
BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were
validated in vivo in samples from vemurafenib-treated HCL patients within a
phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but
not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the
BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene
expression signature, downregulation of the HCL markers CD25, tartrate-resistant
acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface,
and, eventually, apoptosis. Apoptosis was partially blunted by coculture with
bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This
protective effect could be counteracted by combined BRAF and MEK inhibition. Our
results strongly support and inform the clinical use of BRAF and MEK inhibitors
in HCL. MEDICAL HISTORY AND CLINICAL COURSE: A 42-year-old patient with hairy cell
leukemia had been treated for 3 years by a hematologist in private practice.
Initially the patient received 1 course of cladribine upon which the disease
went into complete remission. 6 weeks ago a relapse was diagnosed and
combination therapy with cladibrin and rituximab was initiated. Now the patient
presented to the emergency room with shortness of breath and pain when
breathing.
INVESTIGATIONS, TREATMENT AND COURSE: In the chest x-ray, patchy infiltrates and
pleural effusions were found on both sides. The subsequently performed computed
tomography showed bilateral compactions with an Halo suspicious for fungal
infiltrates. Upon admission to the hospital, an empirical antibiotic therapy
with clarithromycin and piperacillin/tazobactam was initiated, which was later
escalated to meropenem and linezolid. Additionally, an antifungal therapy with
voriconazole was started and later switched to liposomal amphotericin B. At his
admission, a positive aspergillus antigen could be detected in the
microbiological laboratory. Under antimycotic treatment the aspergillus antigen
was repeatedly negative. The patient presented with pronounced cytopenias and
after a switch of therapy to vemurafenib and filgrastim, the hematopoiesis could
only be stimulated insufficiently. The patient was transferred to the intensive
care unit three days after admission with severe respiratory failure. He died on
day 8 after admission. AUTOPSY AND DIAGNOSIS: Diagnosis was consistent with
relapse of hairy cell leukemia with positive BRAF mutation and a bone marrow
infiltration > 80 %. Autopsy revealed a significant hepato-splenomegaly, a lack
of erythro-, granulo- and thrombopoiesis. Clots interspersed with fungal hyphae
were found in both lungs and an infarction of the spleen with evidence of fungal
hyphae was detected. The cultural findings post mortem on yeast or mold were
negative.
CONCLUSION: Patients with refractory hairy cell leukemia and prolonged
neutropenia are at increased risk for systemic fungal infections. Therefore,
prohylactic antimycotic therapy should be considered early in this group of
patients. The therapeutic approach of vemurafenib in treatment-refractory hairy
cell leukemia is promising and offers an additional treatment option. In the
present case, the patient could unfortunately not be stabilized due to the
septic complications. BACKGROUND: BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We
assessed the safety and activity of the oral BRAF inhibitor vemurafenib in
patients with hairy-cell leukemia that had relapsed after treatment with a
purine analogue or who had disease that was refractory to purine analogues.
METHODS: We conducted two phase 2, single-group, multicenter studies of
vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the
United States. The therapy was administered for a median of 16 weeks in the
Italian study and 18 weeks in the U.S. study. Primary end points were the
complete response rate (in the Italian trial) and the overall response rate (in
the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in
April 2013 and is still open (26 of 36 planned patients) in the U.S. trial.
RESULTS: The overall response rates were 96% (25 of 26 patients who could be
evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24)
after a median of 12 weeks in the U.S. study. The rates of complete response
were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively.
In the Italian trial, after a median follow-up of 23 months, the median
relapse-free survival was 19 months among patients with a complete response and
6 months among those with a partial response; the median treatment-free survival
was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the
progression-free survival rate was 73% and the overall survival rate was 91%.
Drug-related adverse events were usually of grade 1 or 2, and the events most
frequently leading to dose reductions were rash and arthralgia or arthritis.
Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50
patients. The frequent persistence of phosphorylated ERK-positive leukemic cells
in bone marrow at the end of treatment suggests bypass reactivation of MEK and
ERK as a resistance mechanism.
CONCLUSIONS: A short oral course of vemurafenib was highly effective in patients
with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione
Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13;
ClinicalTrials.gov number NCT01711632.). |
Which are the inhibitors of histone methyltransferases? | In general, histone methyltransferases (HMTs) have no widely approved high-throughput screening assay format, and therefore reference inhibitors are not available for many of the HMTs. However, there are several selective HMT inhibitors: Trichostatin A (TSA), BIX-01294 and its derivative TM2-115, 2,4-pyridinedicarboxylic acid (2,4-PDCA), 3-deazaneplanocin A (DZNep), Psammaplin A (PsA) and Sulforaphane (SFN). | A copper-mediated aerobic coupling reaction enables direct amidation of
heterocycles or aromatics having weakly acidic C-H bonds with a variety of
nitrogen nucleophiles. These reactions provide efficient access to many
biologically important skeletons, including ones with the potential to serve as
inhibitors of HMTs. Cancer-testis antigens (CTA), such as NY-ESO-1, MAGE-A1, and MAGE-A3, are
immunogenic proteins encoded by genes, which are normally expressed only in male
germ cells but are activated by ill-defined epigenetic mechanisms in human
tumors, including lung cancers. Previously, we reported induction of these CTAs
in cancer cells, but not normal cells, by DNA-demethylating agents and histone
deacetylase inhibitors using clinically achievable exposure conditions. In the
present study, we evaluated chromatin alterations associated with
repression/activation of cancer-testis genes in lung cancer cells to further
develop gene-induction regimens for cancer immunotherapy. Repression of
NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment,
and binding of polycomb-group proteins, and histone heterochromatin
modifications within the promoters of these genes. Derepression coincided with
DNA demethylation, dissociation of polycomb proteins, and presence of
euchromatin marks within the respective promoters. Short hairpin RNAs were used
to inhibit several histone methyltransferases (KMT) and histone demethylases
(KDM) that mediate histone methylation and repress gene expression. Knockdown of
KMT6, KDM1, or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated
activation of these cancer-testis genes in lung cancer cells. DZNep, a
pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6
knockdown. Following DAC-DZNep exposure, lung cancer cells were specifically
recognized and lysed by allogeneic lymphocytes expressing recombit T-cell
receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA-demethylating agents
with compounds, such as DZNep, that modulate histone lysine methylation may
provide a novel epigenetic strategy to augment cancer-testis gene expression as
an adjunct to adoptive cancer immunotherapy. Histone H3-lysine79 (H3K79) methyltransferase DOT1L plays critical roles in
normal cell differentiation as well as initiation of acute leukemia. We used
structure- and mechanism-based design to discover several potent inhibitors of
DOT1L with IC(50) values as low as 38 nM. These inhibitors exhibit only weak or
no activities against four other representative histone lysine and arginine
methyltransferases, G9a, SUV39H1, PRMT1 and CARM1. The X-ray crystal structure
of a DOT1L-inhibitor complex reveals that the N6-methyl group of the inhibitor,
located favorably in a predomitly hydrophobic cavity of DOT1L, provides the
observed high selectivity. Structural analysis shows that it will disrupt at
least one H-bond and/or have steric repulsion for other histone
methyltransferases. These compounds represent novel chemical probes for
biological function studies of DOT1L in health and disease. Histone posttranslational modifications are among the epigenetic mechanisms that
modulate chromatin structure and gene transcription. Histone methylation and
demethylation are dynamic processes controlled respectively by histone
methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have
been implicated in cancer, inflammation, and diabetes, making them attractive
targets for drug therapy. Hence, the discovery of small-molecule modulators for
these two enzyme classes has drawn significant attention from the pharmaceutical
industry. Herein, the authors describe the development and optimization of
homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the
catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3:
SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays
were designed as signal-increase assays using biotinylated peptides derived from
the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated
using full-length histone H3 protein as substrate in the AlphaLISA format.
Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive,
requiring only omolar concentrations of enzyme and substrate. All assays
allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate
that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant
biochemical screening approach toward the identification of small-molecule
inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies. 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. Dynamic methylations and demethylations of histone lysine residues are important
for gene regulation and are facilitated by histone methyltransferases and
histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific
lysine demethylase belonging to the JmjC domain-containing family of histone
demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and
skin cancer, highlighting its potential as a drug target. However, most
inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain
to be explored. Here, we report the expression, purification and
characterization of the catalytic core of recombit KDM5B (ccKDM5B, residues
1-769). We show that ccKDM5B, recombitly expressed in insect cells,
demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed
that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm
for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent
substrate affinity than reported for related HDMs. Despite the presence of a PHD
domain, the catalytic activity was not affected by additional methylation at the
H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and
H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in
vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid
(2,4-PDCA) with a potency similar to that reported for the HDM KDM4C.
Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA
is conserved between KDM4A/C and KDM5B. BACKGROUND: Histone modifications and DNA methylation are two major factors in
epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are
known to exert radiosensitizing effects, there have only been a few studies thus
far concerning the role of DNA methyltransferase (DNMT) inhibitors as
radiosensitizers. The principal objective of this study was to evaluate the
effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines,
and to elucidate the mechanisms relevant to that process.
METHODS: A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to
radiation with or without six DNMT inhibitors (5-azacytidine,
5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and
psammaplin A) for 18 hours prior to radiation, after which cell survival was
evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow
cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via
Western blotting. Expression of γH2AX, a marker of radiation-induced DNA
double-strand break, was examined by immunocytochemistry.
RESULTS: Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine
radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A
increased the sub-G1 fraction of A549 cells, as compared to cells exposed to
radiation alone. Prolongation of γH2AX expression was observed in the cells
treated with DNMT inhibitors prior to radiation as compared with those treated
by radiation alone.
CONCLUSIONS: Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce
radiosensitivity in both A549 and U373MG cell lines, and suggest that this
effect might be associated with the inhibition of DNA repair. Epidemiological studies suggest an important link between obesity and thyroid
cancer. The adipose tissue-derived polypeptide leptin acting via leptin receptor
may modulate cell migration of thyroid cancer cells. Previously we have
demonstrated that leptin receptor is overexpressed in papillary thyroid cancer
and is associated with tumor aggressiveness. The present study was undertaken to
explore the possible regulatory factors which would influence leptin receptor
expression in papillary thyroid cancer cells. We found that DNA
methyltransferase inhibitor (5-Aza-2'-deoxycytidine) and histone deacetylase
inhibitor (trichostatin A) reduced leptin receptor expression. Conversely,
insulin upregulated leptin receptor expression in a time- and dose-dependent
manner. Hypoxia-mimicking agent (cobalt chloride) had no effect on leptin
receptor expression. Taken together, our study provides evidence that epigenetic
events and insulin stimulation take part in regulation of leptin receptor
expression in papillary thyroid cancer cells. BACKGROUND: Histone deacetylase (HDAC) inhibitors are a group of small chemical
molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have
multiple biological effects such as cell cycle arrest, apoptosis, cell
differentiation and auotophagy. At molecular level, HDAC inhibitors cause
histone and nonhistone acetylation and induce gene expression. HDAC inhibitors
are widely used in cancer therapy because of its function of inducing apoptosis.
However, the mechanisms of apoptosis effect are not fully understood. TSA is a
classical HDAC inhibitor and widely used in epigenetic and anti-cancer research.
In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of
HDAC inhibitors apoptotic effect on cancer cells.
METHODS: Cervical cancer cell lines such as Hela, Caski and normal human
keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal
violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay were performed to determine cell number. PARP cleavage and
FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase
(DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western
Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.
RESULTS: HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L
of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC
inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of
normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L
group with untreated control with t-test. There was no significance between 1
µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors
decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of
DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski
cell went apoptosis after deprived of DNMT3B.
CONCLUSIONS: DNMT3B was essential to cervical cancer cell survival.
Down-regulated DNMT3B by HDAC inhibitors may play an important role in the
toxicity of HDAC inhibitors on cervical cancer cells. Epigenetic factors such as histone methylation control the developmental
progression of malaria parasites during the complex life cycle in the human
host. We investigated Plasmodium falciparum histone lysine methyltransferases as
a potential target class for the development of novel antimalarials. We
synthesized a compound library based upon a known specific inhibitor (BIX-01294)
of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its
derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC(50)
values of ~100 nM, values at least 22-fold more potent than their apparent
IC(50) toward two human cell lines and one mouse cell line. These compounds
irreversibly arrested parasite growth at all stages of the intraerythrocytic
life cycle. Decrease in parasite viability (>40%) was seen after a 3-h
incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a
12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain
infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced
parasitemia the following day. Importantly, treatment of P. falciparum parasites
in culture with BIX-01294 or TM2-115 resulted in significant reductions in
histone H3K4me3 levels in a concentration-dependent and exposure time-dependent
manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit
malaria parasite histone methyltransferases, resulting in rapid and irreversible
parasite death. Our data position histone lysine methyltransferases as a
previously unrecognized target class, and BIX-01294 as a promising lead
compound, in a presently unexploited avenue for antimalarial drug discovery
targeting multiple life-cycle stages. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the
Polycomb-repressive complex 2 (PRC2) that epigenetically silences gene
transcription through histone H3 lysine trimethylation (H3K27me3). EZH2 has been
implicated in stem cell maintece and is overexpressed in hematological and
solid maligcie`s including maligt glioma. EZH2 is thought to promote tumor
progression by silencing tumor suppressor genes. Hence pharmacological
disruption of the PRC2 is an attractive therapeutic strategy for cancer
treatment. Here we show that EZH2 is expressed in human glioma and correlates
with maligcy. Silencing of EZH2 reduced glioma cell proliferation and
invasiveness. While we did not observe induction of cell cycle-associated tumor
suppressor genes by silencing or pharmacological inhibition of EZH2, microarray
analyses demonstrated a strong transcriptional reduction of the AXL receptor
kinase. Neither histone nor DNA methylation appeared to be involved in the
positive regulation of AXL by EZH2. Silencing AXL mimicked the antiinvasive
effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas
with high EZH2 expression. Collectively these data suggest that EZH2 drives
glioma invasiveness via transcriptional control of AXL independent of histone or
DNA methylation. Satellite cells function as skeletal muscle stem cells to support postnatal
muscle growth and regeneration following injury or disease. There is great
promise for the improvement of muscle performance in livestock and for the
therapy of muscle pathologies in humans by the targeting of myostatin (MSTN) in
this cell population. Human diet contains many histone deacetylase (HDAC)
inhibitors, such as the bioactive component sulforaphane (SFN), whose epigenetic
effects on MSTN gene in satellite cells are unknown. Therefore, we aimed to
investigate the epigenetic influences of SFN on the MSTN gene in satellite
cells. The present work provides the first evidence, which is distinct from the
effects of trichostatin A (TSA), that SFN supplementation in vitro not only acts
as a HDAC inhibitor but also as a DNA methyltransferase (DNMT) inhibitor in
porcine satellite cells. Compared with TSA and 5-aza-2'-deoxycytidine
(5-aza-dC), SFN treatment significantly represses MSTN expression, accompanied
by strongly attenuated expression of negative feedback inhibitors of the MSTN
signaling pathway. miRNAs targeting MSTN are not implicated in
posttranscriptional regulation of MSTN. Nevertheless, a weakly enriched myoblast
determination (MyoD) protein associated with diminished histone acetylation in
the MyoD binding site located in the MSTN promoter region may contribute to the
transcriptional repression of MSTN by SFN. These findings reveal a new mode of
epigenetic repression of MSTN by the bioactive compound SFN. This novel
pharmacological, biological activity of SFN in satellite cells may thus allow
for the development of novel approaches to weaken the MSTN signaling pathway,
both for therapies of human skeletal muscle disorders and for livestock
production improvement. This study reports the pharmacokinetics and tissue distribution of a novel
histone deacetylase and DNA methyltransferase inhibitor, psammaplin A (PsA), in
mice. PsA concentrations were determined by a validated LC-MS/MS assay method
(LLOQ 2 ng/mL). Following intravenous injection at a dose of 10 mg/kg in mice,
PsA was rapidly eliminated, with the average half-life (t(1/2, λn)) of 9.9 ± 1.4
min and the systemic clearance (CL(s)) of 925.1 ± 570.1 mL/min. The in vitro
stability of PsA was determined in different tissue homogenates. The average
degradation t(1/2) of PsA in blood, liver, kidney and lung was found relatively
short (≤ 12.8 min). Concerning the in vivo tissue distribution characteristics,
PsA was found to be highly distributed to lung tissues, with the lung-to-serum
partition coefficients (K(p)) ranging from 49.9 to 60.2. In contrast, PsA
concentrations in other tissues were either comparable with or less than serum
concentrations. The high and specific lung targeting characteristics indicates
that PsA has the potential to be developed as a lung cancer treatment agent. BACKGROUND: Estrogen receptor-α (ERα)-negative breast cancer is clinically
aggressive and normally does not respond to conventional estrogen
target-directed therapies. The soybean isoflavone, genistein (GE), has been
shown to prevent and inhibit breast cancer and recent studies have suggested
that GE can enhance the anticancer capacity of an estrogen antagonist, tamoxifen
(TAM), especially in ERα-positive breast cancer cells. However, the role of GE
in ERα-negative breast cancer remains unknown.
METHODS: We have evaluated the in vitro and in vivo epigenetic effects of GE on
ERα reactivation by using MTT assay, real-time reverse transcription-polymerase
chain reaction (RT-PCR) assay, western-blot assay, immunoprecipitation (ChIP)
assay, immunohistochemistry and epigenetic enzymatic activity analysis.
Preclinical mouse models including xenograft and spontaneous breast cancer mouse
models were used to test the efficacy of GE in vivo.
RESULTS: We found that GE can reactivate ERα expression and this effect was
synergistically enhanced when combined with a histone deacetylase (HDAC)
inhibitor, trichostatin A (TSA), in ERα-negative MDA-MB-231 breast cancer cells.
GE treatment also re-sensitized ERα-dependent cellular responses to activator
17β-estradiol (E2) and antagonist TAM. Further studies revealed that GE can lead
to remodeling of the chromatin structure in the ERα promoter thereby
contributing to ERα reactivation. Consistently, dietary GE significantly
prevented cancer development and reduced the growth of ERα-negative mouse breast
tumors. Dietary GE further enhanced TAM-induced anti-cancer efficacy due at
least in part to epigenetic ERα reactivation.
CONCLUSIONS: Our studies suggest that soybean genistein can epigenetically
restore ERα expression, which in turn increases TAM-dependent anti-estrogen
therapeutic sensitivity in vitro and in vivo. The results from our studies
reveal a novel therapeutic combination approach using bioactive soybean product
and anti-hormone therapy in refractory ERα-negative breast cancer which will
provide more effective options in breast cancer therapy. |
What is the main characteristic of Amyotrophic Lateral Sclerosis? | Amyotrophic lateral sclerosis (ALS) is a progressive degeneration of upper and lower motor neurons. | There are four main hypotheses about the cause of ALS: excitotoxicity linked to
glutamate receptor overactivation; mutation of the superoxide dismutase gene;
production of autoantibodies to calcium channels; neurofilament accumulation.
The motoneuron degeneration characteristic of ALS could be caused by any one or
a combination of these mechanisms. Future therapeutic approaches should be based
on these mechanisms and given in combination so that different levels of the
degenerative process are targeted. Protection against excitotoxicity could be
achieved with a combination of pharmacologic agents having neuroprotective
activity, such as antiglutamate agents (e.g., riluzole), N-methyl-D-aspartate
(NMDA) and non-NMDA antagonists, free-radical scavengers, calcium-channel
blockers, and neurotrophic factors. Gene transfer is a possible future approach
when causative mutations are identified. Transfer of genes encoding
neuroprotective agents or genetically modified cells stably expressing these
agents is another possible strategy. Amyotrophic lateral sclerosis (ALS) is a progressive degeneration of upper and
lower motor neurons. In the absence of any validated biological marker, the
diagnosis of ALS depends upon recognition of characteristic symptoms and signs
together with supportive electrophysiological findings. The diagnosis of ALS is
easy to recognize in its fully developed form but during the early stages both
false positive and false negative diagnoses are common. In clinical practice,
diagnostic difficulties mostly arise with patients who present either with only
upper motor neuron, or with only lower motor neuron signs. It may be difficult
to distinguish ALS with clinically predomit lower motor neuron involvement
from alternative diagnoses including spinal atrophies of adult onset, Kennedy's
disease, inclusion body myositis and motor neuropathies with conduction blocks.
The diagnosis of ALS related syndromes (progressive muscular atrophy, primary
lateral sclerosis and progressive bulbar palsy) requires the elimination of
alternate diagnoses. This paper reviews the main characteristics of diseases
mimicking ALS and the atypical subsets of ALS. Amyotrophic lateral sclerosis is the most common adult-onset motor neuron
disease and evidence from mice expressing amyotrophic lateral sclerosis-causing
SOD1 mutations suggest that neurodegeneration is a non-cell autonomous process
where microglial cells influence disease progression. However,
microglial-derived neurotoxic factors still remain largely unidentified in
amyotrophic lateral sclerosis. With excitotoxicity being a major mechanism
proposed to cause motor neuron death in amyotrophic lateral sclerosis, our
hypothesis was that excessive glutamate release by activated microglia through
their system [Formula: see text] (a cystine/glutamate antiporter with the
specific subunit xCT/Slc7a11) could contribute to neurodegeneration. Here we
show that xCT expression is enriched in microglia compared to total mouse spinal
cord and absent from motor neurons. Activated microglia induced xCT expression
and during disease, xCT levels were increased in both spinal cord and isolated
microglia from mutant SOD1 amyotrophic lateral sclerosis mice. Expression of xCT
was also detectable in spinal cord post-mortem tissues of patients with
amyotrophic lateral sclerosis and correlated with increased inflammation.
Genetic deletion of xCT in mice demonstrated that activated microglia released
glutamate mainly through system [Formula: see text]. Interestingly, xCT deletion
also led to decreased production of specific microglial
pro-inflammatory/neurotoxic factors including nitric oxide, TNFa and IL6,
whereas expression of anti-inflammatory/neuroprotective markers such as
Ym1/Chil3 were increased, indicating that xCT regulates microglial functions. In
amyotrophic lateral sclerosis mice, xCT deletion surprisingly led to earlier
symptom onset but, importantly, this was followed by a significantly slowed
progressive disease phase, which resulted in more surviving motor neurons. These
results are consistent with a deleterious contribution of microglial-derived
glutamate during symptomatic disease. Therefore, we show that system [Formula:
see text] participates in microglial reactivity and modulates amyotrophic
lateral sclerosis motor neuron degeneration, revealing system [Formula: see
text] inactivation, as a potential approach to slow amyotrophic lateral
sclerosis disease progression after onset of clinical symptoms. |
Which genes are regulated by MEF-2 in the heart? | COX-2, ANF, estrogen receptor (ER)alpha gene, calsequestrin gene, casq2, cTnT, MCK, alpha-cardiac actin, sarco(endo)plasmic reticulum Ca2+-ATPase, SERCA, MLC-2, alpha-cardiac myosin heavy chain gene, phosphoglycerate mutase and PGAM-M are regulated by MEF-2 in the heart | In order to analyze the transcriptional regulation of the muscle-specific
subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes
composed of the upstream region of the PGAM-M gene and the bacterial
chloramphenicol acetyltransferase (CAT) gene were constructed and transfected
into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and
C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted
in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured
cardiac muscle cells, the segment between nucleotides -165 and +41 relative to
the transcription initiation site was sufficient to confer maximal CAT activity.
This region contains two E boxes and one MEF-2 motif. Deletion and substitution
mutation analysis showed that a single MEF-2 motif but not the E boxes had a
substantial effect on skeletal and cardiac muscle-specific enhancer activity and
that the cardiac muscle-specific negative regulatory region was located between
nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected
with MyoD into C3H10T1/2, the profile of CAT activity was similar to that
observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the
nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M
MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M
MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M
MEF-2 oligomer. These observations define the PGAM-M enhancer as the only
cardiac- and skeletal-muscle-specific enhancer characterized thus far that is
mainly activated through MEF-2. Recent studies have identified a conserved 28-bp element (HF-1) within the rat
cardiac MLC-2 gene which confers cardiac muscle-specific and inducible
expression during myocardial cell hypertrophy. Utilizing a combination of
independent experimental approaches, this study characterizes two cardiac
nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A +
T-rich binding factor (HF-1b) which is preferentially expressed in
differentiated cardiac and skeletal muscle cells. The HF-1a binding site is
located in a core region of the 28-bp conserved element, immediately upstream
from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a
number of muscle genes. By a number of separate criteria (gel mobility shift,
competition, and mutagenesis studies), HF-1b and MEF-2 appear to be
indistinguishable and thus are either identical or closely related muscle
factors. Transient assays of luciferase reporter genes containing point
mutations throughout the 28-bp HF-1 regulatory element document the importance
of both the HF-1a and HF-1b sites in transient assays in ventricular muscle
cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E
box had little effect on cardiac muscle specificity, while point mutations in
either the HF-1a or HF-1b binding site significantly reduced promoter activity,
underscoring the importance of both the HF-1a and HF-1b sites in the
transcriptional activation of this cardiac muscle gene. Thus, this study
provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor
(HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific
expression in ventricular cardiac muscle cells. Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC)
gene is regulated by multiple elements in the proximal enhancer/promoter. Within
this region, an M-CAT site and an A-rich site were identified as potential
regulatory elements. Site-specific mutations in each site, individually, reduced
activity from the wild-type promoter by approximately 85% in the adult rat
heart, demonstrating that these sites were positive regulatory elements.
alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites
interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts
that was immunologically related to transcriptional enhancer factor 1, a factor
that binds within the simian virus 40 enhancer. The factor that bound the A-rich
region (ARF) was antigenically related to the RSRF family of proteins, ARF was
distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of
DNA-binding specificity and developmental expression. Like MEF-2, ARF
DNA-binding activity was present in the heart and brain; however, no ARF
activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF
and ARF DNA-binding activities were developmentally regulated with peak levels
in the 1- to 2-day neonatal heart. The activity of both factors increased nearly
fivefold in adult rat hearts subjected to a pressure overload. By comparison,
the levels of alpha-MHC binding factor 2 did not change during hypertrophy.
Binding sites for MCBF and ARF are present in several genes that are upregulated
during cardiac hypertrophy. Our results suggest that these factors participate
in the alterations in gene expression that occur during cardiac development and
hypertrophy. The AT-rich element MEF-2 plays an important role in the maintece of the
muscle-specific expression of a number of cardiac and skeletal muscle genes. In
the MLC-2 gene, an AT-rich element (HF-1b) which contains a consensus MEF-2 site
is required for cardiac tissue-specific expression. The present study reports
the isolation and characterization of a cDNA which encodes a novel C2H2 zinc
finger (HF-1b) that binds in a sequence-specific manner to the HF-1b/MEF-2 site
in the MLC-2 promoter. A number of independent criteria suggest that this HF-1b
zinc finger protein is a component of the endogenous HF-1b/MEF-2 binding
activity in cardiac muscle cells and that it can serve as a transcriptional
activator of the MLC-2 promoter in transient assays. These studies suggest that,
in addition to the previously reported RSRF proteins, structurally divergent
transcriptional factors can bind to MEF-2-like sites in muscle promoters. These
results underscore the complexity of the regulation of the muscle gene program
via these AT-rich elements in cardiac and skeletal muscle. The intergenic region between the mouse alpha-cardiac myosin heavy chain and
beta-myosin heavy chain genes has previously been shown to direct expression of
the bacterial chloramphenicol acetyltransferase reporter gene in transgenic mice
in a tissue-specific manner. Sequence analyses located a putative
myocyte-specific enhancer-binding factor (MEF-2) site situated in the regulatory
region of this gene proximal to the start site of transcription. The role of
this element in directing the cardiac compartment-specific expression of the
transgene was assessed. The polymerase chain reaction was used to perform
substitution mutagenesis of the MEF-2 binding site, and lack of MEF-2 binding
was confirmed by gel retardation assays. The resultant construct was used to
generate transgenic mice. Surprisingly, transgene expression was not
down-regulated, but was significantly increased in the hearts of the MEF-2
mutant mice. In addition, cardiac-specific expression of the transgene was
perturbed with significant levels of ectopic expression occurring in the aorta. The molecular determits that direct gene expression to the ventricles of the
heart are for the most part unknown. Additionally, little data is available on
how the anterior/posterior axis of the heart tube is determined and whether the
left and right atrial and ventricular chambers are assigned as part of this
process. Utilizing myosin light chain-2 ventricular promoter/beta-galactosidase
reporter transgenes, we have determined the minimal cis-acting sequences
required for ventricular-specific gene expression. In multiple independent
transgenic mouse lines, we found that both a 250 base pair myosin light chain-2
ventricular promoter fragment, as well as a dimerized 28 bp sub-element (HF-1)
containing binding sites for HF1a and HF1b/MEF2 factors, directed
ventricular-specific reporter expression from as early as the endogenous gene,
at day 7.5-8.0 post coitum. While the endogenous gene is expressed uniformly
throughout both ventricles, the transgenes were expressed in a right
ventricular/conotruncal domit fashion, suggesting that they contain only a
subset of the elements which respond to positional information in the developing
heart tube. Expression of the transgene was cell autonomous and its
temporospatial characteristics not affected by mouse strain/methylation state of
the genome. To determine whether ventricular-specific expression of the
transgene was dependent upon regulatory genes required for correct ventricular
differentiation, the 250 base pair transgene was bred into both retinoid X
receptoralpha and Nkx2-5 null backgrounds. The transgene was expressed in both
mutant backgrounds, despite the absence of endogenous myosin light chain-2
ventricular transcript in Nkx2-5 null embryos. Ventricular specification, as
judged by transgene expression, appeared to occur normally in both mutants.
Thus, the HF-1 element, directs chamber-specific transcription of a transgene
reporter independently of retinoid X receptoralpha and Nkx2-5, and defines a
minimal combinatorial pathway for ventricular chamber gene expression. The
patterned expression of this transgene may provide a model system in which to
investigate the cues that dictate anterior-posterior (right ventricle/left
ventricle) gradients during mammalian heart development. Thyroid hormone (T3) increases the transcription of the sarcoplasmic reticulum
Ca2+ adenosine triphosphatase (ATPase) gene (SERCA 2) through three thyroid
hormone response elements. The existence of repetitive cis elements with
different configurations is likely to serve specific functions such as
interactions with nuclear transcription factors. In addition, the presence of
different T3 receptor isoforms (T3Rs) may contribute to another level of
complexity in providing specificity for T3 action. In this study, we
investigated T3R alpha 1-vs. T3R beta 1-specific interactions with the myocyte
enhancer-specific factor-2 (MEF-2) on the expression of the SERCA 2 gene in
transient transfection assays in embryonal heart-derived H9c2 cells. MEF-2a in
combination with either T3R alpha 1 or T3R beta 1 isoforms resulted in a
2.5-fold increase in SERCA 2 transgene expression in the absence of T3. Addition
of T3 did not induce any further increase in SERCA 2 expression when T3R alpha 1
and MEF-2a expression vectors were cotransfected. In contrast, in the presence
of T3R beta 1 and MEF-2, the addition of T3 increased chlorampenicol
acetyltransferase activity by an additional 2.2-fold to a total 5.5-fold
increase. The interaction between MEF-2a and T3R is transcription factor
specific because another factor that binds to MEF-2 consensus sites (heart
factor 1b) was not able to interact with T3R. In addition, MEF-2a failed to
interact with other nuclear factors (cAMP response element-binding protein and
Egr-1) that stimulate SERCA 2 gene transcription. In addition, we found that a
single homologous thyroid hormone response element is not able to mediate the
interactions between MEF-2a and T3Rs to increase SERCA 2 gene transcription. Our
findings point to T3R isoform-specific interactions with a cell type-specific
transcription factor (MEF-2) in the regulation of SERCA 2 gene expression. We have characterized the specific DNA regulatory elements responsible for the
function of the human cardiac troponin C gene (cTnC) muscle-specific enhancer in
myogenic cells. We used functional transient transfection assays with deletional
and site-specific mutagenesis to evaluate the role of the conserved sequence
elements. Gel electrophoresis mobility shift assays (EMSA) demonstrated the
ability of the functional sites to interact with nuclear proteins. We
demonstrate that three distinct transcription activator binding sites commonly
found in muscle-specific enhancers (a MEF-2 site, a MEF-3 site, and at least
four redundant E-box sites) all contribute to full enhancer activity but a CArG
box does not. Mutation of either the MEF-2 or MEF-3 sites or deletion of the
E-boxes reduces expression by 70% or more. Furthermore, the MEF-2 site and the
E-boxes specifically bind, respectively, to MEF-2 and myogenic determination
factors derived from nuclear extracts. EMSA assays using a MEF-3 containing
oligonucleotide revealed indistinguishable separation patterns with extracts
from myogenic cells and nonmyogenic cells. These data suggest that expression of
the cTnC gene in slow-twitch skeletal muscle is sustained through complex
interactions at the 3'Ile enhancer between muscle-specific and
nontissue-specific transcription factors: either a myogenic bHLH complex or
MEF-2 can activate transcription but only in the presence of a third
transcriptional activator that appears not to be muscle specific. We conclude
from these observations that the cTnC 3'Ile element is a composite enhancer that
functions through the combined interactions of at least five regulatory elements
and their cognate binding factors: three or four E-boxes, a MEF-2 site, and a
MEF-3 site. The data support the notion that all of these sites contribute to
enhancer function in cell systems in an additive way but that none are
absolutely required for enhancer activity. The data imply that the levels of
transcription of cTnC in myogenic tissues in which the activities of one of the
transcriptional factors is lacking would be partially but not wholly suppressed.
Our data support the critical role of E-box sites in conjunction with the
adjacent elements. Hence, we assign CTnC gene regulation to the "ordinary"
rather than to the "novel" category of transcriptional regulation during
skeletal myogenesis. Expression of the mouse alpha-cardiac actin gene in skeletal and cardiac muscle
is regulated by enhancers lying 5' to the proximal promoter. Here we report the
characterization of a cardiac-specific enhancer located within -2.354/-1.36 kbp
of the gene, which is active in cardiocytes but not in C2 skeletal muscle cells.
In vivo it directs reporter gene expression to the adult heart, where the
proximal promoter alone is inactive. An 85-bp region within the enhancer is
highly conserved between human and mouse and contains a central AT-rich site,
which is essential for enhancer activity. This site binds myocyte enhancer
factor (MEF)2 factors, principally MEF2D and MEF2A in cardiocyte nuclear
extracts. These results are discussed in the context of MEF2 activity and of the
regulation of the alpha-cardiac actin locus. MCAT elements are essential for cardiac gene expression during development.
Avian transcriptional enhancer factor-1 (TEF-1) proteins are muscle-enriched and
contribute to MCAT binding activities. However, direct activation of MCAT-driven
promoters by TEF-1-related proteins has not been uniformly achieved. Divergent
TEF (DTEF)-1 is a unique member of the TEF-1 multigene family with abundant
transcripts in the heart but not in skeletal muscle. Herein we show that DTEF-1
proteins are highly expressed in the heart. Protein expression is activated at
very early stages of chick embryogenesis (Hamburger-Hamilton stage 4, 16-18 h),
after which DTEF-1 becomes abundant in the sinus venosus and is expressed in the
trabeculated ventricular myocardium and ventricular outflow tracts. By chromatin
immunoprecipitation, DTEF-1 interacts with the cardiac troponin T (cTnT)
promoter in vivo. DTEF-1 also interacts with MEF- 2 by coimmunoprecipitation and
independently or cooperatively (with MEF-2) trans-activates the cTnT promoter.
DTEF-1 isoforms do not activate the cTnT promoter in fibroblasts or skeletal
muscle. DTEF-1 expression occurs very early in chick embryogenesis (16-18 h),
preceding sarcomeric protein expression, and it activates cardiac promoters. As
such, DTEF-1 may be an early marker of the myocardial phenotype. DTEF-1
trans-activates the cTnT promoter in a tissue-specific fashion independent of
AT-rich, MEF-2, or GATA sites. The observed spatial pattern suggests decreasing
levels of expression from the cardiac inlet to the ventricular outflow tracts,
which may mark a cardiogenic or differentiation pathway that parallels the
direction of flow through the developing chick heart. To understand the transcriptional regulatory network in eukaryotic cells, it is
essential to identify functional cis-regulatory sequences that interact with
trans-acting factors. A number of algorithms have been developed to predict
common cis-regulatory elements for co-regulated genes with similar expression
patterns. However, previous methods usually deal with disjoint gene groups
partitioned or clustered by arbitrary cutoffs, which might cause information
losses. To preclude the defining step of gene set, we adopted enrichment
analysis and termed the method binding site enrichment analysis (BSEA). BSEA was
first applied for publicly available ChIP-on-chip data of c-MYC, MAX and E2F
transcription factors, identifying significant enrichment for signatures of
corresponding factors and potential co-activators. Using time-scaled expression
profiling of 3T3-L1 adipogenesis, we observed enrichment for signatures of known
adipogenic factors such as C/EBPalpha, C/EBPbeta and PPARgamma, temporally
coincident with previous reports. BSEA was also applied to tissue-specific
expression profiles of human and mouse, identifying well-known tissue-specific
transcription factors such as HNF-4 in liver and MEF-2 in heart along with other
putative tissue-specific regulators. With extended versatility coping with
various kinds of microarray dataset, BSEA can identify key regulators for global
microarray data in which transcriptional regulation plays a major role. As a
generalized method, BSEA would help to elucidate the transcriptional regulatory
networks, the primary challenges in functional genomics. RATIONALE: Mitogen-activated protein kinase (MAPK) pathways provide a critical
connection between extrinsic and intrinsic signals to cardiac hypertrophy.
Extracellular signal-regulated protein kinase (ERK)5, an atypical MAPK is
activated in the heart by pressure overload. However, the role of ERK5 plays in
regulating hypertrophic growth and hypertrophy-induced apoptosis is not
completely understood.
OBJECTIVE: Herein, we investigate the in vivo role and signaling mechanism
whereby ERK5 regulates cardiac hypertrophy and hypertrophy-induced apoptosis.
METHODS AND RESULTS: We generated and examined the phenotypes of mice with
cardiomyocyte-specific deletion of the erk5 gene (ERK5(cko)). In response to
hypertrophic stress, ERK5(cko) mice developed less hypertrophic growth and
fibrosis than controls. However, increased apoptosis together with upregulated
expression levels of p53 and Bad were observed in the mutant hearts.
Consistently, we found that silencing ERK5 expression or specific inhibition of
its kinase activity using BIX02189 in neonatal rat cardiomyocytes (NRCMs)
reduced myocyte enhancer factor (MEF)2 transcriptional activity and blunted
hypertrophic responses. Furthermore, the inhibition of MEF2 activity in NRCMs
using a non-DNA binding mutant form of MEF2 was found to attenuate the
ERK5-regulated hypertrophic response.
CONCLUSIONS: These results reveal an important function of ERK5 in cardiac
hypertrophic remodeling and cardiomyocyte survival. The role of ERK5 in
hypertrophic remodeling is likely to be mediated via the regulation of MEF2
activity. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family
that binds to and activates the EGF receptor. Transactivated by angiotensin II,
ET-1, and various growth factors in cardiomyocytes, HB-EGF is known to induce
cardiac hypertrophy via the PI3K-Akt, MAP kinase, and JAK-STAT pathways.
However, little is known about the potential involvement of the ERK5 pathway in
HB-EGF-induced cardiac hypertrophy. In the present report, we identify and
characterize a novel MEK5-ERK5 pathway that is involved in HB-EGF-induced
cardiomyocyte hypertrophy. HB-EGF (10ng/ml) significantly increased
[(3)H]-leucine incorporation and atrial natriuretic factor (ANF) mRNA expression
in H9c2 cells. In addition, HB-EGF activated a MEK5-ERK5 pathway. Pretreatment
with the EGFR inhibitor AG1478 attenuated the activation of ERK5. Blockade of
MEK5-ERK5 signaling using MEK5 siRNA reduced the ability of HB-EGF to increase
cell size and the expression of ANF mRNA, suggesting the involvement of an
EGFR-ERK5 pathway in HB-EGF-induced cardiomyocyte hypertrophy. We further
analyzed cyclooxygenase-2 (COX-2). HB-EGF enhanced the expression of COX-2, a
response mediated by MEK5-ERK5 signaling, while the COX-2 inhibitor rofecoxib
attenuated HB-EGF-induced ANF mRNA expression, suggesting that COX-2 is also
associated with HB-EGF-induced cardiomyocyte hypertrophy. It has been known that
ERK5 activates the myocyte enhancer factor (MEF) 2 family of transcription
factor, we next tested whether activation of MEF2A contributes to HB-EGF-induced
COX-2 expression. Inhibition of MEF2A using siRNA attenuated HB-EGF-induced
COX-2, ANF expression and cell size. In conclusion, HB-EGF induces cardiomyocyte
hypertrophy through an EGFR-ERK5-MEF2A-COX-2 pathway. Our findings will help us
to better understand the molecular mechanisms behind HB-EGF-induced
cardiomyocyte hypertrophy. The muscle-CAT (M-CAT) promoter element is found on promoters of most
muscle-specific cardiac genes, but its role in cardiac pathology is poorly
understood. Here we studied whether the M-CAT element is involved in
hypertrophic process activated by mechanical stretch, and identified the
intracellular pathways mediating the response. When an in vitro stretch model of
cultured neonatal rat cardiomyocytes and luciferase reporter construct driven by
rat B-type natriuretic peptide (BNP) promoter were used, mutation of M-CAT
element inhibited not only the basal reporter activity (88%), but also the
stretch-activated BNP transcription (58%, p < 0.001). Stretch-induced BNP
promoter activation was associated with an increase in transcriptional enhancer
factor-1 (TEF-1) binding activity after 24 h mechanical stretch (p < 0.05).
Inhibition of mitogen-activated protein kinases ERK, JNK, or p38 attenuated
stretch-induced BNP activation. Interestingly, as opposed to p38 and JNK,
inhibition of ERK had no additional effect on transcriptional activity of BNP
promoter harboring the M-CAT mutation, suggesting a pivotal role for ERK in
regulating stretch-induced BNP transcription via M-CAT binding site. Finally,
immunoprecipitation studies showed that mechanical stretch induced myocyte
enhancer factor-2 (MEF-2) binding to TEF-1. These data suggest a central role
for M-CAT element in regulation of mechanical stretch-induced hypertrophic
response via ERK activation. |
Is there an association between TERT promoter mutation and survival of glioma patients? | Yes, TERT mutation is associated with survival of glioma patients and was suggested as a bio-marker of gliomas. | Frequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and the
promoter of telomerase reverse transcriptase (TERT) represent two significant
discoveries in glioma genomics. Understanding the degree to which these two
mutations co-occur or occur exclusively of one another in glioma subtypes
presents a unique opportunity to guide glioma classification and prognosis. We
analyzed the relationship between overall survival (OS) and the presence of
IDH1/2 and TERT promoter mutations in a panel of 473 adult gliomas. We
hypothesized and show that genetic signatures capable of distinguishing among
several types of gliomas could be established providing clinically relevant
information that can serve as an adjunct to histopathological diagnosis. We
found that mutations in the TERT promoter occurred in 74.2% of glioblastomas
(GBM), but occurred in a minority of Grade II-III astrocytomas (18.2%). In
contrast, IDH1/2 mutations were observed in 78.4% of Grade II-III astrocytomas,
but were uncommon in primary GBM. In oligodendrogliomas, TERT promoter and
IDH1/2 mutations co-occurred in 79% of cases. Patients whose Grade III-IV
gliomas exhibit TERT promoter mutations alone predominately have primary GBMs
associated with poor median OS (11.5 months). Patients whose Grade III-IV
gliomas exhibit IDH1/2 mutations alone predominately have astrocytic
morphologies and exhibit a median OS of 57 months while patients whose tumors
exhibit both TERT promoter and IDH1/2 mutations predominately exhibit
oligodendroglial morphologies and exhibit median OS of 125 months. Analyzing
gliomas based on their genetic signatures allows for the stratification of these
patients into distinct cohorts, with unique prognosis and survival. OBJECTIVE: This study explored the effects of telomerase reverse transcriptase
(TERT) promoter mutations on transcriptional activity of the TERT gene under
hypoxic and temozolomide (TMZ) treatment conditions, and investigated the status
and prognostic value of these mutations in gliomas.
METHODS: The effect of TERT promoter mutations on the transcriptional activity
of the TERT gene under hypoxic and TMZ treatment conditions was investigated in
glioma cells using the luciferase assay. TERT promoter mutations were detected
in 101 glioma samples (grades I-IV) and 49 other brain tumors by sequencing.
TERT mRNA expression in gliomas was examined by real-time PCR. Hazard ratios
from survival analysis of glioma patients were determined relative to the
presence of TERT promoter mutations.
RESULTS: Mutations in the TERT promoter enhanced gene transcription even under
hypoxic and TMZ treatment conditions, inducing upregulation of TERT mRNA
expression. Mutations were detected in gliomas, but not in meningiomas,
pituitary adenomas, cavernomas, intracranial metastases, normal brain tissues,
or peripheral blood of glioma patients. Patients with TERT promoter mutations
had lower survival rates, even after adjusting for other known or potential risk
factors, and the incidence of mutation was correlated with patient age.
CONCLUSION: TERT promoter mutations were specific to gliomas. TERT promoter
mutations maintained its ability of inducing high transcriptional activity even
under hypoxic and TMZ treatment conditions, and the presence of mutations was
associated with poor prognosis in glioma patients. These findings demonstrate
that TERT promoter mutations are novel prognostic markers for gliomas that can
inform prospective therapeutic strategies. Recurrent mutations in the promoter region of telomerase reverse transcriptase
(TERT) have been found in various cancers including diffuse gliomas. Mutations
lead to TERT upregulation and are associated with aggressive clinical behavior
in glioblastomas. However, the clinical significance of TERT promoter mutations
in lower-grade gliomas remains undetermined. The aim of this study is to
evaluate the status of TERT promoter and the respective prognostic significance
in a cohort of 237 lower-grade gliomas comprising grades II and III
astrocytomas, oligodendrogliomas, and oligoastrocytomas. Mutually exclusive
mutations in TERT promoter, C228T and C250T, were identified in 16/105 (15%)
diffuse astrocytomas, 16/63 (25%) anaplastic astrocytomas, 13/18 (72%)
oligodendrogliomas, 3/3 (100%) anaplastic oligodendrogliomas, 17/45 (38%)
oligoastrocytomas, and 2/3 (67%) anaplastic oligoastrocytomas. Mutations
co-occurred with 1p/19q codeletion (P<0.001) and are associated with
oligodendroglial histology (P<0.001). Kaplan-Meier's survival analysis showed
that TERT promoter mutation (P=0.037), Isocitrate dehydrogenase (IDH) mutation
(P<0.001), and 1p/19q codeletion (P<0.001) were associated with favorable
overall survival (OS). In the subset of 116 IDH-mutated lower-grade gliomas
lacking 1p/19q codeletion, 19 TERT promoter-mutated tumors exhibited longer
progression-free survival (PFS) (P=0.027) and OS (P=0.004). Consistent with this
observation, in the subset of 97 IDH-mutated astrocytomas, 14 TERT
promoter-mutated tumors showed longer PFS (P=0.001) and OS (P=0.001). In
contrast, among the subset of 74 IDH wild-type lower-grade gliomas with intact
1p/19q, TERT promoter mutation was associated with shorter PFS (P=0.001) and OS
(P=0.001). Similarly, in the subset of 65 IDH wild-type astrocytomas, 16 TERT
promoter-mutated tumors exhibited unfavorable PFS (P=0.007) and OS (P=0.008).
Our results indicate that when combined with IDH status, TERT promoter mutation
contributes to prognostic subgroups of lower-grade astrocytic tumors or 1p/19q
intact lower-grade gliomas and this may further refine future molecular
classification of lower-grade gliomas. Author information:
(1)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France.
(2)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4] National
Neurological Institute C. Mondino, University of Pavia, 27100 Pavia, Italy.
(3)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4] Institut
du Cerveau et de la Moelle épinière (ICM), Plateforme de Génotypage Séquençage,
Paris 75013, France.
(4)Dipartimento di Neuro Oncologia Molecolare Fondazione I.R.C.C.S. Istituto
Neurologico C. Besta, Milano 20134, Italy.
(5)1] Institut du Cerveau et de la Moelle épinière (ICM), Plateforme de
Génotypage Séquençage, Paris 75013, France [2] Onconeurothèque, Paris 75013,
France.
(6)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4]
Onconeurothèque, Paris 75013, France.
(7)Division of Genetics and Epidemiology, Institute of Cancer Research, Surrey
SM2 5NG, UK.
(8)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4] AP-HP,
Groupe Hospitalier Pitié-Salpêtrière, Service de Neurologie 2, Paris 75013,
France.
(9)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4]
Onconeurothèque, Paris 75013, France [5] AP-HP, Groupe Hospitalier
Pitié-Salpêtrière, Service de Neurologie 2, Paris 75013, France.
(10)1] Sorbonne Universités, UMPC Univ Paris 06, Centre de Recherche de
l'Institut du Cerveau et de la Moelle épinière, Paris 75013, France [2] INSERM U
1127, Paris 75013, France [3] CNRS, UMR 7225, Paris 75013, France [4]
Onconeurothèque, Paris 75013, France [5] AP-HP, Groupe Hospitalier
Pitié-Salpêtrière, Laboratoire de Neuropathologie R. Escourolle, Paris 75013,
France. |
Which JAK (Janus kinase) inhibitor is approved for treatment of rheumatoid arthritis? | Tofacitinib (or CP690.550) is an oral JAK (Janus kinase) inhibitor that is approved for treatment of rheumatoid arthritis. Tofacitinib inhibits JAK family kinase members, in particular JAK1 and JAK3, achieving a broad limitation of inflammation by interfering with several cytokine receptors. Tofacitinib has also a proven efficacy as an immunosuppressive regimen after renal transplantation.
GLPG-0634 and INCB18424 are other JAK kinase inhibitors that are being studied for treatment of rheumatoid arthritis. | Treatment of rheumatoid arthritis (RA) has developed dramatically by the
appearance of biologics. However the development of a new anti-rheumatic drug is
necessary because of its issue on route of administration and expense. Recently,
inhibitors targeting tyrosine kinase known as Janus kinase (Jak) has shown
prominent effect on RA. Jak family is comprised by Jak1, Jak2, Jak3 and Tyk2
which is necessary for signal transduction for inflammatory cytokines. INCB18424
targeting Jak1/2 and CP690,550 targeting Jak3 has been developed and is now on
phase II clinical study for RA. Results from those clinical studies have proven
that these inhibitors can be effective as biologics with few side effects.
However, it has been reported that inhibitors are less specific than it has been
expect and that non-specificity can be important for its effect. Therefore, we
think that the mechanism of inhibitors cannot be explained by its inhibition of
a single kinase. Herein, we describe IL-10 overproduction by Jak3 and Stat6
deficient dendritic cell. We speculate that this is one possible mechanism of
action for CP690,550 although as for its non-specificity we need further
investigation to predict not only its effect but also its side effect in a long
term administration. OBJECTIVE: To determine the efficacy, safety, and tolerability of 3 different
dosages of CP-690,550, a potent, orally active JAK inhibitor, in patients with
active rheumatoid arthritis (RA) in whom methotrexate, etanercept, infliximab,
or adalimumab caused an inadequate or toxic response.
METHODS: Patients (n = 264) were randomized equally to receive placebo, 5 mg of
CP-690,550, 15 mg of CP-690,550, or 30 mg of CP-690,550 twice daily for 6 weeks,
and were followed up for an additional 6 weeks after treatment. The primary
efficacy end point was the American College of Rheumatology 20% improvement
criteria (ACR20) response rate at 6 weeks.
RESULTS: By week 6, the ACR20 response rates were 70.5%, 81.2%, and 76.8% in the
5 mg, 15 mg, and 30 mg twice daily groups, respectively, compared with 29.2% in
the placebo group (P < 0.001). Improvements in disease activity in
CP-690,550-treated patients compared with placebo were seen in all treatment
groups as early as week 1. ACR50 and ACR70 response rates significantly improved
in all treatment groups by week 4. The most common adverse events reported were
headache and nausea. The infection rate in both the 15 mg twice daily group and
the 30 mg twice daily group was 30.4% (versus 26.2% in the placebo group). No
opportunistic infections or deaths occurred. Increases in mean low-density
lipoprotein cholesterol and high-density lipoprotein cholesterol levels, and
increases in mean serum creatinine level (0.04-0.06 mg/dl) were seen in all
CP-690,550 treatment arms.
CONCLUSION: Our findings indicate that CP-690,550 is efficacious in the
treatment of RA, resulting in rapid, statistically significant, and clinically
meaningful reductions in the signs and symptoms of RA. Further studies of
CP-690,550 in RA are warranted. OBJECTIVES: To determine the efficacy of CP-690,550 in improving pain, function
and health status in patients with moderate to severe active rheumatoid
arthritis (RA) and an inadequate response to methotrexate or a tumour necrosis
factor alpha inhibitor.
METHODS: Patients were randomised equally to placebo, CP-690,550 5, 15 or 30 mg
twice daily for 6 weeks, with 6 weeks' follow-up. The patient's assessment of
arthritis pain (pain), patient's assessment of disease activity, Health
Assessment Questionnaire-Disability Index (HAQ-DI) and Short Form-36 (SF-36)
were recorded.
RESULTS: At week 6, significantly more patients in the CP-690,550 5, 15 and 30
mg twice-daily groups experienced a 50% improvement in pain compared with
placebo (44%, 66%, 78% and 14%, respectively), clinically meaningful reductions
in HAQ-DI (> or =0.3 units) (57%, 75%, 76% and 36%, respectively) and clinically
meaningful improvements in SF-36 domains and physical and mental components.
CONCLUSIONS: CP-690,550 was efficacious in improving the pain, function and
health status of patients with RA, from week 1 to week 6. AIMS: To investigate the effects of methotrexate (MTX) on the pharmacokinetics
(PK) of CP-690,550, a novel Janus kinase (JAK) inhibitor in development as a
therapy for rheumatoid arthritis (RA), to determine the effects of multiple
doses of CP-690,550 on the PK of MTX, and to evaluate the short-term safety and
tolerability of co-administration of CP-690,550 and MTX.
METHODS: This was a fixed-dose drug-drug interaction study. Twelve patients
diagnosed with RA for at least 6 months were enrolled in a Phase I, open-label
study of the PK of multiple doses of CP-690,550 (30 mg b.i.d.) and single doses
of MTX (15-25 mg per week).
RESULTS: All patients completed the study and were evaluated for PK and safety.
CP-690,550 exposure was not affected by co-administration with MTX; AUC(12)
ratio (CP-690,550 + MTX/CP-690,550) was 103.06% [90% confidence interval (CI)
99.00, 107.29]. MTX exposure decreased by 10%; AUC(12) ratio (CP-690,550 +
MTX/MTX) was 89.53% (90% CI 77.38, 103.57), which was not considered clinically
significant. Co-administration of CP-690,550 and MTX was safe and well
tolerated. There were no serious adverse events or withdrawals from the study
and there was no trend in the incidence or severity of adverse events across
treatments.
CONCLUSIONS: Co-administration of CP-690,550 and MTX was safe and well
tolerated. There was no clinically significant effect on the PK profile of
either drug. Therefore, dose adjustments should not be required when
co-administering CP-690,550 and MTX. CP-690,550 is an orally active and selective inhibitor of the janus kinase (JAK)
molecules. The molecular pathways through which the JAK moieties function are
described along with the clinical mechanisms associated with their inhibition.
Animal models of JAK inhibition are reviewed as a background for the possible
inhibition of JAK in humans. The pharmacokinetics of CP-690,550 in humans is
described, and the Phase IIA and IIB trials are reviewed in some detail. These
trials were dose-ranging and showed a general dose response with relatively
robust American College of Rheumatology 20 (ACR20) responses. A proof-of-concept
6-week trial in which CP-690,550 was given as monotherapy was associated with
highly efficacious responses at the mid and higher twice-daily dose ranges
employed. A subsequent 24 week dose-ranging trial in which CP-690,550 was
administered in combination with methotrexate showed ACR20 responses, which were
also statistically significant versus placebo interventions. CP-690,550
treatment was associated with side effects, which included headache and nausea.
Infections were more common versus placebo as were elevations in transaminase
enzymes when administered in combination with methotrexate, and increases in
low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol.
Decreases in haemoglobin and white blood cell (WBC) counts were also observed
along with small increases in serum creatinine. Occasional significant decreases
of haemoglobin (>2 g dl(-1)) were observed, although decreases of WBC to less
than 1000 per mm(3) were not seen. Plans for long-term follow-up of the
described trials are described along with the features of five presently ongoing
Phase III trials of the CP-690,550 janus kinase (JAK) inhibitor. Future
directions include completion and publication of these trials along with study
of JAK inhibition for other indications. There is a critical need for safer and more convenient treatments for organ
transplant rejection and autoimmune disorders such as rheumatoid arthritis.
Janus tyrosine kinases (JAK1, JAK3) are expressed in lymphoid cells and are
involved in the signaling of multiple cytokines important for various T cell
functions. Blockade of the JAK1/JAK3-STAT pathway with a small molecule was
anticipated to provide therapeutic immunosuppression/immunomodulation. The
Pfizer compound library was screened against the catalytic domain of JAK3
resulting in the identification of a pyrrolopyrimidine-based series of
inhibitors represented by CP-352,664 (2a). Synthetic analogues of 2a were
screened against the JAK enzymes and evaluated in an IL-2 induced T cell blast
proliferation assay. Select compounds were evaluated in rodent efficacy models
of allograft rejection and destructive inflammatory arthritis. Optimization
within this chemical series led to identification of CP-690,550 1, a potential
first-in-class JAK inhibitor for treatment of autoimmune diseases and organ
transplant rejection. INTRODUCTION: Interleukin (IL)-6-type cytokines exert their effects through
activation of the Janus kinase/signal transducers and activators of
transcription (JAK/STAT) signaling cascade. The JAK/STAT pathways play an
important role in rheumatoid arthritis, since JAK inhibitors have exhibited
dramatic effects on rheumatoid arthritis (RA) in clinical trials. In this study,
we investigated the molecular effects of a small molecule JAK inhibitor,
CP690,550 on the JAK/STAT signaling pathways and examined the role of JAK
kinases in rheumatoid synovitis.
METHODS: Fibroblast-like synoviocytes (FLS) were isolated from RA patients and
stimulated with recombit oncostatin M (OSM). The cellular supernatants were
analyzed using cytokine protein chips. IL-6 mRNA and protein expression were
analyzed by real-time PCR method and ELISA, respectively. Protein
phosphorylation of rheumatoid synoviocytes was assessed by Western blot using
phospho-specific antibodies.
RESULTS: OSM was found to be a potent inducer of IL-6 in FLS. OSM stimulation
elicited rapid phosphorylation of STATs suggesting activation of the JAK/STAT
pathway in FLS. CP690,550 pretreatment completely abrogated the OSM-induced
production of IL-6, as well as OSM-induced JAK/STAT, and activation of
mitogen-activated kinases (MAPKs) in FLS.
CONCLUSIONS: These findings suggest that IL-6-type cytokines contribute to
rheumatoid synovitis through activation of the JAK/STAT pathway in rheumatoid
synoviocytes. Inhibition of these pro-inflammatory signaling pathways by
CP690,550 could be important in the treatment of RA. OBJECTIVE: To compare the efficacy, safety, and tolerability of 5 doses of oral
tofacitinib (CP-690,550) or adalimumab monotherapy with placebo for the
treatment of active rheumatoid arthritis (RA) in patients with an inadequate
response to disease-modifying antirheumatic drugs.
METHODS: In this 24-week, double-blind, phase IIb study, patients with RA (n =
384) were randomized to receive placebo, tofacitinib at 1, 3, 5, 10, or 15 mg
administered orally twice a day, or adalimumab at 40 mg injected subcutaneously
every 2 weeks (total of 6 injections) followed by oral tofacitinib at 5 mg twice
a day for 12 weeks. The primary end point was the responder rate according to
the American College of Rheumatology 20% improvement criteria (ACR20) at week
12.
RESULTS: Treatment with tofacitinib at a dose of ≥3 mg twice a day resulted in a
rapid response with significant efficacy when compared to placebo, as indicated
by the primary end point (ACR20 response at week 12), achieved in 39.2% (3 mg; P
≤ 0.05), 59.2% (5 mg; P < 0.0001), 70.5% (10 mg; P < 0.0001), and 71.9% (15 mg;
P < 0.0001) in the tofacitinib group and 35.9% of patients in the adalimumab
group (P = 0.105), compared with 22.0% of patients receiving placebo.
Improvements were sustained at week 24, according to the ACR20, ACR50, and ACR70
response rates as well as classifications of remission according to the
3-variable Disease Activity Score in 28 joints (DAS28) using C-reactive protein
and the 4-variable DAS28 using the erythrocyte sedimentation rate. The most
common treatment-emergent adverse events (AEs) in patients across all
tofacitinib treatment arms (n = 272) were urinary tract infection (7.7%),
diarrhea (4.8%), headache (4.8%), and bronchitis (4.8%).
CONCLUSION: Tofacitinib monotherapy at ≥3 mg twice a day was efficacious in the
treatment of patients with active RA over 24 weeks and demonstrated a manageable
safety profile. Janus kinases have proved to be essential for many immunological processes but
there is growing evidence that they also play a critical role in pathogenesis of
many diseases including inflammatory diseases and cancer where they promote
multiple steps of tumorigenesis. Several companies are in late stage clinical
programs for the development of JAK kinase inhibitors and the first small
molecule JAK inhibitor, Jakafi® (ruxolitinib) has been just approved for
treatment of myeloproliferative neoplasms. Several other molecules are on the
rise to treat arthritis, psoriasis and multiple types of cancer. This commentary
will provide a review of the JAK kinase field as it pertains to small molecule
inhibition for the treatment of cancer and autoimmune diseases with an emphasis
on JAK2. The use of experimental and clinical inhibitors of JAK will be
discussed for solid tumor and hematological maligcies, lupus, arthritis,
colitis, neurological disorders, pain, diabetes and cardiovascular disease. In
addition, it will review current paradigms in the field and treatment programs
which could be complemented by small molecule inhibitors of Janus kinase. Multiple cytokines play a pivotal role in the pathogenesis of rheumatoid
arthritis (RA). The appropriate intracellular signalling pathways must be
activated via cytokine receptors on the cell surface, and the tyrosine kinases
transduce the first 'outside to in' signals to be phosphorylated after receptor
binding to its ligand. Among them, members of the Janus kinase (JAK) family are
essential for the signalling pathways of various cytokines and are implicated in
the pathogenesis of RA. The in vitro, ex vivo and in vivo effects of a JAK
inhibitor CP-690,550 (tofacitinib) for the treatment of RA are reported. In
vitro experiments indicated that the effects of tofacitinib were mediated
through suppression of interleukin 17 (IL-17) and interferon γ production and
proliferation of CD4 T cells, presumably Th1 and Th17. A treatment study was
conducted in the severe combined immunodeficiency (SCID)-HuRAg mice, an RA
animal model using SCID mice implanted with synovium and cartilage from
patients. Tofacitinib reduced serum levels of human IL-6 and IL-8 in the mice
and also reduced synovial inflammation and invasion into the implanted
cartilage. A phase 2 double-blind study using tofacitinib was carried out in
Japanese patients with active RA and inadequate response to methotrexate (MTX).
A total of 140 patients were randomised to tofacitinib 1, 3, 5, 10 mg or placebo
twice daily and the American College of Rheumatology 20% improvement criteria
(ACR20) response rate at week 12, a primary end point, was significant for all
tofacitinib treatment groups. Thus, an orally available tofacitinib in
combination with MTX was efficacious and had a manageable safety profile.
Tofacitinib at 5 and 10 mg twice a day appears suitable for further evaluation
to optimise the treatment of RA. INTRODUCTION: The JAK family comprises of the four non-receptor tyrosine kinases
JAK1, JAK2, JAK3 and Tyk2, which play key, but differing, roles in cytokine
receptor signal transduction. A non-selective JAK inhibitor, ruxolitinib, has
recently been approved to treat myelofibrosis whereas tofacitinib is poised for
approval to treat rheumatoid arthritis. Selective inhibition of JAK3, JAK1 or
Tyk2 provides the opportunity to achieve clinical efficacy in the treatment of
inflammatory diseases while reducing the risk of dose-limiting effects
attributable to JAK2 inhibition.
AREAS COVERED: This review considers the small number of published patent
filings that claim either selective JAK1 or selective Tyk2 inhibitors. These are
considered in the context of the considerably larger number of disclosures and
patent filings claiming selective JAK2 or JAK3 inhibitors.
EXPERT OPINION: The recent disclosure of the clinical efficacy of a selective
JAK1 inhibitor (GLPG-0634) in rheumatoid arthritis and detailed disclosure of
the some potent and highly selective JAK1 inhibitors provide a clear stimulus
for further activity in this area. The availability of a selective Tyk2
inhibitor will provide the opportunity for better understanding of the
physiological role of this kinase. Recent patent applications indicate that Tyk2
selectivity is achievable and Tyk2 inhibitors have potential in the treatment of
multiple sclerosis. Rheumatoid arthritis (RA) is a representative autoimmune disease characterized
by chronic and destructive inflammatory synovitis. The multiple cytokines play
pivotal roles in RA pathogenesis by inducing intracellular signaling, and
members of the Janus kinase (JAK) family are essential for such signal
transduction. An orally available JAK3 inhibitor, tofacitinib, has been applied
for RA, with satisfactory effects and acceptable safety in multiple clinical
examinations. From phase 2 dose-finding studies, tofacitinib 5 mg and 10 mg
twice a day appear suitable for further evaluation. Subsequently, multiple phase
3 studies were carried out, and tofacitinib with or without methotrexate (MTX)
is efficacious and has a manageable safety profile in active RA patients who are
MTX naïve or show inadequate response to methotrexate (MTX-IR),
disease-modifying antirheumatic drugs (DMARD)-IR, or tumor necrosis factor
(TNF)-inhibitor-IR. The common adverse events were infections, such as
nasopharyngitis; increases in cholesterol, transaminase, and creatinine; and
decreases in neutrophil counts. Although the mode of action of tofacitinib
remains unclear, we clarified that the inhibitory effects of tofacitinib could
be mediated through suppression of interleukin (IL)-17 and interferon (IFN)-γ
production and proliferation of CD4(+) T cells in the inflamed synovium. Taken
together, an orally available kinase inhibitor tofacitinib targeting
JAK-mediated signals would be expected to be a new option for RA treatment. In this review, we describe the current knowledge of the biology of the JAKs.
The JAK family comprises the four nonreceptor tyrosine kinases JAK1, JAK2, JAK3,
and Tyk2, all key players in the signal transduction from cytokine receptors to
transcription factor activation. We also review the progresses made towards the
optimization of JAK inhibitors and the importance of their selectivity profile.
Indeed, the full array of many medicinal chemistry enabling tools (HTS, X-ray
crystallography, scaffold morphing, etc.) has been deployed to successfully
design molecules that discriminate among JAK family and other kinases. While the
first JAK inhibitor was launched in 2011, this review also summarizes the status
of several other small-molecule JAK inhibitors currently in development to treat
arthritis, psoriasis, organ rejection, and multiple cancer types. After two decades of research and development activity focussed on orally active
kinase inhibitors, the first such drug (the JAK inhibitor Xeljanz, tofacitinib)
was approved by the FDA in November 2012 for the treatment of rheumatoid
arthritis (RA). There is an intense activity in many companies both on expanding
the utility of JAK inhibitors in other auto-immune indications and in
discovering inhibitors of the JAK family with different and more selective
profiles. Progress is also being made with orally active Syk inhibitors. One
such inhibitor (fostamatinib) is currently in large-scale phase 3 trials, and
there are others in clinical development. The last two to three years have been
transformative for kinase inhibitors in auto-immune diseases, as several
inhibitors have finally progressed beyond phase 2 trials after so many failures
on other targets. Thus, there are new treatment options for RA patients beyond
existing oral DMARDs and parenteral biologics. OBJECTIVE: To summarise the adverse events (AE) reported in patients with
rheumatoid arthritis (RA) treated with protein kinase inhibitors (PKi), and
identify family and molecule-related AEs.
METHODS: Systematic review of the PKi used in clinical trials (CTs) in RA.
Medline, Embase, Cochrane Library, Web of Knowledge, and international abstracts
of congress were reviewed, (up to 31 October 2012). Search was limited to
interventional studies of PKi used in CTs in RA, written in English, and
reporting frequencies of AE. Diseases with similar comorbidity burden also were
included. Frequency of AE, serious AE (SAE), death and discontinuation due to
AEs (DCAE) were recorded. Risk of bias was assessed. Meta-analysis was carried
using pooled relative risk (RR) with 95% CI as effect measure.
RESULTS: The search produced 4410 hits. Forty-one articles reporting data on 21
PKi of the Janus kinase (JAK), SYK, p38 and cKit families were selected for
detailed analysis. In patients treated with p38 inhibitors, RR for dizziness was
2.36 (1.20 to 4.63), and in patients treated with c-Kit inhibitors, RR for
oedema was 3.43 (1.58 to 7.42). In patients treated with the JAK inhibitor
tofacitinib, RR for hypercholesterolaemia was 1.70 (1.10 to 2.63) that was dose
related. In patients treated with the Syk inhibitor fostamatinib, pooled RR for
hypertransaminasaemia, hypertension, diarrhoea and neutropenia were 2.93 (1.02
to 8.43), 2.80 (1.58 to 5.99), 5.20 (3.19 to 8.49) and 9.24 (2.22 to 38.42),
respectively. Serious infections and maligcies were not significantly more
frequent in PKi-treated patients than in comparator groups.
CONCLUSIONS: Event rates of serious infections and maligcies with PKi are not
different from biologics. In addition, PKi have a unique safety profile related
to target and off-target inhibition of kinases, at times dose related. The advent of anti-Tumor Necrosis Factor (TNF) therapy has changed the way of
treating inflammatory bowel disease (IBD). However, primary and secondary
failure are relatively frequent with all anti-TNF agents, which are available
only as parenteral agents. Tofacitinib is an oral janus kinase (JAK) inhibitor
that inhibits JAK family kinase members, in particular JAK1 and JAK3, achieving
a broad limitation of inflammation by interfering with several cytokine
receptors. It first proved its efficacy as an immunosuppressive regimen after
renal transplantation, and was recently approved by the FDA for rheumatoid
arthritis. First data in IBD are promising, especially in ulcerative colitis.
Ongoing clinical trials in both UC and Crohn's disease (CD) are needed to
further explore its efficacy in CD and to better assess its safety profile. Conventional immunosuppressive therapies have radically transformed patient
survival in systemic lupus erythematosus (SLE), but their use is associated with
considerable toxicity and a substantial proportion of patients remain refractory
to treatment. A more comprehensive understanding of the complexity of SLE
immunopathogenesis has evolved over the past decade and has led to the testing
of several biologic agents in clinical trials. There is a clear need for new
therapeutic agents that overcome these issues, and biologic agents offer
exciting prospects as future SLE therapies.An array of promising new therapies
are currently emerging or are under development including B-cell depletion
therapies, agents targeting B-cell survival factors, blockade of T-cell
co-stimulation and anti-cytokine therapies, such as monoclonal antibodies
against interleukin-6 and interferon-α. OBJECTIVE: Tofacitinib, which is a Janus kinase (JAK) inhibitor, has shown
clinical effects in the treatment of rheumatoid arthritis. JAKs are important
kinases in lymphocyte differentiation; however, their function in dendritic
cells (DCs) is unknown. In this study, the function of JAKs in DCs was
investigated with tofacitinib.
METHODS: The effects of tofacitinib on the maturation of human monocyte-derived
DCs induced by lipopolysaccharide (LPS) stimulation were investigated. In
addition, its effects on T cell stimulatory capability was investigated by
coculturing with naïve CD45RA-positive T cells.
RESULTS: Tofacitinib decreased expression of CD80/CD86 in a
concentration-dependent manner in LPS-stimulated DCs; however, it did not affect
HLA-DR expression. Tofacitinib suppressed tumour necrosis factor, interleukin
(IL)-6 and IL-1β production without affecting transforming growth factor (TGF)-β
and IL-10 production. Meanwhile, CD80/CD86 expression in DCs was enhanced by
type I interferon (IFN) stimulation, and the LPS-induced CD80/CD86 expression
was inhibited by an antibody to type I IFN receptor. Furthermore, tofacitinib
suppressed production of type I IFN and activation of interferon regulatory
factor (IRF)-7, which is a transcription factor involved in CD80/CD86 and type I
IFN expression. Tofacitinib also decreased the T cell stimulatory capability of
DCs and increased expression of indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2.
CONCLUSIONS: Tofacitinib, a JAK1/JAK3 inhibitor, affected the activities of
human DCs. It decreased CD80/CD86 expression and T cell stimulatory capability
through suppression of type I IFN signalling. These results suggest a novel mode
of action for tofacitinib and a pivotal role for JAKs in the differentiation of
DCs. Protein kinase inhibitors represent a novel and promising approach to the
treatment of rheumatoid arthritis (RA). By targeting intracellular signaling
pathways of cytokine-mediated reactions, these substances are able to interfere
with critical immune processes that underly the pathology of RA. With
tofacitinib, the first Janus kinase (JAK) inhibitor has been approved in the
USA, as well as in Switzerland and other countries. Several other substances are
currently undergoing phase II or phase III trials.A crucial question that will
shape the future of these new drugs is whether they are safe and in particular,
whether they are safer than biological therapies. This article provides an
overview on current data concerning the efficacy and safety of the most
promising substances and discusses the potential future role of intracellular
kinase inhibitors. OBJECTIVE: To review the pharmacology, pharmacokinetics, efficacy and safety,
dosage administration, and adverse effects of tofacitinib for rheumatoid
arthritis (RA) treatment.
DATA SOURCES: Primary sources of information were obtained from clinical
studies, which were identified through PubMed (1966 to June 2013) and
International Pharmaceutical Abstracts (1970 to March 2013) using terms:
tofacitinib, tasocitinib, CP-690550, and CP-690,550. Information was used from
tofacitinib package insert, guidelines, and published abstracts from the
American College of Rheumatology (ACR) and the European League Against
Rheumatism.
STUDY SELECTION AND DATA EXTRACTION: Data search was limited to include
publications in English language and from human subjects.
DATA SYNTHESIS: Tofacitinib is the first oral Janus kinase inhibitor indicated
for treatment of moderate to severe RA. Tofacitinib demonstrated efficacy and
safety comparable to other disease-modifying antirheumatic drugs (DMARDs).
Tofacitinib was efficacious in RA patients, indicated by achievements of ACR20,
ACR50, and ACR70 criteria. Similar improvements were observed in patients who
met remission criteria based on the Disease Activity Scores 28 criteria and
quality of life as measured by the Health Assessment Questionnaire-Disability
Index (HAQ-DI). Tofacitinib was associated with infections and maligcies; and
elevations in serum creatinine and lipids were observed. Drug interactions with
inducers and inhibitors of the cytochrome P-450 3A4 and 2C9 isoenzymes were
reported.
CONCLUSIONS: Tofacitinib is an oral treatment option for RA patients who have
inadequate response or intolerance to methotrexate. Postmarket surveillance will
provide further insight to tofacitinib's role in RA therapy, especially in
patients who may require different types of combination therapy with DMARDS. |
Which is the mechanism used for synthesis of a highly functional N-truncated dystrophin isoform that attenuates dystrophinopathy? | Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice | Author information:
(1)1] The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio
State University, Columbus, Ohio, USA. [2].
(2)Section of Microbiology and Medical Genetics, Department of Medical Sciences,
University of Ferrara, Ferrara, Italy.
(3)Department of Proteomics and Nanobiotechnology, School of Biotechnology, KTH
Royal Institute of Technology, Stockholm, Sweden.
(4)Department of Neurology, Washington University School of Medicine, St. Louis,
Missouri, USA.
(5)The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio State
University, Columbus, Ohio, USA.
(6)1] Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.
[2] Department of Molecular and Cellular Biochemistry, The Ohio State
University, Columbus, Ohio, USA.
(7)Department of Neuroscience, University of Messina and Centro Clinico Nemo
Sud, Messina, Italy.
(8)Bambino Gesù Children's Hospital, Rome, Italy.
(9)Centre for Comparative Genomics, Murdoch University, Perth, Western
Australia, Australia.
(10)1] The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio
State University, Columbus, Ohio, USA. [2] Department of Pediatrics, The Ohio
State University, Columbus, Ohio, USA.
(11)Division of Biomedical Informatics, Department of Computer Science,
University of Kentucky Lexington, Kentucky, USA.
(12)Department of Human Genetics, The University of Utah School of Medicine,
Salt Lake City, Utah, USA.
(13)1] The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio
State University, Columbus, Ohio, USA. [2] Department of Pediatrics, The Ohio
State University, Columbus, Ohio, USA. [3] Department of Neurology, The Ohio
State University, Columbus, Ohio, USA. |
Which are the newly identified DNA nucleases that can be used to treat thalassemia? | Thalassemia is genetic diseases of the blood caused by mutations in the globin gene. Main goal for thalassemia treatment is to develop homologous recombination based gene therapy in order to cure these diseases. Zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) are proper targets for the human globin gene. Genome editing using engineered nucleases such as ZFNs and TALENs has become a powerful technology for reverse genetics. | DNA mismatch repair (MMR) greatly contributes to genome integrity via the
correction of mismatched bases that are mainly generated by replication errors.
Postreplicative MMR excises a relatively long tract of error-containing
single-stranded DNA. MutL is a widely conserved nicking endonuclease that
directs the excision reaction to the error-containing strand of the duplex by
specifically nicking the daughter strand. Because MutL apparently exhibits
nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of
MutL has been argued. Recent studies suggest ATP-dependent conformational and
functional changes of MutL, indicating that the regulatory mechanism involves
the ATP binding and hydrolysis cycle. In this study, we investigated the effect
of ATP binding on the structure of MutL. First, a cross-linking experiment
confirmed that the N-terminal ATPase domain physically interacts with the
C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass
spectrometry clarified that the binding of ATP to the N-terminal domain induces
local structural changes at the catalytic sites of MutL C-terminal domain.
Finally, on the basis of the results of the hydrogen/deuterium exchange
experiment, we successfully identified novel regions essential for the
endonuclease activity of MutL. The results clearly show that ATP modulates the
nicking endonuclease activity of MutL via structural rearrangements of the
catalytic site. In addition, several Lynch syndrome-related mutations in human
MutL homolog are located in the position corresponding to the newly identified
catalytic region. Our data contribute toward understanding the relationship
between mutations in MutL homolog and human disease. Induced pluripotent stem (iPS) cell technology holds vast promises for a cure to
the hemoglobinopathies. Constructs and methods to safely insert therapeutic
genes to correct the genetic defect need to be developed. Site-specific
insertion is a very attractive method for gene therapy because the risks of
insertional mutagenesis are eliminated provided that a "safe harbor" is
identified, and because a single set of validated constructs can be used to
correct a large variety of mutations simplifying eventual clinical use. We
report here the correction of α-thalassemia major hydrops fetalis in
transgene-free iPS cells using zinc finger-mediated insertion of a globin
transgene in the AAVS1 site on human chromosome 19. Homozygous insertion of the
best of the 4 constructs tested led to complete correction of globin chain
imbalance in erythroid cells differentiated from the corrected iPS cells. Author information:
(1)is at the Genome Center and the Department of Biochemistry and Molecular
Medicine , University of California, Davis , United States [email protected]. Base excision repair (BER) is a frontline repair system that is responsible for
maintaining genome integrity and thus preventing premature aging, cancer and
many other human diseases by repairing thousands of DNA lesions and strand
breaks continuously caused by endogenous and exogenous mutagens. This
fundamental and essential function of BER not only necessitates tight control of
the continuous availability of basic components for fast and accurate repair,
but also requires temporal and spatial coordination of BER and cell cycle
progression to prevent replication of damaged DNA. The major goal of this review
is to critically examine controversial and newly emerging questions about
mammalian BER pathways, mechanisms regulating BER capacity, BER responses to DNA
damage and their links to checkpoint control of DNA replication. β-Thalassemia (β-Thal) is a group of life-threatening blood disorders caused by
either point mutations or deletions of nucleotides in β-globin gene (HBB). It is
estimated that 4.5% of the population in the world carry β-Thal mutants (1),
posing a persistent threat to public health. The generation of patient-specific
induced pluripotent stem cells (iPSCs) and subsequent correction of the
disease-causing mutations offer an ideal therapeutic solution to this problem.
However, homologous recombination-based gene correction in human iPSCs remains
largely inefficient. Here, we describe a robust process combining efficient
generation of integration-free β-Thal iPSCs from the cells of patients and
transcription activator-like effector nuclease (TALEN)-based universal
correction of HBB mutations in situ. We generated integration-free and
gene-corrected iPSC lines from two patients carrying different types of
homozygous mutations and showed that these iPSCs are pluripotent and have normal
karyotype. We showed that the correction process did not generate TALEN-induced
off targeting mutations by sequencing. More importantly, the gene-corrected
β-Thal iPS cell lines from each patient can be induced to differentiate into
hematopoietic progenitor cells and then further to erythroblasts expressing
normal β-globin. Our studies provide an efficient and universal strategy to
correct different types of β-globin mutations in β-Thal iPSCs for disease
modeling and applications. Tal-effector nucleases (TALENs) are engineered proteins that can stimulate
precise genome editing through specific DNA double-strand breaks. Sickle cell
disease and β-thalassemia are common genetic disorders caused by mutations in
β-globin, and we engineered a pair of highly active TALENs that induce
modification of 54% of human β-globin alleles near the site of the sickle
mutation. These TALENS stimulate targeted integration of therapeutic,
full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells
prior to selection as quantified by single molecule real-time sequencing. We
also developed highly active TALENs to human γ-globin, a pharmacologic target in
sickle cell disease therapy. Using the β-globin and γ-globin TALENs, we
generated cell lines that express GFP under the control of the endogenous
β-globin promoter and tdTomato under the control of the endogenous γ-globin
promoter. With these fluorescent reporter cell lines, we screened a library of
small molecule compounds for their differential effect on the transcriptional
activity of the endogenous β- and γ-globin genes and identified several that
preferentially upregulate γ-globin expression. RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune
system known as CRISPR/Cas, enable targeted genome engineering in cells and
organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a
protein component originated from Streptococcus pyogenes. These enzymes cleave
chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted
manner, producing site-specific DNA double-strand breaks (DSBs), the repair of
which gives rise to targeted genome modifications. Despite broad interest in
RGEN-mediated genome editing, these nucleases are limited by off-target
mutations and unwanted chromosomal translocations associated with off-target DNA
cleavages. Here, we show that off-target effects of RGENs can be reduced below
the detection limits of deep sequencing by choosing unique target sequences in
the genome and modifying both guide RNA and Cas9. We found that both the
composition and structure of guide RNA can affect RGEN activities in cells to
reduce off-target effects. RGENs efficiently discriminated on-target sites from
off-target sites that differ by two bases. Furthermore, exome sequencing
analysis showed that no off-target mutations were induced by two RGENs in four
clonal populations of mutant cells. In addition, paired Cas9 nickases, composed
of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or
nicks on different DNA strands, were highly specific in human cells, avoiding
off-target mutations without sacrificing genome-editing efficiency.
Interestingly, paired nickases induced chromosomal deletions in a targeted
manner without causing unwanted translocations. Our results highlight the
importance of choosing unique target sequences and optimizing guide RNA and Cas9
to avoid or reduce RGEN-induced off-target mutations. Genome editing using engineered nucleases such as transcription activator-like
effector nucleases (TALENs) has become a powerful technology for reverse
genetics. In this study, we have described efficient detection methods for
TALEN-induced mutations at endogenous loci and presented guidelines of TALEN
design for efficient targeted mutagenesis in medaka, Oryzias latipes. We
performed a heteroduplex mobility assay (HMA) using an automated microchip
electrophoresis system, which is a simple and high-throughput method for
evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or
later generations. We found that a specific pattern of mutations is domit for
TALENs harboring several base pairs of homologous sequences in target sequence.
Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is
not essential for genomic DNA cleavage. Our findings provide information that
expands the potential of TALENs and other engineered nucleases as tools for
targeted genome editing in a wide range of organisms, including medaka. Transcription activator-like effectors (TALEs), first identified in Xanthomonas
bacteria, are naturally occurring or artificially designed proteins that
modulate gene transcription. These proteins recognize and bind DNA sequences
based on a variable numbers of tandem repeats. Each repeat is comprised of a set
of ∼ 34 conserved amino acids; within this conserved domain, there are usually
two amino acids that distinguish one TALE from another. Interestingly, TALEs
have revealed a simple cipher for the one-to-one recognition of proteins for DNA
bases. Synthetic TALEs have been used to successfully target genes in a variety
of species, including humans. Depending on the type of functional domain that is
fused to the TALE of interest, these proteins can have diverse biological
effects. For example, after binding DNA, TALEs fused to transcriptional
activation domains can function as robust transcription factors (TALE-TFs),
while fused to restriction endonucleases (TALENs) can cut DNA. Targeted genome
editing, in theory, is capable of modifying any endogenous gene sequence of
interest; this can be performed in cells or organisms, and may be applied to
clinical gene-based therapies in the future. With current technologies, highly
accurate, specific, and reliable gene editing cannot be achieved. Thus,
recognition and binding mechanisms governing TALE biology are currently hot
research areas. In this review, we summarize the major advances in TALE
technology over the past several years with a focus on the interaction between
TALEs and DNA, TALE design and construction, potential applications for this
technology, and unique characteristics that make TALEs superior to zinc finger
endonucleases. In this study, we used zinc finger nuclease-mediated knockout of the aryl
hydrocarbon receptor (AHR) or AHR nuclear translocator (ARNT) in MCF7 and AHR
knockout in MDA-MB-231 human breast cancer cells to investigate cross talk among
AHR, ARNT, and estrogen receptor α (ERα). Knockout of AHR or ARNT prevented the
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent induction of all AHR target
genes examined. Knockout of AHR or ARNT also significantly reduced basal
cytochrome P4501B1 (CYP1B1) expression levels, which were restored with
overexpression of either protein but not with a DNA binding-deficient AHR
mutant. Basal and TCDD-, 17β-estradiol (E2)-, or TCDD + E2-dependent recruitment
of AHR, ARNT, ERα, NCoA3, and RNA polymerase II to CYP1B1 as well as CYP1B1 mRNA
levels were abolished in MCF7-AHR((ko)) and MDA-MB-231 AHR(ko) cells. However,
reduced but significant E2-dependent recruitment of ERα, NCoA3, and RNA
polymerase II to CYP1B1 and weak increases in CYP1B1 mRNA levels were observed
in MCF7 ARNT((ko)) cells. Interestingly, E2-dependent increases in trefoil
factor 1, but not growth regulation by estrogen in breast cancer 1 (GREB1) mRNA
levels, were dependent on ARNT expression. Moreover, the TCDD-dependent
increases in the proteolytic degradation of ERα were prevented by the loss of
AHR or ARNT. Our data show that AHR and ARNT play critical roles in the basal,
TCDD, and E2-induced regulation of CYP1B1 but also reveal distinct roles for
both proteins in ERα transactivation. Over the past 5 years there has been a major transformation in our ability to
precisely manipulate the genomes of animals. Efficiencies of introducing precise
genetic alterations in large animal genomes have improved 100000-fold due to a
succession of site-specific nucleases that introduce double-strand DNA breaks
with a specificity of 10(-9). Herein we describe our applications of
site-specific nucleases, especially transcription activator-like effector
nucleases, to engineer specific alterations in the genomes of pigs and cows. We
can introduce variable changes mediated by non-homologous end joining of DNA
breaks to inactive genes. Alternatively, using homology-directed repair, we have
introduced specific changes that support either precise alterations in a gene's
encoded polypeptide, elimination of the gene or replacement by another unrelated
DNA sequence. Depending on the gene and the mutation, we can achieve 10%-50%
effective rates of precise mutations. Applications of the new precision genetics
are extensive. Livestock now can be engineered with selected phenotypes that
will augment their value and adaption to variable ecosystems. In addition,
animals can be engineered to specifically mimic human diseases and disorders,
which will accelerate the production of reliable drugs and devices. Moreover,
animals can be engineered to become better providers of biomaterials used in the
medical treatment of diseases and disorders. 1. 1. The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system,
a prokaryotic adaptive restriction system against invading nucleic acids, such
as those originating from bacteriophages and plasmids. Recently, this
RNA-directed DNA endonuclease has been harnessed to target DNA sequences of
interest. Here, we review the development of Cas9 as an important tool to not
only edit the genomes of a number of different prokaryotic and eukaryotic
species, but also as an efficient system for site-specific transcriptional
repression or activation. Additionally, a specific Cas9 protein has been
observed to target an RNA substrate, suggesting that Cas9 may have the ability
to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas
subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems
represent an effective and versatile biotechnological tool, which will have
significant impact on future advancements in genome engineering. |
Does ziconotide bind to N-type calcium channels? | Yes, ziconotide/omega-conotoxin MVIIA blocks N-type calcium channels. | Despite their high sequence homology, the peptide neurotoxins omega-conotoxin
MVIIA and MVIIC selectively block N- and P/Q-type calcium channels,
respectively. To study the recognition mechanism of calcium channel subtypes,
two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by
exchanging their N- and C-terminal halves. Binding assay for both N- and
P/Q-type calcium channels showed that amino acid residues restricted to the
N-terminal half are important for the recognition of N-type channels, whereas
essential residues for P/Q-type channel recognition are widely spread over the
whole omega-conotoxin molecule. PURPOSE: Spinal cord ischemia and resulting paraplegia represent a major
complication associated with surgical repair of the thoracoabdominal aorta.
Although the mechanism of spinal neuronal degeneration during ischemia is
unclear, it may involve excessive calcium influx via N-type voltage-sensitive
calcium channels (VSCCs). The neuroprotective capacity of intrathecal (IT)
administration of the selective N-type VSCC blocker ziconotide, previously shown
to be potently analgesic, was studied.
METHODS: In a rat aortic occlusion model, spinal cord ischemia was induced for
8, 9, or 10 minutes by occluding the descending thoracic aorta. Ziconotide was
administered IT as (1) a continuous infusion of 300 or 600 ng/kg/h initiated 24
hours before ischemia and continuing an additional 24 hours or (2) a 0.3
microgram bolus injected 45 minutes before the induction of ischemia. Animals
were allowed to live for 24 hours, and recovery of motor function was evaluated
during this period. Spinal cords were processed using a silver impregnation
technique and microtubule-associated protein type II (MAP2)
immunohistochemistry.
RESULTS: Continuous IT infusion of ziconotide provided significant protection
against 8- and 9-minute occlusions, but not 10-minute occlusions, as indicated
by recovery of motor function, degree of spinal neuronal degeneration, and loss
of MAP2 immunoreactivity. Acute IT pretreatment with ziconotide provided
transient protection during the initial 4 hours of reperfusion; however, this
protective effect was no longer present at 24 hours.
CONCLUSION: These data implicate N-type VSCC activation in spinal neuronal
degeneration caused by transient spinal ischemia, because selective blockade of
this channel by continuous IT infusion of ziconotide was protective against
injurious intervals of spinal ischemia. Based on these findings, ziconotide may
provide both neuroprotection and preemptive analgesia for aortic aneurysm
surgery. Accumulation of calcium following experimental traumatic brain injury (TBI) has
been demonstrated to be a prominent pathophysiological component that can
compromise mitochondrial functioning and threaten cell survival. The
omega-conopeptide SNX-111, also known as Ziconotide, is a potent antagonist of
the voltage-gated N-type calcium channel and has demonstrated significant
neuroprotective effects against ischemia-induced neuronal injury. To determine
whether this compound would be effective in reducing calcium accumulation
associated with TBI, SNX-111 was administered intravenously to rats 1 hour
following a moderate (2.2 to 2.75 atm) lateral fluid-percussion injury (or sham)
at doses of 1 (n = 30), 3 (n = 31), or 5 (n = 30) mg/kg; another group received
0.9% saline solution (n = 35). Brains were processed for calcium 45 (45Ca)
autoradiography at 6, 12, 24, 48, and 96 hours following insult. Optical density
measurements of 20 cortical and subcortical regions were analyzed. Injured
animals administered saline solution exhibited a significant increase in 45Ca
uptake within 12 regions ipsilateral to the site of injury. The most prominent
increases were evident throughout the ipsilateral cerebral cortex. SNX-111
reduced the injury-induced calcium accumulation within the ipsilateral cortex in
a dose-response fashion when measured at 6, 12, and 48 hours after insult. These
drug-induced reductions in calcium accumulation were as high as 75% in the
ipsilateral cerebral cortex, and up to 50% in other ipsilateral regions
(including thalamus and hippocampus). Consequently, the results suggest that
posttraumatic blocking of the voltage-gated N-type calcium channel after injury
reduces prolonged, trauma-induced calcium accumulation. Ziconotide (SNX-111), a selective blocker of neuronal N-type voltage-sensitive
calcium channels, is antinociceptive when it is administered intrathecally. It
is currently under clinical investigation for the treatment of maligt and
non-maligt pain syndromes. The present study was undertaken to compare and
contrast antinociceptive properties of ziconotide, morphine and clonidine in a
rat model of post-operative pain. Post-operative pain was produced by making a
longitudinal incision through the skin, fascia, and muscle of the plantar aspect
of the left hindpaw. This procedure produced immediate (0.5 h after surgery) and
long-lasting (4-7 days post-surgery) heat hyperalgesia and mechanical allodynia
in the injured hindpaw. Pain thresholds in the contralateral hindpaw were
unaffected. Administered one day after incisional surgery, intrathecal
ziconotide blocked established heat hyperalgesia in the injured hindpaw in a
dose-dependent manner yielding an ED(50)4 h) but reversible (<24 h) blockade of
established mechanical allodynia. Administered one day after surgery,
intrathecal bolus injection of morphine dose-dependently blocked heat
hyperalgesia in the injured hindpaw with an ED(50) of 1.6 microg (2.1 nmol) and
heat nociceptive responses in the normal hindpaw with an ED(50) of 2.7 microg
(3.6 nmol). The effects were immediate and short-lasting (</=1 h). Intravenous
bolus injection of 3 mg/kg (1.1 micromol/kg) ziconotide, administered either
before or after incisional surgery, had no effect on thermal pain thresholds
measured in either the injured or normal hindpaw. In contrast, intraperitoneal
injections of 2 mg/kg (2.6 micromol/kg) morphine and 2.5 mg/kg (9.4 micromol/kg)
clonidine blocked heat hyperalgesia in the injured hindpaw; morphine, but not
clonidine, also elevated thermal (heat) nociceptive response thresholds in the
normal hindpaw. The results of this study show that intrathecal ziconotide is
antinociceptive in a rat incisional model of post-operative pain and is more
potent, longer acting, and more specific in its actions than intrathecal
morphine. Ziconotide is a selective, potent and reversible blocker of neuronal N-type
voltage-sensitive calcium channels (VSCCs). Morphine is an agonist of mu-opioid
receptors and inhibits N-type VSCC channels via a G-protein coupling mechanism.
Both agents are antinociceptive when they are administered intrathecally
(spinally). The present study investigated the acute and chronic (7-day)
interactions of intrathecally administered ziconotide and morphine on
nociception in several animal models of pain. In the acute study, intrathecal
bolus injections of morphine and ziconotide alone produced dose-dependent
inhibition of formalin-induced tonic flinch responses and withdrawal responses
to paw pressure. The combination of ziconotide and morphine produced an additive
inhibition of formalin-induced tonic flinch responses and a significant leftward
shift of the morphine dose-response curve in the paw pressure test. After
chronic (7-day) intrathecal infusion, ziconotide enhanced morphine analgesia in
the formalin test. In contrast, chronic intrathecal morphine infusion produced
tolerance to analgesia, but did not affect ziconotide antinociception.
Antinociception produced by ziconotide alone was the same as that observed when
the compound was co-administered with morphine to morphine-tolerant rats. In the
hot-plate and tail immersion tests, chronic intrathecal infusion of morphine
lead to rapid tolerance whereas ziconotide produced sustained analgesia with no
loss of potency throughout the infusion period. Although ziconotide in
combination with morphine produced an apparent synergistic analgesic effects
during the initial phase of continuous infusion, it did not prevent morphine
tolerance to analgesia. These results demonstrate that (1) acute intrathecal
administrations of ziconotide and morphine produce additive or synergistic
analgesic effects; (2) chronic intrathecal morphine infusion results in
tolerance to analgesia but does not produce cross-tolerance to ziconotide; (3)
chronic intrathecal ziconotide administration produces neither tolerance nor
cross-tolerance to morphine analgesia; (4) intrathecal ziconotide does not
prevent or reverse morphine tolerance. BACKGROUND AND OBJECTIVES: Voltage-sensitive calcium channel conductance is
essential for the nervous system to signal a painful event. However, intrathecal
administration of L-type calcium channel blockers does not provide analgesia.
The present investigation was designed to assess the safety and analgesic
efficacy of ziconotide, a new N-type calcium channel blocker, when administered
intrathecally to patients with acute postoperative pain.
METHODS: This randomized, double-blind, pilot study included patients undergoing
elective total abdominal hysterectomy, radical prostatectomy, or total hip
replacement. After intrathecal injection of local anesthetic and before surgical
incision, a continuous intrathecal infusion of either placebo or 1 of 2 doses of
ziconotide (0.7 microg/h or 7.0 microg/h) was started and continued for 48 to 72
hours postoperatively. Primary and secondary efficacy variables were the mean
daily patient controlled analgesia (PCA) morphine equivalent consumption and
visual analog pain intensity (VASPI) scores, respectively.
RESULTS: Thirty patients received study drug; 26 were evaluable for efficacy.
Mean daily PCA morphine equivalent consumption was less in patients receiving
ziconotide than in placebo-treated patients, and the difference was
statistically significant between 24 and 48 hours (P = .040). VASPI scores
during the first 8 hours postoperatively were markedly lower in
ziconotide-treated than in placebo-treated patients. In 4 of 6 patients
receiving the high-dose of ziconotide (7 microg/h), adverse events, such as
dizziness, blurred vision, nystagmus, and sedation contributed to study drug
being discontinued after 24 hours. After ziconotide discontinuation, these
symptoms resolved.
CONCLUSIONS: Ziconotide showed analgesic activity, as shown by decreased PCA
morphine equivalent consumption and lower VASPI scores. Because of a favorable
trend of decreased morphine consumption with an acceptable side-effect profile
in the low-dose ziconotide group, 0.7 microg/h may be closer to the ideal dose
than 7 microg/h. Large-scale studies are required to clarify this issue. OBJECT: Determining the efficacy of a drug used in experimental traumatic brain
injury (TBI) requires the use of one or more outcome measures such as decreased
mortality or fewer neurological and neuropsychological deficits. Unfortunately,
outcomes in these test batteries have a fairly large variability, requiring
relatively large sample sizes, and administration of the tests themselves is
also very time consuming. The authors previously demonstrated that experimental
TBI and human TBI induce mitochondrial dysfunction. Because mitochondrial
dysfunction is easy to assess compared with neurobehavioral endpoints, it might
prove useful as an outcome measure to establish therapeutic time windows and
dose-response curves in preclinical drug testing. This idea was tested in a
model of TBI in rats.
METHODS: Animals treated with the selective N-type voltage-sensitive calcium
channel blocker Ziconotide (also known as SNX-111 and CI-1009) after cortical
impact displayed significant improvement in brain mitochondrial function. When a
single intravenous bolus injection of 4 mg/kg Ziconotide was given at different
time intervals, ranging from 15 minutes before injury to 10 hours after injury,
mitochondrial function was improved at all time points, but more so between 2
and 6 hours postinjury. The authors evaluated the effects on mitochondrial
function of Ziconotide at different doses by administering 0.5 to 6 mg/kg as a
single bolus injection 4 hours after injury, and found 4 mg/kg to be the optimum
dose.
CONCLUSIONS: The authors established these time-window profiles and
dose-response curves on the basis of mitochondrial outcome measures in a total
of 42 rats because there were such low standard deviations in these tests.
Establishing similar time-window profiles and dose-response curves by using
neurobehavioral endpoints would have required using 114 rats in much more
elaborate experiments. We recently reported that amino acid residues contained within a putative EF
hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the
relative barium:calcium permeability of N-type calcium channels (Feng, Z. P.,
Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T.
P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region
partially overlaps with residues previously implicated in block of the channel
by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF
hand domain on channel block by omega-conotoxin GVIA and the structurally
related omega-conotoxin MVIIA. Both of the toxins irreversibly block the
activity of wild type alpha(1B) N-type channels. We find that in addition to
previously identified amino acid residues, residues in positions 1326 and 1332
are important determits of omega-conotoxin GVIA blockade. Substitution of
residue Glu(1332) to arginine slows the time course of development of block.
Point mutations in position Gly(1326) to either arginine, glutamic acid, or
proline dramatically decrease the time constant for development of the block.
Additionally, in the G1326P mutant channel activity was almost completely
recovered following washout. A qualitatively similar result was obtained with
omega-conotoxin MVIIA, suggesting that common molecular determits underlie
block by these two toxins. Taken together the data suggest that residue
Gly(1326) may form a barrier, which controls the access of peptide toxins to
their blocking site within the outer vestibule of the channel pore and also
stabilizes the toxin-channel interaction. Cone snails (Conidae) are marine predators with some extraordinary features.
Their venom contains a hundred or more peptides that target numerous ion
channels and receptors in mammals, including several that are involved in
disease. omega-Conotoxins from fish hunting snails are 24-27 residue peptides
with a rigid 4-loop cysteine framework that target the N-type voltage-gated
calcium channel (VGCC). Two omega-conotoxins, MVIIA and CVID are currently in
clinical development for chronic pain management (Ziconotide or Prialt, and
AM336, respectively). In an attempt to develop small molecule equivalents of
CVID, we defined the Calpha-Cbeta vectors of the residues believed to be
important for binding to the N-type VGCC. Using these vectors, we undertook a
virtual screening of virtual libraries approach to identify compounds that
matched the pharmacophore. Cyclic pentapeptides containing residues of loop 2 of
CVID, with one or more being a D-amino acid were designed and synthesised and
were found to be active at the N-type VGCC (IC50 approximately 20 microM).
Agreeing with the specificity profile of CVID, molecules were inactive at the
P/Q-type VGCC. Ziconotide is a novel peptide that blocks the entry of calcium into neuronal
N-type voltage-sensitive calcium channels, preventing the conduction of nerve
signals. N-type calcium channels are present in the superficial laminae of the
dorsal horn of the spinal cord. In various animal models of pain, intrathecal
administration of ziconotide blocked nerve transmission and nociception. The
United States Food and Drug Administration recently approved ziconotide
intrathecal infusion for the management of severe chronic pain in patients who
require intrathecal therapy and who are intolerant of or refractory to other
treatment, such as systemic analgesics, adjunctive therapies, or intrathecal
morphine. The drug has a narrow therapeutic window and a lag time for the onset
and offset of analgesia and adverse events. In early clinical trials, frequent
and severe psychiatric and central nervous system adverse effects were
associated with rapid intrathecal infusion (0.4 microg/hr) and frequent
up-titration (every 12 hrs). Therefore, patients with psychiatric symptoms are
not candidates for this drug. Drug trials of external intrathecal catheters and
microinfusion devices demonstrated a 3% risk of meningitis. A low initial
infusion rate of 0.1 microg/hour and limiting infusion rate increases to 2-3
times/week are now recommended. Patients responsive to intrathecal ziconotide
require an implanted infusion system to receive long-term therapy. N-type calcium channels located on presynaptic nerve terminals regulate
neurotransmitter release, including that from the spinal terminations of primary
afferent nociceptors. Accordingly, N-type calcium channel blockers may have
clinical utility as analgesic drugs. A selective N-type calcium channel
inhibitor, ziconotide (Prialt), is a neuroactive peptide recently marketed as a
novel nonopioid treatment for severe chronic pain. To develop a small-molecule
N-type calcium channel blocker, the authors developed a 96-well plate
high-throughput screening scintillation proximity assay (SPA) for N-type calcium
channel blockers using [125I]-labeled omega-conotoxin GVIA as a channel-specific
ligand. Assay reagents were handled using Caliper's Allegro automation system,
and bound ligands were detected using a PerkinElmer TopCount. Using this assay,
more than 150,000 compounds were screened at 10 microM and approximately 340
compounds were identified as hits, exhibiting at least 40% inhibition of
[125I]GVIA binding. This is the 1st demonstration of the use of [125I]-labeled
peptides with SPA beads to provide a binding assay for the evaluation of ligand
binding to calcium channels. This assay could be a useful tool for drug
discovery. Ziconotide has been introduced as a new nonopioid treatment for chronic pain.
Structurally, it is a peptide, the synthetic analog of the omega-conotoxin,
derived from the marine snail, Conus magus. N-type voltage-sensitive calcium
channels play a role in the transmission of nociceptive stimuli and also are
involved in the release of neurotransmitters important in pain transmission.
Ziconotide's therapeutic benefit derives from its potent and selective blockade
of neuronal-type voltage-sensitive calcium channels. Blockade of the channels
results in suppression of abnormal ectopic discharges from the injury site or
the dorsal root ganglia, possibly resulting in decreased neuroplasticity, and
decreased synaptic transmission that leads to the generation of chronic pain
syndromes. The advantage of ziconotide is that tolerance does not occur, while
disadvantages associated with ziconotide are the need for intrathecal
administration and significant neurotoxicites associated with its use. When
tested in clinical trials, ziconotide has been shown to have synergistic or
additive value to the effect of morphine. Ziconotide, formerly known also as
SNX- 111, represents a new class of agents, the N-type calcium channel blockers.
These may represent another option for patients with refractory pain and
refractory pain syndromes. Worldwide a large number of patients suffer from severe chronic pain even after
treatment with opioids following the 3-step analgesic ladder developed by the
WHO. Intraspinal agents, including morphine, have been tried as a fourth step.
However, approximately 20% of cases remain refractory. Ziconotide, an
intrathecal analgesic with orphan drug status, is a novel alternative for the
management of chronic intractable pain. Ziconotide is a synthetic peptide based
on the toxin of the fish-hunting marine snail, Conus magus. It is the first
therapeutic agent in a new pharmacological class of "topically" active
analgesics that selectively target neuron-specific (N-type), voltage-gated
calcium channels. Ziconotide produces potent analgesia by interruption of
Ca-dependent primary afferent transmission of pain signals in the spinal cord.
Ziconotide was significantly more effective than placebo in the treatment of
chronic maligt (p < 0.001) and non-maligt pain (p < 0.001). In several
clinical studies morphine dosages could be substituted by ziconotide. The drug
has a lag-time for the onset and offset of analgesia and adverse effects.
Initial doses should therefore be low (2.4 microg/day) and titrated slowly
(increasing up to a maximum of 21.6 microg/day in increases of 2.4 microg/day no
more than twice weekly). The gradual increase in dose helps to reduce the
incidence and severity of adverse events which affect primarily the central
nervous system (e.g. dizziness, nausea, confusion). Ziconotide maintains its
analgesic efficacy over months and does not cause tolerance, dependence or
respiratory depression. Following intrathecal infusion ziconotide is distributed
within the cerebral spinal fluid (CSF) where its clearance (0.38 ml/min)
corresponds to the rate of turnover of the CSF. Negligible amounts of ziconotide
are present in the systemic circulation where it is rapidly degraded by
proteolysis. In conclusion, ziconotide is a new and valuable alternative
analgesic for the acute and long-term treatment of severe pain, especially in
patients refractory to opioids. The N-type calcium channel is a member of the voltage-sensitive calcium channel
family and plays a major role in the regulation of neurotransmitter release in
the central and peripheral nervous systems. Inhibition of the N-type calcium
channel by intrathecal administration of the channel-specific blocker
omega-conotoxin MVIIA (ziconotide) is efficacious in the treatment of severe
chronic pain. While no orally active small molecules that block the N-type
calcium channel are currently available, the discovery of such potentially
valuable therapeutics would benefit from a reliable, high throughput assay.
However, the assay of N-type calcium channel activity by measuring calcium
influx using nonadherent cells in a high throughput fashion has not been
achieved before, likely owing to a number of technical hurdles. For example, the
measurement of calcium levels in nonadherent cells using conventional calcium
indicators, such as Fluo-3 or Fluo-4, requires dyeloading the cells in
suspension and subsequent removal of extracellular dye. This limits plate
throughput and requires constant handling of the cells. To assay the N-type
calcium channel activity using a nonadherent cell line in a high throughput
manner, we investigated the application of no-wash calcium assay kits from
Molecular Devices Corp. (Sunnyvale, CA): FLIPR Calcium, FLIPR Calcium Plus, and
FLIPR Calcium 3. We show here that the FLIPR Calcium 3 assay kit can be used
with nonadherent IMR-32 cells to measure potassium-evoked, omega-conotoxin
MVIIA-reversible calcium flux with high throughput (15,000 data points/day),
high quality (Z approximately 0.6), and minimal handling of the cells. Thus,
this assay can be used to reliably and efficiently screen large compound
libraries in the search for small molecule N-type calcium channel blockers. Ziconotide is a powerful analgesic drug that has a unique mechanism of action
involving potent and selective block of N-type calcium channels, which control
neurotransmission at many synapses. The analgesic efficacy of ziconotide likely
results from its ability to interrupt pain signaling at the level of the spinal
cord. Ziconotide is a peptidic drug and has been approved for the treatment of
severe chronic pain in patients only when administered by the intrathecal route.
Importantly, prolonged administration of ziconotide does not lead to the
development of addiction or tolerance. The current review discusses the various
studies that have addressed the in vitro biochemical and electrophysiological
actions of ziconotide as well as the numerous pre-clinical studies that were
conducted to elucidate its antinociceptive mechanism of action in animals. In
addition, this review considers the pivotal Phase 3 (and other) clinical trials
that were conducted in support of ziconotide's approval for the treatment of
severe chronic pain and tries to offer some insights regarding the future
discovery and development of newer analgesic drugs that would act by a similar
mechanism to ziconotide but which might offer improved safety, tolerability and
ease of use. Spinal neurotransmission plays an important role in the perception of pain
signaling. In the present study, we investigated the spinal anti-nociceptive
mechanism of current standard analgesics in mouse models of tactile allodynia
induced by intrathecal administration of N-methyl-D-aspartic acid (NMDA),
prostaglandin E2 (PGE2), and bicuculline. NMDA-induced allodynia is induced by
postsynaptic NMDA receptor activation, while PGE2-induced allodynia is triggered
by the enhancement of presynaptic glutamate release via EP1 receptor activation.
In contrast, bicuculline induces allodynia by the blockade of gamma-aminobutyric
acid (GABA)A receptor-mediated inhibitory system. As the clinically available
analgesics, pregabalin (alpha2delta-subunit calcium channel ligand), ziconotide
(N-type calcium channel blocker), mexiletine (sodium channel blocker), and
duloxetine (serotonin and norepinephrine reuptake inhibitors) were evaluated in
these neurochemically-induced allodynia models. Pregabalin almost completely
alleviated NMDA-, PGE2-, and bicuculline-induced allodynia. Despite being
classified as an agent with a similar molecular target mechanism, ziconotide
could only alleviate PGE2-induced allodynia, but not NMDA- or
bicuculline-induced allodynia, as did mexiletine and duloxetine. These results
taken together suggest that ziconotide, mexiletine, and duloxetine suppress
spinal hyperactivity via the presynaptic site mechanism. In contrast, pregabalin
could suppress via the downstream step during spinal hyperactivation such as
postsynaptic NMDA activation or dysfunction of GABAergic control in addition to
presynaptic mechanism. In conclusion, present findings provide implication that
the spinal anti-nociceptive mechanistic site of pregabalin is different from
that of ziconotide, mexiletine, and duloxetine, and pregabalin could have a
broader anti-nociceptive mechanism other than N-type calcium channel blockade. BACKGROUND: The authors investigated the role of different voltage-sensitive
calcium channels expressed at presynaptic afferent terminals in substance P
release and on nociceptive behavior evoked by intraplantar formalin by examining
the effects of intrathecally delivered N- (ziconotide), T- (mibefradil), and
L-type voltage-sensitive calcium channel blockers (diltiazem and verapamil).
METHODS: Rats received intrathecal pretreatment with saline or doses of
morphine, ziconotide, mibefradil, diltiazem, or verapamil. The effect of these
injections upon flinching evoked by intraplantar formalin (5%, 50 μl) was
quantified. To assess substance P release, the incidence of neurokinin-1
receptor internalization in the ipsilateral and contralateral lamina I was
determined in immunofluorescent-stained tissues.
RESULTS: Intrathecal morphine (20 μg), ziconotide (0.3, 0.6, and 1 μg),
mibefradil (100 μg, but not 50 μg), diltiazem (500 μg, but not 300 μg), and
verapamil (200 μg, but not 50 and 100 μg) reduced paw flinching in phase 2
compared with vehicle control (P < 0.05), with no effect on phase 1. Ziconotide
(0.3, 0.6, and 1 μg) and morphine (20 μg) significantly inhibited neurokinin-1
receptor internalization (P < 0.05), but mibefradil, diltiazem, and verapamil at
the highest doses had no effect.
CONCLUSION: These results emphasize the role in vivo of N-type but not T- and
L-type voltage-sensitive calcium channel blockers in mediating the
stimulus-evoked substance P release from small primary afferents and suggest
that T- and L-type voltage-sensitive calcium channel blockers exert
antihyperalgesic effects by an action on other populations of afferents or
mechanisms involving postsynaptic excitability. Voltage-gated sodium channels (VGSCs) are large transmembrane proteins that
conduct sodium ions across the membrane and by doing so they generate signals of
communication between many kinds of tissues. They are responsible for the
generation and propagation of action potentials in excitable cells, in close
collaboration with other channels like potassium channels. Therefore, genetic
defects in sodium channel genes can cause a wide variety of diseases, generally
called "channelopathies." The first insights into the mechanism of action
potentials and the involvement of sodium channels originated from Hodgkin and
Huxley for which they were awarded the Nobel Prize in 1963. These concepts still
form the basis for understanding the function of VGSCs. When VGSCs sense a
sufficient change in membrane potential, they are activated and consequently
generate a massive influx of sodium ions. Immediately after, channels will start
to inactivate and currents decrease. In the inactivated state, channels stay
refractory for new stimuli and they must return to the closed state before being
susceptible to a new depolarization. On the other hand, studies with neurotoxins
like tetrodotoxin (TTX) and saxitoxin (STX) also contributed largely to our
today's understanding of the structure and function of ion channels and of VGSCs
specifically. Moreover, neurotoxins acting on ion channels turned out to be
valuable lead compounds in the development of new drugs for the enormous range
of diseases in which ion channels are involved. A recent example of a synthetic
neurotoxin that made it to the market is ziconotide (Prialt(®), Elan). The
original peptide, ω-MVIIA, is derived from the cone snail Conus magus and now
FDA/EMA-approved for the management of severe chronic pain by blocking the
N-type voltage-gated calcium channels in pain fibers. This review focuses on the
current status of research on neurotoxins acting on VGSC, their contribution to
further unravel the structure and function of VGSC and their potential as novel
lead compounds in drug development. Biological, genetic, and clinical evidence provide validation for N-type calcium
channels (Ca(V)2.2) as therapeutic targets for chronic pain. A state-dependent
Ca(V)2.2 inhibitor may provide an improved therapeutic window over ziconotide,
the peptidyl Ca(V)2.2 inhibitor used clinically. Supporting this notion, we
recently reported that in preclinical models, the state-dependent Ca(V)2
inhibitor
(3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one
(TROX-1) has an improved therapeutic window compared with ziconotide. Here we
characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels
are biased toward open/inactivated states by depolarizing the membrane potential
under voltage-clamp electrophysiology, TROX-1 inhibits Ca(V)2.2 channels with an
IC(50) of 0.11 μM. The voltage dependence of Ca(V)2.2 inhibition was examined
using automated electrophysiology. TROX-1 IC(50) values were 4.2, 0.90, and 0.36
μM at -110, -90, and -70 mV, respectively. TROX-1 displayed use-dependent
inhibition of Ca(V)2.2 with a 10-fold IC(50) separation between first (27 μM)
and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx
assay, TROX-1 inhibited Ca(V)2.2 channels with an IC(50) of 9.5 μM under
hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally,
TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC(50)
values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched
conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca(V)2.1, Ca(V)2.2,
and Ca(V)2.3, respectively. Together, these in vitro data support the idea that
a state-dependent, non-subtype-selective Ca(V)2 channel inhibitor can achieve an
improved therapeutic window over the relatively state-independent
Ca(V)2.2-selective inhibitor ziconotide in preclinical models of chronic pain. Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission.
Evidence from knockout animals and localization studies suggest that Ca(V)2.2
channels play a critical role in nociceptive transmission. Additionally,
ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically
used to treat refractory pain. However, the use of ziconotide is limited by its
low therapeutic index, which is believed, at least in part, to be a consequence
of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state.
Subsequent efforts have focused on the discovery of state-dependent inhibitors
that preferentially bind to the inactivated state of Ca(V)2.2 channels in order
to achieve an improved safety profile relative to ziconotide. Much less
attention has been paid to understanding the binding kinetics of these
state-dependent inhibitors. Here, we describe a novel electrophysiology-based
assay on an automated patch platform designed to differentiate Ca(V)2.2
inhibitors based on their combined state dependence and kinetics. More
specifically, this assay assesses inactivated state block, closed state block,
and monitors the kinetics of recovery from block when channels move between
states. Additionally, a use-dependent assay is described that uses a train of
depolarizing pulses to drive channels to a similar level of inactivation for
comparison. This use-dependent protocol also provides information on the
kinetics of block development. Data are provided to show how these assays can be
utilized to screen for kinetic diversity within and across chemical classes. Selective blockers of the N-type calcium channel have proven to be effective in
animal models of chronic pain. However, even though intrathecally delivered
synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been
approved for the treatment of chronic pain in humans, its mode of delivery and
narrow therapeutic window have limited its usefulness. Therefore, the
identification of orally active, small-molecule N-type calcium channel blockers
would represent a significant advancement in the treatment of chronic pain. A
novel series of pyrazole-based N-type calcium channel blockers was identified by
structural modification of a high-throughput screening hit and further optimized
to improve potency and metabolic stability. In vivo efficacy in rat models of
inflammatory and neuropathic pain was demonstrated by a representative compound
from this series. There is strong pharmacological, biological, and genetic evidence supporting the
role of N-type calcium channels (CaV2.2) in nociception. There is also human
validation data from ziconotide, the CaV2.2-selective peptidyl inhibitor used
clinically to treat refractory pain. Unfortunately, ziconotide utility is
limited by its narrow therapeutic window and required intrathecal route of
administration. A major focus has been placed on identifying state-dependent
CaV2.2 inhibitors to improve safety margins. Much less attention, however, has
been given to characterizing the kinetics of CaV2.2 inhibitors as a means to
further differentiate compounds and maximize therapeutic potential. Here we
provide a detailed characterization of the CaV2.2 inhibitor T4 in terms of its
state-dependence, use-dependence, kinetics, and mechanism of inhibition.
Compound T4 displayed a >20-fold difference in potency when measured under
inactivating conditions (IC50=1.1 μM) as compared to closed-state conditions
(IC50=25 μM). At 3 μM, T4 produced a 15-fold hyperpolarizing shift in the
inactivation curve for CaV2.2 while having no effect on channel activation. To
assess the kinetic properties of T4 in a more physiological manner, its
inhibition kinetics were assessed at 32°C using 2 mM Ca(2+) as the charge
carrier. Surprisingly, the repriming rate for CaV2.2 channels at hyperpolarized
potentials was similar in both the presence and absence of T4. This was in
contrast to other compounds which markedly delayed repriming. Furthermore, T4
inhibited CaV2.2 channels more potently when channel inactivation was driven
through a tonic sub-threshold depolarization rather than through a use-dependent
protocol, despite similar levels of inactivation. N-type calcium channels represent a promising target for the treatment of
neuropathic pain. The selective N-type calcium channel blocker ziconotide
ameliorates severe chronic pain but has a narrow therapeutic window and requires
intrathecal administration. We identified tetrahydroisoquinoline derivative 1a
as a novel potent N-type calcium channel blocker. However, this compound also
exhibited potent inhibitory activity against hERG channels. Structural
optimizations led to identification of
(1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone
((S)-1h), which exhibited high selectivity for hERG channels while retaining
potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in
vivo efficacy as an orally available N-type calcium channel blocker in a rat
spinal nerve ligation model of neuropathic pain. |
How is OCT3 associated with serotonin? | OCT3 plays a role in serotonin clearance | The serotonin (5HT) transporter (5HTT) regulates serotonergic neurotransmission
by mediating the reuptake of 5HT from the synaptic cleft. Although lacking the
high affinity and selectivity of the 5HTT, the brain expresses a large number of
other transporters, including the polyspecific organic cation transporters
(OCTs). OCT1 and OCT3, members of the potential-sensitive organic cation
transporter gene family, physiologically transport a wide spectrum of organic
cations. In addition, both transporters mediate low-affinity 5HT transport and,
therefore, may participate in the clearance of excessive 5HT. Because
concentrations of extracellular 5HT are increased in the brain of 5HTT-deficient
mice, they are a model for investigating the role of OCTs in 5HT system
homeostasis. Here, we analyzed OCT1 and OCT3 gene expression in the brain of
5HTT knockout mice by semiquantitative competitive polymerase chain reaction and
in situ hybridization. We demonstrate that, in 5HTT-deficient mice, OCT3 mRNA
concentrations were significantly increased in the hippocampus, but not in other
brain regions, including cortex, striatum, cerebellum, and brainstem. In
contrast, no difference in OCT1 expression was detected between 5HTT knockout
and control mice. Up-regulation of OCT3 expression and enhanced low-affinity 5HT
uptake may limit the adverse effects of elevated extracellular 5HT and may play
a critical role in maintaining 5HT-dependent functions of the hippocampus in the
absence of 5HTT. Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter
that mediates bidirectional, sodium-independent transport of dopamine,
norepinephrine, epinephrine, serotonin, and histamine. OCT3-mediated transport
is directly inhibited by corticosterone, suggesting a potential role for the
transporter in mediating some of the effects of stress and glucocorticoids on
monoaminergic neurotransmission. To elucidate the importance of OCT3 in
clearance of extracellular monoamines in the brain, we used immunohistochemical
techniques to describe the distribution of OCT3-like-immunoreactive (OCT3-ir)
cells throughout the rostrocaudal extent of adult male rat brains. OCT3-ir cell
bodies were widely distributed throughout the brain, with the highest densities
observed in the superior and inferior colliculi, islands of Calleja, subiculum,
lateral septum, lateral and dorsomedial hypothalamic nuclei, and granule cell
layers of the main and accessory olfactory bulbs, the cerebellum, and the
retrosplenial granular cortex. OCT3-ir cells and/or fibers were also observed in
circumventricular organs, and OCT3-ir ependymal cells were observed in the
linings of all cerebral ventricles. The widespread distribution of OCT3-ir cell
bodies, including regions receiving dense monoaminergic projections, suggests an
important role for this transporter in regulating extracellular concentrations
of monoamines in the rat brain and is consistent with the hypothesis that
corticosterone-induced inhibition of OCT3-mediated transport may contribute to
effects of acute stress or corticosterone on monoaminergic neurotransmission. Mood disorders cause much suffering and are the single greatest cause of lost
productivity worldwide. Although multiple medications, along with behavioral
therapies, have proven effective for some individuals, millions of people lack
an effective therapeutic option. A common serotonin (5-HT) transporter
(5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression
in vivo and elevates risk for multiple mood disorders including anxiety,
alcoholism, and major depression. Importantly, this variant is also associated
with reduced responsiveness to selective 5-HT reuptake inhibitor
antidepressants. We hypothesized that a reduced antidepressant response in
individuals with a constitutive reduction in 5-HTT expression could arise
because of the compensatory expression of other genes that inactivate 5-HT in
the brain. A functionally upregulated alternate transporter for 5-HT may prevent
extracellular 5-HT from rising to levels sufficiently high enough to trigger the
adaptive neurochemical events necessary for therapeutic benefit. Here we
demonstrate that expression of the organic cation transporter type 3 (OCT3,
SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with
constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22
diminishes 5-HT clearance and exerts antidepressant-like effects in these mice
but not in WT animals. OCT3 may be an important transporter mediating
serotonergic signaling when 5-HTT expression or function is compromised. The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine
transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters
and is expressed widely across the whole brain. OCTs are a family of
high-capacity, bidirectional, multispecific transporters of organic cations.
These also include serotonin, dopamine and norepinephrine making OCTs attractive
candidates for a variety of neuropsychiatric disorders including anxiety
disorders. OCT3 has been implicated in termination of monoaminergic signalling
in the central nervous system. Interestingly, OCT3 mRNA is however also
significantly up-regulated in the hippocampus of serotonin transporter knockout
mice where it might serve as an alternative reuptake mechanism for serotonin.
The examination of the behavioural phenotype of OCT3 knockout mice thus is
paramount to assess the role of OCT3. We have therefore subjected mice lacking
the OCT3 gene to a comprehensive behavioural test battery. While cognitive
functioning in the Morris water maze test and aggression levels measured with
the resident-intruder paradigm were in the same range as the respective control
animals, OCT3 knockout animals showed a tendency of increased activity and were
significantly less anxious in the elevated plus-maze test and the open field
test as compared to their respective wild-type controls arguing for a role of
OCT3 in the regulation of fear and anxiety, probably by modulating the
serotonergic tone in limbic circuitries. The organic cation transporter (OCT) 3 is widely expressed in various organs in
humans, and involved in the disposition of many exogenous and endogenous
compounds. Several lines of evidence have suggested that OCT3 expressed in the
brain plays an important role in the regulation of neurotransmission. Relative
to wild-type (WT) animals, Oct3 knockout (KO) mice have displayed altered
behavioral and neurochemical responses to psychostimulants such as amphetamine
(AMPH) and methamphetamine. In the present study, both in vitro and in vivo
approaches were utilized to explore potential mechanisms underlying the
disparate neuropharmacological effects observed following AMPH exposure in Oct3
KO mice. In vitro uptake studies conducted in OCT3 transfected cells indicated
that dextroamphetamine (d-AMPH) is not a substrate of OCT3. However, OCT3 was
determined to be a high-capacity and low-affinity transporter for the
neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5-HT).
Inhibition studies demonstrated that d-AMPH exerts relatively weak inhibitory
effects on the OCT3-mediated uptake of DA, NE, 5-HT, and the model OCT3
substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide. The IC(50)
values were determined to be 41.5 +/- 7.5 and 24.1 +/- 7.0 microM for inhibiting
DA and 5-HT uptake, respectively, while 50% inhibition of NE and
4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide uptake was not achieved by
even the highest concentration of d-AMPH applied (100 microM). Furthermore, the
disposition of d-AMPH in various tissues including the brain, liver, heart,
kidney, muscle, intestine, spleen, testis, uterus, and plasma were determined in
both male and female Oct3 KO and WT mice. No significant difference was observed
between either genotypes or sex in all tested organs and tissues. Our findings
suggest that OCT3 is not a prominent factor influencing the disposition of
d-AMPH. Additionally, based upon the inhibitory potency observed in vitro,
d-AMPH is unlikely to inhibit the uptake of monoamines mediated by OCT3 in the
brain. Differentiated neuropharmacological effects of AMPHs noted between Oct3
KO and WT mice appear to be due to the absence of Oct3 mediated uptake of
neurotransmitters in the KO mice. BACKGROUND: Murine basophils can contribute to the T(H)2 polarization of the
immune response by providing rapidly large amounts of IL-4, which suggests that
pharmacologic downregulation of this cytokine might provide a strategy to
attenuate pathologies associated with excessive production.
OBJECTIVE: We examined a number of physiological and pharmacologic ligands of
the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines,
for their inhibitory effect on IL-4 production by basophils, selecting the most
efficient compounds for in vivo evaluation in basophil-dependent experimental
models.
METHODS: IL-4 production by basophils isolated ex vivo or from bone marrow
cultures was assessed in response to various stimuli with or without biogenic
monoamines or pharmacologic analogs. Selected compounds were administered
in vivo to examine their effect on levels of circulating IgE generated during a
basophil-dependent T(H)2 response and on basophil activation in mice receiving
IL-33.
RESULTS: We found a drastic decrease in IL-4 production by stimulated basophils
on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by
basophils through the specific high-affinity transporters serotonin transporter
and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or
Slc22a3) but inhibits their function exclusively through the latter. This
downregulation is likewise observed in vivo in response to 5-HT and other OCT3
ligands, as well as in human basophils sorted from PMBCs of nonatopic donors.
CONCLUSIONS: We provide evidence for a new means of downregulating IL-4
production by basophils, both in vitro and in vivo, through OCT3 targeted by
5-HT and pharmacologic ligands. |
What constitutes an increased risk for individuals with Fanconi anemia? | Fanconi anemia is a rare genetic disorder associated with an increased risk of leukemias and solid tumors. | Three patients with Fanconi's anemia were analyzed for chromosome breaks. T and
B cells were separated and grown in tissue culture with PHA and pokeweed antigen
to ascertain the rates of breakage in these lymphocytic subpopulations. It has
been found that there is no statistically significant difference in breakage
rates in T and B lymphocytes. It is postulated that both T and B cells could be
involved in the development of leukemia in Fanconi's anemia patients, assuming
that chromosome breaks constitute a factor predisposing to the development of
maligcy. Fanconi anemia (FA) is a rare autosomal recessive disease characterized by
skeletal defects, anemia, chromosomal instability and increased risk of
leukemia. At the cellular level FA is characterized by increased sensitivity to
agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been
cloned and interactions among individual FANC proteins have been found. The
FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA
damage and during S-phase, requiring the FANCA, C, E and G proteins to do so.
This finding may reflect a direct role for the BRCA1 protein in double strand
break (DSB) repair and interaction with the FANC proteins. Therefore
interactions between BRCA1 and the FANC proteins were investigated. Among the
known FANC proteins, we find evidence for direct interaction only between the
FANCA protein and BRCA1. The evidence rests on three different tests: yeast
two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and
coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA
and the central part (aa 740-1083) of BRCA1 contain the sites of interaction.
The interaction does not depend on DNA damage, thus FANCA and BRCA1 are
constitutively interacting. The demonstrated interaction directly connects BRCA1
to the FA pathway of DNA repair. Genes of the Fanconi complementation groups [Fanconi anemia (FA) genes] are
suggested to be involved in homologous DNA recombination and produce FA when two
allelic mutations are inherited. BRCA2 is an FA gene and additionally conveys an
inherited risk for breast, ovarian, and pancreatic cancer for individuals
carrying a single mutated allele [N. G. Howlett et al., Science (Wash. DC), 297:
606-609, 2002]. Here we report inherited and somatic mutations of FANCC and
FANCG present in young-onset pancreatic cancer. This may imply a general
involvement of Fanconi genes with an inherited risk of cancer. The known
hypersensitivity of Fanconi cells to mitomycin and other therapeutic agents [M.
S. Sasaki, Nature (Lond.), 257: 501-503, 1975] suggests a therapeutic utility
for a more complete characterization of the DNA repair defects and their
causative genetic mutations in pancreatic cancer. Fanconi anemia is an inherited disease characterized by bone marrow failure,
congenital malformations, and predisposition to cancer. The breast cancer
susceptibility gene BRCA2 was recently found to be associated with Fanconi
anemia complementation group D1 (FA-D1). We examined four kindreds afflicted
with Fanconi anemia for the presence of germline BRCA2 mutations. One kindred,
of Ashkenazi Jewish ancestry, had five members who were diagnosed with breast
cancer and two cousins who were BRCA2*6174delT/C3069X compound heterozygotes and
had Fanconi anemia and brain tumors. In another kindred of Ashkenazi Jewish and
Lithuanian Catholic ancestry, a child with Fanconi anemia and a medulloblastoma
was a BRCA2*6174delT/886delGT compound heterozygote. Two other kindreds each
contained a Fanconi anemia-afflicted child who developed medulloblastoma; one
child was of Latin American ancestry and a compound heterozygote for
BRCA2*I2490T/ 5301insA and the other was African American and a compound
heterozygote for BRCA2*Q3066X/E1308X. Median age of the Fanconi anemia-afflicted
children at brain tumor diagnosis was 3.5 years. The co-occurrence of brain
tumors, Fanconi anemia, and breast cancer observed in one of these kindreds
constitutes a new syndromic association. Individuals who carry a germline BRCA2
mutation and who plan to have children with a partner of Ashkenazi Jewish
descent should consider undergoing genetic counseling. Fanconi anemia (FA) is a rare autosomal recessive disease characterized by a
greatly increased risk of cancer among those diagnosed with the syndrome. The
question as to whether FA heterozygotes are at increased risk for cancer is of
great importance to those at risk for being a carrier. To address this question,
we formed a cohort of grandparents of probands identified through the
International Fanconi Anemia Registry. We obtained informed consent, a short
questionnaire, and either blood or buccal swab DNA. After diagnosis of the
proband was confirmed and complementation studies or DNA sequencing on the
proband were completed, mutation analyses of the putative carriers and
noncarriers was carried out. Standardized incidence ratios (SIR) were calculated
to compare the observed cancer incidence of the grandparents and other relatives
with the expected rates of cancer, using the Surveillance, Epidemiology, and End
Results registries and the Connecticut Cancer registry. In the 944 study
subjects who participated (784 grandparents and 160 other relatives), there was
no suggestion of an increase in overall cancer incidence. On the other hand, a
significantly higher rate of breast cancer than expected was observed among
carrier grandmothers [SIR, 1.7; 95% confidence interval (95% CI), 1.1-2.7].
Among the grandmothers, those who were carriers of FANCC mutations were found to
be at highest risk (SIR, 2.4; 95% CI, 1.1-5.2). Overall, there was no increased
risk for cancer among FA heterozygotes in this study of Fanconi relatives,
although there is some evidence that FANCC mutations are possibly breast cancer
susceptibility alleles. BACKGROUND: Fanconi anemia (FA) is an autosomal recessive DNA repair disorder
with affected individuals having a high risk of developing acute myeloid
leukaemia and certain solid tumours. Thirteen complementation groups have been
identified and the genes for all of these are known (FANCA, B, C, D1/BRCA2, D2,
E, F, G, I, J/BRIP1, L, M and N/PALB2). Previous studies of cancer incidence in
relatives of Fanconi anemia cases have produced conflicting results. A study of
British FA families was therefore carried out to investigate this question,
since increases in cancer risk in FA heterozygotes would have implications for
counselling FA family members, and possibly also for the implementation of
preventative screening measures in FA heterozygotes.
METHODS: Thirty-six families took part and data was collected on 575 individuals
(276 males, 299 females), representing 18,136 person years. In this cohort, 25
males and 30 females were reported with cancer under the age of 85 years, and 36
cancers (65%) could be confirmed from death certificates, cancer registries or
clinical records.
RESULTS: A total of 55 cancers were reported in the FA families compared to an
estimated incidence of 56.95 in a comparable general population cohort, and the
relative risk of cancer was 0.97 (95% C.I. = 0.71-1.23, p = 0.62) for FA family
members. Analysis of relative risk for individual cancer types in each carrier
probability group did not reveal any significant differences with the possible
exception of prostate cancer (RR = 3.089 (95% C.I. = 1.09 - 8.78; Chi2 = 4.767,
p = 0.029).
CONCLUSION: This study has not shown a significant difference in overall cancer
risk in FA families. Fanconi anemia is a genetically heterogeneous disorder associated with
chromosome instability and a highly elevated risk for developing cancer. The
mutated genes encode proteins involved in the cellular response to DNA
replication stress. Fanconi anemia proteins are extensively connected with DNA
caretaker proteins, and appear to function as a hub for the coordination of DNA
repair with DNA replication and cell cycle progression. At a molecular level,
however, the raison d'être of Fanconi anemia proteins still remains largely
elusive. The thirteen Fanconi anemia proteins identified to date have not been
embraced into a single and defined biological process. To help put the Fanconi
anemia puzzle into perspective, we begin this review with a summary of the
strategies employed by prokaryotes and eukaryotes to tolerate obstacles to the
progression of replication forks. We then summarize what we know about Fanconi
anemia with an emphasis on biochemical aspects, and discuss how the Fanconi
anemia network, a late acquisition in evolution, may function to permit the
faithful and complete duplication of our very large vertebrate chromosomes. Fanconi anemia (FA) is an inherited disease characterized by bone marrow
failure, increased cancer risk and hypersensitivity to DNA cross-linking agents,
implying a role for this pathway in the maintece of genomic stability. The
central player of the FA pathway is the multi-subunit E3 ubiquitin ligase
complex activated through a replication- and DNA damage-dependent mechanism. A
consequence of the activation of the complex is the monoubiquitylation of FANCD2
and FANCI, late term effectors in the maintece of genome integrity. The
details regarding the coordination of the FA-dependent response and the DNA
replication process are still mostly unknown. We found, by yeast two-hybrid
assay and co-immunoprecipitation in human cells, that the core complex subunit
FANCF physically interacts with PSF2, a member of the GINS complex essential for
both the initiation and elongation steps of DNA replication. In HeLa cells
depleted for PSF2, we observed a decreased binding to chromatin of the FA core
complex, suggesting that the GINS complex may have a role in either loading or
stabilizing the FA core complex onto chromatin. Consistently, GINS and core
complex bind chromatin contemporarily upon origin firing and PSF2 depletion
sensitizes cells to DNA cross-linking agents. However, depletion of PSF2 is not
sufficient to reduce monoubiquitylation of FANCD2 or its localization to nuclear
foci following DNA damage. Our results suggest a novel crosstalk between DNA
replication and the FA pathway. BACKGROUND: Fanconi anemia (FA) is an autosomal recessive, cancer susceptibility
disorder characterized by diverse clinical features, such as short stature,
skeletal or skin abnormalities, progressive bone marrow (BM) failure, and
increased risk of maligcies. Clonal chromosomal abnormalities are frequently
reported in FA patients transformed to myelodysplastic syndrome (MDS) and acute
myeloid leukemia (AML).
AIM: To study the incidence of maligcy and clonal chromosomal abnormalities
in FA patients.
MATERIALS AND METHODS: Thirty-eight clinically diagnosed FA patients were
studied at the time of diagnosis and the patients were followed-up for a maximum
of 28 months at 3-month intervals. The median duration of follow-up of these
patients was 19.8 months. Chromosomal breakage investigation using mitomycin C
(MMC)- and diepoxybutane (DEB)-induced peripheral blood cultures were stimulated
with phytohemagglutinin. Cytogenetic study was done on the BM cells to detect
clonal chromosomal aberrations.
RESULTS: Eleven (28.95%) out of 38 patients developed maligcies, including 6
(54.54%) MDS, 4 (36.36%) AML, and 1 (2.63%) squamous cell carcinoma. The clonal
chromosomal abnormalities were detected in 5 (45.45%) FA patients who developed
maligcies and the type of chromosomal abnormality detected were monosomies 5,
7, trisomy 10, dup(1)(q12-q24), and inv(7)(p11pter).
CONCLUSION: The FA patients have a high risk of developing maligcies, and
clonal chromosomal abnormalities play an important role in the prognosis of the
disease. Therefore, FA patients need to be followed-up at regular intervals for
early diagnosis and optimal management of the disease. The incidence of esophageal squamous cell carcinoma (ESCC) is very high in
northeastern Iran. Previously, we reported a strong familial component of ESCC
among Turkmens, who constitute approximately one-half of the population of this
region. We hypothesized that the genes which cause Fanconi anemia might be
candidate genes for ESCC. We sequenced the entire coding regions of 12 Fanconi
anemia genes in the germline DNA of 190 Turkmen cases of ESCC. We identified
three heterozygous insertion/deletion mutations: one in FANCD2 (p.Val1233del),
one in FANCE (p.Val311SerfsX2), and one in FANCL (p.Thr367AsnfsX13). All three
patients had a strong family history of ESCC. In addition, four patients (out of
746 tested) were homozygous for the FANCA p.Ser858Arg mutation, compared to none
of 1,373 matched controls (OR = 16.7, 95% CI = 6.2-44.2, P = 0.01). The p.
Lys3326X mutation in BRCA2 (also known as Fanconi anemia gene FANCD1) was
present in 27 of 746 ESCC cases and in 16 of 1,373 controls (OR = 3.38, 95%
CI = 1.97-6.91, P = 0.0002). In summary, both heterozygous and homozygous
mutations in several Fanconi anemia-predisposing genes are associated with an
increased risk of ESCC in Iran. Fanconi anemia (FA) is a rare recessive DNA repair disorder that is clinically
characterized by congenital malformations, progressive bone marrow failure, and
increased incidence of maligcies, especially acute myeloid leukemia and
squamous cell carcinomas of the head and neck (HNSCCs) and the anogenital
regions. On a cellular level, typical features of the disorder are a high degree
of genomic instability and an increased sensitivity to bi-functionally
alkylating agents. So far, germ-line defects in 15 different FA genes have been
identified. Some of these FA genes are also established as tumor susceptibility
genes for familiar cancers.In recent years, the prevention and therapy of HNSCCs
in FA patients has become more important as the percentage of patients surviving
into adulthood is rising. HNSCCs appear in very young FA patients without common
risk factors. Since cisplatin-based chemotherapy in combination with
radiotherapy, essential parts of the standard treatment approach for sporadic
HNSCCs, cannot be used in FA patients due to therapy-associated toxicities and
mortalities even with reduced dosing, surgery is the most important treatment
option for HNSCCs, in FA patients and requires an early and efficient detection
of maligt lesions. So far, no uniform treatment protocol for the management
of HNSCCs in FA patients exists. Therefore, we propose that the information on
affected FA patients should be collected worldwide, practical therapeutic
guidelines developed and national treatment centers established. BACKGROUND: Fanconi anemia (FA) is a rare autosomal recessive genetic disorder
characterized by bone marrow failure and increased risk of cancers including
acute myelogenous leukemia and various solid tumors, especially head and neck
cancer. Management of head and neck cancer in the setting of FA is complicated
by pancytopenia, poor tolerance of chemotherapy, and potentially increased
radiosensitivity. There are limited reports on tolerance of radiotherapy (RT) in
patients with FA.
METHODS: We report a case of a patient with FA who presented with a small oral
tongue cancer that was excised. He rapidly developed extensive locoregional
recurrence and underwent surgical resection followed by postoperative RT with
concurrent cetuximab.
RESULTS: Both RT and cetuximab were well tolerated with manageable toxicities.
Unfortunately, the patient died of early locoregional disease progression.
CONCLUSIONS: RT with concurrent cetuximab was well tolerated and may be an
appropriate option in patients with FA. However, many patients have a poor
prognosis due to aggressive disease. Fanconi anemia (FA) is a heterogeneous disease characterized by spontaneous
chromosomal breaks and abnormal DNA repair. Major clinical problems in FA
include congenital abnormalities, endocrinopathies, early onset bone marrow
failure and increased risk of myelodysplastic syndrome, acute leukemia and solid
tumors. To date, 15 different genes have been shown to cause FA, all of which
have some role in DNA double-strand break repair. Very few strict
genotype-phenotype associations have been identified and clinical manifestations
vary widely from patient to patient, most likely due to modifier genes,
environment and chance effects. Hematopoietic stem cell transplantation is the
only proven cure for the hematopoietic manifestations of FA and aggressive
lifelong surveillance for solid tumors is essential. Fanconi anaemia (FA) is an inherited condition characterised by congenital and
developmental abnormalities and a strong cancer predisposition. In around 3-5%
of cases FA is caused by biallelic mutations in the BRCA2 gene. Individuals
heterozygous for BRCA2 mutations have an increased risk of inherited breast and
ovarian cancer. We reviewed the mutation spectrum in BRCA2-associated FA, and
the spectrum and frequency of BRCA2 mutations in distinct populations. The
rarity of FA due to biallelic BRCA2 mutations supports a fundamental role of
BRCA2 for prevention of maligt transformation during development. The
spectrum of maligcies seen associated with FA support the concept of a tissue
selectivity of BRCA2 mutations for development of FA-associated cancers. This
specificity is illustrated by the distinct FA-associated BRCA2 mutations that
appear to predispose to specific brain or haematological maligcies. For some
populations, the number of FA-patients with biallelic BRCA2 disruption is
smaller than that expected from the carrier frequency, and this implies that
some pregcies with biallelic BRCA2 mutations do not go to term. The apparent
discrepancy between expected and observed incidence of BRCA2 mutation-associated
FA in high-frequency carrier populations has important implications for the
genetic counselling of couples with recurrent miscarriages from high-risk
populations. |
Is stop codon bypass possible? | In 1999, proof-of-concept for treating these disorders was obtained in a mouse model of muscular dystrophy, when administration of aminoglycosides restored protein translation by inducing the ribosome to bypass a PTC.
Aminoglycosides can bypass nonsense mutations and are the prototypic agents for translational bypass therapy (TBT).
Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). | Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain
protein-coding regions in other plant viruses, is mediated by translational
readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco
tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional
base-pairing but recent findings that stop codons can also be bypassed as a
result of extended translocational shifts (tRNA hopping) have encouraged a
re-examination. In light of the alternatives, we investigated the sequences
flanking the leaky UAG codon using an in vivo assay in which bypass of the stop
codon is coupled to the transient expression of beta-glucuronidase (GUS)
reporter genes in tobacco protoplasts. Analysis of GUS constructions in which
codons flanking the stop were altered allowed definition of the minimal sequence
required for read through as UAG-CAA-UUA. The effects of all possible
single-base mutations in the codons flanking the stop indicated that 3' contexts
of the form CAR-YYA confer leakiness and that the 3' context permits read
through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a
major role for the 3' context in the read through process and do not support a
model in which teh UAG is bypassed exclusively as a result of anticodon-codon
interactions. No evidence for tRNA hopping was obtained. The 3' context
apparently represents a unique sequence element that affects translation
termination. Plant RNA viruses commonly exploit leaky translation termination signals in
order to express internal protein coding regions. As a first step to elucidate
the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have
devised a system in which readthrough is coupled to the transient expression of
beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the
stop codons and surrounding nucleotides from the readthrough regions of several
different RNA viruses were constructed and the plasmids were tested for the
ability to direct transient GUS expression. These studies indicated that
ribosomes bypass the leaky termination sites at efficiencies ranging from
essentially 0 to ca. 5% depending upon the viral sequence. The results suggest
that the efficiency of readthrough is determined by the sequence surrounding the
stop codon. We describe improved GUS expression vectors and optimized
transfection conditions which made it possible to assay low-level translational
events. A translational frameshift is necessary in the synthesis of Escherichia coli
release factor 2 (RF-2) to bypass an in-frame termination codon within the
coding sequence. The nucleotide sequence preceding the in-phase stop codon
within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence)
region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that
this pairing triggers the frameshift event. In vitro production of RNA coding
for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing
of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of
eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence)
region. The prokaryotic RF-2 gene and the gene for the other release factor,
RF-1, which does not contain an in-frame stop codon, were subcloned into
transcription vectors such that the RNA transcripts produced in vitro would
resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a
major product of Mr approximately 45,000 when translated in vitro within
reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules.
The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including
those to regions of the protein encoded in the mRNA downstream from the
frameshift site. The putative premature termination product, an oligopeptide of
25 amino acids, was not detected, but a chemically synthesized derivative was
shown to be very unstable within the translation system. Although it was not
possible therefore to calculate an absolute efficiency of frameshifting, the
relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of
that of RF-1 RNA in the reticulocyte system. This was similar to the relative
synthesis of the two proteins in a plasmid-DNA-directed prokaryotic
transcription/translation system. These results show that in vitro on eukaryotic
ribosomes where the Shine-Dalgarno-type interaction is not possible, high
efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can
still occur. Translation of yeast GCN4 mRNA occurs by a reinitiation mechanism that is
modulated by amino acid levels in the cell. Ribosomes which translate the first
of four upstream open reading frames (uORFs) in the mRNA leader resume scanning
and can reinitiate downstream. Under non-starvation conditions reinitiation
occurs at one of the remaining three uORFs and GCN4 is repressed. Under
starvation conditions, in contrast, ribosomes bypass the uORFs and reinitiate at
GCN4 instead. The high frequency of reinitiation following uORF1 translation
depends on an adequate distance to the next start codon and particular sequences
surrounding the uORF1 stop codon. We present evidence that sequences 5' to uORF1
also strongly enhance reinitiation. First, reinitiation was severely inhibited
when uORF1 was transplanted into the position of uORF4, even though the native
sequence environment of the uORF1 stop codon was maintained, and this effect
could not be accounted for by the decreased uORF1-GCN4 spacing. Second,
insertions and deletions in the leader preceding uORF1 greatly reduced
reinitiation at GCN4. Sequences 5' to uORF1 may influence the probability of
ribosome release following peptide termination at uORF1. Alternatively, they may
facilitate rebinding of an initiation factor required for reinitiation prior to
resumption of the scanning process. We isolated from a tomato cDNA library the tomPRO1 locus, which encodes
gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This
locus is unusual among eukaryotic genetic elements because it contains two open
reading frames, and thus resembles prokaryotic polycistronic operons. The first
open reading frame, specifying GK, is terminated by a TAA codon, which is
followed by five nucleotides, an ATG translation initiation codon, and the
second open reading frame, encoding GPR. DNA sequence analysis of fragments
obtained by PCR amplification confirmed that the internal TAA and neighboring
sequences are present in the endogenous tomPRO1 sequence in tomato. We
demonstrated with RNase protection assays that the tomPRO1 locus is transcribed
in tomato tissue culture cells, into a product that contains the internal stop
codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a
33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E.
coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa
product in leaves and roots. These results suggest that in tomato tissues, GPR
is made as part of a longer polypeptide by some translational mechanism that
enables bypass of the internal stop codon, such as frameshifting or ribosome
hopping. The tomPRO1 locus may be the first example of a nuclear genetic element
in plants that encodes two functional enzymes in two distinct open reading
frames. Cytosine deamination to uracil occurs frequently in cellular DNA. In vitro, RNA
polymerase efficiently inserts adenine opposite to uracil, resulting in G to A
base substitutions. In vivo, uracil could potentially alter transcriptional
fidelity, resulting in production of mutant proteins. This study demonstrates
that in nondividing Escherichia coli cells, a DNA template base replaced with
uracil in a stop codon in the firefly luciferase gene results in conversion of
inactive to active luciferase. The level of transcriptional base substitution is
dependent on the capacity to repair uracil. These results provide evidence for a
DNA damage-dependent, transcription-driven pathway for generating mutant
proteins in nondividing cells. Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open
reading frames of bacteriophage T4 gene 60 in order to synthesize a
topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing,
only 50 % resume translation in the second open reading frame. Failure to bypass
is shown here to be independent of the stop codon at the end of the first open
reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to
diminish bypassing efficiency. Unproductive bypassing may result from premature
dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of
translation at inappropriate sites. Assessment of the influence of factors known
to induce drop-off reveals that ribosome recycling factor accounts for a small
fraction of unproductive bypassing products, but none of the other known factors
appear to play a significant role. Resumption of translation at inappropriate
sites appears to be minimal, which suggests that spontaneous release of the
peptidyl-tRNA may account for the remaining unproductive bypassing products and
may be inherent to the gene 60 bypassing mechanism. Stop codons are used to signal the ribosome to terminate the decoding of an mRNA
template. Recent studies on translation termination in the yeast Saccharomyces
cerevisiae have not only enabled the identification of the key components of the
termination machinery, but have also revealed several regulatory mechanisms that
might enable the controlled synthesis of C-terminally extended polypeptides via
stop-codon readthrough. These include both genetic and epigenetic mechanisms.
Rather than being a translation 'error', stop-codon readthrough can have
important effects on other cellular processes such as mRNA degradation and, in
some cases, can confer a beneficial phenotype to the cell. Aminoglycosides can bypass nonsense mutations and are the prototypic agents for
translational bypass therapy (TBT). Initial results demonstrate the need for
more potent drugs and an in vivo model system for quantitative assessment of
TBT. Herein, we present an in vivo system for evaluating the efficacy of
premature stop codon management therapies: in vivo quantitative stop codon
management repli-sampling TBT efficacy assay (IQSCMaRTEA). Application of
IQSCMaRTEA reveals that geneticin is much more efficacious in vivo than
gentamicin. Treatment with geneticin elicits a multiday response, and residual
F9 antigen can be detected after 3 weeks. These data demonstrate the utility of
IQSCMaRTEA for evaluating drugs that bypass nonsense mutations. In addition,
IQSCMaRTEA may be helpful for testing inhibitors of nonsense-mediated decay, as
stop codon management therapy will sometimes require inhibition of
nonsense-mediated decay and translational bypass of the nonsense mutation.
Furthermore, geneticin, its metabolites, or better tolerated analogues should be
evaluated as a general treatment with multiday response for severe genetic
disease caused by nonsense mutation. BACKGROUND: In the C. albicans retrotransposon Tca2, the gag and pol ORFs are
separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is
unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However,
in other retroelements, translational readthrough of the gag stop codon can be
directed by its flanking sequence, including a 3' pseudoknot.
RESULTS: The hypothesis was tested that in Tca2, gag stop codon flanking
sequences direct translational readthrough and synthesis of a gag-pol fusion
protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted
between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual
reporter construct. Although downstream of UGA, luc was expressed, but its
expression was unaffected by inserting additional stop codons at the 3' end of
lacZ. Luc expression was instead being driven by a previously unknown minor
promoter activity within the gag-pol junction region. Evidence together
indicated that junction sequence alone cannot direct UGA readthrough. Using
reporter genes in C. albicans, the activities of this gag-pol junction promoter
and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two
promoters, only the LTR promoter was induced by heat-shock, which also triggers
retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow
detection, was also heat-shock induced, indicating that pol proteins were
expressed from a gag-UGA-pol RNA.
CONCLUSION: This is the first demonstration that the LTR promoter directs Tca2
pol protein expression, and that pol proteins are translated from a gag-pol RNA,
which thus requires a mechanism for stop codon bypass. However, in contrast to
most other retroelement and viral readthrough signals, immediate gag
UGA-flanking sequences were insufficient to direct stop readthrough in S.
cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2. The extensive molecular genetic heterogeneity seen with inherited eye disease is
a major barrier to the development of gene-based therapeutics. The underlying
molecular pathology in a considerable proportion of these diseases however are
nonsense mutations leading to premature termination codons. A therapeutic
intervention targeted at this abnormality would therefore potentially be
relevant to a wide range of inherited eye diseases. We have taken advantage of
the ability of aminoglycoside drugs to suppress such nonsense mutations and
partially restore full-length, functional protein in a zebrafish model of
choroideraemia (chm(ru848); juvenile chorio-retinal degeneration) and in two
models of ocular coloboma (noi(tu29a) and gup(m189); congenital optic fissure
closure defects). In vitro cell-based assays showed significant readthrough with
two drugs, gentamicin and paromomycin, which was confirmed by western blot and
in vitro prenylation assays. The presence of either aminoglycoside during
zebrafish development in vivo showed remarkable prevention of mutant ocular
phenotypes in each model and a reduction in multisystemic defects leading to a
1.5-1.7-fold increase in survival. We also identified a significant reduction in
abnormal cell death shown by TUNEL assay. To test the hypothesis that optic
fissure closure was apoptosis-dependent, the anti-apoptotic agents, curcumin and
zVAD-fmk, were tested in gup(m189) embryos. Both drugs were found to reduce the
size of the coloboma, providing molecular evidence that cell death is required
for optic fissure remodelling. These findings draw attention to the value of
zebrafish models of eye disease as useful preclinical drug screening tools in
studies to identify molecular mechanisms amenable to therapeutic intervention. Several genetic diseases are triggered by nonsense mutations leading to the
formation of truncated and defective proteins. Aminoglycosides have the
capability to mediate a bypass of stop mutations during translation thus
resulting in a rescue of protein expression. So far no attention has been
directed to obesity-associated stop mutations as targets for nonsense
suppression. Herein, we focus on the characterization of the
melanocortin-4-receptor (MC4R) nonsense allele W16X identified in obese
subjects. Cell culture assays revealed a loss-of-function of Mc4r(X16)
characterized by impaired surface expression and defect signaling. The
aminoglycoside G-418 restored Mc4r(X16) function in vitro demonstrating that
Mc4r(X16) is susceptible to nonsense suppression. For the evaluation of nonsense
suppression in vivo, we generated a Mc4r(X16) knock-in mouse line by gene
targeting. Mc4r(X16) knock-in mice developed hyperphagia, impaired glucose
tolerance, severe obesity and an increased body length demonstrating that this
new mouse model resembles typical characteristics of Mc4r deficiency. In a first
therapeutic trial, the aminoglycosides gentamicin and amikacin induced no
amelioration of obesity. Further experiments with Mc4r(X16) knock-in mice will
be instrumental to establish nonsense suppression for Mc4r as an
obesity-associated target gene expressed in the central nervous system. Expression of retroviral replication enzymes (Pol) requires a controlled
translational recoding event to bypass the stop codon at the end of gag. This
recoding event occurs either by direct suppression of termination via the
insertion of an amino acid at the stop codon (readthrough) or by alteration of
the mRNA reading frame (frameshift). Here we report the effects of a host
protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using
a dual luciferase reporter assay, we found that transfection of cells with a
plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent
manner, with a maximal enhancement of nearly twofold. Expression of RPL4
increases recoding of reporters containing retroviral readthrough and frameshift
sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced
enhancement of recoding is cell line specific and appears to be specific to RPL4
among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine
leukemia proviral DNA results in Gag processing defects and a reduction of viral
particle formation, presumably caused by the RPL4-dependent alteration of the
Gag-to-Gag-Pol ratio required for virion assembly and release. Ten percent of inherited diseases are caused by premature termination codon
(PTC) mutations that lead to degradation of the mRNA template and to the
production of a non-functional, truncated polypeptide. In addition, many
acquired mutations in cancer introduce similar PTCs. In 1999, proof-of-concept
for treating these disorders was obtained in a mouse model of muscular
dystrophy, when administration of aminoglycosides restored protein translation
by inducing the ribosome to bypass a PTC. Since, many studies have validated
this approach, but despite the promise of PTC readthrough therapies, the
mechanisms of translation termination remain to be precisely elucidated before
even more progress can be made. Here, we review the molecular basis for PTC
readthrough in eukaryotes and describe currently available compounds with
significant therapeutic potential for treating genetic disorders and cancer. Our goal was to identify evolutionary conserved frame transitions in protein
coding regions and to uncover an underlying functional role of these structural
aberrations. We used the ab initio frameshift prediction program, GeneTack, to
detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete
prokaryotic genomes. We grouped 102 731 fs-genes into 19 430 clusters based on
sequence similarity between protein products (fs-proteins) as well as
conservation of predicted position of the frameshift and its direction. We
identified 4010 pseudogene clusters and 146 clusters of fs-genes apparently
using recoding (local deviation from using standard genetic code) due to
possessing specific sequence motifs near frameshift positions. Particularly
interesting was finding of a novel type of organization of the dnaX gene, where
recoding is required for synthesis of the longer subunit, τ. We selected 20
clusters of predicted recoding candidates and designed a series of genetic
constructs with a reporter gene or affinity tag whose expression would require a
frameshift event. Expression of the constructs in Escherichia coli demonstrated
enrichment of the set of candidates with sequences that trigger genuine
programmed ribosomal frameshifting; we have experimentally confirmed four new
families of programmed frameshifts. |
Is the protein β1-integrin recycled? | Yes, the β1-integrin is recycled. | Integrin trafficking plays an important role in cellular motility and
cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate
the dynamic regulation of cell adhesion. Integrin activity toward the components
of the extracellular matrix is regulated by the ability of these receptors to
switch between active and inactive conformations. Several cellular signalling
pathways have been described in the regulation of integrin traffic under
different conditions. However, the interrelationship between integrin activity
conformations and their endocytic fate have remained incompletely understood.
Here, we have investigated the endocytic trafficking of active and inactive β1
integrins in cancer cells. Both conformers are endocytosed in a clathrin- and
dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is
higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid
recycling back to the plasma membrane via an ARF6- and early endosome antigen
1-positive compartment in an Rab4a- and actin-dependent manner. Owing to these
distinct trafficking routes, the two receptor pools display divergent
subcellular localization. At steady state, the inactive β1 integrin is mainly on
the plasma membrane, whereas the active receptor is predomitly intracellular.
These data provide new insights into the endocytic traffic of integrins and
imply the possibility of a previously unappreciated crosstalk between pathways
regulating integrin activity and traffic. Recycling of internalized integrins is a crucial step in adhesion remodeling and
cell movement. Recently, we determined that the ADP-ribosylation factor-guanine
nucleotide exchange factors (ARF-GEFs) cytohesin 2/ARNO and cytohesin 3/GRP1
have opposing effects on adhesion and stimulated β1 integrin recycling even
though they are very closely related proteins (80% sequence identity). We have
now determined the sequence differences underlying the differential actions of
cytohesin 2/ARNO and cytohesin 3/GRP1. We found that the ability of cytohesins
to promote β1 integrin recycling and adhesion depends upon the presence or
absence of a key glycine residue in their pleckstrin homology (PH) domains. This
glycine residue determines the phosphoinositide specificity and affinity of
cytohesin PH domains. Switching the number of glycines in the PH domains of
cytohesin 2 and cytohesin 3 is sufficient to reverse their effects on adhesion
and spreading and to reverse their subcellular locations. Importantly, we also
find that a mutant form of cytohesin 3/GRP1 that has three rather than two
glycines in its PH domain rescues β1 integrin recycling in cytohesin 2/ARNO
knockdown cells. Conversely, a mutant form of cytohesin 2/ARNO with two glycines
in its PH domain fails to rescue β1 integrin recycling. Therefore, we conclude
that phosphoinositide specificity is the sole functional difference that
determines which cytohesin can promote integrin recycling. Integrin functions are controlled by regulating their affinity for ligand, and
by the efficient recycling of intact integrins through endosomes. Here we
demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain
serves as a molecular switch enabling the sequential binding of two
FERM-domain-containing proteins in different cellular compartments. When β1
integrins are at the plasma membrane, Kindlins control ligand-binding affinity.
However, when they are internalized, Kindlins dissociate from integrins and
sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early
endosomes to prevent β1-integrin degradation, leading to their recycling back to
the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding
protein that interacts with the free Kindlin-binding site in endosomes to
stabilize β1 integrins, resulting in their recycling to the cell surface where
they can be reused. The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of
membrane gangliosides, is crucial in the regulation of cell surface processes,
and has been demonstrated to be significantly up-regulated in renal cell
carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin
trafficking in RCC cells by controlling β1 integrin recycling to the plasma
membrane and controlling activation of the epidermal growth factor receptor
(EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3
silencing in RCC cells increased the membrane ganglioside content, in particular
the GD1a content, and changed the expression of key regulators of the integrin
recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related
protein RAB25, which directs internalized integrins to lysosomes, and
down-regulated the chloride intracellular channel protein 3 (CLIC3), which
induces the recycling of internalized integrins to the plasma membrane. In this
manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked
its recycling and reduced its levels at the plasma membrane, and, consequently,
inhibited EGFR and FAK/AKT. These events had the following effects on the
behavior of RCC cells: they (a) decreased drug resistance mediated by the block
of autophagy and the induction of apoptosis; (b) decreased metastatic potential
mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c)
decreased adhesion to collagen and fibronectin. Therefore, our data identify
NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT
signaling and demonstrate its crucial role in RCC maligcy. Cells inversely adjust the plasma membrane levels of integrins and cadherins
during cell migration and cell-cell adhesion but the regulatory mechanisms that
coordinate these trafficking events remain unknown. Here, we demonstrate that
the small GTPase Rab35 maintains cadherins at the cell surface to promote
cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase
Arf6 to downregulate an Arf6-dependent recycling pathway for β1-integrin and EGF
receptors, resulting in inhibition of cell migration and attenuation of
signaling downstream of these receptors. Importantly, the phenotypes of
decreased cell adhesion and increased cell migration observed following Rab35
knock down are consistent with the epithelial-mesenchymal transition, a feature
of invasive cancer cells, and we show that Rab35 expression is suppressed in a
subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a
key molecular mechanism that efficiently coordinates the inverse intracellular
sorting and cell surface levels of cadherin and integrin receptors for cell
migration and differentiation. Cell migration is dependent on a series of integrated cellular events including
the membrane recycling of the extracellular matrix receptor integrins. In this
paper, we investigate the role of autophagy in regulating cell migration. In a
wound-healing assay, we observed that autophagy was reduced in cells at the
leading edge than in cells located rearward. These differences in autophagy were
correlated with the robustness of MTOR activity. The spatial difference in the
accumulation of autophagic structures was not detected in rapamycin-treated
cells, which had less migration capacity than untreated cells. In contrast, the
knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa
cells. Accordingly, atg3(-/-) and atg5(-/-) MEFs have greater cell migration
properties than their wild-type counterparts. Stimulation of autophagy increased
the co-localization of β1 integrin-containing vesicles with LC3-stained
autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal
degradation of internalized β1 integrins and promoted its membrane recycling.
From these findings, we conclude that autophagy regulates cell migration, a
central mechanism in cell development, angiogenesis, and tumor progression, by
mitigating the cell surface expression of β1 integrins. Integrins have been implicated in various processes of nervous system
development, including proliferation, migration, and differentiation of neuronal
cells. In this study, we show that the serine/threonine kinase Ndr2 controls
integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We
further demonstrate that Ndr2 is able to induce phosphorylation at the activity-
and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC-
and CaMKII-dependent activation of β1-integrins, as well as their exocytosis.
Accordingly, Ndr2 associates with integrin-positive early and recycling
endosomes in primary hippocampal neurons and the surface expression of activated
β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2
in dendritic differentiation is also evident in vivo, because Ndr2-null mutant
mice show arbor-specific alterations of dendritic complexity in the hippocampus.
This indicates a role of Ndr2 in the fine regulation of dendritic growth; in
fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2
knock-down-induced dendritic growth deficits but fails to enhance growth beyond
control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin
3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The
results of this study show that Ndr2-mediated integrin trafficking and
activation are crucial for neurite growth and guidance signals during neuronal
development. Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and
membrane-associated forms, and is implicated in diverse cellular processes,
ranging from ion channel formation to intracellular membrane remodeling. CLIC4
is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and
serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking.
However, the function and subcellular target(s) of CLIC4 remain elusive. Here,
we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix
adhesion, cell spreading and integrin signaling, whereas it increases cell
motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma
membrane and in Rab35-positive endosomes. CLIC4 is required for both the
internalization and the serum- or LPA-induced recycling of β1 integrin, but not
for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35
activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking.
Our results define CLIC4 as a regulator of Rab35 activity and serum- and
LPA-dependent integrin trafficking. Regulated turnover of integrin receptors is essential for cell adhesion and
migration. Pathways selectively regulating β1-integrin recycling are implicated
in cancer invasion and metastasis, yet proteins required for the internalization
of this pro-invasive integrin remain to be identified. Here, we uncover
formin-like 2 (FMNL2) as a critical regulator of β1-integrin internalization
downstream of protein kinase C (PKC). PKCα associates with and phosphorylates
FMNL2 at S1072 within its Diaphanous autoregulatory region, leading to the
release of formin autoinhibition. Phosphorylation of FMNL2 triggers its rapid
relocation and promotes its interaction with the cytoplasmic tails of the
α-integrin subunits for β1-integrin endocytosis. FMNL2 drives β1-integrin
internalization and invasive motility in a phosphorylation-dependent manner,
while a FMNL2 mutant defective in actin assembly interferes with β1-integrin
endocytosis and cancer cell invasion. Our data establish a role for FMNL2 in the
regulation of β1-integrin and provide a mechanistic understanding of the
function of FMNL2 in cancer invasiveness. |
Are BBS mutations involved in syndromic Hirschsprung disease? | In 3 families with Bardet-Biedl syndrome (BBS) and Hirschsprung disease (HSCR), concomitant mutations in BBS genes and regulatory RET elements have been identified. Analysis of the data suggests that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. | Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy
characterized by incomplete innervation along a variable length of the gut. The
pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR
also presents in various syndromes, including Shah-Waardenburg syndrome (WS),
Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR
with concomitant mutations in BBS genes and regulatory RET elements, whose
functionality is tested in physiologically relevant assays. Our data suggest
that BBS mutations can potentiate HSCR predisposing RET alleles, which by
themselves are insufficient to cause disease. We also demonstrate that these
genes interact genetically in vivo to modulate gut innervation, and that this
interaction likely occurs through complementary, yet independent, pathways that
converge on the same biological process. |
Which signaling pathway is activating the dishevelled proteins? | Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling | The Dishevelled protein mediates several diverse biological processes.
Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls
cell fate via canonical Wnt signaling, it also controls cell polarity via the
vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9].
The relationship between subcellular localization of Dishevelled and its
signaling activities remains unclear; conflicting results have been reported
depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new
reagents to sequester wild-type Dishevelled protein either at the cell membrane
or away from the cell membrane. Removal of Dishevelled from the cell membrane
disrupts convergent extension by preventing Rho/Rac activation and mediolateral
cell polarization. By manipulating the subcellular localization of K-->M (dsh1),
we show that this mutation inhibits Dishevelled activation of Rac, regardless of
its subcellular localization. These data demonstrate that membrane localization
of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both
membrane-targeted and cytoplasm-targeted Dishevelled can potently activate
canonical Wnt signaling, suggesting that local concentration of Dishevelled
protein, but not its spatial localization, is central to canonical Wnt
signaling. These results suggest that in vertebrate embryos, subcellular
localization is insufficient to account for the pathway specificity of
Dishevelled in the canonical Wnt versus PCP signaling cascades. Wnt signaling is known to be important for diverse embryonic and post-natal
cellular events and be regulated by the proteins Dishevelled and Axin. Although
Dishevelled is activated by Wnt and involved in signal transduction, it is not
clear how Dishevelled-mediated signaling is turned off. We report that guanine
nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2
inhibited Wnt mediated reporter activity. The inhibition involved reduction of
the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled
was countered by increased expression of Axin. Consistent with these effects in
HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the
formation of secondary axes induced either by XWnt8 or Dishevelled, but not by
beta-catenin. The DEP domain of Dishevelled is necessary for both interaction
with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal
pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of
Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to
downregulate the level of Dishevelled. Activation of Wnt signaling by either of
two methods, increased Frizzled signaling or transient transfection of Wnt, also
led to increased degradation of Dishevelled and the induced Dishevelled loss is
dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with
PLC action and calcium signaling suggested that loss of Dishevelled is mediated
through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca(+2)/PKC
signaling. Together the evidence suggests a novel negative feedback mechanism in
which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled. Dishevelled (DVL) proteins, three of which have been identified in humans, are
highly conserved components of canonical and noncanonical Wnt signaling
pathways. These multifunctional proteins, originally discovered in the fruit
fly, through their different domains mediate complex signal transduction: DIX
(dishevelled, axin) and PDZ (postsynaptic density 95, discs large, zonula
occludens-1) domains serve for canonical beta-catenin signaling, while PDZ and
DEP (dishevelled, Egl-10, pleckstrin) domains serve for non-canonical signaling.
In canonical or beta-catenin signaling, DVL forms large molecular supercomplexes
at the plasma membrane consisting of Wnt-Fz-LRP5/6-DVL-AXIN. This promotes the
disassembly of the beta-catenin destruction machinery, beta-catenin
accumulation, and consequent activation of Wnt signaling. Therefore, DVLs are
considered to be key regulators that rescue cytoplasmic beta-catenin from
degradation. The potential medical importance of DVLs is in both human
degenerative disease and cancer. The overexpression of DVL has been shown to
potentiate the activation of Wnt signaling and it is now apparent that
up-regulation of DVLs is involved in several types of cancer. |
Which is the defective protein causing the lysosomal storage disease Fabry? | Anderson-Fabry disease (referred to as Fabry disease) is an X-linked disorder characterized by a deficiency of the lysosomal enzyme alpha-galactosidase A and the subsequent accumulation in various tissues of globotriaosylceramide (Gb(3)), the main substrate of the defective enzyme. | Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the lysosomal
exoglycosidase responsible for the hydrolysis of terminal alpha-galactosyl
residues from glycoconjugates and is the defective enzyme causing Fabry disease
(McKusick 301500). An unusally elevated level of plasma alpha-Gal A activity (>
2.5 times the normal mean) was detected in two unrelated normal males and the
elevated activities were inherited as X-linked traits in their families.
Sequencing of the alpha-Gal A coding region, intron/exon boundaries and
5'-flanking region from the proband identified a single mutation, a G-->A
transition 30 nt upstream from the initiation of translation codon in exon 1.
The -30G-->A mutation occurred in a putative NF kappa B/Ets consensus binding
site that was recently shown to inhibit protein binding to the 5'-untranslated
region of the gene, providing a possible explanation for its high activity. To
further characterize the mutation, the mRNA and protein expressed by this
variant allele were studied. Purified plasma and lymphoblast alpha-Gal A
activity from individuals with the -30G-->A mutation had normal physical and
kinetic properties. In vitro translation of mRNAs from the cloned normal and
high plasma activity alleles resulted in similar levels of alpha-Gal A protein,
indicating that this mutation did not enhance translation. These findings
suggest that the -30G-->A mutation in the 5'-untranslated region of the
alpha-Gal A gene enhances transcription, presumably by interfering with the
binding of negatively-acting transcription factors which normally decrease
alpha-Gal A expression in various cells. Preliminary studies of the frequency of
the -30G-->A mutation in 395 unrelated normal males of mixed ancestry revealed
two additional unrelated individuals who had high plasma enzymatic activity and
the mutation, confirming the effect of this mutation on enzyme expression and
suggesting that about 0.5% of normal individuals have high plasma alpha-Gal A
activity due to this variant allele. Transgenic mice expressing a human mutant alpha-galactosidase with an R301Q
substitution, which was found in a patient with a variant form of Fabry disease,
were established. The mice transcribed a sufficient amount of
alpha-galactosidase mRNA, but the steady-state levels of the enzyme protein were
decreased in liver, kidney and heart, only residual activity being detected in
these tissues. The mice will be useful for the clarification of the defective
regulation of the structurally altered enzyme protein expressed by the mutant
gene at the organ or individual level as well as for the evaluation of drugs
that stabilize and/or activate the mutant alpha-galactosidase. Sphingolipidoses are a subgroup of lysosomal storage diseases. They are defined
as disorders caused by a genetic defect in catabolism of sphingosine-containing
lipids. Catabolism of these lipids involves enzymes and activator proteins.
After the discovery of lysosomes by de Duve and the demonstration of the first
defective lysosomal enzyme by Hers in 1963, the first enzyme deficiency for
sphingolipidoses was characterized in 1965 and all the defective enzymes were
demonstrated in the last three decades. In 1984, the first activator protein was
found and it expanded the concept of sphingolipidoses. In the following years,
many researches have been undertaken to understand the molecular basis of these
diseases, the mechanism of pathogenesis, the mechanism of lysosomal digestion of
glycosphingolipids (GSLs) and the functional domains of lysosomal enzymes. New
hypotheses and theories have been put forward for the mechanism of lysosomal
digestion and pathogenesis. However, although much has been done, the
pathogenesis of sphingolipidoses has not been fully elucidated. Mouse models of
these diseases have facilitated the elucidation of pathogenesis and the
development of therapeutic strategies for these diseases, which are not
treatable at present except for Fabry and type 1 Gaucher disease. The purpose of
this review is to collect information on the recent researches related to
sphingolipidoses. The review includes the hydrolysis of GSLs in lysosome,
mechanism of hydrolysis, pathogenesis and genetics of sphingolipidoses, a brief
mouse model and therapeutic strategies of these diseases. Lysosomal storage diseases arise from a genetic loss-of-function defect in
enzymes mediating key catabolic steps resulting in accumulation of substrate
within the lysosome. Treatment of several of these disorders has been achieved
by enzyme replacement therapy (ERT), in which a recombit version of the
defective enzyme is expressed in vitro and administered by infusion. However, in
many cases the biodistribution of the administered protein does not match that
of the accumulated substrate due to the glycosylation-mediated clearance of the
enzymes from circulation, resulting in poor or absent substrate clearance from
some tissues. To overcome this limitation, we have evaluated several
peptide-based targeting motifs to redirect recombit human alpha-galactosidase
(rhalphaGal) to specific receptors. A reversible thiol-based PEGylation
chemistry was developed to achieve multivalent peptide display with lysosomal
release. In vitro, cell uptake was peptide dependent and independent of the
normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite
increased plasma half-life and decreased liver uptake, none of the peptide
conjugates showed significantly altered biodistribution in alphaGal-knockout
mice. This suggests that these peptide-based targeting motifs are unlikely to
provide substantial therapeutic benefit likely due to the complexity of factors
affecting PK and biodistribution. The lysosomal storage disorder Fabry disease is characterized by excessive
globotriaosylceramide (Gb3) accumulation in major organs such as the heart and
kidney. Defective lysosomal alpha-galactosidase A (Gla) is responsible for
excessive Gb3 accumulation, and one cell sensitive to the effects of Gb3
accumulation is vascular endothelium. Endothelial dysfunction is associated with
Fabry disease and excessive cellular Gb3. We previously demonstrated that
excessive vascular Gb3 in a mouse model of Fabry disease, the Gla-knockout
(Gla(-/0)) mouse, results in abnormal vascular function, which includes abnormal
endothelium-dependent contractions, a vascular phenomenon known to involve
cyclooxygenase (COX). Therefore, we hypothesized that the vasculopathy in the
Gla knockout mouse may be due to a vasoactive COX-derived product. To test this
hypothesis, vascular reactivity experiments were performed in aortic rings from
wild-type (Gla(+/0)) and Gla(-/0) mice in the presence and absence of specific
and nonspecific COX inhibitors. Specific inhibition of COX1 or COX2 in
endothelium-intact rings from Gla(-/0) mice decreased overall phenylephrine
contractility compared with untreated Gla(-/0) rings, whereas COX inhibitors had
no effect on contractility in endothelium-denuded rings. Nonspecific inhibition
of COX with indomethacin (10 micromol/l) or COX1 inhibition with valeryl
salicylate (3 mmol/l) improved endothelial function in rings from Gla(-/0) mice,
but COX2 inhibition with NS-398 (1 micromol/l) further increased endothelial
dysfunction in rings from Gla(-/0) mice. These results suggest that, in the
Gla(-/0) mice, COX1 and COX2 activity are increased and localized in the
endothelium, producing vasopressor and vasorelaxant products, which contribute
to the Fabry-related vasculopathy. |
Is there any software for automated analysis of immuno-histochemistry images? | In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel ‘hot spots’ has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the ‘hot spots’ assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity | PURPOSE: To develop a fully automated algorithm (AP) to perform a volumetric
measure of the optic disc using conventional stereoscopic optic nerve head (ONH)
photographs, and to compare algorithm-produced parameters with manual
photogrammetry (MP), scanning laser ophthalmoscope (SLO) and optical coherence
tomography (OCT) measurements.
METHODS: One hundred twenty-two stereoscopic optic disc photographs (61
subjects) were analyzed. Disc area, rim area, cup area, cup/disc area ratio,
vertical cup/disc ratio, rim volume and cup volume were automatically computed
by the algorithm. Latent variable measurement error models were used to assess
measurement reproducibility for the four techniques.
RESULTS: AP had better reproducibility for disc area and cup volume and worse
reproducibility for cup/disc area ratio and vertical cup/disc ratio, when the
measurements were compared to the MP, SLO and OCT methods.
CONCLUSION: AP provides a useful technique for an objective quantitative
assessment of 3D ONH structures. INTRODUCTION: Accurate assessment of estrogen receptor (ER), progesterone
receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of
breast cancer. Commercially available image analysis systems are usually bundled
with dedicated analysis hardware and, to our knowledge, no easily installable,
free software for immunostained slide scoring has been described. In this study,
we describe a free, Internet-based web application for quantitative image
analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue
sections.
METHODS: The application, named ImmunoRatio, calculates the percentage of
positively stained nuclear area (labeling index) by using a color deconvolution
algorithm for separating the staining components (diaminobenzidine and
hematoxylin) and adaptive thresholding for nuclear area segmentation.
ImmunoRatio was calibrated using cell counts defined visually as the gold
standard (training set, n = 50). Validation was done using a separate set of 50
ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling
indexes determined by ImmunoRatio were studied for their prognostic value in a
retrospective cohort of 123 breast cancer patients.
RESULTS: The labeling indexes by calibrated ImmunoRatio analyses correlated well
with those defined visually in the test set (correlation coefficient r = 0.98).
Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2
was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown
to adapt to various staining protocols, microscope setups, digital camera
models, and image acquisition settings. The application can be used directly
with web browsers running on modern operating systems (e.g., Microsoft Windows,
Linux distributions, and Mac OS). No software downloads or installations are
required. ImmunoRatio is open source software, and the web application is
publicly accessible on our website.
CONCLUSIONS: We anticipate that free web applications, such as ImmunoRatio, will
make the quantitative image analysis of ER, PR, and Ki-67 easy and
straightforward in the diagnostic assessment of breast cancer specimens. During normal brain development, axons are myelinated by mature oligodendrocytes
(OLGs). Under pathological, demyelinating conditions within the central nervous
system (CNS), axonal remyelination is only partially successful because
oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated
state resulting in a failure to generate myelinating OLGs. Tissue
Transglutaminase (TG2) is a multifunctional enzyme, which amongst other
functions, is involved in cell differentiation. Therefore, we hypothesized that
TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates
remyelination. In vivo studies, using the cuprizone model for de- and
remyelination in TG2(-/-) and wild-type mice, showed that during remyelination
expression of proteolipid protein mRNA, as a marker for remyelination, in the
corpus callosum lags behind in TG2(-/-) mice resulting in less myelin formation
and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies
showed that rat OPCs express TG2 protein and activity which reduces when the
cells have matured into OLGs. Furthermore, when TG2 activity is
pharmacologically inhibited, the differentiation of OPCs into myelin-forming
OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in
remyelination of the CNS, probably through stimulating OPC differentiation into
myelin-forming OLGs. Therefore, manipulating TG2 activity may represent an
interesting new target for remyelination in demyelinating diseases. Zebrafish have emerged as a powerful model organism to study embryo
morphogenesis. Due to their optical clarity, they are uniquely suited for
time-lapse imaging studies, providing insights into the dynamic processes
underlying tissue formation and cell migration. These studies have been
tremendously facilitated by the availability of transgenic zebrafish lines,
labelling distinct embryonic structures, individual cells, or even subcellular
structures, such as the nucleus. Zebrafish studies have revealed that the
migration of several different cell types in the embryo is controlled by
chemokines, small vertebrate-specific proteins. Here, we report methods to
analyze the expression pattern of a given chemokine and its receptor in
transgenic zebrafish using fluorescent in situ hybridization in combination with
an anti-green fluorescent protein (GFP) antibody staining. We furthermore
illustrate how to image migrating cell populations using time-lapse microscopy
in double-transgenic embryos. We show how to investigate cell number and
direction of migration by using a nuclear-localized GFP. The combination of this
transgene with a membrane-targeted red fluorescent protein allows for the
simultaneous determination of changes in cell shape, such as the formation of
filopodial extensions. We exemplify this by describing how a mutation in the
chemokine receptor cxcr4a affects endothelial cell migration and blood vessel
formation. Finally, we provide a method to perform fluorescent angiography to
monitor blood vessel perfusion in chemokine receptor mutants. The basal ganglia are critical for selecting actions and evaluating their
outcome. Although the circuitry for selection is well understood, how these
nuclei evaluate the outcome of actions is unknown. Here, we show in lamprey that
a separate evaluation circuit, which regulates the habenula-projecting globus
pallidus (GPh) neurons, exists within the basal ganglia. The GPh neurons are
glutamatergic and can drive the activity of the lateral habenula, which, in
turn, provides an indirect inhibitory influence on midbrain dopamine neurons. We
show that GPh neurons receive inhibitory input from the striosomal compartment
of the striatum. The striosomal input can reduce the excitatory drive to the
lateral habenula and, consequently, decrease the inhibition onto the
dopaminergic system. Dopaminergic neurons, in turn, provide feedback that
inhibits the GPh. In addition, GPh neurons receive direct projections from the
pallium (cortex in mammals), which can increase the GPh activity to drive the
lateral habenula to increase the inhibition of the neuromodulatory systems. This
circuitry, thus, differs markedly from the "direct" and "indirect" pathways that
regulate the pallidal (e.g., globus pallidus) output nuclei involved in the
control of motion. Our results show that a distinct reward-evaluation circuit
exists within the basal ganglia, in parallel to the direct and indirect
pathways, which select actions. Our results suggest that these circuits are part
of the fundamental blueprint that all vertebrates use to select actions and
evaluate their outcome. LIM homeodomain transcription factors are critical regulators of early
development in multiple systems but have yet to be examined for a role in
circuit formation. The LIM homeobox gene Lhx2 is expressed in cortical
progenitors during development and also in the superficial layers of the
neocortex in maturity. However, analysis of Lhx2 function at later stages of
cortical development has been hampered by severe phenotypes associated with
early loss of function. We identified a particular Cre-recombinase line that
acts in the cortical primordium after its specification is complete, permitting
an analysis of Lhx2 function in neocortical lamination, regionalization, and
circuit formation by selective elimination of Lhx2 in the dorsal telencephalon.
We report a profound disruption of cortical neuroanatomical and molecular
features upon loss of Lhx2 in the cortex from embryonic day 11.5. A unique
feature of cortical circuitry, the somatosensory barrels, is undetectable, and
molecular patterning of cortical regions appears disrupted. Surprisingly,
thalamocortical afferents innervate the mutant cortex with apparently normal
regional specificity. Electrophysiological recordings reveal a loss of responses
evoked by stimulation of individual whiskers, but responses to simultaneous
stimulation of multiple whiskers were present, suggesting that thalamic
afferents are unable to organize the neurocircuitry for barrel formation because
of a cortex-specific requirement of Lhx2. We report that Lhx2 is required for
the expression of transcription factor paired box gene 6, axon guidance molecule
Ephrin A5, and the receptor NMDA receptor 1. These genes may mediate Lhx2
function in the formation of specialized neurocircuitry necessary for
neocortical function. |
Is TALEN being used on stem cells? | Yes, TALEN is being used on stem cells for genome editing. | The rat is the preferred animal model in many areas of biomedical research and
drug development. Genetic manipulation in rats has lagged behind that in mice
due to the lack of efficient gene targeting tools. Previously, we generated a
knockout rat via conventional homologous recombination in rat embryonic stem
(ES) cells. Here, we show that efficient gene targeting in rat ES cells can be
achieved quickly through transcription activator-like effector nuclease
(TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning
technique, we constructed a pair of TALEN targeting vectors for the gene of
interest in 5 days. After gene transfection, the targeted rat ES cell colonies
were isolated, screened, and confirmed by PCR without the need of drug
selection. Our results suggest that TALEN-mediated gene targeting is a superior
means of establishing genetically modified rat ES cell lines with high
efficiency and short turnaround time. Transcription activator-like effector nucleases (TALENs) are a new class of
engineered nucleases that are easier to design to cleave at desired sites in a
genome than previous types of nucleases. We report here the use of TALENs to
rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic
cells or human pluripotent stem cells, the latter for which we differentiated
both the targeted lines and isogenic control lines into various metabolic cell
types. We demonstrate cell-autonomous phenotypes directly linked to
disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy,
motor-neuron death, and hepatitis C infection. We found little evidence of TALEN
off-target effects, but each clonal line nevertheless harbors a significant
number of unique mutations. Given the speed and ease with which we were able to
derive and characterize these cell lines, we anticipate TALEN-mediated genome
editing of human cells becoming a mainstay for the investigation of human
biology and disease. The functional study of Y chromosome genes has been hindered by a lack of mouse
models with specific Y chromosome mutations. We used transcription
activator-like effector nuclease (TALEN)-mediated gene editing in mouse
embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and
insertions in two Y-linked genes--Sry and Uty. TALEN-mediated gene editing is a
useful tool for dissecting the biology of the Y chromosome. Our ability to precisely and efficiently edit mammalian and plant genomes has
been significantly improved in recent years, partially due to increasing use of
designer nucleases that recognize a pre-determined DNA sequence, make a specific
DNA double-strand break, and stimulate gene targeting. A pair of zinc finger
nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs)
that recognize two adjacent unique DNA sequences dimerize through the fused FokI
nuclease domain and cut in the middle of target DNA sequences. We report here
that increasing the length of recognition DNA sequences by TALENs or ZFNs does
not necessarily translate to a higher efficiency or specificity. We also
discover that one subunit of ZFNs and one subunit of TALENs can form a pair of
hybrid nucleases with expanded specificity at two diverse targets, and stimulate
gene targeting in multiple cell types including human induced pluripotent stem
(iPS) cells with improved efficiency. Sickle cell disease (SCD) is the most common human genetic disease which is
caused by a single mutation of human β-globin (HBB) gene. The lack of long-term
treatment makes the development of reliable cell and gene therapies highly
desirable. Disease-specific patient-derived human induced pluripotent stem cells
(hiPSCs) have great potential for developing novel cell and gene therapies. With
the disease-causing mutations corrected in situ, patient-derived hiPSCs can
restore normal cell functions and serve as a renewable autologous cell source
for the treatment of genetic disorders. Here we successfully utilized
transcription activator-like effector nucleases (TALENs), a recently emerged
novel genome editing tool, to correct the SCD mutation in patient-derived
hiPSCs. The TALENs we have engineered are highly specific and generate minimal
off-target effects. In combination with piggyBac transposon, TALEN-mediated gene
targeting leaves no residual ectopic sequences at the site of correction and the
corrected hiPSCs retain full pluripotency and a normal karyotype. Our study
demonstrates an important first step of using TALENs for the treatment of
genetic diseases such as SCD, which represents a significant advance toward
hiPSC-based cell and gene therapies. Safety and reliability of transgene integration in human genome continue to pose
challenges for stem cell-based gene therapy. Here, we report a
baculovirus-transcription activator-like effector nuclease system for AAVS1
locus-directed homologous recombination in human induced pluripotent stem cells
(iPSCs). This viral system, when optimized in human U87 cells, provided a
targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette
and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs,
targeted integration with persistent transgene expression was achieved without
compromising genomic stability. The modified iPSCs continued to express stem
cell pluripotency markers and maintained the ability to differentiate into three
germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in
the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a
Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange.
Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity
associated with chromosomal double-strand breaks was not detectable after
transduction with moderate doses of baculoviral vectors expressing transcription
activator-like effector nucleases. Given high targeted integration efficiency,
flexibility in transgene exchange and low genome toxicity, our baculoviral
transduction-based approach offers great potential and attractive option for
precise genetic manipulation in human pluripotent stem cells. β-Thalassemia (β-Thal) is a group of life-threatening blood disorders caused by
either point mutations or deletions of nucleotides in β-globin gene (HBB). It is
estimated that 4.5% of the population in the world carry β-Thal mutants (1),
posing a persistent threat to public health. The generation of patient-specific
induced pluripotent stem cells (iPSCs) and subsequent correction of the
disease-causing mutations offer an ideal therapeutic solution to this problem.
However, homologous recombination-based gene correction in human iPSCs remains
largely inefficient. Here, we describe a robust process combining efficient
generation of integration-free β-Thal iPSCs from the cells of patients and
transcription activator-like effector nuclease (TALEN)-based universal
correction of HBB mutations in situ. We generated integration-free and
gene-corrected iPSC lines from two patients carrying different types of
homozygous mutations and showed that these iPSCs are pluripotent and have normal
karyotype. We showed that the correction process did not generate TALEN-induced
off targeting mutations by sequencing. More importantly, the gene-corrected
β-Thal iPS cell lines from each patient can be induced to differentiate into
hematopoietic progenitor cells and then further to erythroblasts expressing
normal β-globin. Our studies provide an efficient and universal strategy to
correct different types of β-globin mutations in β-Thal iPSCs for disease
modeling and applications. BACKGROUND: Transcription activator-like effector nucleases (TALENs) have
emerged as a tool for enabling targeted gene editing and disruption in difficult
systems, such as human pluripotent stem cells (hPSCs). The modular architecture
of TAL effectors theoretically enables targeting of any genomic locus and
several cloning systems for custom TALEN assembly have recently been
established. However, there is a lack of versatile TALEN expression systems
applicable to hPSCs.
RESULTS: Here, we extend an existing TALE assembly system by a dual set of
expression vectors for efficient application of TALEN technology in hPSCs. This
is characterized by improved TALEN architecture as well as antibiotic resistance
and fluorescent reporter cassettes, thus enabling enrichment for transfected
cells. Improved functionality of the combined system was demonstrated by
targeted disruption of the HPRT1 gene to create isogenic disease models of
Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1
occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells
both into central nervous system (CNS) as well as into sensory-like neurons
recapitulated previously described phenotypes based on patient-specific induced
PSCs and extended these findings to non-CNS neurons, respectively.
CONCLUSION: The combined vector system allows for flexible and affordable
generation of knock-out hPSCs lines, thus enabling investigation of
developmental processes as well as the generation of isogenic disease models
without the need for patient material. The establishment of embryonic stem cells (ESCs) and gene targeting technologies
in mice has revolutionised the field of genetics. The relative ease with which
genes can be knocked out, and exogenous sequences introduced, has allowed the
mouse to become the prime model for deciphering the genetic code. Not
surprisingly, the lack of authentic ESCs has hampered the livestock genetics
field and has forced animal scientists into adapting alternative technologies
for genetic engineering. The recent discovery of the creation of induced
pluripotent stem cells (iPSCs) by upregulation of a handful of reprogramming
genes has offered renewed enthusiasm to animal geneticists. However, much like
ESCs, establishing authentic iPSCs from the domestic animals is still beset with
problems, including (but not limited to) the persistent expression of
reprogramming genes and the lack of proven potential for differentiation into
target cell types both in vitro and in vivo. Site-specific nucleases comprised
of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs) and clustered regulated interspaced short palindromic repeats (CRISPRs)
emerged as powerful genetic tools for precisely editing the genome, usurping the
need for ESC-based genetic modifications even in the mouse. In this article, in
the aftermath of these powerful genome editing technologies, the role of
pluripotent stem cells in livestock genetics is discussed. MicroRNAs are important gene regulators involved in many biological processes,
including stemness maintece and cellular reprogramming. Current methods used
in loss-of-function studies of microRNAs mainly include locked nucleic acid
(LNA) oligonucleotides and miRZip inhibitors, which have several limitations.
Due to their unique gene structures and small sizes, there is no efficient or
simple strategy to knock down or knock out microRNAs or whole microRNA clusters.
Here, we demonstrate knockdown of the miR-302/367 cluster by using the
Kruppel-associated box repressor domain fused with specific transcription
activator-like effectors (TALEs) designed to bind the miR-302/367 cluster
promoter. We also designed two pairs of TALE nucleases (TALENs) to efficiently
delete the miR-302/367 cluster in primary human fibroblasts and determined that
knockout of the miR-302/367 cluster completely blocked induced pluripotent stem
cell (iPSC) generation. Together, our results demonstrate that TALE-based
transcriptional repressor and TALENs are two promising approaches for
loss-of-function studies of microRNA clusters in somatic cells and pluripotent
stem cells. The development of human embryonic stem cells (ESCs) and induced pluripotent
stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms,
speeds up the process of drug screening, and raises the feasibility of using
cell replacement therapy in clinics. However, the study of genotype-phenotype
relationships in ESCs or iPSCs is hampered by the low efficiency of
site-specific gene editing. Transcription activator-like effector nucleases
(TALENs) spurred interest due to the ease of assembly, high efficiency and
faithful gene targeting. In this study, we optimized the TALEN design to
maximize its genomic cutting efficiency. We showed that using optimized TALENs
in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient
gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb
can be accomplished at high efficiencies. We established human tumor cell lines
and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and
miR-9-2 gene. These cell lines provide a robust platform to dissect the roles
these genes play during cell differentiation and tumorigenesis. We also observed
that the endogenous homologous chromosome can serve as a donor template for gene
editing. Overall, our studies demonstrate the versatility of using ssODN and
TALEN to establish genetically modified cells for research and therapeutic
application. TALEN is one of the most widely used tools in the field of genome editing. It
enables gene integration and gene inactivation in a highly efficient and
specific fashion. Although very attractive, the apparent simplicity and high
success rate of TALEN could be misleading for novices in the field of gene
editing. Depending on the application, specific TALEN designs, activity
assessments and screening strategies need to be adopted. Here we report
different methods to efficiently perform TALEN-mediated gene integration and
inactivation in different mammalian cell systems including induced pluripotent
stem cells and delineate experimental examples associated with these approaches. Generation and precise genetic correction of patient-derived hiPSCs have great
potential in regenerative medicine. Such targeted genetic manipulations can now
be achieved using gene-editing nucleases. Here, we report generation of cystic
fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous
for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase
(GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs.
Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene
cassette at the endogenous CCR5 site of patient-derived disease-specific primary
fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous)
and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was
done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate
site-specific correction of sickle cell disease (SCD) mutations at the
endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous
for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected
hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one
of the HBB alleles, while the other allele remained a mutant phenotype. After
excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines
using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the
wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD
hiPSCs when differentiated into erythroid cells. Furthermore, we also show that
TALEN-mediated generation and genetic correction of disease-specific hiPSCs did
not induce any off-target mutations at closely related sites. Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused
by a mutation in the dystrophin gene. Genetic correction of patient-derived
induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise
for DMD gene therapy; however, the safety of such nuclease treatment must be
determined. Using a unique k-mer database, we systematically identified a unique
target region that reduces off-target sites. To restore the dystrophin protein,
we performed three correction methods (exon skipping, frameshifting, and exon
knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most
effective approach. We further investigated the genomic integrity by
karyotyping, copy number variation array, and exome sequencing to identify
clones with a minimal mutation load. Finally, we differentiated the corrected
iPSCs toward skeletal muscle cells and successfully detected the expression of
full-length dystrophin protein. These results provide an important framework for
developing iPSC-based gene therapy for genetic disorders using programmable
nucleases. |
Is there an association between bruxism and reflux | There is an association between bruxism and reflux. | The purpose of this study was to examine the relationships among nocturnal jaw
muscle activities, decreased esophageal pH, and sleep positions. Twelve adult
volunteers, including 4 bruxism patients, participated in this study. Portable
pH monitoring, electromyography of the temporal muscle, and audio-video
recordings were conducted during the night in the subjects' homes. Rhythmic
masticatory muscle activity (RMMA) episodes were observed most frequently, with
single short-burst episodes the second most frequent. The frequencies of RMMA,
single short-burst, and clenching episodes were significantly higher during
decreased esophageal pH episodes than those during other times. Both the
electromyography and the decreased esophageal pH episodes were most frequently
observed in the supine position. These results suggest that most jaw muscle
activities, ie, RMMA, single short-burst, and clenching episodes, occur in
relation to gastroesophageal reflux mainly in the supine position. Bruxism is a pathological activity of the stomatognathic system that involves
tooth grinding and clenching during parafunctional jaw movements. Clinical signs
of bruxism are mostly related to dental wear and muscular and joint discomforts,
but a large number of etiological factors can be listed, as local, systemic,
psychological and hereditary factors. The association between bruxism, feeding
and smoking habits and digestive disorders may lead to serious consequences to
dental and related structures, involving dental alterations (wear, fractures and
cracks), periodontal signs (gingival recession and tooth mobility) and
muscle-joint sensitivity, demanding a multidisciplinary treatment plan. This
paper presents a case report in which bruxism associated with acid feeding,
smoking habit and episodes of gastric reflow caused severe tooth wear and great
muscular discomfort with daily headache episodes. From the diagnosis, a
multidisciplinary treatment plan was established. The initial treatment approach
consisted of medical follow up with counseling on diet and smoking habits and
management of the gastric disorders. This was followed by the installation of an
interocclusal acrylic device in centric relation of occlusion (CRO) for
reestablishment of the occlusal stability, vertical dimension of occlusion,
anterior guides and return to normal muscle activity (90-day use approximately).
After remission of initial symptoms, oral rehabilitation was implemented in CRO
by means of full resin composite restorations and new interocclusal device for
protection of restorations. Satisfactory esthetics, improved function and
occlusal stability were obtained after oral rehabilitation. The patient has
attended annual follow-ups for the past 2 years. The multidisciplinary treatment
seems to be the key for a successful rehabilitation of severe cases of dental
wear involving the association of different health disorders. INTRODUCTION: Chronic regurgitation of gastric acids in patients with
gastroesophageal reflux disease may cause dental erosion, which can lead in
combination with attrition or bruxism to extensive loss of coronal tooth tissue.
CASE PRESENTATION: This clinical report describes treatment of severe tooth wear
of a gastroesophageal reflux disease patient who is 54-year-old Turkish male
patient. After his medical treatment, severe tooth wear, bruxism and decreased
vertical dimensions were determined. The vertical dimension was re-established
and maxillary and mandibular anterior and posterior teeth were prepared for
metal-ceramic restorations. Metal-ceramic fixed partial dentures were fabricated
as full mouth restorations for both maxillary and mandibular arches because of
splinting all teeth. And then maxillary stabilization splint was fabricated for
his bruxism history.
CONCLUSION: Significant loss of coronal tooth structure must taken into
consideration. Gastroesophageal reflux disease by itself or in combination with
attrition, abrasion or bruxism may be responsible for the loss. An extensive
diagnostic evaluation is essential for the medical and dental effects of the
problem. The aim of this cross-over, randomized, single-blinded trial was to examine
whether intra-esophageal acidification induces sleep bruxism (SB).
Polysomnography with electromyogram (EMG) of masseter muscle, audio-video
recording, and esophageal pH monitoring were performed in a sleep laboratory.
Twelve healthy adult males without SB participated. Intra-esophageal infusions
of 5-mL acidic solution (0.1 N HCl) or saline were administered. The frequencies
of EMG bursts, rhythmic masticatory muscle activity (RMMA) episodes, grinding
noise, and the RMMA/microarousal ratio were significantly higher in the
20-minute period after acidic infusion than after saline infusion. RMMA episodes
including SB were induced by esophageal acidification. This trial is registered
with the UMIN Clinical Trials Registry, UMIN000002923.
ABBREVIATIONS: ASDA, American Sleep Disorders Association; EMG, electromyogram;
GER, gastroesophageal reflux; LES, lower esophageal sphincter; NREM, non-rapid
eye movement; REM, rapid eye movement; RMMA, rhythmic masticatory muscle
activity; SB, sleep bruxism; SD, standard deviation; UES, upper esophageal
sphincter. Gastroesophageal reflux disease (GERD) is a gastrointestinal disorder in which
stomach acids are chronically regurgitated into the esophagus and oral cavity.
Continual exposure of the teeth to these acids can cause severe tooth wear.
Dentists are often the first healthcare professionals to diagnose dental erosion
in patients with GERD. This article presents a case report of a 27-year-old male
smoker with tooth wear and dentin sensitivity caused by GERD associated with
bruxism. After diagnosis, a multidisciplinary treatment plan was established.
The initial treatment approach consisted of medical follow-up with counseling on
dietary and smoking habits, as well as management of the gastric disorders with
medication. GERD management and the dental treatment performed for the eroded
dentition are described, including diagnosis, treatment planning, and
restorative therapy. BACKGROUND: There are few large-scale epidemiologic studies examining the
associations between sleep problems, gastroesophageal reflux disease (GERD)
symptoms, lifestyle and food habits and problem behaviors (PB) in adolescents.
The aim of this study was to evaluate the associations among these factors in
Japanese adolescents.
METHODS: A cross-sectional survey of 1840 junior high school students was
carried out using questionnaires. The subjects were classified into PB or normal
behavior (NB) groups using the Pediatric Symptom Checklist (PSC). The scores of
the sleep-related factors, sleep bruxism, lifestyle and food habits, and GERD
symptoms were compared. Logistic regression analysis was used to determine the
factors related to PB.
RESULTS: Mean subject age was 13.3 ± 1.8 years. The PB group had significantly
longer sleep latency and higher GERD symptom score (P < 0.001). Furthermore, the
PB group was significantly more likely to experience absence of the mother at
dinner time, skip breakfast, and have <30 min of conversation among family at
dinner time. The PB group had significantly higher frequencies of sleep bruxism,
difficulty falling asleep within 30 min, nightmares, feeling of low sleep
quality, daytime somnolence, and daytime lack of motivation. Feelings of low
sleep quality had the strongest association with PB, with an adjusted odds ratio
of 12.88 (95% confidence interval: 8.99-18.46).
CONCLUSIONS: PB in adolescents are associated with sleep problems, including
sleep bruxism, as well as lifestyle and food habits and GERD symptoms. STATEMENT OF PROBLEM: Rhythmic masticatory muscle activity, including sleep
bruxism (SB), can be induced in healthy individuals by experimental esophageal
acidification, which plays an important role in the pathogenesis of
gastroesophageal reflux disease (GERD). However, no robust evidence supports the
association between SB and GERD.
PURPOSE: The purpose of this study was to investigate the association between SB
and GERD.
MATERIAL AND METHODS: Forty-five individuals were eligible to participate in
this observational transversal study at the Gastroenterology Service of the
Clinical Hospital of Porto Alegre, Brazil. The participants were classified into
2 groups, those with and without GERD, according to the Montreal Criteria and
pH-metry/endoscopy findings. The diagnosis of SB was not assessed in a sleep
laboratory but was based on self-report plus clinical inspection, according to
the minimal diagnostic criteria of the American Academy of Sleep Medicine. The
Lipp Stress Symptom Inventory was used to evaluate self-perceived stress.
Univariate and multiple logistic regression analyses were performed with SB as
dependent variable and GERD, sex, age, body mass index, and stress as predictors
(α=.05; 90% power).
RESULTS: The study population included individuals with SB without GERD (13.3%)
and individuals with SB with GERD (31.1%). In participants with GERD, the
prevalence of SB was 73.7%. Only the variable GERD was significantly associated
with SB (P=.017; odds ratio 6.58; 95% confidence interval 1.40-30.98), although
adjusted for stress and age.
CONCLUSIONS: Sleep bruxism is prevalent in GERD patients, and GERD is highly
associated with SB. |
what is the role of IGF-1 in cardiac regeneration after myocardial infarction? | Ischemia-reperfusion injury is a strong stimulus for both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of IGF-1. Furthermore, in an animal model of myocardial infarction, intracoronary administration of IGF-1 is shown to reduce pathological cardiac remodeling, induce myocardial regeneration, and improve ventricular function. IGF-1 is a potent modulator of stem cell replication, commitment to the myocyte lineage, and myocyte differentiation. In another study, the dual delivery of IGF-1 and HGF from affinity-binding alginate biomaterial prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry and increased the incidence of GATA-4-positive cell clusters. The addition of nanofiber-mediated IGF-1 delivery to Cardiac Progenitor Cells therapy improved in part the recovery of myocardial structure and function after infarction. IGF-1 promotes proliferation and survival of CPCs. The strategy of IGF-1 transgene expression has shown to induce massive stem cell mobilization via SDF-1alpha signaling and culminated in extensive angiomyogenesis in the infarcted heart. | To determine whether IGF-1 opposes the stimulation of myocyte death in the
surviving myocardium after infarction, transgenic mice overexpressing human
IGF-1B in myocytes (FVB.Igf+/-) and wild-type littermates at 1.5 and 2.5 mo of
age were subjected to coronary ligation and killed 7 d later. Myocardial
infarction involved an average 50% of the left ventricle, and produced cardiac
failure. In the region proximate to infarction, myocyte apoptosis increased 4.
2-fold and 2.1-fold in nontransgenics at 1.5 and 2.5 mo, respectively.
Corresponding increases in myocyte necrosis were 1. 8-fold and 1.6-fold. In
contrast, apoptotic and necrotic myocyte death did not increase in FVB.Igf+/-
mice at either age after infarction. In 2.5-mo-old infarcted nontransgenics,
functional impairment was associated with a 29% decrease in wall thickness, 43%
increase in chamber diameter, and a 131% expansion in chamber volume.
Conversely, the changes in wall thickness, chamber diameter, and cavitary volume
were 41, 58, and 48% smaller in infarcted FVB.Igf+/- than in nontransgenics. The
differential response to infarction of FVB.Igf+/- mice resulted in an attenuated
increase in diastolic wall stress, cardiac weight, and left and right
ventricular weight-to-body wt ratios. In conclusion, constitutive overexpression
of IGF-1 prevented activation of cell death in the viable myocardium after
infarction, limiting ventricular dilation, myocardial loading, and cardiac
hypertrophy. Cardiac stem cells and early committed cells (CSCs-ECCs) express c-Met and
insulin-like growth factor-1 (IGF-1) receptors and synthesize and secrete the
corresponding ligands, hepatocyte growth factor (HGF) and IGF-1. HGF mobilizes
CSCs-ECCs and IGF-1 promotes their survival and proliferation. Therefore, HGF
and IGF-1 were injected in the hearts of infarcted mice to favor, respectively,
the translocation of CSCs-ECCs from the surrounding myocardium to the dead
tissue and the viability and growth of these cells within the damaged area. To
facilitate migration and homing of CSCs-ECCs to the infarct, a growth factor
gradient was introduced between the site of storage of primitive cells in the
atria and the region bordering the infarct. The newly-formed myocardium
contained arterioles, capillaries, and functionally competent myocytes that with
time increased in size, improving ventricular performance at healing and long
thereafter. The volume of regenerated myocytes was 2200 microm3 at 16 days after
treatment and reached 5100 microm3 at 4 months. In this interval, nearly 20% of
myocytes reached the adult phenotype, varying in size from 10,000 to 20,000
microm3. Moreover, there were 43+/-13 arterioles and 155+/-48 capillaries/mm2
myocardium at 16 days, and 31+/-6 arterioles and 390+/-56 capillaries at 4
months. Myocardial regeneration induced increased survival and rescued animals
with infarcts that were up to 86% of the ventricle, which are commonly fatal. In
conclusion, the heart has an endogenous reserve of CSCs-ECCs that can be
activated to reconstitute dead myocardium and recover cardiac function. Strategies for cardiac repair include injection of cells, but these approaches
have been hampered by poor cell engraftment, survival, and differentiation. To
address these shortcomings for the purpose of improving cardiac function after
injury, we designed self-assembling peptide ofibers for prolonged delivery of
insulin-like growth factor 1 (IGF-1), a cardiomyocyte growth and differentiation
factor, to the myocardium, using a "biotin sandwich" approach. Biotinylated
IGF-1 was complexed with tetravalent streptavidin and then bound to biotinylated
self-assembling peptides. This biotin sandwich strategy allowed binding of IGF-1
but did not prevent self-assembly of the peptides into ofibers within the
myocardium. IGF-1 that was bound to peptide ofibers activated Akt, decreased
activation of caspase-3, and increased expression of cardiac troponin I in
cardiomyocytes. After injection into rat myocardium, biotinylated ofibers
provided sustained IGF-1 delivery for 28 days, and targeted delivery of IGF-1 in
vivo increased activation of Akt in the myocardium. When combined with
transplanted cardiomyocytes, IGF-1 delivery by biotinylated ofibers decreased
caspase-3 cleavage by 28% and increased the myocyte cross-sectional area by 25%
compared with cells embedded within ofibers alone or with untethered IGF-1.
Finally, cell therapy with IGF-1 delivery by biotinylated ofibers improved
systolic function after experimental myocardial infarction, demonstrating how
engineering the local cellular microenvironment can improve cell therapy. BACKGROUND: Myoblast transplantation (Tx) is promising for the improvement of
cardiac function in ischemic cardiomyopathy. Insulin-like growth factor-1
(IGF-1) has anti-apoptotic and angiogenic effects, and induces myocyte
hypertrophy. Our hypothesis is that topical and slow-release IGF-1 enhances the
efficacy of Tx through its multiple functions.
METHODS: Four weeks after coronary artery ligation, Lewis rats were divided into
four groups: (1) IGF-1+Tx, injection of 6 x 10(6) myoblasts into the infarcted
area with placement of an IGF-1-impregnated sheet on the left ventricular (LV)
free wall; (2) Tx, Tx alone; (3) IGF-1, IGF-1 sheet alone; and (4) control. We
measured cardiac function and performed immunohistochemical examinations.
RESULTS: At 4 weeks after treatment, LV diastolic dimension was the smallest,
end-systolic elastance was the highest, and tau was the smallest in the IGF-1+Tx
group. The graft volume in the IGF-1+Tx group was 3-fold larger than in the Tx
group. One day after transplantation, TUNEL-positive donor cells were fewer in
the IGF-1+Tx than in the Tx group. Western blot analysis demonstrated that the
phosphorylation of Akt increased and the expression of Bax decreased in the
transplanted area of IGF-1+Tx rats compared with Tx rats. The vascular density
in the peri-infarcted area was larger in IGF-1+Tx than in Tx rats. The mean
diameter of graft-derived myotubes was larger in IGF-1+Tx than in Tx animals.
CONCLUSIONS: IGF-1 increases the graft volume and enhances the efficacy of Tx in
the chronic myocardial infarction model due to its multiple effects of
preventing apoptosis, inducing angiogenesis, and promoting myoblast growth. Ischemic heart disease is characterized chronically by a healed infarct, foci of
myocardial scarring, cavitary dilation, and impaired ventricular performance.
These alterations can only be reversed by replacement of scarred tissue with
functionally competent myocardium. We tested whether cardiac progenitor cells
(CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated
locally by hepatocyte growth factor and insulin-like growth factor-1 invade the
scarred myocardium and generate myocytes and coronary vessels improving the
hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful
chemoattractant of CPCs, and insulin-like growth factor-1 promotes their
proliferation and survival. Injection of CPCs or growth factors led to the
replacement of approximately 42% of the scar with newly formed myocardium,
attenuated ventricular dilation and prevented the chronic decline in function of
the infarcted heart. Cardiac repair was mediated by the ability of CPCs to
synthesize matrix metalloproteinases that degraded collagen proteins, forming
tunnels within the fibrotic tissue during their migration across the scarred
myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes,
excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for
cardiac repair in patients with chronic heart failure. CPCs may be isolated from
myocardial biopsies and, following their expansion in vitro, administered back
to the same patients avoiding the adverse effects associated with the use of
nonautologous cells. Alternatively, growth factors may be delivered locally to
stimulate resident CPCs and promote myocardial regeneration. These forms of
treatments could be repeated over time to reduce progressively tissue scarring
and expand the working myocardium. We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like
growth factor (IGF)-1 showed improved survival and engraftment in the infarcted
heart and promoted stem cell recruitment through paracrine release of stromal
cell-derived factor (SDF)-1alpha. Rat bone marrow-derived MSCs were used as
nontransduced ((Norm)MSCs) or transduced with adenoviral-null vector
((Null)MSCs) or vector encoding for IGF-1 ((IGF-1)MSCs). (IGF-1)MSCs secreted
higher IGF-1 until 12 days of observation (P<0.001 versus (Null)MSCs). Molecular
studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl.xL and
inhibition of glycogen synthase kinase 3beta besides release of SDF-1alpha in
parallel with IGF-1 expression in (IGF-1)MSCs. For in vivo studies, 70 muL of
DMEM without cells (group 1) or containing 1.5x10(6) (Null)MSCs (group 2) or
(IGF-1)MSCs (group 3) were implanted intramyocardially in a female rat model of
permanent coronary artery occlusion. One week later, immunoblot on rat heart
tissue (n=4 per group) showed elevated myocardial IGF-1 and phospho-Akt in group
3 and higher survival of (IGF-1)MSCs (P<0.06 versus (Null)MSCs) (n=6 per group).
SDF-1alpha was increased in group 3 animal hearts (20-fold versus group 2), with
massive mobilization and homing of ckit(+), MDR1(+), CD31(+), and CD34(+) cells
into the infarcted heart. Infarction size was significantly reduced in cell
transplanted groups compared with the control. Confocal imaging after
immunostaining for myosin heavy chain, actinin, connexin-43, and von Willebrand
factor VIII showed extensive angiomyogenesis in the infarcted heart. Indices of
left ventricular function, including ejection fraction and fractional
shortening, were improved in group 3 as compared with group 1 (P<0.05). In
conclusion, the strategy of IGF-1 transgene expression induced massive stem cell
mobilization via SDF-1alpha signaling and culminated in extensive
angiomyogenesis in the infarcted heart. BACKGROUND: Cardiac progenitor cells (CPCs) possess the insulin-like growth
factor-1 (IGF-1)-IGF-1 receptor system, and IGF-1 can be tethered to
self-assembling peptide ofibers (NF-IGF-1), leading to prolonged release of
this growth factor to the myocardium. Therefore, we tested whether local
injection of clonogenic CPCs and NF-IGF-1 potentiates the activation and
differentiation of delivered and resident CPCs enhancing cardiac repair after
infarction.
METHODS AND RESULTS: Myocardial infarction was induced in rats, and untreated
infarcts and infarcts treated with CPCs or NF-IGF-1 only and CPCs and NF-IGF-1
together were analyzed. With respect to infarcts exposed to CPCs or NF-IGF-1
alone, combination therapy resulted in a greater increase in the ratio of left
ventricular mass to chamber volume and a better preservation of +dP/dt, -dP/dt,
ejection fraction, and diastolic wall stress. Myocardial regeneration was
detected in all treated infarcts, but the number of newly formed myocytes with
combination therapy was 32% and 230% higher than with CPCs and NF-IGF-1,
respectively. Corresponding differences in the volume of regenerated myocytes
were 48% and 115%. Similarly, the length density of newly formed coronary
arterioles with both CPCs and NF-IGF-1 was 73% and 83% greater than with CPCs
and NF-IGF-1 alone, respectively. Importantly, activation of resident CPCs by
paracrine effects contributed to cardiomyogenesis and vasculogenesis.
Collectively, CPCs and NF-IGF-1 therapy reduced infarct size more than CPCs and
NF-IGF-1 alone.
CONCLUSIONS: The addition of ofiber-mediated IGF-1 delivery to CPC therapy
improved in part the recovery of myocardial structure and function after
infarction. Although most medicines have historically been small molecules, many newly
approved drugs are derived from proteins. Protein therapies have been developed
for treatment of diseases in almost every organ system, including the heart.
Great excitement has now arisen in the field of regenerative medicine,
particularly for cardiac regeneration after myocardial infarction. Every year,
millions of people suffer from acute myocardial infarction, but the adult
mammalian myocardium has limited regeneration potential. Regeneration of the
heart after myocardium infarction is therefore an exciting target for protein
therapeutics. In this review, we discuss different classes of proteins that have
therapeutic potential to regenerate the heart after myocardial infarction.
Protein candidates have been described that induce angiogenesis, including
fibroblast growth factors and vascular endothelial growth factors, although thus
far clinical development has been disappointing. Chemotactic factors that
attract stem cells, e.g., hepatocyte growth factor and stromal cell-derived
factor-1, may also be useful. Finally, neuregulins and periostin are proteins
that induce cell-cycle reentry of cardiomyocytes, and growth factors like IGF-1
can induce growth and differentiation of stem cells. As our knowledge of the
biology of regenerative processes and the role of specific proteins in these
processes increases, the use of proteins as regenerative drugs could develop as
a cardiac therapy. Proper spatio-temporal delivery of multiple therapeutic proteins represents a
major challenge in therapy strategies aimed at inducing myocardial regeneration
after myocardial infarction (MI). We hypothesized that the dual delivery of
insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) by
injectable affinity-binding alginate biomaterial would maximize their
therapeutic effects, leading to a more favorable course of tissue restoration
after acute MI. A sequential release of IGF-1 followed by HGF was attained from
affinity-binding alginate biomaterial, which also protected the proteins from
proteolysis (shown by mass spectroscopy). The released factors retained
bioactivity, as judged by their capability to activate their respective
signaling pathways and to prevent cardiomyocyte apoptosis in vitro. In a rat
model of acute MI, an intramyocardial injection of the dual IGF-1/HGF
affinity-bound alginate biomaterial preserved scar thickness, attenuated infarct
expansion and reduced scar fibrosis after 4 weeks, concomitantly with increased
angiogenesis and mature blood vessel formation at the infarct. Furthermore, this
treatment prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry
and increased the incidence of GATA-4-positive cell clusters. The dual delivery
of IGF-1 and HGF from affinity-binding alginate biomaterial represents a useful
strategy to treat MI. It showed a marked therapeutic efficacy at various tissue
levels, as well as potential to induce endogenous regeneration of cardiac
muscle. RATIONALE: Age and coronary artery disease may negatively affect the function of
human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for
autologous cell transplantation in the failing heart.
OBJECTIVE: Insulin-like growth factor (IGF)-1, IGF-2, and angiotensin II (Ang
II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in
c-kit(+) hCSCs to establish whether these systems would allow us to separate
hCSC classes with different growth reserve in the aging and diseased myocardium.
METHODS AND RESULTS: C-kit(+) hCSCs were collected from myocardial samples
obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac
surgery for coronary artery disease. The expression of IGF-1R in hCSCs
recognized a young cell phenotype defined by long telomeres, high telomerase
activity, enhanced cell proliferation, and attenuated apoptosis. In addition to
IGF-1, IGF-1R(+) hCSCs secreted IGF-2 that promoted myocyte differentiation.
Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R,
identified senescent hCSCs with impaired growth reserve and increased
susceptibility to apoptosis. The ability of IGF-1R(+) hCSCs to regenerate
infarcted myocardium was then compared with that of unselected c-kit(+) hCSCs.
IGF-1R(+) hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of
IGF-1R(+) hCSCs with IGF-2 resulted in the formation of more mature myocytes and
superior recovery of ventricular structure.
CONCLUSIONS: hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which
are potent modulators of stem cell replication, commitment to the myocyte
lineage, and myocyte differentiation, which points to this hCSC subset as the
ideal candidate cell for the management of human heart failure. To explore how cardiac regeneration and cell turnover adapts to disease,
different forms of stress were studied for their effects on the cardiac
progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5,
and mast cells. Adult female rats were examined during pregcy, after
myocardial infarction and ischemia-reperfusion injury with/out insulin like
growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac
sub-domains were analyzed at one and two weeks post-intervention, both at the
mRNA and protein levels. While pregcy and myocardial infarction up-regulated
Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion
injury induced the strongest up-regulation which occurred globally throughout
the entire heart and not just around the site of injury. This response seems to
be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary
to c-Kit, Isl1 was not up-regulated by pregcy or myocardial infarction while
ischemia-reperfusion injury induced not a global but a focal up-regulation in
the outflow tract and also in the peri-ischemic region, correlating with the
up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the
endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1.
c-Kit expression was not further influenced by the exogenous growth factors.
This indicates that there is a spatial mismatch between on one hand c-Kit and
Nkx2.5 expression and on the other hand Isl1 expression. In conclusion,
ischemia-reperfusion injury was the strongest stimulus with both global and
focal cardiomyocyte progenitor cell marker up-regulations, correlating to the
endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregcy
induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes
throughout the heart. Utilization of these pathways could provide new strategies
for the treatment of cardiac disease. |
What is known about prostate cancer screening in the UK | Screening for early disease has been available for many years, but there is still no national screening programme established in the United Kingdom. Two systematic reviews have concluded that screening should not be carried out. In general, this recommendation has been accepted in the United Kingdom. | OBJECTIVES: To review evidence regarding the potential introduction of prostate
cancer screening programmes and highlight issues pertinent to the management of
screen-detected prostate cancer.
METHODS: Screening for prostate cancer is a controversial health care issue in
general and urological practice. A PubMed database search was performed,
followed by a systematic review of the literature, to examine the evidence base
underlying prostate cancer screening.
RESULTS: A prostate cancer screening programme should satisfy several key
postulates prior to its introduction. To date, several of these postulates have
not been satisfied, and the evidence available for prostate cancer screening is
currently insufficient to warrant its introduction as a public health policy.
The natural history of screen-detected prostate cancer remains poorly
understood, and recent evidence suggests that a screening programme may detect a
large number of men with indolent disease who may be subsequently overtreated.
Several randomised clinical trials are currently in progress and it is hoped
that they will provide robust evidence to inform future practice.
CONCLUSIONS: National systematic prostate cancer screening programmes outside
randomised clinical trial settings have not been implemented to date owing to
lack of robust evidence that such programmes would improve survival and/or
quality of life in men with screen-detected disease. Forthcoming results of
clinical trials and the application of appropriate risk stratification to
prevent overtreatment of indolent prostate cancer are likely to change practice
in coming years. Making informed decisions about breast and prostate cancer screening requires
knowledge of its benefits. However, country-specific information on public
knowledge of the benefits of screening is lacking. Face-to-face
computer-assisted personal interviews were conducted with 10,228 persons
selected by a representative quota method in nine European countries (Austria,
France, Germany, Italy, the Netherlands, Poland, Russia, Spain, and the United
Kingdom) to assess perceptions of cancer-specific mortality reduction associated
with mammography and prostate-specific antigen (PSA) screening. Participants
were also queried on the extent to which they consulted 14 different sources of
health information. Correlation coefficients between frequency of use of
particular sources and the accuracy of estimates of screening benefit were
calculated. Ninety-two percent of women overestimated the mortality reduction
from mammography screening by at least one order of magnitude or reported that
they did not know. Eighty-nine percent of men overestimated the benefits of PSA
screening by a similar extent or did not know. Women and men aged 50-69 years,
and thus targeted by screening programs, were not substantially better informed
about the benefits of mammography and PSA screening, respectively, than men and
women overall. Frequent consulting of physicians (r = .07, 95% confidence
interval [CI] = 0.05 to 0.09) and health pamphlets (r = .06, 95% CI = 0.04 to
0.08) tended to increase rather than reduce overestimation. The vast majority of
citizens in nine European countries systematically overestimate the benefits of
mammography and PSA screening. In the countries investigated, physicians and
other information sources appear to have little impact on improving citizens'
perceptions of these benefits. Prostate specific antigen (PSA) was introduced as a prostate cancer screening
tool more than 20 years ago. However, there is continuing debate regarding its
utility in screening for prostate cancer. Mass screening is costly, may result
in the diagnosis and treatment of prostate cancers that never become clinically
significant, and the evidence of a subsequent reduction in mortality is
inconclusive. In addition to its role in screening, PSA is also used to monitor
the progression of the disease, both localized and metastatic. Although the
evidence is contradictory, PSA is still an important tool for monitoring patient
progression following treatment of definitive localized prostate cancer.
However, its use in monitoring castrate-resistant prostate cancer (CRPC) is more
controversial, particularly in the context of novel targeted treatments, which
may have little impact on PSA levels. These issues highlight the urgent need to
identify prostate cancer biomarkers that will improve early disease detection,
increase accuracy of diagnosis, determine the aggressiveness of disease, and
monitor treatment efficacy, particularly in late-stage disease. This review
discusses the key issues associated with the use of PSA as an early screening
tool for prostate cancer, as a prognostic marker to measure disease progression
in both early- and late-stage prostate cancer, and as a surrogate endpoint in
clinical trials with new agents. BACKGROUND: Web-based decision aids are known to have an effect on knowledge,
attitude, and behavior; important components of informed decision making. We
know what decision aids achieve in randomized controlled trials (RCTs), but we
still know very little about how they are used and how this relates to the
informed decision making outcome measures.
OBJECTIVE: To examine men's use of an online decision aid for prostate cancer
screening using website transaction log files (web-logs), and to examine
associations between usage and components of informed decision making.
METHODS: We conducted an observational web-log analysis of users of an online
decision aid, Prosdex. Men between 50 and 75 years of age were recruited for an
associated RCT from 26 general practices across South Wales, United Kingdom. Men
allocated to one arm of the RCT were included in the current study. Time and
usage data were derived from website log files. Components of informed decision
making were measured by an online questionnaire.
RESULTS: Available for analysis were 82 web-logs. Overall, there was large
variation in the use of Prosdex. The mean total time spent on the site was 20
minutes. The mean number of pages accessed was 32 (SD 21) out of a possible 60
pages. Significant associations were found between increased usage and increased
knowledge (Spearman rank correlation [rho] = 0.69, P < .01), between increased
usage and less favorable attitude towards PSA testing (rho = -0.52, P < .01),
and between increased usage and reduced intention to undergo PSA testing (rho =
-0.44, P < .01). A bimodal distribution identified two types of user: low access
and high access users.
CONCLUSIONS: Increased usage of Prosdex leads to more informed decision making,
the key aim of the UK Prostate Cancer Risk Management Programme. However,
developers realistically have roughly 20 minutes to provide useful information
that will support informed decision making when the patient uses a web-based
interface. Future decision aids need to be developed with this limitation in
mind. We recommend that web-log analysis should be an integral part of online
decision aid development and analysis.
TRIAL REGISTRATION: ISRCTN48473735;
http://www.controlled-trials.com/ISRCTN48473735 (Archived by WebCite at
http://www.webcitation.org/5pqeF89tS). OBJECTIVE: To evaluate the role of targeted prostate cancer screening in men
with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of
Men with a genetic predisposition to ProstAte Cancer: Targeted screening in
BRCA1/2 mutation carriers and controls), was established. This is the first
multicentre screening study targeted at men with a known genetic predisposition
to prostate cancer. A preliminary analysis of the data is reported.
PATIENTS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2
mutations were offered annual prostate specific antigen (PSA) testing, and those
with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men
age-matched (± 5 years) who were negative for the familial mutation.
RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116
BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0%
(21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten
individuals, a prevalence of 3.3%. The positive predictive value of PSA
screening in this cohort was 47·6% (10/21). One prostate cancer was diagnosed at
year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers,
two in controls, and eight were clinically significant.
CONCLUSIONS: The present study shows that the positive predictive value of PSA
screening in BRCA mutation carriers is high and that screening detects
clinically significant prostate cancer. These results support the rationale for
continued screening in such men. INTRODUCTION: Prostate cancer is the commonest cancer in men and a major health
issue worldwide. Screening for early disease has been available for many years,
but there is still no national screening programme established in the United
Kingdom.
OBJECTIVE: To assess the latest evidence regarding prostate cancer screening and
whether it meets the necessary requirements to be established as a national
programme for all men.
METHODS: Electronic databases and library catalogues were searched
electronically and manual retrieval was performed. Only primary research results
were used for the analysis.
RESULTS: In recent years, several important randomised controlled trials have
produced varied outcomes. In Europe the largest study thus far concluded that
screening reduced prostate cancer mortality by 20%. On the contrary, a large
American trial found no reduction in mortality after 7-10 years follow-up. Most
studies comment on the adverse effects of screening - principally those of
overdiagnosis and subsequent overtreatment.
DISCUSSION: Further information about the natural history of prostate cancer and
accuracy of screening is needed before a screening programme can be truly
justified. In the interim, doctors and patients should discuss the risks,
benefits and sequelae of taking part in voluntary screening for prostate cancer. |
Can a peptide aptamer be used as protein inhibitor? | Yes, peptide aptamers can be used as inhibitors. | The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This
critical regulatory pathway controls the G1/S transition of the mammalian cell
cycle by positive and negative regulation of E2F-responsive genes required for
DNA replication. To assess the value of the transcription factors E2Fs as
targets for antiproliferative strategies, we have initiated a program aiming to
develop inhibitors targeting specifically these proteins in vitro and in vivo.
The cellular activity of E2F is the result of the heterodimeric association of
two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two
hybrid approach to isolate from combinatorial libraries peptide aptamers that
specifically interact with E2Fs DNA binding and dimerization domains. One of
these is a potent inhibitor of E2F binding activity in vitro and in mammalian
fibroblasts, blocks cells in G1, and the free variable region from this aptamer
has the same effect. Our experiments argue that the variable region of this
aptamer is structured, and that it functions by binding E2F with a motif that
resembles a DP heterodimerization region, and blocking E2F's association with
DP. These results show that cell proliferation can be inhibited using
genetically-selected synthetic peptides that specifically target protein-protein
interaction motifs within cell cycle regulators. These results also emphasize
the critical role of the E2F pathway for cell proliferation and might allow the
design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F
pathway. The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin
cytoskeleton via their Rho-GTPase targets and affect numerous physiological
processes such as transformation and cell motility. They are therefore
attractive targets to design specific inhibitors that may have therapeutic
applications. Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate
the Rac and RhoA pathways, respectively. Here we have used a genetic screen in
yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that
could inhibit GEFD2 activity. One aptamer, TRIAPalpha (TRio Inhibitory APtamer),
specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding
peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in
intact cells and specifically reverts the neurite retraction phenotype induced
by TrioGEFD2 in PC12 cells. Thus TRIPalpha is the first Rho-GEF inhibitor
isolated so far, and represents an important step in the design of inhibitors
for the expanding family of Rho-GEFs. Peptide aptamers are peptides constrained and presented by a scaffold protein
that are used to study protein function in cells. They are able to disrupt
protein-protein interactions and to constitute recognition modules that allow
the creation of a molecular toolkit for the intracellular analysis of protein
function. The success of peptide aptamer technology is critically dependent on
the performance of the scaffold. Here, we describe a rational approach to the
design of a new peptide aptamer scaffold. We outline the qualities that an ideal
scaffold would need to possess to be broadly useful for in vitro and in vivo
studies and apply these criteria to the design of a new scaffold, called STM.
Starting from the small, stable intracellular protease inhibitor stefin A, we
have engineered a biologically neutral scaffold that retains the stable
conformation of the parent protein. We show that STM is able to present peptides
that bind to targets of interest, both in the context of known interactors and
in library screens. Molecular tools based on our scaffold are likely to be used
in a wide range of studies of biological pathways, and in the validation of drug
targets. FLASH protein is a component of death-inducing signaling complex and might be
involved in death receptor-mediated extrinsic apoptosis. Here we developed the
peptide aptamer against death effecter domain recruiting domain (DRD) of FLASH
protein and showed that the peptide bound to FLASH protein in vitro.
Intracellular expression of the DRD-binding peptide aptamer specifically
suppressed receptor-mediated extrinsic apoptosis but not intrinsic pathway,
which was recapitulated by the antisense oligonucleotides for FLASH. These data
suggest that DRD-binding peptide is not only a novel inhibitor modulating
receptor-mediated apoptosis but also a tool for elucidating the roles of FLASH
in apoptosis. In order to efficiently select aptamers that bind to and inhibit proteins, we
developed a method that involves screening DNA aptamers based on their
inhibitory activities using an evolution-mimicking algorithm after the
pre-selection by SELEX. The value of this method was demonstrated by the
identification of an inhibitor of Taq DNA polymerase in a unique single-stranded
DNA library, which was expected to form a G-quartet structure. This method
consists of selection via an inhibition assay, sequence shuffling, and mutation
in silico. After six rounds of selection, the inhibitory activities of the
aptamers had evolved significantly. This demonstrates the utility of this
strategy for screening aptamers based on their inhibitory actions. Amyloid-beta peptide-binding alcohol dehydrogenase (ABAD) inhibiting peptide, as
a specific inhibitor between ABAD and amyloid-beta (Abeta), has been
demonstrated to effectively inhibit Abeta peptide cytotoxicity. However, a major
drawback is its short half-life, which results in the need for multiple
applications and high synthesis costs. To overcome this, we established a
lentiviral expression system that allowed the stable expression of the small
ABAD-inhibiting peptide by fusion with cytosolic thioredoxin-1 (TRX). The fusion
peptide, TA aptamer, was observed within PC12 cytoplasm and maintained both
Abeta-binding ability and antioxygenic property similar to TRX. Our data showed
that overexpression of both TRX and TA aptamer could protect PC12 cells from
intracellular Abeta cytotoxicity. The present study suggests that TRX, as a
cytosolic protein and a fusion motif, could not only assist ABAD-inhibiting
peptide expression, cytoplasmic localization, but rebalance the disturbed "redox
equilibrium" caused by intracellular Abeta in PC12 cells. Proteases are involved in various biological functions. Thus, inhibition of
their activities is scientifically interesting and medically important. However,
there is no systematic method established to date to generate endopeptidase
inhibitory peptides. Here, we report a general system to identify endopeptidase
inhibitory peptides based on the use of in vitro evolution. Using this system,
we generated peptides that inhibit cathepsin E (CE) specifically at a
submicromolar IC(50). This system generates protease inhibitor peptides
utilizing techniques of cDNA display, selection-by-function, Y-ligation-based
block shuffling, and others. We further demonstrated the importance and
effectiveness of a secondary library for obtaining small-sized and active
peptides. CE inhibitory peptides generated by this method were characterized by
a small size (8 to 12 aa) and quite different sequences, suggesting that they
bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained
here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of
pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally
similar to CE). The general applicability of this system suggests that it may be
useful to identify inhibitory peptides for various kinds of proteases and that
it may therefore contribute to protein science and drug discovery. The peptide
binding to a protein is discussed in comparison with the antibody binding to an
antigen. Inhibitors of differentiation or DNA binding (Id) proteins have been shown to be
involved in tumor growth, invasiveness, metastasis, and angiogenesis.
Overexpression of Id proteins, especially Id1, correlates with unfavorable
clinical prognosis. Thus, they are attractive molecular targets for anticancer
therapy. Overexpression of Id proteins mediates breast cancer metastasis to
lung. Targeting Id1 and Id3 expression in breast cancer cells reduces breast
cancer metastasis in animal models. Different breast tumors failed to grow
and/or metastasize in Id1 (+/-) Id3 (-/-) mice. Id1 and Id3 preferentially
dimerize with the key regulatory E-proteins which inhibit the expression of
different tumor suppressor genes. Nevertheless, the inhibition of tumorigenic
activities of Id1 and Id3 at protein level has never been studied. Here, we
isolated a novel peptide aptamer, Id1/3-PA7, specifically interacting with Id1
and Id3 from randomized combinatorial expression library using yeast and
mammalian two-hybrid systems. Intracellular delivered Id1/3-PA7 co-localized to
Id1 and Id3 and interfered with their functions. It repressed E47 protein
sequestration by Id1 and Id3, activated the E-box promoter and increased the
expression level of cyclin-dependent kinase inhibitors (CDKN1A and CDKN1B) in a
dose-dependent fashion, paralleled by the cleavage of poly ADP ribose polymerase
(PARP). These effects were counteracted by ectopically overexpressed Id1 and
Id3. Peptide aptamer Id1/3-PA7 induced cell cycle arrest and apoptosis in breast
cancer cells MCF7 and MDA-MB-231. In conclusion, Id1/3-PA7 could represent a
nontoxic exogenous agent that can significantly provoke antiproliferative and
apoptotic effects in breast cancer cells, which are associated with deregulated
expression of Id1 and Id3. BACKGROUND: Inhibitors of DNA-binding proteins (Id1-4), lacking the basic
DNA-binding domain, function as domit inhibitors of cell-cycle regulators.
Overexpression of Id proteins promotes cancer cell proliferation and resistance
against apoptosis. Level of Id protein expression, especially of Id1, correlates
with poor differentiation, enhanced maligt potential and more aggressive
clinical behaviour of ovarian tumours. Although overexpression of Ids has been
found and shown to correlate with poor clinical outcome, their inhibition at
protein level has never been studied.
METHODS: A peptide aptamer, Id1/3-PA7, targeting Id1 and Id3, was isolated from
a randomised combinatorial expression library using yeast and mammalian
two-hybrid systems. Id1/3-PA7 was fused, expressed and purified with a
cell-penetrating protein transduction domain.
RESULTS: Intracellular-delivered Id1/3-PA7 colocalised to Id1 and Id3. It
induced cell-cycle arrest and apoptosis in ovarian cancer cells ES-2 and PA-1.
It activated the E-box promoter and increased the expression level of
cyclin-dependent kinase inhibitor (CDKN2A) in a dose-dependent manner that is
paralleled by the cleavage of poly-ADP ribose polymerase. These effects were
counteracted by ectopically overexpressed Id1 and Id3.
CONCLUSION: Id1/3-PA7 could represent an exogenous anti-tumour agent that can
significantly trigger cell-cycle arrest and apoptosis in ovarian cancer. The eukaryotic DNA recombination repair protein BRCA2 is functional in the
parasitic protozoan Trypanosoma brucei. The mechanism of the involvement of
BRCA2 in homologous recombination includes its interaction with the DNA
recombinase proteins of the RAD51 family. BRCA2 is known to interact with RAD51
through its unique and essential BRC sequence motifs. T. brucei BRCA2 homolog
(TbBRCA2) has fifteen repeating BRC motifs as compared to mammalian BRCA2 that
has only eight. We report here our yeast 2-hybrid analysis studies on the
interactions of TbBRCA2 BRC motifs with five different RAD51 paralogues of T.
brucei. Our study revealed that a single BRC motif is sufficient to bind to
these RAD51 paralogues. To test the possibility whether a single 44 amino acid
long repeating unit of the TbBRCA2 BRC motif may be exploited as an inhibitor of
T. brucei growth, we ectopically expressed this peptide segment in the procyclic
form of the parasite and evaluated its effects on cell survival as well as the
sensitivity of these cells to the DNA damaging agent methyl methane sulfonate
(MMS). Expression of a single BRC motif led to MMS sensitivity and inhibited
cellular proliferation in T. brucei. Among the many abnormally expressed proteins in ovarian cancer, the prominent
cancer in women, ID1 (inhibitors of DNA binding protein 1) is a potential one
among other several targets. Interaction of ID1 with ETS-1 (transcriptional
activator of p16(INK4a)) suppresses the transcription of p16(INK4a) and causes
abnormal cell proliferation. A peptide aptamer (ID1/3-PA7) has been designed to
prevent this interaction and thereby leading to the transcription of p16(INK4a).
However, the structural basis behind the molecular interaction of ID1 with ETS-1
(agonist) and ID1/3-PA7 (antagonist) is poorly understood. In order to
understand this structural recognition and their interaction mechanism, in
silico methods were used. From this interaction analysis, the residues of ETS-1
involved in interaction with the p16(INK4a) promoter were found to be targeted
by ID1. Subsequently, ETS-1 binding residues of ID1 were found to be targeted by
its aptamer- ID1/3-PA7. These results suggest that both ETS-1 and ID1/3-PA7
binds at the same region harbored by the residues-H97, D100, R103, D104, L107,
A144, C145, D149, D150 and C154 of ID1. All these observations correlate with
the experimental reports, suggesting that the identified residues might play a
crucial role in promulgating the oncogenic effects of ID1. In silico alanine
scanning mutagenesis also confirms the role of identified hot spot residues in
p16(INK4a) regulation. Finally, the molecular dynamic simulation studies reveal
the prolonged stability of the aforementioned interacting complexes. The
obtained results throw light on the structure and residues of ID1 involved in
transcriptional regulation of p16(INK4a). Many advanced tumors produce excessive amounts of Transforming Growth Factor-β
(TGF-β) which, in normal epithelial cells, is a potent growth inhibitor.
However, in oncogenically activated cells, the homeostatic action of TGF-β is
often diverted along alternative pathways. Hence, TGF-β signaling elicits
protective or tumor suppressive effects during the early growth-sensitive stages
of tumorigenesis. However, later in tumor development when carcinoma cells
become refractory to TGF-β-mediated growth inhibition, the tumor cell responds
by stimulating pathways with tumor progressing effects. At late stages of
maligcy, tumor progression is driven by TGF-β overload. The tumor
microenvironment is a target of TGF-β action that stimulates tumor progression
via pro-tumorigenic effects on vascular, immune, and fibroblastic cells. Bone is
one of the richest sources of TGF-β in the body and a common site for
dissemination of breast cancer metastases. Osteoclastic degradation of bone
matrix, which accompanies establishment and growth of metastases, triggers
further release of bone-derived TGF-β. This leads to a vicious positive feedback
of tumor progression, driven by ever increasing levels of TGF-β released from
both the tumor and bone matrix. It is for this reason, that pharmaceutical
companies have developed therapeutic agents that block TGF-β signaling.
Nonetheless, the choice of drug design and dosing strategy can affect the
efficacy of TGF-β therapeutics. This review will describe pre-clinical and
clinical data of four major classes of TGF-β inhibitor, namely i) ligand traps,
ii) antisense oligonucleotides, iii) receptor kinase inhibitors and iv) peptide
aptamers. Long term dosing strategies with TGF-β inhibitors may be ill-advised,
since this class of drug has potentially highly pleiotropic activity, and
development of drug resistance might potentiate tumor progression. Current
paradigms for the use of TGF-β inhibitors in oncology have therefore moved
towards the use of combinatorial therapies and short term dosing, with
considerable promise for the clinic. Accumulating work over the past decade has shown that peptide aptamer screening
represents a valid strategy for inhibitor identification that can be applied to
a variety of different targets. Because of the screening method in cells and the
highly combinatorial libraries available, this approach yields rapidly highly
specific candidate inhibitors. Once a hit peptide has been identified, its
interaction strength and affinity towards its target protein can be optimized
even more, in order to increase its inhibition efficiency when subsequently
applied in vivo. A condition to a successful optimization is that gain of
inhibition strength should not result in loss of specificity. Here we present a
simple method for peptide aptamer optimization, which can be achieved by
PCR-based random mutagenesis combined with a selection screen in yeast using a
strong selective drug. The rationale of this approach, which has proven valid
and efficient, is that stronger interaction in yeast will also lead to stronger
inhibition. Our optimization method is effective, without loss of specificity,
which is of a great importance for the discovery of inhibitors that target
specific protein-protein interactions. To identify critical host factors necessary for human immunodeficiency virus 1
(HIV-1) replication, large libraries of short-peptide-aptamers were expressed
retrovirally. The target of one inhibitor peptide, Pep80, identified in this
screen was determined to be Snapin, a protein associated with the soluble
N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that
is critical for calcium-dependent exocytosis during neurotransmission. Pep80
inhibited Ca²⁺ release from intracellular stores and blocked downstream
signaling by direct interruption of the association between Snapin and an
intracellular calcium release channel, the ryanodine receptor (RyR). NFAT
signaling was preferentially abolished by Pep80. Expression of Snapin overcame
Pep80-mediated inhibition of Ca²⁺/NFAT signaling and HIV-1 replication.
Furthermore, Snapin induced HIV-1 replication in primary CD4⁺ T cells. Thus,
through its interaction with RyR, Snapin is a critical regulator of Ca²⁺
signaling and T cell activation. Use of the genetically selected intracellular
aptamer inhibitors allowed us to define unique mechanisms important to HIV-1
replication and T cell biology. Peptide aptamers of LIM-only protein 2 (Lmo2) were previously used to
successfully treat Lmo2-induced tumours in a mouse model of leukaemia. Here we
show that the Lmo2 aptamer PA207, either as a free peptide or fused to
thioredoxin Trx-PA207, causes purified Lmo2 to precipitate rather than binding
to a defined surface on the protein. Stabilisation of Lmo2 through interaction
with LIM domain binding protein 1 (Ldb1), a normal binding partner of Lmo2,
abrogates this effect. The addition of free zinc causes Trx-PA207 to self
associate, suggesting that PA207 destabilises Lmo2 by modulating normal
zinc-coordination in the LIM domains. GST-pulldown experiments with other Lmo
and Gata proteins indicates that PA207 can bind to a range of zinc finger
proteins. Thus, PA207 and other cysteine-containing peptide aptamers for Lmo2
may form a class of general zinc finger inhibitors. |
List inflammatory caspase proteins? | caspase-1
caspase-4
caspase-5 | INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a worldwide burden
and a major cause of death. The disease is accompanied by chronic inflammation
and increased cellular turnover that is partly due to an overwhelming induction
of apoptosis. In this study, we hypothesized that systemic markers of apoptosis
are altered in patients with mild-to-severe COPD.
MATERIALS AND METHODS: A total number of 64 patients and controls were enrolled
in the study. Lung function parameters of all groups (nonsmoker, healthy smoker,
COPD GOLD I&II, COPD GOLD III&IV) were evaluated at the time of inclusion.
Enzyme-linked immunosorbent assays were used to quantify protein levels in serum
samples.
RESULTS: Serum contents of apoptotic end-products caspase-cleaved cytokeratin-18
and histone-associated-DNA-fragments were increased in patients with COPD,
whereas anti-inflammatory soluble ST2 showed a peak in patients with COPD I&II
(P=0.031) compared to healthy smokers. Levels of pro-inflammatory caspase-1/ ICE
correlated significantly with the number of pack years (R=0.337; P=0.007).
DISCUSSION: Our results indicate a systemic release of apoptosis-specific
proteins as markers for increased cellular turnover accompanied by progression
of COPD. Furthermore, soluble ST2 seems to have a critical role in the
anti-inflammatory regulatory mechanism at early stages of the disease. Host protection against fungi depends on intact innate and adaptive immune
responses. Consistently, fungal infections can cause systemic life-threatening
diseases in immunocomprimised individuals, suffering e.g. from cancer or AIDS.
Recent work has uncovered essential roles for the spleen tyrosine kinase (SYK)
and the cytosolic NLRP3 inflammasome for Interleukin-1beta (IL-1beta) production
in innate antifungal immunity. Upon fungal infection, SYK is activated by
several C-type lectin pattern recognition receptors on myeloid cells.
Subsequently, SYK signals for the production of reactive oxygen species and for
gene transcription to induce pro-inflammatory factors, including pro-IL-1beta to
initiate antifungal responses. Mature IL-1beta production additionally requires
cleavage of the pro-IL-1beta precursor protein by the inflammatory caspase-1
which is controlled within the NLRP3 inflammasome. Here, we discuss how SYK
signaling cooperates with the NLRP3 inflammasome for IL-1beta production in
antifungal immunity. INTRODUCTION: Infections with fungi can cause systemic life-threatening diseases
in immunocompromised individuals like cancer or AIDS patients. Recent work has
uncovered essential roles for C-type lectin pattern recognition receptors,
spleen tyrosine kinase (SYK) and the cytosolic NLRP3 inflammasome in innate
antifungal immunity. Upon fungal infection, SYK is activated by several
ITAM-containing or ITAM-coupled C-type lectin receptors on myeloid cells leading
to the production of pro-inflammatory cytokines including IL-1beta to initiate
antifungal responses. Mature IL-1beta production requires in addition to the
synthesis of pro-IL-1beta a cleavage of the precursor protein by the
inflammatory Caspase-1 which is controlled within the NLRP3 inflammasome.
SCOPE: Here, we discuss how ITAM receptor signaling and NLRP3 cooperate for the
induction of antifungal immunity. Although it is conventionally regarded as an inflammatory caspase, recent
studies have shown that caspase-4 plays a role in induction of apoptosis by
endoplasmic reticulum (ER) stress. We report here that activation of caspase-4
is also involved in induction of apoptosis by TNF-related apoptosis-inducing
ligand (TRAIL) in human melanoma cells. Treatment with TRAIL resulted in
activation of caspase-4. This appeared to be mediated by caspase-3, in that
caspase-4 was activated later than caspase-8, -9, and -3, and that inhibition of
caspase-3 blocked TRAIL-induced caspase-4 activation. Notably, TRAIL triggered
ER stress in melanoma cells as shown by up-regulation of the GRP78 protein and
the spliced form of XBP-1 mRNA. This seemed to be necessary for activation of
caspase-4, as activation of caspase-3 by agents that did not trigger ER stress
did not cause activation of caspase-4. Importantly, inhibition of caspase-4 also
partially blocked caspase-3 activation, suggesting that activation of caspase-4
may be positive feed-back mechanism to further enhance caspase-3 activation.
Collectively, these results show that activation of caspase-4 contributes to
TRAIL-induced apoptosis and is associated with induction of ER stress by TRAIL
in melanoma cells, and may have important implications for improving therapeutic
efficacies of TRAIL in melanoma. INTRODUCTION: When apoptosis is disrupted, the transformed cells can survive,
proliferate, and evolve into a maligcy. The strictly conserved caspase genes
and the reliable experimental data clearly show that some caspases play a
crucial role in apoptosis even if some of them have no apoptotic activity and
others exhibit both apoptotic and nonapoptotic properties. Although caspase-2
belongs to initiator caspases, its normal role remains unclear. Experimental
studies have shown that it is primarily necessary for the execution of apoptosis
in mutagenic cells. Human caspase-5 is classified as an inflammatory caspase,
although its substrate has not been identified yet. In this research, the
activities of caspase-2 and caspase-5 have been estimated during the progression
of human cervical maligcy.
METHODS: The experimental material includes human cervical tissue samples
(normal and pathological) and blood serum samples of the corresponding tissue
donors, where enzyme activities have been measured colorimetrically.
RESULTS: Both caspases' activities showed the highest increase, statistically
significant (P < 0.01, by t test) compared with the controls, in the low-grade
squamous intraepithelial lesion tissues. Caspase-2 of all pathological tissues
was proved more active than the controls. Serum caspases' activities were
significantly lower than those of the tissues. Serum caspase-2's activity in
patients with low-grade squamous intraepithelial lesion stage showed no
statistically significant increase compared with the controls. Serum caspase-5's
activity of all patients with maligcy stages was presented elevated, whereas
that of the serum of patients with cervical cancer had the highest activity (P <
0.01, by t test).
CONCLUSIONS: The changes of caspase-2 and caspase-5 activities could be
indicative of their involvement in the cervical maligcy mechanisms. Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed
pathways: survival-inflammation and cell death. An additional switch decides,
depending on the cellular context, between caspase-dependent apoptosis and RIP
kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the
contribution of both cell death pathways in TNF-induced systemic inflammatory
response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3
or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion
of RIPK3 conferred complete protection against lethal SIRS and reduced the
amounts of circulating damage-associated molecular patterns. Pretreatment with
the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These
results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives
mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal
ligation and puncture, underscoring the clinical relevance of RIPK kinase
inhibition in sepsis and identifying components of the necroptotic pathway that
are potential therapeutic targets for treatment of SIRS and sepsis. Inflammasomes are cytosolic multiprotein complexes assembled by intracellular
nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they
initiate innate immune responses to invading pathogens and danger signals by
activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and
release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, as well
as lytic inflammatory cell death known as pyroptosis. Recently, a new
non-canonical inflammasome was described that activates caspase-11, a
pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This
study also highlighted that previously generated caspase-1 knockout mice lack a
functional allele of Casp11 (also known as Casp4), making them functionally
Casp1 Casp11 double knockouts. Previous studies have shown that these mice are
more susceptible to infections with microbial pathogens, including the bacterial
pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the
individual contributions of caspase-1 and caspase-11 to this phenotype are not
known. Here we show that non-canonical caspase-11 activation contributes to
macrophage death during S. typhimurium infection. Toll-like receptor 4
(TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-β
(TRIF)-dependent interferon-β production is crucial for caspase-11 activation in
macrophages, but is only partially required for pro-caspase-11 expression,
consistent with the existence of an interferon-inducible activator of
caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to
infection with S. typhimurium than mice lacking both pro-inflammatory caspases
(Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial
counts, the formation of extracellular bacterial microcolonies in the infected
tissue and a defect in neutrophil-mediated clearance. These results indicate
that caspase-11-dependent cell death is detrimental to the host in the absence
of caspase-1-mediated innate immunity, resulting in extracellular replication of
a facultative intracellular bacterial pathogen. Inflammasomes are cytosolic multiprotein complexes that assemble in response to
a variety of infectious and noxious insults. Inflammasomes play a critical role
in the initiation of innate immune responses, primarily by serving as platforms
for the activation of inflammatory caspase proteases. One such caspase,
CASPASE-1 (CASP1), initiates innate immune responses by cleaving pro-IL-1β and
pro-IL-18, leading to their activation and release. CASP1 and another
inflammatory caspase termed CASP11 can also initiate a rapid and inflammatory
form of cell death termed pyroptosis. Several distinct inflammasomes have been
described, each of which contains a unique sensor protein of the NLR
(nucleotide-binding domain, leucine-rich repeat-containing) superfamily or the
PYHIN (PYRIN and HIN-200 domain-containing) superfamily. Here we describe the
surprisingly diverse mechanisms by which NLR/PYHIN proteins sense bacteria and
initiate innate immune responses. We conclude that inflammasomes represent a
highly adaptable scaffold ideally suited for detecting and initiating rapid
innate responses to diverse and rapidly evolving bacteria. Human beings are constantly exposed to pathogens. The innate immune system is
the first line of defense against microbes. It has evolved to recognize
conserved microbial motifs (PAMP or pathogen-associated molecular patterns)
thanks to a limited array of receptors termed pattern recognition receptors
(PRR). Upon activation, most PRR trigger a transcriptional response leading to
neosynthesis of hundreds of genes. In contrast, engagement of various PRR in the
recently identified inflammasome complexes lead to activation of a cysteine
protease, caspase-1. This inflammatory caspase has a dual activity: it triggers
the release of very potent proinflammatory cytokines IL-1β and IL-18 and, an
hyperinflammatory cell death termed pyroptosis. In this review, we describe the
inflammasome receptors and their ligands, the molecular mechanisms leading to
the assembly of this innate immune platform and the role of the inflammasome
during viral and bacterial infections. |
List invertebrates where ultraconserved elements have been identified. | Ultraconserved elements have been identified in the following genomes of invertebrates: tunicates, diptera, worm and yeast. | Recently, we identified a large number of ultraconserved (uc) sequences in
noncoding regions of human, mouse, and rat genomes that appear to be essential
for vertebrate and amniote ontogeny. Here, we used similar methods to identify
ultraconserved genomic regions between the insect species Drosophila
melanogaster and Drosophila pseudoobscura, as well as the more distantly related
Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects
appear to occur primarily in intergenic and intronic sequences, and at
intron-exon junctions. The sequences are significantly associated with genes
encoding developmental regulators and transcription factors, but are less
frequent and are smaller in size than in vertebrates. The longest identical,
nongapped orthologous match between the three genomes was found within the
homothorax (hth) gene. This sequence spans an internal exon-intron junction,
with the majority located within the intron, and is predicted to form a highly
stable stem-loop RNA structure. Real-time quantitative PCR analysis of different
hth splice isoforms and Northern blotting showed that the conserved element is
associated with a high incidence of intron retention in hth pre-mRNA, suggesting
that the conserved intronic element is critically important in the
post-transcriptional regulation of hth expression in Diptera. We have conducted a comprehensive search for conserved elements in vertebrate
genomes, using genome-wide multiple alignments of five vertebrate species
(human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been
performed with multiple alignments of four insect species (three species of
Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven
species of Saccharomyces. Conserved elements were identified with a computer
program called phastCons, which is based on a two-state phylogenetic hidden
Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by
maximum likelihood, subject to constraints designed to calibrate the model
across species groups, and then predicting conserved elements based on this
model. The predicted elements cover roughly 3%-8% of the human genome (depending
on the details of the calibration procedure) and substantially higher fractions
of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans
(18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to
vertebrates, in order of increasing genome size and general biological
complexity, increasing fractions of conserved bases are found to lie outside of
the exons of known protein-coding genes. In all groups, the most highly
conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases
long. These elements share certain properties with ultraconserved elements, but
they tend to be longer and less perfectly conserved, and they overlap genes of
somewhat different functional categories. In vertebrates, HCEs are associated
with the 3' UTRs of regulatory genes, stable gene deserts, and megabase-sized
regions rich in moderately conserved noncoding sequences. Noncoding HCEs also
show strong statistical evidence of an enrichment for RNA secondary structure. We have explored the distributions of fully conserved ungapped blocks in
genome-wide pair-wise alignments of recently completed species of Drosophila: D.
melanogaster, D. yakuba, D. aassae, D. pseudoobscura, D. virilis, and D.
mojavensis. Based on these distributions we have found that nearly every
functional sequence category possesses its own distinctive conservation pattern,
sometimes independent of the overall sequence conservation level. In the coding
and regulatory regions, the ungapped blocks were longer than in introns, UTRs,
and nonfunctional sequences. At the same time, the blocks in the coding regions
carried a 3N + 2 signature characteristic of synonymous substitutions in the
third-codon position. Larger block sizes in transcription regulatory regions can
be explained by the presence of conserved arrays of binding sites for
transcription factors. We also have shown that the longest ungapped blocks, or
"ultraconserved" sequences, are associated with specific gene groups, including
those encoding ion channels and components of the cytoskeleton. We discuss how
restraining conservation patterns may help in mapping functional sequence
categories and improve genome annotation. In a recent study that identified highly evolutionary conserved sequences in
three genomes of Diptera species we described an ultraconserved element found at
an internal exon-intron junction of the Drosophila melanogaster homothorax (hth)
gene that appeared to be involved in the control of hth pre-mRNA splicing. We
also discussed a possible role of RNA secondary structure at this site in the
regulation of hth pre-mRNA splicing. In this report we identify a shorter
evolutionary conserved intronic element within the hth gene that is located
downstream of the first element and has sequence complementarity to it. We
demonstrate that intramolecular interactions between these two elements would
give rise to alternative RNA secondary structures, which in turn may result in
differential control of homothorax pre-mRNA splicing. We also provide additional
comparative genomic data from several newly available insect genomes supporting
our original conclusion that these conserved elements are important in the
post-transcriptional regulation of homothorax gene expression in Diptera. Aligning and comparing genomic sequences enables the identification of conserved
sequence signatures and can enrich for coding and noncoding functional regions.
In vertebrates, the comparison of human and rodent genomes and the comparison of
evolutionarily distant genomes, such as human and pufferfish, have identified
specific sets of 'ultraconserved' sequence elements associated with the control
of early development. However, is this just the tip of a 'conservation iceberg'
or do these sequences represent a specific class of regulatory element? Studies
on the zebrafish phox2b gene region and the ENCODE project suggest that many
regulatory elements are not highly conserved, posing intriguing questions about
the relationship between noncoding sequence conservation and function and the
evolution of regulatory sequences. BACKGROUND: Ultraconserved elements of DNA have been identified in vertebrate
and invertebrate genomes. These elements have been found to have diverse
functions, including enhancer activities in developmental processes. The
evolutionary origins and functional roles of these elements in cellular systems,
however, have not yet been determined.
RESULTS: Here, we identified a wide range of ultraconserved elements common to
distant species, from primitive aquatic organisms to terrestrial species with
complicated body systems, including some novel elements conserved in fruit fly
and human. In addition to a well-known association with developmental genes,
these DNA elements have a strong association with genes implicated in essential
cell functions, such as epigenetic regulation, apoptosis, detoxification, innate
immunity, and sensory reception. Interestingly, we observed that ultraconserved
elements clustered by sequence similarity. Furthermore, species composition and
flanking genes of clusters showed lineage-specific patterns. Ultraconserved
elements are highly enriched with binding sites to developmental transcription
factors regardless of how they cluster.
CONCLUSION: We identified large numbers of ultraconserved elements across
distant species. Specific classes of these conserved elements seem to have been
generated before the divergence of taxa and fixed during the process of
evolution. Our findings indicate that these ultraconserved elements are not the
exclusive property of higher modern eukaryotes, but rather transmitted from
their metazoan ancestors. Co-option of cis-regulatory modules has been suggested as a mechanism for the
evolution of expression sites during development. However, the extent and
mechanisms involved in mobilization of cis-regulatory modules remains elusive.
To trace the history of non-coding elements, which may represent candidate
ancestral cis-regulatory modules affirmed during chordate evolution, we have
searched for conserved elements in tunicate and vertebrate (Olfactores) genomes.
We identified, for the first time, 183 non-coding sequences that are highly
conserved between the two groups. Our results show that all but one element are
conserved in non-syntenic regions between vertebrate and tunicate genomes, while
being syntenic among vertebrates. Nevertheless, in all the groups, they are
significantly associated with transcription factors showing specific functions
fundamental to animal development, such as multicellular organism development
and sequence-specific DNA binding. The majority of these regions map onto
ultraconserved elements and we demonstrate that they can act as functional
enhancers within the organism of origin, as well as in cross-transgenesis
experiments, and that they are transcribed in extant species of Olfactores. We
refer to the elements as 'Olfactores conserved non-coding elements'. |
Which gene is most commonly associated with severe congenital and cyclic neutropenia? | Neutrophil elastase gene (ELANE) mutations are responsible for the majority of cases of severe congenital neutropenia (SCN) and cyclic neutropenia (CN). | Severe neutropenia disorders are characterized by extremely low levels of
peripheral blood neutrophils, a maturation block of bone marrow progenitor cells
and recurring severe bacterial and fungal infections. Recent reports indicated
that severe neutropenia is a consequence of an impaired survival and abnormal
cell cycle progression of myeloid progenitor cells in both cyclic and severe
congenital neutropenia. Mutations in the neutrophil elastase gene were
identified in all patients with cyclic neutropenia and most of the patients with
severe congenital neutropenia. We hypothesize that expression of mutant
neutrophil elastase protein results in deregulation of intracellular activity
and premature cell death of myeloid-committed progenitor cells in these
disorders, resulting in the lack of peripheral blood neutrophils. The potential
molecular mechanisms of mutant-protein-mediated neutropenia is discussed. Two forms of inherited deficiency of neutrophil numbers are cyclic hematopoiesis
and severe congenital neutropenia. In cyclic hematopoiesis, neutrophil counts
oscillate opposite monocytes in a 3-week cycle. Severe congenital neutropenia
consists of static neutropenia and a predisposition to myelodysplasia and acute
myelogenous leukemia. All cases of cyclic neutropenia and most cases of severe
congenital neutropenia result from heterozygous germline mutations in the gene
encoding neutrophil elastase, ela2. Recent work extends the list of neutropenia
genes to include WASp, Gfi-1, adaptin, and tafazzin. Studies of mosaic patients
suggest that ela2 mutations act in a cell-autonomous fashion. A hypothetical
feedback circuit potentially interconnects these genes. Genetic dissection of
signaling in model organisms along with experimental hematology implicate
C/EPBepsilon, RUNX1/AML1, Notch family members, LEF1, and Cdc42 as additional
nodes in this pathway. The authors propose that neutrophil elastase acts as an
inhibitor of myelopoiesis, substantiating a chalone hypothesis proposed many
years ago. Heterozygous mutations of the gene encoding neutrophil elastase (ELA2) have been
associated with cyclic neutropenia (CN) and severe congenital neutropenia (SCN).
To date, 30 different mutations have been reported, but no correlation has been
found with the degree of neutropenia. To address this issue, we analyzed the
clinical, hematologic, and molecular characteristics of 81 unrelated patients
with SCN (n = 54) or CN (n = 27). We identified mutations in 31 patients, two
thirds of whom had sporadic forms. Familial cases were consistent with domit
inheritance. Seventeen novel mutations were identified, showing that the
mutational spectrum encompasses not only the region encoding the mature enzyme
but also the prodomains and promoter region. Genotype-phenotype analysis
strongly suggested that ELA2 mutations correlate with more severe expression of
neutropenia, specifically in patients diagnosed with SCN. This study underlines
the importance of ELA2 molecular screening to identify patients who may be at
particular risk of severe bacterial infections and/or acute myeloid
leukemia/myelodysplasia. By phenotypic analysis of affected relatives and
carriers of the same ELA2 mutations, we showed that the expression of
neutropenia in CN and SCN may be either homogeneous or variable according to the
type of mutations, suggesting different pathogenetic mechanisms. Mutations in ELA2 encoding the neutrophil granule protease, neutrophil elastase
(NE), are the major cause of the 2 main forms of hereditary neutropenia, cyclic
neutropenia and severe congenital neutropenia (SCN). Genetic evaluation of other
forms of neutropenia in humans and model organisms has helped to illuminate the
role of NE. A canine form of cyclic neutropenia corresponds to human
Hermansky-Pudlak syndrome type 2 (HPS2) and results from mutations in AP3B1
encoding a subunit of a complex involved in the subcellular trafficking of
vesicular cargo proteins (among which NE appears to be one). Rare cases of SCN
are attributable to mutations in the transcriptional repressor Gfi1 (among whose
regulatory targets also include ELA2). The ultimate biochemical consequences of
the mutations are not yet known, however. Gene targeting of ELA2 has thus far
failed to recapitulate neutropenia in mice. The cycling phenomenon and origins
of leukemic transformation in SCN remain puzzling. Nevertheless, mutations in
all 3 genes are capable of causing the mislocalization of NE and may also induce
the unfolded protein response, suggesting that there might a convergent
pathogenic mechanism focusing on NE. Proteases and their serpin inhibitors are abundantly expressed in haemopoietic
and peripheral blood cells. There is, however, relatively little information
about the role played by serpins in the control of protease activity within
these cells and in the pericellular region. The observation that mutations in
the neutrophil elastase gene, which cause cyclic and severe congenital
neutropenia, are associated with protease maldistribution gives some clue as to
the potential importance of inhibitor proteins. To begin to address the role of
protease/inhibitor balance in blood cells we used reverse transcription
polymerase chain reaction to examine protease and serpin gene expression in
mature peripheral blood cells, differentiating haemopoietic progenitors,
leukaemic blasts and haemopoietic cell lines. The results demonstrate
stage-specific expression of proteases together with widespread expression of
intra- and extra-cellular serpins. The elastase inhibitors monocyte neutrophil
elastase inhibitor (MNEI) and antitrypsin (AT) showed overlapping expression.
MNEI is predomitly expressed in early haemopoietic progenitors while
antitrypsin is mainly expressed in more mature myeloid precursors, peripheral
blood granulocytes and mononuclear cells. Our results give an overall picture of
serpin and protease gene expression and draws attention to the potential
importance of elastase regulators at all stages of myelopoiesis. Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis.
Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for
most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of
granulopoiesis. However, the mechanisms by which these mutations disrupt
granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the
production of misfolded NE protein, activation of the unfolded protein response
(UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant
NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA
splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a
specific ELA2 mutation correlated with its associated clinical phenotype.
Consistent with the UPR model, expression of mutant NE in primary human
granulocytic precursors increased expression of CHOP (DDITS) and induced
apoptosis in a protease-independent fashion. Most strikingly, UPR activation and
decreased NE protein expression were detected in primary granulocytic precursors
from SCN patients. Collectively, these data provide strong support for a UPR
model of SCN disease pathogenesis and place SCN in a growing list of human
diseases caused by misfolded proteins. PURPOSE OF REVIEW: Severe congenital neutropenia has been a well known
hematological condition for over 50 years. Over this long period of time, the
variable genetic causes and associated sequelae of the disease have been
ascertained, and successful treatment strategies developed. Over the past 2
years, however, new studies have added greatly to our understanding of the
molecular basis of the disease, details of which are presented in this review.
RECENT FINDINGS: Recent studies have elucidated a role for the unfolded protein
response in mediating the pathogenic effects of ELA2 mutations, the most common
mutation in severe congenital neutropenia (SCN) as well as cyclic neutropenia.
Genetic lesions in HAX1 have also been identified in the original Kostmann
pedigree representing the autosomal recessive form of SCN. An emerging theme is
the convergence of these and other genetic lesions underlying SCN in enhancing
neutrophil apoptosis. Other studies have revealed the importance of multiple
independent mutations in these and other genes in SCN. Finally, the key role for
signal transducer and activator of transcription 5 in mediating the effects of
granulocyte colony-stimulating factor receptor truncation mutations in the
development of myelodysplastic syndrome/acute myeloid leukemia following SCN has
been elucidated.
SUMMARY: As the full spectrum of molecular mutations causing neutropenia
emerges, it is becoming possible to differentiate patients into subtypes with
different prognoses, for whom tailored therapies are indicated. PURPOSE: Three familial cases of each of severe congenital neutropenia (SCN) and
cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for
neutrophil elastase (Ela2) gene mutation. The contents of the
neutrophil-specific granule proteins cathelicidin antimicrobial peptide and
neutrophil gelatinase-associated lipocalin were also analyzed in SCN.
METHODS: Genomic DNA was extracted from the patients' peripheral blood or bone
marrow, and the coding sequence of the Ela2 gene was amplified by polymerase
chain reaction and subjected to direct sequencing. The contents of antimicrobial
peptides were analyzed by flow cytometry.
RESULTS: Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN
(W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have
heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case
was a novel mutation of Ela2. Prophylactic treatment for growth factors or
antibiotic prophylaxis against bacterial infection was useful for lowering the
frequency of infectious episodes. Adult patients tended to have less frequent
infections compared with minors in the same family. The contents of both
cathelicidin antimicrobial peptide and neutrophil gelatinase-associated
lipocalin were significantly reduced in SCN compared with healthy controls.
CONCLUSIONS: Prophylaxis by growth factor or antibiotics is useful for
decreasing risks of bacterial infections in SCN and CN. Adults were likely to
have less frequent infections than children in familial cases of SCN and CN with
the same mutation of Ela2. BACKGROUND: Cyclic neutropenia (CN) and severe congenital neutropenia (SCN) are
disorders of neutrophil production that differ markedly in disease severity.
Mutations of the ELANE gene (the symbol recently replacing ELA2) are considered
largely responsible for most cases of CN and SCN, but specific mutations are
typically associated with one or the other.
PROCEDURE: We performed ELANE genotyping on all individuals and paternal sperm
in an SCN kindred with eight SCN progeny of a sperm donor and six different
mothers.
RESULTS: One patient with CN had the same S97L ELANE mutation as seven patients
with the SCN phenotype. The mutant allele was detected in the donor's
spermatozoa, representing 18% of the ELANE gene pool, but not in DNA from his
lymphocytes, neutrophils, or buccal mucosa, indicating gonadal mosaicism.
CONCLUSIONS: The coexistence of CN and SCN phenotypes in this kindred with a
shared paternal haplotype strongly suggests both a role for modifying genes in
determination of congenital neutropenia disease phenotypes, and the
classification of CN and SCN within a spectrum of phenotypes expressing varying
degrees of the same disease process. Neutrophil elastase gene (ELANE) mutations are responsible for the majority of
cases of severe congenital neutropenia (CN) and cyclic neutropenia (CyN). We
screened CN (n = 395) or CyN (n = 92) patients for ELANE mutations and
investigated the impact of mutations on mRNA expression, protein expression, and
activity. We found 116 different mutations in 162 (41%) CN patients and 26 in 51
(55%) CyN patients, 69 of them were novel. CyN-associated mutations were
predicted to be more benign than CN-associated mutations, but the mutation
severity largely overlapped. The frequency of acquired CSF3R mutations,
maligt transformation, and the need for hematopoietic stem cell
transplantation was significantly higher in CN patients with ELANE mutation than
in ELANE mutation negative patients. Cellular elastase activity was reduced in
neutrophils from CN/CyN patients, irrespective of the mutation status. In CN,
enzymatic activity was significantly lower in patients with ELANE mutations
compared with those with wild-type ELANE. Despite differences in the spectrum of
mutations in CN or CyN, type or localization of mutation only partially
determine the clinical phenotype. Specific ELANE mutations have limited
predictive value for leukemogenesis; the risk for leukemia was correlated with
disease severity rather than with occurrence of an ELANE mutation. |
what is the role of TGFbeta in cardiac regeneration after myocardial injury? | TGFβ is activated in the myocardium in response to injury and plays a crucial role in cardiac repair by suppressing inflammation while promoting myofibroblast phenotypic modulation and extracellular matrix deposition. In fact, upregulation of TGF-beta signaling promotes the formation of a myofibroblast-like phenotype. TGF-beta interacts with bone morphogenic protein and Wnt pathways to form a complex signaling network that is critical in regulating the fate choices of both stromal and tissue-specific resident stem cells, determining whether functional regeneration or the formation of scar tissue follows an injury. In addition, TGF-beta enhances the formation of cardiospheres and could potentially enhance the regenerative potential of adult cardiac progenitor cells. | Myocardial infarction is the most common cause of cardiac injury and results in
acute loss of a large number of myocardial cells. Because the heart has
negligible regenerative capacity, cardiomyocyte death triggers a reparative
response that ultimately results in formation of a scar and is associated with
dilative remodeling of the ventricle. Cardiac injury activates innate immune
mechanisms initiating an inflammatory reaction. Toll-like receptor-mediated
pathways, the complement cascade and reactive oxygen generation induce nuclear
factor (NF)-kappaB activation and upregulate chemokine and cytokine synthesis in
the infarcted heart. Chemokines stimulate the chemotactic recruitment of
inflammatory leukocytes into the infarct, while cytokines promote adhesive
interactions between leukocytes and endothelial cells, resulting in
transmigration of inflammatory cells into the site of injury. Monocyte subsets
play distinct roles in phagocytosis of dead cardiomyocytes and in granulation
tissue formation through the release of growth factors. Clearance of dead cells
and matrix debris may be essential for resolution of inflammation and transition
into the reparative phase. Transforming growth factor (TGF)-beta plays a crucial
role in cardiac repair by suppressing inflammation while promoting myofibroblast
phenotypic modulation and extracellular matrix deposition. Myofibroblast
proliferation and angiogenesis result in formation of highly vascularized
granulation tissue. As the healing infarct matures, fibroblasts become apoptotic
and a collagen-based matrix is formed, while many infarct neovessels acquire a
muscular coat and uncoated vessels regress. Timely resolution of the
inflammatory infiltrate and spatial containment of the inflammatory and
reparative response into the infarcted area are essential for optimal infarct
healing. Targeting inflammatory pathways following infarction may reduce
cardiomyocyte injury and attenuate adverse remodeling. In addition,
understanding the role of the immune system in cardiac repair is necessary in
order to design optimal strategies for cardiac regeneration. The regulation of valve interstitial cell (VIC) function in response to tissue
injury and valve disease is not well understood. Because transforming growth
factor-beta (TGF-beta) has been implicated in tissue repair, we tested the
hypothesis that TGF-beta is a regulator of VIC activation and associated cell
responses that occur during early repair processes. We used a well-characterized
wound model that was created by mechanical denudation of a confluent VIC
monolayer to study activation and repair 24 hours after wounding. VIC activation
was demonstrated by immunofluorescent localization of alpha-smooth muscle actin
(alpha-SMA), and alpha-SMA mRNA levels were quantified by real-time polymerase
chain reaction. Proliferation and apoptosis were quantified by bromodeoxyuridine
staining and terminal deoxynucleotidyl transferase dUTP nick end labeling,
respectively. Repair was quantified by measuring VIC extension into the wound,
and TGF-beta expression was shown by immunofluorescent localization of
intracellular TGF-beta. Compared with nonwounded monolayers, VICs at the wound
edge showed alpha-SMA staining, increased alpha-SMA mRNA content, elongation
into the wound with stress fibers, proliferation, and apoptosis. VICs at the
wound edge also showed increased TGF-beta and pSmad2/3 staining with
co-expression of alpha-SMA. Addition of TGF-beta neutralizing antibody to the
wound decreased VIC activation, alpha-SMA mRNA content, proliferation,
apoptosis, wound closure rate, and stress fibers. Conversely, exogenous addition
of TGF-beta to the wound increased VIC activation, proliferation, wound closure
rate, and stress fibers. Thus, wounding activates VICs, and TGF-beta signaling
modulates VIC response to injury. Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps)
represent a promising candidate for cardiac regenerative therapy. A better
understanding of the origin and mechanisms underlying human CSps formation and
maturation is undoubtedly required to enhance their cardiomyogenic potential.
Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that
is implicated in the acquisition of stem cell-like properties in different adult
tissues, and it is activated in the epicardium after ischemic injury to the
heart. We investigated whether EMT is involved in the formation and
differentiation of human CSps, revealing that an up-regulation of the expression
of EMT-related genes accompanies CSps formation that is relative to primary
explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps
formation is enhanced in the presence of transforming growth factor β1 (TGFβ1)
and drastically blocked by the type I TGFβ-receptor inhibitor SB431452,
indicating that TGFβ-dependent EMT is essential for the formation of these
niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium
in response to injury, our data suggest that CSps formation mimics an adaptive
mechanism that could potentially be enhanced to increase in vivo or ex vivo
regenerative potential of adult CPCs. Fibroblasts are the predomit cell type in the cardiac interstitium. As the
main matrix-producing cells in the adult mammalian heart, fibroblasts maintain
the integrity of the extracellular matrix network, thus preserving geometry and
function. Following myocardial infarction fibroblasts undergo dynamic phenotypic
alterations and direct the reparative response. Due to their strategic location,
cardiac fibroblasts serve as sentinel cells that sense injury and activate the
inflammasome secreting cytokines and chemokines. During the proliferative phase
of healing, infarct fibroblasts undergo myofibroblast transdifferentiation
forming stress fibers and expressing contractile proteins (such as α-smooth
muscle actin). Mechanical stress, transforming growth factor (TGF)-β/Smad3
signaling and alterations in the composition of the extracellular matrix induce
acquisition of the myofibroblast phenotype. In the highly cellular and growth
factor-rich environment of the infarct, activated myofibroblasts produce matrix
proteins, proteases and their inhibitors regulating matrix metabolism. As the
infarct matures, "stress-shielding" of myofibroblasts by the cross-linked matrix
and growth factor withdrawal may induce quiescence and ultimately cause
apoptotic death. Because of their critical role in post-infarction cardiac
remodeling, fibroblasts are promising therapeutic targets following myocardial
infarction. However, the complexity of fibroblast functions and the
pathophysiologic heterogeneity of post-infarction remodeling in the clinical
context discourage oversimplified approaches in clinical translation. This
article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac
Pathways of Differentiation, Metabolism and Contraction. Adult stem cells are activated to proliferate and differentiate during normal
tissue homeostasis as well as in disease states and injury. This activation is a
vital component in the restoration of function to damaged tissue via either
complete or partial regeneration. When regeneration does not fully occur,
reparative processes involving an overproduction of stromal components ensure
the continuity of tissue at the expense of its normal structure and function,
resulting in a "reparative disorder". Adult stem cells from multiple organs have
been identified as being involved in this process and their role in tissue
repair is being investigated. Evidence for the participation of mesenchymal
stromal cells (MSCs) in the tissue repair process across multiple tissues is
overwhelming and their role in reparative disorders is clearly demonstrated, as
is the involvement of a number of specific signaling pathways. Transforming
growth factor beta, bone morphogenic protein and Wnt pathways interact to form a
complex signaling network that is critical in regulating the fate choices of
both stromal and tissue-specific resident stem cells (TSCs), determining whether
functional regeneration or the formation of scar tissue follows an injury. A
growing understanding of both TSCs, MSCs and the complex cascade of signals
regulating both cell populations have, therefore, emerged as potential
therapeutic targets to treat reparative disorders. This review focuses on recent
advances on the role of these cells in skeletal muscle, heart and lung tissues. |
List two chemotherapeutic agents that are used for treatment of Subependymal Giant Cell Astrocytoma | Everolimus and rapamycin are chemotherapeutic agents that are used for treatment of Subependymal Giant Cell Astrocytoma. | The authors present a 21-year-old woman who has been receiving rapamycin for 5
months for bilateral subependymal giant cell astrocytomas. The patient was
started at a dose of 0.2 mg/kg/day. Levels were maintained between 11 and 13
ng/mL. Magnetic resoce imaging of the brain 2(1/2) months after initiating
rapamycin demonstrated a decrease in size of both astrocytomas (11 to 7.5 mm on
the right and 8 to 5 mm on the left). Further studies are needed with prolonged
observation to confirm these findings, determine the length of necessary
treatment, and evaluate recurrence risk after discontinuation of rapamycin. BACKGROUND: Neurosurgical resection is the standard treatment for subependymal
giant-cell astrocytomas in patients with the tuberous sclerosis complex. An
alternative may be the use of everolimus, which inhibits the mammalian target of
rapamycin, a protein regulated by gene products involved in the tuberous
sclerosis complex.
METHODS: Patients 3 years of age or older with serial growth of subependymal
giant-cell astrocytomas were eligible for this open-label study. The primary
efficacy end point was the change in volume of subependymal giant-cell
astrocytomas between baseline and 6 months. We gave everolimus orally, at a dose
of 3.0 mg per square meter of body-surface area, to achieve a trough
concentration of 5 to 15 ng per milliliter.
RESULTS: We enrolled 28 patients. Everolimus therapy was associated with a
clinically meaningful reduction in volume of the primary subependymal giant-cell
astrocytoma, as assessed on independent central review (P<0.001 for baseline vs.
6 months), with a reduction of at least 30% in 21 patients (75%) and at least
50% in 9 patients (32%). Marked reductions were seen within 3 months and were
sustained. There were no new lesions, worsening hydrocephalus, evidence of
increased intracranial pressure, or necessity for surgical resection or other
therapy for subependymal giant-cell astrocytoma. Of the 16 patients for whom
24-hour video electroencephalography data were available, seizure frequency for
the 6-month study period (vs. the previous 6-month period) decreased in 9, did
not change in 6, and increased in 1 (median change, -1 seizure; P=0.02). The
mean (±SD) score on the validated Quality-of-Life in Childhood Epilepsy
questionnaire (on which scores can range from 0 to 100, with higher scores
indicating a better quality of life) was improved at 3 months (63.4±12.4) and 6
months (62.1±14.2) over the baseline score (57.8±14.0). Single cases of grade 3
treatment-related sinusitis, pneumonia, viral bronchitis, tooth infection,
stomatitis, and leukopenia were reported.
CONCLUSIONS: Everolimus therapy was associated with marked reduction in the
volume of subependymal giant-cell astrocytomas and seizure frequency and may be
a potential alternative to neurosurgical resection in some cases, though
long-term studies are needed. (Funded by Novartis; ClinicalTrials.gov number,
NCT00411619.). INTRODUCTION: Better understanding of aberrantly active molecular pathways in
tumors offers potential to develop more specific and less toxic therapies.
Abnormal mammalian target of rapamycin (mTOR) complex signaling and defects in
TSC1 and TSC2 have been associated with the development of subependymal giant
cell astrocytomas (SEGAs) in tuberous sclerosis complex (TSC) patients.
Recently, mTOR inhibitors such as everolimus have shown encouraging benefit for
patients with SEGAs.
AREAS COVERED: The authors discuss a molecular genetic pathway linked with TSC,
specifically the role of two proteins whose functional absence is responsible
for most SEGA tumors that arise in TSC patients. The authors also examine the
rationale for targeted agents against this pathway therapeutically and describe
the clinical evidence underlying the FDA approval of everolimus for patients
with inoperable SEGAs.
EXPERT OPINION: Everolimus (Afinitor) selectively targets a molecular defect of
SEGAs in TSC patients. Although surgery is effective, most SEGAs recur. An agent
that inhibits an underlying molecular abnormality represents a particularly
attractive therapeutic option for patients with inoperable or recurrent tumors.
Studies are also underway to assess everolimus in treating other sequelae of
TSC, and other gliomas. Finally, additional research aimed at better
understanding aberrant cell signaling pathways may lead to the development of
more effective therapeutics. BACKGROUND: Tuberous sclerosis complex is a genetic disorder leading to
constitutive activation of mammalian target of rapamycin (mTOR) and growth of
benign tumours in several organs. In the brain, growth of subependymal giant
cell astrocytomas can cause life-threatening symptoms--eg, hydrocephalus,
requiring surgery. In an open-label, phase 1/2 study, the mTOR inhibitor
everolimus substantially and significantly reduced the volume of subependymal
giant cell astrocytomas. We assessed the efficacy and safety of everolimus in
patients with subependymal giant cell astrocytomas associated with tuberous
sclerosis complex.
METHODS: In this double-blind, placebo-controlled, phase 3 trial, patients (aged
0-65 years) in 24 centres in Australia, Belgium, Canada, Germany, the UK, Italy,
the Netherlands, Poland, Russian Federation, and the USA were randomly assigned,
with an interactive internet-response system, in a 2:1 ratio to oral everolimus
4·5 mg/m(2) per day (titrated to achieve blood trough concentrations of 5-15
ng/mL) or placebo. Eligible patients had a definite diagnosis of tuberous
sclerosis complex and at least one lesion with a diameter of 1 cm or greater,
and either serial growth of a subependymal giant cell astrocytoma, a new lesion
of 1 cm or greater, or new or worsening hydrocephalus. The primary endpoint was
the proportion of patients with confirmed response--ie, reduction in target
volume of 50% or greater relative to baseline in subependymal giant cell
astrocytomas. Analysis was by intention to treat. This study is registered with
ClinicalTrials.gov, number NCT00789828.
FINDINGS: 117 patients were randomly assigned to everolimus (n=78) or placebo
(n=39). 27 (35%) patients in the everolimus group had at least 50% reduction in
the volume of subependymal giant cell astrocytomas versus none in the placebo
group (difference 35%, 95% CI 15-52; one-sided exact Cochran-Mantel-Haenszel
test, p<0·0001). Adverse events were mostly grade 1 or 2; no patients
discontinued treatment because of adverse events. The most common adverse events
were mouth ulceration (25 [32%] in the everolimus group vs two [5%] in the
placebo group), stomatitis (24 [31%] vs eight [21%]), convulsion (18 [23%] vs
ten [26%]), and pyrexia (17 [22%] vs six [15%]).
INTERPRETATION: These results support the use of everolimus for subependymal
giant cell astrocytomas associated with tuberous sclerosis. Additionally,
everolimus might represent a disease-modifying treatment for other aspects of
tuberous sclerosis.
FUNDING: Novartis Pharmaceuticals. Everolimus (RAD001), a mTOR inhibitor, was initially used as an
immunosuppressant in organ transplant patients; however, it also has significant
antineoplastic properties. In patients with subependymal giant cell astrocytomas
(SEGAs) associated with tuberous sclerosis complex who are not candidates for
surgery, single-agent everolimus has demonstrated the ability to significantly
reduce SEGA volume with good tolerability. In the Phase III, randomized,
placebo-controlled trial, everolimus was associated with a SEGA response rate of
35% compared with 0% in the placebo group. The most common adverse events in
clinical trials were stomatitis/mouth ulceration and upper respiratory tract
infections, and most adverse events were grade 1 or 2; grade 4 events were rare. Tuberous sclerosis complex (TSC) is an autosomal domit genetic disorder
caused by inactivating mutations in either the TSC1 or TSC2 genes. It is
characterized by the development of multiple, benign tumors in several organs
throughout the body. Lesions occur in the brain, kidneys, heart, liver, lungs,
and skin and result in seizures and epilepsy, mental retardation, autism, and
renal and pulmonary organ system dysfunction, as well as other complications.
Elucidation of the molecular pathways and etiological factors responsible for
causing TSC has led to a paradigm shift in the management and treatment of the
disease. TSC1 or TSC2 mutations lead to constitutive upregulation of the
mammalian target of rapamycin pathway, which affects many cellular processes
involved in tumor growth. By targeting mammalian target of rapamycin with
everolimus, an orally active rapamycin derivative, clinically meaningful and
statistically significant reductions in tumor burden have been achieved for the
main brain (subependymal giant cell astrocytoma) and renal manifestations
(angiomyolipoma) associated with TSC. This review provides an overview of TSC,
everolimus, and the clinical trials that led to its approval for the treatment
of TSC-associated subependymal giant cell astrocytoma and renal angiomyolipoma. BACKGROUND: Brain subependymal giant cell astrocytomas (SEGAs) in patients with
tuberous sclerosis have been reported to respond to everolimus.
METHODS: A 15-year-old male patient with intractable seizures and multiple SEGAs
of the brain developed leptomeningeal enhancement and multiple metastatic,
histologically confirmed SEGAs of the spinal cord. He received daily everolimus
at a dose of 3 mg/m for 6 weeks, which was then increased to 6 mg/m.
RESULTS: Magnetic resoce image of the brain and spine showed significant
reduction in the size of SEGAs after 6 weeks of treatment. The patient has
remained free of progression for 24 months. Additional benefits included:
excellent seizure control, decrease in the size of cardiac rhabdomyomas, and
improved quality of life.
CONCLUSIONS: We describe a rare case of metastatic SEGA, which was successfully
treated with everolimus. We are reporting on a 13.5-year-old girl with tuberous sclerosis complex (TSC)
who was treated with everolimus because of giant cell astrocytoma and bilateral
angiomyolipoma. She suffered from pharmacoresistant partial epilepsy with
clusters of tonic and tonic-clonic seizures. Treatment with carbamazepine and
sulthiame had led to a stable situation for more than 2.5 years. The dosage of
everolimus had to be increased and refractory status epilepticus followed after
12 days. In the absence of any other possible cause, we believe that the status
epilepticus was provoked by everolimus. So far, only a few cases of possible
seizure aggravation by everolimus have been reported. The clinical relevance of
possible negative effects in epileptic patients remains unclear. Similar
observations should be documented and reported. OBJECTIVES: To evaluate the efficacy and side effects of oral mammalian target
of rapamycin (mTOR) inhibitors in children and adolescents with tuberous
sclerosis complex (TSC) and intractable epilepsy or subependymal giant cell
astrocytoma (SEGA).
STUDY DESIGN: Single-center series of 13 children and adolescents with TSC who
received sirolimus or everolimus (mTOR inhibitors). The anticonvulsant response
was evaluated in 7 patients with TSC and refractory seizures. Six patients with
SEGAs were treated with either sirolimus or everolimus for nonsurgical
management. SEGA volumes were assessed longitudinally using 1.5-T magnetic
resoce imaging.
RESULTS: Of the intractable seizure group (7 patients), 1 patient had >90%
reduction, 4 had 50%-90% reduction, and 2 had <50% reduction. Three reported
subjective improvements in learning. By 12 months of treatment, there were
statistically significant reductions in the SEGA volumes in 4 patients who
received mTOR inhibitors (P < .04). The mean SEGA volume after 6 months of
treatment was 2.18 cm(3), which represents 33% reduction in the mean baseline
volume of 3.26 cm(3). The mTOR inhibitors were well tolerated. Adverse effects
include dyslipidaemia (3 of 13), gingivitis (1 of 13), anorexia (1 of 13), and
mild gastrointestinal side effects (1 of 13).
CONCLUSION: This case series suggests that mTOR inhibitors can improve seizures
in those with TSC and refractory epilepsy. They are also an effective treatment
for reducing the volume of SEGAs in patients with TSC not amenable to surgery
with an acceptable side effect profile. BACKGROUND: Everolimus, an orally administered rapamycin analogue, inhibits the
mammalian target of rapamycin (mTOR), a highly conserved intracellular
serine-threonine kinase that is a central node in a network of signaling
pathways controlling cellular metabolism, growth, survival, proliferation,
angiogenesis, and immune function. Everolimus has demonstrated substantial
clinical benefit in randomized, controlled, phase III studies leading to
approval for the treatment of advanced renal cell carcinoma, advanced
neuroendocrine tumors of pancreatic origin, renal angiomyolipoma and
subependymal giant-cell astrocytoma associated with tuberous sclerosis complex,
as well as advanced hormone-receptor-positive (HR(+)) and human epidermal growth
factor receptor-2-negative advanced breast cancer.
MATERIALS AND METHODS: We discuss clinically relevant everolimus-related adverse
events from the phase III studies, including stomatitis, noninfectious
pneumonitis, rash, selected metabolic abnormalities, and infections, with focus
on appropriate clinical management of these events and specific considerations
in patients with breast cancer.
RESULTS: The majority of adverse events experienced during everolimus therapy
are of mild to moderate severity. The safety profile and protocols for toxicity
management are well established. The class-effect adverse event profile observed
with everolimus plus endocrine therapy in breast cancer is (as expected)
distinct from that of endocrine therapy alone, but is similar to that observed
with everolimus in other solid tumors. Information gained from the experience in
other carcinomas on prompt diagnosis and treatments to optimize drug exposure,
treatment outcomes, and patients' quality of life also applies to the patient
population with advanced breast cancer.
CONCLUSIONS: As with all orally administered agents, education of both
physicians and patients in the management of adverse events for patients
receiving everolimus is critical to achieving optimal exposure and clinical
benefit. Active monitoring for early identification of everolimus-related
adverse events combined with aggressive and appropriate intervention should lead
to a reduction in the severity and duration of the event. BACKGROUND: Tuberous sclerosis complex (TSC) is characterized by benign tumours
in multiple organs, including the brain, kidneys, skin, lungs and heart. Our
objective was to evaluate everolimus, an mTOR inhibitor, in the treatment of
angiomyolipoma in patients with subependymal giant cell astrocytoma (SEGA)
associated with TSC.
METHODS: EXamining everolimus In a Study of Tuberous Sclerosis Complex-1
(NCT00789828), a prospective, double-blind, randomized, placebo-controlled,
Phase 3 study, examined everolimus in treating SEGA associated with TSC.
Patients with serial SEGA growth from pre-baseline to baseline scans were
randomly assigned (2:1) to receive 4.5 mg/m(2)/day everolimus (target blood
trough: 5-15 ng/mL; n = 78) or placebo (n = 39). Angiomyolipoma response rates
were analysed in patients (n = 44) with target baseline angiomyolipoma lesions
(≥1 angiomyolipoma; longest diameter ≥1.0 cm). An angiomyolipoma response rate,
defined as the proportion of patients with confirmed angiomyolipoma response,
was assessed by kidney CT or MRI screening at baseline, at 12, 24 and 48 weeks
and annually.
RESULTS: Angiomyolipoma response rates were 53.3% (16/30) and 0% (0/14) for
everolimus- and placebo-treated patients, respectively. Angiomyolipoma
reductions ≥50% in the sum of volumes of all target lesions were seen only in
everolimus-treated patients (56.5, 78.3 and 80.0%) compared with placebo-treated
patients (0% at each time point) at Weeks 12, 24 and 48, respectively. Greater
percentages of everolimus-treated patients had angiomyolipoma reductions ≥30% at
these same time points (82.6, 100 and 100% versus 8.3, 18.2 and 16.7% for
everolimus versus placebo, respectively).
CONCLUSIONS: Everolimus showed efficacy in reducing angiomyolipoma lesion volume
in patients with SEGA associated with TSC.The trial is registered with
ClinicalTrials.gov, number NCT00789828;
http://clinicaltrials.gov/ct2/show/NCT00789828?term=EXIST-1&rank=1. |
Are integrins part of the extracellular matrix? | Yes, integrins are a central family of extracellular matrix receptors. | Mechanosensitive ion channels (MSCs) have long been the only established
molecular class of cell mechanosensors; however, in the last decade, a variety
of non-channel type mechanosensor molecules have been identified. Many of them
are focal adhesion-associated proteins that include integrin, talin, and actin.
Mechanosensors must be non-soluble molecules firmly interacting with relatively
rigid cellular structures such as membranes (in terms of lateral stiffness),
cytoskeletons, and adhesion structures. The partner of MSCs is the membrane in
which MSC proteins efficiently transduce changes in the membrane tension into
conformational changes that lead to channel opening. By contrast, the integrin,
talin, and actin filament form a linear complex of which both ends are typically
anchored to the extracellular matrices via integrins. Upon cell deformation by
forces, this structure turns out to be a portion that efficiently transduces the
generated stress into conformational changes of composite molecules, leading to
the activation of integrin (catch bond with extracellular matrices) and talin
(unfolding to induce vinculin bindings). Importantly, this structure also serves
as an "active" mechanosensor to detect substrate rigidity by pulling the
substrate with contraction of actin stress fibers (SFs), which may induce talin
unfolding and an activation of MSCs in the vicinity of integrins. A recent study
demonstrates that the actin filament acts as a mechanosensor with unique
characteristics; the filament behaves as a negative tension sensor in which
increased torsional fluctuations by tension decrease accelerate ADF/cofilin
binding, leading to filament disruption. Here, we review the latest progress in
the study of those non-channel mechanosensors and discuss their activation
mechanisms and physiological roles. We have previously reported a unique response of traction force generation for
cells grown on mature cardiac ECM, where traction force was constant over a
range of stiffnesses. In this study we sought to further investigate the role of
the complex mixture of ECM on this response and assess the potential mechanism
behind it. Using traction force microscopy, we measured cellular traction forces
and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a
range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM,
different singular ECM proteins including collagen I, fibronectin, and laminin,
or ECM mimics comprised of varying amounts of collagen I, fibronectin, and
laminin. We also measured the expression of integrins on these different
substrates as well as probed for β1 integrin binding. There was no significant
change in traction force generation for cells grown on the adult ECM, as
previously reported, whereas cells grown on singular ECM protein substrates had
increased traction force generation with an increase in substrate stiffness.
Cells grown on ECM mimics containing collagen I, fibronectin and laminin were
found to be reminiscent of the traction forces generated by cells grown on
native ECM. Integrin expression generally increased with increasing stiffness
except for the β1 integrin, potentially implicating it as playing a role in the
response to adult cardiac ECM. We inhibited binding through the β1 integrin on
cells grown on the adult ECM and found that the inhibition of β1 binding led to
a return to the typical response of increasing traction force generation with
increasing stiffness. Our data demonstrates that cells grown on the mature
cardiac ECM are able to circumvent typical stiffness related cellular behaviors,
likely through β1 integrin binding to the complex composition. Integrin receptors connect the extracellular matrix to the cell cytoskeleton to
provide essential forces and signals. To examine the contributions of the β1
integrin cytoplasmic tail to adhesive forces, we generated cell lines expressing
wild-type and tail mutant β1 integrins in β1-null fibroblasts. Deletion of β1
significantly reduced cell spreading, focal adhesion assembly, and adhesive
forces, and expression of human β1 (hβ1) integrin in these cells restored
adhesive functions. Cells expressing a truncated tail mutant had impaired
spreading, fewer and smaller focal adhesions, reduced integrin binding to
fibronectin, and lower adhesion strength and traction forces compared to
hβ1-expressing cells. All these metrics were equivalent to those for β1-null
cells, demonstrating that the β1 tail is essential to these adhesive functions.
Expression of the constitutively-active D759A hβ1 mutant restored many of these
adhesive functions in β1-null cells, although with important differences when
compared to wild-type β1. Even though there were no differences in
integrin-fibronectin binding and adhesion strength between hβ1- and
hβ1-D759A-expressing cells, hβ1-D759A-expressing cells assembled more but
smaller adhesions than hβ1-expressing cells. Importantly, hβ1-D759A-expressing
cells generated lower traction forces compared to hβ1-expressing cells. These
differences between hβ1- and hβ1-D759A-expressing cells suggest that regulation
of integrin activation is important for fine-tuning cell spreading, focal
adhesion assembly, and traction force generation. Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determit of
spindle orientation. However, the signaling pathways that couple integrins to
spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1),
a member of the cyclin-dependent kinases (CDKs) whose function is poorly
characterized, plays an essential role in this process. PCTK1 regulates spindle
orientation in a kinase-dependent manner. Phosphoproteomic analysis together
with an RNA interference screen revealed that PCTK1 regulates spindle
orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of
protein kinase A (PKA). This phosphorylation is dispensable for KAP0
dimerization and for PKA binding but is necessary for its interaction with
myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of
myosin X and enhances the association of myosin X-FERM with β1 integrin. This
interaction between myosin X-FERM and β1 integrin appeared to be crucial for
spindle orientation control. We propose that PCTK1-KAP0-myosin X-β1 integrin is
a functional module providing a link between ECM and the actin cytoskeleton in
the ECM-dependent control of spindle orientation. Breast cancer is the second leading cause of maligt death among women. A
crucial feature of metastatic cancers is their propensity to lose adhesion to
the underlying basement membrane as they transition to a motile phenotype and
invade surrounding tissue. Attachment to the extracellular matrix is mediated by
a complex of adhesion proteins, including integrins, signaling molecules, actin
and actin-binding proteins, and scaffolding proteins. Focal adhesion kinase
(FAK) is pivotal for the organization of focal contacts and maturation into
focal adhesions, and disruption of this process is a hallmark of early cancer
invasive potential. Our recent work has revealed that myoferlin (MYOF) mediates
breast tumor cell motility and invasive phenotype. In this study we demonstrate
that noninvasive breast cancer cell lines exhibit increased cell-substrate
adhesion and that silencing of MYOF using RNAi in the highly invasive human
breast cancer cell line MDA-MB-231 also enhances cell-substrate adhesion. In
addition, we detected elevated tyrosine phosphorylation of FAK (FAK(Y397)) and
paxillin (PAX(Y118)), markers of focal adhesion protein activation. Morphometric
analysis of PAX expression revealed that RNAi-mediated depletion of MYOF
resulted in larger, more elongated focal adhesions, in contrast to cells
transduced with a control virus (MDA-231(LVC) cells), which exhibited smaller
focal contacts. Finally, MYOF silencing in MDA-MB-231 cells exhibited a more
elaborate ventral cytoskeletal structure near focal adhesions, typified by
pronounced actin stress fibers. These data support the hypothesis that MYOF
regulates cell adhesions and cell-substrate adhesion strength and may account
for the high degree of motility in invasive breast cancer cells. The extracellular matrix component periostin is a secreted protein that
functions as both a cell attachment protein and an autocrine or paracrine factor
that signals through the cell adhesion molecule integrins αvβ3 and αvβ5.
Periostin participates in normal physiological activities such as cardiac
development, but is also involved in pathophysiological processes in vascular
diseases, wound repair, bone formation, and tumor development. It is of
increasing interest in tumor biology because it is frequently overexpressed in a
variety of epithelial carcinomas and is functionally involved in multiple steps
of metastasis progression. These include the maintece of stemness, niche
formation, EMT, the survival of tumor cells, and angiogenesis, all of which are
indispensable for gastric cancer metastasis. Periostin has been reported to
activate the PI-3K/AKT, Wnt, and FAK-mediated signaling pathways to promote
metastasis. Therefore, periostin represents a potentially promising candidate
for the inhibition of metastasis. In this review article, we summarize recent
advances in knowledge concerning periostin, its antagonist PNDA-3, and their
influence on such key processes in cancer metastasis as maintece of stemness,
niche formation, epithelial-to-mesenchymal transition, tumor cell survival, and
angiogenesis. In particular, we focus our attention on the role of periostin in
gastric cancer metastasis, speculate as to the usefulness of periostin as a
therapeutic and diagnostic target for gastric cancer metastasis, and consider
potential avenues for future research. BACKGROUND: The growth properties and self-renewal capacity of embryonic stem
(ES) cells are regulated by their immediate microenvironment such as the
extracellular matrix (ECM). Integrins, a central family of cellular ECM
receptors, have been implicated in these processes but their specific role in ES
cell self-renewal remains unclear.
RESULTS: Here we have studied the effects of different ECM substrates and
integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF)
using short-term assays as well as long-term cultures. Removal of LIF from ES
cell culture medium induced morphological differentiation of ES cells into
polarized epistem cell-like cells. These cells maintained epithelial morphology
and expression of key stemness markers for at least 10 passages in the absence
of LIF when cultured on laminin, fibronectin or collagen IV substrates. The
specific functional roles of α6-, αV- and β1-integrin subunits were dissected
using stable lentivirus-mediated RNAi methodology. β1-integrins were required
for ES cell survival in long-term cultures and for the maintece of stem cell
marker expression. Inhibition of α6-integrin expression compromised self-renewal
on collagen while αV-integrins were required for robust ES cell adhesion on
laminin. Analysis of the stemness marker expression revealed subtle differences
between α6- and αV-depleted ES cells but the expression of both was required for
optimal self-renewal in long-term ES cell cultures.
CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem
cell-like epithelial phenotype and retain the expression of multiple stem cell
markers. Long-term maintece of such self-renewing cultures depends on the
expression of β1-, α6- and αV-integrins. During development, growth factors (GFs) such as bone morphogenetic proteins
(BMPs) exert important functions in several tissues by regulating signaling for
cell differentiation and migration. In vivo, the extracellular matrix (ECM) not
only provides support for adherent cells, but also acts as reservoir of GFs.
Several constituents of the ECM provide adhesive cues, which serve as binding
sites for cell trans-membrane receptors, such as integrins. In conveying
adhesion-mediated signaling to the intracellular compartment, integrins do not
function alone but rather crosstalk and cooperate with other receptors, such as
GF receptors. Here, we present a strategy for the immobilization of BMP-2 onto
cellular fibronectin (cFN), a key protein of the ECM, to investigate GF-mediated
signaling and migration. Following biotinylation, BMP-2 was linked to
biotinylated cFN using NeutrAvidin as cross-linker. Characterization with quartz
crystal microbalance with dissipation monitoring and enzyme-linked immunosorbent
assay confirmed the efficient immobilization of BMP-2 on cFN over a period of
24 h. To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2), we
investigated short- and long-term responses of C2C12 myoblasts, which are an
established in vitro model for BMP-2 signaling, in comparison to soluble BMP-2
(sBMP-2) or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5
phosphorylation and translocation of the complex to the nucleus, corresponding
to the activation of BMP-mediated Smad-dependent pathway. Additionally,
successful suppression of myotube formation was observed after 6 days in sBMP-2
and iBMP-2. We next implemented this approach in the fabrication of cFN
micropatterned stripes by soft lithography. These stripes allowed cell-surface
interaction only on the patterned cFN, since the surface in between was
passivated, thus serving as platform for studies on directed cell migration.
During a 10-h observation time, the migratory behavior, especially the cells'
net displacement, was increased in presence of BMP-2. As such, this versatile
tool retains the bioactivity of GFs and allows the presentation of ECM adhesive
cues. The aim of the present review is to survey the accumulated knowledge on the
extracellular matrix (ECM) of tumors referring to its putative utility as
therapeutic target. Following the traditional observation on the extensive
morphological alteration in the tumor-affected tissue, the well-documented
aberrant cellular regulation indicated that ECM components have an active role
in tumor progression. However, due to the diverse functions and variable
expression of proteoglycans, matrix proteins, and integrins, it is rather
difficult to identify a comprehensive therapeutic target among ECM components.
At present, the elevated level of heparanase and the prominent expression of
αvβ5 integrin are considered as promising therapeutic targets. The inhibition of
glycosaminoglycan offers another promising approach in the treatment of those
tumors which are stimulated by proteoglycans. It can be ascertained that a
selective ECM inhibitor would be a great asset to control metastasis driven by
ECM-mediated signaling. The diverse morphologies of animal tissues are underlain by different
configurations of adherent cells and extracellular matrix (ECM). Here, we
elucidate a cross-scale mechanism for tissue assembly and ECM remodeling
involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5.
Fluorescence cross-correlation spectroscopy within the zebrafish paraxial
mesoderm mesenchyme reveals a physical association between Integrin α5 on
adjacent cell membranes. This Integrin-Integrin complex correlates with
conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin
association and inactive Integrin conformation. Thus, Integrin repression within
the adherent mesenchymal interior of the tissue biases Fibronectin
fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent
somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with
levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the
active conformation and then commences ECM assembly. This cross-scale regulation
of Integrin activation organizes a stereotypic pattern of ECM necessary for
vertebrate body elongation and segmentation. |
Is Calcium homeostasis important in cardiac physiology and pathophysiology? | Calcium homeostasis is very important in cardiac physiology and pathophysiology. Maintenance of cellular calcium homeostasis is critical to regulating cardiac contraction. Abnormalities in calcium homeostasis underlie cardiac arrhythmia, contractile dysfunction and cardiac remodelling. | OBJECTIVE: To compare clinically relevant pharmacokinetic, pharmacodynamic and
toxico logical characteristics of calcium-modulating compounds used in ischemic
heart disease.
DATA SOURCES: A MEDLINE search (1990 pt B to 1991 pt A revised for 1993; 1991 pt
B to 1992 revised for 1993; and January to May 1993) combining the search
phrases 'calcium channel blockers', 'myocardial ischemia', 'pharmacodynamics'
and 'pharmacokinetics', and a search in Compact Cambridge Drug Information
Source vol-6 (revised 1992, fourth quarter) using the search phrase 'calcium
antagonists' and medical subject headings (MeSH) 'pharmacokinetics' and
'pharmacodynamics' were used to obtain title and abstract information on
available current literature.
STUDY SELECTION: Review articles, proceedings and studies published in English
and available within the University of Saskatchewan library system, as they
appeared to relate closely to the objective, were obtained for closer
evaluation. In addition, primary references were examined, and journal reprints
were selected from the authors' files.
DATA EXTRACTION: The focus was on studies and objective reviews that profiled
one or more representative compounds in a manner suitable for deriving
background and comparative information pertaining to the objective. Data from
multiple studies, or from studies that employed multiple methodological
approaches, were preferentially extracted and summarized for presentation.
DATA SYNTHESIS: The role of calcium in cardiac and vascular smooth muscle
physiology was reviewed, highlighting the major mechanisms responsible for
maintaining calcium homeostasis in these cells. With a focus on verapamil,
diltiazem and 1,4-dihydropyridines currently employed in the treatment of
cardiovascular disorders, a general survey of their sites of action, tissue
selective pharmacodynamics, pharmacokinetic properties and side effects was
undertaken in a comparative context.
CONCLUSIONS: Calcium antagonists are employed in the treatment of angina,
certain cardiac arrhythmias and hypertension. They are a chemically and
pharmacologically heterogeneous group of compounds that act principally to
inhibit the influx of calcium across certain voltage-dependent membrane
channels. Concepts pertaining to calcium mobilization in the pathophysiology of
myocardial ischemia, particularly at the molecular level, have evolved
remarkably over the past decade. The repertoire of agents having
calcium-regulating properties has expanded in parallel. The task of integrating
new knowledge in both of these areas requires further attention in order to
determine optimal approaches to treatment. Ion channels underlie the electrical activity of cells. Calcium channels have a
unique functional role, because not only do they participate in this activity,
they form the means by which electrical signals are converted to responses
within the cell. Calcium channels play an integral role in excitation in the
heart and shaping the cardiac action potential. In addition, calcium influx
through calcium channels is responsible for initiating contraction.
Abnormalities in calcium homeostasis underlie cardiac arrhythmia, contractile
dysfunction and cardiac remodelling. Reactive oxygen species participate in the
development of pathology by altering the redox state of regulatory proteins.
There is now good evidence that reactive oxygen species regulate the function of
calcium channels. In this mini-review, the evidence for regulation of calcium
channels by reactive oxygen species and implications with respect to pathology
are presented. Calcium channels may represent a target for intervention during
hypoxic trigger of arrhythmia or chronic pathological remodelling. Central to controlling intracellular calcium concentration ([Ca(2+)](i)) are a
number of Ca(2+) transporters and channels with the L-type Ca(2+) channel,
Na(+)-Ca(2+) exchanger and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) being of
particular note in the heart. This review concentrates on the regulation of
[Ca(2+)](i) in cardiac muscle and the homeostatic mechanisms employed to ensure
that the heart can operate under steady-state conditions on a beat by beat
basis. To this end we discuss the relative importance of various sources and
sinks of Ca(2+) responsible for initiating contraction and relaxation in cardiac
myocytes and how these can be manipulated to regulate the Ca(2+) content of the
major Ca(2+) store, the sarcoplasmic reticulum (SR). We will present a simple
feedback system detailing how such control can be achieved and highlight how
small perturbations to the steady-state operation of the feedback loop can be
both beneficial physiologically and underlie changes in systolic Ca(2+) in
ageing and heart disease. In addition to manipulating the amplitude of the
normal systolic Ca(2+) transient, the tight regulation of SR Ca(2+) content is
also required to prevent the abnormal, spontaneous or diastolic release of
Ca(2+) from the SR. Such diastolic events are a major factor contributing to the
genesis of cardiac arrhythmias in disease situations and in recently identified
familial mutations in the SR Ca(2+) release channel (ryanodine receptor, RyR).
How such diastolic release arises and potential mechanisms for controlling this
will be discussed. BACKGROUND: The cardiac sodium-calcium exchanger (NCX1) is a key sarcolemmal
protein for the maintece of calcium homeostasis in the heart. Because heart
failure is associated with increased expression of NCX1, heterozygous (HET) and
homozygous (HOM) transgenic mice overexpressing NCX1 were developed and
evaluated.
METHODS AND RESULTS: The NCX1 transgenic mice display 2.3-fold (HET) and
3.1-fold (HOM) increases in exchanger activity from wild-type (WT) mice.
Functional information was obtained by echocardiography and catheterizations
before and after hemodynamic stress from pregcy, treadmill exercise or
transaortic constriction (TAC). HET and HOM mice exhibited hypertrophy and
blunted responses with beta-adrenergic stimulation. Postpartum mice from all
groups were hypertrophied, but only the HOM mice exhibited premature death from
heart failure. HOM mice became exercise intolerant after 6 weeks of daily
treadmill running. After 21 days TAC, HET, and HOM mice exhibited significant
contractile dysfunction and 15% to 40% mortality with clinical evidence of heart
failure.
CONCLUSIONS: Hemodynamic stress results in a compensated hypertrophy in WT mice,
but NCX1 transgenic mice exhibit decreased contractile function and heart
failure in proportion to their level of NCX1 expression. Thus exchanger
overexpression in mice leads to abnormal calcium handling and a decompensatory
transition to heart failure with stress. With aging, the heart develops myocyte hypertrophy associated with impaired
relaxation indices. To define the cellular basis of this adaptation, we examined
the physiological changes that arise in calcium handling in the aging heart and
contrasted the adaptations that occur following the imposition of a stimulus
that alters calcium homeostasis in a young and an old heart. We utilized a
cardiac-specific conditional transgenic approach to "switch on" protein kinase
(PKC)-beta II expression in mice at different stages of adult life (3 and 12
months) and characterized alterations in ICa and calcium release in wild-type
(WT) and PKC-beta II-expressing cells. Amplitude or voltage dependence of ICa
were not significantly altered by expression of PKC-beta II at any age. No
significant differences in calcium-release properties were seen with age. Upon
activation of PKC-beta II, the amplitude of the calcium transient was larger,
and the calcium spark frequency was greater in PKC-beta II mice compared to WT
at both 3 and 12 months. Spark amplitude increased only in the 12-month PKC-beta
II mice. These changes occurred in parallel with an increase in cell size (as
determined by capacitance measurements) in the 12-month PKC-beta II mice but not
the 3-month PKC-beta II mice. These data suggest that alterations in the
calcium-handling machinery of the cardiocyte differ in the context of age and as
such may predispose the older heart to the development of a hypertrophic
phenotype. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a primary
electrical myocardial disease characterized by exercise- and stress-related
ventricular tachycardia manifested as syncope and sudden death. The disease has
a heterogeneous genetic basis, with mutations in the cardiac Ryanodine Receptor
channel (RyR2) gene accounting for an autosomal-domit form (CPVT1) in
approximately 50% and mutations in the cardiac calsequestrin gene (CASQ2)
accounting for an autosomal-recessive form (CPVT2) in up to 2% of CPVT cases.
Both RyR2 and calsequestrin are important participants in the cardiac cellular
calcium homeostasis. We review the physiology of the cardiac calcium
homeostasis, including the cardiac excitation contraction coupling and myocyte
calcium cycling. The pathophysiology of cardiac arrhythmias related to myocyte
calcium handling and the effects of different modulators are discussed. The
putative derangements in myocyte calcium homeostasis responsible for CPVT, as
well as the clinical manifestations and therapeutic options available, are
described. Calcium ions are the most ubiquitous and versatile signaling molecules in
eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both
the normal growth of the budding yeast Saccharomyces cerevisiae and the
intricate working of the mammalian heart. In this paper, we make a detailed
comparison between the calcium homeostasis/signaling networks in yeast cells and
those in mammalian cardiac myocytes. This comparison covers not only the
components, structure and function of the networks but also includes existing
knowledge on the measured and simulated network dynamics using mathematical
models. Surprisingly, most of the factors known in the yeast calcium
homeostasis/signaling network are conserved and operate similarly in mammalian
cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is
a simple organism that affords powerful genetic and genomic tools. Thus,
exploring and understanding the calcium homeostasis/signaling system in yeast
can provide a shortcut to help understand calcium homeostasis/signaling systems
in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat
relevant human diseases such as pathological cardiac hypertrophy and heart
failure. Important insights into the molecular basis of hypertrophic cardiomyopathy and
related diseases have been gained by studying families with inherited cardiac
hypertrophy. Integrated clinical and genetic investigations have demonstrated
that different genetic defects can give rise to the common phenotype of cardiac
hypertrophy. Diverse pathways have been identified, implicating perturbations in
force generation, force transmission, intracellular calcium homeostasis,
myocardial energetics, and cardiac metabolism in causing disease. Although not
fully elucidated, the fundamental mechanisms linking gene mutations to clinical
disease are being characterized. Further advances will allow a better
understanding of pathogenesis, diagnosis, and treatment, not just of relatively
rare inherited cardiomyopathies, but potentially also of relevance to more
common acquired forms of hypertrophic remodeling. Energy metabolism and Ca(2+) handling serve critical roles in cardiac physiology
and pathophysiology. Peroxisome proliferator-activated receptor gamma
coactivator 1 alpha (PGC-1 alpha) is a multi-functional coactivator that is
involved in the regulation of cardiac mitochondrial functional capacity and
cellular energy metabolism. However, the regulation of PGC-1 alpha in cardiac
Ca(2+) signaling has not been fully elucidated. To address this issue, we
combined confocal line-scan imaging with off-line imaging processing to
characterize calcium signaling in cultured adult rat ventricular myocytes
expressing PGC-1 alpha via adenoviral transduction. Our data shows that
overexpressing PGC-1 alpha improved myocyte contractility without increasing the
amplitude of Ca(2+) transients, suggesting that myofilament sensitivity to
Ca(2+) increased. Interestingly, the decay kinetics of global Ca(2+) transients
and Ca(2+) waves accelerated in PGC-1 alpha-expressing cells, but the decay rate
of caffeine-elicited Ca(2+) transients showed no significant change. This
suggests that sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), but not
Na(+)/Ca(2+) exchange (NCX) contribute to PGC-1 alpha-induced cytosolic Ca(2+)
clearance. Furthermore, PGC-1 alpha induced the expression of SERCA2a in
cultured cardiac myocytes. Importantly, overexpressing PGC-1 alpha did not
disturb cardiac Ca(2+) homeostasis, because SR Ca(2+) load and the propensity
for Ca(2+) waves remained unchanged. These data suggest that PGC-1 alpha can
ameliorate cardiac Ca(2+) cycling and improve cardiac work output in response to
physiological stress. Unraveling the PGC-1 alpha-calcium handling pathway sheds
new light on the role of PGC-1 alpha in the therapy of cardiac diseases. Our understanding of the molecular processes which regulate cardiac function has
grown immeasurably in recent years. Even with the advent of β-blockers,
angiotensin inhibitors and calcium modulating agents, heart failure (HF) still
remains a seriously debilitating and life-threatening condition. Here, we review
the molecular changes which occur in the heart in response to increased load and
the pathways which control cardiac hypertrophy, calcium homeostasis, and immune
activation during HF. These can occur as a result of genetic mutation in the
case of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) or as
a result of ischemic or hypertensive heart disease. In the majority of cases,
calcineurin and CaMK respond to dysregulated calcium signaling and adrenergic
drive is increased, each of which has a role to play in controlling blood
pressure, heart rate, and left ventricular function. Many major pathways for
pathological remodeling converge on a set of transcriptional regulators such as
myocyte enhancer factor 2 (MEF2), nuclear factors of activated T cells (NFAT),
and GATA4 and these are opposed by the action of the natriuretic peptides ANP
and BNP. Epigenetic modification has emerged in recent years as a major
influence cardiac physiology and histone acetyl transferases (HATs) and histone
deacetylases (HDACs) are now known to both induce and antagonize hypertrophic
growth. The newly emerging roles of microRNAs in regulating left ventricular
dysfunction and fibrosis also has great potential for novel therapeutic
intervention. Finally, we discuss the role of the immune system in mediating
left ventricular dysfunction and fibrosis and ways this can be targeted in the
setting of viral myocarditis. Cardiac calcium (Ca(2+)) handling subsumes the mechanisms maintaining the
myocardial Ca(2+) homeostasis that contribute essentially to cardiac
performance. This review addresses the interaction of transplasmalemmal and
transsarcoplasmic Ca(2+) flux, its potential modifications due to β-adrenergic
stimulation and its implications on cardiac action potential. Calcium is an important mediator in cardiac excitation and disorders in cardiac
Ca(2+) homeostasis have great influence on the cardiac action potential.
Therefore dysfunction in regulatory proteins that are involved in Ca(2+)
handling can lead to the occurrence of severe arrhythmia. Although mutations in
Ca(2+) regulating proteins are quite rare, they can offer general insights into
arrhythmogenesis. Here, we briefly review some important aspects of
arrhythmia-associated mutations in Ca(2+) regulating proteins with special
emphasis to its associated pathophysiology. Ca(2+) is a universal carrier of biological information: it controls cell life
from its origin at fertilization to its end in the process of programmed cell
death. Ca(2+) is a conventional diffusible second messenger released inside
cells by the interaction of first messengers with plasma membrane receptors.
However, it can also penetrate directly into cells to deliver information
without the intermediation of first or second messengers. Even more
distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma
membrane receptor to set in motion intracellular signaling pathways that involve
Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its
ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes
an agent that mediates cell distress, or even (toxic) cell death, if its
concentration and movements inside cells are not carefully tuned. Ca(2+) is
controlled by reversible complexation to specific proteins, which could be pure
Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its
signal to pass it on to targets. The most important actors in the buffering of
cell Ca(2+) are proteins that transport it across the plasma membrane and the
membrane of the organelles: some have high Ca(2+) affinity and low transport
capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+)
uptake system of mitochondria). Between the initial event of fertilization, and
the terminal event of programmed cell death, the Ca(2+) signal regulates the
most important activities of the cell, from the expression of genes, to heart
and muscle contraction and other motility processes, to diverse metabolic
pathways involved in the generation of cell fuels. Maintece of cellular calcium homeostasis is critical to regulating
mitochondrial ATP production and cardiac contraction. The ion channel known as
the L-type calcium channel is the main route for calcium entry into cardiac
myocytes. The channel associates with cytoskeletal proteins that assist with the
communication of signals from the plasma membrane to intracellular organelles,
including mitochondria. This article explores the roles of calcium and the
cytoskeleton in regulation of mitochondrial function in response to alterations
in L-type calcium channel activity. Direct activation of the L-type calcium
channel results in an increase in intracellular calcium and increased
mitochondrial calcium uptake. As a result, mitochondrial NADH production, oxygen
consumption and reactive oxygen species production increase. In addition the
L-type calcium channel is able to regulate mitochondrial membrane potential via
cytoskeletal proteins when conformational changes in the channel occur during
activation and inactivation. Since the L-type calcium channel is the initiator
of contraction, a functional coupling between the channel and mitochondria via
the cytoskeleton may represent a synchronised process by which mitochondrial
function is regulated in addition to calcium influx to meet myocardial energy
demand on a beat to beat basis. |
List available databases containing information about conserved noncoding elements. | Ancora and TFCONES. | BACKGROUND: Transcription factors (TFs) regulate gene transcription and play
pivotal roles in various biological processes such as development, cell cycle
progression, cell differentiation and tumor suppression. Identifying
cis-regulatory elements associated with TF-encoding genes is a crucial step in
understanding gene regulatory networks. To this end, we have used a comparative
genomics approach to identify putative cis-regulatory elements associated with
TF-encoding genes in vertebrates.
DESCRIPTION: We have created a database named TFCONES (Transcription Factor
Genes & Associated COnserved Noncoding ElementS) (http://tfcones.fugu-sg.org)
which contains all human, mouse and fugu TF-encoding genes and conserved
noncoding elements (CNEs) associated with them. The CNEs were identified by
gene-by-gene alignments of orthologous TF-encoding gene loci using MLAGAN. We
also predicted putative transcription factor binding sites within the CNEs. A
significant proportion of human-fugu CNEs contain experimentally defined binding
sites for transcriptional activators and repressors, indicating that a majority
of the CNEs may function as transcriptional regulatory elements. The TF-encoding
genes that are involved in nervous system development are generally enriched for
human-fugu CNEs. Users can retrieve TF-encoding genes and their associated CNEs
by conducting a keyword search or by selecting a family of DNA-binding proteins.
CONCLUSION: The conserved noncoding elements identified in TFCONES represent a
catalog of highly prioritized putative cis-regulatory elements of TF-encoding
genes and are candidates for functional assay. Metazoan genomes contain arrays of highly conserved noncoding elements (HCNEs)
that span developmental regulatory genes and define regulatory domains. We
describe Ancora http://ancora.genereg.net, a web resource that provides data and
tools for exploring genomic organization of HCNEs for multiple genomes. Ancora
includes a genome browser that shows HCNE locations and features novel HCNE
density plots as a powerful tool to discover developmental regulatory genes and
distinguish their regulatory elements and domains. |
What was the purpose of the FANTOM5 project? | The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. The FANTOM5 and ENCODE projects represent two independent large scale efforts to map regulatory and transcriptional features to the human genome. | BACKGROUND: Deciphering the most common modes by which chromatin regulates
transcription, and how this is related to cellular status and processes is an
important task for improving our understanding of human cellular biology. The
FANTOM5 and ENCODE projects represent two independent large scale efforts to map
regulatory and transcriptional features to the human genome. Here we investigate
chromatin features around a comprehensive set of transcription start sites in
four cell lines by integrating data from these two projects.
RESULTS: Transcription start sites can be distinguished by chromatin states
defined by specific combinations of both chromatin mark enrichment and the
profile shapes of these chromatin marks. The observed patterns can be associated
with cellular functions and processes, and they also show association with
expression level, location relative to nearby genes, and CpG content. In
particular we find a substantial number of repressed inter- and intra-genic
transcription start sites enriched for active chromatin marks and Pol II, and
these sites are strongly associated with immediate-early response processes and
cell signaling. Associations between start sites with similar chromatin patterns
are validated by significant correlations in their global expression profiles.
CONCLUSIONS: The results confirm the link between chromatin state and cellular
function for expressed transcripts, and also indicate that active chromatin
states at repressed transcripts may poise transcripts for rapid activation
during immune response. Collaborators: Forrest AR, Kawaji H, Rehli M, Baillie JK, de Hoon MJ, Haberle V,
Lassmann T, Kulakovskiy IV, Lizio M, Itoh M, Andersson R, Mungall CJ, Meehan TF,
Schmeier S, Bertin N, Jørgensen M, Dimont E, Arner E, Schmid C, Schaefer U,
Medvedeva YA, Plessy C, Vitezic M, Severin J, Semple CA, Ishizu Y, Young RS,
Francescatto M, Alam I, Albanese D, Altschuler GM, Arakawa T, Archer JA, Arner
P, Babina M, Rennie S, Balwierz PJ, Beckhouse AG, Pradhan-Bhatt S, Blake JA,
Blumenthal A, Bodega B, Bonetti A, Briggs J, Brombacher F, Burroughs AM,
Califano A, Cannistracti CV, Carbajo D, Chen Y, Chierici M, Ciani Y, Clevers HC,
Dalla E, Davis CA, Detmar M, Diehl AD, Dohi T, Drabløs F, Edge AS, Edinger M,
Ekwall K, Endoh M, Enomoto H, Fagiolini M, Fairbairn L, Fang H, Farach-Carson
MC, Faulkner GJ, Favorov AV, Fisher ME, Frith MC, Fujita R, Fukuda S, Furlanello
C, Furuno M, Furusawa J, Geijtenbeek TB, Gibson AP, Gingeras T, Goldowitz D,
Gough J, Guhl S, Guler R, Gustincich S, Ha TJ, Hamaguchi M, Hara M, Harbers M,
Harshbarger J, Hasegawa A, Hasegawa Y, Hashimoto T, Herlyn M, Hitchens KJ, Ho
Sui SJ, Hofman OM, Hoof I, Hori F, Huminiecki L, Iida K, Ikawa T, Jankovic BR,
Jia H, Joshi A, Jurman G, Kaczkowski B, Kai C, Kaida K, Kaiho A, Kajiyama K,
Kanamori-Katayama M, Kasianov AS, Kasukawa T, Katayama S, Kato S, Kawaguchi S,
Kawamoto H, Kawamura YI, Kawashima T, Kempfle JS, Kenna TJ, Kere J, Khachigian
LM, Kitamura T, Klinken SP, Knox AJ, Kojima M, Kojima S, Kondo N, Koseki H,
Koyasu S, Krampitz S, Kubosaki A, Kwon AT, Laros JF, Lee W, Lennartsson A, Li K,
Lilje B, Lipovich L, Mackay-Sim A, Manabe R, Mar JC, Marchand B, Mathelier A,
Mejhert N, Meynert A, Mizuno Y, de Lima Morais DA, Morikawa H, Morimoto M, Moro
K, Motakis E, Motohashi H, Mummery CL, Murata M, Nagao-Sato S, Nakachi Y,
Nakahara F, Nakamura T, Nakamura Y, Nakazato K, van Nimwegen E, Ninomiya N,
Nishiyori H, Noma S, Nozaki T, Ogishima S, Ohkura N, Ohmiya H, Ohno H, Onshima
M, Okada-Hatakeyama M, Okazaki Y, Orlando V, Ovchinnikov DA, Pain A, Passier R,
Patrikakis M, Persson H, Piazza S, Prendergast JG, Rackham OJ, Ramilowski JA,
Rashid M, Ravasi T, Rizzu P, Roncador M, Roy S, Rye MB, Saijyo E, Sajantila A,
Saka A, Sakaguchi S, Sakai M, Sato H, Satoh H, Savvi S, Saxena A, Schneider C,
Schultes EA, Schultz-Tanzil GG, Schwegmann A, Sengstag T, Sheng G, Shimoji H,
Shimoni Y, Shin JW, Simon C, Sugiyama D, Sugiyama T, Suzuki M, Suzuki N, Swoboda
RK, 't Hoen PA, Tagami M, Takahashi N, Takai J, Tanaka H, Tatsukawa H, Tatum Z,
Thompson M, Toyoda H, Toyodo T, Valen E, van de Wetering M, van den Berg LM,
Verardo R, Vijayan D, Vorontsov IE, Wasserman WW, Watanabe S, Wells CA,
Winteringham LN, Wolvetang E, Wood EJ, Yamaguchi Y, Yamamoto M, Yoneda M,
Yonekura Y, Yoshida S, Zabierowski SE, Zhang PG, Zhao X, Zucchelli S, Summers
KM, Suzuki H, Daub CO, Kawai J, Heutink P, Hide W, Freeman TC, Lenhard B, Bajic
VB, Taylor MS, Makeev VJ, Sandelin A, Hume DA, Carninci P, Hayashizaki Y. FANTOM Consortium and the RIKEN PMI and CLST (DGT); Forrest AR, Kawaji H, Rehli
M, Baillie JK, de Hoon MJ, Haberle V, Lassmann T, Kulakovskiy IV, Lizio M, Itoh
M, Andersson R, Mungall CJ, Meehan TF, Schmeier S, Bertin N, Jørgensen M, Dimont
E, Arner E, Schmidl C, Schaefer U, Medvedeva YA, Plessy C, Vitezic M, Severin J,
Semple C, Ishizu Y, Young RS, Francescatto M, Alam I, Albanese D, Altschuler GM,
Arakawa T, Archer JA, Arner P, Babina M, Rennie S, Balwierz PJ, Beckhouse AG,
Pradhan-Bhatt S, Blake JA, Blumenthal A, Bodega B, Bonetti A, Briggs J,
Brombacher F, Burroughs AM, Califano A, Cannistraci CV, Carbajo D, Chen Y,
Chierici M, Ciani Y, Clevers HC, Dalla E, Davis CA, Detmar M, Diehl AD, Dohi T,
Drabløs F, Edge AS, Edinger M, Ekwall K, Endoh M, Enomoto H, Fagiolini M,
Fairbairn L, Fang H, Farach-Carson MC, Faulkner GJ, Favorov AV, Fisher ME, Frith
MC, Fujita R, Fukuda S, Furlanello C, Furino M, Furusawa J, Geijtenbeek TB,
Gibson AP, Gingeras T, Goldowitz D, Gough J, Guhl S, Guler R, Gustincich S, Ha
TJ, Hamaguchi M, Hara M, Harbers M, Harshbarger J, Hasegawa A, Hasegawa Y,
Hashimoto T, Herlyn M, Hitchens KJ, Ho Sui SJ, Hofmann OM, Hoof I, Hori F,
Huminiecki L, Iida K, Ikawa T, Jankovic BR, Jia H, Joshi A, Jurman G, Kaczkowski
B, Kai C, Kaida K, Kaiho A, Kajiyama K, Kanamori-Katayama M, Kasianov AS,
Kasukawa T, Katayama S, Kato S, Kawaguchi S, Kawamoto H, Kawamura YI, Kawashima
T, Kempfle JS, Kenna TJ, Kere J, Khachigian LM, Kitamura T, Klinken SP, Knox AJ,
Kojima M, Kojima S, Kondo N, Koseki H, Koyasu S, Krampitz S, Kubosaki A, Kwon
AT, Laros JF, Lee W, Lennartsson A, Li K, Lilje B, Lipovich L, Mackay-Sim A,
Manabe R, Mar JC, Marchand B, Mathelier A, Mejhert N, Meynert A, Mizuno Y, de
Lima Morais DA, Morikawa H, Morimoto M, Moro K, Motakis E, Motohashi H, Mummery
CL, Murata M, Nagao-Sato S, Nakachi Y, Nakahara F, Nakamura T, Nakamura Y,
Nakazato K, van Nimwegen E, Ninomiya N, Nishiyori H, Noma S, Noma S, Noazaki T,
Ogishima S, Ohkura N, Ohimiya H, Ohno H, Ohshima M, Okada-Hatakeyama M, Okazaki
Y, Orlando V, Ovchinnikov DA, Pain A, Passier R, Patrikakis M, Persson H, Piazza
S, Prendergast JG, Rackham OJ, Ramilowski JA, Rashid M, Ravasi T, Rizzu P,
Roncador M, Roy S, Rye MB, Saijyo E, Sajantila A, Saka A, Sakaguchi S, Sakai M,
Sato H, Savvi S, Saxena A, Schneider C, Schultes EA, Schulze-Tanzil GG,
Schwegmann A, Sengstag T, Sheng G, Shimoji H, Shimoni Y, Shin JW, Simon C,
Sugiyama D, Sugiyama T, Suzuki M, Suzuki N, Swoboda RK, 't Hoen PA, Tagami M,
Takahashi N, Takai J, Tanaka H, Tatsukawa H, Tatum Z, Thompson M, Toyodo H,
Toyoda T, Valen E, van de Wetering M, van den Berg LM, Verado R, Vijayan D,
Vorontsov IE, Wasserman WW, Watanabe S, Wells CA, Winteringham LN, Wolvetang E,
Wood EJ, Yamaguchi Y, Yamamoto M, Yoneda M, Yonekura Y, Yoshida S, Zabierowski
SE, Zhang PG, Zhao X, Zucchelli S, Summers KM, Suzuki H, Daub CO, Kawai J,
Heutink P, Hide W, Freeman TC, Lenhard B, Bajic VB, Taylor MS, Makeev VJ,
Sandelin A, Hume DA, Carninci P, Hayashizaki Y. |
Is the gene DUX4 epigenetically regulated in somatic cells? | The human double-homeodomain retrogene DUX4 is expressed in the testis and epigenetically repressed in somatic tissues. Recent studies provide evidence that DUX4 is expressed in the human germline and then epigenetically silenced in somatic cells. | Each unit of the D4Z4 macrosatellite repeat contains a retrotransposed gene
encoding the DUX4 double-homeobox transcription factor. Facioscapulohumeral
dystrophy (FSHD) is caused by deletion of a subset of the D4Z4 units in the
subtelomeric region of chromosome 4. Although it has been reported that the
deletion of D4Z4 units induces the pathological expression of DUX4 mRNA, the
association of DUX4 mRNA expression with FSHD has not been rigorously
investigated, nor has any human tissue been identified that normally expresses
DUX4 mRNA or protein. We show that FSHD muscle expresses a different splice form
of DUX4 mRNA compared to control muscle. Control muscle produces low amounts of
a splice form of DUX4 encoding only the amino-terminal portion of DUX4. FSHD
muscle produces low amounts of a DUX4 mRNA that encodes the full-length DUX4
protein. The low abundance of full-length DUX4 mRNA in FSHD muscle cells
represents a small subset of nuclei producing a relatively high abundance of
DUX4 mRNA and protein. In contrast to control skeletal muscle and most other
somatic tissues, full-length DUX4 transcript and protein is expressed at
relatively abundant levels in human testis, most likely in the germ-line cells.
Induced pluripotent (iPS) cells also express full-length DUX4 and
differentiation of control iPS cells to embryoid bodies suppresses expression of
full-length DUX4, whereas expression of full-length DUX4 persists in
differentiated FSHD iPS cells. Together, these findings indicate that
full-length DUX4 is normally expressed at specific developmental stages and is
suppressed in most somatic tissues. The contraction of the D4Z4 repeat in FSHD
results in a less efficient suppression of the full-length DUX4 mRNA in skeletal
muscle cells. Therefore, FSHD represents the first human disease to be
associated with the incomplete developmental silencing of a retrogene array
normally expressed early in development. Autosomal domit facioscapulohumeral muscular dystrophy (FSHD) has an unusual
pathogenic mechanism. FSHD is caused by deletion of a subset of D4Z4
macrosatellite repeat units in the subtelomere of chromosome 4q. Recent studies
provide compelling evidence that a retrotransposed gene in the D4Z4 repeat,
DUX4, is expressed in the human germline and then epigenetically silenced in
somatic tissues. In FSHD, the combination of inefficient chromatin silencing of
the D4Z4 repeat and polymorphisms on the FSHD-permissive alleles that stabilize
the DUX4 mRNAs emanating from the repeat result in inappropriate DUX4 protein
expression in muscle cells. FSHD is thereby the first example of a human disease
caused by the inefficient repression of a retrogene in a macrosatellite repeat
array. BACKGROUND: The epigenome refers to marks on the genome, including DNA
methylation and histone modifications, that regulate the expression of
underlying genes. A consistent profile of gene expression changes in end-stage
cardiomyopathy led us to hypothesize that distinct global patterns of the
epigenome may also exist.
METHODS AND RESULTS: We constructed genome-wide maps of DNA methylation and
histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and
normal human hearts. More than 506 Mb sequences per library were generated by
high-throughput sequencing, allowing us to assign methylation scores to ≈28
million CG dinucleotides in the human genome. DNA methylation was significantly
different in promoter CpG islands, intragenic CpG islands, gene bodies, and
H3K36me3-enriched regions of the genome. DNA methylation differences were
present in promoters of upregulated genes but not downregulated genes. H3K36me3
enrichment itself was also significantly different in coding regions of the
genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus
correlated to differential DNA methylation and H3K36me3 enrichment. In vitro,
Dux gene expression was responsive to a specific inhibitor of DNA
methyltransferase, and Dux siRNA knockdown led to reduced cell viability.
CONCLUSIONS: Distinct epigenomic patterns exist in important DNA elements of the
cardiac genome in human end-stage cardiomyopathy. The epigenome may control the
expression of local or distal genes with critical functions in myocardial stress
response. If epigenomic patterns track with disease progression, assays for the
epigenome may be useful for assessing prognosis in heart failure. Further
studies are needed to determine whether and how the epigenome contributes to the
development of cardiomyopathy. In most cases facioscapulohumeral muscular dystrophy (FSHD) is caused by
contraction of the D4Z4 repeat in the 4q subtelomere. This contraction is
associated with local chromatin decondensation and derepression of the DUX4
retrogene. Its complex genetic and epigenetic cause and high clinical
variability in disease severity complicate investigations on the pathogenic
mechanism underlying FSHD. A validated cellular model bypassing the considerable
heterogeneity would facilitate mechanistic and therapeutic studies of FSHD.
Taking advantage of the high incidence of somatic mosaicism for D4Z4 repeat
contraction in de novo FSHD, we have established a clonal myogenic cell model
from a mosaic patient. Individual clones are genetically identical except for
the size of the D4Z4 repeat array, being either normal or FSHD sized. These
clones retain their myogenic characteristics, and D4Z4 contracted clones differ
from the noncontracted clones by the bursts of expression of DUX4 in sporadic
nuclei, showing that this burst-like phenomenon is a locus-intrinsic feature.
Consequently, downstream effects of DUX4 expression can be observed in D4Z4
contracted clones, like differential expression of DUX4 target genes. We also
show their participation to in vivo regeneration with immunodeficient mice,
further expanding the potential of these clones for mechanistic and therapeutic
studies. These cell lines will facilitate pairwise comparisons to identify
FSHD-specific differences and are expected to create new opportunities for
high-throughput drug screens. Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of
the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded
DUX4 gene in skeletal muscle. The more common form, autosomal domit FSHD1, is
caused by contraction of the D4Z4 array, whereas the genetic determits and
inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here, we
show that mutations in SMCHD1 (encoding structural maintece of chromosomes
flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein
levels and segregate with genome-wide D4Z4 CpG hypomethylation in human
kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and
a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4
expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4
contraction-independent DUX4 expression. Our study identifies SMCHD1 as an
epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic
determit of FSHD2 and possibly other human diseases subject to epigenetic
regulation. Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder
linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric
region. This deletion induces epigenetic modifications that affect the
expression of several genes located in the vicinity. In each D4Z4 element, we
identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription
factor that plays a major role in the development of FSHD through the initiation
of a large gene dysregulation cascade that causes myogenic differentiation
defects, atrophy and reduced response to oxidative stress. Because miRNAs
variably affect mRNA expression, proteomic approaches are required to define the
dysregulated pathways in FSHD. In this study, we optimized a differential
isotope protein labeling (ICPL) method combined with shotgun proteomic analysis
using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56(+)
FSHD myoblasts were found to fuse into myotubes presenting various proportions
of an atrophic or a disorganized phenotype. To better understand the FSHD
myogenic defect, our improved proteomic procedure was used to compare
predomitly atrophic or disorganized myotubes to the same matching healthy
control. FSHD atrophic myotubes presented decreased structural and contractile
muscle components. This phenotype suggests the occurrence of atrophy-associated
proteolysis that likely results from the DUX4-mediated gene dysregulation
cascade. The skeletal muscle myosin isoforms were decreased while non-muscle
myosin complexes were more abundant. In FSHD disorganized myotubes, myosin
isoforms were not reduced, and increased proteins were mostly involved in
microtubule network organization and myofibrillar remodeling. A common feature
of both FSHD myotube phenotypes was the disturbance of several caveolar
proteins, such as PTRF and MURC. Taken together, our data suggest changes in
trafficking and in the membrane microdomains of FSHD myotubes. Finally, the
adjustment of a nuclear fractionation compatible with mass spectrometry allowed
us to highlight alterations of proteins involved in mRNA processing and
stability. Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused
by decreased epigenetic repression of the D4Z4 macrosatellite repeats and
ectopic expression of DUX4, a retrogene encoding a germline transcription factor
encoded in each repeat. Unaffected individuals generally have more than 10
repeats arrayed in the subtelomeric region of chromosome 4, whereas the most
common form of FSHD (FSHD1) is caused by a contraction of the array to fewer
than 10 repeats, associated with decreased epigenetic repression and variegated
expression of DUX4 in skeletal muscle. We have generated transgenic mice
carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice
recapitulate important epigenetic and DUX4 expression attributes seen in
patients and controls, respectively, including high DUX4 expression levels in
the germline, (incomplete) epigenetic repression in somatic tissue, and
FSHD-specific variegated DUX4 expression in sporadic muscle nuclei associated
with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to
activate similar functional gene groups in mouse muscle cells as it does in
human muscle cells. These transgenic mice therefore represent a valuable animal
model for FSHD and will be a useful resource to study the molecular mechanisms
underlying FSHD and to test new therapeutic intervention strategies. The human double-homeodomain retrogene DUX4 is expressed in the testis and
epigenetically repressed in somatic tissues. Facioscapulohumeral muscular
dystrophy (FSHD) is caused by mutations that decrease the epigenetic repression
of DUX4 in somatic tissues and result in mis-expression of this transcription
factor in skeletal muscle. DUX4 binds sites in the human genome that contain a
double-homeobox sequence motif, including sites in unique regions of the genome
as well as many sites in repetitive elements. Using ChIP-seq and RNA-seq on
myoblasts transduced with DUX4 we show that DUX4 binds and activates
transcription of mammalian apparent LTR-retrotransposons (MaLRs), endogenous
retrovirus (ERVL and ERVK) elements, and pericentromeric satellite HSATII
sequences. Some DUX4-activated MaLR and ERV elements create novel promoters for
genes, long non-coding RNAs, and antisense transcripts. Many of these novel
transcripts are expressed in FSHD muscle cells but not control cells, and thus
might contribute to FSHD pathology. For example, HEY1, a repressor of
myogenesis, is activated by DUX4 through a MaLR promoter. DUX4-bound motifs,
including those in repetitive elements, show evolutionary conservation and some
repeat-initiated transcripts are expressed in healthy testis, the normal
expression site of DUX4, but more rarely in other somatic tissues. Testis
expression patterns are known to have evolved rapidly in mammals, but the
mechanisms behind this rapid change have not yet been identified: our results
suggest that mobilization of MaLR and ERV elements during mammalian evolution
altered germline gene expression patterns through transcriptional activation by
DUX4. Our findings demonstrate a role for DUX4 and repetitive elements in
mammalian germline evolution and in FSHD muscular dystrophy. Although the pathophysiology of facioscapulohumeral dystrophy (FSHD) has been
controversial over the last decades, progress in recent years has led to a model
that incorporates these decades of findings and is gaining general acceptance in
the FSHD research community. Here we review how the contributions from many labs
over many years led to an understanding of a fundamentally new mechanism of
human disease. FSHD is caused by inefficient repeat-mediated epigenetic
repression of the D4Z4 macrosatellite repeat array on chromosome 4, resulting in
the variegated expression of the DUX4 retrogene, encoding a double-homeobox
transcription factor, in skeletal muscle. Normally expressed in the testis and
epigenetically repressed in somatic tissues, DUX4 expression in skeletal muscle
induces expression of many germline, stem cell, and other genes that might
account for the pathophysiology of FSHD. Although some disagreements regarding
the details of mechanisms remain in the field, the coalescing agreement on a
central model of pathophysiology represents a pivot-point in FSHD research,
transitioning the field from discovery-oriented studies to translational studies
aimed at developing therapies based on a sound model of disease pathophysiology. |
What is the function of the mammalian gene Irg1? | Human IRG1 and mouse Irg1 mediates antiviral and antimicrobial immune responses, without its exact role having been elucidated. Irg1 has been suggested to have a role in apoptosis and to play a significant role in embryonic implantation. Irg1 is reported as the mammalian ortholog of methylcitrate dehydratase. | Implantation of the developing blastocyst is regulated by multiple effectors,
such as steroid hormones, growth factors, and cytokines. To understand how these
diverse signaling pathways interact to modulate uterine gene expression, we
employed a gene expression screen technique to identify the molecules that are
induced in the periimplantation rat uterus. Here we report the isolation of a
complementary DNA representing a novel gene, interferon-regulated gene 1 (IRG1).
This gene exhibits significant homology to interferon (IFN)-alpha/beta-inducible
human genes p27 and 6-16, indicating that these genes may belong to the same
family. Consistent with this finding, expression of IRG1 messenger RNA (mRNA) in
rat uterus increased about 20-fold in response to IFNalpha. Uterine expression
of IRG1 was also stimulated by estrogen and was partially inhibited by an
antiestrogen, ICI 182,780. In pregt rats, IRG1 expression was high on day 1,
but declined on days 2 and 3. The level of IRG1 mRNA again rose transiently on
day 4 immediately preceding implantation. In situ hybridization analysis
localized the IRG1 mRNA expression in the endometrial epithelium and the
surrounding stroma. Interestingly, the expression of p27, which shows high
homology to IRG1, was strongly enhanced in human endometrium during the
midsecretory phase of the menstrual cycle, overlapping the putative window of
implantation. Both IRG1 and p27 mRNAs are therefore induced in the endometrium
in an implantation stage-specific manner. We also observed a synergistic
interaction between IFNalpha and estrogen receptor signaling pathways that led
to maximal induction of p27 mRNA in Ishikawa cells. Although the functional
roles of IRG1 and p27 remain unclear, we describe for the first time,
identification of a gene family regulated by IFNalpha in both rodent and human
uteri. More importantly, our studies reveal that a complex interplay between the
steroid hormone and IFN pathways regulates the expression of these genes in the
endometrium at the time of implantation. Implantation requires coordination between development of the blastocyst and the
sex steroid hormone-regulated differentiation of the uterus. Under the influence
of these hormones, the uterine luminal epithelium becomes receptive to
attachment of the hatched blastocyst. In this study we sought to identify genes
regulated by progesterone (P4) in the uterine epithelium. This resulted in the
identification of one novel P4-regulated gene that had been previously found in
lipopolysaccharide-stimulated macrophages and called immune response gene-1
(Irg1) and which is the mammalian ortholog of the bacterial gene encoding
methylcitrate dehydratase. In adult mice Irg1 expression was limited to the
uterine luminal epithelium where it is expressed only during pregcy with a
peak coinciding with implantation. Irg1 mRNA expression is regulated
synergistically by P4 and estradiol (E2) but not by E2 alone. In macrophages
Irg1 is induced by lipopolysaccharide through a protein kinase C (PKC)-regulated
pathway. Now we demonstrate that the PKC pathway is induced in the uterine
epithelium at implantation by the synergistic action of P4 and E2 and is
responsible for the hormone induction of Irg1. These results suggest that the
PKC pathway plays an important role in modulating steroid hormone responsiveness
in the uterine luminal epithelium during the implantation window and that Irg1
will be an important marker of this window and may play an important role in
implantation. The steroid hormone progesterone (P) is a critical regulator of uterine
receptivity during blastocyst implantation. The hormone acts through nuclear P
receptors (PRs) to modulate the expression of specific gene networks in various
uterine cell types. To identify the P-regulated pathways underlying uterine
receptivity, we previously used oligonucleotide microarrays to analyze uterine
mRNA profiles at the time of implantation in response to RU486, a PR antagonist.
We reported that the mRNA corresponding to the immune-responsive gene 1 (Irg1),
a previously described lipopolysaccharide-inducible gene, is one of the several
mRNAs that are markedly down-regulated by RU486 in the preimplantation uterus.
In the present study, we performed in situ hybridization to show that P
stimulates Irg1 mRNA synthesis in the luminal epithelial cells of uteri of
ovariectomized wild-type but not PR knockout mice. We also report that Irg1 mRNA
was induced in the luminal epithelium of pregt uterus between d 3 and 5,
overlapping the window of implantation. To investigate the function of Irg1
during implantation, we administered sense or antisense oligodeoxynucleotides
into preimplantation mouse uteri. Treatment with antisense oligodeoxynucleotides
led to suppression in Irg1 mRNA expression without affecting unrelated mRNAs in
the pregt uterus. This intervention was also accompanied by impairment in
embryo implantation, indicating that the phenotype is linked to the suppression
of Irg1 mRNA. Collectively, our studies identified Irg1 as a novel target of PR
in the pregt uterus and also revealed that it is a critical regulator of the
early events leading to implantation. Activation of the receptors for leukemia inhibitory factor (LIF) and IL-11 is
essential for embryo attachment and decidualization in mice. Both receptors
induce activation of the Stat family of signal transducers via the Jak/Stat
pathway. Here, we aimed to establish whether activation of Stat3 in maternal
endometrium is essential for successful implantation. Functional blockade of
Stat3 before implantation, by injection into the uterine lumen of a
cell-permeable Stat3 peptide inhibitor, reduced embryo implantation specifically
by 70% (P < 0.001). Stat3 is phosphorylated in the luminal epithelium (LE) in
response to LIF, and this phosphorylation was significantly reduced both in
vitro and in vivo by the Stat3 inhibitor. The inhibitor also blocked induction
by LIF of several LIF-regulated genes in the LE including Irg1, which has been
shown previously to be essential for implantation. Successful implantation is
therefore dependent on phosphorylation and activation of Stat3 in the
endometrium before implantation. This finding provides a target for
contraceptive development, based on selective blockade of signal transduction
pathways essential for implantation. This study demonstrates that cell-permeable
peptide inhibitors can be used effectively to target intracellular signaling
pathways in the uterine LE. Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis
in rumits. In addition, MAP is presently the most favored pathogen linked to
Crohn's disease. In this study, we were interested in dissecting the molecular
mechanisms of macrophage activation or deactivation after infection with MAP. By
subtractive hybridization of cDNAs, we identified the immune-responsive gene 1
(IRG1), which was expressed substantially higher in lipopolysaccharide
(LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear
run-on transcription assay revealed that the IRG1 gene was activated
transcriptionally in LPS-stimulated and MAP-infected macrophages with higher
expression in LPS-stimulated cells. Analysis of post-transcriptional regulation
demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in
MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages
infected with the nonpathogenic Mycobacterium smegmatis differed from those of
LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M.
smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h
postinfection, it increased nearly to the level in LPS-stimulated macrophages.
Transient transfection experiments revealed similar IRG1 promoter activities in
MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased
IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA
stabilization was p38 mitogen-activated protein kinase-independent. Inhibition
of protein synthesis revealed that constitutively expressed factors seemed to be
responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that
transcriptional and post-transcriptional mechanisms are responsible for a
differential IRG1 gene expression in murine macrophages treated with LPS, MAP,
and M. smegmatis. Interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) are essential
cytokines for successful clearance of microbial infections. Activation of
macrophages by synergistic effects of these cytokines leads to induction of
antimicrobial effector systems like reactive oxygen and reactive nitrogen
intermediates. Strikingly, IFN-gammaR(-/-) and TNFRp55(-/-) mice are
considerably more susceptible to infections than inducible nitric oxide
synthase(-/-) and p47phox(-/-) mice. Thus we applied transcriptome-profiling
studies to identify genes synergistically upregulated by IFN-gamma and TNF in
macrophages which are potentially involved in the defense against intracellular
pathogens. From a total of 234 regulated genes we found 35 genes that were
upregulated by combined effects of IFN-gamma and TNF and were at least 2-fold
induced. The majority of these genes are involved in signal transduction and
transcriptional regulation. However, we found several genes were poorly
characterized with regard to immunological functions. As a prototypic TNF- and
IFN-gamma-coregulated gene we characterized the expression and the subcellular
localization of immunoresponsive gene 1 (IRG1) in murine macrophages. IRG1 is
highly upregulated in murine ANA-1 macrophages by several proinflammatory
cytokines and Toll-like receptor (TLR) agonists, as well as in spleen and lung
of Listeria monocytogenes or Toxoplasma gondii infected mice, respectively.
Furthermore, this study identifies 35 genes that constitute the
IFN-gamma/TNF-triggered effector program in innate immunity. Murine immune-responsive gene 1 (IRG1) plays significant roles in embryonic
implantation and neurodegeneration. The expression pattern of the human IRG1
gene, however, has not yet been established, and the predicted gene sequence has
been revised several times according to computed expressed sequence tags (ESTs).
To determine the human IRG1 gene expression profile, human fetal tissue samples,
peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the
human leukemia cell lines THP-1 and K-562 challenged with lipopolysaccharide
(LPS) were subjected to RT-PCR using degenerate primers. The results indicated
that the IRG1 gene is differentially expressed in human fetal PBMCs and
LPS-stimulated adult PBMCs. The amplified gene fragment was cloned into the
pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system.
After affinity chromatography, human IRG1h fusion proteins were isolated by
SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen
to immunize rabbits. The titer and specificity of the purified rabbit antiserum
were sufficient to measure human IRG1 gene expression in various tissues and
cultures. This purified polyclonal antiserum will allow us to initiate studies
to elucidate the biological roles of the human IRG1 gene. Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in
vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates
these inflammatory responses still remains elusive. We hypothesize that IL-10
inhibits effector functions of multiple genes induced by B. burgdorferi in
macrophages to control concomitantly elicited inflammation. Because macrophages
are essential in the initiation of inflammation, we used mouse J774 macrophages
and live B. burgdorferi spirochetes as the model target cell and stimulant,
respectively. First, we employed transcriptome profiling to identify genes that
were induced by stimulation of cells with live spirochetes and that were
perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly
induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10
inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h
genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes.
Prominent among the novel identified IL-10-inhibited genes also validated by
quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2,
IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using
a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10
modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2
(MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor
(G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha
(TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were
obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show
that IL-10 alters effectors induced by B. burgdorferi in macrophages to control
concomitantly elicited inflammatory responses. Moreover, for the first time,
this study provides global insight into potential mechanisms used by IL-10 to
control Lyme disease inflammation. |
Have hESC been tested for the treatment of age-related macular degeneration? | Yes, human embryonic stem cell (hESC) therapies are being assessed for age-related macular degeneration (AMD). | Age-related macular degeneration (AMD) is characterized by the loss or
dysfunction of retinal pigment epithelium (RPE) and is the most common cause of
vision loss among the elderly. Stem-cell-based strategies, using human embryonic
stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide
an abundant donor source for generating RPE cells in cell replacement therapies.
Despite a significant amount of research on deriving functional RPE cells from
various stem cell sources, it is still unclear whether stem-cell-derived RPE
cells fully mimic primary RPE cells. In this report, we demonstrate that
functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with
varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like
morphology, transcripts, proteins and phagocytic function similar to human fetal
RPE (fRPE) cells. In addition, we performed global gene expression profiling of
stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated
hESCs and fibroblasts to determine the differentiation state of
stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells
closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a
unique differentiation state. Furthermore, we identified a set of 87 signature
genes that are unique to human fRPE and a majority of these signature genes are
shared by stem-cell-derived RPE cells. These results establish a panel of
molecular markers for evaluating the fidelity of human pluripotent stem cell to
RPE conversion. This study contributes to our understanding of the utility of
hESC/hiPSC-derived RPE in AMD therapy. Age-related macular degeneration (AMD) is the leading cause of vision loss in
older adults and ultimately leads to the death of photoreceptor cells in the
macular area of the neural retina. Currently, treatments are only available for
patients with the wet form of AMD. In this review, we describe recent approaches
to develop cell-based therapies for the treatment of AMD. Recent research has
focused on replacing the retinal pigment epithelium (RPE), a monolayer of cells
vital to photoreceptor cell health. We discuss the various methods used to
differentiate and purify RPE from human embryonic stem cells (HESC), and
describe the surgical approaches being used to transplant these cells in
existing and forthcoming clinical trials. |
What disease in Loxapine prominently used for? | The best indication of loxapine is paranoid schizophrenia. | Since the beginning of the neuroleptics in 1952, French psychiatrists have
proposed a classification of neuroleptics, taking into account the
pharmacological and therapeutic differences between these drugs. They
distinguished 3 different clinical effects of neuroleptics: sedative effects,
effects on the positive symptoms of schizophrenia and effects on the negative
symptoms. The effect of some neuroleptics on negative symptoms is recognized by
the international community, which considers clozapine to be effective. In
France, in most cases, the indication of clozapine is still refractory paranoid
schizophrenia. The effect of this atypical neuroleptic on other types of
schizophrenic patient is not well known. Remoxipride appears to be as effective
in treating psychotic symptoms and to have fewer side effects than haloperidol.
Remoxipride is effective for both positive and negative symptoms. Loxapine has
been prescribed in France since 1980. Its pharmacological profile is close to
that of clozapine: it has dopamine (D2), histamine (H1), serotonin (5-HT2) and
adrenergic (alpha 1)-blocking activities. Its best indication seems to be
paranoid schizophrenia, although some data suggest bipolar action. The bipolar
action of some new neuroleptics is illustrated by amisulpride, a substitute
benzamide derivative. The originality of this molecule lies in its two opposite
actions at two distinct doses. Doses of 600-1200 mg/day are effective against
positive symptoms; 50-150 mg/day improves negative symptoms. This latter effect
could be mediated by activation of the dopamine system.(ABSTRACT TRUNCATED AT
250 WORDS) |
What is the systemic nickel allergy syndrome? | A severe form of this allergy is the Systemic nickel allergy syndrome, clinically characterized by cutaneous manifestions (contact dermatitis, pompholyx, hand dermatitis dyshydrosis, urticaria) with chronic course and systemic symptoms (headache, asthenia, itching, and gastrointestinal disorders related to histopathological alterations of gastrointestinal mucosa, borderline with celiac disease). | Nickel sensitization can not only induce allergic contact dermatitis (ACD), but
also can induce an overlapping disease referred to as "systemic nickel allergy
syndrome" (SNAS), characterized by urticaria/angioedema and gastrointestinal
symptoms correlated to the ingestion of nickel-containing foods. This study was
designed to determine if oxidative stress occurs in patients with nickel
allergy. Thirty-one female patients (mean age 31.26 + 13.04 years, range 16-64
years) with confirmed nickel CD underwent oral nickel challenge because of
clinically suspected SNAS; serum concentrations of protein carbonyl groups
(PCGs) and nitrosylated proteins (NPs; biomarkers of oxidative stress) were
measured before and after oral nickel challenge as well as in healthy female
controls. Twenty-three of these 31 patients were diagnosed with SNAS because
they had a positive reaction to the oral nickel challenge, and 8 patients had no
reaction and therefore were classified as patients with contact nickel allergy
only. Although both nickel-allergic patients and controls presented similar
serum levels of PCGs, NP values in nickel-allergic patients appeared higher than
in controls and tended to decrease after the challenge; furthermore, serum
levels of NPs in patients affected by SNAS were higher (although not
significantly) than in patients with nickel ACD only. The involvement of
specific biomarkers of oxidative stress such as NPs and the lack of involvement
of other biomarkers such as PCGs may help to better understand the alteration of
the redox homeostasis occurring in nickel ACD and particularly in SNAS. Some patients with nickel (Ni) allergic contact dermatitis suffer from systemic
(intestinal or cutaneous) symptoms after ingestion of Ni-rich foods and
experience symptoms reduction with low-Ni diet, a condition termed Systemic Ni
Allergy Syndrome (SNAS). We aimed at evaluating whether oral administration of
low nickel doses improved clinical conditions and modulated immunological
aspects of SNAS, without significant side effects. Thirty-six SNAS patients were
enrolled. Treatment started after 1-month of low-Ni diet and consisted in an
incremental oral NiOH dose phase (0.3ng to 1.5 microg/week) followed by a
12-months maintece phase (1.5 microg/week). Randomly, twenty-four patients
added Ni therapy to low-Ni diet and 12 remained with diet alone. All patients
were allowed rescue medications (antihistamines and topical steroids). After 4
months, Ni-rich foods were gradually reintroduced. In vitro allergen-driven
IL13, IL5 and IFN-gamma release by peripheral blood mononuclear cells was
evaluated before and after treatment. Twenty-three patients receiving NiOH and
the 12 control patients completed the study. Evaluation of SNAS clinical
severity (by VAS and drug consumption) showed a significant difference in favor
of NiOH-treated patients compared to controls. Twenty of 23 patients in the NiOH
group and none in the control group tolerated Ni-rich food reintroduction.
Release of all studied cytokines in culture supernatants was significantly lower
after NiOH treatment. In conclusion NiOH is effective in reducing symptoms and
drug consumption in SNAS and is able to modulate inflammatory parameters. Nickel is a very common metal contained in many everyday objects and is the
leading cause of ACD (Allergic Contact Dermatitis). Nickel is present in most of
the constituents of a normal diet, but some food groups are usually considered
to be richer. However, the nickel content of specific food can vary widely,
depending on many factors. Thus, the daily intake of nickel is also highly
variable both among different populations and in a single individual, in
different seasons and even in different days. Measuring precisely the daily
intake of nickel from food and drinks is extremely difficult, if not impossible.
The relationship between ACD and contact with nickel is undisputed and widely
confirmed in literature. The situation is different for systemic nickel allergy
syndrome (SNAS). The SNAS can have cutaneous signs and symptoms (Systemic
Contact Dermatitis or SCD) or extracutaneous signs and symptoms
(gastrointestinal, respiratory, neurological, etc.).The occurrence of SCD as a
systemic reaction to the nickel normally assumed in the daily diet is very
controversial. A rigorous demonstration of the relationship between SCD and
nickel is extremely difficult. In particular, further and larger studies are
needed to assess the reality and the prevalence of nickel urticaria. With
respect to nickel-related gastrointestinal symptoms, as well as chronic fatigue
syndrome, fibromyalgia, headache, recurring cold sores and recurrent infections
in general, the data available in literature are not conclusive and the studies
lack the support of clear, first-hand evidence. With respect to respiratory
disorders, the role of food nickel and the effectiveness of a dietary treatment
have been assumed but not proven. In fact, the usefullness of a therapeutic
low-nickel diet is controversial: rare, if not exceptional, and limited to very
sporadic cases of SCD. Additionally, the quantitative and qualitative
composition of a low-nickel diet presents few certainties and many
uncertainties. The low-nickel diets suggested in literature are highly variable,
both in the extension of the restrictions and in their details--and the
differences are not marginal.
CONCLUSION: an evaluation of the data presented by medical literature about SNAS
and its relationship with oral nickel does not allow to draw final conclusions.
In the absence of genuine certainty we can only conclude that further and
broader studies, more rigorously conducted, are needed. Some patients affected by nickel-contact allergy present digestive symptoms in
addition to systemic cutaneous manifestations, falling under the condition known
as systemic nickel allergy syndrome (SNAS). A nickel-related pro-inflammatory
status has been documented at intestinal mucosal level. The aim of the present
study is to evaluate the prevalence of lactose intolerance in patients affected
by SNAS compared to a healthy population. Consecutive patients affected by SNAS
referring to our departments were enrolled. The control population consisted of
healthy subjects without gastrointestinal symptoms. All subjects enrolled
underwent lactose breath test under standard conditions. One hundred and
seventy-eight SNAS patients and 60 healthy controls were enrolled. Positivity of
lactose breath test occurred in 74.7% of the SNAS group compared to 6.6% of the
control group. Lactose intolerance is highly prevalent in our series of patients
affected by SNAS. Based on our preliminary results, we can hypothesize that in
SNAS patients, the nickel-induced pro-inflammatory status could temporarily
impair the brush border enzymatic functions, resulting in hypolactasia. Further
trials evaluating the effect of a nickel-low diet regimen on lactase activity,
histological features and immunological pattern are needed. Some patients with nickel (Ni) allergic contact dermatitis (ACD) suffer from
systemic symptoms after ingestion of Ni-rich foods, a condition termed Systemic
Nickel Allergy Syndrome (SNAS). The aim of this study is to investigate in
children the relationship between Ni ACD and lymphocyte subsets or
susceptibility to infections. Nineteen children with Ni ACD and 18 controls
matched for sex and age were enrolled. All participants underwent patch test,
skin prick test and clinical assessment. Serum immunoglobulins and flow
cytometry for lymphocyte subset study were also evaluated. In children with Ni
ACD a higher incidence of recurrent upper respiratory tract infections and
recurrent otitis media were detected. Serum levels of immunoglobulins and
lymphocyte subsets did not show significant changes (p>0.05) between the two
groups studied. We can hypothesize that in children with Ni ACD the risk of
recurrent infections is increased. Although the clinical manifestations of SNAS
are still controversial, we can suppose that recurrent infections may be
considered a clinical symptom of this syndrome. Nowadays nickel is a global problem, related to occupations (mainly
metalworkers) and to life habits. The dietary intake, more than environments, is
the source of a basic immune identification and also of possible complications
after occupational inhalation or contact. Nickel insoluble compounds are proved
to be cancerogenic. Nickel ions are very reactive with proteins and oxidant,
then also potential irritant for the airways. Metallic nickel and soluble
compounds are sensitizers, causing dermatitis, rhinitis and asthma. Recently a
Systemic Nickel Allergy Syndrome (SNAS) has been identified in allergic
subjects, with a clinical picture of urticaria, general hitching, headache,
gastrointestinal troubles. SNAS may affect allergic occupational or
non-occupational ones. Studies are in progress aimed to an oral treatment
inducing immunotolerance. Nickel in urine seems to be the best tool for
monitoring nickel absorption. Subjects with Nickel sensitization proved by patch test may suffer of contact
eczema, but also of a Sistemic Nickel Allergy Syndrome (SNAS) consisting of
urticaria-like troubles, itch, erythema, cutaneous rush, headache, intestinal
symptoms, recurrent vesicular palmar dermatitis. 160 subjects (130 F, 30 M) were
classified into three groups and underwent dosage of Nickel in urines (U-Ni) and
blood (B-Ni). The two groups with SNAS showed an higher indicators of Nickel
absorption, while the only-eczema group did not. 95 subjects with SNAS were
enrolled for a Nickel-scanty diet: most of them improved. 24 ones again
symptomatic were admitted to an experimental treatment, by a schedule of oral
increasing microdose (ograms) of Nickel sulphate: all of them improved. In
conclusion Nickel pathology is changing, allergy seems to be due to different
mechanism, dietary intake is important, an immune-tolerance can be induced. Nickel ingested with food can elicit either systemic cutaneous or
gastrointestinal symptoms causing a systemic nickel allergy syndrome (SNAS) that
can be treated with tolerance by oral ingestion of the metal. It has been
suggested that interleukins 2 (IL-2) and 10 (IL-10) are involved in the
mechanisms underlying oral tolerance. We evaluated the clinical efficacy of oral
desensitization therapy in SNAS consisting in the administration of nickel
sulphate. Because nickel allergy prevalently affects women, only female subjects
(N = 22) were recruited. Oral nickel desensitizing therapy was associated with
low-nickel diet for three months. Before and after therapy, clinical conditions
were evaluated, and circulating cytokines IL-2 and IL-10 were measured. After
the two-year treatment, visual analogue scale (VAS) scores for symptoms were
significantly reduced (P less than 0.001). Patients were released by either
cutaneous or gastrointestinal symptoms and by tolerating nickel-containing food.
At the end of the treatment, nickel oral challenge test was negative in 18
patients, and IL-2 level in the serum was significantly reduced while IL-10 was
increased, although this datum was not statistically significant. Our study
confirms the clinical efficacy of nickel oral immunotherapy and focuses on the
mechanisms triggered by oral tolerance indicating that reduction of IL-2 can be
associated with success of oral nickel desensitizing therapy. Systemic (gastrointestinal and skin) reactions to ingestion of nickel rich foods
in patients with nickel allergic contact dermatitis characterize Systemic Nickel
Allergy Syndrome (SNAS). The objective of the study was to describe the
nosologic framework of the syndrome and to compare sensibility and specificity
for SNAS diagnosis between two different low nickel diets - BraMa-Ni and the
usually prescribed list of forbidden foods - along with patient adherence to
diet. One hundred forty-five patients with suspected SNAS (by history and
benefit from nickel dietary restrictions) were selected and orally challenged
with nickel for a definite diagnosis. Specificity and sensibility of the diets
were calculated in relation to the results of nickel challenges. The nosologic
framework of SNAS was deduced from the clinical pictures of 98 patients with
positive nickel challenge and characterized essentially by skin and
gastrointestinal symptoms, whereas all other symptoms (dizziness, headache etc.)
were never elicited by the oral nickel challenge. The specificity and
sensibility of BraMa-Ni in detecting SNAS were significantly higher than the
forbidden food list diet, with an excellent patient adherence. Therefore,
BraMa-Ni diet can be prescribed for the treatment of the syndrome other than for
the diagnosis, the gold standard of which remains the oral nickel challenge. BACKGROUND: This is the first randomized, double-blind, placebo-controlled trial
(EUDRACT No. 2009-013923-43) evaluating nickel oral hyposensitizing treatment
(NiOHT) in patients with "systemic nickel allergy syndrome" (SNAS),
characterized by Ni-allergic contact dermatitis and systemic reactions after
eating Ni-rich food.
METHODS: Adults with positive Ni-patch test, who reported symptoms suggesting
SNAS, which improved after Ni-poor diet, and were positive to Ni-oral challenge
were eligible. Patients were randomly assigned to three treatments (1.5 μg, 0.3
μg, or 30 ng Ni/week) or placebo for a year, with progressive reintroduction of
Ni-rich foods form the 5(th) month. Out of 141 patients randomized, 113
completed the trial. Endpoints were efficacy and tolerability of treatment.
RESULTS: During Ni-rich food re-introduction, the 1.5 μg Ni/week group had a
mean VAS score significantly higher than placebo (p = 0.044), with significant
improvement of gastrointestinal symptoms (p = 0.016;) and significantly fewer
rescue medications. Cutaneous manifestations also improved but without reaching
statistical significance. After the treatment, oral challenge with higher Ni
doses than at baseline were needed to cause symptoms to flare-up in
significantly more patients given 1.5 μg Ni/week than placebo (p = 0.05).
Patients reported no side-effects.
CONCLUSIONS: NiOHT is effective in SNAS, in particular on gastrointestinal
manifestations, with trend toward improvement of cutaneous symptoms. |
Which antibodies cause Riedel thyroiditis? | Riedel thyroiditis (Immunoglobulin G4-related thyroid disease) is caused by IgG4 antibodies. It is part of the spectrum of Ig4-related sclerosing disease.
It is associated with fibrosis and inflammation of the thyroid gland. | Patients with IgG4-related disease (IgG4-RD) typically have elevated serum
concentrations of IgG4 and share histopathologic features that are similar
across affected organ(s). IgG4-RD patients frequently require prolonged
treatment with glucocorticoids and are often unable to taper these medications.
Traditional disease-modifying antirheumatic drugs (DMARDs) are generally
ineffective. We assessed the clinical and serologic responses to B lymphocyte
depletion therapy in 10 consecutive patients with steroid- and DMARD-refractory
IgG4-RD.Ten patients with IgG4-RD were treated with rituximab (RTX) (2 infusions
of 1000 mg, 15 days apart). Clinical improvement was assessed by monitoring the
patient's ability to taper prednisone to discontinuation and to stop DMARDs; by
serial measurements of total IgG and IgG subclasses; and by follow-up radiologic
assessments guided by the patient's particular pattern of organ involvement. We
also developed and retrospectively applied the IgG4-RD Disease Activity Index
and Flare Tool.Organ involvement included the pancreas, biliary tree, aorta,
salivary glands (submandibular and parotid), lacrimal glands, lymph nodes,
thyroid gland, and retroperitoneum. Nine of 10 patients demonstrated striking
clinical improvement within 1 month of starting RTX. One patient with advanced
thyroid fibrosis associated with Riedel thyroiditis and a history of disease in
multiple other organ systems did not have improvement in the thyroid gland, but
the disease did not progress to involve new organs. All 10 patients were able to
discontinue prednisone and DMARDs following RTX therapy. Significant decreases
in IgG concentrations were observed for the IgG4 subclass only. Four patients
were re-treated with RTX after 6 months because of either symptom recurrence and
increasing IgG4 concentration at the time of peripheral B cell reconstitution (n
= 2) or because of physician discretion (n = 2). Repeated courses of RTX
maintained their effectiveness and resulted in further decreases in IgG4
concentrations. In patients who had an increased IgG4 concentration at the time
of presentation, the level of serum IgG4 appeared to be a reliable measure of
disease activity.IgG4-RD is an idiopathic, multiorgan inflammatory disease in
which diverse organ manifestations are linked by characteristic histopathologic
and immunohistochemical features. Treatment with RTX led to prompt clinical and
serologic improvement in refractory IgG4-RD in all patients with active
inflammation. Serial treatments with RTX may lead to progressive declines in
serum IgG4 concentrations and better disease control. Serum IgG4 concentrations
may remain low, and clinical disease activity may remain quiescent even after B
cell reconstitution in a significant proportion of patients. More than a century has passed since the first description of Hashimoto
thyroiditis (HT) as a clinicopathologic entity. HT is an autoimmune disease in
which a breakdown of immune tolerance is caused by interplay of a variety of
immunologic, genetic, and environmental factors. Thyrocyte injury resulting from
environmental factors results in expression of new or hidden epitopes that leads
to proliferation of autoreactive T and B cells. Infiltration of thyroid by these
cells results in HT. In addition to the usual type of HT, several variants such
as the fibrous type and Riedal thyroiditis are also recognized. The most
recently recognized variant is immunoglobulin G4(+) HT, which may occur as
isolated thyroid limited disease or as part of a generalized Ig4-related
sclerosing disease. The relationship between HT and Riedel thyroiditis remains
unclear; however, recent evidence seems to suggest that it may also be part of
the spectrum of Ig4-related sclerosing disease. HT is frequently associated with
papillary thyroid carcinoma and may indeed be a risk factor for developing this
type of cancer. The relationship between thyroid lymphoma and HT on the other
hand appears well established. Hakaru Hashimoto described 4 patients with a hitherto unknown cause for goitre,
struma lymphomatosa, a century ago. He was careful to distinguish this from
Riedel thyroiditis but it has become clear that fibrosis and atrophy of the
thyroid are indeed components of Hashimoto thyroiditis, and in rare cases
IgG4-related sclerosing disease may be an outcome. Although the cause of the
lymphocytic infiltration was unknown to Hashimoto, we now know through the
pioneering studies of N.R. Rose and E. Witebsky [J Immunol 1956;76:417-427] that
this condition is the archetype for autoimmune destruction as a disease
mechanism. In the last two decades in particular, there has been huge interest
in unravelling the genetic basis for this and related autoimmune disorders. The
list of polymorphisms associated with autoimmune thyroid disease grows each
year, and in the case of vitiligo, which is frequently found in association with
thyroid autoimmunity, we know that 27 separate susceptibility loci account for
less than 20% of the heritability of this condition. Environmental and
existential factors may turn out to be just as complex in number and in
interactions. We can thus imagine a 'Swiss cheese' model for the causation of
autoimmune thyroid disease, in which the effects of cumulative weaknesses line
up - like the holes in slices of cheese - to allow the catastrophic event of
autoimmune destruction to occur. Immunoglobulin G4-related sclerosing disease (IgG4-RSD) represents a recently
identified inflammatory disorder in which infiltration of IgG4 plasma cells
causes fibrosis in organs. While IgG4-RSD is well documented in the pancreas and
other organs, it is poorly characterized in the thyroid gland. We report a case
of a 48-year-old female with a fibrotic thyroid mass associated with a
retroperitoneal fibrosis. Diagnosed early as Riedel disease, the high serum
IgG4, immunohistopathology and decreased fibrosis with corticosteroid therapy,
finally confirm for the first time, the origin of IgG4-RSD fibrosis of the
thyroid. Immunoglobulin (Ig)G4-related sclerosing disease (IgG4-RSD) is a new disease
entity first proposed with regard to autoimmune pancreatitis. A 67-year-old male
patient was examined because of weight loss and an abdominal pain. Based on the
clinical characteristics, laboratory parameters and ultrasound features, we
identified the diagnosis of the IgG4-related systemic disease (IgG4-RSD), that
was confirmed by the histopathological analysis after the biopsy of the head of
pancreas. After confirmation, we started with the corticosteroid therapy with a
good clinical, biochemical and morphological response. During the previous
therapy, the disturbance of glucoregulation appeared, so we had to change the
modality of treatment. We decided to add Azathioprine to the therapy in a dose
of 150 mg/day. We achieved a stable phase of the disease with IgG 4.37 g/l and
IgG4 0.179 g/l, and with no side effects from the therapy.
LEARNING POINTS: There are potential clinical applications of identifying
subsets of patients with IgG4 thyroiditis (FVHT and Riedel thyroiditis).A trial
of immunosuppressive therapy should be included if a resection is deemed
inadvisable.In particular, cases of FVHT that mimic maligcy, tissue and serum
IgG4 may provide supportive diagnostic information. |
What are the effects of ILK ablation? | Depending on the tissue or cell where ILK is ablated we see different effects:
Ablation of ILK in heart results in dilated cardiomyopathy and spontaneous heart failure
Ablation of ILK in fibroblasts leads to impaired healing due to a severe reduction in the number of myofibroblasts
Ablation of ILK in osteoclasts inhibits bone resorption
Ablation of ILK in liver results in enhanced and prolonged cell proliferation and hepatomegaly after phenobarbital administration and in abnormal histology
Ablation of ILK in podocytes caused an aberrant distribution of nephrin and alpha-actinin-4 | Integrin-linked kinase (ILK) has been implicated in the pathogenesis of
proteinuria and congenital nephrotic syndrome. However, the function of ILK in
glomerular podocyte in a physiologic setting remains unknown. In this study, a
mouse model was generated in which ILK gene was selectively disrupted in
podocytes by using the Cre-LoxP system. Podocyte-specific ablation of ILK
resulted in heavy albuminuria, glomerulosclerosis, and kidney failure, which led
to animal death beginning at 10 wk of age. Podocyte detachment and apoptosis
were not observed at 4 wk of age, when albuminuria became prominent, indicating
that they are not the initial cause of proteinuria. Electron microscopy revealed
an early foot process effacement, as well as morphologic abnormality, in
ILK-deficient podocytes. ILK deficiency caused an aberrant distribution of
nephrin and alpha-actinin-4 in podocytes, whereas the localization of podocin
and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated
that ILK physically interacted with nephrin to form a ternary complex, and
alpha-actinin-4 participated in ILK/nephrin complex formation. Therefore, ILK
plays an essential role in specifying nephrin and alpha-actinin-4 distribution
and in maintaining the slit diaphragm integrity and podocyte architecture. These
results also illustrate that the integrin and slit diaphragm signals in
podocytes are intrinsically coupled through an ILK-dependent mechanism. A requirement for integrin-mediated adhesion in cardiac physiology is revealed
through targeted deletion of integrin-associated genes in the murine heart. Here
we show that targeted ablation of the integrin-linked kinase (ILK) expression
results in spontaneous cardiomyopathy and heart failure by 6 wk of age. Deletion
of ILK results in disaggregation of cardiomyocytes, associated with disruption
of adhesion signaling through the beta1-integrin/FAK (focal adhesion kinase)
complex. Importantly, the loss of ILK is accompanied by a reduction in cardiac
Akt phosphorylation, which normally provides a protective response against
stress. Together, these results suggest that ILK plays a central role in
protecting the mammalian heart against cardiomyopathy and failure. During development, Schwann cells (SCs) interpret different extracellular cues
to regulate their migration, proliferation, and the remarkable morphological
changes associated with the sorting, ensheathment, and myelination of axons.
Although interactions between extracellular matrix proteins and integrins are
critical to some of these processes, the downstream signaling pathways they
control are still poorly understood. Integrin-linked kinase (ILK) is a focal
adhesion protein that associates with multiple binding partners to link
integrins to the actin cytoskeleton and is thought to participate in integrin
and growth factor-mediated signaling. Using SC-specific gene ablation, we report
essential functions for ILK in radial sorting of axon bundles and in
remyelination in the peripheral nervous system. Our in vivo and in vitro
experiments show that ILK negatively regulates Rho/Rho kinase signaling to
promote SC process extension and to initiate radial sorting. ILK also
facilitates axon remyelination, likely by promoting the activation of downstream
molecules such as AKT/protein kinase B. Following liver regeneration after partial hepatectomy, liver grows back
precisely to its original mass and does not exceed it. The mechanism regulating
this "hepatostat" is not clear and no exceptions have been found to date.
Although pathways initiating liver regeneration have been well studied,
mechanisms involved in the termination of liver regeneration are unclear. Here,
we report that integrin-linked kinase (ILK) (involved in transmission of the
extracellular matrix [ECM] signaling by way of integrin receptors) and/or
hepatic adaptations that ensue following ILK hepatocyte-targeted removal are
critical for proper termination of liver regeneration. Following partial
hepatectomy (PHx), mice with a liver-specific ILK ablation (ILK-KO-Liver)
demonstrate a termination defect resulting in 58% larger liver than their
original pre-PHx mass. This increase in post-PHx liver mass is due to sustained
cell proliferation driven in part by increased signaling through hepatocyte
growth factor (HGF), and the beta-catenin pathway and Hippo kinase pathways.
CONCLUSION: The data indicate that ECM-mediated signaling by way of ILK is
essential in proper termination of liver regeneration. This is the first
evidence of a defect leading to impaired termination of regeneration and
excessive accumulation of liver weight following partial hepatectomy. Wound healing crucially relies on the mechanical activity of fibroblasts
responding to TGFβ1 and to forces transmitted across focal adhesions.
Integrin-linked kinase (ILK) is a central adapter recruited to integrin β1 tails
in focal adhesions mediating the communication between cells and extracellular
matrix. Here, we show that fibroblast-restricted inactivation of ILK in mice
leads to impaired healing due to a severe reduction in the number of
myofibroblasts, whereas inflammatory infiltrate and vascularization of the
granulation tissue are unaffected. Primary ILK-deficient fibroblasts exhibit
severely reduced levels of extracellular TGFβ1, α-smooth muscle actin (αSMA)
production and myofibroblast conversion, which are rescued by exogenous TGFβ1.
They are further characterized by elevated RhoA and low Rac1 activities,
resulting in abnormal shape and reduced directional migration. Interference with
RhoA-ROCK signaling largely restores morphology, migration and TGFβ1 levels. We
conclude that, in fibroblasts, ILK is crucial for limiting RhoA activity, thus
promoting TGFβ1 production, which is essential for dermal repair following
injury. |
Does d-tubocurarine (d-TC) induces irreversible inhibition of nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction? | The d-tubocurarine is a nondepolarizing neuromuscular blocking agent (nondepolarizing muscle relaxant - NDMR). The nondepolarizing muscle relaxants act by blocking the nicotinic acetylcholine receptors of the neuromuscular junction. The neuromuscular blocking action of tubocurarine is reversible and concentration-dependent. The inhibition of acetylcholine-induced currents by d-tubocurarine can be reversed by anticholinesterase agents, such as edrophonium and methamidophos. | 1. Although (+)-tubocurarine (Tc) is classically considered to be a competitive
antagonist at the neuromuscular junction, kinetic details of the interaction
remain unclear. 2. We studied the competitive action of Tc on the nicotinic
receptor at the frog neuromuscular junction using a quantitative analysis of the
generation phase of miniature endplate currents (m.e.p.cs) recorded in Ringer
solution (20 degrees C) under voltage clamp (-90 mV) in the absence or presence
of 1-5 microM Tc. Under control conditions four neurotransmission parameters
were estimated by non-linear regression using a mathematical model of synaptic
transmission incorporating transmitter release, diffusion, hydrolysis, receptor
binding and channel gating. These parameters were then used in a further
regression to estimate binding rate constants for Tc at the same endplate.
Allowance was made for open channel block by Tc, which under the conditions of
this study was only a small component of total blockade. 3. The results suggest
that Tc binds to the two agonist recognition sites on the nicotinic receptor
with equal affinity (stoichiometric KDs of 2.2 and 8.8 microM), and that most of
the functional blockade at concentrations up to 5 microM is due to occupancy of
only one site. 4. The association rate constant for Tc binding to sites on the
nicotinic acetylcholine receptor appears to be very fast (k+D = 8.9 x 10(8) M-1
s-1) and comparable to that for acetylcholine (ACh). 5. In the brief time during
which an m.e.p.c. is generated (approximately 200 microseconds, reversal of Tc
blockade by transiently high concentrations of ACh seems to be kinetically
limited. Studies were conducted to ascertain the temporal and dose-dependent effects of
nicotinic ligand exposure on functional activity of different nicotinic
acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat
pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR)
clonal line. Chronic (3-72-h) agonist (nicotine or carbamylcholine) treatment of
cells led to a complete (TE671) or nearly complete (PC12) loss of functional
nAChR responses, which is referred to as "functional inactivation." Some
inactivation of nAChR function was also observed for the nicotinic ligands
d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal
inactivation of nAChR function was observed within 3 min for TE671 cells and
within 10 min for PC12 cells treated with inactivating ligands. Functional
inactivation occurred with dose dependencies that could not always be reconciled
with those obtained for acute agonist activation of nAChR function or for acute
inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment
of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium
alone had no effect on levels of expression of functional nAChRs. However,
evidence was obtained that either of these antagonists protected TE671 cell
muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation
on long-term treatment with agonists. Recovery of TE671 cell nAChR function
following treatment with carbamylcholine, nicotine, or d-TC occurred with
half-times of 1-3 days whether cells were maintained in situ or harvested and
replated after removal of ligand. By contrast, 50% recovery of functional nAChRs
on PC12 cells occurred within 2-6 h after drug removal. In either case the time
course for recovery from nAChR functional inactivation is much slower than
recovery from nAChR "functional desensitization," which is a reversible process
that occurs on shorter-term (0-5-min) agonist exposure of cells. These results
indicate that ganglia-type and muscle-type nAChRs are similar in their
sensitivities to functional inactivation by nicotinic ligands but differ in
their rates of recovery from and onset of those effects. The ability of drugs
such as the agonists d-TC, decamethonium, and mecamylamine to induce functional
inactivation may relate to their activities as partial/full agonists, channel
blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and
alcuronium are likely to reflect simple competitive inhibition of primary ligand
binding at functional activation sites.(ABSTRACT TRUNCATED AT 400 WORDS) The pharmacological diversity of the different isoforms of the nicotinic
acetylcholine receptor arises from the diversity of the subunits that assemble
to form the native receptors. The aim of this study was to investigate the
actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on
different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle,
mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression
system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits
(the native foetal muscle subunit combination), or with cRNAs for alpha, beta,
epsilon, delta subunits (the native adult muscle subunit combination), or with
cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination).
Acetylcholine had a similar potency at all three subunit combinations (EC50
11.6, 17.4 and 19.1 microM, respectively). At all three receptor types,
d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine
in a reversible manner. Furthermore, the inhibition of the acetylcholine
currents for the foetal and adult nicotinic acetylcholine receptor by
pancuronium and d-tubocurarine was independent of the holding voltage over the
range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic
acetylcholine receptors the inhibition of the current in response to 100 microM
acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7),
and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P
> 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic
acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both
more effective at blocking the response to 100 microM acetylcholine compared to
the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P
< 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the
developmental switch from the gamma to the epsilon subunit alters the antagonism
of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine.
Vecuronium was more potent than pancuronium. One nM vecuronium reduced the
response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/-
5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2
neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a
more effective antagonist of the response to 100 microM acetylcholine (69 +/-
6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to
pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine
receptor, where pancuronium and d-tubocurarine were equieffective. The
expression of the beta2 subunit with muscle alpha, epsilon and delta subunits
formed a functional receptor which was blocked by pancuronium and d-tubocurarine
in a similar manner to the alphabeta1epsilondelta subunit consistent with the
hypothesis that the beta subunit is not a major determit in the action of
this drug at the adult muscle nicotinic acetylcholine receptor. Equilibrium conditions of neurotransmitter concentration and receptor binding
are never achieved during synaptic transmission at the neuromuscular junction.
Thus, it is important to determine the binding kinetics of drugs that act this
synapse. Previous determinations of the dissociation rate of (+)-tubocurarine
have produced inconsistent results ranging from 0.1 to 4000/s. Here, we used a
direct approach to measure association (l(on)) and dissociation (l(on)) rates
for two competitive antagonists (clinically used as nondepolarizing muscle
relaxants), pancuronium and (+)-tubocurarine, at nicotinic acetylcholine
receptors (nAChR). We made macroscopic current recordings from outside-out
patches of BC3H-1 cells expressing embryonic mouse muscle nAChR. We used a
three-tube rapid perfusion system to make timed applications of antagonists and
acetylcholine to the patch. We made independent measurements of the equilibrium
inhibition (IC(50)) and the kinetics of onset and recovery of antagonist
inhibition at 20 to 23 degrees C. Rate constants were calculated from the
predictions of a single (high-affinity) site model of competitive inhibition.
For pancuronium: IC(50) = 5.5 +/- 0.5 nM (mean +/- S.D.), l(on) = 2.7 +/- 0.9 x
10(8) M(-1) s(-1), l(off) = 2.1 +/- 0.7/s [corrected] x 10(8)/s. For
(+)-tubocurarine: IC(50) = 41 +/- 2 nM, l(on) = 1.2 +/- 0.2 x 10(8) M(-1) s(-1),
l(off) = 5.9 +/- 1.3/s. The kinetic results are consistent with the equilibrium
results in that l(off)/l(on) is in good agreement with the IC(50) values. All
differences between the antagonists are significant at the p < 0.001 level. The
higher affinity of pancuronium is caused by a faster association rate (2.2-fold)
coupled with a slower dissociation rate (2.8-fold). The association rates of
both antagonists are comparable with or greater than the association rate for
acetylcholine binding to nAChR. We studied the inhibition of fetal (gamma-nAChR) and adult (epsilon-nAChR)
muscle-type nicotinic acetylcholine receptors by the two new nondepolarizing
muscle relaxants (NDMRs) rocuronium and rapacuronium, the metabolite 3-desacetyl
rapacuronium (Org 9488), and five other, longer-used NDMRs (pancuronium,
vecuronium, mivacurium, d-tubocurarine, and gallamine). Receptors were expressed
in Xenopus laevis oocytes by cytoplasmic injection of subunit complementary
RNAs. Functional channels were activated with 10 microM acetylcholine, alone or
in combination with various concentrations of the NDMRs. Currents were recorded
with a whole-cell two-electrode voltage clamp technique. All NDMRs reversibly
inhibited acetylcholine-activated currents in a dose-dependent fashion.
Potencies of rapacuronium and Org 9488 were not statistically different at
either gamma-nAChR (half-maximal response = 58.2 and 36.5 nM, respectively) or
epsilon-nAChR (half-maximal response = 80.3 and 97.7 nM, respectively). The rank
order of potencies at the epsilon-nAChR (pancuronium > vecuronium similar
mivacurium > rocuronium similar d-tubocurarine > rapacuronium similar Org 9488 >
gallamine) correlated highly with the clinical doses needed to produce 50%
twitch depression at the adductor pollicis muscle in adults. Neuromuscular
blockade by rapacuronium may be enhanced by its metabolite Org 9488. Different
drug-receptor affinities of the tested NDMRs contribute to the differences in
clinical dose requirements of these drugs needed to achieve appropriate muscle
relaxation.
IMPLICATIONS: Potencies of nondepolarizing muscle relaxants, studied at muscle
nicotinic acetylcholine receptors expressed in a recombit expression system,
correlate highly with the clinical doses needed in adults to produce 50% twitch
depression at the adductor pollicis muscle. Volatile anesthetics enhance the neuromuscular blockade produced by
nondepolarizing muscle relaxants (NDMRs). The neuromuscular junction is a
postulated site of this interaction. We tested the hypothesis that volatile
anesthetic enhancement of muscle relaxation is the result of combined drug
effects on the nicotinic acetylcholine receptor. The adult mouse muscle
nicotinic acetylcholine receptor (alpha(2), beta, delta, epsilon) was
heterologously expressed in Xenopus laevis oocytes. Concentration-effect curves
for the inhibition of acetylcholine-induced currents were established for
vecuronium, d-tubocurarine, isoflurane, and sevoflurane. Subsequently,
inhibitory effects of NDMRs were studied in the presence of the volatile
anesthetics at a concentration equivalent to half the concentration producing a
50% inhibition alone. All individually tested compounds produced rapid and
readily reversible concentration-dependent inhibition. The calculated 50%
inhibitory concentration values were 9.9 nM (95% confidence interval [CI],
8.4-11.4 nM), 43.4 nM (95% CI, 33.6-53.3 nM), 897 microM (95% CI, 699-1150
microM), and 818 microM (95% CI, 685-1001 microM) for vecuronium,
d-tubocurarine, isoflurane, and sevoflurane, respectively. Coapplication of
either isoflurane or sevoflurane significantly enhanced the inhibitory effects
of vecuronium and d-tubocurarine, especially so at small concentrations of
NDMRs. Volatile anesthetics increase the potency of NDMRs, possibly by enhancing
antagonist affinity at the receptor site. This effect may contribute to the
clinically observable enhancement of neuromuscular blockade by volatile
anesthetics.
IMPLICATIONS: Isoflurane and sevoflurane enhance the receptor blocking effects
of nondepolarizing muscle relaxants on nicotinic acetylcholine receptors. BACKGROUND: Nondepolarizing neuromuscular blocking agents (NMBAs) are
extensively used in the practice of anesthesia and intensive care medicine.
Their primary site of action is at the postsynaptic nicotinic acetylcholine
receptor (nAChR) in the neuromuscular junction, but their action on neuronal
nAChRs have not been fully evaluated. Furthermore, observed adverse effects of
nondepolarizing NMBAs might originate from an interaction with neuronal nAChRs.
The aim of this study was to examine the effect of clinically used
nondepolarizing NMBAs on muscle and neuronal nAChR subtypes.
METHODS: Xenopus laevis oocytes were injected with messenger RNA encoding for
the subunits included in the human alpha1beta1epsilondelta, alpha3beta2,
alpha3beta4, alpha4beta2, and alpha7 nAChR subtypes. The interactions between
each of these nAChR subtypes and atracurium, cisatracurium, d-tubocurarine,
mivacurium, pancuronium, rocuronium, and vecuronium were studied using an
eight-channel two-electrode voltage clamp setup. Responses were measured as peak
current and net charge.
RESULTS: All nondepolarizing NMBAs inhibited both muscle and neuronal nAChRs.
The neuronal nAChRs were reversibly and concentration-dependently inhibited in
the low micromolar range. The mechanism (i.e., competitive vs. noncompetitive)
of the block at the neuronal nAChRs was dependent both on subtype and the NMBA
tested. The authors did not observe activation of the nAChR subtypes by any of
the NMBAs tested.
CONCLUSIONS: The authors conclude that nondepolarizing NMBAs
concentration-dependently inhibit human neuronal nAChRs. The inhibition of the
presynaptic alpha3beta2 nAChR subtype expressed at the motor nerve ending
provides a possible molecular explanation for the tetanic and train-of-four fade
seen during a nondepolarizing neuromuscular block. AIMS: The aim of this study was to investigate the mechanism for the reversal
effect of NF449 (a suramin analogue) on the neuromuscular block induced by
d-tubocurarine (d-TC).
MAIN METHODS: Nerve-stimulated muscle contractions and end-plate potentials were
performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine
(ACh)-induced muscle contractions were performed in the chick biventer cervicis
preparations. Presynaptic nerve terminal waveform recordings were performed in
mouse triangularis sterni preparations.
KEY FINDINGS: Amongst the suramin analogues in this study, only the NF449 and
suramin were able to reverse the blockade effect produced by d-TC on
nerve-stimulated muscle contractions. Each of these suramin analogues (NF007,
NF023, NF279 and NF449) alone has no significant effect on the amplitude of
nerve-stimulated muscle contractions. NF449 and suramin also showed the
antagonising effects on the inhibition of end-plate potentials induced by d-TC.
Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in
ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a
greater rightward shift of the dose-response inhibition curve for d-TC than did
suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and
NF279, do not have the reverse effects on the neuromuscular blockade of d-TC,
the effect of NF449 seems irrelevant to inhibition of P2X receptors.
SIGNIFICANCE: These data suggest that NF449 was able to compete with the binding
of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent
than suramin in reducing the inhibition of d-TC. The structure of NF449 may
provide useful information for designing potent antidotes against neuromuscular
toxins. |
Which receptors are bound by Tasimelteon? | Tasimelteon (HETLIOZ™) is an orally bioavailable agonist of the melatonin MT1 and MT2 receptors that has been approved in the US for the treatment of non-24-hour sleep-wake disorder. | Tasimelteon, developed by Vanda Pharmaceuticals Inc under license from
Bristol-Myers Squibb Co, is a melatonin receptor agonist. Because of the high
density of melatonin receptors in the circadian pacemaker, the suprachiasmatic
nucleus, melatonergic actions can phase-shift circadian rhythms and promote
sleep. Tasimelteon was effective in reducing sleep onset latency (in phase II
and III clinical trials) and in resetting the circadian melatonin rhythm (in
phase II trials), which indicated its potential suitability as treatment for jet
lag, shift work and circadian rhythm sleep disorders. Statistically significant
improvements in sleep maintece have also been observed with the drug.
Tasimelteon has been claimed to be useful in the treatment of depression, and
preclinical evidence in this respect is to be confirmed in a phase II clinical
trial, which was ready to be initiated at the time of publication. It is
plausible that the drug may be effective in the treatment of depressive
disorders, at least those that are related to circadian dysfunction, and that it
may attenuate sleep problems in depressed patients of different subtypes. A
general suitability in mitigating other symptoms of major depressive disorder
cannot be deduced from the actions of tasimelteon via the melatonin receptors
MT1 and MT2. The drug is well tolerated, does not induce impairment of next-day
functioning or dependence, and seems to be safe in short-term treatment;
however, toxicological data would be required for assessing its long-term
safety. Several melatonin receptors agonists (ramelteon, prolonged-release melatonin,
agomelatine and tasimelteon) have recently become available for the treatment of
insomnia, depression and circadian rhythms sleep-wake disorders. The efficacy
and safety profiles of these compounds in the treatment of the indicated
disorders are reviewed. Accumulating evidence indicates that sleep-wake
disorders and co-existing medical conditions are mutually exacerbating. This
understanding has now been incorporated into the new Diagnostic and Statistical
Manual of Mental Disorders, 5th Edition (DSM-5). Therefore, when evaluating the
risk/benefit ratio of sleep drugs, it is pertinent to also evaluate their
effects on wake and comorbid condition. Beneficial effects of melatonin receptor
agonists on comorbid neurological, psychiatric, cardiovascular and metabolic
symptomatology beyond sleep regulation are also described. The review underlines
the beneficial value of enhancing physiological sleep in comorbid conditions. |
Is zyxin a focal adhesion protein? | Yes, zyxin is a focal adhesion protein. | BACKGROUND: SIRT1 is a mammalian homologue of NAD+-dependent deacetylase sirtuin
family. It regulates longevity in several model organisms and is involved with
cell survival, differentiation, metabolism among other processes in mammalian
cells. SIRT1 modulates functions of various key targets via deacetylation.
Recent studies have revealed SIRT1 protects neurons from axonal degeneration or
neurodegeneration. Further, SIRT1 null mice exhibit growth retardation and
developmental defects, suggesting its critical roles in neurons and development.
RESULTS: To identify novel binding partners for SIRT1 in the central nervous
system, we performed yeast two-hybrid screening on human fetal brain cDNA
library and found that zyxin is a possible binding partner. SIRT1 and zyxin
transcript were both preferentially expressed in developmental mouse brain.
Zyxin accumulates in the nucleus where it is co-localized with SIRT1 after
treatment with leptomycin B in COS-7 cells. Furthermore, SIRT1 deacetylates
zyxin, suggesting SIRT1 could interact with nuclear-accumulated zyxin and
modulate its function through deacetylation.
CONCLUSION: Zyxin could be a novel interacting partner of SIRT1. Zyxin is an
adaptor protein at focal adhesion plaque, regulating cytoskeletal dynamics and
signal transduction to convey signal from the ECM (extracellular matrix) to the
nucleus. Our results raise the possibility that SIRT1 regulates signal
transmission from ECM to the nucleus by modulating the functions of zyxin via
deacetylation. Synemin is a unique cytoplasmic intermediate filament protein for which there is
limited understanding of its exact cellular functions. The single human synemin
gene encodes at least two splice variants named alpha-synemin and beta-synemin,
with the larger alpha-synemin containing an additional 312 amino acid insert
within the C-terminal tail domain. We report herein that, by using the entire
tail domain of the smaller beta-synemin as the bait in a yeast two-hybrid screen
of a human skeletal muscle cDNA library, the LIM domain protein zyxin was
identified as an interaction partner for human synemin. The synemin binding site
in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain
repeats, whereas the binding site for zyxin within beta-synemin is within the
C-terminal 332 amino acid region (SNbetaTII) at the end of the long tail domain.
Transient expression of SNbetaTII within mammalian cells markedly reduced zyxin
protein level, blocked localization of zyxin at focal adhesion sites and
resulted in decreased cell adhesion and increased motility. Knockdown of synemin
expression with siRNAs within mammalian cells resulted in significantly
compromised cell adhesion and cell motility. Our results suggest that synemin
participates in focal adhesion dynamics and is essential for cell adhesion and
migration. Focal adhesion proteins link cell surface integrins and intracellular actin
stress fibers and therefore play an important role in mechanotransduction and
cell motility. When endothelial cells are subjected to cyclic mechanical strain,
time-lapse imaging revealed that cells underwent significant morphological
changes with their resultant long axes aligned away from the strain direction.
To explore how this response is regulated by focal adhesion-associated proteins
the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were
knocked down using gene silencing techniques. In addition, rescue of endogenous
and two mutant zyxins were used to investigate the specific role of zyxin
interactions. Cells with decreased zyxin expression levels and rescue with the
mutant lacking zyxin/alpha-actinin binding exhibited lower orientation angles
after comparable times of stretching as compared to normal and control cells.
However, knockdown of the expression levels of paxillin and FAK and rescue with
the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding
did not significantly affect the degree of cell orientation. In addition, wound
closure speed and cell-substratum adhesive strength were observed to be
significantly reduced only for cells with zyxin depletion and the mutation
lacking zyxin/alpha-actinin binding. These results suggest that zyxin and its
interaction with alpha-actinin are important in the regulation of endothelial
cell adhesive strength, motility and orientation response to mechanical
stretching. We report the development and application of photoactivatable Green Cherry
(G(PA)C), the first genetically encoded "continuously red-photoactivatable
green" two-color probe for live cell imaging. G(PA)C is unique in that it
enables real-time tracking of selected subpopulations of proteins and organelles
in the cell or of cells within tissues and whole organisms, with constant
reference to the entire population of the probe. Using G(PA)C-zyxin as proof of
utility, we obtained new insights into the dynamic movement of the cytoskeletal
protein zyxin. We show that zyxin is continuously and rapidly recruited from the
cytosol into established focal adhesions. It can also move rapidly within a
given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the
dynamic nature of proteins within these structures. The in vivo utility of
G(PA)C is exemplified by tracking hemocyte movements using a versatile
transgenic Drosophila model engineered to express G(PA)C in tissues and cells of
interest under the control of the GAL4-inducible promoter. Cellular responses to mechanical perturbation are vital to cell physiology. In
particular, migrating cells have been shown to sense substrate stiffness and
alter cell morphology and speed. Zyxin is a focal adhesion protein that responds
to external mechanical forces; however, the mechanisms of zyxin recruitment at
force-bearing sites are unknown. Using force-sensing microfabricated substrates,
we simultaneously measured traction force and zyxin recruitment at force-bearing
sites. GFP-tagged zyxin accumulates at force-bearing sites at the leading edge,
but not at the trailing edge, of migrating epithelial cells. Zyxin recruitment
at force-bearing sites depends on Rho-kinase and myosin II activation,
suggesting that zyxin responds not only to the externally applied force, as
previously shown, but also to the internally generated actin-myosin force. Zyxin
in turn recruits vasodilator-stimulated phosphoprotein, a regulator of actin
assembly, to force-bearing sites. To dissect the domains of zyxin that are
essential for this unique force-dependent accumulation, we generated two zyxin
truncation mutants: one lacking the LIM domain (ΔLIM) and one containing only
the LIM domain with all three LIM motifs (LIM). GFP-tagged ΔLIM does not
localize to the force-bearing sites, but GFP-tagged zyxin LIM-domain is
sufficient for the recruitment to and dynamics at force-bearing focal adhesions.
Furthermore, one or two LIM motifs are not sufficient for force-dependent
accumulation, suggesting that all three LIM motifs are required. Therefore, the
LIM domain of zyxin recruits zyxin to force-bearing sites at the leading edge of
migrating cells. Tubulobulbar complexes (TBCs) are actin-related double-membrane invaginations
formed at intercellular junctions in the seminiferous epithelium of mammalian
testis. They occur at basal junction complexes between neighboring Sertoli cells
and at apical junctions between Sertoli cells and spermatids. They are proposed
to internalize intercellular junctions during the translocation of spermatocytes
from basal to adluminal compartments of the seminiferous epithelium, and during
sperm release from Sertoli cells. Although TBCs are specific to the seminiferous
epithelium, they morphologically resemble podosomes in osteoclasts. Previously,
we have reported that a key group of proteins consisting of N-WASp, Arp2/3,
cortactin and dynamin that occur at podosomes also is present at TBCs. Here we
explore the prediction that zyxin, a focal adhesion protein known to be present
at podosomes, also is present at apical TBCs. A rabbit polyclonal anti-zyxin
antibody (B71) was used to label fixed fragments and frozen sections of testis.
In both fragments and sections, B71 labeled tubular regions of TBCs at apical
sites of attachment between Sertoli cells and spermatids, in addition to being
localized at actin related intercellular adhesion junctions termed ectoplasmic
specializations. Although the function of zyxin at TBCs has yet to be
determined, the protein is known to interact with the cytoplasmic domain of
integrins at focal adhesions, and integrins are known to be present in TBCs. The ability to sense mechanical forces is vital to cell physiology. Yet, the
molecular basis of mechano-signaling remains unclear. Previous studies have
shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites
on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing
protein candidate. Furthermore, zyxin accumulation at force-bearing sites
requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain
consists of three LIM motifs, each containing two zinc-binding sites. Since
individual LIM motifs do not accumulate at focal adhesions or force-bearing
sites, we hypothesize that multiple zyxin LIM domains increase force
sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we
quantified the relationship between single, tandem dimer and trimer LIM protein
localization and traction forces. While the presence of extra LIM domains
affected VASP recruitment to focal adhesions, force sensitivity was not enhanced
over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a
single LIM domain, while additional LIM domains fail to enhance force
sensitivity. Altered cell motility is considered to be a key factor in determining tumor
invasion and metastasis. Epidermal growth factor (EGF) signaling has been
implicated in this process by affecting cytoskeletal organization and dynamics
in multiple ways. To sort the temporal and spatial regulation of EGF-dependent
cytoskeletal re-organization in relation to a cell's motile behavior time-lapse
microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells
co-expressing different fluorescently labeled cytoskeletal filaments and focal
adhesion components in various combinations. The experiments showed that EGF
almost instantaneously induces a considerable increase in membrane ruffling and
lamellipodial activity that can be inhibited by Cetuximab EGF receptor
antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells.
The transient cell extensions are rich in actin but lack microtubules and
keratin intermediate filaments. We show that this EGF-induced increase in
membrane motility can be measured by a simple image processing routine.
Microtubule plus-ends subsequently invade growing cell extensions, which start
to accumulate focal complexes at the lamellipodium-lamellum junction. Such
paxillin-positive complexes mature into focal adhesions by tyrosine
phosphorylation and recruitment of zyxin. These adhesions then serve as
nucleation sites for keratin filaments which are used to enlarge the neighboring
peripheral keratin network. Focal adhesions are either disassembled or give rise
to stable zyxin-rich fibrillar adhesions which disassemble in the presence of
EGF to support formation of new focal adhesion sites in the cell periphery.
Taken together the results serve as a basis for modeling the early cytoskeletal
EGF response as a tightly coordinated and step-wise process which is relevant
for the prediction of the effectiveness of anti-EGF receptor-based tumor
therapy. Focal adhesions are large protein complexes organized at the basal surface of
cells, which physically connect the extracellular matrix to the cytoskeleton and
have long been speculated to mediate cell migration. However, whether clustering
of these molecular components into focal adhesions is actually required for
these proteins to regulate cell motility is unclear. Here we use quantitative
microscopy to characterize descriptors of focal adhesion and cell motility for
mouse embryonic fibroblasts and human fibrosarcoma cells, across a wide range of
matrix compliance and following genetic manipulations of focal adhesion proteins
(vinculin, talin, zyxin, FAK, and paxilin). This analysis reveals a tight,
biphasic gaussian relationship between mean size of focal adhesions (not their
number, surface density, or shape) and cell speed. The predictive power of this
relationship is comprehensively validated by disrupting nonfocal adhesion
proteins (α-actinin, F-actin, and myosin II) and subcellular organelles
(mitochondria, nuclear DNA, etc.) not known to affect either focal adhesions or
cell migration. This study suggests that the mean size of focal adhesions
robustly and precisely predicts cell speed independently of focal adhesion
surface density and molecular composition. Zyxin is a focal adhesion protein that has been implicated in the modulation of
cell adhesion and motility, and is hypothesized to be a mechano-sensor in
integrin-mediated responses to mechanical force. To test the functional role of
zyxin in the mechanotransduction of microvascular smooth muscle cells (VSMC), we
utilized atomic force microscopy (AFM) to apply localized pulling forces to VSMC
through a fibronectin (FN) focal adhesion induced by a FN-coated bead on cell
surface. Application of force with the AFM induced an increase of zyxin
accumulation at the site of the FN-bead focal adhesion that accompanied the VSMC
contractile response. Whereas, reduction of zyxin expression by using a
zyxin-shRNA construct abolished the VSMC contractile response to AFM pulling
forces, even though the zyxin-silenced VSMCs displayed increased adhesion to FN
in both AFM adhesion assays and cell adhesion assays. The reduced zyxin
expression significantly impaired cell spreading and reorganization of the actin
cytoskeleton that could indicate a possible underlying reason for the loss of a
contractile response to mechanical force. Consistent with these observations, in
zyxin-silenced VSMC, we also observed a reduced expression of Rac1, which plays
an important role in the actin reorganization in VSMC, but increased thyroid
receptor-interacting proteins (TRIP6) and FAK expression, the latter being a
major protein that promote cell adhesion. In conclusion, these data support an
important enabling role for zyxin in VSMCs ability to mechanically respond to
applied force. Cells continuously sense and respond to external mechanical forces through their
cytoskeleton. Here we show that only a small subset of actin fibers, those
forming the perinuclear actin cap that wraps around the nucleus, form in
response to low physiological mechanical stresses in adherent fibroblasts. While
conventional basal stress fibers form only past a threshold shear stress of 0.5
dyn/cm(2), actin-cap fibers are formed at shear stresses 50 times lower and
orders-of-magnitude faster than biochemical stimulation. This fast differential
response is uniquely mediated by focal adhesion protein zyxin at low shear
stress and actomyosin fibers of the actin cap. We identify additional roles for
lamin A/C of the nuclear lamina and linkers of nucleus to cytoskeleton (LINC)
molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the
nucleus. These results suggest an interconnected physical pathway for
mechanotransduction, from the extracellular milieu to the nucleus. During heart valve development, epithelial-mesenchymal transformation (EMT) is a
key process for valve formation. EMT leads to the generation of mesenchymal
cells that will eventually become the interstitial cells (fibroblasts) of the
mature valve. During EMT, cell architecture and motility change markedly;
significant changes are also observed in various signaling pathways. Here we
systematically examined the expression, localization, and function of zyxin, a
focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis.
Expression and localization studies showed that zyxin was expressed in the AV
canal region during crucial stages of valve development. An in vitro 3D collagen
gel culture system was used to determine zyxin function either after siRNA gene
knockdown or after overexpression. Our studies revealed that zyxin
overexpression inhibited endocardial cell migration and cell differentiation and
also led to a decrease in the number of migrating mesenchymal cells. Moreover,
correlative cytoskeletal changes were apparent in response to both
overexpression and knockdown treatments. Thus, zyxin appears to play a role as a
regulator of cell migration and differentiation during EMT in chicken AV valve
formation. Treatment of cultured cells with inhibitors of actomyosin contractility induces
rapid deterioration of stress fibers, and disassembly of the associated focal
adhesions (FAs). In this study, we show that treatment with the Rho kinase
inhibitor Y-27632, which blocks actomyosin contractility, induces disarray in
the FA-associated actin bundles, followed by the differential dissociation of
eight FA components from the adhesion sites. Live-cell microscopy indicated that
the drug triggers rapid dissociation of VASP and zyxin from FAs (τ values of 7-8
min), followed by talin, paxillin and ILK (τ ~16 min), and then by FAK, vinculin
and kindlin-2 (τ = 25-28 min). Examination of the molecular kinetics of the
various FA constituents, using Fluorescence Recovery After Photobleaching
(FRAP), in the absence of or following short-term treatment with the drug,
revealed major changes in the kon and koff values of the different proteins
tested, which are in close agreement with their differential dissociation rates
from the adhesion sites. These findings indicate that mechanical,
actomyosin-generated forces differentially regulate the molecular kinetics of
individual FA-associated molecules, and thereby modulate FA composition and
stability. Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond
to cyclic strain by a varying degree of focal adhesion assembly and actin
reorganization. We speculated that the individual shape of the cells, which is
linked to cytoskeletal structure and pre-stress, might tune these
integrin-dependent mechanotransduction events. To this aim, fibronectin circles,
squares and rectangles of identical surface area (2000μm(2)) were micro-contact
printed onto elastomeric substrates. Fibroblasts plated on these patterns
occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to
patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesions
were quantified after fluorescence staining. After strain, megakaryocytic
leukemia-1 protein translocated to the nucleus in most cells, indicating
efficient RhoA activation independently of cell shape. However, circular and
square cells (with radial symmetry) showed a significantly greater increase in
the number of actin stress fibers and vinculin-positive focal adhesions after
cyclic strain than rectangular (bipolar) cells of identical size. Conversely,
cyclic strain induced larger changes in pY397-FAK positive focal complexes and
zyxin relocation from focal adhesions to stress fibers in bipolar compared to
symmetric cells. Thus, radially symmetric cells responded to cyclic strain with
a larger increase in assembly, whereas bipolar cells reacted with more
pronounced reorganization of actin stress fibers and matrix contacts. We
conclude that integrin-mediated responses to external mechanical strain are
differentially modulated in cells that have the same spreading area but
different geometries, and do not only depend on mere cell size. |
What is the role of ELMO1 gene in cell migration? | ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck.The ELMO1/DOCK180 complex then forms a guanine nucleotide exchange factor for Rac1, regulating its activation during cell migration in different biological systems. Rac activation by the ELMO.Dock180 complex at discrete intracellular locations is mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration. | The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs
crkII, dock180, and rac1, mediate cytoskeletal rearrangements during
phagocytosis of apoptotic cells and cell motility. Here, we describe an
additional member of this signaling pathway, ced-12, and its mammalian homologs,
elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells
and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with
CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1
binds directly to CED-5/Dock180; this evolutionarily conserved complex
stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal
rearrangements. These studies identify CED-12/ELMO as an upstream regulator of
Rac1 that affects engulfment and cell migration from C. elegans to mammals. Cell migration is essential throughout embryonic and adult life. In numerous
cell systems, the small GTPase Rac is required for lamellipodia formation at the
leading edge and movement ability. However, the molecular mechanisms leading to
Rac activation during migration are still unclear. Recently, a mammalian
superfamily of proteins related to the prototype member Dock180 has been
identified with homologues in Drosophila and Caenorhabditis elegans. Here, we
addressed the role of Dock180 and ELMO1 proteins, which function as a complex to
mediate Rac activation, in mammalian cell migration. Using mutants of Dock180
and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans
mutant lacking CED-5 (Dock180 homologue), we identified specific regions of
Dock180 and ELMO1 required for migration in vitro and in a whole animal model.
In both systems, the Dock180.ELMO1 complex formation and the ability to activate
Rac were required. We also found that ELMO1 regulated multiple Dock180
superfamily members to promote migration. Interestingly, deletion mutants of
ELMO1 missing their first 531 or first 330 amino acids that can still bind and
cooperate with Dock180 in Rac activation failed to promote migration, which
correlated with the inability to localize to lamellipodia. This finding suggests
that Rac activation by the ELMO.Dock180 complex at discrete intracellular
locations mediated by the N-terminal 330 amino acids of ELMO1 rather than
generalized Rac activation plays a role in cell migration. RATIONALE: Angiogenesis is regulated by the small GTPase Rac1. The ELMO1/DOCK180
complex forms a guanine nucleotide exchange factor for Rac1, regulating its
activation during cell migration in different biological systems.
OBJECTIVE: To investigate the function of ELMO1/DOCK180 in vascular development.
METHODS AND RESULTS: In situ hybridization studies for elmo1 identified a
vascular and neuronal expression in zebrafish. Morpholino-based expression
silencing of elmo1 severely impaired the formation of the vasculature, including
intersomitic vessels, the dorsal longitudinal anastomotic vessel, the
parachordal vessel, and the development of the thoracic duct in tg(fli1:EGFP)
embryos. Mechanistically, we identified Netrin-1 and its receptor Unc5B as
upstream activators of the ELMO1/DOCK180 complex, regulating its functional
interaction and leading to Rac1 activation in endothelial cells and vessel
formation in zebrafish.
CONCLUSIONS: Our data have identified a novel signaling cascade regulating
vasculature formation in zebrafish. Apoptotic cell death is essential for development and tissue homeostasis.
Failure to clear apoptotic cells can ultimately cause inflammation and
autoimmunity. Apoptosis has primarily been studied by staining of fixed tissue
sections, and a clear understanding of the behavior of apoptotic cells in living
tissue has been elusive. Here, we use a newly developed technique to track
apoptotic cells in real time as they emerge and are cleared from the zebrafish
brain. We find that apoptotic cells are remarkably motile, frequently migrating
several cell diameters to the periphery of living tissues. F-actin remodeling
occurs in surrounding cells, but also within the apoptotic cells themselves,
suggesting a cell-autonomous component of motility. During the first 2 days of
development, engulfment is rare, and most apoptotic cells lyse at the brain
periphery. By 3 days postfertilization, most cell corpses are rapidly engulfed
by macrophages. This engulfment requires the guanine nucleotide exchange factor
elmo1. In elmo1-deficient macrophages, engulfment is rare and may occur through
macropinocytosis rather than directed engulfment. These findings suggest that
clearance of apoptotic cells in living vertebrates is accomplished by the
combined actions of apoptotic cell migration and elmo1-dependent macrophage
engulfment. OBJECTIVE: Engulfment and cell motility 1 (Elmo1) has been reported to cooperate
with dedicator of cytokinesis 1 (Dock180) and to be linked to the invasive
phenotype of cancer cells through activating small G-protein Rac. We aimed to
study the role of Elmo1 in the maligt migration of ovarian cancer.
METHODS: Engulfment and cell motility 1 expression was evaluated in specimens
from 93 patients with serous ovarian cancer (SOC) by immunohistochemical
staining. Next, Elmo1-RNAi cells were established by validated small
interference RNAs. Cell proliferation and cell motility were observed and
compared with Dock180-RNAi cells. To confirm their synergetic contribution to
forming focal adhesion and activating Rac1, Rac1-GTP level was measured by GST
pull-down assay and immunofluorescence was used to observe focal adhesion
formation both in Elmo1-RNAi and Dock180-RNAi cells.
RESULTS: Engulfment and cell motility 1 was mainly overexpressed in high-grade
SOC tissues. Western blot analysis demonstrated that both Elmo1 and Dock180
expressions were hampered in Elmo1-RNAi cells. Compared with the negative
control, decreased colony formation and cell invasion were observed in
Elmo1-RNAi cells and Dock180-RNAi cells. Consistently, both exhibited reduced
Rac1-GTP level and inhibited focal adhesion formation.
CONCLUSIONS: Engulfment and cell motility 1 presents with synergetic action in
helping Dock180 to activate Rac1 and promote cell motility, and thus promote
untoward expansion and aggressiveness of SOC. Elmo1 and Elmo2 are highly homologous cytoplasmic adaptor proteins that interact
with Dock family guanine nucleotide exchange factors to promote activation of
the small GTPase Rac. In T lymphocytes, Dock2 is essential for CCR7- and
CXCR4-dependent Rac activation and chemotaxis, but the role of Elmo proteins in
regulating Dock2 function in primary T cells is not known. In this article, we
show that endogenous Elmo1, but not Elmo2, interacts constitutively with Dock2
in mouse and human primary T cells. CD4(+) T cells from Elmo1(-/-) mice were
profoundly impaired in polarization, Rac activation, and chemotaxis in response
to CCR7 and CXCR4 stimulation. Transfection of full-length Elmo1, but not Elmo2
or a Dock2-binding mutant of Elmo1, rescued defective migration of Elmo1(-/-) T
cells. Interestingly, Dock2 protein levels were reduced by 4-fold in Elmo1(-/-)
lymphocytes despite normal levels of Dock2 mRNA. Dock2 polyubiquitination was
increased in Elmo1(-/-) T cells, and treatment with proteasome inhibitors
partially restored Dock2 levels in Elmo1(-/-) T cells. Finally, we show that
Dock2 is directly ubiquitinated in CD4(+) T cells and that Elmo1 expression in
heterologous cells inhibits ubiquitination of Dock2. Taken together, these
findings reveal a previously unknown, nonredundant role for Elmo1 in controlling
Dock2 levels and Dock2-dependent T cell migration in primary lymphocytes.
Inhibition of Dock2 has therapeutic potential as a means to control recruitment
of pathogenic lymphocytes in diseased tissues. This work provides valuable
insights into the molecular regulation of Dock2 by Elmo1 that can be used to
design improved inhibitors that target the Elmo-Dock-Rac signaling complex. |
What is the biological role of K-48 linked protein ubiquitination? | The proteasome, which identifies and destroys unwanted proteins rapidly, plays a vital role in maintaining cellular protein homeostasis. Proteins that are destined for proteasome-mediated degradation are usually tagged with a chain of ubiquitin linked via lysine (K) 48 that targets them to the proteolytic machinery. K(48)-linked polyubiquitinated proteins are degraded by the proteasomes to elevate cellular levels of amino acids needed for intracellular proliferation. NF-κB and ubiquitylation initially became linked when it was recognised that lysine (K)48-linked ubiquitin chains are involved in the processing of NF-κB precursors and the degradation of inhibitor of kappa B (IκB) proteins. | Proteins tagged with lysine (Lys, K) 48 polyubiquitins chains are destined for
degradation by the 26S proteasomal system. Impairment of the ubiquitin
proteasome system (UPS) function culminates in the accumulation of
polyubiquitinated proteins in many neurodegenerative conditions including
Parkinson's disease (PD). Nevertheless, the cellular mechanisms underlying cell
death induced by an impaired UPS are still not clear. Intriguingly, recent
studies indicate that several proteins associated with familial PD are capable
of promoting the assembly of Lys-63 polyubiquitin chains. Therefore, the
objective of this study was to examine the role of K48 and K63 ubiquitination in
mitochondria-mediated apoptosis in in vitro models of dopaminergic degeneration.
Exposure of the widely used proteasome inhibitor MG-132 to dopaminergic neuronal
cell line (N27) induced a rapid accumulation of polyubiquitinated proteins in
the mitochondria. This appears to result in the preferential association of
ubiquitin conjugates in the outer membrane and polyubiquitination of outer
membrane proteins. Interestingly, the ubiquitin(K48R) mutant effectively rescued
cells from MG-132-induced mitochondrial apoptosis without altering the
antioxidant status of cells; whereas the ubiquitin(K63R) mutant augmented the
proapoptotic effect of MG-132. Herein, we report a novel conclusion that
polyubiquitinated proteins, otherwise subjected to proteasomal degradation,
preferentially accumulate in the mitochondria during proteolytic stress; and
that polyubiquitination of Lys-48 and Lys-63 are key determits of
mitochondria-mediated cell death during proteasomal dysfunction. Together, these
findings yield novel insights into a crosstalk between the UPS and mitochondria
in dopaminergic neuronal cells. Upon entry of Legionella pneumophila into amoebas and macrophages, host-mediated
farnesylation of the AnkB effector enables its anchoring to the
Legionella-containing vacuole (LCV) membrane. On the LCV, AnkB triggers docking
of K(48)-linked polyubiquitinated proteins that are degraded by the host
proteasomes to elevate cellular levels of amino acids needed for intracellular
proliferation. Interference with AnkB function triggers L. pneumophila to
exhibit a starvation response and differentiate into the nonreplicative phase in
response to the basal levels of cellular amino acids that are not sufficient to
power intracellular proliferation of L. pneumophila. Therefore, we have
determined whether the biological function of AnkB is temporally and spatially
triggered upon bacterial attachment to the host cell to circumvent a
counterproductive bacterial differentiation into the nonreplicative phase upon
bacterial entry. Here, we show that upon attachment of L. pneumophila to human
monocyte-derived macrophages (hMDMs), the host farnesylation and ubiquitination
machineries are recruited by the Dot/Icm system to the plasma membrane
exclusively beneath sites of bacterial attachment. Transcription and injection
of ankB is triggered by attached extracellular bacteria followed by rapid
farnesylation and anchoring of AnkB to the cytosolic side of the plasma membrane
beneath bacterial attachment, where K(48)-linked polyubiquitinated proteins are
assembled and degraded by the proteasomes, leading to a rapid rise in the
cellular levels of amino acids. Our data represent a novel strategy by an
intracellular pathogen that triggers rapid nutritional remodeling of the host
cell upon attachment to the plasma membrane, and as a result, a gratuitous
surplus of cellular amino acids is generated to support proliferation of the
incoming pathogen. |
Could DNA (cytosine-5-)-methyltransferases serve as tumour markers? | Yes. It has been demonstrated in a number of experimental studies that DNA (Cytosine-5-)-methyltransferases (DNMT1, DNMT3A and DNMT3B) are deregulated in several types of cancer (invasive cervical cancer, colon cancer, esophageal squamous cell carcinoma (ESCC), gastric cancer, embryonal carcinoma, cervical cancer, adenoma, adenoid cystic carcinoma, salivary gland neoplasms). Moreover, three single nucleotide polymorphisms (SNPs) of the DNMT3B promoter region have been reported to be stratification markers that can predict an individual's susceptibility to cancers. Therefore, DNA (Cytosine-5-)-methyltransferases can serve as tumour markers. | We evaluated the significance of aberrant DNA methyltransferase 1 (DNMT1)
protein expression during gastric carcinogenesis. The protein expression of
DNMT1, Muc2, human gastric mucin, E-cadherin, and proliferating cell nuclear
antigen was examined immunohistochemically in gastric cancers and corresponding
noncancerous mucosae from 134 patients. The DNA methylation status of the CpG
islands of the p16, human MutL homologue 1 (hMLH1), E-cadherin, and
thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT)-1, -2, -12,
and -31 clones was examined by methylation-specific polymerase chain reaction
and combined bisulfite restriction enzyme analysis. Epstein-Barr virus (EBV)
infection was detected by in situ hybridization. Nuclear immunoreactivity for
DNMT1 was not detected in any of the noncancerous epithelia, except in
proliferative zones (positive internal control), but was found in 97 (72%) of
the gastric cancers. DNMT1 overexpression correlated significantly with poorer
tumor differentiation (P < 0.001), but not with the phenotype (gastric type
versus intestinal type) of the cancer cells. It also correlated significantly
with DNA hypermethylation of the CpG islands of the hMLH1 (P = 0.024) and THBS-1
genes (P = 0.043), and with the CpG island methylator phenotype in the gastric
cancers (P = 0.007). Reduced E-cadherin expression correlated significantly with
poorer tumor differentiation (P = 0.002), DNA hypermethylation of the E-cadherin
gene (P < 0.001) and DNMT1 overexpression (P = 0.014). DNMT1 overexpression was
also associated with EBV infection (a potential etiological factor in gastric
carcinogenesis) but not with the proliferative activity of the cancer cells as
indicated by the proliferating cell nuclear antigen-labeling index. These
results suggest that DNMT1 overexpression may not be just a secondary effect of
increased cancer cell proliferative activity, but may be associated with EBV
infection and other etiological factors during gastric carcinogenesis.
Furthermore, DNMT1 may play a significant role in the development of poorly
differentiated gastric cancers by inducing frequent DNA hypermethylation of
multiple CpG islands. DNA (cytosine-5-)-methyltransferase 1 (DNMT1) plays an important role in the
maintece of DNA methylation patterns via complicated networks including
signaling pathways and transcriptional factors, relating to cell differentiation
or carcinogenesis. In the present study, we designed an antisense
oligodeoxynucleotide of DNMT1 (AS/MT: 5'-CGGTAC GCGCCGGCATCT-3') and
demonstrated successful inhibition of DNMT1 expression by AS/MT at the protein
level, using gastric cancer cell lines in vitro. E-cadherin protein expression
was increased, and both cyclin D1 and PCNA were decreased by AS/MT treatment.
AS/MT also induced suppression of cell growth as determined by BrDU uptake
incorporation, in a dose-dependent manner, suggesting specificity of AS/MT.
Simultaneously, morphological alterations were observed in both TMK-1 and MKN-45
cells after 24 h incubation with 2 micro M of AS/MT. The cells changed shape
from their original forms to dispersed, fibroblast-like cells with neurite-like
processes, accompanied by an increased adhesive potential of the cells. An in
vivo model of peritoneal dissemination using the nude mouse system showed an
increased maligt potential of AS/MT treated TMK-1 cells as demonstrated by a
greater number of peritoneal tumor nodules in the AS/MT as compared to the NS/MT
treated group, 34.8+/-4.3 vs. 22.4+/-3.0 nodules, respectively (p=0.0039). The
total wet tumor weight in the AS/MT group (350+/-47.4 g) was significantly
greater than that in the NS/MT group (248+/-41.5 g) (p=0.0065). In conclusion,
the inhibition of DNA methylation by DNMT1 by an antisense oligodeoxynucleotide
influences cell morphology and adhesion, as well as cell growth in gastric
cancer cells in vitro. Moreover, these alterations in the characteristics of
cancer cells resulted in an increased ability to attach onto the peritoneum in
the nude mouse system in vivo, suggesting that strict clinical guidelines will
be necessary to utilize such a DNA methylation inhibitor, since it does not
always mean a therapeutic antitumor strategy. Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has
been hypothesized to result in aberrant hypermethylation of genes required for
cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and
human cell lines decreased clonogenicity. By frame-shift and deletion
constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino
acid domain, which mediates interaction with proliferating cell nuclear antigen
(PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies.
Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded
weaker effects on clonogenicity. Following introduction of the toxic domain, no
significant effects on apoptosis, replication, or overall DNA methylation were
observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed
in cell lines lacking wild-type p53, p21(CIP1), or p16(INK4A). Suppression of
clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against
carcinogenicity of sustained Dnmt1 overexpression. DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA
methylation patterns in a mammalian genome during replication. Dnmt1 is targeted
to replication foci, interacts with PCNA, and favors methylating the
hemimethylated form of CpG sites. To understand the underlying mechanism of its
maintece function, we purified recombit forms of full-length Dnmt1, a
truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA
and examined their processivity using a series of long unmethylated and
hemimethylated DNA substrates. Direct analysis of methylation patterns using
bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length
Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of
over 95%, but unmethylated DNA with much less processivity. The truncated form
of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the
N-terminal 290-amino acid residue region of Dnmt1 is not required for
preferential activity toward hemimethylated sites or for processivity of the
enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates
hemimethylated CpG sites on one strand of double-stranded DNA during a single
processive run. Our findings suggest that these inherent enzymatic properties of
Dnmt1 play an essential role in the faithful and efficient maintece of
methylation patterns in the mammalian genome. Aberrant DNA methylation has been shown to play an important role during
multistage carcinogenesis in various human organs. The aim of the present study
was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein
expression during pancreatic carcinogenesis. Immunohistochemical analysis of
DNMT1 in 48 peripheral pancreatic duct epithelia showing no remarkable
histological findings without an inflammatory background (DE), 54 peripheral
pancreatic duct epithelia with an inflammatory background (DEI), 188 pancreatic
intraepithelial neoplasias (PanIN), and 220 areas of invasive ductal carcinoma
from surgical specimens resected from 100 patients, was carried out. The average
incidence of DNMT1 immunoreactivity increased progressively from DE to DEI (P =
0.003), from DE and DEI to PanIN (P < 0.0001), among PanIN with different grades
of dysplasia (from PanIN I to PanIN II, P = 0.0012), from PanIN to invasive
ductal carcinomas (P < 0.0001) and among invasive ductal carcinomas with
different grades of histological differentiation (from well or moderately to
poorly differentiated adenocarcinomas, P < 0.0001). High-level DNMT1 protein
expression in invasive ductal carcinomas was correlated significantly with an
advanced t category (P = 0.0224) and an advanced stage (P = 0.0294). Moreover,
patients with invasive ductal carcinomas showing high-level DNMT1 protein
expression had a poorer outcome (P = 0.0469). These data suggest that increased
DNMT1 protein expression participates in multistage pancreatic carcinogenesis
from the precancerous stage to maligt progression of ductal carcinomas and
may be a biological predictor of poor prognosis. DNA methylation patterns in genome are maintained during replication by a DNA
methyltransferase Dnmt1. Mouse Dnmt1 is a 180 kDa protein comprising the
N-terminal regulatory domain, which covers 2/3 of the molecule, and the rest
C-terminal catalytic domain. In the present study, we demonstrated that the
limited digestion of full-length Dnmt1 with different proteases produced a
common N-terminal fragment, which migrated along with Dnmt1 (1-248) in
SDS-polyacrylamide gel electrophoresis. Digestion of the N-terminal domains
larger than Dnmt1 (1-248) with chymotrypsin again produced the fragment
identical to the size of Dnmt1 (1-248). These results indicate that the
N-terminal domain of 1-248 forms an independent domain. This N-terminal domain
showed DNA binding activity, and the responsible sequence was narrowed to the 79
amino acid residues involving the proliferating cell nuclear antigen (PCNA)
binding motif. The DNA binding activity did not distinguish between DNA
methylated and non-methylated states, but preferred to bind to the minor groove
of AT-rich sequence. The DNA binding activity of the N-terminal domain competed
with the PCNA binding. We propose that DNA binding activity of the N-terminal
domain contributes to the localization of Dnmt1 to AT-rich sequence such as Line
1, satellite, and the promoter of tissue-specific silent genes. We assessed expression of the BRCA1, CTCF and DNMT3b methyltransferase genes
along with BRCA1 promoter methylation to better define the epigenetic events
involved in BRCA1 inactivation in sporadic breast cancer. These gene expression
patterns were determined in 54 sporadic breast tumours by immunohistochemistry
and the methylation status of the BRCA1 promoter was evaluated using
methylation-specific PCR. We observed significant DNMT3b expression in 80% of
the tumours and that 43% of tumours exhibited novel cytoplasmic CTCF expression.
Pairwise analyses of gene expression patterns showed that 28/32 tumours lacked
BRCA1 expression and also exhibited cytoplasmic CTCF staining, while 24/32 of
these tumours also overexpressed DNMT3b. Furthermore, 86% of the BRCA1
low-expressing tumours were methylated at the BRCA1 promoter and a subset of
these tumours displayed both cytoplasmic CTCF and increased DNMT3b expression.
Thus, tumour subsets exist that display concurrent decreased BRCA1 expression,
BRCA1 promoter methylation, cytoplasmic CTCF expression and with DNMT3b
over-expression. We suggest that these altered CTCF and DNMT3b expression
patterns represent (a) critical events responsible for the epigenetic
inactivation of BRCA1 and (b) a diagnostic signature for epigenetic inactivation
of other tumour suppressor genes in sporadic breast tumours. Aberrant DNA methylation has been shown to play important roles during
multistage carcinogenesis in various human organs. The aim of this study was to
evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression
during cervical carcinogenesis. We carried out an immunohistochemical
examination for DNMT1 in 34 samples of histologically normal squamous
epithelium, 36 samples of low-grade cervical intraepithelial neoplasia (CIN), 61
samples of higher-grade CIN and 30 samples of squamous cell carcinoma of the
uterine cervix. The DNMT1 protein expression score, reflecting the intensity and
incidence of DNMT1 nuclear immunoreactivity, was increased even in low-grade CIN
(P<0.0001) in comparison with histologically normal squamous epithelium and was
further increased in higher-grade CIN (P<0.0001 compared to low-grade CIN). The
DNMT1 protein expression score remained at a plateau in microinvasive carcinoma
(Stage IA, P=0.0690 compared to higher-grade CIN) and then decreased with cancer
invasion (Stage IB or more, P=0.0176 compared to Stage IA), whereas the
proliferating cell nuclear antigen (PCNA) labeling index did not decrease with
cancer invasion (P=0.8259 between Stage IA and Stage IB or more). Thus, the
DNMT1 protein expression score and the PCNA labeling index were not mutually
correlated in squamous cell carcinoma of the uterine cervix (P=0.2304). These
data suggest that progressively increasing expression of DNMT1 protein is not
entirely a secondary result of increased cell proliferative activity, but is
associated with an early step of multistage cervical carcinogenesis. Epigenetic events play a prominent role during cancer development. This is
evident from the fact that almost all cancer types show aberrant DNA
methylation. These abnormal DNA methylation levels are not restricted to just a
few genes but affect the whole genome. Previous studies have shown genome-wide
DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most
cancers. Molecules like DNA methyltransferase act as effectors of epigenetic
reprogramming. In the present study we have examined the possibility that the
reprogramming genes themselves undergo epigenetic modifications reflecting their
changed transcriptional status during cancer development. Comparison of DNA
methylation status between the normal and cervical cancer samples was carried
out at the promoters of a few reprogramming molecules. Our study revealed
statistically significant DNA methylation differences within the promoter of
DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L
promoter was found to have lost DNA methylation to varying levels in 14 out of
15 cancer cervix samples analysed. The present study highlights the importance
of DNA methylation profile at DNMT3L promoter not only as a promising biomarker
for cervical cancer, which is the second most common cancer among women
worldwide, but also provides insight into the possible role of DNMT3L in cancer
development. Overexpression of the DNA methyltransferase 3B (DNMT3B) gene and its effect on
carcinogenesis has been demonstrated for various types of cancer. Recently,
three single nucleotide polymorphisms (SNPs) of the DNMT3B promoter region,
C46359T (-149C>T), -283T>C, and -579G>T have also been reported to be
stratification markers that can predict an individual's susceptibility to
cancers. In this study, we analyzed expression of DNMT3B in nasopharyngeal
carcinoma (NPC) specimens and did not find elevated levels of DNMT3B in tumors
using cDNA microarray analysis and RT-PCR. Meanwhile, 259 NPC patients and 250
controls were genotyped for the above three SNPs using a MALDI-TOF based
mini-sequencing method. For C46359T (-149C>T), only the T/T genotype was found
to be present in both patient and control groups (100% frequency). The frequency
of the genotypes, -283CC, -283CT and -283TT, amongst NPC patients versus
controls was, respectively, 86.1% versus 84.0%, 13.5% versus 15.6%, and 0.4%
versus 0.4% (P=0.589). The allele frequency, -597TT, -597GT and -597GG, for
patients versus controls was, respectively, 87.3% versus 84.8%, 12.0% versus
15.2%, and 0.8% versus 0 (P=0.501). The distribution of SNPs among cancer
patients either featuring or not featuring cervical metastasis also did not
reveal any significant difference. In conclusion, our data indicate that neither
overexpression of DNMT3B nor the presence of three DNMT3B SNPs are associated
with NPC, which suggests that DNMT3B might not play a role in hypermethylation
of many tumor suppressor genes during carcinogenesis of NPC. Genomic hypomethylation is a consistent finding in both human and animal tumors
and mounting experimental evidence suggests a key role for epigenetic events in
tumorigenesis. Furthermore, it has been suggested that early changes in DNA
methylation and histone modifications may serve as sensitive predictive markers
in animal testing for carcinogenic potency of environmental agents. Alterations
in metabolism of methyl donors, disturbances in activity and/or expression of
DNA methyltransferases, and presence of DNA single-strand breaks could
contribute to the loss of cytosine methylation during carcinogenesis; however,
the precise mechanisms of genomic hypomethylation induced by chemical
carcinogens remain largely unknown. This study examined the mechanism of DNA
hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators
WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP
(di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of
action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5
months), the level of genomic hypomethylation increased by approximately 2-fold,
as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5
months) DNA methylation did not change. Global DNA hypomethylation in livers
from WY-14,643 group was accompanied by the accumulation of DNA single-strand
breaks, increased cell proliferation, and diminished expression of DNA
methyltransferase 1, while the metabolism of methyl donors was not affected. In
contrast, none of these parameters changed significantly in rats fed DEHP. Since
WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent
of loss of DNA methylation may be related to the carcinogenic potential of the
chemical agent, and that accumulation of DNA single-strand breaks coupled to the
increase in cell proliferation and altered DNA methyltransferase expression may
explain genomic hypomethylation during peroxisome proliferator-induced
carcinogenesis. OBJECTIVE: Salivary gland neoplasms pathogenesis has not been well established.
DNA methylation occurs when methyl groups are added to cytosine nucleotides in
specific areas of the gene by the enzyme DNA methyltransferase (DNMT). This
chemical modification can alter gene expression without altering DNA sequence.
While DNMT3a is mostly involved in de novo methylation, DNMT1 acts as a
maintece methyltransferase. We aimed to investigate the immunoexpression of
DNMT3a and DNMT1 in minor salivary gland neoplasms, comparing it with normal
tissue.
MATERIAL: Forty-four formalin-fixed and paraffin-embedded samples of pleomorphic
adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and polymorphous
low-grade adenocarcinoma were included in the study. The DNMT1 and DNMT3a
proteins were identified by using a highly sensitive polymer-based system.
RESULTS: Positive nuclear and cytoplasmic labeling for DNMT1 was observed in all
samples, including the controls. Positive nuclear labeling for DNMT3a was found
only in few neoplasms: 1 pleomorphic adenoma (9.0%), 2 adenoid cystic carcinoma
(16.6%) and 1 mucoepidermoid (9.0%) cases.
CONCLUSION: Our results were not able to demonstrate a clear correlation between
DNMT1 and DNMT3a immunoexpression and salivary gland neoplasms development. 5-Azactydine inhibits cell growth by direct cytotoxic action as well as by
inhibition of DNA methyl transferase enzyme. Inhibitors of DNMT have been
reported to potentiate the therapeutic activity of cisplatin in vitro. Dose
dependent bone marrow toxicity, neurotoxicity and nephrotoxicity are the major
side effects of cisplatin, limiting its use as an effective chemotherapeutic
agent. The present study was aimed to reduce the nephrotoxic potential of
cisplatin without compensating its potency. To best of our knowledge, this is
the first report which shows that the combination of 5-azacytidine with
cisplatin leads to remarkable reduction in nephrotoxicity, by involving
inhibition of cisplatin induced metallothionein expression. 5-Azacytidine
treatment with cisplatin leads to maximum reduction in tumor size in DMH induced
colon cancer and tumor volume in DMBA induced breast cancer bearing SD rats.
This combination regimen prevents phosphorylation and acetylation of histone H3
which may be involved in inhibition of aberrant gene expression in colon tumors.
Further, 5-azacytidine potentiated cisplatin induced antitumor activity by
involving decreased expression of pAKT, DNMT1 and an increased expression of p38
in colon tumors. Thus, combination of 5-azactydine with cisplatin attenuates the
cisplatin induced nephrotoxicity and potentiates the anti-cancer activity which
can have profound clinical implications. BACKGROUND: A hallmark of cancer cells is hypermethylation of CpG islands
(CGIs), which probably arises from upregulation of one or more DNA
methyltransferases. The purpose of this study was to identify the targets of
DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer.
METHODOLOGY/PRINCIPAL FINDINGS: Chromatin immunoprecipitation with DNMT3B
specific antibody followed by CGI microarray identified genes with or without
CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis
showed that the majority of the target genes including P16, DCC, DISC1, SLIT1,
CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI
in their promoters, were methylated in multiple colon cancer cell lines but not
in normal colon epithelial cells. Further, these genes were reactivated in RKO
cells after treatment with 5-aza-2'-deoxycytidine, a DNA hypomethylating agent.
COBRA showed that the CGIs encompassing the promoter and/or coding region of
DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in
matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY
analysis demonstrated that the CGI located approximately 4.5 kb upstream of
HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal
cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated
in DNMT1(-/-) HCT cells but was almost methylation free in cells lacking both
DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly
methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited
growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13
abolished tumor growth in nude mice.
CONCLUSIONS/SIGNIFICANCE: This is the first report that identifies several
important tumor suppressors and transcription factors as direct DNMT3B targets
in colon cancer and as potential biomarkers for this cancer. Further, this study
shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer. PURPOSE: Testicular germ cell tumors (TGCT) have a unique epigenetic profile
distinct from that of other types of cancer. Elucidation of these properties has
a potential to identify novel markers for TGCTs.
EXPERIMENTAL DESIGN: We conducted comprehensive analysis of DNA
methyltransferase (DNMT) gene expression in TGCTs. Based on the expression
profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human
DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of
two embryonal carcinoma (EC) cell lines with a small interfering RNA system.
Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT
tissues. We also compared the patterns of DNMT3L immunohistochemistry with those
of CD30 and SOX2.
RESULTS: Among the DNMT genes, we found that mRNA for DNMT3L was specifically
expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells
of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell
lines, but not in the cell lines of somatic tissue origin. Transfection of small
interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted
in growth suppression and apoptosis in EC cells. Immunohistochemical analysis
showed that DNMT3L protein was present only in EC cells, but not in the other
types of TGCT components and cancer cells of somatic tissue origin. DNMT3L
staining was more prominent and specific than CD30 or SOX2 staining for
detecting EC cells.
CONCLUSION: DNMT3L is a novel marker and is essential for the growth of human
embryonal carcinoma. Global DNA hypomethylation is a hallmark of cancer cells, but its molecular
mechanisms have not been elucidated. Here, we show that the disruption of
Dnmt1/PCNA/UHRF1 interactions promotes a global DNA hypomethylation in human
gliomas. We then demonstrate that the Dnmt1 phosphorylations by Akt and/or PKC
abrogate the interactions of Dnmt1 with PCNA and UHRF1 in cellular and acellular
studies including mass spectrometric analyses and the use of primary cultured
patient-derived glioma. By using methylated DNA immunoprecipitation, methylation
and CGH arrays, we show that global DNA hypomethylation is associated with genes
hypomethylation, hypomethylation of DNA repeat element and chromosomal
instability. Our results reveal that the disruption of Dnmt1/PCNA/UHRF1
interactions acts as an oncogenic event and that one of its signatures (i.e. the
low level of mMTase activity) is a molecular biomarker associated with a poor
prognosis in GBM patients. We identify the genetic and epigenetic alterations
which collectively promote the acquisition of tumor/glioma traits by human
astrocytes and glial progenitor cells as that promoting high proliferation and
apoptosis evasion. DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA
methylation. We describe a previously unknown mode of regulation of DNMT1
protein stability through the coordinated action of an array of DNMT1-associated
proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60,
which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1
for proteasomal degradation. In contrast, DNMT1 was stabilized by histone
deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus-associated
ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as
well as the association between HAUSP and DNMT1, suggested that during the cell
cycle the initiation of DNMT1 degradation was coordinated with the end of DNA
replication and the need for DNMT activity. In human colon cancers, the
abundance of DNMT1 correlated with that of HAUSP. HAUSP knockdown rendered colon
cancer cells more sensitive to killing by HDAC inhibitors both in tissue culture
and in tumor xenograft models. Thus, these studies provide a mechanism-based
rationale for the development of HDAC and HAUSP inhibitors for combined use in
cancer therapy. This study was designed to determine the significance of DNA methyltransferases
(DNMTs) in DNA hypermethylation in esophageal squamous cell carcinoma (ESCC) and
to identify DNA methylation markers in serum for the early diagnosis of ESCC. A
promoter methylation profile of 12 tumor-related genes was assessed using
methylation-specific PCR in ESCC and paired non-tumor tissue samples from 47
patients. Expression levels of DNMTs were examined by real-time reverse
transcription-PCR and immunohistochemistry. Using MethyLight, the methylation
status of 5 genes was analyzed in serum samples from 45 patients and 15 healthy
individuals. A total of 46 (97.9%) of 47 ESCC samples showed methylation in at
least one of the examined genes, and methylation was most frequent for RAR-β
(46.8%), DAPK (46.8%), p16 (44.7%), and CDH1 (42.6%). Methylation of RASSF1A was
significantly correlated with the poorly differentiated tumors and the early
pathologic tumor classification (P=0.035 and P=0.046, respectively). Tumoral
DNMT3b mRNA up-regulation was significantly correlated with hypermethylation of
multiple tumor-related genes (P=0.021). In addition, hypermethylation of
cell-free serum DNA was common in ESCC patients, and diagnostic accuracy was
increased when methylation of multiple genes (RAR-β, DAPK, CDH1, p16 and
RASSF1A) were analyzed in combination (ROC AUC 0.911, 82.2% sensitivity and 100%
specificity). The present study suggests that hypermethylation of multiple
tumor-related genes may be involved in the pathogenesis of ESCC and mediated by
the increase of DNMT3b expression. A cluster of multiple methylated genes in
serum DNA has the potential as a novel biomarker for ESCC diagnosis. Candidate gene-based studies have identified a handful of aberrant CpG DNA
methylation events in prostate cancer. However, DNA methylation profiles have
not been compared on a large scale between prostate tumor and normal prostate,
and the mechanisms behind these alterations are unknown. In this study, we
quantitatively profiled 95 primary prostate tumors and 86 benign adjacent
prostate tissue samples for their DNA methylation levels at 26,333 CpGs
representing 14,104 gene promoters by using the Illumina HumanMethylation27
platform. A 2-class Significance Analysis of this data set revealed 5912 CpG
sites with increased DNA methylation and 2151 CpG sites with decreased DNA
methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set
identified 87 CpGs that are the most predictive diagnostic methylation
biomarkers of prostate cancer. By integrating available clinical follow-up data,
we also identified 69 prognostic DNA methylation alterations that correlate with
biochemical recurrence of the tumor. To identify the mechanisms responsible for
these genome-wide DNA methylation alterations, we measured the gene expression
levels of several DNA methyltransferases (DNMTs) and their interacting proteins
by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in
tumors. Subsequent transient transfection assays in cultured primary prostate
cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased
methylation of a substantial subset of CpG sites that showed tumor-specific
increased methylation. Changes in DNA methylation patterns is a prominent characteristic of human
tumors. Tumor cells display reduced levels of genomic DNA methylation and
site-specific CpG island hypermethylation. Methylation of CpG dinucleotides is
catalyzed by the enzyme family of DNA methyltransferases (DNMTs). In this
review, the role of DNA methylation and DNMTs as key determits of
carcinogenesis is further elucidated. The chromatin modifying proteins that are
known to interact with DNMTs are also described. Finally, the role of DNMTs as
potential therapeutic targets is addressed. Vorinostat (suberoylanilide hydroxamic acid), a class of histone deacetylase
inhibitors, represents an emerging class of anticancer agents currently
progressing in clinical trials. It causes cell growth inhibition,
differentiation, and apoptosis of many tumor types in vitro and in vivo.
Recently, it was reported that hTERT is one of the targets for cancer therapy in
cancer cells. Telomerase repeat amplification protocol assay was used to analyze
the expression of hTERT after vorinostat treatment in the A549 lung cancer
cells. Vorinostat inhibited telomerase activity by reducing the expression of
human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells.
The epigenetic regulation mechanism is responsible for the repression of hTERT
by vorinostat, analyzed through the methylation-specific PCR and bisulfite
sequencing of the hTERT promoter. Vorinostat induced the demethylation of
site-specific CpGs on the promoter region of hTERT, which was caused by the
down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and
DNMT3b) were also decreased in vorinostat-treated A549 cancer cells.
Furthermore, chromatin immunoprecipitation analysis of the hTERT promoter
revealed that vorinostat decreased the level of inactive chromatin markers
dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The
novel insights showed that vorinostat down-regulated telomerase via epigenetic
alteration in lung cancer to vorinostat-mediated cancer-specific therapies. Epigenetic modifications are a driving force in carcinogenesis. However, their
role in cancer metastasis remains poorly understood. The present study
investigated the role of DNA methylation in the cervical cancer metastasis.
Here, we report evidence of the overexpression of DNA methyltransferases 3B
(DNMT3B) in invasive cervical cancer and of the inhibition of metastasis by
DNMT3B interference. Using methyl-DNA immunoprecipitation coupled with
microarray analysis, we found that the protein tyrosine phosphatase receptor
type R (PTPRR) was silenced through DNMT3B-mediated methylation in the cervical
cancer. PTPRR inhibited p44/42 MAPK signaling, the expression of the
transcription factor AP1, human papillomavirus (HPV) oncogenes E6/E7 and DNMTs.
The methylation status of PTPRR increased in cervical scrapings (n=358) in
accordance with disease severity, especially in invasive cancer. Methylation of
the PTPRR promoter has an important role in the metastasis and may be a
biomarker of invasive cervical cancer. The prevalence, the prognostic effect, and interaction with other molecular
markers of DNMT3A mutations was studied in 415 patients with acute myeloid
leukemia (AML) younger than 60 years. We show mutations in DNMT3A in 96 of 415
patients with newly diagnosed AML (23.1%). Univariate Cox regression analysis
showed that patients with DNMT3A(mutant) AML show significantly worse overall
survival (OS; P = .022; hazard ratio [HR], 1.38; 95% confidence interval [CI],
1.04-1.81), and relapse-free survival (RFS; P = .005; HR, 1.52; 95% CI,
1.13-2.05) than DNMT3A(wild-type) AMLs. In a multivariable analysis, DNMT3A
mutations express independent unfavorable prognostic value for OS (P = .003; HR,
1.82; 95% CI, 1.2-2.7) and RFS (P < .001; HR, 2.2; 95% CI, 1.4-3.3). In a
composite genotypic subset of cytogenetic intermediate-risk AML without FLT3-ITD
and NPM1 mutations, this association is particularly evident (OS: P = .013; HR,
2.09; 95% CI, 1.16-3.77; RFS: P = .001; HR, 2.65; 95% CI, 1.48-4.89). The effect
of DNMT3A mutations in human AML remains elusive, because DNMT3A(mutant) AMLs
did not express a methylation or gene expression signature that discriminates
them from patients with DNMT3A(wild-type) AML. We conclude that DNMT3A mutation
status is an important factor to consider for risk stratification of patients
with AML. Aberrant DNA methylation plays a relevant role in multiple myeloma (MM)
pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that
recently emerged as master regulator of gene expression by targeting
protein-coding mRNAs. However, miRNAs involvement in the regulation of the
epigenetic machinery and their potential use as therapeutics in MM remain to be
investigated. Here, we provide evidence that the expression of de novo DNA
methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that
miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation in MM
cells. In vitro transfection of MM cells with synthetic miR-29b mimics
significantly impairs cell cycle progression and also potentiates the
growth-inhibitory effects induced by the demethylating agent 5-azacitidine. Most
importantly, in vivo intratumor or systemic delivery of synthetic miR-29b
mimics, in two clinically relevant murine models of human MM, including the
SCID-synth-hu system, induces significant anti-tumor effects. All together, our
findings demonstrate that aberrant DNMTs expression is efficiently modulated by
tumor suppressive synthetic miR-29b mimics, indicating that methyloma modulation
is a novel matter of investigation in miRNA-based therapy of MM. It has been recently shown that DNA methyl transferase overexpression is
correlated with unfavourable prognosis in human maligcies while methylation
deregulation remains a hallmark that defines acute myeloid leukemia (AML). The
oncogenic transcription factor EVI1 is involved in methylation deregulation and
its overexpression plays a major role for predicting an adverse outcome.
Moreover, the identification of DNMT3A mutations in AML patients has recently
been described as a poor prognostic indicator. In order to clarify relationship
between these key actors in methylation mechanisms and their potential impact on
patient outcomes, we analysed 195 de novo AML patients for the expression of
DNMT3A, 3B (and its non-catalytic variant 3B(NC)) and their correlations with
the outcome and the expression of other common prognostic genetic biomarkers
(EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of
DNMT3B/3B(NC) is (i) significantly correlated with a shorter overall survival,
and (ii) inversely significantly correlated with event-free survival and DNMT3A
expression level. Moreover, multivariate analysis showed that a high expression
level of DNMT3B/3B(NC) is statistically a significant independent poor
prognostic indicator. This study represents the first report showing that the
overexpression of DNMT3B/3B(NC) is an independent predictor of poor survival in
AML. Its quantification should be implemented to the genetic profile used to
stratify patients for therapeutical strategies and should be useful to identify
patients who may benefit from therapy based on demethylating agents. |
Which RNA polymerase is used for the replication of viroids? | DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies | Analysis by molecular hybridization of the RNAs transcribed by a cell-free
fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated
the presence of newly synthesized viroid-specific sequences, most of which were
of the same polarity as the mature infectious viroid RNA. Treatment of the
cell-free fraction with DNase reduced the total synthesis of RNA considerably,
but it did not influence that of the ASBV-specific RNAs, indicating that the
latter were transcribed on an RNA template. Inhibition studies with
alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected
by concentrations of 1 and 200 micrograms/ml of the drug, which typically
inhibit RNA polymerase II and III, respectively, from most animal and plant
systems. These results suggest that either RNA polymerase I or an unidentified
RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template,
plays a role in the replication of ASBV, whereas for the rest of the viroids
studied so far it appears that RNA polymerase II is involved. Analysis by
polycrylamide gel electrophoresis under partially and fully denaturing
conditions of the ASBV-specific RNAs synthesized in vitro showed that they
contain unit and longer than unit length viroid strands, probably associated in
complexes with single- and double-stranded regions. The structural properties of
these complexes are similar to those of the RNAs accumulating in vivo in
viroid-infected tissues, which are the postulated replicative intermediates of
the rolling-circle mechanism proposed for viroid synthesis. The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro
into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA
polymerase, respectively, from Escherichia coli. In vitro synthesis of
complementary RNA produces distinct transcripts larger than unit length thus
reflecting the in vivo mechanism of viroid replication. The influence of varying
experimental conditions on the transcription process is studied; actinomycin D
is found to drastically reduce complementary RNA synthesis from the PSTV RNA
template by RNA polymerase. RNA-dependent RNA polymerase from healthy tomato plant tissue accepts potato
spindle tuber viroid (PSTV) RNA as a template for the in vitro synthesis of
full-length RNA copies of the PSTV genome. Viroid transcription requires the
presence of Mn2+ and /or Mg2+ ions and is not inhibited by concentrations of
10(-5) M alpha-amanitin. This is the first report of a well-defined product
synthesized in vitro by an RNA-dependent RNA polymerase from healthy plants. DNA-dependent RNA polymerase II purified from healthy plant tissue is capable of
synthesizing linear (-)-viroid RNA copies of full length from (+)-viroid RNA
templates in vitro. Together with the specific alpha-amanitin sensitivity of
viroid replication observed in vivo, these findings suggest that viroids
replicate by an entirely novel mechanism in which infecting viroid RNA molecules
are copied by the host enzyme which is normally responsible for the synthesis of
nuclear precursors to messenger RNA. An active replication complex of citrus exocortis viroid (CEV) was isolated as a
chromatin-enriched fraction of infected tomato leaf with CEV RNA synthesis
activity. This activity was solubilised from the chromatin with ammonium
sulphate, but not with sarkosyl. Nucleoprotein complexes in the soluble fraction
which bound to a monoclonal antibody to the carboxy terminal domain of the
largest subunit of RNA polymerase II (8WG16) were affinity purified and
contained plus- and minus-sense CEV RNA. The results support a role for RNA
polymerase II in viroid replication and provide the first direct evidence of an
association in vivo between host RNA polymerase II and CEV. RNA polymerase II is implicated in the RNA-templated RNA synthesis during
replication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA
template nor protein factor requirements for this process are well defined. We
have developed an in vitro transcription system based on HeLa cell nuclear
extract (NE), in which a segment of antigenomic RNA corresponding to the
left-hand tip region of the HDV rod-like structure serves as a template for
efficient and highly specific RNA synthesis. Accumulation of the unique RNA
product is highly sensitive to alpha-amanitin in HeLa NE and only partially
sensitive to this drug in NE from PMG cells that contain an allele of the
alpha-amanitin-resistant subunit of pol II, strongly suggesting pol II
involvement in this reaction. Detailed analysis of the RNA product revealed that
it represents a chimeric molecule composed of a newly synthesized transcript
covalently attached to the 5' half of the RNA template. Selection of the start
site for transcription is remarkably specific and depends on the secondary
structure of the RNA template, rather than on its primary sequence. Some
features of this reaction resemble the RNA cleavage-extension process observed
for pol II-arrested complexes in vitro. A possible involvement of the described
reaction in HDV replication is discussed. Pospiviroidae, with their main representative potato spindle tuber viroid
(PSTVd), are replicated via a rolling circle mechanism by the host-encoded
DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand
circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet
it is not known whether transcription is initiated by promotors at specific
start sites or is distributed non-specifically over the whole circle. An in
vitro transcription extract was prepared from a non-infected potato cell culture
which exhibited transcriptional activity using added circular PSTVd (+)-strand
RNA as template. In accordance with pol II activity, transcription could be
inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two
different start sites and primer extension identified these as nucleotides
A(111) and A(325). The sequences of the first 7 nt transcribed are very similar,
(105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of
15 and 16 nt upstream, respectively, in the native viroid structure, which may
act to facilitate initiation. The GC-boxes may have a similar function to the
GC-rich hairpin II in the (-)-strand intermediate, as described previously. The
results are compared with the corresponding features of avocado sunblotch
viroid, which belongs to a different family of viroids and exhibits different
transcription initiation properties. Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate
autonomously in their host plants but do not code for a protein. Viroids of the
family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type
strain, are replicated by the host's DNA-dependent RNA polymerase II in the
nucleus. To analyze the initiation site of transcription from the (+)-stranded
circles into (-)-stranded replication intermediates, we used a nuclear extract
from a non-infected cell culture of the host plant S. tuberosum. The
(-)-strands, which were de novo-synthesized in the extract upon addition of
circular (+)-PSTVd, were purified by affinity chromatography. This purification
avoided contamination by host nucleic acids that had resulted in a misassignment
of the start site in an earlier study. Primer-extension analysis of the de
novo-synthesized (-)-strands revealed a single start site located in the hairpin
loop of the left terminal region in circular PSTVd's secondary structure. This
start site is supported further by analysis of the infectivity and replication
behavior of site-directed mutants in planta. Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations
from infected Gynura aurantiaca was optimum at 18-24 degrees C. Incubation of
reaction mixtures at higher temperatures (30-36 degrees C) resulted in an
increase of CEV linear molecules and the recovery of incomplete or nicked newly
synthesized RNA species. Although the Mg(2+) optimum (2.5-5 mM) for CEV
synthesis was lower than that for total [(32)P]CMP incorporation (10 mM), the
response to Mn(2+) ion was distinctly different. Whereas maximum total activity
was observed in 1 mM Mn(2+) with a pronounced reduction (80%) in 5 mM Mn(2+),
CEV synthesis was maintained in 1-15 mM Mn(2+). Inhibition of
alpha-amanitin-sensitive CEV synthesis in 200 mM (NH(4))(2)SO(4) resembles the
reaction of RNA polymerase II on a free nucleic acid template. However,
detection of trace levels of alpha-amanitin-resistant CEV synthesis activity
inhibited by low (NH(4))(2)SO(4) concentrations (25 mM) suggests the possible
involvement of RNA polymerase I- and/or III-like activity. RNA-templated RNA replication is essential for viral or viroid infection, as
well as for regulation of cellular gene expression. Specific RNA motifs likely
regulate various aspects of this replication. Viroids of the Pospiviroidae
family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in
the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the
role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication.
A tertiary-structural model of this motif, inferred by comparative sequence
analysis and comparison with nuclear magnetic resoce and X-ray crystal
structures of loop E motifs in other RNAs, is presented in which core
non-Watson-Crick base pairs are precisely specified. Isostericity matrix
analysis of these base pairs showed that the model accounts for the reported
natural sequence variations and viable experimental mutations in loop E motifs
of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us
to design disruptive, as well as compensatory, mutations of PSTVd loop E.
Functional analyses of such mutants by in vitro and in vivo experiments
demonstrated that loop E structural integrity is crucial for replication,
specifically during transcription. Our results suggest that the PSTVd loop E
motif exists and functions in vivo and provide loss-of-function genetic evidence
for the essential role of a viroid RNA three-dimensional motif in rolling-circle
replication. The use of isostericity matrix analysis of non-Watson-Crick base
pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro
and in vivo functional assays of mutants offers a novel, comprehensive approach
to elucidate the tertiary-structure-function relationships for RNA motifs of
general biological significance. Potato spindle tuber viroid (PSTVd) is a small, single-stranded, circular,
non-coding RNA pathogen. Host DNA-dependent RNA polymerase II (RNAP II) was
proposed to be critical for its replication, but no interaction site for RNAP II
on the PSTVd RNA genome was identified. Using a co-immunoprecipitation strategy
involving a mAb specific for the conserved heptapeptide (i.e. YSPTSPS) located
at the carboxy-terminal domain of the largest subunit of RNAP II, we established
the interaction of tomato RNAP II with PSTVd RNA and showed that RNAP II
associates with the left terminal domain of PSTVd (+) RNA. RNAP II did not
interact with any of several PSTVd (-) RNAs tested. Deletion and site-directed
mutagenesis of a shortened model PSTVd (+) RNA fragment were used to identify
the role of specific nucleotides and structural motifs in this interaction. Our
results provide evidence for the interaction of a RNAP II complex from a natural
host with the rod-like conformation of the left terminal domain of PSTVd (+)
RNA. |
List Parkin binding partners | HSP90
CDC37
GRP75
HSP60
LRPPRC
TUFM
PICK1
PSMA7
Pael receptor | Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations of the
parkin gene. Parkin is an E3 ubiquitin ligase that specifically recognizes its
substrate protein, promoting its ubiquitination and subsequent degradation.
Accordingly, we hypothesized that AR-JP may be caused by accumulation of an
unidentified neurotoxic protein, which is a substrate of parkin. Based on this
hypothesis, we cloned parkin-binding protein using a yeast two-hybrid system and
identified a putative G protein-coupled receptor protein,which we named the Pael
receptor (Pael-R). When overexpressed in cells, this receptor became unfolded,
insoluble, and ubiquitinated. Accumulation of the insoluble Pael-R subsequently
led to endoplasmic reticulum (ER) stress-induced cell death. Parkin specifically
ubiquitinates the unfolded Pael-R and promotes its degradation, resulting in
suppression of cell death induced by the accumulation of unfolded Pael-R.
Moreover, insoluble Pael-R accumulates in the brains of AR-JP patients. It is
highly expressed by the dopaminergic neurons of the substantia nigra, strongly
suggesting that accumulation of unfolded Pael-R may lead to selective death of
dopaminergic neurons in AR-JP.Recently, we identified Hsp70 and its co-chaperone
CHIP as novel parkin-binding partners. We found that CHIP enhanced
parkinmediated ubiquitination of Pael-R. In concert with Hsp70, CHIP also
enhanced the ability of parkin to inhibit cell death induced by Pael-R,
indicating that CHIP and Hsp70 are both co-factors of parkin. Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a
frequent cause of autosomal recessive parkinsonism and are also involved in
sporadic Parkinson's disease. Loss of Parkin function is thought to compromise
the polyubiquitylation and proteasomal degradation of specific substrates,
leading to their deleterious accumulation. Several studies have analyzed the
effects of parkin gene mutations on the biochemical properties of the protein.
However, the absence of a cell-free system for studying intrinsic Parkin
activity has limited the interpretation of these studies. Here we describe the
biochemical characterization of Parkin and 10 pathogenic variants carrying
amino-acid substitutions throughout the sequence. Mutations in the RING fingers
or the ubiquitin-like domain decreased the solubility of the protein in
detergent and increased its tendency to form visible aggregates. None of the
mutations studied compromised the binding of Parkin to a series of known protein
partners/substrates. Moreover, only two variants with substitutions of conserved
cysteine residues of the second RING finger were inactive in a purely in vitro
ubiquitylation assay, demonstrating that loss of ligase activity is a minor
pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed
the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein
ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly,
in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate
p38, rather than its polyubiquitylation. Thus, Parkin may mediate
polyubiquitylation or proteasome-independent monoubiquitylation depending on the
protein context. The discovery of monoubiquitylated Parkin species in cells
hints at a novel post-translational modification potentially involved in the
regulation of Parkin function. CASK, which belongs to the family of membrane-associated guanylate kinase
(MAGUK) proteins, is recognized as a multidomain scaffolding protein highly
expressed in the mammalian nervous system. MAGUK proteins generally target to
neuronal synapses and regulate trafficking, targeting, and signaling of ion
channels. However, CASK is a unique MAGUK protein in several respects. It not
only plays a role in synaptic protein targeting but also contributes to neural
development and regulation of gene expression. Several CASK-interacting proteins
have been identified from yeast two-hybrid screening and biochemical isolation.
These proteins, whose interactions with CASK are reviewed here, include the
Parkinson's disease molecule parkin, the adhesion molecule neurexin, syndecans,
calcium channel proteins, the cytoplasmic adaptor protein Mint1,
Veli/mLIN-7/MALS, SAP97, caskin and CIP98, transcription factor Tbr-1, and
nucleosome assembly protein CINAP. More important, CASK may form different
complexes with different binding partners and perform different functions. Among
these interactions, CASK, Tbr-1, and CINAP can form a transcriptional complex
regulating gene expression. Reelin and NMDAR subunit 2b (NR2b) genes have been
identified as Tbr-1 target genes. Reelin is critical for neural development.
NR2b is an important subunit of NMDAR, which plays important roles in neural
function and neurological diseases. Regulation of reelin and NR2b expression
suggests the potential roles of the Tbr-1-CASK-CINAP complex in neural activity,
development, and disease. The functions of these CASK protein complexes are also
discussed in detail in this review. p193/CUL7 is an E3 ubiquitin ligase initially identified as an SV40 Large T
Antigen binding protein. Expression of a domit interfering variant of mouse
p193/CUL7 (designated 1152stop) conferred resistance to MG132- and
etoposide-induced apoptosis in U2OS cells. Immune precipitation/Western analyses
revealed that endogenous p193/CUL7 formed a complex with Parc (a recently
identified parkin-like ubiquitin ligase) and p53. Apoptosis resistance did not
result from 1152stop-mediated disruption of the endogenous p193/CUL7 binding
partners. Moreover, 1152stop molecule did not directly bind to endogenous
p193/CUL7, Parc or p53. These data suggested a role for p193/CUL7 in the
regulation of apoptosis independently of p53 and Parc activity. Dysregulation of genes that control cell-cycle progression and DNA repair is a
hallmark of tumorigenesis. It is becoming increasingly apparent, however, that
these defects also contribute to degeneration of post-mitotic neurons under
certain conditions. The gene for ataxia-telangiectasia mutated (ATM) is a
prototype for this dual mechanism of action, with loss-of-function mutations
causing not only selective degeneration of cerebellar neurons but also increased
susceptibility to breast cancer and hematologic maligcy. Increased dosage of
amyloid precursor protein in Down syndrome (trisomy 21) predisposes to dementia
of Alzheimer type and may also contribute to acute leukemia and transient
myeloproliferative disorder. The gene parkin, loss-of-function mutations in
which account for about half of cases of early-onset Parkinson disease, has been
identified as a candidate tumor suppressor gene by several groups. Parkin is
deleted or downregulated in several tumor types, and its re-expression
sensitizes derivative cell lines to inhibitors of cell-cycle progression. The
overlap of molecular pathways implicated in cancer and neurodegeneration
challenges long-held notions about differentiated cellular states and may open
the door to novel therapeutic approaches to both groups of disorders. Mutations in the parkin gene are responsible for a common inherited form of
Parkinson's disease (PD). Parkin is a RING-type E3 ubiquitin ligase with an
N-terminal ubiquitin-like domain (Ubl). We report here that the parkin Ubl binds
SH3 domains from endocytic BAR proteins such as endophilin-A with an affinity
comparable to proline-rich domains (PRDs) from well-established SH3 partners.
The NMR structure of the Ubl-SH3 complex identifies the PaRK extension, a unique
C-terminal motif in the parkin Ubl required for SH3 binding and for
parkin-mediated ubiquitination of endophilin-A in vitro. In nerve terminals,
conditions that promote phosphorylation enhance the interaction between parkin
and endophilin-A and increase the levels of ubiquitinated proteins within
PRD-associated synaptic protein complexes in wild-type but not parkin knockout
brain. The findings identify a pathway for the recruitment of synaptic
substrates to parkin with the potential to explain the defects in synaptic
transmission observed in recessive forms of PD. Recent publications suggest that the Parkinson's disease- (PD-) related
PINK1/Parkin pathway promotes elimination of dysfunctional mitochondria by
autophagy. We used tandem affinity purification (TAP), SDS-PAGE, and mass
spectrometry as a first step towards identification of possible substrates for
PINK1. The cellular abundance of selected identified interactors was
investigated by Western blotting. Furthermore, one candidate gene was sequenced
in 46 patients with atypical PD. In addition to two known binding partners
(HSP90, CDC37), 12 proteins were identified using the TAP assay; four of which
are mitochondrially localized (GRP75, HSP60, LRPPRC, and TUFM). Western blot
analysis showed no differences in cellular abundance of these proteins comparing
PINK1 mutant and control fibroblasts. When sequencing LRPPRC, four exonic
synonymous changes and 20 polymorphisms in noncoding regions were detected. Our
study provides a list of putative PINK1 binding partners, confirming previously
described interactions, but also introducing novel mitochondrial proteins as
potential components of the PINK1/Parkin mitophagy pathway. |
What is generic name of drug Adempas? | Riociguat is generic name of drug Adempas. It is a soluble guanylate cyclase stimulator that was approved for the treatment of patients with chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension. | Duavee, an oral contraceptive; riociguat (Adempas) for two types of pulmonary
hypertension; and macitentan (Opsumit) for pulmonary arterial hypertension. Pulmonary hypertension (PH) is a progressive disease that is accompanied by a
poor prognosis. Pulmonary vasoconstriction is facilitated through multiple
pathways and results in increased pulmonary vascular pressure leading to cell
proliferation, vascular remodeling, right ventricular hypertrophy/failure, and
ultimately death. Until recently, just six medications were approved -all for
one subclass of PH. On October 8, 2013, riociguat (Adempas®) became the first
medication approved for multiple etiologies of PH. Preclinical studies have
demonstrated safety and efficacy with significant clinical trials supporting its
advancement into phase IV trials. Although long-term safety and efficacy and
place in therapy remain to be established, riociguat presents as an exciting new
option for the treatment of PH and potentially has additional indications in the
near future. Riociguat (Adempas(®)), a soluble guanylate cyclase stimulator, is a new,
first-in-class drug approved for the treatment of patients with chronic
thromboembolic pulmonary hypertension (CTEPH) [inoperable or
persistent/recurrent following surgery] or pulmonary arterial hypertension
(PAH). It has been designated an orphan medicine by the European Medicines
Agency and the US FDA. This article reviews the available pharmacological
properties of oral riociguat and its clinical efficacy and tolerability in
adults with CTEPH or PAH. Riociguat is effective and well tolerated in patients
with inoperable CTEPH or persistent/recurrent CTEPH following pulmonary
endarterectomy, and in patients with PAH. It has a positive result on exercise
capacity and pulmonary haemodynamics, and improves WHO functional class. Most
adverse events can be attributed to the vasodilatory mechanism of riociguat;
however, there is a potential for serious bleeding and fetal harm, and riociguat
use is contraindicated in pregt patients. Pulmonary endarterectomy remains
the first treatment of choice for CTEPH, as it is potentially curative.
Head-to-head trials comparing riociguat with the approved phosphodiesterase type
5 inhibitors in patients with PAH would be of value for the placement of
riociguat in the management of this disease. Riociguat is a promising addition
to the treatment options for patients with CTEPH or PAH. |
What is known about Vancomycin dosing in neonates? | Staphylococcus epidermis, including methicillin-resistant strains, are inhibited by vancomycin concentrations of 1-4 µg/ml.
Staphylococcus pyogenes, Streptococcus pneumonia, and Streptococcus viridans are susceptible to 2 µg/ml vancomycin.
Bacillus spp. are inhibited by 2 µg/ml, Corynebacterium spp. by 0.04-3.1 µg/ml and Clostridium spp. by 0.39-6 µg/ml vancomycin.
Peak and trough concentrations of vancomycin should be 40 μg/ml and 10 μg/ml, respectively, to both be effective and avoid oto- or nephrotoxicity in adults. There is no ideal pattern of vancomycin dosing; vancomycin dosages must be individualized. Because vancomycin activity is primarily time-dependent, the 24-h area under the curve (AUC0-24h) divided by the minimum inhibitor concentration (MIC) value (AUC0-24h/MIC) is a better predictor of efficacy. In adults with MIC values less than 1 μg/ml, trough concentrations greater than 10 µg/ml result in AUC0-24h/MIC values 400
Compared with adults, neonates have a higher extracellular fluid volume and a limited renal elimination capacity resulting in different pharmacokinetics subject to maturation stage. Infants weighing less than 1,000 gm had significantly larger volumes of drug distribution and consequently longer drug half-lives than larger premature infants, regardless of postconceptual or actual age. These differences alter the vancomycin dosing recommendations in these two groups of premature infants.
Vancomycin-associated nephrotoxicity is rare in neonates, even with serum peak concentrations 40 microg/mL. Vancomycin is associated with ototoxicity.
There is no consensus on vancomycin dosing in newborns and young infants, which leads to significant variation in vancomycin dosing regimens and TDM guidance across neonatal units. The development of standardized, evidence-based protocols should be prioritized. | Multidose pharmacokinetics of vancomycin were studied in 15 infants with
gestational age less than 36 weeks and suspected or confirmed Staphylococcus
epidermidis infections. Postconceptional age (PCA) at the time of the study
ranged from 26 to 44 weeks. Vancomycin individual doses ranged from 6.7 to 10.6
mg/kg and were infused over 60 min. Five postinfusion samples were obtained in
13 infants, while 4 samples were obtained in 2 patients. Vancomycin
pharmacokinetic parameters were determined by fitting the data to a
two-compartment model using a weighted least-squares nonlinear regression
method. Mean vancomycin body clearance (CL), volume of distribution (Vdss) and
terminal elimination half-life were 1.37 ml/min, 0.58 liters and 5.6 h,
respectively. When standardized for patient weight, the CL and Vdss values were
1.07 ml/min/kg and 0.48 liters/kg, respectively. The CL (ml/min/kg) was strongly
inversely correlated with the serum creatinine (r = -0.82), while a weaker but
significant association was noted with PCA (r = 0.41). These data suggest that
in sick infants, in addition to the PCA, serum creatinine should be considered
when determining the initial vancomycin dosing regimen. Vancomycin pharmacokinetics were studied in nine premature infants. Infants
weighing less than 1,000 gm had significantly larger volumes of drug
distribution and consequently longer drug half-lives than larger premature
infants, regardless of postconceptual or actual age. These differences alter the
vancomycin dosing recommendations in these two groups of premature infants. We
recommend initial dosage regimens consisting of a loading dose of vancomycin of
25 mg/kg followed by doses of 15 mg/kg every 12 hours for infants with weights
less than 1,000 gm. Infants weighing over 1,000 gm should receive 10 mg/kg every
12 hours, with a loading dose of 12.5 mg/kg. Serum vancomycin concentration
should be monitored, however, for final optimization of therapy. OBJECTIVE: The purpose of this study was to compare the incidence of
nephrotoxicity, defined as doubling of baseline serum creatinine concentration,
in newborn infants with peak vancomycin serum concentrations </=40 microg/mL at
steady state to infants with peak vancomycin serum concentrations >40 microg/mL.
A secondary objective was to correlate concomitant disease states and
potentially nephrotoxic drug therapy with rises in serum creatinine in
vancomycin recipients.
METHODS: Newborn infants with culture-proven Staphylococcus aureus or
coagulase-negative staphylococcal septicemia who received vancomycin therapy for
>3 days between 1985 and 1995 were identified from an existing database and a
review of medical record. All 69 patients included in the study had serial serum
creatinine determinations, including a baseline value within 48 hours of
starting treatment with vancomycin, and serum vancomycin concentrations
determined after at least three doses, with peak and trough concentrations
determined 1 hour after a 60-minute infusion and 15 to 30 minutes before a dose,
respectively. Infants with congenital renal or cardiac anomalies were excluded.
Demographic characteristics, vancomycin dosing regimen, serum vancomycin
concentrations and sample times, concomitant drug therapy, and disease states
were recorded. Patients were divided into group A (peak vancomycin concentration
</=40 microg/mL) and group B (peak vancomycin concentration >40 microg/mL). The
change in serum creatinine concentration between the start and end of vancomycin
therapy was determined. Nephrotoxicity was identified if serum creatinine
doubled at any time from the start to the end of vancomycin therapy. Alternative
definitions of nephrotoxicity (any rise in serum creatinine to >0.6 mg/dL or new
abnormalities of urine sediment) were used in additional analyses.
RESULTS: A total of 69 evaluable patients (gestational age, 28.9 +/- 3.0 weeks;
birth weight, 1219 +/- 516 g) were identified, 61 in group A and 8 in group B.
Six patients in group A underwent doubling of serum creatinine concentration
during vancomycin therapy, whereas none in group B did so. Serum creatinine
doubled to >0.6 mg/dL in only 3 infants (all in group A). Any increase in serum
creatinine to >0.6 mg/dL was seen in 10 infants, 9 of whom were in group A. No
confounding variable, including previous or concomitant underlying disease
states associated with renal dysfunction or treatment with other potentially
nephrotoxic agents, were associated with a significant rise in serum creatinine.
CONCLUSION: Vancomycin-associated nephrotoxicity is rare in neonates, even with
serum peak concentrations >40 microg/mL. BACKGROUND: Recently the value of vancomycin therapeutic drug monitoring, as
well as the required therapeutic range, has been subject of debate, resulting in
new recommendations. This study was performed to incorporate these new insights
in an up-to-date dosing scheme for neonates of various gestational ages.
METHODS: In this retrospective study with prospective validation, 108 newborns
with suspected central line-related septicemia during the first month of life
received 30 mg/kg/day vancomycin divided into two doses regardless of
gestational or postconceptional age. Trough and peak vancomycin serum
concentrations were determined before and after the third dose. Vancomycin data
were analyzed according to a one-compartment open model with use of NONMEM
population pharmacokinetic software. Model parameters were evaluated and then
used to simulate vancomycin dosing for different dose and dose interval
combinations. Targets were a trough concentration between 5 and 15 mg/L and a
peak below 40 mg/L. In the prospective study, the optimal scheme was tested in
22 patients.
RESULTS: Of the 108 patients, 34.3% of measured trough concentrations and 17.6%
of peak concentrations were outside the desired therapeutic range. The model
that best fitted the data included clearance and volume per kilogram and was
independent of gestational age. Simulation of various dosing schemes showed that
a dosing schedule of 30 mg/kg/day, irrespective of gestational age, in three
doses was optimal, and this scheme was prospectively tested. Mean trough
concentrations before the second dose were 8.2 +/- 2.2 mg/L versus a predicted
trough of 8.9 +/- 2.5 mg/L. No peak levels higher than 40 mg/L were found.
CONCLUSIONS: The use of the proposed schedule leads to adequate vancomycin
trough serum concentrations, and there is no need for routine monitoring of peak
serum concentrations. BACKGROUND: There is no consensus on vancomycin dosing in newborns and young
infants.
OBJECTIVE: The first objective was to assess the efficiency of a simplified
dosing regimen with a cohort study. The secondary objective was to examine
pharmacokinetic data to determine how this simplified dosing could be improved.
METHODS: All neonates admitted to our intensive care unit and treated with
vancomycin were included in the pharmacokinetic study (PK group, 83 treatments,
156 measurements). The vancomycin dosing regimen consisted of a loading dose of
7 mg/kg, followed by a constant continuous dose of 30 mg/kg/day. The target
serum vancomycin concentration ranged from 10 mg/l to 30 mg/l. Data from
patients whose medications followed the scheduled dosing without modifications
or prescription errors (actual dosing group: 62 treatments, 108 measurements)
were analysed separately. A population pharmacokinetic analysis was performed
(PK group) to simulate several vancomycin dosings.
RESULTS: Prescription errors were found in 10 of 83 treatments (12%). In the
actual dosing group, 89.2% of vancomycin measurements were within the target
range. Serum creatinine remained stable throughout treatment. Vancomycin
concentrations varied widely. The modified regimen for a target vancomycin
concentration of 25 mg/l consisted of a bolus of 20 mg/kg followed by continuous
infusion of 30 mg/kg.
CONCLUSION: Our pharmacokinetic data and bedside results suggest that a
simplified schedule of vancomycin can achieve the targeted drug concentrations
in most patients while avoiding secondary renal toxicity. The proposed new
dosing scheme should be validated in a drug survey, but due to pharmacokinetic
variability, still requires therapeutic drug monitoring. BACKGROUND: Aminoglycoside-induced ototoxicity has been reported in neonates but
its incidence is poorly defined, whereas vancomycin-induced ototoxicity has not
been reported in neonates.
OBJECTIVE: To compare hearing test results in infants in a neonatal intensive
care unit (NICU) who were or were not treated with extended interval gentamicin
dosing and/or standard vancomycin dosing.
METHOD: A database of otoacoustic emissions (OAE), over a 5-year period of NICU
admissions, was combined with databases of gentamicin and vancomycin dosing to
compare patients treated or not treated with these antibiotics.
RESULTS: A total of 2,347 OAE results was available. OAE failure rates were: no
gentamicin and no vancomycin (noGnoV), 7% (85/1,233); gentamicin but no
vancomycin (GnoV), 4% (42/949); vancomycin but no gentamicin (VnoG), 22% (9/41)
and gentamicin and vancomycin (GandV), 14% (17/124). Compared to noGnoV there
was a decreased risk of OAE failure in GnoV (p = 0.022, OR 0.64, 95% CI
0.44-0.94) and an increased risk in VnoG (p = 0.003, OR 3.46, 95% CI 1.54-7.75)
and GandV, (p = 0.006, OR 2.20, 95% CI 1.26-3.83).
CONCLUSIONS: Gentamicin, as used and evaluated in this audit, showed no evidence
of an increased risk of ototoxicity; what was observed was a statistically
significant decrease in OAE failure rate. Vancomycin, by contrast, was
associated with ototoxicity. STUDY OBJECTIVE: To compare four common dosing regimens for vancomycin in
preterm and term neonates by assessing the probability that each regimen would
achieve the widely used therapeutic target serum trough concentrations of 5-15
mg/L and the newly suggested target of 15-20 mg/L.
DESIGN: Retrospective population pharmacokinetic analysis using therapeutic drug
monitoring data obtained from 1990-2007, with a subsequent simulation study
performed on the pharmacokinetic model.
SETTING: Tertiary-care children's hospital.
PATIENTS: One hundred thirty-four preterm (66%) and term (34%) neonates, with a
postnatal age of 1-121 days and postmenstrual age of 24.6-44 weeks.
MEASUREMENTS AND MAIN RESULTS: Therapeutic drug monitoring data for vancomycin
were used to develop a population pharmacokinetic model in the target
population. Parameter estimates for the derived pharmacostatistical model were
used to perform Monte Carlo simulations for four recommended dosing regimens: a
standard dose for all neonates, postmenstrual age-based dosing, postmenstrual
and postnatal age-based dosing, and serum creatinine-based dosing. Multivariate
age-weight distributions were established for term and preterm neonates using
Centers for Disease Control and Prevention growth charts and intrauterine and
postnatal growth charts from the literature, respectively. Each dosing regimen
was treated as a separate scenario in which 200 replicates with 100
patients/replicate were simulated. The 5-15-mg/L target trough serum
concentration was achieved in 34% (90% confidence interval [CI] 20-53%), 42%
(90% CI 31-55%), 52% (90% CI 43-60%), and 63% (90% CI 54-72%) of patients
receiving the standard dose, postmenstrual age-based dose, postmenstrual and
postnatal age-based dose, and serum creatinine-based dose, respectively. Serum
creatinine-based dosing produced trough concentrations predomitly in the
5-15-mg/L target range, with the smallest variability in both term and preterm
neonates. As expected, when the target range was narrow and higher (15-20 mg/L),
only 13-21% of patients were within the range across the four dosing regimens.
CONCLUSION: Monte Carlo simulations based on our population pharmacokinetic
model suggest that vancomycin dosing guidelines based on serum creatinine
concentration have a greater likelihood of achieving trough concentrations in
the 5-15-mg/L range compared with other evaluated dosing regimens. None of the
four dosing regimens is suitable to produce target trough concentration of 15-20
mg/L in an acceptable number of patients. Neonatal sepsis is common and is a major cause of morbidity and mortality.
Vancomycin is the preferred treatment of several neonatal staphylococcal
infections. The aim of this study was to review published data on vancomycin
pharmacokinetics in neonates and to provide a critical analysis of the
literature. A bibliographic search was performed using PubMed and Embase, and
articles with a publication date of August 2011 or earlier were included in the
analysis. Vancomycin pharmacokinetic estimates, which are different in neonates
compared with adults, also exhibit extensive inter-neonatal variability. In
neonates, several vancomycin dosing schedules have been proposed, mainly based
on age (i.e., postmenstrual and postnatal), body weight or serum creatinine
level. Other covariates [e.g., extracorporeal membrane oxygenation (ECMO),
indomethacin or ibuprofen, and growth restriction] of vancomycin
pharmacokinetics have been reported in neonates. Finally, vancomycin penetrates
cerebrospinal fluid (range = 7-42%). Renal function drives vancomycin
pharmacokinetics. Because either age or weight is the most relevant covariate of
renal maturation, these covariates should be considered first in neonatal
vancomycin dosing guidelines and further adjusted by renal dysfunction
indicators (e.g., ECMO and ibuprofen/indomethacin). In addition to the
prospective validation of available dosing guidelines, future studies should
focus on the relevance of therapeutic drug monitoring and on the value of
continuous vancomycin administration in neonates. |
Is single guide RNA part of the CRISPR/Cas9 tool or an inhibitor of its function? | Single guide RNA is part of the CRISPR/Cas9 system. | Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome
modifications across a range of eukaryotes. Here we engineer this system to
enable RNA-guided genome regulation in human cells by tethering transcriptional
activation domains either directly to a nuclease-null Cas9 protein or to an
aptamer-modified single guide RNA (sgRNA). Using this functionality we developed
a transcriptional activation-based assay to determine the landscape of
off-target binding of sgRNA:Cas9 complexes and compared it with the off-target
activity of transcription activator-like (TALs) effectors. Our results reveal
that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and
18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches,
respectively. By engineering a requirement for cooperativity through offset
nicking for genome editing or through multiple synergistic sgRNAs for robust
transcriptional activation, we suggest methods to mitigate off-target phenomena.
Our results expand the versatility of the sgRNA:Cas9 tool and highlight the
critical need to engineer improved specificity. Genetic modification, including plant breeding, has been widely used to improve
crop yield and quality, as well as to increase disease resistance. Targeted
genome engineering is expected to contribute significantly to future varietal
improvement, and genome editing technologies using zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases (TALENs), and clustered
regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA
(sgRNA) have already been successfully used to genetically modify plants.
However, to date, there has been no reported use of any of the current genome
editing approaches in sweet orange, an important fruit crop. In this study, we
first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing
transient protein expression in sweet orange leaves. We then successfully
employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a
synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing
confirmed that the CsPDS gene was mutated at the target site in treated sweet
orange leaves. The mutation rate using the Cas9/sgRNA system was approximately
3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA
sequences in our study. This is the first report of targeted genome modification
in citrus using the Cas9/sgRNA system-a system that holds significant promise
for the study of citrus gene function and for targeted genetic modification. Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in
part due to the availability of excellent genetic tools. Although reverse
genetic tools are available in a few widely utilized laboratory strains, they
rely on special genetic backgrounds that are not easily implemented in natural
isolates. Recent progress in modifying CRISPR (clustered regularly interspaced
short palindromic repeats), a system of DNA recognition used as a defense
mechanism in bacteria and archaea, has led to extremely efficient gene
disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based
system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9
provided an extremely efficient system for targeted gene disruption and for
site-specific insertion of selectable markers through homologous recombination.
CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology,
thus increasing the utility of this approach over existing technology. We then
tested whether CRISPR/CAS9 would enable efficient transformation of a natural
isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18
knockouts and complemented lines in the type I GT1 strain, which has been used
for forward genetic crosses but which remains refractory to reverse genetic
approaches. Assessment of their phenotypes in vivo revealed that ROP18
contributed a greater proportion to acute pathogenesis in GT1 than in the
laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic
techniques to diverse isolates of T. gondii, allowing exploration of a much
wider spectrum of biological diversity.
IMPORTANCE: Genetic approaches have proven very powerful for studying the
biology of organisms, including microbes. However, ease of genetic manipulation
varies widely among isolates, with common lab isolates often being the most
amenable to such approaches. Unfortunately, such common lab isolates have also
been passaged frequently in vitro and have thus lost many of the attributes of
wild isolates, often affecting important traits, like virulence. On the other
hand, wild isolates are often not amenable to standard genetic approaches, thus
limiting inquiry about the genetic basis of biological diversity. Here we
imported a new genetic system based on CRISPR/CAS9, which allows high efficiency
of targeted gene disruption in natural isolates of T. gondii. This advance
promises to bring the power of genetics to bear on the broad diversity of
T. gondii strains that have been described recently. CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly
efficient targeting of DNA sequences complementary to their RNA guide strands.
However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA
sequences containing mismatches to the guide strand. A better understanding of
the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large
mammalian genomes. Here we show that genomic sites could be cleaved by
CRISPR/Cas9 systems when DNA sequences contain insertions ('DNA bulge') or
deletions ('RNA bulge') compared to the RNA guide strand, and Cas9 nickases used
for paired nicking can also tolerate bulges in one of the guide strands.
Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as
model systems, and their cleavage activities were quantified at different
positions with 1- to 5-bp bulges. We further investigated 114 putative genomic
off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each
harboring a single-base bulge and one to three mismatches to the guide strand.
Our results strongly indicate the need to perform comprehensive off-target
analysis related to DNA and sgRNA bulges in addition to base mismatches, and
suggest specific guidelines for reducing potential off-target cleavage. BACKGROUND: The type II clustered, regularly interspaced, short palindromic
repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular
tool for site-specific genome modification. The CRISPR-Cas9 system was recently
introduced into plants by transient or stable transformation.
FINDINGS: Here, we report gene targeting in rice via the Agrobacterium
tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed
to pair with diverse sites followed by the protospacer adjacent motif (PAM) of
the rice herbicide resistance gene BEL. After integrating the single-guide RNA
(sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants
harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable
transformation. By analyzing the targeting site on the genome of corresponding
transgenic plants, the mutations were determined. The mutagenesis efficiency was
varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the
biallelic mutated transgenic plant was sensitive to bentazon.
CONCLUSIONS: Our results indicate that the agricultural trait could be purposely
modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be
exploited as a powerful tool for trait improvements in crop breeding. CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against
infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which
use small RNA guides (the crRNAs) to specify a cleavage site within the genome
of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving
activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using
synthetic single RNA guides, Cas9 can be reprogrammed to create specific
double-stranded DNA breaks in the genomes of a variety of organisms, ranging
from human cells to bacteria, and thus constitutes a powerful tool for genetic
engineering. Here we describe recent advancements in our understanding of type
II CRISPR-Cas immunity and how these studies led to revolutionary genome editing
applications. Engineered DNA-binding proteins that manipulate the human genome and
transcriptome have enabled rapid advances in biomedical research. In particular,
the RNA-guided CRISPR/Cas9 system has recently been engineered to create
site-specific double-strand breaks for genome editing or to direct targeted
transcriptional regulation. A unique capability of the CRISPR/Cas9 system is
multiplex genome engineering by delivering a single Cas9 enzyme and two or more
single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can
be used to simultaneously create multiple DNA breaks or to target multiple
transcriptional activators to a single promoter for synergistic enhancement of
gene induction. To address the need for uniform and sustained delivery of
multiplex CRISPR/Cas9-based genome engineering tools, we developed a single
lentiviral system to express a Cas9 variant, a reporter gene and up to four
sgRNAs from independent RNA polymerase III promoters that are incorporated into
the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently
expressed and can mediate multiplex gene editing and sustained transcriptional
activation in immortalized and primary human cells. This delivery system will be
significant to enabling the potential of CRISPR/Cas9-based multiplex genome
engineering in diverse cell types. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9
system has become a powerful and precise tool for targeted gene modification
(e.g., gene knockout and gene replacement) in numerous eukaryotic organisms.
Initial attempts to apply this technology to a model, the single-cell alga,
Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To
determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C.
reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one
of four different sgRNAs to cause targeted gene disruption during a 24-h period
immediately following transformation. All three exogenously supplied gene
targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C.
reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as
determined by DNA sequencing of cloned PCR amplicons of the target site region.
Success in transient expression of Cas9 and sgRNA genes contrasted with the
recovery of only a single rapamycin-resistant colony bearing an appropriately
modified FKB12 target site in 16 independent transformation experiments
involving >10(9) cells. Failure to recover transformants with intact or
expressed Cas9 genes following transformation with the Cas9 gene alone (or even
with a gene encoding a Cas9 lacking nuclease activity) provided strong
suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C.
reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA
genes function properly in C. reinhardtii to cause targeted gene modifications
and point to the need for a focus on development of methods to properly stem
Cas9 production and/or activity following gene editing. Author information:
(1)Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084,
China.
(2)Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084,
China; Tsinghua Fly Center, Tsinghua University, Beijing 100084, China; School
of Basic Medical Sciences, Wuhan University, Wuhan 430071, China; College of
Bioengineering, Hubei University of Technology, Wuhan 430068, China.
(3)Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084,
China; Sichuan Academy of Grassland Science, Chengdu 611731, China.
(4)Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
(5)Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
(6)Department of Genetics, Stanford University, Stanford, CA 94305, USA.
(7)Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084,
China; Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
(8)Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M
Health Science Center, College Station, TX 77843, USA.
(9)Department of Genetics, Stanford University, Stanford, CA 94305, USA.
Electronic address: [email protected].
(10)Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing
100084, China. Electronic address: [email protected]. Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced
short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool
for genome editing, transcriptional regulation, and cellular imaging
applications. Structures of Streptococcus pyogenes Cas9 alone or bound to
single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture
that undergoes major conformational changes upon guide RNA and DNA binding. To
investigate the molecular determits and relevance of the interlobe
rearrangement for target recognition and cleavage, we designed a split-Cas9
enzyme in which the nuclease lobe and α-helical lobe are expressed as separate
polypeptides. Although the lobes do not interact on their own, the sgRNA
recruits them into a ternary complex that recapitulates the activity of
full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified
sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the
development of an inducible dimerization system. We propose that split-Cas9 can
act as a highly regulatable platform for genome-engineering applications. As a powerful genome-editing tool, the clustered regularly interspaced short
palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic
repeats-associated protein 9 (Cas9) system has been quickly developed into a
large-scale function-based screening strategy in mammalian cells. This new type
of genetic library is constructed through the lentiviral delivery of
single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with
effectors to interrogate gene function or regulate gene transcription in
targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system
demonstrates much higher levels of effectiveness and reliability with respect to
both loss-of-function and gain-of-function screening. Unlike the RNA
interference strategy, a CRISPR-Cas9 library can target both protein-coding
sequences and regulatory elements, including promoters, enhancers and elements
transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will
undoubtedly accelerate the mechanistic discovery of various biological
processes. In this mini review, we summarize the general procedure of
CRISPR-Cas9 library mediated functional screening, system optimization
strategies and applications of this new genetic toolkit. |
What is the target protein of the drug Idelalisib? | Idelalisib represents a first-in-class specific inhibitor of the phosphoinositol-3 kinase (PI3K) delta isoform. | Normal B lymphocytes receive signals from B-cell antigen receptor (BCR) that are
triggered by binding of the BCR to an external antigen. Tonic signaling through
the BCR provides growth and signals to chronic lymphocytic leukemia (CLL) cells,
and plays an important role in the pathogenesis and progression of the disease.
Antigen engagement of BCR is followed by intracellular recruitment and
activation of BCR-associated kinases including spleen tyrosine kinase (Syk),
Bruton's tyrosine kinase (Btk) and phosphatidylinositol 3-kinases (PI3K).
Inhibition of signaling pathways downstream of the BCR induces disruption of
chemokine-mediated CLL cell migration and cell killing. BCR signal transduction
inhibitors represent a promising new strategy for targeted CLL treatment. A
number of therapeutic agents have recently been developed with significant
activity in CLL. The compounds that are currently investigated in patients with
CLL include ibrutinib -inhibitor of Btk, fostamatinib-inhibitor of Syk and
idelalisib (GS-1101) -a specific isoform of the PI3K (PI3K) inhibitor. The
clinical activity of ibrutinib, GS-1101 and fostamatinib in patients with CLL is
associated with marked lymphocytosis due to release of tumor cells from the
lymph nodes into the peripheral blood. Further studies are ongoing with single
agents and their combinations with other targeted and conventional therapies.
This article will review the preclinical rationale of BCR signaling inhibitors
in the treatment of CLL, and the clinical evidence supporting the use of these
agents in CLL patients. Membrane antigens are critical to the pathogenesis of chronic lymphocytic
leukemia (CLL) as they facilitate microenvironment homing, proliferation, and
survival. Targeting the CLL membrane and associated signaling patterns is a
current focus of therapeutic development. Many tumor membrane targets are
simultaneously targeted by humoral immunity, thus forming recognizable
immunoglobulin responses. We sought to use this immune response to identify
novel membrane-associated targets for CLL. Using a novel strategy, we
interrogated CLL membrane-specific autologous immunoglobulin G reactivity. Our
analysis unveiled lymphocyte cytosolic protein 1 (LCP1), a lymphocyte-specific
target that is highly expressed in CLL. LCP1 plays a critical role in B-cell
biology by crosslinking F-actin filaments, thereby solidifying cytoskeletal
structures and providing a scaffold for critical signaling pathways. Small
interfering RNA knockdown of LCP1 blocked migration toward CXCL12 in transwell
assays and to bone marrow in an in vivo xenotransplant model, confirming a role
for LCP1 in leukemia migration. Furthermore, we demonstrate that the Bruton's
tyrosine kinase inhibitor ibrutinib or the PI3K inhibitor idelalisib block
B-cell receptor induced activation of LCP1. Our data demonstrate a novel
strategy to identify cancer membrane target antigens using humoral anti-tumor
immunity. In addition, we identify LCP1 as a membrane-associated target in CLL
with confirmed pathogenic significance. This clinical trial was registered at
clinicaltrials.gov; study ID number: OSU-0025 OSU-0156. The B cell antigen receptor (BCR) and its downstream pathways are pivotal in the
pathogenesis of chronic lymphocytic leukemia (CLL). Recently, inhibitors of
kinases in the BCR pathway have shown promising clinical activity in CLL. Based
upon these results, the treatment paradigm for CLL will likely undergo major
changes. The kinases essential for BCR signal transduction, which are emerging
as targets for CLL treatment, and the specific inhibitors under development are
the focus of this chapter. In particular, the BTK inhibitor ibrutinib and the
PI3K inhibitor idelalisib (GS-1101) are two evolving targeted therapies with the
most mature clinical data. The B-cell receptor (BCR) is essential for normal B-cell development and
maturation. In an increasing number of B-cell maligcies, BCR signaling is
implicated as a pivotal pathway in tumorigenesis. Mechanisms of BCR activation
are quite diverse and range from chronic antigenic drive by microbial or viral
antigens to autostimulation of B-cells by self-antigens to activating mutations
in intracellular components of the BCR pathway. Hepatitis C virus infection can
lead to the development of splenic marginal zone lymphoma, while Helicobacter
pylori infection is associated with the development of mucosa-associated
lymphoid tissue lymphomas. In some of these cases, successful treatment of the
infection removes the inciting antigen and results in resolution of the
lymphoma. Chronic lymphocytic leukemia has been recognized for decades as a
maligcy of auto-reactive B-cells and its clinical course is in part
determined by the differential response of the maligt cells to BCR
activation. In a number of B-cell maligcies, activating mutations in signal
transduction components of the BCR pathway have been identified; prominent
examples are activated B-cell-like (ABC) diffuse large B-cell lymphomas (DLBCL)
that carry mutations in CD79B and CARD11 and display chronic active BCR
signaling resulting in constitutive activation of the NF-κB pathway. Despite
considerable heterogeneity in biology and clinical course, many mature B-cell
maligcies are highly sensitive to kinase inhibitors that disrupt BCR
signaling. Thus, targeted therapy through inhibition of BCR signaling is
emerging as a new treatment paradigm for many B-cell maligcies. Here, we
review the role of the BCR in the pathogenesis of B-cell maligcies and
summarize clinical results of the emerging class of kinase inhibitors that
target this pathway. Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse
cellular processes including proliferation, adhesion, survival, and motility.
Dysregulated PI3K pathway signaling occurs in one-third of human tumors.
Aberrantly activated PI3K signaling also confers sensitivity and resistance to
conventional therapies. PI3K has been recognized as an attractive molecular
target for novel anti-cancer molecules. In the last few years, several classes
of potent and selective small molecule PI3K inhibitors have been developed, and
at least fifteen compounds have progressed into clinical trials as new
anticancer drugs. Among these, idelalisib has advanced to phase III trials in
patients with advanced indolent non-Hodgkin's lymphoma and mantle cell lymphoma.
In this review, we summarized the major molecules of PI3K signaling pathway, and
discussed the preclinical models and clinical trials of potent small-molecule
PI3K inhibitors. DISEASE OVERVIEW: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma
characterized by involvement of the lymph nodes, spleen, blood, and bone marrow
with a short remission duration to standard therapies and a median overall
survival of 4-5 years.
DIAGNOSIS: Diagnosis is based on lymph node, bone marrow, or tissue morphology
of centrocytic lymphocytes, small cell type, or blastoid variant cells. A
chromosomal translocation t(11:14) is the molecular hallmark of MCL, resulting
in the overexpression of cyclin D1. Cyclin D1 is detected by
immunohistochemistry in 98% of cases. The absence of SOX-11 or a low Ki-67 may
correlate with a more indolent form of MCL. The differential diagnosis of MCL
includes small lymphocytic lymphoma, marginal zone lymphoma, and follicular
lymphoma.
RISK STRATIFICATION: The Mantle Cell Lymphoma International Prognostic Index
(MIPI) is the prognostic model most often used and incorporates ECOG performance
status, age, leukocyte count, and lactic dehydrogenase. A modification of the
MIPI also adds the Ki-67 proliferative index if available. The median overall
survival (OS) for the low risk group was not reached (5-year OS of 60%). The
median OS for the intermediate risk group was 51 months and 29 months for the
high risk group.
RISK-ADAPTED THERAPY: For selected indolent, low MIPI MCL patients, initial
observation may be appropriate therapy. For younger patients with intermediate
or high risk MIPI MCL, aggressive therapy with a cytarabine containing regimen ±
autologous stem cell transplantation should be considered. For older MCL
patients with intermediate or high risk MIPI, combination chemotherapy with
R-CHOP, R-Bendamustine, or a clinical trial should be considered. At the time of
relapse, agents directed at activated pathways in MCL cells such as bortezomib
(NFkB inhibitor) or lenalidamide (anti-angiogenesis) are approved agents.
Clinical trials with Ibruitinib (Bruton's Tyrosine Kinase inhibitor) or
Idelalisib (PI3K inhibitor) have demonstrated excellent clinical activity in MCL
patients. Autologous or allogeneic stem cell transplantation can also be
considered in young patients. Interactions between maligt B lymphocytes and the tissue microenvironment
play a major role in the pathogenesis of chronic lymphocytic leukemia (CLL) and
other B-cell maligcies. The coexistence and coevolution of CLL cells with
their tissue neighbors provided the basis for discovery of critical cellular and
molecular drivers of the disease and identification of new therapeutic targets.
Bone marrow stromal cells (BMSC), monocyte-derived nurselike cells (NLC), and T
cells are key players in the CLL microenvironment, which activate and protect
CLL cells within the tissues. CLL surface molecules, such as the B-cell antigen
receptor (BCR), chemokine receptors, adhesion molecules, and TNF receptor
superfamily members (e.g., CD40, BCMA, and BAFF-R) engage in cross-talk with
respective tissue ligands. This cross-talk results in survival and expansion of
the CLL clone, and protects CLL cells from conventional cytotoxic drugs.
Inhibiting these pathways represents an alternative therapeutic strategy to more
conventional chemoimmunotherapy. Here, we review central components of the CLL
microenvironment, with a particular emphasis on BCR signaling, and we summarize
the most relevant clinical advances with inhibitors that target the
BCR-associated spleen tyrosine kinase/SYK (fostamatinib), Bruton's tyrosine
kinase/BTK (ibrutinib), and PI3Kδ (idelalisib). |
Which antiepileptic drug is most strongly associated with spina bifida? | Phenytoin is not used in pregnancy as it is associated with a severe fetal deformation. From the other anticonvulsants most studies report the higher association between use during pregnancy and spin bifida to occur with Valproate. | The distribution of anticonvulsant drug therapy was studied in 318 malformed
infants with known histories of maternal epilepsy. Data on the infants was
collected from six birth defect monitoring programs in Europe and South America.
Use of specific types of anticonvulsants varies widely among reporting
countries. Heterogeneity of drug-malformation distribution, was analyzed to
determine whether use of specific drugs were linked to specific malformations. A
significant association was seen between maternal use of valproic acid and spina
bifida, and a weaker, non-significant one between carbamazepine and spina
bifida. Facial clefts were associated with both diphenylhydantoin and
phenobarbitone use and also with polytherapy. These differences indicate that
the actual drug used is significant for the teratogenic process. The technique
may be useful in analysis of other drug-related teratogenic questions. A case control study on the association between maternal epilepsy,
anticonvulsants use during pregcy and birth defects was carried out in the
Italian Multicentric Registry of Birth Defects (IPIMC). In the period 1980-1983,
7,607 malformed babies out of 439,717 total births (still + live) were
registered. Fourty-one malformed babies with maternal epilepsy were identified
(5.39 X 1,000). The overall relative risk of having a malformed baby among
pregt epileptic women was 1.87. Spina Bifida, Congenital Heart Defects,
Clefts, Diaphragmatic Hernia and Trisomy 18 were more frequent than expected
among babies with maternal epilepsy. The different therapeutic regimens were
also tested to identify the possible independent teratogenic effect of
anticonvulsants. A statistically significant association between Spina Bifida
and Valproic Acid (odds ratio 22.7; Fisher p value = 0.0364) was observed: no
other anticonvulsant tested showed any association with any type of
malformation. Women with epilepsy giving birth during 1973 to 1991 were identified by record
linkage of Swedish health registries. Among 3,625 identified infants, 9 had
spina bifida. A nested case-control study was performed, comparing drugs used in
early pregcy in the 9 cases and in 18 controls, matched for year of delivery,
maternal age, and parity. Six of the spina bifida mothers had used carbamazepine
and two had used valproic acid. Among the controls, 5 women used carbamazepine
and one valproic acid. There is an apparent excess risk for spina bifida after
use of either of these two drugs, but it is not statistically significant when
the analysis is restricted to drug-using women. The findings support earlier
reports in the literature of an association between carbamazepine and spina
bifida. PURPOSE: The study goal was to assess teratogenic effects of antiepileptic drugs
(AEDs) through the use of a surveillance system (MADRE) of infants with
malformations.
METHODS: Information on all malformed infants (1990-1996) with maternal
first-trimester drug exposure was collected by the International Clearinghouse
for Birth Defects and Monitoring Systems (ICBDMS). Cases were defined as infants
presenting with a specific malformation, and controls were defined as infants
presenting with any other birth defect. Exposure was defined by the use of AEDs
during the first trimester of pregcy. The association of AEDs with
malformations was then estimated by calculating the odds ratios with 95%
confidence intervals and testing their homogeneity among registries.
RESULTS: Among 8005 cases of malformations, 299 infants were exposed in utero to
AEDs. Of those exposed to monotherapy, 65 were exposed to phenobarbital, 10 to
methylphenobarbital, 80 to valproic acid, 46 to carbamazepine, 24 to phenytoin,
and 16 to other AEDs. Associations were found for spina bifida with valproic
acid. Infants exposed to phenobarbital and to methylphenobarbital showed an
increased risk of oral clefts. Cardiac malformations were found to be associated
with phenobarbital, methylphenobarbital, valproic acid, and carbamazepine.
Hypospadias was associated with valproic acid. Porencephaly and other specified
anomalies of brain, anomalies of face, coarctation of aorta, and limb reduction
defects were found to be associated with valproic acid.
CONCLUSIONS: Using the MADRE system, we confirmed known teratogenic effects of
AEDs. We also found increased risks for malformations that had never been
reported associated with AEDs or for which the association was suggested by case
reports. Valproic acid, a drug commonly used to treat seizures and other psychiatric
disorders, causes neural tube defects (NTDs) in exposed fetuses at a rate 20
times higher than in the general population. Failure of the neural tube to close
during development results in exencephaly or anencephaly, as well as spina
bifida. In mice, nonspecific activation of the maternal immune system can reduce
fetal abnormalities caused by diverse etiologies, including diabetes-induced
NTDs. We hypothesized that nonspecific activation of the maternal immune system
with interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating
factor (GM-CSF) could reduce valproic acid (VA)-induced defects as well. Female
CD-1 mice were given immune stimulant prebreeding: either IFN-gamma or GM-CSF.
Approximately half of the control and immune-stimulated pregt females were
then exposed to 500 mg/kg VA on the morning of gestational day 8. The incidence
of developmental defects was determined on gestational day 17 from at least
eight litters in each of the following treatment groups: control, VA only,
IFN-gamma only, IFN-gamma+VA, GM-CSF only, and GM-CSF+VA. The incidence of NTDs
was 18% in fetuses exposed to VA alone, compared to 3.7% and 2.9% in fetuses
exposed to IFN-gamma+VA, or GM-CSF+VA respectively. Ocular defects were also
significantly reduced from 28.0% in VA exposed groups to 9.8% in IFN-gamma+VA
and 12.5% in GM-CSF+VA groups. The mechanisms by which maternal immune
stimulation prevents birth defects remain unclear, but may involve maternal or
fetal production of cytokines or growth factors which protect the fetus from the
dysregulatory effects of teratogens. PURPOSE: To investigate pregcy, delivery, and child outcome in an unselected
population of women with both treated and untreated epilepsy.
METHODS: In the compulsory Medical Birth Registry of Norway, all 2,861
deliveries by women with epilepsy recorded from 1999-2005 were compared to all
369,267 nonepilepsy deliveries in the same period.
RESULTS: The majority (66%, n = 1900) in the epilepsy group did not use
antiepileptic drugs (AEDs) during pregcy. A total of 961 epilepsy-pregcies
were exposed to AEDs. Compared to nonepilepsy controls, AED-exposed infants were
more often preterm (p = 0.01), and more often had birth weight <2,500 g (p <
0.001), head circumference <2.5 percentile (p < 0.001), and low Apgar score (p =
0.03). Small-for-gestational-age (SGA) infants (<10 percentile) occurred more
frequently in both AED-exposed (p = 0.05) and unexposed (p = 0.02)
epilepsy-pregcies. Frequency of major congenital malformations (MCMs) was
2.8% (n = 81) in the epilepsy group versus 2.5% in controls (p = 0.3). Increased
risk for MCMs could be demonstrated only for exposure to valproate (5.6%, p =
0.005) and AED polytherapy (6.1%, p = 0.02). Neonatal spina bifida was not
significantly increased, but was a major indication for elective pregcy
termination among women with epilepsy. Cesarean section was performed more often
in maternal epilepsy, regardless of AED-exposure (p < 0.001).
DISCUSSION: Adverse pregcy and birth outcome in women with epilepsy is mainly
confined to AED-exposed pregcies, although some risks are associated also
with untreated epilepsy. The risk for congenital malformations was lower than
previously reported. This could be due to a shift in AED selection, folic acid
supplement, or possibly reflect the true risks in an unselected epilepsy
population. Selected antiepileptic drugs (AEDs) increase the risk of birth defects. To
assess the impact of influencing AED prescribing practices on spina bifida and
cleft palate we searched the literature for estimates of the association between
valproic acid or carbamazepine use during pregcy and these defects and
summarized the associations using meta-analyses. We estimated distributions of
the prevalence of valproic acid and carbamazepine use among women of
childbearing age based on analyses of four data sets. We estimated the
attributable fractions and the number of children born with each defect that
could be prevented annually in the United States if valproic acid and
carbamazepine were not used during pregcy. The summary odds ratio estimate
for the association between valproic acid and spina bifida was 11.9 (95%
uncertainty interval (UI): 4.0-21.2); for valproic acid and cleft palate 5.8
(95% UI: 3.3-9.5); for carbamazepine and spina bifida 3.6 (95% UI: 1.3-7.8); and
for carbamazepine and cleft palate 2.4 (95% UI: 1.1-4.5) in the United States.
Approximately 40 infants (95% UI: 10-100) with spina bifida and 35 infants (95%
UI: 10-70) with cleft palate could be born without these defects each year if
valproic acid were not used during pregcy; 5 infants (95% UI: 0-15) with
spina bifida and 5 infants (95% UI: 0-15) with cleft palate could be born
without these defects each year if carbamazepine were not used during pregcy.
This modeling approach could be extended to other medications to estimate the
impact of translating pharmacoepidemiologic data to evidence-based prenatal care
practice. The teratogenicity of antiepilepsy drug valproic acid (VPA) mostly is found in
genetic and somatic levels, causing teratogenesis involving neurotubular defects
(NTDs), anencephaly, lumbosacral meningomyelocele, and leg dysfunction due to
spina bifida aperta. A diversity of nutraceutics have been tried to alleviate
the risk of VPA-teratogenicity. The effect was varying. In order to promote the
preventive prescription, to find out its action mechanism can be rather crucial.
We used chicken embryo model to try the effect of folic acid (FA), ascorbic acid
(AA), and N-acetyl cysteine (NAC). VPA at 30mM showed the higher malformation
rate (66.7%) with the least mortality (22.2%). Pathological findings indicated
that the cervical muscle was more susceptible to VPA injury than the ankle
muscle. VPA downregulated levels of superoxide dismutase (SOD), glutathione
(GSH), histone deacetylase (HDAC) and folate, and upregulated H(2)O(2) and
homocysteine. FA, AA, and NAC significantly upregulated SOD, but only AA alone
activated GSH. AA and NAC downregulated H(2)O(2), while FA was totally
ineffective. All three nutraceutics comparably rescued HDAC with simultaneously
suppressed homocysteine accumulation and folate re-elevation, although less
effectively by NAC. Based on these data, we conclude VPA possesses "Multiple
Point Action Mechanism". In addition to affecting the cited transcription and
translation levels, we hypothesize that VPA competitively antagonize the
glutamic acid to couple with pteroic acid in biosynthesis of dihydrofolic acid
(DHFA). H(2)O(2) directly destroyed the NADPH reducing system at dihydrofolate
reductase (DHFR) and methylene tetrahydrofolate reductase (MTHFR) levels, while
completely restored by AA, an implication in preservation of intact apoenzymes.
In addition, the GSH-GSSG system is sandwiched between the reducing systems
NADPH/NADP and DHA-AA, its net balance is highly dependent on in situ in vivo
Redox state, hence folic acid transformation is varying. To rescue the
VPA-induced teratogenicity, simultaneous multiple prescriptions are suggested. Fetal hydantoin syndrome is a rare disorder that is believed to be caused by
exposure of a fetus to the anticonvulsant drug phenytoin. The classic features
of fetal hydantoin syndrome include craniofacial anomalies, prenatal and
postnatal growth deficiencies, underdeveloped nails of the fingers and toes, and
mental retardation. Less frequently observed anomalies include cleft lip and
palate, microcephaly, ocular defects, cardiovascular anomalies, hypospadias,
umbilical and inguinal hernias, and significant developmental delays.
Anaesthesia for incidental surgery in such a patient poses unique challenges for
the anesthesiologist. We report the successful management of a 4-year-old male
child with fetal hydantoin syndrome, cleft palate, spina bifida, atrial septal
defect, and dextrocardia for tibialis anterior lengthening under subarachnoid
block. |
Which bacteria caused plague? | Yersinia pestis is the causative bacteria of the plague. | Protein tyrosine phosphatases (PTPases) and kinases coregulate the critical
levels of phosphorylation necessary for intracellular signalling, cell growth
and differentiation. Yersinia, the causative bacteria of the bubonic plague and
other enteric diseases, secrete an active PTPase, Yop51, that enters and
suppresses host immune cells. Though the catalytic domain is only approximately
20% identical to human PTP1B, the Yersinia PTPase contains all of the invariant
residues present in eukaryotic PTPases, including the nucleophilic Cys 403 which
forms a phosphocysteine intermediate during catalysis. We present here
structures of the unliganded (2.5 A resolution) and tungstate-bound (2.6 A)
crystal forms which reveal that Cys 403 is positioned at the centre of a
distinctive phosphate-binding loop. This loop is at the hub of several
hydrogen-bond arrays that not only stabilize a bound oxyanion, but may activate
Cys 403 as a reactive thiolate. Binding of tungstate triggers a conformational
change that traps the oxyanion and swings Asp 356, an important catalytic
residue, by approximately 6 A into the active site. The same anion-binding loop
in PTPases is also found in the enzyme rhodanese. Plague is still an endemic disease in different regions of the world. Increasing
reports of incidence, the discovery of antibiotic resistance strains, and
concern about a potential use of the causative bacteria Yersinia pestis as an
agent of biological warfare have highlighted the need for a safe, efficacious,
and rapidly producible vaccine. The use of F1 and V antigens and the derived
protein fusion F1-V has shown great potential as a protective vaccine in animal
studies. Plants have been extensively studied for the production of
pharmaceutical proteins as an inexpensive and scalable alternative to common
expression systems. In the current study the recombit plague antigens F1, V,
and fusion protein F1-V were produced by transient expression in Nicotiana
benthamiana by using a deconstructed tobacco mosaic virus-based system that
allowed very rapid and extremely high levels of expression. All of the
plant-derived purified antigens, administered s.c. to guinea pigs, generated
systemic immune responses and provided protection against an aerosol challenge
of virulent Y. pestis. |
The drug JTV519 is derivative of which group of chemical compounds? | The 1,4-benzothiazepine derivative JTV-519 is a new type of calcium ion channel modulator.JTV-519, which has potential use as an antiarrhythmic [285800]. The drug is a novel cardioprotectant derivative of 1,4-benzothiazepine for which phase I trials were completed in the third quarter of 1998 | BACKGROUND: A new 1,4-benzothiazepine derivative, JTV519, has a strong
protective effect against Ca(2+) overload-induced myocardial injury. We
investigated the effect of JTV519 on ischemia/reperfusion injury in isolated rat
hearts.
METHODS AND RESULTS: At 30 minutes of reperfusion after 30-minute global
ischemia, the percent recovery of left ventricular developed pressure was
improved, and the creatine phosphokinase and lactate dehydrogenase leakage was
reduced in a concentration-dependent manner when JTV519 was administered in the
coronary perfusate both at 5 minutes before the induction of ischemia and at the
time of reperfusion. The myocardial protective effect of JTV519 was completely
blocked by pretreatment of the heart with GF109203X, a specific protein kinase C
(PKC) inhibitor. In contrast, the effect of JTV519 was not affected by
alpha(1)-, A(1)-, and B(2)-receptor blockers that couple with PKC in the
cardiomyocyte. Both immunofluorescence images and immunoblots of JTV519-treated
left ventricular myocardium and isolated ventricular myocytes demonstrated that
this agent induced concentration-dependent translocation of the delta-isoform
but not the other isoforms of PKC to the plasma membrane.
CONCLUSIONS: The mechanism of cardioprotection by JTV519 against
ischemia/reperfusion injury involves isozyme-specific PKC activation through a
receptor-independent mechanism. This agent may provide a novel pharmacological
approach for the treatment of patients with acute coronary diseases via a
subcellular mechanism mimicking ischemic preconditioning. BACKGROUND: JTV519 is know to protect cardiomyocytes from calcium
overloading-induced damage. The aim of this study was to investigate the
potential protective effect of JTV519 on myocardium subjected to prolonged
ischemia and the underlying mechanism of such protection. The effect of JTV519
was also compared with that of diltiazem, a 1,5-benzothiazepine derivative.
METHODS: Isolated rat hearts were randomly divided into three groups. Control
hearts were arrested with histidine-tryptophan-ketoglutarat (HTK) cardioplegic
solution alone. In the JTV519 group of hearts, cardiac arrest was achieved with
JTV519 (10(-3) mmol/L) in the HTK solution. Hearts in the diltiazem group were
arrested with diltiazem (0.5 mmol/L) in the HTK solution. All the hearts were
then subjected to 6-hour storage in HTK solution at 4 degrees C.
RESULTS: After a 30-minute reperfusion, the left ventricular developed pressure
in the JTV519 and diltiazem groups were improved significantly compared with the
control group. There was a significantly lower left ventricular end-diastolic
pressure level and higher recovery of coronary flow in the JTV519 group than in
the control group. The postischemic intracellular calcium concentration was
attenuated by adding JTV519 or diltiazem to HTK cardioplegia.
CONCLUSION: As an adjunct to cardioplegia, JTV519 showed a significant
protective effect on myocardium undergoing 6 hours of ischemia. The beneficial
protective effects of JTV519 are correlated with its ability to inhibit the
postischemic rise in intracellular calcium. A new 1,4-benzothiazepine derivative, JTV519 (JTV), has strong protective
effects against isoproterenol-induced myocardial injury. We investigated the
effects of JTV on Ca2+ overload and on functional recovery during
ischemia/reperfusion in isolated coronary-perfused rat hearts. After 30 minutes
of reperfusion following 30 min of global ischemia, the % recovery of LV
developed pressure was improved in a concentration-dependent manner when JTV
(0.3-3.0 microM) was administered either 5 min before induction of ischemia or
for 5 min at the time of reperfusion only JTV showed a negative inotropic effect
only at concentrations above 3.0 microM. In indol-loaded isolated heart
preparations, 0.3 microM JTV did not affect the preischemic systolic or
diastolic Ca2+ levels of the Ca2+ transient as measured by the ratio of
2-wavelength fluormetry (R405/500). In contrast, it significantly reduced the
increase in the ratio in the postischemic reperfusion period (% change of
R405/500 from baseline: JTV(-), by 42.7 +/- 3.2%; JTV(+), by 18.4 +/- 9.1%, p <
0.05). In isolated rat ventricular myocytes with a standard patch-clamp method,
we further tested the interaction of JTV with the L-type Ca2+ channel (I(Ca)).
The % inhibition of the peak current of I(Ca) was 6.2 +/- 0.8% at 0.3 microM (p
= n.s.), 22.0 +/- 3.3% at 1.0 microM (p < 0.05), and 59.6 +/- 1.4% at 3.0 microM
(p < 0.01). Thus, the marked cardioprotection due to JTV at 0.3 microM may not
be solely attributed to its inhibitory effect on the transsarcolemmal Ca2+
influx through I(Ca). In conclusion, JTV519 is a novel pharmacological agent
that has been demonstrated for the first time to have clinical potential for the
treatment of acute coronary syndrome by its efficacy in administration at the
time of reperfusion, by its suppression of reperfusion-related intracellular
Ca2+ overload with no significant interaction with I(Ca), and by its subsequent
ability of strong myocardial protection. The 1,4-benzothiazepine derivative JTV-519 is a new type of calcium ion channel
modulator. We examined the modulatory effect of JTV-519 on the antitumor
activity of several platinum compounds (cisplatin, carboplatin, and nedaplatin)
in a human cancer cell line resistant to cisplatin (PC-14 / CDDP) in vitro.
PC-14 / CDDP cells showed 8-fold resistance to cisplatin compared with the
parental PC-14 cells as determined by dye formation
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] assay. In
PC-14 / CDDP, but not PC-14 cells, augmentation of cytotoxicity was observed
when a nontoxic concentration (10 mM) of JTV-519 was combined with the platinum
compounds. Increased intracellular cisplatin accumulation was observed in PC-14
/ CDDP cells in the presence of JTV-519 as measured by atomic absorption assay.
Therefore, increased cisplatin accumulation was considered to be a possible
mechanism underlying the reversing effect of JTV-519 on cisplatin resistance.
These results suggest that JTV-519 is a potent agent reversing cisplatin
resistance. A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl)
propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride
(JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and
multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in
K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the
resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16),
adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their
resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in
K562/MDR cells to the level in parental K562 cells and inhibited the efflux of
ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was
almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also
partially reversed the resistance of KB/MRP cells to VCR and at 500 microM
partially inhibited the photoaffinity labeling of MRP1 with
(125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that
JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1
overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1,
and competitively inhibiting transport of the anti-cancer agents. Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with
normal hearts and in those with underlying disease such as heart failure. In
animals with heart failure and in patients with inherited forms of
exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2
(FKBP12.6) from the ryanodine receptor-calcium release channel (RyR2) complex
causes an intracellular Ca2+ leak that can trigger fatal cardiac arrhythmias. A
derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2
for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak
that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may
be a therapeutic strategy for common ventricular arrhythmias. K201 (JTV519) is a 1,4-benzothiazepine derivative that exhibits a strong
cardioprotective action and acts as a multiple-channel blocker, including as a
K+ channel blocker. An experimental model of prolongation of the QT interval and
torsades de pointes can be induced in rabbits by treatment with clofilium in the
presence of the alpha1-adrenoreceptor agonist methoxamine. In this study we
examined the effects of K201 with and without methoxamine on the QT and QTc
intervals, and determined whether K201 inhibits clofilium-induced torsades de
pointes in the presence of methoxamine (15 microg/kg/min) in rabbits (n=74).
Administration of K201 (0, 40, 100, 200 and 400 microg/kg/min) with and without
methoxamine prolonged the QT interval in a dose-dependent manner, and torsades
de pointes did not occur in any animals. However, clofilium (50 microg/kg/min)
with methoxamine induced torsades de pointes in all animals (6/6). Torsades de
pointes occurred at rates of 100%, 67%, 40% and 0% at K201 concentrations of 0,
50, 200 and 400 microg/kg/min, respectively, in the clofilium-infused torsades
de pointes model. Therefore, 400 microg/kg/min of K201 completely inhibited
clofilium-induced torsades de pointes and attenuated the increase of
repolarization caused by clofilium; the inhibitory effects of K201 may be
related to its pharmacological properties as an alpha1-adrenoceptor blocker.
Overall, our results show that K201 causes prolongation of the QT and QTc
intervals, but does not induce torsades de pointes, with and without
alpha1-adrenoceptor stimulation. Furthermore, K201 inhibits clofilium-induced
torsades de pointes, despite QT prolongation, suggesting that QT prolongation
alone is not a proarrhythmic signal. K201 (JTV519), a benzothiazepine derivative, has been shown to possess
anti-arrhythmic and cardioprotective properties, but the mechanism of its action
is both complex and controversial. It is believed to stabilize the closed state
of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the
FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest,
Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292-296]. In the
present study, we investigated the effect of K201 on spontaneous Ca2+ release
induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human
embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action
of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac
myocytes in a concentration-dependent manner. Treating ventricular myocytes with
FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of
spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress
spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and
FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells
expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same
potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt
(wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden
death, N4104K, in the absence of FKBP12.6. These observations demonstrate that
FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+
release. Our results also suggest that suppression of spontaneous Ca2+ release
and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic
properties of K201. Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in
the rapid release of Ca(2+) from the sarcoplasmic reticulum and muscle
contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12)
increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine
derivative JTV519, and the more specific derivative S107
(2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to
improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here,
we report a high degree of nonspecific and specific low affinity [(3)H]S107
binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [(3)H]S107 per
RyR1 tetramer with EC(50) ∼52 µM and Hillslope ∼2. The effects of S107 and
FKBP12 on RyR1 were examined under conditions that altered the redox state of
RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of
reduced glutathione and the NO-donor NOC12, with no effect in the presence of
oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12
dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12
dissociation was observed in skeletal muscle homogenates that contained 0.43 µM
myoplasmic FKBP12 and was attenuated by S107. In single channel measurements
with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented
the FKBP12-mediated decrease in channel activity. The data suggest that S107 can
reverse the harmful effects of redox active species on SR Ca(2+) release in
skeletal muscle by binding to RyR1 low affinity sites. |
Is the long non- coding RNA malat-1 up or downregulated in cancer? | Malat-1 expression is upregulated in several tumor types | In early-stage non-small-cell lung cancer (NSCLC), a substantial proportion of
patients can be cured by surgery. Development of distant metastases is the most
frequent cause of therapeutic failure. The possibility to accurately predict a
patient's risk for developing distant metastasis would help to identify patients
that are candidates for further intervention such as conventional adjuvant
chemotherapy or experimental drugs. Current molecular biology techniques allow
the genome-wide screening for differentially expressed genes; and adequate
bioinformatics approaches are developed at a rapid pace to improve prognosis
prediction. Genes associated with metastasis do not necessarily play a role in
disease pathogenesis but rather reflect the activation of specific
signal-transduction pathways that are associated with enhanced migration and
invasion capability. In our own work, we have identified several genes (e.g.
thymosin beta-4, elF4A1), including a novel non-coding RNA (MALAT-1) to be
expressed at significantly higher levels in stage-I and stage-II NSCLC primary
tumours that subsequently metastasised. As a consequence, patients with
high-level expression of these genes were shown to have significantly worse
survival compared to patients with low-level expression of these genes. These
data support the hypothesis that gene-expression patterns in primary tumours
determine the tumours' likelihood to metastasise. In the near future, this
information will be used for tailored therapy approaches for patients with
early-stage NSCLC. Endometrial stromal sarcoma (ESS) of the uterus is a rare uterine maligcy
that has not been characterized in detail. To characterize the phenotype of ESS
of the uterus, we extracted RNA from ESS and the stroma of normal endometrium
using a tissue microdissection system and compared the expression profiles in
the two tissues. After suppression subtractive hybridization and differential
screening, we detected the metastasis-associated lung adenocarcinoma transcript
1 (MALAT-1) gene as one of the major genes upregulated in ESS, and a full-length
placental cDNA clone (CS0DI066YJ10) as one of the major genes downregulated. The
results were confirmed by in situ hybridization in four resected specimens of
ESS and 36 biopsy specimens of normal endometrial tissue. All ESS (4/4) and all
cases of endometrial stromal cells in the proliferative phase (13/13) were
positive for MALAT-1, but samples of normal stroma in the secretory phase and
menopausal state included some that were negative or weakly positive for MALAT-1
(5/13 and 3/10, respectively). In contrast, all ESS and 12 of 13 cases of
stromal cells in the proliferative phase were negative for the full-length
placental cDNA clone but 10 of 13 cases of endometrial stromal cells in the
secretory phase were positive for transcripts of the gene (P < 0.05). These
results indicated that endometrial stromal cells have different phenotypic
characteristics between proliferative and secretory phases and the tumor cells
of ESS have the phenotypic character of endometrial stromal cells in the
proliferative phase. Recent studies with tiling arrays have revealed more genomic transcription than
previously anticipated. Whole new groups of non-coding transcripts (NCTs) have
been detected. Some of these NCTs, including miRNAs, can regulate gene
expression. To date, most known NCTs studied have been relatively short, but
several important regulatory NCTs, including XIST, MALAT-1, BC1 and BC200, are
considerably larger in length and represent a novel class of long, non-coding
RNA species. Whole-genome tiling arrays were utilized to identify novel long
NCTs across the entire human genome. Our results have identified a new group of
long (>400 nt), abundantly expressed NCTs and have found that a subset of these
are also highly evolutionarily conserved. In this report, we have begun to
characterize 15 long, conserved NCTs. Quantitative real-time RT-PCR was used to
analyze their expression in different normal human tissue and also in breast and
ovarian cancers. We found altered expression of many of these NCTs in both
cancer types. In addition, several of these NCTs have consistent mutations when
sequences of normal samples were compared with a panel of cancer-derived cell
lines. One NCT was found to be consistently mutated in a panel of endometrial
cancers compared with matched normal blood. These NCTs were among the most
abundantly expressed transcripts detected. There are probably many long,
conserved NCTs, albeit with lower levels of expression. Although the function of
these NCTs is currently unknown, our study indicates that they may play an
important function in both normal cells and in cancer development. RNA processing is vital for the high fidelity and diversity of eukaryotic
transcriptomes and the encoded proteomes. However, control of RNA processing is
not fully established. Σ RNA is a class of conserved large non-coding RNAs
(murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense
technology, we identified that RNA post-transcriptional modification is the most
significant global function of Σ RNA. Specifically, processing of the pre-mRNAs
of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ
RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a
regulatory molecule exerting roles in RNA post-transcriptional modification. Metastasis-associated lung adenocarcinoma transcript 1(MALAT1), a long
non-coding RNA (lncRNA), is up-regulated in many solid tumors and associated
with cancer metastasis and recurrence. However, its role in hepatocellular
carcinoma (HCC) remains poorly understood. In the present study, we evaluated
the expression of MALAT1 by quantitative real-time PCR in 9 liver cancer cell
lines and 112 HCC cases including 60 cases who received liver transplantation
(LT) with complete follow-up data. Moreover, small interfering RNA (siRNA) was
used to inhibit MALAT1 expression to investigate its biological role in tumor
progression. We found that MALAT1 was up-regulated in both cell lines and
clinical tissue samples. Patients with high expression level of MALAT1 had a
significantly increased risk of tumor recurrence after LT, particularly in
patients who exceeded the Milan criteria. On multivariate analysis, MALAT1 was
an independent prognostic factor for predicting HCC recurrence (hazard ratio,
3.280, P = 0.003).In addition, inhibition of MALAT1 in HepG2 cells could
effectively reduce cell viability, motility, invasiveness, and increase the
sensitivity to apoptosis. Our data suggest that lncRNA MALAT1 play an important
role in tumor progression and could be a novel biomarker for predicting tumor
recurrence after LT and serve as a promising therapeutic target. INTRODUCTION: The functions of large noncoding RNAs (ncRNAs) have remained
elusive in many cases. Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1
(MALAT-1) is an ncRNA that is highly expressed in several tumor types.
METHODS: Overexpression and RNA interference (RNAi) approaches were used for the
analysis of the biological functions of MALAT-1 RNA. Tumor growth was studied in
nude mice. For prognostic analysis, MALAT-1 RNA was detected on
paraffin-embedded non-small cell lung cancer (NSCLC) tissue probes (n = 352)
using in situ hybridization.
RESULTS: MALAT-1 was highly expressed in several human NSCLC cell lines. MALAT-1
expression was regulated by an endogenous negative feedback loop. In A549
NSCLCs, RNAi-mediated suppression of MALAT-1 RNA suppressed migration and
clonogenic growth. Forced expression of MALAT-1 in NIH 3T3 cells significantly
increased migration. Upon injection into nude mice, NSCLC xenografts with
decreased MALAT-1 expression were impaired in tumor formation and growth. In
situ hybridization on paraffin-embedded lung cancer tissue probes revealed that
high MALAT-1 RNA expression in squamous cell carcinoma of the lung was
associated with a poor prognosis. On genetic level, MALAT-1 displays the
strongest association with genes involved in cancer like cellular growth,
movement, proliferation, signaling, and immune regulation.
CONCLUSIONS: These data indicate that MALAT-1 expression levels are associated
with patient survival and identify tumor-promoting functions of MALAT-1. Recent studies reveal that long non-coding RNAs (lncRNAs) have been shown to
have important regulatory roles in cancer biology, and lncRNA MALAT-1 expression
is upregulated in some tumors. However, the contributions of MALAT-1 to bladder
cancer metastasis remain largely unknown. In the present study we evaluated
MALAT-1 expression in bladder cancer tissues by real-time PCR, and defined its
biological functions. We verified that MALAT-1 levels were upregulated in
bladder cancer tissues compared with adjacent normal tissues, and MALAT-1
expression was remarkably increased in primary tumors that subsequently
metastasized, when compared to those primary tumors that did not metastasize.
SiRNA-mediated MALAT-1 silencing impaired in vitro bladder cancer cell
migration. Downregulation of MALAT-1 resulted in a decrease of the
epithelial-mesenchymal transition (EMT)-associated ZEB1, ZEB2 and Slug levels,
and an increase of E-cadherin levels. We further demonstrated that MALAT-1
promoted EMT by activating Wnt signaling in vitro. These data suggest an
important role for MALAT-1 in regulating metastasis of bladder cancer and the
potential application of MALAT-1 in bladder cancer therapy. The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long
non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer
metastasis. It is highly abundant, its expression is strongly regulated in many
tumor entities including lung adenocarcinoma and hepatocellular carcinoma as
well as physiological processes, and it is associated with many RNA binding
proteins and highly conserved throughout evolution. The nuclear transcript
MALAT-1 has been functionally associated with gene regulation and alternative
splicing and its regulation has been shown to impact proliferation, apoptosis,
migration and invasion. Here, we have developed a human and a mouse knockout
system to study the loss-of-function phenotypes of this important ncRNA. In
human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases.
Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation
nor cell cycle progression nor nuclear architecture in human lung or liver
cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not
give rise to any obvious phenotype or histological abnormalities in Malat1-null
compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA
MALAT1 is compatible with cell viability and normal development. 1. Examining plasma RNA is an emerging non-invasive diagnosis technique. However,
whether tumour-derived long non-coding RNAs (lncRNAs) in plasma can be used as a
novel approach to detect human prostate cancer (PCa) has not yet been
established. The study was divided into three parts: (1) the characteristics of
PCa-related lncRNA fragments were systematically studied in the plasma or serum
of 25 patients; (2) the source of the circulating lncRNA fragments was explored
in vitro and in vivo; and (3) the diagnostic performance of metastasis
associated in lung adenocarcinoma transcript 1 (MALAT-1) derived (MD) miniRNA
was validated in an independent cohort of 192 patients. The expression levels of
lncRNAs were measured by quantitative real time polymerase chain reaction
(qRT-PCR). The MD-miniRNA copies were calculated using a standard curve in an
area under the ROC curve (AUC)-receiver operating characteristic (ROC) analysis.
Genome-wide profiling revealed that MALAT-1 and prostate cancer gene 3 (PCA3)
are overexpressed in PCa tissues. Plasma lncRNAs probably exist in the form of
fragments in a stable form. MD-miniRNA enters cell culture medium at measurable
levels, and MD-miniRNA derived from human PCa xenografts actually enters the
circulation in vivo and can be measured to distinguish xenografted mice from
controls. In addition, plasma MD-miniRNA levels are significantly elevated in
PCa patients compared to non-PCa patients (p<0.001). At a cut-off of 867.8
MD-miniRNA copies per microlitre of plasma, the sensitivity is 58.6%, 58.6% and
43.5% and the specificity is 84.8%, 84.8% and 81.6% for discriminating PCa from
non-PCa, positive biopsy from negative biopsy and positive biopsy from negative
biopsy, respectively. We conclude that MD-miniRNA can be used as a novel
plasma-based biomarker for PCa detection and can improve diagnostic accuracy by
predicting prostate biopsy outcomes. Further large-scale studies are needed to
confirm our findings. Although metastasis-associated lung adenocarcinoma transcript (MALAT)-1 is known
to be consistently upregulated in several epithelial maligcies, little is
known about its function or regulation. We therefore examined the relationship
between MALAT-1 expression and candidate modulators such as DNA tumor virus
oncoproteins human papillomavirus (HPV)-16 E6 and E7, BK virus T antigen
(BKVTAg), mouse polyoma virus middle T antigen (MPVmTAg) and tumor suppressor
genes p53 and pRb. Using suppressive subtractive hybridization (SSH) and
real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays,
MALAT-1 was shown to be increased in viral oncongene-expressing salivary gland
biopsies from humans and mice. The results also indicated that MALAT-1
transcripts and promoter activity were increased in vitro when viral
oncongene-expressing plasmids were introduced into different cell types. These
same viral oncogenes in addition to increasing MALAT-1 transcription have also
been shown to inhibit p53 and/or pRb function. In p53 mutant or inactive cell
lines MALAT-1 was also shown to be highly upregulated. We hypothesize that there
is a correlation between MALAT-1 over-expression and p53 deregulation. In
conclusion, we show that disruption of p53, by both polyoma and papilloma
oncoproteins appear to play an important role in the up-regulation of MALAT-1.
MALAT-1 might therefore represent a biomarker for p53 deregulation within
maligcies. |
Oxantel is used for periodontitis treatment. How does it work? | Oxantel, a cholinergic anthelmintic and fumarate reductase inhibitor, significantly inhibited biofilm formation by P. gingivalis and disrupted established biofilms. | Bacterial pathogens commonly associated with chronic periodontitis are the
spirochete Treponema denticola and the Gram-negative, proteolytic species
Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex
anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been
shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria
and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that
oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration
(IC50) of 2.2 μM and planktonic growth of T. forsythia with a MIC of 295 μM, but
it had no effect on the growth of T. denticola. Oxantel treatment caused the
downregulation of six P. gingivalis gene products and the upregulation of 22
gene products. All of these genes are part of a regulon controlled by heme
availability. There was no large-scale change in the expression of genes
encoding metabolic enzymes, indicating that P. gingivalis may be unable to
overcome Frd inhibition. Oxantel disrupted the development of polymicrobial
biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a
concentration-dependent manner. In these biofilms, all three species were
inhibited to a similar degree, demonstrating the synergistic nature of biofilm
formation by these species and the dependence of T. denticola on the other two
species. In a murine alveolar bone loss model of periodontitis oxantel addition
to the drinking water of P. gingivalis-infected mice reduced bone loss to the
same level as the uninfected control. |
What is apelin? | Apelin, a small regulatory peptide, is the endogenous ligand for the apelin receptor (APJ) receptor. | The adipocytokine apelin is a peptide, Apelin and its receptor are abundantly
expressed in the nervous and cardiovascular systems. Previous studies had found
apelin-13 reduces brain injuries and postischemic cerebral edema through
blocking programmed cell death, Apelin-13 is also able to inhibit glucose
deprivation induced cardiomyocyte autophagy in a concentration dependent
fashion. To observe the effect of Apelin-13 on the brain injury induced by
traumatic brain injury (TBI), and explore the effect of Apelin-13 on autophagy
in TBI, We performed The neurological test, and the numbers of TBI-induced
neural cell death were also counted by propidium iodide labeling. At last, the
autophagy associated proteins LC3, Beclin-1, Bcl-2, p62 were also assessed with
western-blotting. Compared with saline vehicle groups, the neural cell death,
lesion volume, and neural dysfunction were attenuated by apelin-13 after TBI. In
additionally, Apelin-13 also reversed TBI induced downregulation of LC3,
Beclin-1, Bcl-2, p62 expression, compared with saline vehicle groups, at 24 and
48 h post TBI. Apelin-13 attenuates TBI induced brain damage by suppressing
autophagy. All these results revealed that Apelin-13 suppressed autophagy. The
autophagy may be involved in the mechanism of Apelin-13 rescue the subsequent
damaged neuron in TBI. The aim of this study was to determine the levels of regulatory peptides apelin,
glucagon-like peptide (GLP-1) and visfatin in hypercholesterolemic and
hyperhomocysteinemic state and to examine their relation with nitric oxide (NO)
metabolism. 32 Male guinea pigs were divided into four groups and each group was
fed as follows: (a) commercial chow, (b) cholesterol (chol)-rich diet, (c)
methionine (meth)-rich diet, and (d) chol + meth-rich diet. Blood samples were
drawn at the end of 10 weeks, and abdominal aorta was dissected for
histopathological examination. Serum insulin, GLP-1, apelin, visfatin, and
nitrotyrosine concentrations were measured by the manufacturer's kits based on
ELISA; asymmetric dimethylarginine (ADMA) and arginine levels were measured by
the high performance liquid chromatography. Homocysteine level was measured by
the chemiluminescence immunoassay; glucose, total chol and triglyceride levels
were measured by the autoanalyzer. The microscopic examination of aorta
indicated varying degrees of vascular disturbance in chol- and chol + meth-fed
groups. High levels of chol and homocysteine, accompanied with significantly low
levels of apelin and GLP-1 were detected in the plasma. Visfatin, ADMA, and
nitrotyrosine levels both in chol- and chol + meth-fed groups were significantly
higher than those in control animals, whereas arginine and arginine/ADMA ratio
were lower. This study indicated that circulating levels of apelin, GLP-1, and
visfatin are markedly altered during the development of atherosclerotic changes
in close association with chol, homocysteine, NO, and ADMA levels. The
measurements of these peptides in serum may help for the diagnosis and follow-up
of vascular dysfunction. Children with sickle cell disease (SCD) have painful vaso-occlusive episodes
(VOEs), which often reoccur across the individual's lifespan. Vaso-constrictive
and vaso-dilatory molecules have been hypothesized to play a role in VOEs.
Endothelin-1 (ET-1) is a potent vasoconstrictor that is released during VOEs and
is correlated with pain history. Apelin is a vaso-dilatory peptide that also has
a modulatory role in pain processing. We hypothesize that the ratio between
vaso-dilatory and vaso-constrictive tone in children with SCD may be a marker of
pain sensitization and vaso-occlusion. Plasma endothelin and apelin levels were
measured in 47 children with SCD. Procedural pain and baseline pain were
assessed via child- and caregiver-reports and observational distress. Pain
history was assessed using retrospective chart review. Plasma apelin was related
to age, with decreased levels in older children. The ratio between apelin and
ET-1 was negatively correlated to observational baseline pain. The ratio between
apelin and Big ET was negatively correlated to caregiver ratings of baseline
pain and positively correlated to history of VOEs, which is possibly due to
hydroxyurea treatment. These results suggest that an imbalance in the apelin and
endothelin systems may contribute to an increasing number of VOEs and baseline
pain in children with SCD. BACKGROUND: C-terminal fragments of adipokine apelin are able to attenuate
myocardial ischemia-reperfusion (I/R) injury, but whether their effects are
manifested during cardioplegic arrest remain obscure. This study was designed to
evaluate the efficacy of natural apelin-12
(H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12) and its novel
structural analogs
(H-(N(α)Me)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, AI, and
N(G)-Arg(N(G)NO2)-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-NH2, AII) as
additives to crystalloid cardioplegia and explore benefits of early reperfusion
with these peptides.
METHODS: Isolated working rat hearts subjected to normothermic global ischemia
and further reperfusion were used. St. Thomas' Hospital cardioplegic solution
No.2 (STH2) containing 140 μM A12, AI, or AII was infused for 5 min at 25 °C
before ischemia. In separate series, peptide administration was used for 5 min
after ischemia. Metabolic state of the hearts was evaluated by myocardial
content of high energy phosphates and lactate. Lactate dehydrogenase (LDH)
leakage was assessed in myocardial effluent on early reperfusion.
RESULTS: Addition of the peptides to STH2 enhanced functional and metabolic
recovery of reperfused hearts compared with those of control (STH2 without
additives). Cardioplegia with analog AII was the most effective and accompanied
by a reduction of postischemic LDH leakage. Infusion of A12, AI, or AII after
ischemia improved the majority indices of cardiac function and metabolic state
of the heart by the end of reperfusion. However, the overall protective effect
of the peptides was less than when they were added to STH2.
CONCLUSIONS: Enhancement of apelin bioavailability may minimize myocardial I/R
damage during cardiac surgery. Structural analogs of A12 are promising
components of clinical cardioplegic solutions. Obesity is a major health problem worldwide. The prevalence of obesity is
increasing in both developed and developing countries. In the UK, for example,
60% of adults are overweight and 25% are obese. Obesity is associated with many
pathological complications including respiratory, cardiovascular and endocrine,
but it also affects fertility and is associated with many reproductive
complications. This has led us and others to investigate links between women
with high BMI, pregcy outcome and uterine function. These studies in turn
have led investigators to ask how obesity can have such an impact on
reproduction and, as part of this, to consider the role of the adipokines
released from adipose tissues. Our focus in this short review is on adipokines
and myometrial activity, and for completeness we overview their effects on other
smooth muscles. To date four adipokines (leptin, visfatin, apelin and ghrelin)
have been investigated and all affect myometrial contractility, but some more
potently than others. We consider the possible mechanisms involved in how
adipokines may modify uterine contractility, and discuss the potential impact on
labor and delivery. Protection and replenishment of a functional pancreatic β-cell mass (BCM) are
key goals of all diabetes therapies. Apelin, a small regulatory peptide, is the
endogenous ligand for the apelin receptor (APJ) receptor. The apelin-APJ
signaling system is expressed in rodent and human islet cells. Apelin exposure
has been shown to inhibit and to stimulate insulin secretion. Our aim was to
assess the influence of a selective APJ deletion in pancreatic islet cells on
islet homeostasis and glucose tolerance in mice. Cre-LoxP strategy was utilized
to mediate islet APJ deletion. APJ deletion in islet cells (APJ(Δislet))
resulted in a significantly reduced islet size, density and BCM. An ip glucose
tolerance test showed significantly impaired glucose clearance in APJ(Δislet)
mice. APJ(Δislet) mice were not insulin resistant and in vivo glucose-stimulated
insulin secretion was reduced modestly. In vitro glucose-stimulated insulin
secretion showed a significantly reduced insulin secretion by islets from
APJ(Δislet) mice. Glucose clearance in response to ip glucose tolerance test in
obese APJ(Δislet) mice fed a chronic high-fat (HF) diet, but not pregt
APJ(Δislet) mice, was impaired significantly. In addition, the obesity-induced
adaptive elevations in mean islet size and fractional islet area were reduced
significantly in obese APJ(Δislet) mice when compared with wild-type mice.
Together, these findings demonstrate a stimulatory role for the islet cell
apelin-APJ signaling axis in regulation of pancreatic islet homeostasis and in
metabolic induced β-cell hyperplasia. The results indicate the apelin-APJ system
can be exploited for replenishment of BCM. Apelin is a novel bioactive peptide as the endogenous ligand for APJ. Apelin and
APJ have also been identified in the testis, hypothalamic nuclei such as
arcuate, supraoptic and paraventricular nuclei, implicating roles in the control
of reproduction. Therefore, the present study was designed to investigate the
effects of chronic central infusion of apelin-13 on LH, FSH and testosterone
levels and testis morphology. 21 Wistar-Albino male rats received continuous
intracerebroventricular infusion via Alzet osmotic mini pumps filled artificial
cerebrospinal fluid (vehicle) or apelin-13 at concentrations of 1 or 10 nmol (10
μl/h) for seven days. At the last 90 min of the infusion period, the blood
samples were collected at 15 min intervals (0-90 min) for LH and FSH analyses.
At the last sampling point, the blood samples were analyzed for testosterone
levels. Infusion of high dose apelin-13 significantly suppressed LH release
compared with the vehicle values at 30, 60 and 75 min (p<0.05). However, FSH
levels did not significantly differ among the groups. Serum testosterone levels
in high dose apelin-13 group were statistically lower than the control group
(p<0.05). In addition, histological examination showed that infusion of high
dose apelin-13 significantly decreased the number of Leydig cells compared with
the control and lower dose apelin-13 groups (p<0.05, p<0.01). Our results
suggest that apelin-13 may play a role in the central regulation and decreases
testosterone release by suppressing LH secretion. Thus, antagonists of the
apelin receptor may, therefore, be useful for pharmaceuticals in the treatment
of infertility. Pericytes are a population of cells that participate in normal vessel
architecture and regulate permeability. Apelin, as the endogenous ligand of G
protein-coupled receptor APJ, participates in a number of physiological and
pathological processes. To date, the effect of apelin on pericyte is not clear.
Our study aimed to investigate the potential protection mechanisms of apelin,
with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence
staining revealed that pericytes colocalized with APJ in the fibrovascular
membranes dissected from proliferative diabetic retinopathy patients. In the in
vitro studies, we first demonstrated that the expression of apelin/APJ was
upregulated in pericytes under hypoxia, and apelin increased pericytes
proliferation and migration. Moreover, knockdown of apelin in pericyte was
achieved via lentivirus-mediated RNA interference. After the inhibition of
apelin, pericytes proliferation was inhibited significantly in hypoxia culture
condition. Furthermore, exogenous recombit apelin effectively prevented
hypoxia-induced apoptosis through downregulating active-caspase 3 expression and
increasing the ratio of B cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein
(Bax) in pericytes. These results suggest that apelin suppressed hypoxia-induced
pericytes injury, which indicated that apelin could be a potential therapeutic
target for retinal angiogenic diseases. |
What is the function of the protein encoded by the gene PABPC4? | The main function of PABPC4 is in mRNA stability and translation initiation. PABPC4 may also play a role in chronic inflammation and in the pathogenesis of colorectal cancer. | In testis mRNA stability and translation initiation are extensively under the
control of poly(A)-binding proteins (PABP). Here we have cloned a new human
testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical
residues to the ubiquitous PABP1. A northern blot of multiple human tissues
hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two
PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2
kb) was present in all tested tissues. In human adult testis, PABP3 mRNA
expression was restricted to round spermatids, whereas PABP1 was expressed in
these cells as well as in pachytene spermatocytes. PABP3-specific antibodies
identified a protein of 70 kDa in human testis extracts. This protein binds
poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3
gene is intronless with a transcription start site 61 nt upstream from the
initiation codon. A sequence of 256 bp upstream from the transcription start
site drives the promoter activity of PABP3 and its tissue-specific expression.
The expression of PABP3 might be a way to bypass PABP1 translational repression
and to produce the amount of PABP needed for active mRNA translation in
spermatids. BACKGROUND: C-reactive protein (CRP) is a heritable marker of chronic
inflammation that is strongly associated with cardiovascular disease. We sought
to identify genetic variants that are associated with CRP levels.
METHODS AND RESULTS: We performed a genome-wide association analysis of CRP in
66 185 participants from 15 population-based studies. We sought replication for
the genome-wide significant and suggestive loci in a replication panel
comprising 16 540 individuals from 10 independent studies. We found 18
genome-wide significant loci, and we provided evidence of replication for 8 of
them. Our results confirm 7 previously known loci and introduce 11 novel loci
that are implicated in pathways related to the metabolic syndrome (APOC1, HNF1A,
LEPR, GCKR, HNF4A, and PTPN2) or the immune system (CRP, IL6R, NLRP3, IL1F10,
and IRF1) or that reside in regions previously not known to play a role in
chronic inflammation (PPP1R3B, SALL1, PABPC4, ASCL1, RORA, and BCL7B). We found
a significant interaction of body mass index with LEPR (P<2.9×10(-6)). A
weighted genetic risk score that was developed to summarize the effect of risk
alleles was strongly associated with CRP levels and explained ≈5% of the trait
variance; however, there was no evidence for these genetic variants explaining
the association of CRP with coronary heart disease.
CONCLUSIONS: We identified 18 loci that were associated with CRP levels. Our
study highlights immune response and metabolic regulatory pathways involved in
the regulation of chronic inflammation. Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of
mRNA translation and stability, both of which are crucial for a wide variety of
cellular processes. Although generally a diffuse cytoplasmic protein, it can be
found in discrete foci such as stress and neuronal granules. Mammals encode
several additional cytoplasmic PABPs that remain poorly characterised, and with
the exception of PABP4, appear to be restricted in their expression to a small
number of cell types. We have found that PABP4, similarly to PABP1, is a
diffusely cytoplasmic protein that can be localised to stress granules. However,
UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear
relocalisation of PABPs was accompanied by a reduction in protein synthesis but
was not linked to apoptosis. In examining the mechanism of PABP relocalisation,
we found that it was related to a change in the distribution of poly(A) RNA
within cells. Further investigation revealed that this change in RNA
distribution was not affected by PABP knockdown but that perturbations that
block mRNA export recapitulate PABP relocalisation. Our results support a model
in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a
block in this process following UV exposure leads to accumulation of cytoplasmic
PABPs in the nucleus. These data also provide mechanistic insight into reports
that transcriptional inhibitors and expression of certain viral proteins cause
relocation of PABP to the nucleus. Cytoplasmic poly(A) binding protein 4 (PABPC4) is an RNA-processing protein that
plays an important role in the regulation of gene expression. The aim of this
study was to investigate the expression pattern and identify the potential
clinical significance of PABPC4 in colorectal cancer. Immunohistochemical
analysis revealed that 26.7% (27/101 patients) of primary colorectal tumors and
60.5% (23/38 patients) of corresponding adjacent, normal tissues showed high
cytoplasmic expression of PABPC4, whereas expression was absent in 98% (43/44
patients) of distant, normal tissues. Using Kaplan-Meier analysis, we observed
that the expression of PABPC4 was significantly correlated with disease-free
survival and overall survival in patients with stage II and stage III colorectal
cancer (P=0.022 and P=0.020, respectively). PABPC4 expression was positively
associated with survival outcome, and may have predictive value in the prognosis
of patients with colorectal cancer. Taken together, our findings indicate that
PABPC4 may play a role in the pathogenesis of colorectal cancer. Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and
translation. Although predomitly localized in the cytoplasm, PABP proteins
also cycle through the nucleus. Recent work has established that their
steady-state localization can be altered by cellular stresses such as
ultraviolet (UV) radiation, and infection by several viruses, resulting in
nuclear accumulation of PABPs. Here, we present further evidence that their
interaction with and release from mRNA and translation complexes are important
in determining their sub-cellular distribution and propose an integrated model
for regulated nucleo-cytoplasmic transport of PABPs. Spermiogenesis is a postmeiotic process that drives development of round
spermatids into fully elongated spermatozoa. Spermatid elongation is largely
controlled post-transcriptionally after global silencing of mRNA synthesis from
the haploid genome. Here, rats that differentially express EGFP from a
lentiviral transgene during early and late steps of spermiogenesis were used to
flow sort fractions of round and elongating spermatids. Mass-spectral analysis
of 2D gel protein spots enriched >3-fold in each fraction revealed a
heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL,
hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2,
TDRD6, TDRD7) abundantly expressed in round spermatids prior to their
elongation. Notably, each protein within this ontology cluster regulates
alternative splicing, sub-cellular transport, degradation and/or translational
repression of mRNAs. In contrast, elongating spermatid fractions were enriched
with glycolytic enzymes, redox enzymes and protein synthesis factors.
Retrogene-encoded proteins were over-represented among the most abundant
elongating spermatid factors identified. Consistent with these biochemical
activities, plus corresponding histological profiles, the identified RNA
processing factors are predicted to collectively drive post-transcriptional
expression of an alternative exome that fuels finishing steps of sperm
maturation and fitness. |
What kind of enzyme is encoded by the proto-oncogene ABL1? | ABL-family proteins comprise one of the best conserved branches of the tyrosine kinases. Each ABL protein contains an SH3-SH2-TK (Src homology 3-Src homology 2-tyrosine kinase) domain cassette, which confers autoregulated kinase activity and is common among nonreceptor tyrosine kinases. This cassette is coupled to an actin-binding and -bundling domain, which makes ABL proteins capable of connecting phosphoregulation with actin-filament reorganization. Two vertebrate paralogs, ABL1 and ABL2, have evolved to perform specialized functions. | The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine
kinase (c-Abl) that has been implicated in processes of cell differentiation,
cell division, cell adhesion and stress response. Alterations of ABL1 by
chromosomal rearrangement or viral transduction can lead to maligt
transformation. Activity of the c-Abl protein is negatively regulated by its SH3
domain through an unknown mechanism, and deletion of the SH3 domain turns ABL1
into an oncogene. We present evidence for an intramolecular inhibitory
interaction of the SH3 domain with the catalytic domain and with the linker
between the SH2 and catalytic domain (SH2-CD linker). Site-directed mutations in
each of these three elements activate c-Abl. Mutations in the linker cause a
conformational change of the molecule and increase binding of the SH3 domain to
peptide ligands. Individual mutation of two charged residues in the SH3 and
catalytic domain activates c-Abl, while inhibition is restored in the double
reciprocal mutant. We propose that regulators of c-Abl will have opposite
effects on its activity depending on their ability to favour or disrupt these
intramolecular interactions. Agrin signals through the muscle-specific receptor tyrosine kinase (MuSK) to
cluster acetylcholine receptors (AChRs) on the postsynaptic membrane of the
neuromuscular junction (NMJ). This stands as the prevailing model of synapse
induction by a presynaptic factor, yet the agrin-dependent MuSK signaling
cascade is largely undefined. Abl1 (previously known as Abl) and the
Abl1-related gene product Abl2 (previously known as Arg) define a family of
tyrosine kinases that regulate actin structure and presynaptic axon guidance.
Here we show that the Abl kinases are critical mediators of postsynaptic
assembly downstream of agrin and MuSK. In mouse muscle, Abl kinases were
localized to the postsynaptic membrane of the developing NMJ. In cultured
myotubes, Abl kinase activity was required for agrin-induced AChR clustering and
enhancement of MuSK tyrosine phosphorylation. Moreover, MuSK and Abl kinases
effected reciprocal tyrosine phosphorylation and formed a complex after agrin
engagement. Our findings suggest that Abl kinases provide the developing synapse
with the kinase activity required for signal amplification and the intrinsic
cytoskeletal regulatory capacity required for assembly and remodeling. Constitutively activated mutants of the non-receptor tyrosine kinases (TK) ABL1
(Abelson murine leukemia viral (v-abl) homolog (1) protein) and JAK2 (JAnus
Kinase 2 or Just Another Kinase 2) play a central role in the pathogenesis of
clinically and morphologically distinct chronic myeloproliferative disorders but
are also found in some cases of de novo acute leukemia and lymphoma.
Ligand-independent activation occurs as a consequence of point mutations or
insertions/deletions within functionally relevant regulatory domains (JAK2) or
the creation of TK fusion proteins by balanced reciprocal translocations,
insertions or episomal amplification (ABL1 and JAK2). Specific abnormalities are
correlated with clinical phenotype, although some are broad and encompass
several World Health Organization-defined entities. TKs are excellent drug
targets as exemplified by the activity of imatinib in BCR-ABL1-positive disease,
particularly chronic myeloid leukemia. Resistance to imatinib is seen in a
minority of cases and is often associated with the appearance of secondary point
mutations within the TK domain of BCR-ABL1. These mutations are highly variable
in their sensitivity to increased doses of imatinib or alternative TK inhibitors
such as nilotinib or dasatinib. Selective and non-selective inhibitors of JAK2
are currently being developed, and encouraging data from pre-clinical
experiments and initial phase-I studies regarding efficacy and potential
toxicity of these compounds have already been reported. ABL family tyrosine kinases are tightly regulated by autoinhibition and
phosphorylation mechanisms. These kinases maintain an inactive conformation
through intramolecular interactions involving SH3 and SH2 domains. RIN1, a
downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates
ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a
large decrease in Km and a small increase in Vmax toward an ABL consensus
substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than
5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation
of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique
protein structures and distinct target sequences. Importantly RIN1-mediated
stimulation of ABL kinase activity was independent of activation by SRC-mediated
phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and
this occurred in the presence or absence of ABL regulatory domains outside the
SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic
site mutation associated with broad drug resistance, ABL1T315I, remains
responsive to stimulation by RIN1. These findings are consistent with an
allosteric kinase activation mechanism by which RIN1 binding promotes a more
accessible ABL catalytic site through relief of autoinhibition. Direct
disruption of RIN1 binding may therefore be a useful strategy to suppress the
activity of normal and oncogenic ABL, including inhibitor-resistant mutants that
confound current therapeutic strategies. Stimulation through derepression may be
applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2
domains. Stimulation of the T-cell antigen receptor (TCR) leads to the activation of
signaling pathways that are essential for T-cell development and the response of
mature T cells to antigens. The TCR has no intrinsic catalytic activity, but TCR
engagement results in tyrosine phosphorylation of downstream targets by
non-receptor tyrosine kinases. Three families of tyrosine kinases have long been
recognized to play critical roles in TCR-dependent signaling. They are the Src,
zeta-associated protein of 70 kDa, and Tec families of kinases. More recently,
the Abelson (Abl) tyrosine kinases have been shown to be activated by TCR
engagement and to be required for maximal TCR signaling. Using T-cell
conditional knockout mice deficient for Abl family kinases, Abl (Abl1) and
Abl-related gene (Arg) (Abl2), it was recently shown that loss of Abl kinases
results in defective T-cell development and a partial block in the transition to
the CD4(+)CD8(+) stage. Abl/Arg double null T cells exhibit impaired TCR-induced
signaling, proliferation, and cytokine production. Moreover, conditional
knockout mice lacking Abl and Arg in T cells exhibit impaired CD8(+) T-cell
expansion in vivo upon Listeria monocytogenes infection. Thus, Abl kinase
signaling is required for both T-cell development and mature T-cell function. Protein tyrosine kinases form a large family of signaling proteins implicated in
both normal and maligt cell signaling. The aim of this study was to identify
protein tyro-sine kinases that can transform hematopoietic cells to growth
factor independent proliferation when constitutively activated by
homodimerization. We used a modified retroviral insertion mutagenesis screen
with a retroviral vector containing the homodimerization domain of ETV6 followed
by an artificial splice donor site. Integration of this retroviral vector within
a gene of the host genome would generate a fusion transcript containing the
dimerization domain and part of the disrupted gene. Using this strategy with the
IL3 dependent Ba/F3 cell line, we identified 8 different protein tyrosine
kinases (Abl1, Fgfr1, Hck, Jak2, Lck, Mertk, Mst1r, Tnk1) that transformed the
cells. These results characterize HCK, MERTK, MST1R and TNK1 as potential
oncogenes and describe a method to identify gain-of-function fusion genes using
a retroviral insertion screen. ABL-family proteins comprise one of the best conserved branches of the tyrosine
kinases. Each ABL protein contains an SH3-SH2-TK (Src homology 3-Src homology
2-tyrosine kinase) domain cassette, which confers autoregulated kinase activity
and is common among nonreceptor tyrosine kinases. This cassette is coupled to an
actin-binding and -bundling domain, which makes ABL proteins capable of
connecting phosphoregulation with actin-filament reorganization. Two vertebrate
paralogs, ABL1 and ABL2, have evolved to perform specialized functions. ABL1
includes nuclear localization signals and a DNA binding domain through which it
mediates DNA damage-repair functions, whereas ABL2 has additional binding
capacity for actin and for microtubules to enhance its cytoskeletal remodeling
functions. Several types of posttranslational modifications control ABL
catalytic activity, subcellular localization, and stability, with consequences
for both cytoplasmic and nuclear ABL functions. Binding partners provide
additional regulation of ABL catalytic activity, substrate specificity, and
downstream signaling. Information on ABL regulatory mechanisms is being mined to
provide new therapeutic strategies against hematopoietic maligcies caused by
BCR-ABL1 and related leukemogenic proteins. Chromosomal rearrangements involving the ABL1 gene, leading to a BCR-ABL1 fusion
gene, have been mainly associated with chronic myeloid leukemia and B-cell acute
lymphoblastic leukemia (ALL). At present, six other genes have been shown to
fuse to ABL1. The kinase domain of ABL1 is retained in all chimeric proteins
that are also composed of the N-terminal part of the partner protein that often
includes a coiled-coil or a helix-loop-helix domain. These latter domains allow
oligomerization of the protein that is required for tyrosine kinase activation,
cytoskeletal localization, and neoplastic transformation. Fusion genes that have
a break in intron 1 or 2 (BCR-ABL1, ETV6-ABL1, ZMIZ1-ABL1, EML1-ABL1, and
NUP214-ABL1) have transforming activity, although NUP214-ABL1 requires
amplification to be efficient. The NUP214-ABL1 gene is the second most prevalent
fusion gene involving ABL1 in maligt hemopathies, with a frequency of 5% in
T-cell ALL. Both fusion genes (SFPQ-ABL1 and RCSD1-ABL1) characterized by a
break in intron 4 of ABL1 are associated with B-cell ALL, as the chimeric
proteins lacked the SH2 domain of ABL1. Screening for ABL1 chimeric genes could
be performed in patients with ALL, more particularly in those with T-cell ALL
because ABL1 modulates T-cell development and plays a role in cytoskeletal
remodeling processes in T cells. The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced
"spontaneous" DNA damage and unfaithful repair in Philadelphia chromosome
positive leukemia cells. Here, we investigated the changes of protein profile in
H2O2-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a
proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE)
coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially
expressed and 13 proteins were identified with further MS analysis. Two
essential proteins, Proto-oncogene tyrosine-protein kinase ABL1 (c-ABL) and Heat
shock 70kDa protein 4 (Apg-2), were confirmed by Western blot and showed
consistent changes with proteomic results. Moreover, functional analysis
demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but
also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL,
BaF3-BCR/ABL(T315I)). We also proved that Apg-2 inhibition aggravated H2O2
induced damage in BCR/ABL positive cells, and enhanced the sensitivity of
BaF3-BCR/ABL(T315I) to STI571. Taken together, the findings in this work provide
us with some clues to a better understanding of the molecular mechanisms
underlying BCR/ABL in the DNA damage/repair processes and demonstrated that
Apg-2 would be a valid target for anti-leukemia drug development. |
What is the mode of inheritance of Romano Ward long QT syndrome? | The Romano Ward long QT syndrome (LQTS) has an autosomal dominant mode of inheritance. | A family with the Romano-Ward syndrome is presented. This family showed typical
features of this syndrome with QT prolongation, torsades de pointes ventricular
tachycardia, sudden death and an autosomal domit inheritance pattern. The
index case presented with an exacerbation of torsades de pointes ventricular
tachycardia from diuretic induced hypokalaemia, and responded to diuretic
withdrawal and beta blocker therapy. Loss of consciousness in childhood may be due to cardiovascular causes, and the
Long Q-T syndromes can present with seizures. The Romano-Ward syndrome is of
autosomal domit inheritance, and the Jervell and Lange-Nielson syndrome, with
associated deafness, of autosomal recessive inheritance. The diagnosis is often
delayed, but a careful history can avoid this. The syndromes can appear to be
due to an imbalance in the sympathetic nerve to the ventricular myocardium, and
precipitating causes such as stress suggest a CNS influence on this. The
electrocardiogram can confirm the prolonged Q-T interval, but this is not always
present, at least without an exercise test. Treatment with beta-blockers is
often successful. If a wrong diagnosis of epilepsy is made a chance may be
missed of avoiding sudden death, quite apart from all the medical, and social
consequences that can result from such a diagnosis. The Romano-Ward long QT syndrome, an autosomal domit inherited disorder, is
an increasingly recognized cause of sudden cardiac death in children and adults.
Sudden death may or may not be preceded by a history of "seizures" and recurrent
syncope; the diagnosis relies on electrocardiographic evidence, including
prolongation of the QT interval corrected for heart rate, T wave abnormalities,
sinus bradycardia, and polymorphous ventricular tachycardia (torsades de
pointes). Recently, a gene responsible for causing long QT syndrome was
localized (using molecular genetic methods) to the short arm of chromosome 11
(11p15.5) in the region near the Harvey ras-1 locus. The purpose of this report
is to describe the current molecular genetic understanding of long QT syndrome,
including information regarding gene mapping, genetic heterogeneity, and
prenatal or presymptomatic diagnosis. Romano-Ward syndrome is an autosomal domit long-QT syndrome (LQTS) that
predisposes affected individuals to sudden death from tachyarrhythmias. We
investigated the molecular basis of LQTS in a Taiwanese kindred. Clinical and
genetic analyses revealed that a mutation was linked to the human
ether-a-go-go-related gene (HERG). The coding sequences and exon-intron borders
of HERG were amplified by means of polymerase chain reaction and subjected to
single-strand conformation polymorphism (SSCP) analysis. An exon with an
aberrant SSCP pattern was cloned and sequenced to study the molecular lesion. A
C-->T transition in codon 614, leading to substitution of a valine for an
alanine residue in the pore region of the HERG protein, was identified. Analysis
with Bsp12861 endonuclease digestion showed the mutation to be present in all
affected family members. Given that an unaffected paternal uncle had inherited
the same allele from the grandfather as the proband's father, a de novo mutation
had apparently occurred in the father and was transmitted to his offspring. In
addition to offering presymptomatic genetic diagnosis, identification of the
disease-causing mutation may suggest new therapeutic approaches for treatment
and prevention of this cardiovascular disease. OBJECTIVE: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous
disease characterized by prolonged QT intervals and an increased risk for
ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated
potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is
associated with two different entities of LQTS, the autosomal-domit
Romano-Ward syndrome (RWS), and the autosomal-recessive Jervell and
Lange-Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to
cardiac arrhythmias. In this study, we investigate and discuss domit-negative
I(Ks) current reduction by a KCNQ1 deletion mutation identified in a RWS family.
METHODS: Single-strand conformation polymorphism analysis and direct sequencing
were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were
heterologously expressed in Xenopus oocytes, and potassium currents were
recorded using the two-microelectrode voltage clamp technique.
RESULTS: A heterozygous deletion of three nucleotides (CTT) identified in the
KCNQ1 gene caused the loss of a single phenylalanine residue at position 339
(KCNQ1-deltaF339). Electrophysiological measurements in the presence and absence
of the regulatory beta-subunit KCNE1 revealed that mutant and wild type forms of
an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger
domit-negative current reduction than the mutant form of the full-length
KCNQ1 subunit (isoform 1).
CONCLUSION: This study highlights the functional relevance of the truncated
KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in
long QT syndrome. In the RWS family presented here, the autosomal-domit trait
is caused by multiple domit-negative effects provoked by heteromultimeric
channels formed by wild type and mutant KCNQ1-isoforms in combination with
KCNE1. Long QT and short QT syndromes (LQTS and SQTS) are cardiac repolarization
abnormalities that are characterized by length perturbations of the QT interval
as measured on electrocardiogram (ECG). Prolonged QT interval and a propensity
for ventricular tachycardia of the torsades de pointes (TdP) type are
characteristic of LQTS, while SQTS is characterized by shortened QT interval
with tall peaked T-waves and a propensity for atrial fibrillation. Both
syndromes represent a high risk for syncope and sudden death. LQTS exists as a
congenital genetic disease (cLQTS) with more than 700 mutations described in 12
genes (LQT1-12), but can also be acquired (aLQTS). The genetic forms of LQTS
include Romano-Ward syndrome (RWS), which is characterized by isolated LQTS and
an autosomal domit pattern of inheritance, and syndromes with LQTS in
association with other conditions. The latter includes Jervell and Lange-Nielsen
syndrome (JLNS), Andersen syndrome (AS), and Timothy syndrome (TS). The genetics
are further complicated by the occurrence of double and triple heterozygotes in
LQTS and a considerable number of nonpathogenic rare polymorphisms in the
involved genes. SQTS is a very rare condition, caused by mutations in five genes
(SQTS1-5). The present mutation update is a comprehensive description of all
known LQTS- and SQTS-associated mutations. |
Which histone modifications have been associated to alternative splicing? | H3K36m3 has been systematically associated to exon inclusion in almost all published studies. Other marks have been associated as well in specific studies to exon expression, but it can not be concluded that the effect of these marks in exon expression it is not a consequence of their effect in gene expression. | Variation in patterns of methylations of histone tails reflects and modulates
chromatin structure and function. To provide a framework for the analysis of
chromatin function in Caenorhabditis elegans, we generated a genome-wide map of
histone H3 tail methylations. We find that C. elegans genes show distributions
of histone modifications that are similar to those of other organisms, with
H3K4me3 near transcription start sites, H3K36me3 in the body of genes and
H3K9me3 enriched on silent genes. We also observe a novel pattern: exons are
preferentially marked with H3K36me3 relative to introns. H3K36me3 exon marking
is dependent on transcription and is found at lower levels in alternatively
spliced exons, supporting a splicing-related marking mechanism. We further show
that the difference in H3K36me3 marking between exons and introns is
evolutionarily conserved in human and mouse. We propose that H3K36me3 exon
marking in chromatin provides a dynamic link between transcription and splicing. The genomes of higher organisms are packaged in nucleosomes with functional
histone modifications. Until now, genome-wide nucleosome and histone
modification studies have focused on transcription start sites (TSSs) where
nucleosomes in RNA polymerase II (RNAPII) occupied genes are well positioned and
have histone modifications that are characteristic of expression status. Using
public data, we here show that there is a higher nucleosome-positioning signal
in internal human exons and that this positioning is independent of expression.
We observed a similarly strong nucleosome-positioning signal in internal exons
of Caenorhabditis elegans. Among the 38 histone modifications analyzed in man,
H3K36me3, H3K79me1, H2BK5me1, H3K27me1, H3K27me2, and H3K27me3 had evidently
higher signals in internal exons than in the following introns and were clearly
related to exon expression. These observations are suggestive of roles in
splicing. Thus, exons are not only characterized by their coding capacity, but
also by their nucleosome organization, which seems evolutionarily conserved
since it is present in both primates and nematodes. Alternative splicing of pre-mRNA is a prominent mechanism to generate protein
diversity, yet its regulation is poorly understood. We demonstrated a direct
role for histone modifications in alternative splicing. We found distinctive
histone modification signatures that correlate with the splicing outcome in a
set of human genes, and modulation of histone modifications causes splice site
switching. Histone marks affect splicing outcome by influencing the recruitment
of splicing regulators via a chromatin-binding protein. These results outline an
adaptor system for the reading of histone marks by the pre-mRNA splicing
machinery. How are short exonic sequences recognized within the vast intronic oceans in
which they reside? Despite decades of research, this remains one of the most
fundamental, yet enigmatic, questions in the field of pre-mRNA splicing
research. For many years, studies aiming to shed light on this process were
focused at the RNA level, characterizing the manner by which splicing factors
and auxiliary proteins interact with splicing signals, thereby enabling,
facilitating and regulating splicing. However, we increasingly understand that
splicing is not an isolated process; rather it occurs co-transcriptionally and
is presumably also regulated by transcription-related processes. In fact,
studies by our group and others over the past year suggest that DNA structure in
terms of nucleosome positioning and specific histone modifications, which have a
well established role in transcription, may also have a role in splicing. In
this review we discuss evidence for the coupling between transcription and
splicing, focusing on recent findings suggesting a link between chromatin
structure and splicing, and highlighting challenges this emerging field is
facing. While much attention has been focused on chromatin at promoters and exons, human
genes are mostly composed of intronic sequences. Analyzing published surveys of
nucleosomes and 41 chromatin marks in humans, we identified histone
modifications specifically associated with 5' intronic sequences,
distinguishable from promoter marks and bulk nucleosomes. These intronic marks
were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3),
typically transitioning near internal exons. Several marks transitioned near
bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we
examined whether splicing affects histone marking. Even with considerable
changes in regulated alternative splicing, histone marks were stable. Notably,
these findings are consistent with exon definition influencing histone marks. In
summary, we show that the location of many intragenic marks in humans can be
distilled into a simple organizing principle: association with 5' intronic or 3'
exonic regions. Recent genome-wide studies have revealed a remarkable correspondence between
nucleosome positions and exon-intron boundaries, and several studies have
implicated specific histone modifications in regulating alternative splicing. In
addition, recent progress in cracking the 'splicing code' shows that sequence
motifs carried on the nascent RNA molecule itself are sufficient to accurately
predict tissue-specific alternative splicing patterns. Together, these studies
shed light on the complex interplay between RNA sequence, DNA sequence, and
chromatin properties in regulating splicing. Alternative splicing affects more than 90% of human genes. Coupling between
transcription and splicing has become crucial in the complex network underlying
alternative splicing regulation. Because chromatin is the real template for
nuclear transcription, changes in its structure, but also in the "reading" and
"writing" of the histone code, could modulate splicing choices. Here, we discuss
the evidence supporting these ideas, from the first proposal of chromatin
affecting alternative splicing, performed 20 years ago, to the latest findings
including genome-wide evidence that nucleosomes are preferentially positioned in
exons. We focus on two recent reports from our laboratories that add new
evidence to this field. The first report shows that a physiological stimulus
such as neuron depolarization promotes intragenic histone acetylation (H3K9ac)
and chromatin relaxation, causing the skipping of exon 18 of the neural cell
adhesion molecule gene. In the second report, we show how specific histone
modifications can be created at targeted gene regions as a way to affect
alternative splicing: Using small interfering RNAs (siRNAs), we increased the
levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the
human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA)
through a mechanism that recalls RNA-mediated transcriptional gene silencing. Splicing is a complex process regulated by sequence at the classical splice
sites and other motifs in exons and introns with an enhancing or silencing
effect. In addition, specific histone modifications on nucleosomes positioned
over the exons have been shown to correlate both positively and negatively with
exon expression. Here, we trained a model of "IF … THEN …" rules to predict exon
inclusion levels in a transcript from histone modification patterns.
Furthermore, we showed that combinations of histone modifications, in particular
those residing on nucleosomes preceding or succeeding the exon, are better
predictors of exon inclusion levels than single modifications. The resulting
model was evaluated with cross validation and had an average accuracy of 72% for
27% of the exons, which demonstrates that epigenetic signals substantially mark
alternative splicing. MOTIVATION: Chromatin structure, including post-translational modifications of
histones, regulates gene expression, alternative splicing and cell identity.
ChIP-seq is an increasingly used assay to study chromatin function. However,
tools for downstream bioinformatics analysis are limited and are only based on
the evaluation of signal intensities. We reasoned that new methods taking into
account other signal characteristics such as peak shape, location and
frequencies might reveal new insights into chromatin function, particularly in
situation where differences in read intensities are subtle.
RESULTS: We introduced an analysis pipeline, based on linear predictive coding
(LPC), which allows the capture and comparison of ChIP-seq histone profiles.
First, we show that the modeled signal profiles distinguish differentially
expressed genes with comparable accuracy to signal intensities. The method was
robust against parameter variations and performed well up to a signal-to-noise
ratio of 0.55. Additionally, we show that LPC profiles of activating and
repressive histone marks cluster into distinct groups and can be used to predict
their function.
AVAILABILITY AND IMPLEMENTATION:
http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP A Matlab
implementation along with usage instructions and an example input file are
available from: http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP. BACKGROUND: While alternative splicing (AS) contributes greatly to protein
diversities, the relationship between various types of AS and epigenetic factors
remains largely unknown.
RESULTS: In this study, we discover that a number of epigenetic features,
including DNA methylation, nucleosome occupancy, specific histone modifications
and protein features, are strongly associated with AS. To further enhance our
understanding of the association between these features and AS, we cluster our
investigated features based on their association patterns with each AS type into
four groups, with H3K36me3, EGR1, GABP, SRF, SIN3A and RNA Pol II grouped
together and showing strongest association with AS. In addition, we find that
the AS types can be classified into two general classes, namely the exon
skipping related process (ESRP), and the alternative splice site selection
process (ASSP), based on their association levels with the epigenetic features.
CONCLUSION: Our analysis thus suggests that epigenetic features are likely to
play important roles in regulating AS. Pre-mRNA splicing is a complex process involving combinatorial effects of cis-
and trans-elements. Here, we focused on histone modifications as typical
trans-regulatory elements and performed systematic analyses of associations
between splicing patterns and histone modifications by using publicly available
ChIP-Seq, mRNA-Seq, and exon-array data obtained in two human cell lines. We
found that several types of histone modifications including H3K36me3 were
associated with the inclusion or exclusion of alternative exons. Furthermore, we
observed that the levels of H3K36me3 and H3K79me1 in the cell lines were well
correlated with the differences in alternative splicing patterns between the
cell lines. |
Is phospholamban phosphorylated by Protein kinase A? | Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that is phosphorylated at Ser16 by PKA. Phosphorylation of PLB by PKA reverses the inhibitory action of PLB. | Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated
by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase,
and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of
which remains to be defined. The aim of this study was therefore to characterize
the latter kinase, called phospholamban kinase. Phospholamban kinase was
purified approximately 42-fold with a yield of 11%. The purified fraction
exhibits a specific activity of 6.5 nmol of phosphate incorporated into
exogenous phospholamban per minute per milligram of protein. Phospholamban
kinase appears to be a high molecular weight enzyme and presents a broad
substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin
regulatory light chain being the best substrates. Phospholamban kinase
phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum
pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data
suggest that phospholamban kinase might be an isoenzyme of the multifunctional
Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr
50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum
proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of
Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration
velocity of the protein. In addition, this protein that binds calmodulin was
always found to be present in fractions containing phospholamban kinase
activity. This Mr 56 000 protein is therefore a good candidate for being a
subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding
protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two
distinct entities. The phosphorylation-induced mobility shift of the high molecular weight form of
phospholamban (24,500 daltons) in the cardiac sarcoplasmic reticulum produced on
3',5'-cyclic AMP (cAMP)-dependent phosphorylation with 5 mM ATP was resolved
into five clear steps on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), and on Ca2+-calmodulin-dependent phosphorylation
into ten steps. The mobility shift of the low molecular weight form of
phospholamban (less than 14,400 daltons) in these reactions occurred in one step
and two steps, respectively. With the two protein kinase activities, the
electrophoretic pattern of the mobility shifts of the high and low molecular
weight forms of phospholamban was similar to that obtained with
Ca2+-calmodulin-dependent protein kinase alone. The results of pulse-chase
experiments involving the centrifuge column method suggested that the site(s) of
phosphorylation by cAMP- and Ca2+-calmodulin-dependent protein kinase activities
are on the same phospholamban molecule. Two-dimensional tryptic peptide maps of
phosphorylated phospholamban indicated that cAMP-dependent protein kinase
phosphorylates at a single site, A, and Ca2+-calmodulin-dependent protein kinase
phosphorylates at sites C1 and C2 in the low molecular weight form, where A is
different from C1 but may be the same as C2. The high molecular weight form of
phospholamban is suggested to be a pentamer of identical monomers (low molecular
weight form) having one phosphorylation site for cAMP-dependent protein kinase
and two for Ca2+-calmodulin-dependent protein kinase. Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that
occurs in ischemia-reperfused hearts. Because SR function is regulated by
phosphorylation of phospholamban (PLB), a SR protein phosphorylated by
cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent
protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during
ischemia and reperfusion was examined in Langendorff-perfused rat hearts.
Ser(16)phosphorylation increased significantly after 20 min of ischemia from
2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific
phosphorylation and decreased to control values immediately after reperfusion.
Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min
of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in
Ser(16)phosphorylation was significantly diminished in hearts from
catecholamine-depleted animals and/or after beta-blockade and abolished in the
presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of
ischemia was blunted by nifedipine, whereas at R1 it was significantly
diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the
Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit
CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half
relaxation time. The results demonstrated a dissociation between the
phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the
beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on
Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation
of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with
Ca(2+)overload, transiently favoring the recovery of relaxation during early
reperfusion. The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused
hearts remains controversial. Although a decrease in the phosphorylation of both
PLB residues (Ser16, PKA site, and Thr17, CaMKII site) was previously reported,
experiments from our laboratory failed to detect this decrease. In an attempt to
elucidate the cause for this discrepancy, experiments were performed in
Langendorff-perfused rat hearts with two main goals: (1) To determine whether
keeping pacing during ischemia, a protocol followed in other
ischemia-reperfusion models, decreases the phosphorylation of PLB residues,
below pre-ischemic values; (2) To investigate whether a maximal beta-adrenergic
challenge allows to detect a decrease in the ability of PLB to be phosphorylated
in ischemia-reperfused hearts. Hearts were submitted to a global
ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during
ischemia, and phosphorylation of PLB residues was assessed by immunodetection.
The recovery of contractility upon reperfusion was lower in P vs. NP hearts.
Ser16 of PLB, was phosphorylated at the end of ischemia in NP hearts. This
increase appeared earlier in P hearts and was significantly diminished by
catecholamine depletion and beta-blockade. Thr17 site was phosphorylated at the
beginning of ischemia and the onset of reperfusion. The ischemia-induced
phosphorylation of Thr17 was higher and more sustained in P vs. NP hearts, and
inhibited by the calcium channel blocker, nifedipine, whereas the
reperfusion-induced increase in Thr17 phosphorylation was similar in P and NP
hearts and was significantly diminished by the Na+/Ca2+ exchanger inhibitor
KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at
any time during ischemia and reperfusion. However, the phosphorylation,
inotropic and lusitropic response to beta-adrenergic stimulation was
significantly decreased both in P and NP hearts. Although dilated cardiomyopathy (DCM) is known to result in cardiac contractile
dysfunction, the underlying mechanisms are unclear. The sarcoplasmic reticulum
(SR) is the main regulator of intracellular Ca2+ required for cardiac
contraction and relaxation. We therefore hypothesized that abnormalities in both
SR function and regulation will contribute to cardiac contractile dysfunction of
the J2N-k cardiomyopathic hamster, an appropriate model of DCM.
Echocardiographic assessment indicated contractile dysfunction, because the
ejection fraction, fractional shortening, cardiac output, and heart rate were
all significantly reduced in J2N-k hamsters compared with controls. Depressed
cardiac function was associated with decreased cardiac SR Ca2+ uptake in the
cardiomyopathic hamsters. Reduced SR Ca2+ uptake could be further linked to a
decrease in the expression of the SR Ca(2+)-ATPase and cAMP-dependent protein
kinase (PKA)-mediated phospholamban (PLB) phosphorylation at serine-16.
Depressed PLB phosphorylation was paralleled with a reduction in the activity of
SR-associated PKA, as well as an elevation in protein phosphatase activity in
J2N-k hamster. The results of this study suggest that an alteration in SR
function and its regulation contribute to cardiac contractile dysfunction in the
J2N-k cardiomyopathic hamster. The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac
contractility and its function is abnormal in the diabetic heart. However, the
mechanisms underlying SR dysfunction in the diabetic heart are not clear.
Because protein phosphorylation regulates SR function, this study examined the
phosphorylation state of phospholamban, a key SR protein that regulates SR
calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley
rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals
were humanely killed after 6 weeks and cardiac SR function was examined.
Depressed cardiac performance was associated with reduced SR Ca2+-uptake
activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent
with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a)
protein. The level of phospholamban (PLB) protein was also decreased, however,
the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR
Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the
Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein
kinase (PKA). Although the activities of the SR-associated
Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase
(PKA) were increased in the diabetic heart, depressed phosphorylation of PLB
could partly be attributed to an increase in the SR-associated protein
phosphatase activities. These results suggest that there is increased inhibition
of SERCA2a by PLB and this appears to be a major defect underlying SR
dysfunction in the diabetic heart. Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when
phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity
of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR
function, myocardial relaxation and myocardial contractility. The present study
was undertaken to examine the status of PLB phosphorylation after ischemia and
reperfusion and to provide evidence about the possible role of the
phosphorylation of Thr17 PLB residue on the recovery of contractility and
relaxation after a period of ischemia. Experiments were performed in Langendorff
perfused hearts from Wistar rats. Hearts were submitted to a protocol of global
normothermic ischemia and reperfusion. The results showed that (1) the
phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the
ischemia and the onset of reperfusion, respectively. The increase in Thr17
phosphorylation was associated with a recovery of relaxation to preischemic
values. This recovery occurred in spite of the fact that contractility was
depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was
dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur
by the coupled activation of the Na+ / H+ exchanger and the Na+ / Ca2+ exchanger
working in the reverse mode, since phosphorylation of Thr17 was decreased by
inhibition of these exchangers and not affected by blockade of the L-type Ca2+
channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased
Thr17 phosphorylation. This decrease was associated with an impairment of
myocardial relaxation. The present study suggests that the phosphorylation of
Thr17 of PLB upon reflow, may favor the full recovery of relaxation after
ischemia. Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by
beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban
(PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate
the mechanism of SERCA2a activation, we compared the kinetic properties of
SERCA2a expressed with (+) and without (-) PLB in High Five insect cell
microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle
SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity
(10-50 microM) activation of pre-steady-state catalytic site dephosphorylation
by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and
a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast,
SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the
high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and
exhibited low steady-state levels of E1P. The results indicate that the kinetic
differences in Ca2+ transport between skeletal and cardiac SR are due to the
presence of phospholamban in cardiac SR, and not due to isoform-dependent
differences between SERCA1 and SERCA2a. Therefore, the results are discussed in
terms of a model in which PLB interferes with SERCA2a oligomeric interactions,
which are important for the mechanism of Ca2+ transport in skeletal muscle
SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry
43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules
during catalytic cycling is obligatory for the changes in Ca2+ transport
activity that accompany the relief of PLB inhibition of the cardiac SR
Ca2+-ATPase. We previously found that a canine model of selective surgical ventricular
denervation (VD), which does not permit increased sympathetic tone during the
pathogenesis of heart failure (HF), tolerated the development of HF better than
controls. To investigate the cellular mechanisms, we examined cellular
contraction and L-type Ca(2+) channel currents (I(Ca)) and their responses to
beta-adrenergic receptor (beta-AR) stimulation in left ventricular myocytes from
1) control, 2) VD, 3) HF induced by rapid pacing, and 4) HF induced in VD
(VD-HF) dogs. The magnitude of myocyte contraction and rate of relaxation in VD
were similar to control but were depressed in both HF and VD-HF. These changes
were associated with reduced protein expression of sarcoplasmic reticulum
Ca(2+)-ATPase (SERCA2a) and protein kinase A phosphorylated phospholamban (PLB),
which was reduced in HF, but essentially abolished in VD-HF. beta-AR kinase
(GRK2) was increased in HF but reduced in VD-HF. Basal I(Ca) density did not
differ among control, VD, and HF groups, but VD-HF myocytes showed a markedly
reduced I(Ca) density (approximately 40%). Compared to controls, the sensitivity
of I(Ca) to isoproterenol (ISO), was significantly higher in VD, but reduced in
HF. While I(Ca) responses to ISO in VD-HF were maintained at control levels, the
amplitude of the ISO-stimulated I(Ca) was significantly smaller (approximately
50%) compared with HF myocytes. The relative decrease in Ca(2+) influx due to
downregulation of I(Ca) density may contribute to the cardioprotective effects
in VD-HF hearts by preventing Ca(2+) overload during the development of HF.
These findings, in combination with the virtual abolition of phosphorylated PLB
in VD-HF and the decrease in GRK2, may explain, in part, why VD dogs tolerate
the development of HF better than control dogs. Oligomeric interactions between Ca-ATPase polypeptide chains and their
modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic
reticulum (SR) microsomes. Progressive modification of Lys(514) with fluorescein
5-isothiocyanate (FITC), which physically blocks access to the nucleotide
binding site by ATP, demonstrates that Ca-ATPase active sites function
independently of one another prior to the phosphorylation of PLB. However, upon
cAMP-dependent protein kinase (PKA) phosphorylation of PLB, a second-order
dependence between residual enzyme activity and the fraction of active sites is
observed, consistent with a dimeric functional complex. Complementary distance
measurements were made using FITC or 5-iodoacetamidofluorescein (IAF) bound to
Cys(674) within the N- or P-domains, respectively, to detect structural coupling
within oligomeric complexes. Accompanying the phosphorylation of PLB,
neighboring Ca-ATPase polypeptide chains exhibit a 4 +/- 2 A decrease in the
proximity between FITC sites within the N-domain and a 9 +/- 3 A increase in the
proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB
induces spatial rearrangements between the N- and P-domain elements of proximal
Ca-ATPase polypeptide chains which restore functional interactions between
neighboring polypeptide chains and, in turn, result in increased rates of
catalytic turnover. These results are interpreted in terms of a structural
model, calculated through optimization of shape complementarity, desolvation,
and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase
polypeptide chains through the proximal association of N-domains that
accommodates interaction with PLB. We suggest that the phosphorylation of PLB
acts to release constraints involving interdomain subunit interactions that
enhance catalytically important N-domain motions. |
Are there telemedicine applications for chronic pain management? | Yes, telemedicine is feasible and cost-effective for education and therapy of patients with chronic pain. | OBJECTIVES: Telemedicine has been used extensively in various settings,
including monitoring patient treatment response and counseling. However, there
are few data on the application of telemedicine to chronic pain patients. The
present study was the first pilot project to determine whether telemedicine
technology for chronic pain consultation was feasible, cost-saving, and
satisfactory to patients and pain physicians.
METHODS: A prospective pilot study was conducted on chronic pain patients
requiring follow-up consultations using telemedicine technology. Patients were
interviewed by phone following the consultation.
RESULTS: Eleven telemedicine anesthesia consultations involving eight patients
(age 42+/-9 years; six men, two women) were performed. All were follow-up
consultations. The average distance from patients' home to the clinic was
314+/-170 km. The reasons for consultation were for update of patient progress
(10/11), medication change (6/11), and counseling (3/11). The time to complete
the consultation was 24.5+/-9.5 minutes. The data for the time and the cost that
the patient spent on the consultation are presented as median and 25% to 75%
interquartile range. Patients having telemedicine consultations spent 0.9 hours
(0.83-1) and Canadian dollar 3 (dollar 2-4) versus an estimate of 8 hours (6-8)
and Canadian dollar 80 (dollar 46-260) for a conventional consultation (both
P<0.005). Telemedicine consultation was found to be highly satisfactory to the
patient and the consulting and attending anesthesiologists.
CONCLUSIONS: This pilot study indicates that telemedicine follow-up
consultations for chronic pain patients are feasible and cost-saving. Patients
and anesthesiologists were highly satisfied with telemedicine consultation.
Patients reported a significant saving in time and cost compared with a
conventional consultation. The under treatment of pain has been well documented. Contributing to this is
the limited availability of pain management specialists in many geographic
areas. The use of technology to provide care to underserved areas is gaining
momentum. We chose to study whether stable patients and staff in chronic pain
clinic were satified with the use of a videoconferencing format in care
delivery. Our goals were to determine whether patients and staff could
successfully operate the extant videoconferencing equipment, was the equipment
dependably functional, was the use of a videoconferencing format an acceptable
method of healthcare delivery for both patients and staff, whether patients and
staff were satisfied with the process, and whether this was a cost-effective
mode of care delivery. Thirty-six patients were enrolled over 29 months.
Questionnaires were administered to staff and patients. Routine pain clinic
patient assessment tools were administered. Results showed the use of
videoconferencing for this group of patients is useable and satisfactory for
both patients and staff, that the patients save time and money, and that for a
system where videoconferencing equipment is already in use, it is also cost
effective. Staff were able to identify new patient problems. Some patients would
prefer to be seen in person but find that the savings in time and money override
this preference. Hearing impaired patients have difficulty using this medium.
Dependable equipment and phone connections are needed. A videoconferencing
clinic format is a clinically acceptable and cost effective method for follow-up
of stable patients with chronic pain. OBJECTIVE: Adapting therapeutic practice from traditional face-to-face exchange
to remote technology-based delivery presents challenges for the therapist,
patient, and technical writer. This article documents the process of therapy
adaptation and the resultant specification for the SMART2 project-a
technology-based self-management system for assisting long-term health
conditions, including chronic pain.
MATERIALS AND METHODS: Focus group discussions with healthcare professionals and
patients were conducted to inform selection of therapeutic objectives and
appropriate technology.
RESULTS: Pertinent challenges are identified, relating to (1) reduction and
definition of therapeutic objectives, and (2) how to approach adaptation of
therapy to a form suited to technology delivery. The requirement of the system
to provide dynamic and intelligent responses to patient experience and behavior
is also emphasized.
CONCLUSION: Solutions to these challenges are described in the context of the
SMART2 technology-based intervention. More explicit discussion and documentation
of therapy adaptation to technology-based delivery within the literature is
encouraged. Pain is generally undertreated in the United States, owing to a number of
barriers including geographic distance from specialty treatment providers;
functional disability that limits mobility; treatment-related stigma; economic
limitations; and educational barriers. Pain undertreatment exacerbates pain
chronicity and emotional disruption that can significantly erode a pain
patient's quality of life, and there is widespread agreement that pain care must
evolve to address this significant problem. The growing field of telehealth
(defined for the purposes of this paper as technology that allows for distance
interaction between providers and/or patients) offers a novel opportunity to
expand pain assessment, consultation, and treatment services beyond the walls of
the specialty pain clinic, but there is limited availability of resources
describing how to best use this technology to improve access to care. A recent
literature review (September 2011) using universally endorsed MeSH search
criteria revealed only 32 MEDLINE references focusing on telehealth for pain.
This is surprising in light of the very large number of references covering
telehealth (14,164 references) and pain (104,564 references), respectively. Of
the studies available, there are very few randomized trials of telehealth pain
care and only one general overview of e-health and chronic pain, which dedicates
just a few paragraphs to telehealth. This manuscript represents one of the first
comprehensive reviews of the current state of telehealth and pain management
research and practice. The goals are to provide a rationale for the potential
benefit of telehealth-based pain management services; describe the various
applications of telehealth technology for pain management; orient the reader to
cost models for telehealth; present examples of services in place; and offer
recommendations for future research based on the current state of knowledge. BACKGROUND: Internet-based interventions using cognitive behavioral approaches
can be effective in promoting self-management of chronic pain conditions.
Web-based programs delivered via smartphones are increasingly used to support
the self-management of various health disorders, but research on smartphone
interventions for persons with chronic pain is limited.
OBJECTIVE: The aim of this trial was to study the efficacy of a 4-week
smartphone-delivered intervention with written diaries and therapist feedback
following an inpatient chronic pain rehabilitation program.
METHODS: A total of 140 women with chronic widespread pain who participated in a
4-week inpatient rehabilitation program were randomized into 2 groups: with or
without a smartphone intervention after the rehabilitation. The smartphone
intervention consisted of 1 face-to-face session and 4 weeks of written
communication via a smartphone. Participants received 3 smartphone diary entries
daily to support their awareness of and reflection on pain-related thoughts,
feelings, and activities. The registered diaries were immediately available to a
therapist who submitted personalized written feedback daily based on cognitive
behavioral principles. Both groups were given access to a noninteractive website
after discharge to promote constructive self-management. Outcomes were measured
with self-reported questionnaires. The primary outcome measure of
catastrophizing was determined using the pain catastrophizing scale (score range
0-52). Secondary outcomes included acceptance of pain, emotional distress,
functioning, and symptom levels.
RESULTS: Of the 140 participants, 112 completed the study: 48 in the
intervention group and 64 in the control group. Immediately after the
intervention period, the intervention group reported less catastrophizing (mean
9.20, SD 5.85) than the control group (mean 15.71, SD 9.11, P<.001), yielding a
large effect size (Cohen's d=0.87) for study completers. At 5-month follow-up,
the between-group effect sizes remained moderate for catastrophizing (Cohen's
d=0.74, P=.003), acceptance of pain (Cohen's d=0.54, P=.02), and functioning and
symptom levels (Cohen's d=0.75, P=.001).
CONCLUSIONS: The results suggest that a smartphone-delivered intervention with
diaries and personalized feedback can reduce catastrophizing and prevent
increases in functional impairment and symptom levels in women with chronic
widespread pain following inpatient rehabilitation.
TRIAL REGISTRATION: Clinicaltrials.gov NCT01236209;
http://www.clinicaltrials.gov/ct2/show/NCT01236209 (Archived by WebCite at
http://www.webcitation.org/6DUejLpPY). OBJECTIVE: Access to care has become a priority for the Veterans Administration
(VA) health care system as a significant number of veterans enrolled in the VA
health care system reside in rural areas. The feasibility and effects of a novel
clinical intervention that combined group therapy and biofeedback training was
evaluated on women veterans living in rural areas.
METHODS: The study was conducted at selected community-based outpatient clinics
(CBOCs) in Texas. Thirty four women veterans with chronic pain and comorbid
depression and/or posttraumatic stress disorder (PTSD) were recruited. Five
sessions of education/therapy were delivered via telemedicine in combination
with daily home practice of a portable biofeedback device (Stress Eraser®,
Helicor, New York, NY, USA). Participants responded to self-report
questionnaires at baseline, at posttreatment, and at 6-week follow-up. Daily
practice logs were also maintained by participants.
RESULTS: The clinical protocol was acceptable, easy to administer, and
associated with statistically significant decreases in self-reported pain
unpleasantness, pain interference, depressive symptoms, PTSD symptoms, and sleep
disturbance at posttreatment. Improvements were maintained at 6-week follow-up.
Qualitative analyses indicated that many participants 1) wished to continue to
meet as a support group in their respective CBOCs and 2) felt less isolated and
more empowered to cope with their problems of daily living as a result of the
treatment.
CONCLUSIONS: It is feasible to provide treatment to women veterans living in
rural areas by utilizing video-teleconferencing technology between larger VA
medical centers and facilities at CBOCs in more rural settings. A controlled
trial of the intervention is warranted. |
What is a P-body (processing body)? | Processing bodies (P bodies, PB) are cytoplasmic protein complexes involved in degradation and translational arrest of mRNA. | Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine
(SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA
and export of mature mRNA from the nucleus. Although its role in nuclear mRNA
processing is well understood, the mechanism by which it alters the fate of
cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3
not only regulates the alternative splicing pattern of programmed cell death 4
(PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm
by lowering translation levels. We observed a marked increase in PDCD4 mRNA in
translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic
overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal
fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs),
where translationally silenced mRNAs are deposited, and this localization was
abrogated upon SRSF3 silencing. Furthermore, using two different reporter
systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates
translational repression by binding to the 5'-untranslated region (5'-UTR). In
summary, our data suggest that the oncogenic potential of SRSF3 might be
realized, in part, through the translational repression of PDCD4 mRNA. Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing
non-translating mRNAs and proteins from the mRNA decay and silencing
machineries. The mechanism of P-body assembly has been typically addressed by
depleting P-body components. Here we apply a complementary approach and
establish an automated cell-based assay platform to screen for molecules
affecting P-body assembly. From a unique library of compounds derived from
myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA),
a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also
inhibited, under stress conditions, phosphorylation of eIF2α and stress granule
formation. Other hits uncovered interesting novel links between P-body assembly,
lipid metabolism, and internal organelle physiology. The obtained results
provide a chemical toolbox to manipulate P-body assembly and function. Processing (P)-bodies are cytoplasmic RNA protein aggregates responsible for the
storage, degradation, and quality control of translationally repressed messenger
RNAs in eukaryotic cells. In mammals, P-body-related RNA and protein exchanges
are actomyosin dependent, whereas P-body movement requires intact microtubules.
In contrast, in plants, P-body motility is actin based. In this study, we show
the direct interaction of the P-body core component DECAPPING PROTEIN1 (DCP1)
with the tails of different unconventional myosins in Arabidopsis (Arabidopsis
thaliana). By performing coexpression studies with AtDCP1, domit-negative
myosin fragments, as well as functional full-length myosin XI-K, the association
of P-bodies and myosins was analyzed in detail. Finally, the combination of
mutant analyses and characterization of P-body movement patterns showed that
myosin XI-K is essential for fast and directed P-body transport. Together, our
data indicate that P-body movement in plants is governed by myosin XI members
through direct binding to AtDCP1 rather than through an adapter protein, as
known for membrane-coated organelles. Interspecies and intraspecies interaction
approaches with mammalian and yeast protein homologs suggest that this mechanism
is evolutionarily conserved among eukaryotes. Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces
cerevisiae. In logarithmically growing cells, only 0-1 P bodies per cell are
detectable. However, the number and appearance of P bodies change once the cell
encounters stress. Here, we show that the polysome-associated mRNA-binding
protein Scp160 interacts with P body components, such as the decapping protein
Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either
Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive
structures. These Dcp2-positive foci contained mRNA, because their formation was
inhibited by the presence of cycloheximide. In addition, Scp160 was required for
proper P body formation because only a subset of bona fide P body components
could assemble into the Dcp2-positive foci in Δscp160 cells. In either Δbfr1 or
Δscp160 cells, P body formation was uncoupled from translational attenuation as
the polysome profile remained unchanged. Collectively, our data suggest that
Bfr1 and Scp160 prevent P body formation under normal growth conditions. During cell stress, the transcription and translation of immediate early genes
are prioritized, while most other messenger RNAs (mRNAs) are stored away in
stress granules or degraded in processing bodies (P-bodies). TIA-1 is an
mRNA-binding protein that needs to translocate from the nucleus to seed the
formation of stress granules in the cytoplasm. Because other stress granule
components such as TDP-43, FUS, ATXN2,SMN, MAPT, HNRNPA2B1, and HNRNPA1 are
crucial for the motor neuron diseases amyotrophic lateral sclerosis (ALS)/spinal
muscular atrophy (SMA) and for the frontotemporal dementia(FTD), here we studied
mouse nervous tissue to identify mRNAs with selective dependence on Tia1
deletion. Transcriptome profiling with oligonucleotide microarrays in comparison
of spinal cord and cerebellum, together with independent validation in
quantitative reverse transcriptase PCR and immunoblots demonstrated several
strong and consistent dysregulations. In agreement with previously reported TIA1
knock down effects, cell cycle and apoptosis regulators were affected markedly
with expression changes up to +2-fold, exhibiting increased levels for Cdkn1a,
Ccnf, and Tprkb vs.decreased levels for Bid and Inca1 transcripts. Novel and
surprisingly strong expression alterations were detected for fat storage and
membrane trafficking factors, with prominent +3-fold upregulations of Plin4,
Wdfy1, Tbc1d24, and Pnpla2 vs. a −2.4-fold downregulation of Cntn4 transcript,
encoding an axonal membrane adhesion factor with established
haploinsufficiency.In comparison, subtle effects on the RNA processing machinery
included up to 1.2-fold upregulations of Dcp1b and Tial1. The effect on lipid
dynamics factors is noteworthy, since also the gene deletion of Tardbp (encoding
TDP-43) and Atxn2 led to fat metabolism phenotypes in mouse. In conclusion,
genetic ablation of the stress granule nucleator TIA-1 has a novel major effect
on mRNAs encoding lipid homeostasis factors in the brain, similar to the fasting
effect. mRNA accumulation is tightly regulated by diverse molecular pathways. The
identification and characterization of enzymes and regulatory proteins involved
in controlling the fate of mRNA offers the possibility to broaden our
understanding of posttranscriptional gene regulation. Processing bodies (P
bodies, PB) are cytoplasmic protein complexes involved in degradation and
translational arrest of mRNA. Composition and dynamics of these subcellular
structures have been studied in animal systems, yeasts and in the model plant
Arabidopsis. Their assembly implies the aggregation of specific factors related
to decapping, deadenylation, and exoribonucleases that operate synchronously to
regulate certain mRNA targets during development and adaptation to stress.
Although the general function of PB along with the flow of genetic information
is understood, several questions still remain open. This review summarizes data
on the composition, potential molecular roles, and biological significance of PB
and potentially related proteins in Arabidopsis. In eukaryotic cells, components of the 5' to 3' mRNA degradation machinery can
undergo a rapid phase transition. The resulting cytoplasmic foci are referred to
as processing bodies (P-bodies). The molecular details of the self-aggregation
process are, however, largely undetermined. Herein, we use a bottom-up approach
that combines NMR spectroscopy, isothermal titration calorimetry, X-ray
crystallography, and fluorescence microscopy to probe if mRNA degradation
factors can undergo phase transitions in vitro. We show that the
Schizosaccharomyces pombe Dcp2 mRNA decapping enzyme, its prime activator Dcp1,
and the scaffolding proteins Edc3 and Pdc1 are sufficient to reconstitute a
phase-separation process. Intermolecular interactions between the Edc3 LSm
domain and at least 10 helical leucine-rich motifs in Dcp2 and Pdc1 build the
core of the interaction network. We show that blocking of these interactions
interferes with the clustering behavior, both in vitro and in vivo. Author information:
(1)Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch,
France.
(2)Luxembourg Centre for Systems Biomedicine, University of Luxembourg,
Esch-sur-Alzette, Luxembourg; National Center for Microscopy and Imaging
Research, University of California San Diego, La Jolla, California, United
States of America.
(3)Institute for Systems Biology, Seattle, Washington, United States of America.
(4)Tampere University of Technology, Pori, Finland; BioMediTech, University of
Tampere, Tampere, Finland.
(5)Physics and Bioengineering, University of Washington, Seattle, Washington,
United States of America.
(6)Centre for High-Throughput Biology, Department of Physics and Astronomy,
University of British Columbia, Vancouver, British Columbia, Canada.
(7)Institute for Systems Biology, Seattle, Washington, United States of America;
Molecular and Cellular Biology Program, University of Washington, Seattle,
Washington, United States of America.
(8)Molecular and Cellular Biology Program, University of Washington, Seattle,
Washington, United States of America; Pacific Northwest Diabetes Research
Institute, Seattle, Washington, United States of America. In eukaryotic cells, non-translating mRNAs can accumulate into cytoplasmic mRNP
(messenger ribonucleoprotein) granules such as P-bodies (processing bodies) and
SGs (stress granules). P-bodies contain the mRNA decay and translational
repression machineries and are ubiquitously expressed in mammalian cells and
lower eukaryote species including Saccharomyces cerevisiae, Drosophila
melanogaster and Caenorhabditis elegans. In contrast, SGs are only detected
during cellular stress when translation is inhibited and form from aggregates of
stalled pre-initiation complexes. SGs and P-bodies are related to NGs (neuronal
granules), which are essential in the localization and control of mRNAs in
neurons. Importantly, RNA granules are linked to the cytoskeleton, which plays
an important role in mediating many of their dynamic properties. In the present
review, we discuss how P-bodies, SGs and NGs are linked to cytoskeletal networks
and the importance of these linkages in maintaining localization of their RNA
cargoes. The localization of mRNA forms a key facet of the post-transcriptional control
of gene expression and recent evidence suggests that it may be considerably more
widespread than previously anticipated. For example, defined mRNA-containing
granules can be associated with translational repression or activation.
Furthermore, mRNA P-bodies (processing bodies) harbour much of the mRNA decay
machinery and stress granules are thought to play a role in mRNA storage. In the
present review, we explore the process of mRNA localization in the yeast
Saccharomyces cerevisiae, examining connections between organellar mRNA
localization and the response to stress. We also review recent data suggesting
that even where there is a global relocalization of mRNA, the specificity and
kinetics of this process can be regulated. The decapping enzymes DCP1 and DCP2 are components of a decapping complex that
degrades mRNAs. DCP2 is the catalytic core and DCP1 is an auxiliary subunit. It
has been assumed that DCP1 and DCP2 are consistently co-localized in cytoplasmic
RNA granules called processing bodies (P-bodies). However, it has not been
confirmed whether DCP1 and DCP2 co-localize in Arabidopsis thaliana. In this
study, we generated DCP1-green fluorescent protein (GFP) and DCP2-GFP transgenic
plants that complemented dcp1 and dcp2 mutants, respectively, to see whether
localization of DCP2 is identical to that of DCP1. DCP2 was present throughout
the cytoplasm, whereas DCP1 formed P-body-like foci. Use of DCP1-GFP/DCP2-red
fluorescent protein (RFP) or DCP1-RFP/DCP2-GFP plants showed that heat treatment
induced DCP2 assembly into DCP1 foci. In contrast, cold treatment did not induce
DCP2 assembly, while the number of DCP1 foci increased. These changes in DCP1
and DCP2 localization during heat and cold treatments occurred without changes
in DCP1 and DCP2 protein abundance. Our results show that DCP1 and DCP2 respond
differently to environmental changes, indicating that P-bodies have diverse DCP1
and DCP2 proportions depending on environmental conditions. The localization
changes of DCP1 and DCP2 may explain how specific mRNAs are degraded during
changes in environmental conditions. The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not
only allows localized protein production but also determines the fate of mRNAs.
For instance, translationally repressed mRNAs localize to P-bodies and stress
granules where their decay and storage, respectively, are directed. Here, we
find that several mRNAs are localized to granules in unstressed, actively
growing cells. These granules play a key role in the stress-dependent formation
of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per
cell, which aggregate during P-body formation. Such aggregation is still
observed under conditions or in mutants where P-bodies do not form. In
unstressed cells, the mRNA granules appear associated with active translation;
this might enable a coregulation of protein expression from the same pathways or
complexes. Parallels can be drawn between this coregulation and the advantage of
operons in prokaryotic systems. Author information:
(1)Division of Rheumatology, Immunology, and Allergy, Brigham and Women's
Hospital, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School,
Boston, MA 02115, USA. Electronic address: [email protected].
(2)Division of Rheumatology, Immunology, and Allergy, Brigham and Women's
Hospital, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School,
Boston, MA 02115, USA.
(3)Division of Rheumatology, Immunology, and Allergy, Brigham and Women's
Hospital, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School,
Boston, MA 02115, USA. Electronic address: [email protected]. The mammalian target of rapamycin complex 1 (mTORC1) is involved in the cellular
transcription and translation processes. The undertaken study characterized the
enhancer of mRNA decapping protein 4 (Edc4) as mTORC1 interacting protein. Human
T lymphoblast (CCRF-CEM) cells were used for mTORC1 purification.
Co-immunoprecipitation coupled with immunoblotting analysis was used to confirm
the interaction of Edc4 in mTORC1 specific purifications. Further assays were
incorporated to conclude the role of mTORC1 in mRNA decapping via Edc4. Edc4 was
identified as a new interacting protein with mTORC1 in both the endogenous and
myc-tag raptor component mTORC1 specific purifications. Quantitative
co-localization using confocal microscopy demonstrated that raptor component of
mTORC1 coexists with Edc4 in processing (P) bodies, a site for mRNA degradation.
Incubation of cells with rapamycin, a known inhibitor of mTOR kinase activity,
increased the total Edc4 protein expression but at the same time decreased the
Edc4 interaction with mTORC1. Moreover, rapamycin treatment resulted in a
significant decrease in total serine phosphorylated Edc4 protein signal and the
total 5'-capped mRNA. These findings provide the first evidence for the pivotal
role of mTORC1 in Edc4 regulation. Further in-depth studies are required to get
a complete understanding of molecular crosstalk between mTORC1 signaling and
mRNA decapping pathway. |
What is the mechanism of action of decitabine? | Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We also discuss the following questions: What is the best administration schedule of decitabine in solid tumors? Is there tumor type specificity for decitabine-based epigenetic therapy? We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. | Decitabine is a potent demethylating agent that exhibits clinical activity
against myeloid maligcies. Numerous genes silenced by hypermethylation are
reactivated by decitabine through a mechanism involving promoter demethylation
with subsequent release of histone deacetylases (HDACs) and accumulation of
acetylated histones. Recent studies indicating that decitabine also induces
regional chromatin remodeling of some unmethylated genes suggest additional
mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML
cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has
not been studied in detail and decitabine effects on p21WAF1 chromatin
remodeling have not been reported. We found that p21WAF1 mRNA was undetectable
in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no
evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in
AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased
acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects
on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor
trichostatin A and were independent of wild type p53. Our findings indicate that
decitabine can relieve p21WAF1 repression in AML by a mechanism that involves
release of HDAC1 without requiring promoter demethylation. Furthermore, our
study provides evidence that combined decitabine and HDAC inhibitor treatment
can enhance chromatin remodeling and reactivation of an unmethylated tumor
suppressor gene. This latter finding is of relevance to the clinical use of
these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo. During the last 10 years, three European phase II studies were performed to
investigate the treatment of elderly patients with myelodysplastic syndrome
(MDS) with low-dose 5-aza-2'-deoxycytidine (decitabine, DAC). All these European
trial data were reviewed on the basis of the International Prognostic Scoring
System (IPSS) risk criteria and the response criteria as recently published by
an international working group. To investigate the results in a larger cohort of
patients and to determine risk factors, all data were pooled with some
observations from the PCH 95-06 US phase II study. The response rate in the 177
patients evaluated (median age 70 years) was 49%. The median response duration
was 36 weeks, and the median survival was 15 months. Analysis of the data
according to sex, age, French-American-British classification, percentage of
blasts in the bone marrow, IPSS risk group, lactate dehydrogenase and
cytogenetics did not reveal any factor predictive of response. Overall, 69% of
patients benefited, including those with stable disease during therapy. Response
duration was significantly shorter with increasing risk (according to the IPSS
classification). Haemoglobin level and neutrophil count showed an inverse
correlation to the IPSS classification. Univariate analysis showed a
significantly inferior survival for elderly patients (>75 years of age) and for
those with high levels of serum lactate dehydrogenase (LDH) (more than two times
the normal values). Patients with high-risk cytogenetic abnormalities according
to the IPSS risk criteria showed better overall survival than those with
intermediate-risk abnormalities. When analysed according to the IPSS risk
classification, high-risk patients had worse survival prospects following
decitabine therapy than those with intermediate risk; however, compared to the
originally reported IPPS outcomes for high-risk patients, they probably showed
better survival. During the treatment period, 18% of the patients progressed
towards acute leukaemia. Decitabine showed a rather low toxicity profile in this
elderly patient group. In conclusion, low-dose decitabine is an active drug for
the treatment of MDS patients, even for those older than 75 years with bad
prognostic characteristics. Recent evidence demonstrates that epigenetic silencing of genes is associated
with myelodysplasia and that a worse prognosis may be correlated with
hypermethylation of certain genes, such as the cyclin-dependent kinase inhibitor
p15. 5-Aza-2'-deoxycytidine (decitabine, DAC) is a nucleoside analog, which, at
low doses, acts as a hypomethylating agent and is fivefold to tenfold more
active than 5-azacytidine (azacitidine, Vidaza)--currently the only approved
drug for treatment of myelodysplastic syndrome (MDS). Clinical studies have
demonstrated that decitabine has activity in patients with MDS. Preliminary
results of a phase III multicenter North American trial comparing low-dose
decitabine to supportive care verified that therapy with decitabine resulted in
higher response rates, improved quality of life, and prolonged time to leukemic
transformation and/or death. However, further elucidation of its mechanism of
action is required, as clinical response to decitabine does not correlate with
demethylation of the p15 gene promoter or the repetitive DNA element LINE.
Decitabine appears to upregulate both hypermethylated and nonmethylated genes.
Ongoing studies aim to determine the optimal dose, schedule, and route of
administration of decitabine, and to evaluate whether efficacy can be improved
by using it in combination with other agents, such as histone deacetylase
inhibitors. Progress on several unresolved issues in cancer epigenetics will benefit from
rapid and standardized methods for profiling DNA methylation genome-wide. In the
area of epigenetic therapy, the demethylating drug decitabine
(5-aza-2'-deoxycytidine) is increasingly used to treat acute myelogenous
leukemia and myelodysplastic syndrome, but the mechanisms of its anticancer
activity have remained unclear. Given the clinical efficacy of decitabine and
the uncertainties about its mode of action, it will be useful to optimize
methods for following DNA methylation as a biochemical response in individual
patients. Here, we describe a single nucleotide polymorphism (SNP) chip-based
method (MSNP) for profiling DNA methylation. Using this procedure, the extent of
demethylation in bone marrow aspirates from patients with leukemia receiving
decitabine can be assessed genome-wide using commercially available (Affymetrix)
SNP chips. We validated the accuracy of MSNP by comparing the results with
combined bisulfite restriction analysis and by sequencing cloned PCR products
from bisulfite-converted DNA. We further validated MSNP in a Wilms' tumor/normal
kidney comparison, comparing the results with methylation-sensitive Southern
blotting. MSNP simultaneously detects aberrations in DNA copy number and loss of
heterozygosity, making it a generally useful approach for combined genetic and
epigenetic profiling in tissue samples from cancer patients. We have previously demonstrated proteasomal degradation of DNMT1 in mammalian
cells following treatment with several DNA hypomethylating agents. Here, we
demonstrate dose-dependent degradation of Dnmt1 in mouse embryonic stem (ES)
cells expressing catalytic site mutant (cys-ser), confirming that the covalent
bond formation between Dnmt1 and decitabine-incorporated DNA is not essential
for this process. DNMT1o, the oocyte-specific isoform that lacks the N-terminal
118-amino acid domain, did not undergo decitabine-mediated degradation, which
further proves the requirement of multiple domains including nuclear
localization signal, KEN box, and BAH domains for this process. Analysis of
glycerol density gradient fractions of micrococcal nuclease-digested nuclei
showed that both nucleosomal and nucleoplasmic DNMT1 are degraded upon
decitabine treatment. Among different inhibitors tested, the inhibitors of the
proteasomal pathway and several protein kinases impeded decitabine-induced DNMT1
degradation. The maximal effect caused by inhibiting protein kinase C (PKC)
persuaded us to investigate further its role in decitabine-mediated DNMT1
degradation. Blockage of the degradation process after treatment with rottlerin,
an inhibitor of PKCδ, or after siRNA-mediated depletion of PKCδ, indicated that
this protein kinase is involved in decitabine-mediated depletion of DNMT1. PKCδ
interacted with and phosphorylated DNMT1 in vitro. Moreover, rottlerin inhibited
both basal and decitabine-induced phosphorylation of DNMT1. These studies
provide substantial evidence that decitabine-induced degradation of the
maintece methyltransferase DNMT1 does not require covalent bond formation
with the substrate and also elucidate its underlying molecular mechanism. BACKGROUND: Cancer testis antigens (CTAs) are a novel group of tumor associated
antigens. Demethylating agent decitabine was reported to be able to up-regulate
CTAs through its hypomethylation mechanism, thus enhance the immunogenicity of
leukemia cells. However, few researches have ever focused on the questions that
whether this immunostimulatory effect of decitabine could induce autologous CTA
specific cytotoxic T lymphocytes (CTLs) in vivo, and if so, whether this effect
contributes to disease control. In this study, we aimed to show that decitabine
could induce specific autologous CTLs against some mouse CTAs in leukemia cells
in vitro and in vivo.
METHODS: Several mouse CTAs were screened by RT-PCR. CTL specific to one of the
CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry.
The activity of specific CTLs was measured by real time RT-PCR.
RESULTS: We firstly screened expression of some CTAs in mouse leukemia cells
before and after decitabine treatment and found that decitabine treatment did
up-regulate expression of many CTAs. Then we measured the CTLs' activity
specific to a mouse CTA P1A in vivo and showed that this activity increased
after decitabine treatment. Finally, we sorted these in vivo induced P1A
specific CTLs by flow cytometry and demonstrated their cytotoxicity against
decitabine treated leukemia cells.
CONCLUSIONS: Our study showed the autologous immune response induced by
decitabine in vivo. And more importantly, we firstly proved that this response
may contribute to disease control. We believe that this immunostimulatory effect
is another anti-cancer mechanism of decitabine, and this special effect would
inspire new applications of decitabine in the field of leukemia treatment in the
future. Decitabine, a demethylating drug, is the first-line treatment for
myelodysplastic syndromes and gains better overall survival, which is based on
epigenetic mechanism. Activated by promoter demethylation, melanoma-associated
antigens-A (MAGE-A), cancer-testis antigens are attractive targets for
immunotherapy. Our purpose was to investigate whether decitabine could show
anti-tumor effects for esophageal cancer and explore its mechanism. In addition,
we aimed to examine its modulation for most MAGE-A members. The results showed
the baseline expression were MAGE-A2, -3,-9, and -10 in Eca109 cells and
decitabine (0.5 μM) could induce MAGE-A8 and -A4 whereas reduce MAGE-A9 and
-A10. Moreover, decitabine (0.5 μM) inhibited cell proliferation, migration and
invasive ability by 15%, 34% and 47.2%, respectively and decreased expressions
of NF-κB2 and MMP2. Our results demonstrated that low-dose decitabine induced
the expression of MAGE-A8 and -A4, and inhibited cell invasion through
decreasing expression of MMP2 and NF-κB2, which provides possibilities for
combing decitabine with immunotherapy targeting MAGE-A to treat advanced
esophageal squamous cell carcinoma. |
Which gene is associated with the Mitchell-Riley syndrome? | Mutations in the gene coding for the transcription factor RFX6 (regulatory factor X,6) have been described as the cause of the Mitchell-Riley syndrome. | Recently, bi-allelic mutations in the transcription factor RFX6 were described
as the cause of a rare condition characterized by neonatal diabetes with
pancreatic and biliary hypoplasia and duodenal/jejunal atresia. A male infant
developed severe hyperglycemia (446 mg/dL) within 24 h of birth. Acute abdominal
concerns by day five necessitated exploratory surgery that revealed duodenal
atresia, gallbladder agenesis, annular pancreas and intestinal malrotation. He
also exhibited chronic diarrhea and feeding intolerance, cholestatic jaundice,
and subsequent liver failure. He died of sepsis at four months old while
awaiting liver transplantation. The phenotype of neonatal diabetes with
intestinal atresia and biliary agenesis clearly pointed to RFX6 as the causative
gene; indeed, whole exome sequencing revealed a novel homozygous RFX6 mutation
c.779A>C; p.Lys260Thr (K260T). This missense mutation also changes the consensus
5' splice donor site before intron 7 and is thus predicted to cause disruption
in splicing. Both parents, who were not known to be related, were heterozygous
carriers. Targeted genetic testing based on consideration of phenotypic features
may reveal a cause among the many genes now associated with heterogeneous forms
of monogenic neonatal diabetes. Our study demonstrates the feasibility of using
modern sequencing technology to identify one such rare cause. Continued research
is needed to determine the possible cost-effectiveness of this approach,
especially when clear phenotypic clues are absent. Further study of patients
with RFX6 mutations should clarify its role in pancreatic, intestinal and
enteroendocrine cellular development and explain features such as the diarrhea
exhibited in our case. |
Is paramyxovirus involved in human subacute thyroiditis? | There is no evidence that paramyxovirus are involved in etiology of subacute thyroiditis. | New data on viruses isolated from patients with subacute thyroiditis de Quervain
are reported. Characteristic morphological, cytological, some physico-chemical
and biological features of the isolated viruses are described. A possible role
of these viruses in human and animal health disorders is discussed. The isolated
viruses remain unclassified so far. Virological and serological methods were used in examination of 28 patients
suffering from subacute thyroiditis de Quervain. Attempts to isolate a presumed
viral agent from 8 patients were performed by inoculation of serum, urine, and
aspiration biopsies of thyroid glands taken at different stages of the illness,
into tissue cultures of different types of human and animal cells. Recovery of a
cytopathic viral agent on cells of a rabbit lung continuous line was successful
in 5 cases. Serological cross reactions exist between the isolated viruses and
patient serum but not with serum of healthy people. Cases with the acquired
illness and positive antibodies against the isolated viruses who had been in
close and prolonged contact with patients suffering from subacute thyroiditis de
Quervain were also investigated. The author experienced a case of subacute thyroiditis (de Quervain) in a
36-year-old female. Electron microscopic examination of the thyroid tissue of
the patient revealed virus-like particles (VLP) in the degenerated follicular
epithelium Judging from the size, the VLP corresponded to the influenza or mumps
virus. The etiology of subacute granulomatous thyroiditis (SAT) is obscure, although it
is postulated to be associated with viral infections and genetic factors. In the
present study, the possibility of an infectious etiology was prospectively
studied in 27 consecutive patients with SAT. Special emphasis was put on the
role of enteroviruses. Coupled sera (interval one month) were taken from all
patients and single sera from 29 control subjects for virus antibody
determinations. Stool samples were collected for virus isolation and fine-needle
aspiration samples from thyroid gland for the detection of enterovirus RNA using
RT-PCR were taken from SAT patients. Enteroviral antibodies were tested using
three different methods: indirect EIA, heavy chain capture RIA, and standard
complement fixation (CF) test. Antibodies against other common viral pathogens,
including enteroviruses, were screened using the CF test and those against
Mycoplasma pneumoniae and Chlamydia pneumoniae using EIA and
microimmunofluorescence techniques, respectively. Common respiratory viruses
were also screened from nasopharyngeal suction samples by antigen detection EIA.
Based on serological findings, one patient had acute Cytomegalovirus infection.
All other patients were negative in antibody tests, virus isolation, RT-PCR, and
antigen detection. Enterovirus RNA was not detected by PCR in the thyroid tissue
in any of the fine-needle aspiration samples. There was no evidence of recent
enteroviral infections in SAT patients. The results suggest that SAT is not
usually associated with acute infections. No evidence was obtained to support
the proposed role of enteroviruses as an important etiologic agent of SAT. Subacute thyroiditis is a rare disease in childhood and is considered postviral
in origin. Epstein-Barr virus (EBV) involvement has been suspected, but just
once demonstrated in an adult female. We report a case of subacute thyroiditis
during infectious mononucleosis in a 3-year-old girl. The diagnosis of
infectious mononucleosis was supported, in addition to the typical clinical
symptoms, by presence of EBV DNA both in plasma and leukocytes. Subacute
thyroiditis was diagnosed based on elevated thyroid hormone levels with
thyrotropin (TSH) suppression, high inflammation markers and almost absent
(99)TC uptake by thyroid. Moreover after 3 months from diagnosis thyroid
function went back to normal as well as the (99)TC uptake. de Quervain thyroiditis, also known as subacute thyroiditis, is a self-limited
inflammatory disease of the thyroid gland. It is extremely rare in children. The
hallmarks for diagnosis are painful thyroid enlargement, elevated inflammatory
markers, and decreased uptake of the thyroid gland on thyroid scintigraphy.
Viral infection has been proposed to be associated with de Quervain thyroiditis.
Coxsackie virus has been reported to be one of the viruses associated with the
disease. To our knowledge, childhood de Quervain thyroiditis associated with
hand-foot-mouth disease caused by coxsackie infection has never been reported.
We report a 2.7-year-old boy who presented with typical features of de Quervain
thyroiditis following hand-foot-mouth disease caused by coxsackie B4 infection.
He had a brief thyrotoxic phase initially, followed by transient hypothyroid
phase and euthyroidism thereafter. His thyroid scintigraphy showed a typical
faint uptake at the diagnosis, and an improvement of the thyroid scan and uptake
was shown 8 weeks later. He was treated with prednisolone and nearly complete
resolution was documented within 2 months. Careful evaluation of the patient led
to the correct diagnosis and appropriate management. We describe a case of quick recovery of myocardium damage in a 15-year-old
adolescent with subacute thyroiditis. After 1 week of admission, his
cardiovascular status began to show signs of improvement accompanied by the
recovery of electrocardiogram and indicators of myocardial damage. We speculate
that myocardium damage associated with subacute thyroiditis is a complication of
common virus, although we did not detect any abnormal virus antibody and
deoxyribonucleic acid in the patient's serum. 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. |
What are the mobile applications fields of use for patients ? | Weight-loss mobile applications
pediatric obesity prevention and treatment, healthy eating, and physical activity promotion
A total of 229 dermatology-related apps were identified in the following categories: general dermatology reference (61 [26.6%]), self-surveillance/diagnosis (41 [17.9%]), disease guide (39 [17.0%]), educational aid (20 [8.7%]), sunscreen/UV recommendation (19 [8.3%]), calculator (12 [5.2%]), teledermatology (8 [3.5%]), conference (6 [2.6%]), journal (6 [2.6%]), photograph storage/sharing (5 [2.2%]), dermoscopy (2 [0.9%]), pathology (2 [0.9%]), and other (8 [3.5%]). The most reviewed apps included Ultraviolet ~ UV Index (355 reviews), VisualDx (306), SPF (128), iSore (61), and SpotMole (50)
mobile health and fitness app
alcohol-use behavior change or recovery
More than 17,000 mHealth apps now are available for smart phones and other devices, and they do everything from monitoring urine flow for patients with enlarged prostates to reminding people prone to kidney stones to drink more water. | BACKGROUND: Interactive and mobile technologies (i.e., smartphones such as
Blackberries, iPhones, and palm-top computers) show promise as an efficacious
and cost-effective means of communicating health-behavior risks, improving
public health outcomes, and accelerating behavior change. The present study was
conducted as a "needs assessment" to examine the current available mobile
smartphone applications (e.g., apps) that utilize principles of ecological
momentary assessment (EMA)-daily self-monitoring or near real-time
self-assessment of alcohol-use behavior-to promote positive behavior change,
alcohol harm reduction, psycho-education about alcohol use, or abstinence from
alcohol.
METHODS: Data were collected and analyzed from iTunes for Apple iPhone(©) . An
inventory assessed the number of available apps that directly addressed alcohol
use and consumption, alcohol treatment, or recovery, and whether these apps
incorporated empirically based components of alcohol treatment.
RESULTS: Findings showed that few apps addressed alcohol-use behavior change or
recovery. Aside from tracking drinking consumption, a minority utilized
empirically based components of alcohol treatment. Some apps claimed they could
serve as an intervention; however, no empirical evidence was provided.
CONCLUSIONS: More studies are needed to examine the efficacy of mobile
technology in alcohol intervention studies. The large gap between availability
of mobile apps and their use in alcohol treatment programs indicates several
important future directions for research. Smartphones and tablets are slowly but steadily changing the way we look after
our health and fitness. Today, many high quality mobile apps are available for
users and health professionals and cover the whole health care chain, i.e.
information collection, prevention, diagnosis, treatment and monitoring. Our
team has developed a mobile health and fitness app called myFitnessCompanion®
which has been available via Android market since February 2011. The objective
of this paper is to share our experience with rolling out a mobile health and
fitness app. We discuss the acceptance of health apps by end-users and
healthcare industry. We discuss how mobile health apps will be distributed in
the near future, the use of Personal Health Record (PHR) systems such as
Microsoft HealthVault and the impact of regulations (FDA) on the future of
mobile health apps. The paper is based on seven years of experience by the
authors as mobile health and fitness application developers and we discuss the
challenges and opportunities for app developers in the health industry. IMPORTANCE: With advancements in mobile technology, cellular phone-based mobile
applications (apps) may be used in the practice and delivery of dermatologic
care.
OBJECTIVE: To identify and categorize the variety of current mobile apps
available in dermatology for patients and providers.
DESIGN, SETTING, AND PARTICIPANTS: Dermatology-related search terms were queried
in the online app stores of the most commonly used mobile platforms developed by
Apple, Android, Blackberry, Nokia, and Windows. Applications were assigned to
categories based on description. Popularity, price, and reviews were recorded
and target audiences were determined through websites offering online mobile
apps.
MAIN OUTCOMES AND MEASURES: Number, type, and price of mobile apps in
dermatology.
RESULTS: A total of 229 dermatology-related apps were identified in the
following categories: general dermatology reference (61 [26.6%]),
self-surveillance/diagnosis (41 [17.9%]), disease guide (39 [17.0%]),
educational aid (20 [8.7%]), sunscreen/UV recommendation (19 [8.3%]), calculator
(12 [5.2%]), teledermatology (8 [3.5%]), conference (6 [2.6%]), journal (6
[2.6%]), photograph storage/sharing (5 [2.2%]), dermoscopy (2 [0.9%]), pathology
(2 [0.9%]), and other (8 [3.5%]). The most reviewed apps included
Ultraviolet ~ UV Index (355 reviews), VisualDx (306), SPF (128), iSore (61), and
SpotMole (50). There were 209 unique apps, with 17 apps existing on more than 1
operating system. More than half of the apps were offered free of charge (117
[51.1%]). Paid apps (112 [48.9%]) ranged from $0.99 to $139.99 (median, $2.99).
Target audiences included patient (117 [51.1%]), health care provider (94
[41.0%]), and both (18 [7.9%]).
CONCLUSIONS AND RELEVANCE: The widespread variety and popularity of mobile apps
demonstrate a great potential to expand the practice and delivery of
dermatologic care. Mobile applications (apps) offer a novel way to engage children in behavior
change, but little is known about content of commercially available apps for
this population. We analyzed the content of apps for iPhone/iPad for pediatric
weight loss, healthy eating (HE), and physical activity (PA). Fifty-seven apps
were downloaded and tested by two independent raters. Apps were coded for:
inclusion of the Expert Committee for Pediatric Obesity Prevention's (ECPOP)
eight recommended strategies (e.g., set goals) and seven behavioral targets
(e.g., do ≥1 h of PA per day), utilization of gaming elements, and general
characteristics. Most apps lacked any expert recommendations (n = 35, 61.4 %).
The mean number of recommendations among apps that used recommendations was
3.6 ± 2.7 out of 15, 56.1 % (n = 32) apps were classified as games, and mean
price per app was $1.05 ± 1.66. Most apps reviewed lacked expert recommendations
and could be strengthened by addition of comprehensive information about health
behavior change and opportunities for goal setting. BACKGROUND: Physicians have limited time for weight-loss counseling, and there
is a lack of resources to which they can refer patients for assistance with
weight loss. Weight-loss mobile applications (apps) have the potential to be a
helpful tool, but the extent to which they include the behavioral strategies
included in evidence-based interventions is unknown.
PURPOSE: The primary aims of the study were to determine the degree to which
commercial weight-loss mobile apps include the behavioral strategies included in
evidence-based weight-loss interventions, and to identify features that enhance
behavioral strategies via technology.
METHODS: Thirty weight-loss mobile apps, available on iPhone and/or Android
platforms, were coded for whether they included any of 20 behavioral strategies
derived from an evidence-based weight-loss program (i.e., Diabetes Prevention
Program). Data on available apps were collected in January 2012; data were
analyzed in June 2012.
RESULTS: The apps included on average 18.83% (SD=13.24; range=0%-65%) of the 20
strategies. Seven of the strategies were not found in any app. The most common
technology-enhanced features were barcode scanners (56.7%) and a social network
(46.7%).
CONCLUSIONS: Weight-loss mobile apps typically included only a minority of the
behavioral strategies found in evidence-based weight-loss interventions.
Behavioral strategies that help improve motivation, reduce stress, and assist
with problem solving were missing across apps. Inclusion of additional
strategies could make apps more helpful to users who have motivational
challenges. |
Is there any link between the aurora B kinase and the polycomb protein ring1B? | Yes. The aurora B kinase and the polycomb protein ring1B combine to regulate active promoters in quiescent lymphocytes. | Reversible cellular quiescence is critical for developmental processes in
metazoan organisms and is characterized by a reduction in cell size and
transcriptional activity. We show that the Aurora B kinase and the polycomb
protein Ring1B have essential roles in regulating transcriptionally active genes
in quiescent lymphocytes. Ring1B and Aurora B bind to a wide range of active
promoters in resting B and T cells. Conditional knockout of either protein
results in reduced transcription and binding of RNA Pol II to promoter regions
and decreased cell viability. Aurora B phosphorylates histone H3S28 at active
promoters in resting B cells as well as inhibiting Ring1B-mediated
ubiquitination of histone H2A and enhancing binding and activity of the USP16
deubiquitinase at transcribed genes. Our results identify a mechanism for
regulating transcription in quiescent cells that has implications for epigenetic
regulation of the choice between proliferation and quiescence. |
Which are the genes responsible for Dyskeratosis Congenita? | To date, 8 genes have been associated with Dyskeratosis Congenita development. These are DKC1, TERC, TERT, NOP10, NHP2, TIN2, C16orf57, and TCAB1. Seven of these are important in telomere maintenance, because either they encode components of the telomerase enzyme complex (DKC1, TERC, TERT, NOP10, NHP2, and TCAB1) or the shelterin complex (TINF2). | Dyskeratosis congenita (DC) is a rare inherited disorder characterised by the
early onset of reticulate skin pigmentation, nail dystrophy, and mucosal
leucoplakia. In over 80% of cases bone marrow failure develops and this is the
main cause of early mortality. The DC1 gene responsible for the X linked form
(MIM 305000) of dyskeratosis congenita has been mapped to Xq28. In order to
narrow the candidate gene region, genetic linkage analysis was performed in
eight X linked pedigrees using a set of markers spanning Xq28. A maximum lod
score of 5.31 with no recombinations was achieved with marker DXS1073. Two
recombination events were identified; one of these uses X chromosome
inactivation pattern analysis to determine carrier status and haplotype analysis
to fine map the recombination breakpoint. The fine mapping of these
recombination events has enabled the candidate gene region for X linked
dyskeratosis congenita to be defined as the 1.4 Mb interval between Xq3274 and
DXS1108. Dyskeratosis congenita (DC) is an inherited disorder characterized by skin
pigmentation, nail dystrophy and mucosal leucoplakia. In 1995 a Dyskeratosis
Congenita Registry was established at the Hammersmith Hospital. In the 46
families recruited, 76/83 patients were male, suggesting that the major form of
DC is X-linked. As well as a variety of noncutaneous abnormalities, the majority
(93%) of patients had bone marrow (BM) failure and this was the principal cause
(71%) of early mortality. In addition to BM hypoplasia, some patients also
developed myelodysplasia and acute myelod leukaemia. Pulmonary abnormalities
were present in 19% of patients. In affected females the phenotype was less
severe. Some female carriers of X-linked DC had clinical features. Carriers of
X-linked DC showed skewed X-chromosome inactivation patterns (XCIPs), suggesting
that cells expressing the normal DC allele have a growth/survival advantage over
cells that express the mutant allele. Linkage analysis in multiplex families
confirmed that the DKC1 gene, responsible for the X-linked form of DC, is
located within Xq28 and facilitated its positional cloning. The high incidence
of BM failure in association with a wide range of somatic abnormalities together
with the ubiquitous expression of DKC1 suggest that, as well as having a
critical role in normal haemopoiesis, this gene has a key role in normal cell
biology. Mutations in the DKC1 gene are responsible for causing X-linked recessive
dyskeratosis congenita (DKC) and a more severe allelic variant of the disease,
Hoyeraal-Hreidarsson syndrome. Both diseases are characterized by progressive
and fatal bone marrow failure. The nucleolar protein dyskerin is the
pseudouridine synthase component of the box H+ACA snoRNAs and also interacts
with the RNA component (human telomerase, hTR) of the telomerase complex.
Dyskerin is therefore thought to function in the processing of pre-rRNA and of
the hTR, strengthening the notion that the underlying mechanism of DKC is a
premature senescence of cells, especially of the rapidly dividing epithelial and
hemopoietic cells. To examine the functions of dyskerin in vivo, it will be
necessary to generate mouse models. As a first step, we here provide the genomic
structure of the mouse Dkc1 gene and expression analysis of the transcript.
Northern hybridizations revealed the tissue-specific expression of an
alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript.
RNA in situ hybridizations on day 10.5-18.5 postconception embryos showed a
ubiquitous expression of Dkc1 with a notably higher level of expression confined
to the epithelial tissues. In addition, higher level Dkc1 expression was
confined to embryonic neural tissues as well as to specific neurons in the
cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult
brain. In adult testis, elevated expression was limited to the Leydig cells. The
results indicate that some of the pertinent functions of dyskerin may be more
tissue-specific than previously thought and are not limited to rapidly dividing
cells. Dyskeratosis congenita (DC) is a multi-system disorder which in its classical
form is characterised by abnormalities of the skin, nails and mucous membranes.
In approximately 80% of cases, it is associated with bone marrow dysfunction. A
variety of other abnormalities (including bone, brain, cancer, dental, eye,
gastrointestinal, immunological and lung) have also been reported. Although
first described almost a century ago it is the last 10 years, following the
identification of the first DC gene (DKC1) in 1998, in which there has been
rapid progress in its understanding. Six genes have been identified, defects in
which cause different genetic subtypes (X-linked recessive, autosomal domit,
autosomal recessive) of DC. The products of these genes encode components that
are critical for telomere maintece; either because they are core constituents
of telomerase (dyskerin, TERC, TERT, NOP10 and NHP2) or are part of the
shelterin complex that protects the telomeric end (TIN2). These advances have
also highlighted the connection between the more "cryptic/atypical" forms of the
disease including aplastic anaemia and idiopathic pulmonary fibrosis. Equally,
studies on this disease have demonstrated the critical importance of telomeres
in human cells (including stem cells) and the severe consequences of their
dysfunction. In this context DC and related diseases can now be regarded as
disorders of "telomere and stem cell dysfunction". Dyskeratosis congenita (DC) is a multisystem inherited syndrome exhibiting
marked clinical and genetic heterogeneity. In its classic form, it is
characterized by mucocutaneous abnormalities, BM failure, and a predisposition
to cancer. BM failure is the principal cause of premature mortality. Studies
over the last 15 years have led to significant advances, with 8 DC genes (DKC1,
TERC, TERT, NOP10, NHP2, TIN2, C16orf57, and TCAB1) having been characterized.
Seven of these are important in telomere maintece either because they encode
components of the telomerase enzyme complex (DKC1, TERC, TERT, NOP10, NHP2, and
TCAB1) or the shelterin complex (TINF2). DC is therefore principally a disease
of defective telomere maintece and patients usually have very short
telomeres. The genetic advances have led to the unification of DC with several
other disorders, including the severe multisystem disorders Hoyeraal-Hreidarsson
and Revesz syndromes, as well as a subset of patients with aplastic anemia,
myelodysplasia, leukemia, and idiopathic pulmonary fibrosis. This wide spectrum
of diseases ranging from classic DC to aplastic anemia can be regarded as
disorders of defective telomere maintece-"the telomereopathies." These
advances have increased our understanding of normal hematopoiesis and
highlighted the important role of telomerase and telomeres in human biology.
They are also facilitating the diagnosis (especially when presentation is
atypical) and management of DC. |
Is CD99 encoded by MIC2 gene? | CD99 is a 32-kDa transmembrane glycoprotein that is encoded by the MIC2 gene | Monoclonal antibody (MAb) HBA71, which was raised against Ewing's sarcoma cells,
recognizes a cell-surface glycoprotein, p30/32MIC2, that is encoded by the MIC2
gene in the pseudoautosomal region of human chromosomes X and Y. This
immunohistochemical study evaluates the specificity and sensitivity of MAb HBA71
for tumor diagnosis. Frozen and paraffin-embedded tissues of more than 300
tumors of diverse histologic type, including more than 100 small round cell
tumors of childhood and adolescence, were tested with this MAb by the
avidin-biotin immunoperoxidase procedure. The authors found HBA71
immunoreactivity in 61 of 63 Ewing's sarcomas studied and 9 of 11 primitive
neuroectodermal tumors and peripheral neuroepitheliomas. HBA71-negative tumors
included neuroblastomas (0 of 24), melanomas (0 of 13), an
esthesioneuroblastoma, small cell osteosarcomas (0 of 2), a maligt
ectomesenchymoma, desmoplastic SRCT (0 of 5), and medulloblastomas (0 of 5).
Heterogeneous expression of HBA71 immunostaining was found in some embryonal
rhabdomyosarcomas (3 of 14) and astrocytomas (4 of 7), and in a few
neuroendocrine tumors (4 of 26), carcinomas (3 of 94), and lymphomas (6 of 30).
Because Ewing's sarcomas are consistently HBA71 positive, the authors searched
for antigen-positive normal cells that may represent precursors for these
tumors; however, no obvious candidate for the elusive cell of origin for Ewing's
sarcoma was identified in the normal fetal tissues tested. Their findings
indicate that HBA71 is a highly restricted cell-surface antigen of Ewing's
sarcomas and primitive neuroectodermal tumors, and immunohistochemistry
employing this antibody may be of value in the differential diagnosis of
selected small round cell tumors in childhood and adolescence. Monoclonal antibodies (mAbs) directed against E2, a 32-kDa transmembrane protein
encoded by the MIC2 gene located in the pseudoautosomal region, induce a
transbilayer movement of phosphatidylserine and, to a lesser extent,
phosphatidylethanolamine in human thymocytes and a Jurkat T lymphocytes. The
translocation of phosphatidylserine has been evidenced by using either
derivatization of anionic phospholipids with trinitrobenzenesulfonate (TNBS) or
cytofluorimetry after labeling of cells with antiphosphatidylserine antibodies.
The perturbation of membrane phospholipids induced by anti-E2 mAbs was further
evidenced by labeling the cells with merocyanine 540. The specificity of
anti-E2-induced perturbations of membrane asymmetry was tested by using a number
of mAbs able to activate T cells, including CD3 and CD2. The results strongly
suggest that anti-E2-induced changes in PtdSer are related to cell aggregation
since the same mAbs specifically induce the aggregation of both thymocytes and
Jurkat cells and since the E2 molecule has been previously implicated in the
adhesive properties of human T cells with erythrocytes. In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell
antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as
VAP21) which is efficiently incorporated into the rabies virion. By using these
MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids
from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was
assumed to encode a full-length sequence of VAP21 antigen: i) expression of the
cDNA in animal cells resulted in the production of a polypeptide recognized by
the two MAbs, and its electrophoretic mobility was the same as that of authentic
VAP21 antigen; and ii) immunization with the products from the cDNA-transformed
E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa
polypeptide in the virion. From the deduced amino acid sequence, it is suggested
that the VAP21 antigen has a molecular structure of type-I transmembrane protein
containing characteristic proline-rich and glycine-rich regions in its
ectodomain. Homology searches resulted in finding homologous sequences (totally
about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T
lymphocytes. These results suggest that the VAP21 antigen in the rabies virion
is a cellular CD99-related transmembrane protein. The Ewing family of tumors (EFT) is characterized by high MIC2/CD99 expression
and specific EWS/ETS gene rearrangements, resulting in different chimeric
transcripts. Further division into peripheral primitive neuroectodermal tumors
and Ewing's sarcoma is still debated and, in the absence of distinct
morphological parameters, has been based on the reactivity with neuroglial
markers (NgM). We investigated 44 EFT in terms of a possible correlation between
the type of EWS chimeric transcripts and reactivity with the following NgM:
polyclonal and monoclonal neuron-specific enolase (NSE), S-100, chromogranin A,
synaptophysin, Leu-7, glial fibrillary acid protein, and neurofilament. EWS/Fli1
fusion type 1 was detected in 30 of 44 and type 2 in 11 of 44 tumors. Three
tumors, presenting with an uncommon morphology, carried rare chimeric
transcripts. Our results indicate an association of lack of NgM staining with
type 1 EWS/Fli1 translocations, found in 16 of 18 tumors with no NgM expression
as detectable by any of the antibodies we applied. Using the monoclonal NSE
antibody, 21 of 26 tumors without NgM staining expressed type 1 EWS/FLI1chimeric
RNA, whereas in the groups with 1 or more and 2 or more NgM, only 9 of 17 and 1
of 5 tumors, respectively, carried type 1 EWS/Fli1 fusion transcripts. Despite
this association of increased NgM expression with a non-type 1 EWS/Fli1 gene
fusion, a strict correlation between the extent of NgM expression and certain
EWS fusion types was not evident. This fortifies the concept to consider EFT as
a spectrum of tumors and suggests the type of EWS fusion transcripts as one, but
not the only parameter influencing the extent of differentiation. CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative
polymorphism coregulated with the polymorphism of the XG blood group gene. As a
preliminary study of this phenomenon, human XG and CD99 recombit proteins
were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins
were expressed independently and at a similar level in single and double
transfectants. Immunoprecipitation and Western blot analysis, using the murine
monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd
(CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also
detected, as was a 26-kd species after neuraminidase treatment of
CD99-expressing cells. No evidence of association or complex formation between
XG and CD99 proteins could be proven, either on transfected RAG cells or on
human erythrocytes. These results were confirmed using somatic hybrids between
single transfectants. These findings suggest that the phenotypic relationship
between XG and CD99 is mostly regulated at the transcriptional level, but they
do not formally exclude some posttranscriptional effect. Studies on the tissue
specificity of XG expression showed that surface expression of the XG protein
could not be restored in somatic hybrids between B-lymphoblastoid cell lines
from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR
analysis of the transcripts revealed the existence of an XG mRNA in each cell
line, suggesting that the tissue-specific regulation of cell surface XG
expression occurs either at a quantitative transcriptional level or is a
posttranscriptional event. By Northern blot analysis, XG transcripts were
detected in erythroid tissues and several nonerythroid tissues. (Blood.
2000;95:1819-1826) Two cases of extraosseous Ewing's sarcoma/peripheral neuroectodermal tumor
arising in unusual, superficial sites are reported. One tumor involved the
vaginal wall of a 35-year-old woman, and the other neoplasm arose in the dermis
of the vulva in a 28-year-old woman. The tumors showed characteristic
microscopic features of Ewing's sarcoma/peripheral neuroectodermal tumor with
nodular monotonous proliferations of undifferentiated, small, round,
hyperchromatic cells with a low mitotic index. Rare rosette-like formations were
apparent only in the vulvar neoplasm. The tumors displayed intense
immunoreactivity in a membranous pattern for CD99, the cell surface glycoprotein
encoded by the MIC2 gene. Genetically, the tumors expressed the EWS/FLI-1
chimeric transcript, derived from the t(11;22)(q24;q12) chromosomal
translocation. Both patients had localized disease treated with wide local
excision; one received postoperative chemotherapy, and the other received
chemotherapy and radiotherapy. To date, 18 and 19 months after diagnosis,
neither patient has had clinical evidence of local recurrence or metastasis. To
our knowledge, these are the first reported cases of vaginal and vulvar Ewing's
sarcoma/peripheral neuroectodermal tumor, confirmed with molecular genetic
analysis, in the English literature. CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative
polymorphism co-regulated with the Xga blood group polymorphism. The
co-expression of X-linked MIC2 and XG genes is presumably controlled at the
transcriptional level by a single XGR locus in the pseudoautosomal region of
sexual chromosomes. This locus is composed of two alleles, XGR(low) and
XGR(high), which determine low or high CD99 levels (CD99-L, CD99-H) and the
Xg(a-)/ Xg(a+) status. To test this hypothesis, the phenotypic relationship
between Xga and CD99 antigens on human RBCs was investigated by quantitative
flow cytometry using NBL-1 (anti-Xga) and 12E7 (anti-CD99) monoclonal antibodies
and semi-quantitative estimate of membrane proteins and RNA by Western blot and
Northern blot, respectively. The antibody binding capacity of RBCs, which is an
estimation of the antigen density, was determined for 118 blood donors including
60 males and 58 females. Xg(a+) RBCs, which all belong to the group of CD99-H
expressors, carry 159+/-13 and 960+/-50 copies of Xga and CD99 molecules/cell,
respectively. Xg(a-) RBCs have no Xga antigen, but are subdivided into CD99-H
(all male) and CD99-L expressors carrying 747+/-28 and 200+/-22 CD99
copies/cell, respectively, with identical CD99 levels between CD99-L males and
females. However, among males, the CD99 expression was higher in Xg(a+) than in
Xg(a-)/CD99-H individuals (P<0.01). In addition, CD99-H expressors in Xg(a+)
males could be clearly subdivided into two categories, high and super high
expressors, which are presumably heterozygous and homozygous for the XGR(high)
allele, which fits the above hypothesis. This was not the case for Xg(a+)
females where CD99-H subcategories were not found. Quantitative differences were
confirmed by Western blot analysis of red cell membrane preparations from
individuals of different Xga and CD99 phenotypes and by Northern blot analysis
showing that the reticulocytes from CD99-L individuals expressed a reduced level
of MIC2 transcripts compared to CD99-H donors. These findings further support
the hypothesis of a single genetic control of CD99 and Xga expression by the XGR
locus. Although considered a specific marker for Ewing's sarcoma/peripheral
neuroectodermal tumour, the MIC2 gene product (CD99) has been immunolocalised in
a variety of human tumours. The present study evaluated immunohistochemically
the prevalence of CD99 expression in a series of 68 neuroendocrine tumours of
different gastrointestinal and pulmonary sites. We now report on membrane and/or
granular cytoplasmic immunoreactivity in 25% of these tumours, independent of
their anatomical sites. In lung neuroendocrine tumours, CD99 was preferentially
confined to typical carcinoids (P=0.009). A statistically significant
relationship was observed between the number of CD99 positive cells but not the
immunostaining patterns and the presence of local invasion and/or distant
metastases (P<0.001). Moreover, there was a tendency for CD99-reactive tumours
to show a reduced proliferative activity expressed by a Ki67 index of 2%
(P=0.119). The number of CD99 immunoreactive cells or patterns of
immunoreactivity did not correlate with the presence of associated clinical
syndrome or particular hormonal immunostaining. Although the molecular basis
underlying CD99 expression in neuroendocrine tumours is still poorly understood,
our data suggest that CD99 may be involved in cell-to-cell adhesion of
neuroendocrine tumour cells and in downregulation of their proliferative
activity. Human CD99, which is encoded by the mic2 gene, is a ubiquitous 32 kDa
transmembrane protein. Its major cellular functions are related to homotypic
cell adhesion, apoptosis, vesicular protein transport, and differentiation of
thymocytes or T cells. Recent reports have suggested that expression of a splice
variant of CD99 increases the invasiveness of human breast cancer cells. In
order to determine the structural basis of CD99 function, we have initiated
structural studies on the human CD99 Type I cytoplasmic domain (hCD99cytoI)
using circular dichroism and multi-dimensional NMR spectroscopy. The solution
structure of hCD99cytoI shows that it has a hairpin shape anchored by two
flexible loops. Consequently, hCD99cytoI does not have any regular secondary
structural element; however, the NMR and CD data indicate that it possesses an
intrinsic helical nature. The leukocyte surface molecule CD99 is an integral membrane glycoprotein encoded
by the E2/MIC2 gene. This molecule is broadly expressed on cells of the
hematopoietic system and displays two surface forms, a long 32 kDa form and a
short 28 kDa form. While the complete function of the CD99 molecule is unclear,
it has been reported to be involved in regulation of cell adhesion, migration,
and apoptosis. Thus, several CD99 monoclonal antibodies (MAbs) have been
generated for biochemical and functional studies of the CD99 molecule. In the
present study two CD99 MAbs, MT99/1 and MT99/2, were produced. The MAbs
recognized different epitopes of the CD99 molecule. MAb MT99/1, but not MT99/2,
was appropriate for biochemical characterization. Binding of MAb MT99/1 with its
epitope led to the induction of cell adhesion and apoptosis. The generated MAbs
can be used for future study of the function and mechanism of the CD99 molecule,
including its role in the immune system, and may have application in tumor
diagnosis and treatment. CD99 is a 32-kDa transmembrane glycoprotein that is encoded by the MIC2 gene.
Our study was carried out to examine the role of CD99 in tumor progression of
classical Hodgkin lymphoma (cHL). Here, we showed that lowly expressed CD99
protein in cHL cell lines and primary cHL cases correlates with the deficient
expression of the positive regulatory domain 1 (PRDM1/BLIMP1). In addition, cHL
cell lines showed high levels of miR-9 expression. We determined that the
upregulation of CD99 induced expression of transcription factor PRDM1, a master
regulator of plasma-cell differentiation, which is also a target for
miR-9-mediated downregulation. Indeed, inhibition of miR-9 also triggered
upregulation of PRDM1 expression. Furthermore, overexpression of CD99 resulted
in changed growth features and reorganization of actin cytoskeleton. As
upregulation of CD99 led to a decrease in cHL diagnosis marker CD30 and CD15 and
an increase in plasma-cell differentiation marker CD38 and the restoration of
B-cell makers PAX5, CD79α and CD19, we suggest that downregulated CD99 leads to
the prevention of plasma-cell differentiation in Hodgkin/Reed-Sternberg (H/RS)
cells. Furthermore, these data indicate that CD99 may control miR-9 expression,
which directly targets PRDM1. Altogether, these results reveal a
CD99-miR-9-PRDM1 molecule axis in lymphomagenesis of cHL and suggest that
upregulation of CD99 in H/RS cells induces terminal B-cell differentiation,
which may provide a novel therapeutic strategies for cHL. The XG blood group system is best known for its contributions to the fields of
genetics and chromosome mapping. This system comprises two antigens, Xg(a) and
CD99, that are not antithetical but that demonstrate a unique phenotypic
relationship. XG is located on the tip of the short arm of the X chromosome with
exons 1 to 3 present in the pseudoautosomal region of the X (and Y)
chromosome(s) and exons 4 to 10 located only on the X chromosome. Xg(a)
demonstrates a clear X-linked pattern of inheritance. MIC2, the gene encoding
the CD99 antigen, is found in the pseudoautosomal region of both the X and Y
chromosomes. Anti-Xg(a) is comparatively rare, and only two examples of
anti-CD99 have ever been identified. Alloanti-Xg(a) is considered clinically
insignificant; only one example of autoanti-Xg(a) has been reported, but it
resulted in severe hemolytic anemia. Insufficient data exist to determine the
clinical significance of anti-CD99. Linkage of XG to several X-borne genes
encoding inherited disorders has been demonstrated. CD99 is an adhesion
molecule, and high levels are associated with some types of cancer. CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple
cellular events including cell adhesion and migration, apoptosis, cell
differentiation and regulation of protein trafficking either in physiological or
pathological conditions. In osteosarcoma, CD99 is expressed at low levels and
functions as a tumour suppressor. The full-length protein (CD99wt) and the
short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have
been associated with distinct functional outcomes with respect to tumour
maligcy. In this study, we especially evaluated modulation of cell-cell
contacts, reorganisation of the actin cytoskeleton and modulation of signalling
pathways by comparing osteosarcoma cells characterised by different metastasis
capabilities and CD99 expression, to identify molecular mechanisms responsible
for metastasis. Our data indicate that forced expression of CD99wt induces
recruitment of N-cadherin and β-catenin to adherens junctions. In addition,
transfection of CD99wt inhibits the expression of several molecules crucial to
the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A,
Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as
ezrin, an ezrin/radixin/moesin family member that has been clearly associated
with tumour progression and metastatic spread in osteosarcoma. Functional
studies point to ROCK2 as a crucial intracellular mediator regulating
osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt
inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane
while N-cadherin and β-catenin translocate to the plasma membrane and function
as main molecular bridges for actin cytoskeleton. Taken together, we propose
that the re-expression of CD99wt, which is generally present in osteoblasts but
lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to
increase contact strength and reactivate stop-migration signals that counteract
the otherwise domit promigratory action of ezrin in osteosarcoma cells. We report a rare case of Ewing's sarcoma (ES)/primitive neuroectodermal tumor
(PNET) arising from the adrenal gland. A 17-year-old Japanese woman presented
with left upper abdominal pain and high fever. Computed tomography and magnetic
resoce imaging revealed a 15 × 10 cm tumor replacing the adrenal gland.
Preoperative diagnosis was an adrenocortical carcinoma. Resection of the tumor
was performed. We obtained the final diagnosis of ES/PNET by immunohistochemical
molecular study with positive staining for the MIC2 gene product (CD99) and a
Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangement. Local recurrence
was observed one month after the surgery. The patient was then treated with
systemic chemotherapy and localized radiotherapy. Ewing sarcoma is the third most common sarcoma in children and young adults. Its
characteristic chromosomal rearrangement results in a chimerical EWSR1-ETS
transcription factor. Secondary genetic alterations are very common. Membranous
expression of CD99 is seen in almost all tumors. We report 2 unusual cytogenetic
findings in a pediatric Ewing sarcoma, an insertion of the MIC2 gene encoding
CD99 from Xp to 10p and a submicroscopic deletion of the well-known tumor
supressor gene KLF6. The latter has not been described previously in pediatric
neoplasms. Molecular pathways in tumorigenesis and genetic complexity in cancer
are discussed. |
Name five programs for transcript quantification from RNASeq experiments | Popular programs for transcript quantification from RNASeq experiments include: Cufflinks, RSEM, Flux Capacitor, Mitie, Miso, Tigar, Montebello, Drut, Traph, Pome, IsoformEx, Neuma, | We propose a novel, efficient and intuitive approach of estimating mRNA
abundances from the whole transcriptome shotgun sequencing (RNA-Seq) data. Our
method, NEUMA (Normalization by Expected Uniquely Mappable Area), is based on
effective length normalization using uniquely mappable areas of gene and mRNA
isoform models. Using the known transcriptome sequence model such as RefSeq,
NEUMA pre-computes the numbers of all possible gene-wise and isoform-wise
informative reads: the former being sequences mapped to all mRNA isoforms of a
single gene exclusively and the latter uniquely mapped to a single mRNA isoform.
The results are used to estimate the effective length of genes and transcripts,
taking experimental distributions of fragment size into consideration.
Quantitative RT-PCR based on 27 randomly selected genes in two human cell lines
and computer simulation experiments demonstrated superior accuracy of NEUMA over
other recently developed methods. NEUMA covers a large proportion of genes and
mRNA isoforms and offers a measure of consistency ('consistency coefficient')
for each gene between an independently measured gene-wise level and the sum of
the isoform levels. NEUMA is applicable to both paired-end and single-end
RNA-Seq data. We propose that NEUMA could make a standard method in quantifying
gene transcript levels from RNA-Seq data. BACKGROUND: Comprehensive annotation and quantification of transcriptomes are
outstanding problems in functional genomics. While high throughput mRNA
sequencing (RNA-Seq) has emerged as a powerful tool for addressing these
problems, its success is dependent upon the availability and quality of
reference genome sequences, thus limiting the organisms to which it can be
applied.
RESULTS: Here, we describe Rnnotator, an automated software pipeline that
generates transcript models by de novo assembly of RNA-Seq data without the need
for a reference genome. We have applied the Rnnotator assembly pipeline to two
yeast transcriptomes and compared the results to the reference gene catalogs of
these organisms. The contigs produced by Rnnotator are highly accurate (95%) and
reconstruct full-length genes for the majority of the existing gene models
(54.3%). Furthermore, our analyses revealed many novel transcribed regions that
are absent from well annotated genomes, suggesting Rnnotator serves as a
complementary approach to analysis based on a reference genome for comprehensive
transcriptomics.
CONCLUSIONS: These results demonstrate that the Rnnotator pipeline is able to
reconstruct full-length transcripts in the absence of a complete reference
genome. BACKGROUND: mRNA-Seq technology has revolutionized the field of transcriptomics
for identification and quantification of gene transcripts not only at gene level
but also at isoform level. Estimating the expression levels of transcript
isoforms from mRNA-Seq data is a challenging problem due to the presence of
constitutive exons.
RESULTS: We propose a novel algorithm (IsoformEx) that employs weighted
non-negative least squares estimation method to estimate the expression levels
of transcript isoforms. Validations based on in silico simulation of mRNA-Seq
and qRT-PCR experiments with real mRNA-Seq data showed that IsoformEx could
accurately estimate transcript expression levels. In comparisons with published
methods, the transcript expression levels estimated by IsoformEx showed higher
correlation with known transcript expression levels from simulated mRNA-Seq
data, and higher agreement with qRT-PCR measurements of specific transcripts for
real mRNA-Seq data.
CONCLUSIONS: IsoformEx is a fast and accurate algorithm to estimate transcript
expression levels and gene expression levels, which takes into account short
exons and alternative exons with a weighting scheme. The software is available
at http://bioinformatics.wistar.upenn.edu/isoformex. BACKGROUND: RNA-Seq is revolutionizing the way transcript abundances are
measured. A key challenge in transcript quantification from RNA-Seq data is the
handling of reads that map to multiple genes or isoforms. This issue is
particularly important for quantification with de novo transcriptome assemblies
in the absence of sequenced genomes, as it is difficult to determine which
transcripts are isoforms of the same gene. A second significant issue is the
design of RNA-Seq experiments, in terms of the number of reads, read length, and
whether reads come from one or both ends of cDNA fragments.
RESULTS: We present RSEM, an user-friendly software package for quantifying gene
and isoform abundances from single-end or paired-end RNA-Seq data. RSEM outputs
abundance estimates, 95% credibility intervals, and visualization files and can
also simulate RNA-Seq data. In contrast to other existing tools, the software
does not require a reference genome. Thus, in combination with a de novo
transcriptome assembler, RSEM enables accurate transcript quantification for
species without sequenced genomes. On simulated and real data sets, RSEM has
superior or comparable performance to quantification methods that rely on a
reference genome. Taking advantage of RSEM's ability to effectively use
ambiguously-mapping reads, we show that accurate gene-level abundance estimates
are best obtained with large numbers of short single-end reads. On the other
hand, estimates of the relative frequencies of isoforms within single genes may
be improved through the use of paired-end reads, depending on the number of
possible splice forms for each gene.
CONCLUSIONS: RSEM is an accurate and user-friendly software tool for quantifying
transcript abundances from RNA-Seq data. As it does not rely on the existence of
a reference genome, it is particularly useful for quantification with de novo
transcriptome assemblies. In addition, RSEM has enabled valuable guidance for
cost-efficient design of quantification experiments with RNA-Seq, which is
currently relatively expensive. MOTIVATION: Alternative splicing (AS) is a pre-mRNA maturation process leading
to the expression of multiple mRNA variants from the same primary transcript.
More than 90% of human genes are expressed via AS. Therefore, quantifying the
inclusion level of every exon is crucial for generating accurate transcriptomic
maps and studying the regulation of AS.
RESULTS: Here we introduce SpliceTrap, a method to quantify exon inclusion
levels using paired-end RNA-seq data. Unlike other tools, which focus on
full-length transcript isoforms, SpliceTrap approaches the expression-level
estimation of each exon as an independent Bayesian inference problem. In
addition, SpliceTrap can identify major classes of alternative splicing events
under a single cellular condition, without requiring a background set of reads
to estimate relative splicing changes. We tested SpliceTrap both by simulation
and real data analysis, and compared it to state-of-the-art tools for transcript
quantification. SpliceTrap demonstrated improved accuracy, robustness and
reliability in quantifying exon-inclusion ratios.
CONCLUSIONS: SpliceTrap is a useful tool to study alternative splicing
regulation, especially for accurate quantification of local exon-inclusion
ratios from RNA-seq data.
AVAILABILITY AND IMPLEMENTATION: SpliceTrap can be implemented online through
the CSH Galaxy server http://cancan.cshl.edu/splicetrap and is also available
for download and installation at http://rulai.cshl.edu/splicetrap/.
CONTACT: [email protected].
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. MOTIVATION: RNA sequencing (RNA-Seq) is a powerful new technology for mapping
and quantifying transcriptomes using ultra high-throughput next-generation
sequencing technologies. Using deep sequencing, gene expression levels of all
transcripts including novel ones can be quantified digitally. Although extremely
promising, the massive amounts of data generated by RNA-Seq, substantial biases
and uncertainty in short read alignment pose challenges for data analysis. In
particular, large base-specific variation and between-base dependence make
simple approaches, such as those that use averaging to normalize RNA-Seq data
and quantify gene expressions, ineffective.
RESULTS: In this study, we propose a Poisson mixed-effects (POME) model to
characterize base-level read coverage within each transcript. The underlying
expression level is included as a key parameter in this model. Since the
proposed model is capable of incorporating base-specific variation as well as
between-base dependence that affect read coverage profile throughout the
transcript, it can lead to improved quantification of the true underlying
expression level.
AVAILABILITY AND IMPLEMENTATION: POME can be freely downloaded at
http://www.stat.purdue.edu/~yuzhu/pome.html.
CONTACT: [email protected]; [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. MOTIVATION: Quantification of sequence abundance in RNA-Seq experiments is often
conflated by protocol-specific sequence bias. The exact sources of the bias are
unknown, but may be influenced by polymerase chain reaction amplification, or
differing primer affinities and mixtures, for example. The result is decreased
accuracy in many applications, such as de novo gene annotation and transcript
quantification.
RESULTS: We present a new method to measure and correct for these influences
using a simple graphical model. Our model does not rely on existing gene
annotations, and model selection is performed automatically making it applicable
with few assumptions. We evaluate our method on several datasets, and by
multiple criteria, demonstrating that it effectively decreases bias and
increases uniformity. Additionally, we provide theoretical and empirical results
showing that the method is unlikely to have any effect on unbiased data,
suggesting it can be applied with little risk of spurious adjustment.
AVAILABILITY: The method is implemented in the seqbias R/Bioconductor package,
available freely under the LGPL license from http://bioconductor.org
CONTACT: [email protected]
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. Accurately mapping RNA-Seq reads to the reference genome is a critical step for
performing downstream analysis such as transcript assembly, isoform detection
and quantification. Many tools have been developed; however, given the huge size
of the next generation sequencing datasets and the complexity of the
transcriptome, RNA-Seq read mapping remains a challenge with the ever-increasing
amount of data. We develop Omicsoft sequence aligner (OSA), a fast and accurate
alignment tool for RNA-Seq data. Benchmarked with existing methods, OSA improves
mapping speed 4-10-fold with better sensitivity and less false positives.
AVAILABILITY: OSA can be downloaded from http://omicsoft.com/osa. It is free to
academic users. OSA has been tested extensively on Linux, Mac OS X and Windows
platforms. The RNA transcriptome varies in response to cellular differentiation as well as
environmental factors, and can be characterized by the diversity and abundance
of transcript isoforms. Differential transcription analysis, the detection of
differences between the transcriptomes of different cells, may improve
understanding of cell differentiation and development and enable the
identification of biomarkers that classify disease types. The availability of
high-throughput short-read RNA sequencing technologies provides in-depth
sampling of the transcriptome, making it possible to accurately detect the
differences between transcriptomes. In this article, we present a new method for
the detection and visualization of differential transcription. Our approach does
not depend on transcript or gene annotations. It also circumvents the need for
full transcript inference and quantification, which is a challenging problem
because of short read lengths, as well as various sampling biases. Instead, our
method takes a divide-and-conquer approach to localize the difference between
transcriptomes in the form of alternative splicing modules (ASMs), where
transcript isoforms diverge. Our approach starts with the identification of ASMs
from the splice graph, constructed directly from the exons and introns predicted
from RNA-seq read alignments. The abundance of alternative splicing isoforms
residing in each ASM is estimated for each sample and is compared across sample
groups. A non-parametric statistical test is applied to each ASM to detect
significant differential transcription with a controlled false discovery rate.
The sensitivity and specificity of the method have been assessed using simulated
data sets and compared with other state-of-the-art approaches. Experimental
validation using qRT-PCR confirmed a selected set of genes that are
differentially expressed in a lung differentiation study and a breast cancer
data set, demonstrating the utility of the approach applied on experimental
biological data sets. The software of DiffSplice is available at
http://www.netlab.uky.edu/p/bioinfo/DiffSplice. The paper addresses the problem of how to use RNA-Seq data for transcriptome
reconstruction and quantification, as well as novel transcript discovery in
partially annotated genomes. We present a novel annotation-guided general
framework for transcriptome discovery, reconstruction and quantification in
partially annotated genomes and compare it with existing annotation-guided and
genome-guided transcriptome assembly methods. Our method, referred as Discovery
and Reconstruction of Unotated Transcripts (DRUT), can be used to enhance
existing transcriptome assemblers, such as Cufflinks, as well as to accurately
estimate the transcript frequencies. Empirical analysis on synthetic datasets
confirms that Cufflinks enhanced by DRUT has superior quality of reconstruction
and frequency estimation of transcripts. As next generation sequencing technologies are getting more efficient and less
expensive, RNA-Seq is becoming a widely used technique for transcriptome
studies. Computational analysis of RNA-Seq data often starts with the mapping of
millions of short reads back to the genome or transcriptome, a process in which
some reads are found to map equally well to multiple genomic locations
(multimapping reads). We have developed the Minimum Unique Length Tool (MULTo),
a framework for efficient and comprehensive representation of mappability
information, through identification of the shortest possible length required for
each genomic coordinate to become unique in the genome and transcriptome. Using
the minimum unique length information, we have compared different uniqueness
compensation approaches for transcript expression level quantification and
demonstrate that the best compensation is achieved by discarding multimapping
reads and correctly adjusting gene model lengths. We have also explored
uniqueness within specific regions of the mouse genome and enhancer mapping
experiments. Finally, by making MULTo available to the community we hope to
facilitate the use of uniqueness compensation in RNA-Seq analysis and to
eliminate the need to make additional mappability files. The recent development of various deep sequencing techniques has led to the most
powerful transcript profiling method available to date, RNA sequencing or
RNA-Seq. Besides the identification of new genes and new splice variants of
known genes, RNA-Seq allows to compare the whole transcriptome of any organism
under two or more experimental conditions, such as before and after jasmonate
treatment. However, the vast amounts of data generated during RNA-Seq
experiments require complex computational methods for read mapping and
expression quantification. Here, we describe a detailed protocol for the
analysis of deep sequencing data, starting from the raw RNA-Seq reads. First, a
quality check is performed on the raw reads to assess the quality of the
sequencing. Subsequently, adapters and low-quality sequences are trimmed off the
raw reads. The resulting processed reads are mapped to the reference genome, and
the mapped reads are counted to generate expression data for the annotated genes
for each sample. This method can be used for the analysis of RNA-Seq data of any
organism for which a reference genome is available. BACKGROUND: Through transcription and alternative splicing, a gene can be
transcribed into different RNA sequences (isoforms), depending on the
individual, on the tissue the cell is in, or in response to some stimuli. Recent
RNA-Seq technology allows for new high-throughput ways for isoform
identification and quantification based on short reads, and various methods have
been put forward for this non-trivial problem.
RESULTS: In this paper we propose a novel radically different method based on
minimum-cost network flows. This has a two-fold advantage: on the one hand, it
translates the problem as an established one in the field of network flows,
which can be solved in polynomial time, with different existing solvers; on the
other hand, it is general enough to encompass many of the previous proposals
under the least sum of squares model. Our method works as follows: in order to
find the transcripts which best explain, under a given fitness model, a splicing
graph resulting from an RNA-Seq experiment, we find a min-cost flow in an offset
flow network, under an equivalent cost model. Under very weak assumptions on the
fitness model, the optimal flow can be computed in polynomial time.
Parsimoniously splitting the flow back into few path transcripts can be done
with any of the heuristics and approximations available from the theory of
network flows. In the present implementation, we choose the simple strategy of
repeatedly removing the heaviest path.
CONCLUSIONS: We proposed a new very general method based on network flows for a
multiassembly problem arising from isoform identification and quantification
with RNA-Seq. Experimental results on prediction accuracy show that our method
is very competitive with popular tools such as Cufflinks and IsoLasso. Our tool,
called Traph (Transcrips in gRAPHs), is available at:
http://www.cs.helsinki.fi/gsa/traph/. RNA sequencing is a recent technology which has seen an explosion of methods
addressing all levels of analysis, from read mapping to transcript assembly to
differential expression modeling. In particular the discovery of isoforms at the
transcript assembly stage is a complex problem and current approaches suffer
from various limitations. For instance, many approaches use graphs to construct
a minimal set of isoforms which covers the observed reads, then perform a
separate algorithm to quantify the isoforms, which can result in a loss of
power. Current methods also use ad-hoc solutions to deal with the vast number of
possible isoforms which can be constructed from a given set of reads. Finally,
while the need of taking into account features such as read pairing and sampling
rate of reads has been acknowledged, most existing methods do not seamlessly
integrate these features as part of the model. We present Montebello, an
integrated statistical approach which performs simultaneous isoform discovery
and quantification by using a Monte Carlo simulation to find the most likely
isoform composition leading to a set of observed reads. We compare Montebello to
Cufflinks, a popular isoform discovery approach, on a simulated data set and on
46.3 million brain reads from an Illumina tissue panel. On this data set
Montebello appears to offer a modest improvement over Cufflinks when considering
discovery and parsimony metrics. In addition Montebello mitigates specific
difficulties inherent in the Cufflinks approach. Finally, Montebello can be
fine-tuned depending on the type of solution desired. MOTIVATION: RNA-Seq technology is promising to uncover many novel alternative
splicing events, gene fusions and other variations in RNA transcripts. For an
accurate detection and quantification of transcripts, it is important to resolve
the mapping ambiguity for those RNA-Seq reads that can be mapped to multiple
loci: >17% of the reads from mouse RNA-Seq data and 50% of the reads from some
plant RNA-Seq data have multiple mapping loci. In this study, we show how to
resolve the mapping ambiguity in the presence of novel transcriptomic events
such as exon skipping and novel indels towards accurate downstream analysis. We
introduce ORMAN ( O ptimal R esolution of M ultimapping A mbiguity of R N A-Seq
Reads), which aims to compute the minimum number of potential transcript
products for each gene and to assign each multimapping read to one of these
transcripts based on the estimated distribution of the region covering the read.
ORMAN achieves this objective through a combinatorial optimization formulation,
which is solved through well-known approximation algorithms, integer linear
programs and heuristics.
RESULTS: On a simulated RNA-Seq dataset including a random subset of transcripts
from the UCSC database, the performance of several state-of-the-art methods for
identifying and quantifying novel transcripts, such as Cufflinks, IsoLasso and
CLIIQ, is significantly improved through the use of ORMAN. Furthermore, in an
experiment using real RNA-Seq reads, we show that ORMAN is able to resolve
multimapping to produce coverage values that are similar to the original
distribution, even in genes with highly non-uniform coverage.
AVAILABILITY: ORMAN is available at http://orman.sf.net |
What are the main characteristics/symptoms of the "Brugada" syndrome | In 1992, Brugada and Brugada first described a new entity, which became known as Brugada syndrome, that is associated with a high risk of ventricular arrhythmias and sudden cardiac death in patients without structural heart disease. This syndrome is characterized by a distinct electrocardiographic phenotype, type 1 Brugada pattern, consisting of a coved ST-segment elevation (≥0.2 mV) followed by a negative T wave in more than one right precordial lead. The typical Brugada electrocardiogram (ECG) phenotype is often concealed in affected population. Brugada syndrome is a genetically determined familial disease with autosomal dominant transmission and variable penetrance, conferring a predisposition to sudden cardiac death due to ventricular arrhythmias. Brugada syndrome (BrS)is considered to be a primary inherited channelopathy often involving the inward sodium current and the diagnosis has traditionally required the exclusion of overt structural heart disease. Brugada syndrome (BrS) is an inherited cardiac disease characterized by ST segment elevation in V1-V3 ECG leads. Mutations SCN5A gene encoding for the cardiac voltage-gated Na(+) channel are found in some BrS patients, but also in family members with isolated conduction disturbances. | In 1992 we described a new syndrome consisting of syncopal episodes and/or
sudden death in patients with a structurally normal heart and a characteristic
electrocardiogram displaying a pattern resembling right bundle branch block with
an ST segment elevation in leads V1 to V3. In 1998 it was described that the
disease is genetically determined with an autosomal domit pattern of
transmission. Three different mutations have been identified. All three
mutations affect the structure and the function of the sodium channel SCN5A. Two
mutations result in total loss of function of the sodium channel. The other
mutation results in acceleration of the recovery of the sodium channel from
inactivation. The disease causes 4 to 10 sudden deaths per 10,000 inhabitants
per year in areas like Thailand and Laos. Up to 50% of the yearly sudden deaths
in patients with a normal heart might be caused by this syndrome. The diagnosis
is easily made by means of the electrocardiogram (ECG). The presence of
concealed and intermittent forms, however, makes the diagnosis difficult in some
patients. The ECG can be modulated by changes in autonomic balance and the
administration of antiarrhythmic drugs. Beta-adrenergic stimulation normalises
the ECG, while i.v. ajmaline, flecainide or procainamide accentuate the ST
segment elevation and are capable of unmasking concealed and intermittent forms
of the disease. The prognosis is poor for patients who do not receive an
implantable cardioverter-defibrillator. Antiarrhythmic drugs like amiodarone or
beta-blockers do not prevent sudden death in symptomatic or asymptomatic
individuals. Brugada's syndrome is one of the main causes of sudden death in young adults
without a structural heart disease. This is an electrical cardiac illness
secondary to a mutation of SCN5A gene of chromosome 3 that has a domit
autosomic transmission pattern. This mutation implies the dysfunction of the
sodium channel that increases the Ito, loosing the dome of the epicardiac action
potential phase two. An "all or none" repolarization pattern ensues and gives
rise to a phase two reentry. This kind of reentry is responsible for the
initiation and perpetuation of maligt ventricular arrhythmias among these
patients. The clinical characteristics of the syndrome are the right bundle
branch block, ST segment elevation from V1 to V3 leads and sudden death or
syncope. In some patients, a pharmacological test must be done with ajmaline or
procainamide to unmask the electrocardiographic changes. At present, the only
effective treatment is the implantable cardioverter defibrillator (ICD). This
device has the capability to reduce mortality from 40% annually to 0% at ten
years. Pharmacological treatment is not useful. Brugada syndrome is a major cause of sudden death in young adults. Fever has
been described to induce a Brugada-type electrocardiogram in asymptomatic
patients with a negative family history, to disclose Brugada syndrome and to
increase the risk of death and induce T wave alters in patients with
diagnosed Brugada syndrome. Risk stratification is challenging and demands a
careful evaluation. Here we present 2 case reports and review the literature. Brugada syndrome (BrS), one of the most frequently diagnosed inherited
arrhythmogenic syndromes, is responsible for more than 4% of all sudden deaths
and at least 20% of sudden deaths in patients with structurally normal hearts.
The sudden death is often the first symptom of BrS and appears most often
already during the fourth decade of life of BrS patients. Implantation of
cardioverter--defibrillator was proved to be the only effective treatment, i.e.
prevention of the sudden death, in BrS. Thus, it is uniquely determined to be
used in case of symptomatic BrS patients. On the contrary, the individual risk
of life-threatening arrhythmias has to be thoroughly considered in case of
asymptomatic BrS patients due to substantial side effects of implantation of
cardioverter-defibrillator. This review first provides a summary of factors
recommended for the risk stratification in BrS patients in 2005 including their
support or rejection in the following studies. Subsequently, we focused on the
most important risk factors newly suggested after 2005. AIMS: Brugada syndrome (BrS) is an inherited cardiac disease characterized by ST
segment elevation in V1-V3 ECG leads. Mutations SCN5A gene encoding for the
cardiac voltage-gated Na(+) channel are found in some BrS patients, but also in
family members with isolated conduction disturbances. However, some patients
show coved ST elevation in the inferior or lateral leads whose association with
SCN5A and familial conduction disturbances are poorly known.
METHODS AND RESULTS: Two novel SCN5A mutations, D1430N and Q1476X, were
identified in two unrelated families comprising patients with Brugada-like ST
elevation located in the inferior leads or isolated conduction disturbances.
Wild-type (WT) and D1430N mutant channels were expressed in tsA201 cells. Patch
clamp electrophysiological experiments revealed total absence of Na(+) current
resulting from Nav1.5 mutant when compared to WT channels. Treatments known to
restore trafficking defect (incubation at low temperature, with mexiletine or
lidocaine) did not restore Na(+) current supporting that Nav1.5 mutation is not
a defective trafficking mutation. Furthermore, immunocytolabelling indicates the
membrane localisation of both WT and mutant channels confirming what we observed
in our patch clamp experiments. This suggests that the mutation may induce a
complete block of Na(+) permeation. The nonsense mutation Q1476X was leading to
a premature stop codon and was not expressed.
CONCLUSION: Brugada-like ST elevation in the inferior ECG leads or isolated
conduction disturbances were found in two unrelated families and associated with
two novel SCN5A mutations. The missense and nonsense mutations are both
resulting in a complete loss of ventricular Na(+) current explaining the
phenotypes. AIMS: Brugada syndrome is characterized by typical ECG features, ventricular
arrhythmias and sudden cardiac death (SCD), more frequent during nighttime.
Autonomic cardiovascular control has been implicated in triggering the
ventricular arrhythmias. Sleep-disordered breathing (SDB) elicits marked
autonomic changes during sleep and is also associated with an increased risk of
nighttime SCD. Brugada patients may have a higher likelihood of SDB compared to
controls. However, no data are available on cardiac autonomic control in Brugada
patients, particularly with regard to the comorbidity of SDB.
METHODS: We evaluated autonomic cardiovascular control in Brugada patients with
SDB (BRU-SDB, n=9), without SDB (BRU, n=9), in controls (CON, n=8) and in
non-Brugada patients with SDB (n=6), during wakefulness and sleep (N2, N3 and
REM). Linear spectral and entropy-derived measures of heart rate variability
(HRV) were performed during apnea-free stable breathing epochs.
RESULTS: Total HRV was attenuated in BRU-SDB compared to CON and BRU. During N2
and REM, in BRU-SDB patients sympathetic modulation decreased compared to BRU
and CON, while during REM, they showed an increased parasympathetic modulation,
compared to the other two groups. BRU-SDB and SDB were similar in terms of
spectral components. Entropy-derived indices showed preserved dynamic changes in
Brugada patients compared to controls through the different sleep stages.
CONCLUSION: Brugada syndrome per se does not appear associated with an altered
autonomic cardiovascular control during wakefulness and sleep. The comorbidity
with SDB may contribute to disrupted autonomic cardiovascular regulation during
sleep, possibly predisposing to the increased likelihood of sleep-related
ventricular tachyarrhythmias and SCD. Brugada syndrome is an inherited arrhythmia syndrome predisposing to sudden
cardiac death. Six years after its initial description as a clinical entity, the
first mutations in SCN5A encoding the cardiac sodium channel Nav1.5 were
reported. Over 300 mutations in SCN5A have since been described in addition to
mutations in genes encoding Nav1.5 auxiliary units, potassium and calcium
channels. This review summarizes the current knowledge on the genetics of
Brugada syndrome, focusing on SCN5A, and discusses its use as a biomarker for
diagnosis, prognosis and treatment. Our understanding of Brugada syndrome (BrS) has evolved since the syndrome was
first described in 1992. BrS is considered to be a primary inherited
channelopathy often involving the inward sodium current and the diagnosis has
traditionally required the exclusion of overt structural heart disease. In view
of recently published observations about BrS, we propose that the term BrS may
actually encompass a heterogeneous group of disorders with a variety of genetic
and clinical phenotypes. This disease has classically been described as a
primary electrical disorder involving the sodium channel leading to the
characteristic electrocardiogram (ECG) changes of BrS. We challenge the current
understanding and propose that patients with structurally normal hearts, family
history of sudden cardiac death, with associated genetic abnormalities only
account for a subset of patients with the "Brugada pattern" ECG. There may also
be some patients with a diagnosis of BrS who may also have features which
overlap with arrhythmogenic right ventricular cardiomyopathy. In these patients
there may be an underlying structural abnormality. In this context, it is
possible that catheter ablation may abolish the "Brugada pattern" ECG changes as
well as abolishing the risk of life threatening arrhythmias in these patients.
Given the recent developments in the field, we propose a novel comprehensive
multimodality model for risk stratification and assessment of patients with BrS.
Identification of variations of diseases may facilitate more specific risk
stratification models and management paradigms in patients with Brugada ECG
pattern. Brugada syndrome is a genetically determined familial disease with autosomal
domit transmission and variable penetrance, conferring a predisposition to
sudden cardiac death due to ventricular arrhythmias. The syndrome is
characterized by a typical electrocardiographic pattern in the right precordial
leads. This article will focus on the new electrocardiographic features recently
agreed on by expert consensus helping to identify this infequent
electrocardiographic pattern. Brugada syndrome is a rare cardiac arrhythmia characterized by
electrocardiographic right bundle branch block and persistent ST-segment
elevation in the right precordial leads. It is associated with ventricular
fibrillation and a high risk for sudden cardiac death, predomitly in younger
males with structurally normal hearts. Patients can remain asymptomatic, and
electrocardiographic patterns can occur both spontaneously or after
pharmacological induction. So far, several pathogenic genes have been identified
as associated with the disease, but SCN5A is the most prevalent one. Two
consensus reports to define the diagnostic criteria, risk stratification, and
management of patients have been published in the last few years. This brief
review focuses on the recent clinical diagnosis, genetic basis, and advances in
pharmacological treatment of Brugada syndrome. Brugada syndrome predisposes individuals to ventricular arrhythmias and sudden
cardiac death, in the absence of structural heart disease. The typical Brugada
electrocardiogram (ECG) phenotype is often concealed in affected population, and
the existing genetic testing is capable of detecting just about 20% of cases.
Therefore, the diagnosis largely requires various pharmacological provocative
agents like class I antiarrhythmic drugs to unmask the unique features of
Brugada ECG phenotype. We report an unusual case of "unmasking" of Brugada ECG
pattern with ventricular tachycardia brought out by amiodarone infusion. In 1992, Brugada and Brugada first described a new entity, which became known as
Brugada syndrome, that is associated with a high risk of ventricular arrhythmias
and sudden cardiac death in patients without structural heart disease. This
syndrome is characterized by a distinct electrocardiographic phenotype, type 1
Brugada pattern, consisting of a coved ST-segment elevation (≥0.2 mV) followed
by a negative T wave in more than one right precordial lead. This pattern is
dynamic, and can be spontaneous or concealed, but is unmasked under certain
circumstances, like febrile states. The authors report a case in which the
diagnosis of Brugada syndrome was made in the course of etiologic investigation
of recurrent syncope in a febrile state. |
What is the main component of the Lewy bodies? | Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of Lewy bodies (LB) in a proportion of the remaining neurones. Alpha-synuclein has been identified as the main component of the Lewy bodies. | alpha-Synuclein and ubiquitin are two Lewy body protein components that may play
antagonistic roles in the pathogenesis of Lewy bodies. We examined the
relationship between alpha-synuclein, ubiquitin, and lipids in Lewy bodies of
fixed brain sections or isolated from cortical tissues of dementia with Lewy
bodies. Lewy bodies exhibited a range of labeling patterns for alpha-synuclein
and ubiquitin, from a homogeneous pattern in which alpha-synuclein and ubiquitin
were evenly distributed and overlapped across the inclusion body to a concentric
pattern in which alpha-synuclein and ubiquitin were partially segregated, with
alpha-synuclein labeling concentrated in the peripheral domain and ubiquitin in
the central domain of the Lewy body. Lipids represented a significant component
in both homogeneous and concentric Lewy bodies. These results suggest that Lewy
bodies are heterogeneous in their subregional composition. The segregation of
alpha-synuclein to Lewy body peripheral domain is consistent with the hypothesis
that alpha-synuclein is continually deposited onto Lewy bodies. The term alpha-synucleinopathy is used to name a group of disorders having in
common the abnormal deposition of alpha-synuclein in the cytoplasm of neurons or
glial cells, as well as in extracellular deposits of amyloid. In Parkinson's
disease and Lewy body dementia, alpha-synuclein is the main component of Lewy
bodies and dystrophic neurites; alpha-synuclein also accumulates in the
cytoplasm of glial cells. In multiple system atrophy, alpha-synuclein conforms
the cytoplasmic oligodendroglial inclusions and the neuronal inclusions which
are the hallmark of this disease. Finally, the amyloidogenic fragment 61-95
amino acids of alpha-synuclein is the non-Abeta component of senile plaque
amyloid in Alzheimer disease. Accumulations of alpha-synuclein in all these
disorders have in common a fibrilar configuration, but they differ in the
binding of alpha-synuclein to distinct proteins with the exception of ubiquitin
whose binding to alpha-synuclein is common to all alpha-synuclein inclusions.
The mechanisms leading to alpha-synuclein fragmentation and aggegation into
extracellular amyloid are not known, although alpha-synuclein fragment and
betaA4 aggregates are the result of abnormal cleavage of large precursors. On
the other hand, several studies have shown that alpha-synuclein may adopt a
fibrilar conformation and give rise to insoluble forms and high molecular weight
aggregates in vitro. Similar complexes have also been observed in
alpha-synucleinopathies. Although studies in vitro and in vivo have shown toxic
effects of alpha-synuclein, the consequence of alpha-synuclein deposition on
cell survival in alpha-synucleinopathies is not known. Amyloid precursor protein (APP) is involved in the accumulation of
alpha-synuclein, the main component of Lewy bodies. It is currently unknown,
however, whether any of the APP isoforms is instrumental in alpha-synuclein
deposition in dementia with Lewy bodies (DLB). Using real-time RT-PCR, we have
studied relative mRNA expression levels of APP isoforms in frozen postmortem
frontal cortices of DLB patients, Alzheimer disease (AD) patients, and control
subjects. Of the three main APP isoforms, the two with a Kunitz protease
inhibitory (KPI) motif (APP770 and APP751) were found to be specifically
overexpressed in the frontal cortices of DLB patients when compared with
controls and AD patients. These findings suggest a specific role of APP isoforms
containing Kunitz protease inhibitor in DLB pathogenesis. Dementia with Lewy bodies (DLB) is characterized by the widespread presence of
Lewy bodies (LBs) in the brain. alpha-Synuclein, the main component of LBs, is
expressed as two main isoforms (112 and 140), but little is known about their
differential expression in the brain. We compared alpha-synuclein 112 and
alpha-synuclein 140 expression levels in the prefrontal cortices of six DLB
patients, eight Alzheimer disease (AD) patients, and six control subjects.
Relative alpha-synuclein 112 and alpha-synuclein 140 expression levels were
determined by real-time polymerase chain reaction with competimer technology
using a LightCycler System. Whereas total alpha-synuclein levels were just
marginally elevated in DLB in comparison with the other groups, alpha-synuclein
112 was seen to be markedly increased in DLB compared with AD cases and
controls. In contrast, alpha-synuclein 140 levels were significantly diminished
in both neurodegenerative disorders in comparison with controls. These results
show differential overexpression of alpha-synuclein 112 in DLB, a finding that
could be of importance in DLB pathogenesis. Alpha-synuclein, a main component of Lewy bodies in synucleinopathies and senile
plaques in Alzheimer disease, is centrally involved in neurodegeneration. Three
different isoforms (alpha-synuclein 112, 126, and 140) resulting from
alternative splicing have been described so far. The present study explores
alpha-synuclein 126 mRNA expression levels in the prefrontal cortex of six
patients with dementia with Lewy bodies, eight patients with Lewy body variant
of Alzheimer disease, eight patients with Alzheimer disease, and 10 controls.
Relative alpha-synuclein 126 expression levels were determined by real-time
polymerase chain reaction with competimer technology. Alpha-synuclein 126 mRNA
expression was markedly decreased in the three dementias in comparison with
controls, suggesting an important role of this alpha-synuclein isoform in the
normal brain. A common finding in many neurodegenerative diseases is the presence of inclusion
bodies made of aggregated proteins in neurons of affected brain regions. In
Parkinson's disease, the inclusion bodies are referred to as Lewy bodies and
their main component is alpha-synuclein. Although many studies have suggested
that inclusion bodies may be cell protective, it is still not clear whether Lewy
bodies promote or inhibit dopaminergic cell death in Parkinson's disease.
Synphilin-1 interacts with alpha-synuclein and is present in Lewy bodies.
Accumulation of ubiquitylated synphilin-1 leads to massive formation of
inclusion bodies, which resemble Lewy bodies by their ability to recruit
alpha-synuclein. We have recently isolated an isoform of synphilin-1,
synphilin-1A, that spontaneously aggregates in cells, and is present in
detergent-insoluble fractions of brain protein samples from
alpha-synucleinopathy patients. Synphilin-1A displays marked neuronal toxicity
and, upon proteasome inhibition, accumulates into ubiquitylated inclusions with
concomitant reduction of its intrinsic toxicity. The fact that alpha-synuclein
interacts with synphilin-1A, and is recruited to synphilin-1A inclusion bodies
in neurons together with synphilin-1, further indicates that synphilin-1A cell
model is relevant for research on Parkinson's disease. Synphilin-1A cell model
may help provide important insights regarding the role of inclusion bodies in
Parkinson's disease and other neurodegenerative disorders. The pre-synaptic protein alpha-synuclein is the main component of Lewy bodies
and Lewy neurites, the defining neuropathological characteristics of Parkinson's
disease and dementia with Lewy bodies. Mutations in the alpha-synuclein gene
cause familial forms of Parkinson's disease and dementia with Lewy bodies. We
previously described a transgenic mouse line expressing truncated human
alpha-synuclein(1-120) that develops alpha-synuclein aggregates, striatal
dopamine deficiency and reduced locomotion, similar to Parkinson's disease. We
now show that in the striatum of these mice, as in Parkinson's disease, synaptic
accumulation of alpha-synuclein is accompanied by an age-dependent
redistribution of the synaptic SNARE proteins SNAP-25, syntaxin-1 and
synaptobrevin-2, as well as by an age-dependent reduction in dopamine release.
Furthermore, the release of FM1-43 dye from PC12 cells expressing either human
full-length alpha-synuclein(1-140) or truncated alpha-synuclein(1-120) was
reduced. These findings reveal a novel gain of toxic function of alpha-synuclein
at the synapse, which may be an early event in the pathogenesis of Parkinson's
disease. A shared neuropathological feature of idiopathic Parkinson's disease, dementia
with Lewy bodies, and multiple system atrophy is the development of
intracellular aggregates of α-synuclein that gradually engage increasing parts
of the nervous system. The pathogenetic mechanisms underlying these
neurodegenerative disorders, however, are unknown. Several studies have
highlighted similarities between classic prion diseases and these neurological
proteinopathies. Specifically, identification of Lewy bodies in fetal
mesencephalic neurons transplanted in patients with Parkinson's disease raised
the hypothesis that α-synuclein, the main component of Lewy bodies, could be
transmitted from the host brain to a graft of healthy neurons. These results and
others have led to the hypothesis that a prion-like mechanism might underlie
progression of synucleinopathy within the nervous system. We review experimental
findings showing that misfolded α-synuclein can transfer between cells and, once
transferred into a new cell, can act as a seed that recruits endogenous
α-synuclein, leading to formation of larger aggregates. This model suggests that
strategies aimed at prevention of cell-to-cell transfer of α-synuclein could
retard progression of symptoms in Parkinson's disease and other
synucleinopathies. Parkinson disease (PD) is the most important movement disorder and about 50% of
patients develop dementia over the time. PD belongs to the group of Lewy body
disorders. Alpha-synuclein (AS) is the main component of Lewy bodies and its
aggregation is a key event in the pathogenesis of PD. Beta-synuclein (BS)
inhibits AS aggregation in vitro and in vivo and has been shown to interact
directly with AS regulating its functionality and preventing its
oligomerization. Recently, we have described a molecular subgroup of DLB
characterized by the drastic BS reduction in cortical areas. In this study we
have analyzed the expression of two BS transcripts and the main AS transcript
SNCA140, in frozen samples of three brain areas, temporal cortex, caudate
nucleus and pons, from patients with PD and PDD in comparison with controls.
Relative mRNA expression was determined by real-time PCR with SybrGreen,
neuron-specific-enolase as housekeeping gene and the deltadeltaCt method. The
most important difference in BS and AS mRNA expression between PD and PDD was
found in the caudate nucleus, where BS mRNA was overexpressed in PD and AS mRNA
diminished in PDD. Our findings provide new insights into the pathogenesis of
dementia in PD, indicating that differential BS and AS expression in the caudate
nucleus may represent one of the molecular mechanisms involved in these complex
diseases. α-Synuclein is a major component of Lewy bodies in Parkinson disease (PD) and
dementia with Lewy bodies (DLB). We recently showed that abnormal α-synuclein
with resistance to proteinase K (PK) is deposited at presynapses of distinct
brain anatomic regions from the early stages of PD and DLB. NUB1, a
synphilin-1-binding protein, also accumulates in Lewy bodies, but it is not
known whether abnormal α-synuclein is associated with NUB1. Here, we demonstrate
that, in the brain of patients with PD and DLB, NUB1 accumulates in the
presynapses in the hippocampus, cerebral neocortex, and substantia nigra in
which PK-resistant α-synuclein is deposited. Endogenous NUB1 also accumulated
with PK-resistant α-synuclein in the presynapses of transgenic mice that express
human α-synuclein with an A53T mutation. Immunoelectron microscopy showed that
NUB1 is localized to presynaptic nerve terminals where no abnormal filaments are
seen. Biochemical analyses showed that NUB1 coexists with abnormal α-synuclein
in the brain of DLB patients. These findings suggest that NUB1 along with
abnormal α-synuclein is involved in the pathogenesis of Lewy body disease. Fibrillar α-synuclein (α-Syn) is the principal component of Lewy bodies, which
are evident in individuals affected by Parkinson disease (PD). This
neuropathologic form of α-Syn plays a central role in PD progression as it has
been shown to propagate between neurons. Tools that interfere with α-Syn
assembly or change the physicochemical properties of the fibrils have potential
therapeutic properties as they may be sufficient to interfere with and/or halt
cell-to-cell transmission and the systematic spread of α-Syn assemblies within
the central nervous system. Vertebrate molecular chaperones from the
constitutive/heat-inducible heat shock protein 70 (Hsc/p70) family have been
shown to hinder the assembly of soluble α-Syn into fibrils and to bind to the
fibrils and very significantly reduce their toxicity. To understand how Hsc70
family members sequester soluble α-Syn, we set up experiments to identify the
molecular chaperone-α-Syn surface interfaces. We cross-linked human Hsc70 and
its yeast homologue Ssa1p and α-Syn using a chemical cross-linker and mapped the
Hsc70- and Ssa1p-α-Syn interface. We show that the client binding domain of
Hsc70 and Ssa1p binds two regions within α-Syn similar to a tweezer, with the
first spanning residues 10-45 and the second spanning residues 97-102. Our
findings define what is necessary and sufficient for engineering Hsc70- and
Ssa1p-derived polypeptide with minichaperone properties with a potential as
therapeutic agents in Parkinson disease through their ability to affect α-Syn
assembly and/or toxicity. Parkinson's disease (PD) is characterized by the progressive degeneration of
substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of
Lewy bodies (LB) in a proportion of the remaining neurones. α-synuclein is the
main component of LB, but the pathological mechanisms that lead to
neurodegeneration associated with LB formation remain unclear. Three pivotal
elements have emerged in the development of PD: α-synuclein, mitochondria and
protein degradation systems. We previously reported a unique model, created by
conditional genetic depletion of 26S proteasomes in the SNpc of mice, which
mechanistically links these three elements with the neuropathology of PD:
progressive neurodegeneration and intraneuronal inclusion formation. Using this
model, we tested the hypothesis that α-synuclein was essential for the formation
of inclusions and neurodegeneration caused by 26S proteasomal depletion. We
found that both of these processes were independent of α-synuclein. This
provides an important insight into the relationship between the proteasome,
α-synuclein, inclusion formation and neurodegeneration. We also show that the
autophagy-lysosomal pathway is not activated in 26S proteasome-depleted
neurones. This leads us to suggest that the paranuclear accumulation of
mitochondria in inclusions in our model may reflect a role for the ubiquitin
proteasome system in mitochondrial homeostasis and that neurodegeneration may be
mediated through mitochondrial factors linked to inclusion biogenesis. Parkinson's disease (PD) is one of the most common neurodegenerative diseases.
Majority of PD are sporadic, for which genetic causes remain largely unknown.
Alpha-synuclein, the main component of Lewy bodies, plays a central role in the
PD pathogenesis. Macroautophagy is a highly conserved cellular process that
digests dysfunctional macromolecules and damaged organelles. Accumulating
evidence indicates that macroautophagy (hereafter referred to as autophagy) is
involved in alpha-synuclein degradation. Dysregulation of autophagy has been
observed in the brain tissues from PD patients and animal models. We
hypothesized that change expression levels of autophagy-related genes (ATG),
including ATG5, may contribute to PD. In this study, we genetically and
functionally analyzed the ATG5 gene promoter in groups of sporadic PD patients
and ethnic-matched healthy controls. A novel heterozygous variant, 106774459T>A,
was identified in one female patient, but in none of controls, which
significantly enhanced transcriptional activities of the ATG5 gene promoter.
Furthermore, ATG5 gene expression level in the PD patient was significantly
elevated than that in controls. Four novel heterozygous variants, 106774423C>A,
106774418C>A, 106774382C>A and 106774206G>A, were only found in controls. The
variant, 106774464C>T, and SNP-106774030A>G (rs510432) were found in PD patients
and controls with similar frequencies. Collectively, the variant identified in
PD patient may change ATG5 protein levels and alter autophagy activities,
contributing to PD onset as a risk factor. We proposed the term 'Lewy body disease' (LBD) in 1980. Subsequently, we
classified LBD into three types according to the distribution pattern of Lewy
bodies: a brainstem type, a transitional type and a diffuse type. Later, we
added the cerebral type. As we have proposed since 1980, LBD has recently been
used as a generic term, including Parkinson's disease, Parkinson's disease with
dementia and dementia with Lewy bodies. LBD has neuropathological
characteristics whereby numerous Lewy bodies are present in the central and
sympathetic nervous systems, and it is a type of alpha-synucleinopathy because
the main component of Lewy body is alpha-synuclein. In this paper we explain the
most recent concept of LBD from the historical viewpoint. |
How does thyroid hormone regulate mitochondrial biogenesis in the myocardium? | T4 increases myocardial mitochondrial bioenergetic capacity, oxygen consumption and markers of mitochondrial biogenesis. The marked, parallel increases in PPARalpha levels suggest its potential involvement in mediating myocardial-specific remodeling of mitochondria in response to T4. T3 induces mitochondrial biogenesis. In fact, T3 treatment for 72h increases activity of respiratory complexes II, IV, V and citrate synthase (CS), levels of mitochondrial enzyme subunits (e.g. COXI, COXIV) and nuclear-encoded transcription factors, involved in mitochondrial biogenesis (e.g. PGC-1, mtTFA and PPAR-alpha). Furthermore, L-T3 increases the expression of factors involved in mitochondrial DNA transcription and biogenesis, such as hypoxic inducible factor-1α, mitochondrial transcription factor A and peroxisome proliferator activated receptor γ coactivator-1α, in the LV peri-infarct zone.
The activation of TFAM and TFB2M expression is shown to be required for the induction of mtDNA biogenesis by T3. Truncated forms of the nuclear receptor TRα1, with molecular weights of 43 kDa (p43) and 28 Kda have been previously identified in mitochondria. P43 is a mitochondrial T3 receptor which stimulates mitochondrial transcription and protein synthesis in the presence of T3. p43 depletion in mice decreases mitochondrial DNA replication and respiratory chain activity. | The energy relationships between cytosolic and mitochondrial metabolism were
studied in the hearts from euthyroid, hypothyroid, and hyperthyroid rats.
Isolated mitochondria showed high respiratory control ratios and impermeability
to exogenous NADH. Hypo- and hyperthyroidism, respectively, resulted in lower
and higher contents of both cytochromes per mitochondrion and mitochondrial
protein per gram of wet weight of heart without changes in the ratio of
cytochrome c to cytochrome aa3. In isolated perfused heart, the hyperthyroid
state led to an increase in work rate and thereby an elevation of Vo2, which
resulted in an increase oxidation-reduction turnover number for the cytochromes.
An agreement was found between [ATP]/[ADP][Pi] of cytosolic free adenine
nucleotides and the value calculated from a mathematical model of mitochondrial
respiration. This implies that mitochondrial respiration is controlled at the
cytochrome oxidase reaction and that oxidative phosphorylation in intact tissue
is tightly coupled irrespective of thyroid state. It is concluded that thyroid
hormone causes an increase in the mitochondrial mass, mitochondrial cytochrome
content, and respiratory rate, and consequently expands the capacity of
oxidative metabolism without an uncoupling effect on oxidative phosphorylation. Thyroid hormone is one of the few known physiological regulators of mammalian
mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it
does so by regulating the expression of a limited number of unidentified
mitochondrial proteins. We have investigated these hormone-regulated proteins in
rat liver. Hormone injection induced a 30-fold increase in the levels of
cytochrome-c1 mRNA after 3 d. In addition, the mRNA for the growth-activated
adenine-nucleotide translocator, ANT2, was increased 13-fold and that for the
ATPase N,N'-dicyclohexylcarbodiimide-binding protein increased 4-5-fold.
Mitochondrial transcripts of cytochrome-oxidase subunit I also increased. No
changes were found in the mRNA levels for the F1-ATPase beta-subunit or
cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid
increases in cytochrome-c1 and ANT2 mRNA species which parallel changes in the
activity of the hormone-responsive malic enzyme, but are earlier than other
mitochondrial biogenetic events. These data strengthen the view that thyroid
hormone regulates synthesis of specific components within each respiratory-chain
complex and that these products apparently play key roles in inner-membrane
biogenesis and assembly. The significance of ANT2 induction is also discussed
with respect to the rapid respiratory response induced by thyroid hormone. T3 and GH have been implicated in the regulation of mitochondrial biogenesis.
Since thyroid hormone promotes the synthesis of growth hormone, its control of
human mitochondrial biogenesis could arise through a permissive action on GH
biosynthesis. This was studied in hypophysectomized rats treated with T3 and/or
human GH by the continuous infusion of hormone for 6 days from mini-infusion
pumps implanted sc. Increases in mitochondrial respiration, enzyme activities,
and protein synthesis were found in isolated liver mitochondria from rats
receiving T3. In contrast, GH alone had no effect, nor did it increase the
response to T3. Since it has been argued that mitochondrial biogenesis results
from a direct interaction (binding) of GH with mitochondria, GH-specific binding
sites were measured with 125I-bGH, a specific somatogenic receptor ligand, in
isolated mitochondrial membranes in vitro. In addition, the intracellular
endocytic uptake of 125I-bGH injected in vivo was compared in purified
subcellular membrane fractions and mitochondria. No evidence in favour of
specific GH interaction on mitochondrial membranes was found by either test. It
is concluded that T3 exerts a direct, rather than permissive, effect on
mitochondrial biogenesis, and that high affinity binding sites for GH are not
present in rat liver mitochondria. In order to analyze the role of thyroid hormones in mitochondrial biogenesis, we
have studied the expression pattern of the beta subunit of the mitochondrial
ATP-synthase complex in liver and in isolated mitochondria during postnatal
development of hypothyroid rats. Chemically induced hypothyroidism promoted a
significant reduction in body and liver masses at all stages of development.
Furthermore, plasma 3,5,3'-triiodo-L-thyronine (T3) and
3,5,3',5'-tetraiodo-L-thyronine (T4) concentrations were significantly reduced
in hypothyroid animals when compared to euthyroid animals. Remarkably,
steady-state beta-F1-ATPase mRNA levels in livers of hypothyroid animals showed
an approximately 50% reduction when compared to age-matched euthyroid rats at
all stages of development. The relative amounts of beta-F1-ATPase protein
determined in isolated mitochondria of 1-day-old and adult hypothyroid animals
were similar to those determined in mitochondria of age-matched euthyroids,
indicating that hypothyroidism does not affect organelle differentiation in the
liver of suckling and adult rats. In contrast, the relative amount of
beta-F1-ATPase protein in liver homogenates varied (0-30% reduction) due to the
hypothyroid condition during development. These findings suggest the existence
of compensatory mechanisms operating at the translational and/or
post-translational levels which promote proliferation of mitochondria in the
hypothyroid liver. However, when the liver mass was considered, hypothyroidism
significantly reduced overall mitochondrial proliferation in rat liver.
Interestingly, the effects of thyroid hormones on the biogenesis of the ATP
synthase complex at latter stages of development provide an example in which the
hypothyroid condition limits the expression of the nuclear-encoded gene with no
apparent effect on the expression of the mitochondrial-encoded genes (ATP
synthase subunits 6-8). Thyroid hormone [3,5,3'-triiodo-l-thyronine (T(3))] induces phenotypic
alterations in cardiac mitochondria, in part by influencing protein import and
the expression of the import motor mitochondrial heat shock protein (mtHsp70).
Here we examined the adaptability of translocases of the inner membrane (Tim)
proteins, as well as the outer membrane receptor Tom34, to T(3). Administration
of T(3) to rats for 5 days increased cardiac Tim23 and Tim44 mRNA levels by 55
and 50%, respectively, but had no effect on Tim17. T(3) treatment also induced a
45% increase in Tom34 mRNA, with no accompanying changes at the protein level,
suggesting regulation at the posttranscriptional level. In H9c2 cardiac cells,
Tim17 mRNA was elevated by 114% by 9 days of differentiation, whereas Tim23 and
Tim44 declined by 25 and 29%, respectively. To determine the functional
consequences of these T(3)-induced changes, malate dehydrogenase (MDH) import
rates were measured in H9c2 cells stably overexpressing Tim44 and mtHsp70,
either alone or in combination. MDH import remained unaltered in cells
overexpressing Tim44 or in cells overexpressing both Tim44 and mtHsp70. However,
when mtHsp70 was overexpressed alone, a 13% (P < 0.05) increase in MDH import
rate was observed. These findings indicate that import machinery components are
differentially regulated in response to stimuli that induce mitochondrial
biogenesis, like T(3) and differentiation. In addition, the induction of an
import machinery component in response to T(3) may not necessarily result in
functional changes in protein import during mitochondrial biogenesis. Finally,
mtHsp70 may play a regulatory role in the import process that is independent of
its interaction with Tim44. The transcriptional coactivator the peroxisome proliferator-activated receptor
gamma coactivator-1alpha (PGC-1alpha) has been identified as an important
mediator of mitochondrial biogenesis based on its ability to interact with
transcription factors that activate nuclear genes encoding mitochondrial
proteins. The induction of PGC-1alpha protein expression under conditions that
provoke mitochondrial biogenesis, such as contractile activity or thyroid
hormone (T(3)) treatment, is not fully characterized. Thus we related PGC-1alpha
protein expression to cytochrome c oxidase (COX) activity in 1) tissues of
varying oxidative capacities, 2) tissues from animals treated with T(3), and 3)
skeletal muscle subject to contractile activity both in cell culture and in
vivo. Our results demonstrate a strong positive correlation (r = 0.74; P < 0.05)
between changes in PGC-1alpha and COX activity, used as an index of
mitochondrial adaptations. The highest constitutive levels of PGC-1alpha were
found in the heart, whereas the lowest were measured in fast-twitch white muscle
and liver. T(3) increased PGC-1alpha content similarly in both fast- and
slow-twitch muscle, as well as in the liver, but not in heart. T(3) also induced
early (6 h) increases in AMP-activated protein kinase (AMPKalpha) activity, as
well as later (5 day) increases in p38 MAP kinase activity in slow-twitch, but
not in fast-twitch, muscle. Contractile activity provoked early increases in
PGC-1alpha, coincident with increases in mitochondrial transcription factor A
(Tfam), and nuclear respiratory factor-1 (NRF-1) protein expression, suggesting
that PGC-1alpha is physiologically important in coordinating the expression of
the nuclear and mitochondrial genomes. Ca(2+) ionophore treatment of muscle
cells led to an approximately threefold increase in PGC-1alpha protein, and
contractile activity induced rapid and marked increases in both p38 MAP kinase
and AMPKalpha activities. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside
(AICAR) treatment of muscle cells also led to parallel increases in AMPKalpha
activity and PGC-1alpha protein levels. These data are consistent with
observations that indicate that increases in PGC-1alpha protein are affected by
Ca(2+) signaling mechanisms, AMPKalpha activity, as well as posttranslational
phosphorylation events that increase PGC-1alpha protein stability. Our data
support a role for PGC-1alpha in the physiological regulation of mitochondrial
content in a variety of tissues and suggest that increases in PGC-1alpha
expression form part of a unifying pathway that promotes both T(3)- and
contractile activity-induced mitochondrial adaptations. Changes in thyroid status are associated with profound alterations in
biochemical and physiological functioning of cardiac muscle impacting metabolic
rate, contractility and structural hypertrophy. Using an in vivo model of
chronic treatment with thyroid hormone (T4, 0.3 mg/kg/day), we evaluated how
mitochondria are regulated in response to T4, and assessed the relationship of
T4-induced mitochondrial biogenesis and bioenergetics to overall cardiac
hypertrophy. The role of thyroid hormone in cardiac bioenergetic remodeling was
addressed in rats treated with T4 for 5, 10 and 15 days. Over that time,
myocardial oxygen consumption substantially increased as did cardiac
hypertrophy. Myocardial levels of mitochondrial enzyme activities, mitochondrial
DNA (mtDNA), specific proteins and transcript were assessed. Activity levels of
respiratory complexes I-V and citrate synthase significantly increased with 15
but not with 5 or 10-day T4 treatment. Myocardial levels of mtDNA, mitochondrial
proteins (e.g. cytochrome c, cytochrome b, ATPase subunits, MnSOD) and the
global transcription factor PPARalpha were significantly elevated with 15-day
T4. Transcript analysis revealed increased expression of transcription factors
and cofactors involved in mitochondrial biogenesis including PPARalpha, mtTFA,
ErbAalpha and PGC-1alpha. Our findings indicate parallel increases in myocardial
mitochondrial bioenergetic capacity, oxygen consumption and markers of
mitochondrial biogenesis with 15-day T4; these changes were not present with
10-day T4 even with significant cardiac hypertrophy. The marked, parallel
increases in PPARalpha levels suggest its potential involvement in mediating
myocardial-specific remodeling of mitochondria in response to T4. Thyroid hormone (TH) induces marked changes in the biochemical and physiological
functioning of cardiac muscle affecting its bioenergetics, contractility and
structure. Using a time-course analysis of in vitro treatment of neonatal rat
cardiomyocytes with triiodothyronine (T3), mitochondrial biogenesis, functional
bioenergetics and cardiomyocyte hypertrophic phenotype were assessed. Activity
of respiratory complexes II, IV, V and citrate synthase (CS), levels of
mitochondrial enzyme subunits (e.g. COXI, COXIV) and nuclear-encoded
transcription factors, involved in mitochondrial biogenesis (e.g. PGC-1, mtTFA
and PPAR-alpha), were significantly elevated with 72 h T3 treatment. A
time-course analysis showed an early increase (between 3 and 12 h) in activity
and levels of subunits of complex IV and V, mitochondrial Ca2+ accumulation and
a late increase (at 72 h) in complex II and CS activities, mitochondrial protein
content and mitochondrial respiration. Based on overall protein content and
specific peptide levels (e.g. actin or myosin) only mild cardiomyocyte
hypertrophy was detected. T3 mediates an early stimulation of enzymes containing
mtDNA encoded subunits (e.g. complex IV and V) in contrast to a different
regulatory pattern for the entirely nuclear-encoded enzymes (e.g. CS and complex
II). T3-regulation was similar in both neonatal and young adult cardiomyocytes
(ARCM) but absent in the senescent cardiomyocytes. This model offer an
opportunity to study the rapid timing of events involved in myocardial cell
signaling, bioenergetics and growth dynamics in a timeframe not available with
whole animal studies. Thyroid hormone (T(3)) regulates the function of many tissues within the body.
The effects of T(3) have largely been attributed to the modulation of thyroid
hormone receptor-dependent gene transcription. However, nongenomic actions of
T(3) via the initiation of signaling events are emerging in a number of cell
types. This study investigated the ability of short-term T(3) treatment to
phosphorylate and, therefore, activate signaling proteins in rat tissues in
vivo. The kinases investigated included p38, AMP-activated protein kinase
(AMPK), and extracellular signal-regulated kinase (ERK) 1/2. Following 2 h of
T(3) treatment, p38 and AMPK phosphorylation was increased in both the
slow-twitch soleus and the fast-twitch plantaris muscles. In contrast, ERK1/2
was not activated in either muscle type. Neither p38 nor AMPK was affected in
heart. However, AMPK activation was decreased by T(3) in liver. ERK1/2
activation was decreased by T(3) in heart, but increased in liver. Possible
downstream consequences of T(3)-induced kinase phosphorylation were investigated
by measuring cAMP response element binding protein (CREB) and thyroid hormone
receptor DNA binding, as well as peroxisome proliferator-activated
receptor-alpha coactivator-1 mRNA levels. Protein DNA binding to the cAMP or
thyroid hormone response elements was unaltered by T(3). However, peroxisome
proliferator-activated receptor-alpha coactivator-1 mRNA expression was
increased following 12 h of T(3) treatment in soleus. These data are the first
to characterize the effects of T(3) treatment on kinase phosphorylation in vivo.
We show that T(3) rapidly modifies kinase activity in a tissue-specific fashion.
Moreover, the T(3)-induced phosphorylation of p38 and AMPK in both slow- and
fast-twitch skeletal muscles suggests that these events may be important in
mediating hormone-induced increases in mitochondrial biogenesis in skeletal
muscle. Mitochondrial (mt)DNA mutations contribute to various disease states
characterized by low ATP production. In contrast, thyroid hormone
[3,3',5-triiodothyronine (T(3))] induces mitochondrial biogenesis and enhances
ATP generation within cells. To evaluate the role of T(3)-mediated mitochondrial
biogenesis in patients with mtDNA mutations, three fibroblast cell lines with
mtDNA mutations were evaluated, including two patients with Leigh's syndrome and
one with hypertrophic cardiomyopathy. Compared with control cells, patient
fibroblasts displayed similar levels of mitochondrial mass, peroxisome
proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha),
mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2)
protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS
production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold
elevation in mitochondrial membrane potential, and 30% less complex V activity
compared with control cells. Patient cells also displayed 20-25% reductions in
both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with
control cells. After T(3) treatment of patient cells, ROS production was
decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was
increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the
absence of a change in mitochondrial mass. There were no significant alterations
in the protein expression of PGC-1alpha, Tfam, or UCP2 in either T(3)-treated
patient or control cells. However, T(3) restored the mitochondrial membrane
potential, complex V activity, and levels of MnSOD to normal values in patient
cells and elevated MnSOD levels by 21% in control cells. These results suggest
that T(3) acts to reduce cellular oxidative stress, which may help attenuate
ROS-mediated damage, along with improving mitochondrial function and energy
status in cells with mtDNA defects. In previous studies, we characterized a new hormonal pathway involving a
mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor.
In in vitro and in vivo studies, we have shown that p43 increases mitochondrial
transcription and mitochondrial biogenesis. In addition, p43 overexpression in
skeletal muscle stimulates mitochondrial respiration and induces a shift in
metabolic and contractile features of muscle fibers which became more
oxidative.Here we have studied the influence of p43 overexpression in skeletal
muscle of mice during aging. We report that p43 overexpression initially
increased mitochondrial mass. However, after the early rise in mitochondrial DNA
occurring at 2 months of age in transgenic mice, we observed a progressive
decrease of mitochondrial DNA content which became 2-fold lower at 23 months of
age relatively to control animals. Moreover, p43 overexpression induced an
oxidative stress characterized by a strong increase of lipid peroxidation and
protein oxidation in quadriceps muscle, although antioxidant enzyme activities
(catalase and superoxide dismutase) were stimulated. In addition, muscle atrophy
became detectable at 6 months of age, probably through a stimulation of the
ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3,
Atrogin-1/MAFbx and MuRF1.Taken together, these results demonstrate that a
prolonged stimulation of mitochondrial activity induces muscle atrophy. In
addition, these data underline the importance of a tight control of p43
expression and suggest that a deregulation of the direct T3 mitochondrial
pathway could be one of the parameters involved in the occurrence of sarcopenia. 3,5,3'-Levo-triiodothyronine (L-T3) is essential for DNA transcription,
mitochondrial biogenesis and respiration, but its circulating levels rapidly
decrease after myocardial infarction (MI). The main aim of our study was to test
whether an early and sustained normalization of L-T3 serum levels after MI
exerts myocardial protective effects through a mitochondrial preservation.
Seventy-two hours after MI induced by anterior interventricular artery ligation,
rats were infused with synthetic L-T3 (1.2 μg/kg/day) or saline over 4 weeks.
Compared to saline, L-T3 infusion restored FT3 serum levels at euthyroid state
(3.0 ± 0.2 versus 4.2 ± 0.3 pg/ml), improved left ventricular (LV) ejection
fraction (39.5 ± 2.5 versus 65.5 ± 6.9%), preserved LV end-systolic wall
thickening in the peri-infarct zone (6.34 ± 3.1 versus 33.7 ± 6.21%) and reduced
LV infarct-scar size by approximately 50% (all P < 0.05). Moreover, L-T3
significantly increased angiogenesis and cell survival and enhanced the
expression of nuclear-encoded transcription factors involved in these processes.
Finally, L-T3 significantly increased the expression of factors involved in
mitochondrial DNA transcription and biogenesis, such as hypoxic inducible
factor-1α, mitochondrial transcription factor A and peroxisome proliferator
activated receptor γ coactivator-1α, in the LV peri-infarct zone. To further
explore mechanisms of L-T3 protective effects, we exposed isolated neonatal
cardiomyocytes to H(2)O(2) and found that L-T3 rescued mitochondrial biogenesis
and function and protected against cell death via a mitoKATP dependent pathway.
Early and sustained physiological restoration of circulating L-T3 levels after
MI halves infarct scar size and prevents the progression towards heart failure.
This beneficial effect is likely due to enhanced capillary formation and
mitochondrial protection. Hypothyroidism during early mammalian brain development is associated with
decreased expression of various mitochondrial encoded genes along with evidence
for mitochondrial dysfunction. However, in-spite of the similarities between
neurological disorders caused by perinatal hypothyroidism and those caused by
various genetic mitochondrial defects we still do not know as to how thyroid
hormone (TH) regulates mitochondrial transcription during development and
whether this regulation by TH is nuclear mediated or through mitochondrial TH
receptors? We here in rat cerebellum show that hypothyroidism causes reduction
in expression of nuclear encoded genes controlling mitochondrial biogenesis like
PGC-1alpha, NRF-1alpha and Tfam. Also, we for the first time demonstrate a
mitochondrial localization of thyroid hormone receptor (mTR) isoform in
developing brain capable of binding a TH response element (DR2) present in
D-loop region of mitochondrial DNA. These results thus indicate an integrated
nuclear-mitochondrial cross talk in regulation of mitochondrial transcription by
TH during brain development. Exogenous thyroid hormones are regulators of cellular metabolism that involves,
along with other cell structures, mitochondria. Mechanisms of the influence of
thyroid hormones on the biogenesis of mtDNA are not fully understood due to
their pleiotropic nature. Different ways of regulation of mitochondrial
biogenesis by thyroid hormones are discussed in literature, but thyroid
receptors, localized in both the nucleus and mitochondria, are the main elements
of most pathways. Data on events occurring after receptor activation are rather
contradictory. We investigated the degree of involvement of mitochondrial
transcription factors in the biogenesis of mtDNA induced by triiodothyronine.
The contribution of TFAM, TFB2M, and helicase Twinkle in thyroid-induced mtDNA
biogenesis was assessed. The activation of TFAM and TFB2M expression is shown to
be required for the induction of mtDNA biogenesis. The role of helicase Twinkle,
the expression induction of which is also observed after triiodothyronine
addition, remains unclear. The analysis of factors that activate TFAM and TFB2M
expression showed that NRF-1 is the determinative regulator: deficiency of this
factor leads to complete collapse of mtDNA biogenesis. However, lack of
transcriptional coactivator PGC-1α did not lead to significant reduction in
thyroid-induced biogenesis, whereas literature data point to its key role in the
biogenesis of mitochondria. Thus, in this study the role of key transcription
factors in mtDNA biogenesis induced by triiodothyronine was demonstrated for the
first time in a model system. Thyroid hormone is a major determit of energy expenditure and a key regulator
of mitochondrial activity. We have previously identified a mitochondrial
triiodothyronine receptor (p43) that acts as a mitochondrial transcription
factor of the organelle genome, which leads, in vitro and in vivo, to a
stimulation of mitochondrial biogenesis. Here we generated mice specifically
lacking p43 to address its physiological influence. We found that p43 is
required for normal glucose homeostasis. The p43(-/-) mice had a major defect in
insulin secretion both in vivo and in isolated pancreatic islets and a loss of
glucose-stimulated insulin secretion. Moreover, a high-fat/high-sucrose diet
elicited more severe glucose intolerance than that recorded in normal animals.
In addition, we observed in p43(-/-) mice both a decrease in pancreatic islet
density and in the activity of complexes of the respiratory chain in isolated
pancreatic islets. These dysfunctions were associated with a down-regulation of
the expression of the glucose transporter Glut2 and of Kir6.2, a key component
of the K(ATP) channel. Our findings establish that p43 is an important regulator
of glucose homeostasis and pancreatic β-cell function and provide evidence for
the first time of a physiological role for a mitochondrial endocrine receptor. In vertebrates, skeletal muscle myofibers display different contractile and
metabolic properties associated with different mitochondrial content and
activity. We have previously identified a mitochondrial triiodothyronine
receptor (p43) regulating mitochondrial transcription and mitochondrial
biogenesis. When overexpressed in skeletal muscle, it increases mitochondrial
DNA content, stimulates mitochondrial respiration, and induces a shift in the
metabolic and contractile features of muscle fibers toward a slower and more
oxidative phenotype. Here we show that a p43 depletion in mice decreases
mitochondrial DNA replication and respiratory chain activity in skeletal muscle
in association with the induction of a more glycolytic muscle phenotype and a
decrease of capillary density. In addition, p43(-/-) mice displayed a
significant increase in muscle mass relative to control animals and had an
improved ability to use lipids. Our findings establish that the p43
mitochondrial receptor strongly affects muscle mass and the metabolic and
contractile features of myofibers and provides evidence that this receptor
mediates, in part, the influence of thyroid hormone in skeletal muscle. |
What is the link between HOT regions and RNA polymerase recruitment? | Most HOT regions co-localize with RNA polymerase II binding sites, but many are not near the promoters of annotated genes. At HOT promoters, TF occupancy is strongly predictive of transcription preinitiation complex recruitment and moderately predictive of initiating Pol II recruitment, but only weakly predictive of elongating Pol II and RNA transcript abundance. | BACKGROUND: High-occupancy target (HOT) regions are compact genome loci occupied
by many different transcription factors (TFs). HOT regions were initially
defined in invertebrate model organisms, and we here show that they are a
ubiquitous feature of the human gene-regulation landscape.
RESULTS: We identified HOT regions by a comprehensive analysis of ChIP-seq data
from 96 DNA-associated proteins in 5 human cell lines. Most HOT regions
co-localize with RNA polymerase II binding sites, but many are not near the
promoters of annotated genes. At HOT promoters, TF occupancy is strongly
predictive of transcription preinitiation complex recruitment and moderately
predictive of initiating Pol II recruitment, but only weakly predictive of
elongating Pol II and RNA transcript abundance. TF occupancy varies
quantitatively within human HOT regions; we used this variation to discover
novel associations between TFs. The sequence motif associated with any given
TF's direct DNA binding is somewhat predictive of its empirical occupancy, but a
great deal of occupancy occurs at sites without the TF's motif, implying
indirect recruitment by another TF whose motif is present.
CONCLUSIONS: Mammalian HOT regions are regulatory hubs that integrate the
signals from diverse regulatory pathways to quantitatively tune the promoter for
RNA polymerase II recruitment. |
List variants of the MC1R gene. | V60L
D84E
V92M
R151C
R160W
R163Q
D294H | Several MC1R variants are associated with increased risk of maligt melanoma
(MM) in a variety of populations. We aim to examine the influence of the MC1R
variants (RHC: D84E, R151C, R160W; NRHC: V60L, R163Q and the synonymous
polymorphism T314T) on the MM risk in a population from the Canary Islands.
Overall, 1,046 Caucasian individuals were included in the study. A thousand of
them were genotyped for MC1R variants: 509 were sporadic MM patients and 491
were healthy control subjects from general population. The analysis was adjusted
for age, sex, hair colour, eye colour, skin phototype and ancestry. We found
that carriers of the R151C and R163Q variants were at an increased risk for
melanoma OR 2.76 (1.59-4.78) and OR 5.62 (2.54-12.42), respectively. The risk of
carrying RHC variants was 3.04 (1.90-4.86). Current study confirms the increased
MM risk for R151C carriers. It also supports the association between R163Q
variant and MM risk in the population on the Canary Islands, as opposed to
reported on northern populations. These results highlight the importance of the
sample population selection in this kind of studies. The MC1R gene is a key regulator of skin pigmentation. We aimed to evaluate the
association between MC1R variants and the risk of sporadic cutaneous melanoma
(CM) within the M-SKIP project, an international pooled-analysis on MC1R, skin
cancer and phenotypic characteristics. Data included 5,160 cases and 12,119
controls from 17 studies. We calculated a summary odds ratio (SOR) for the
association of each of the nine most studied MC1R variants and of variants
combined with CM by using random-effects models. Stratified analysis by
phenotypic characteristics were also performed. Melanoma risk increased with
presence of any of the main MC1R variants: the SOR for each variant ranged from
1.47 (95%CI: 1.17-1.84) for V60L to 2.74 (1.53-4.89) for D84E. Carriers of any
MC1R variant had a 66% higher risk of developing melanoma compared with
wild-type subjects (SOR; 95%CI: 1.66; 1.41-1.96) and the risk attributable to
MC1R variants was 28%. When taking into account phenotypic characteristics, we
found that MC1R-associated melanoma risk increased only for darker-pigmented
Caucasians: SOR (95%CI) was 3.14 (2.06-4.80) for subjects with no freckles, no
red hair and skin Type III/IV. Our study documents the important role of all the
main MC1R variants in sporadic CM and suggests that they have a direct effect on
melanoma risk, independently on the phenotypic characteristics of carriers. This
is of particular importance for assessing preventive strategies, which may be
directed to darker-pigmented Caucasians with MC1R variants as well as to lightly
pigmented, fair-skinned subjects. The melanocortin 1 receptor (MC1R) gene encodes for a seven-pass transmembrane
receptor primarily expressed on melanocytes and melanoma cells. Single
nucleotide polymorphisms (SNPs, also termed variants) in MC1R frequently cause
red hair, fair skin and are associated with melanoma and keratinocyte-derived
skin cancer development. Activation of wild-type (WT) MC1R in skin assists
cutaneous photoprotection whereas reduced MC1R signalling, seen with MC1R
variants, impairs ultraviolet radiation (UVR)-protective responses. As ancestral
humans migrated out of Africa, the evolutionary advantage of MC1R variants may
have related to improved cutaneous vitamin D synthesis and higher birthweight
reported with certain MC1R variants. Reduced photoprotection secondary to MC1R
dysfunction involves pigmentary and non-pigmentary mechanisms (reduced DNA
repair, effects on cell proliferation and possibly immunological parameters),
leading to clonal expansion of mutated cells within skin and subsequent
carcinogenesis. Recent investigations suggest an association between MC1R
genotype and vitiligo, with preliminary evidence that a MC1R agonist,
[Nle4-D-Phe7]-alpha-MSH, in combination with UVB, assists repigmentation. Future
development of compounds to correct defective MC1R responses secondary to MC1R
variants could result in photoprotective benefits for fair-skinned individuals
and reduce their skin cancer risk. Author information:
(1)Division of Epidemiology and Biostatistics, European Institute of Oncology,
Via Ripamonti 435, Milan 20141, Italy.
(2)Department of Dermatology, University of L'Aquila, 47100 L'Aquila, Italy.
(3)Department of Forensic Molecular Biology, Erasmus MC University Medical
Center, 3000 DR Rotterdam, The Netherlands.
(4)Department of Dermatology, Erasmus MC University Medical Center, 3000 DR
Rotterdam, The Netherlands.
(5)1] Department of Dermatology, Brigham and Women's Hospital and Harvard
Medical School, Boston, MA 02115, USA [2] Channing Laboratory, Department of
Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA
02115, USA [3] Department of Epidemiology, Harvard School of Public Health,
Boston, MA 02115, USA.
(6)Division of Molecular Genetic Epidemiology, German Cancer Research Center,
D-69120 Heidelberg, Germany.
(7)Department of Dermatology, Leiden University Medical Center, 2300 RC Leiden,
The Netherlands.
(8)Department of Chronic Disease Epidemiology, Yale School of Public Health,
Yale Cancer Center, New Haven, CT 06520-8034, USA.
(9)Institute of Forensic Research, 31-033 Krakow, Poland.
(10)Murdoch Childrens Research Institute, Royal Children's Hospital, Victoria
3052, Australia.
(11)Menzies Research Institute Tasmania, University of Tasmania, Hobart, 7001
Australia.
(12)Department of Pathology, Oslo University Hospital, N-0027 Oslo, Norway.
(13)International Prevention Research Institute, Lyon 69006, France.
(14)Department of Biochemistry, Molecular Biology and Immunology, University of
Murcia, 30100 Murcia, Spain.
(15)Department of Cancer Epidemiology, Moffitt Cancer Center, Tampa, FL 33612,
USA.
(16)Division of Cancer Epidemiology and Genetics, National Cancer Institute,
NIH, Bethesda, MD 20892-7236, USA.
(17)School of Epidemiology, Public Health and Preventive Medicine, University of
Ottawa, Ottawa, Canada ON K1N 6N5.
(18)Section of Epidemiology and Biostatistics, Institute of Cancer and
Pathology, University of Leeds, Leeds LS9 7TF, UK.
(19)UCL Institute of Child Health, London WC1N 1EH, UK. |
Which is the enzyme that degrades decapped mRNAs? | The removal of the 5'-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5'-to-3' exonucleolytic degradation by XRN1 | |
Which phenotypes are associated with heterozygous mutations of the BSCL2 gene? | Heterozygous mutations in the Berardinelli-Seip congenital lipodystrophy (BSCL2) gene have been associated with different clinical phenotypes including Silver syndrome/spastic paraplegia 17, distal hereditary motor neuropathy type V, and Charcot-Marie-Tooth disease type 2 (CMT2) with predominant hand involvement. | Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy
(OMIM #182960) is a heterogeneous group of disorders characterized by an almost
exclusive degeneration of motor nerve fibers, predomitly in the distal part
of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary
spastic paraparesis mapped to chromosome 11q12-q14 (SPG17) in which spasticity
of the legs is accompanied by amyotrophy of the hands and occasionally also the
lower limbs. Silver syndrome and most forms of dHMN are autosomal domitly
inherited with incomplete penetrance and a broad variability in clinical
expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed
linkage to the locus SPG17, which was confirmed in 16 additional families with a
phenotype characteristic of dHMN or Silver syndrome. After refining the critical
region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy
(BSCL2) and identified two heterozygous missense mutations resulting in the
amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes
the protein seipin, were previously shown to be associated with autosomal
recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show
that seipin is an integral membrane protein of the endoplasmic reticulum (ER).
The amino acid substitutions N88S and S90L affect glycosylation of seipin and
result in aggregate formation leading to neurodegeneration. Silver syndrome is a rare autosomal domit neurodegenerative disorder
characterized by marked amyotrophy and weakness of small hand muscles and
spasticity in the lower limbs. The locus for Silver syndrome (SPG17) was
assigned to a 13 cM region on chromosome 11q12-q14 in a single large pedigree.
We recently found heterozygous mutations in the Berardinelli-Seip congenital
lipodystrophy (BSCL2, seipin) gene causing SPG17 and distal hereditary motor
neuropathy type V (distal HMN V). Here we report the clinical features of two
families with heterozygous BSCL2 mutations. Interestingly, both families show a
clinical phenotype different from classical Silver syndrome, and in some
patients the phenotype is also different from distal HMN V. Patients in the
first family had marked spasticity in the lower limbs and very striking distal
amyotrophy that always started in the legs. Patients in the second family had
distal amyotrophy sometimes starting and predominating in the legs, but no
pyramidal tract signs. These observations broaden the clinical phenotype of
disorders associated with BSCL2 mutations, having consequences for molecular
genetic testing. OBJECTIVE: Heterozygous mutations in the Seipin/BSCL2 gene have recently been
identified in two autosomal domit motor neuron diseases, distal hereditary
motor neuropathy type V and Silver's syndrome. Seipin protein is reportedly a
transmembrane protein localized in the endoplasmic reticulum (ER). N88S and S90L
mutations of this protein disrupt its glycosylation, resulting in its
aggregation, but the mechanism of neurodegeneration remains unclear. To clarify
the molecular pathogenesis of seipin-related motor neuron diseases, we expressed
wild-type and mutant seipin proteins in neuronal and nonneuronal cells.
METHODS AND RESULTS: Coexpression of human seipin and ubiquitin showed that
seipin is polyubiquitinated and its ubiquitination is enhanced by mutation.
Treatment of cells with a proteasome inhibitor increased the amounts of mutant
seipin in the cells, suggesting that they are degraded through the ER-associated
degradation pathway. Immunoprecipitation studies showed that mutant seipin
stably binds to the ER chaperone calnexin, indicating accumulation of unfolded
mutant seipin in the ER. Furthermore, expression of mutant seipin increased the
level of ER stress-mediated molecules and induced apoptosis in cultured cells.
INTERPRETATION: These findings demonstrate that seipin/BSCL2-related motor
neuron diseases are novel conformational diseases, and we suspect that they are
tightly associated with ER stress-mediated cell death. In 2004, heterozygous mutations (N88S, S90L) in the Seipin/BSCL2 gene were
identified in two autosomal domit motor neuron diseases, distal hereditary
motor neuropathy type V (OMIM #182960) and Silver syndrome (OMIM #270685). The
Seipin/BSCL2 gene was originally identified as a candidate gene for congenital
generalized lipodystrophy type 2 (CGL2) (OMIM #269700). Individuals with
homozygous null mutations in seipin have severe lipoatrophy, insulin resistance,
hypertriglyceridemia, and mental retardation without any abnormality of the
motor neurons. Recent phenotype analyses of the N88S and S90L mutations have
revealed a wide spectrum of Seipin/BSCL2-related motor neuron diseases,
including Silver syndrome, distal hereditary motor neuropathy type V, variants
of Charcot-Marie-Tooth disease type 2, and spastic paraplegia 17; therefore,
these diseases should be termed "seipinopathies". Seipin is a transmembrane
protein that is localized in the endoplasmic reticulum (ER). Interestingly, the
N88S and S90L mutations both disturb the N-glycosylation motif, suggesting that
improper glycosylation of seipin is closely associated with the pathogenesis of
motor neuron diseases. Our recent study demonstrated that seipin is
proteolytically cleaved into N and C-terminal fragments and then
polyubiquitinated. The N88S and S90L mutations enhance ubiquitination and
degradation by UPS, and N88S and S90L mutants appear to be improperly folded,
resulting in their accumulation in the ER. Furthermore, expression of mutant
seipin in cultured cells activates UPR stress and induces ER stress-mediated
apoptosis. Our findings suggest that seipin-related motor neuron diseases,
seipinopathies are novel conformational diseases, and we propose that the
pathological process of these diseases is tightly associated with ER
stress-mediated cell death. BACKGROUND: Distal hereditary motor neuropathy type V (dHMN-V) and Silver
syndrome are rare phenotypically overlapping diseases which can be caused by
mutations in the Berardinelli-Seip Congenital Lipodystrophy 2 (BSCL2) gene or
Seipin.
AIM: To report the first Serbian family with a BSCL2 mutation showing variable
expression within the family.
PATIENTS AND METHODS: A 55-year-old woman presented with weakness of both hands
at the age of 45. At age 47, she noticed distal muscle weakness and atrophy in
her legs. Physical examination revealed atrophy and weakness of small hand
muscles and mild atrophy and weakness of the lower limbs. There was generalized
hyperreflexia with the exception of ankle reflexes which were diminished. Her
25year-old son had only stiffness of both legs at the age of 22. Physical
examination revealed only generalized hyporeflexia. The third affected member in
this family was her 55year-old cousin who showed a more prominent involvement of
leg muscles with mild asymmetrical weakness of hand muscles and no pyramidal
tract features.
RESULTS: In all three patients sensory nerve conduction velocities (NCV) were
normal in all extremities. Compound muscle action potential (CMAP) amplitudes
were markedly reduced in all patients. Concentric needle EMG showed evidence of
chronic denervation in distal muscles. DNA sequencing of BSCL2 was performed and
a heterozygous N88S missense mutation in BSCL2 gene was detected in all three
patients.
CONCLUSION: This report is further confirmation of phenotypic heterogenity due
to the N88S mutation of BSCL2 gene in the same family. |
Which is the target protein of the drug nivolumab? | Nivolumab was developed as a monoclonal antibody against programmed death receptor-1, an immune checkpoint inhibitor which negatively regulates T-cell proliferation and activation. | Author information:
(1)Scott N. Gettinger and Mario Sznol, Yale Cancer Center, New Haven, CT; Leora
Horn, David P. Carbone, and Jeffrey A. Sosman, Vanderbilt University Medical
Center; David R. Spigel, Sarah Cannon Research Institute/Tennessee Oncology,
Nashville, TN; Leena Gandhi, David M. Jackman, and F. Stephen Hodi, Dana-Farber
Cancer Institute; Rebecca S. Heist and Lecia V. Sequist, Massachusetts General
Hospital Cancer Center; David F. McDermott, Beth Israel Deaconess Medical
Center, Boston, MA; Scott J. Antonia and Mary C. Pinder-Schenck, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL; Naiyer A. Rizvi, Richard D.
Carvajal, and Matthew D. Hellmann, Memorial Sloan Kettering Cancer Center, New
York, NY; John D. Powderly, Carolina BioOncology Institute, Huntersville, NC;
David C. Smith, University of Michigan, Ann Arbor, MI; Philip Leming, Christ
Hospital Cancer Center, Cincinnati, OH; Suzanne L. Topalian, Drew M. Pardoll,
and Julie R. Brahmer, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins,
Baltimore, MD; and Vindira Sankar, Christoph M. Ahlers, Mark Salvati, Jon M.
Wigginton, Georgia D. Kollia, and Ashok K. Gupta, Bristol-Myers Squibb,
Princeton, NJ. [email protected].
(2)Scott N. Gettinger and Mario Sznol, Yale Cancer Center, New Haven, CT; Leora
Horn, David P. Carbone, and Jeffrey A. Sosman, Vanderbilt University Medical
Center; David R. Spigel, Sarah Cannon Research Institute/Tennessee Oncology,
Nashville, TN; Leena Gandhi, David M. Jackman, and F. Stephen Hodi, Dana-Farber
Cancer Institute; Rebecca S. Heist and Lecia V. Sequist, Massachusetts General
Hospital Cancer Center; David F. McDermott, Beth Israel Deaconess Medical
Center, Boston, MA; Scott J. Antonia and Mary C. Pinder-Schenck, H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL; Naiyer A. Rizvi, Richard D.
Carvajal, and Matthew D. Hellmann, Memorial Sloan Kettering Cancer Center, New
York, NY; John D. Powderly, Carolina BioOncology Institute, Huntersville, NC;
David C. Smith, University of Michigan, Ann Arbor, MI; Philip Leming, Christ
Hospital Cancer Center, Cincinnati, OH; Suzanne L. Topalian, Drew M. Pardoll,
and Julie R. Brahmer, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins,
Baltimore, MD; and Vindira Sankar, Christoph M. Ahlers, Mark Salvati, Jon M.
Wigginton, Georgia D. Kollia, and Ashok K. Gupta, Bristol-Myers Squibb,
Princeton, NJ. BACKGROUND: Nivolumab (a programmed death 1 [PD-1] checkpoint inhibitor) and
ipilimumab (a cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] checkpoint
inhibitor) have been shown to have complementary activity in metastatic
melanoma. In this randomized, double-blind, phase 3 study, nivolumab alone or
nivolumab plus ipilimumab was compared with ipilimumab alone in patients with
metastatic melanoma.
METHODS: We assigned, in a 1:1:1 ratio, 945 previously untreated patients with
unresectable stage III or IV melanoma to nivolumab alone, nivolumab plus
ipilimumab, or ipilimumab alone. Progression-free survival and overall survival
were coprimary end points. Results regarding progression-free survival are
presented here.
RESULTS: The median progression-free survival was 11.5 months (95% confidence
interval [CI], 8.9 to 16.7) with nivolumab plus ipilimumab, as compared with 2.9
months (95% CI, 2.8 to 3.4) with ipilimumab (hazard ratio for death or disease
progression, 0.42; 99.5% CI, 0.31 to 0.57; P<0.001), and 6.9 months (95% CI, 4.3
to 9.5) with nivolumab (hazard ratio for the comparison with ipilimumab, 0.57;
99.5% CI, 0.43 to 0.76; P<0.001). In patients with tumors positive for the PD-1
ligand (PD-L1), the median progression-free survival was 14.0 months in the
nivolumab-plus-ipilimumab group and in the nivolumab group, but in patients with
PD-L1-negative tumors, progression-free survival was longer with the combination
therapy than with nivolumab alone (11.2 months [95% CI, 8.0 to not reached] vs.
5.3 months [95% CI, 2.8 to 7.1]). Treatment-related adverse events of grade 3 or
4 occurred in 16.3% of the patients in the nivolumab group, 55.0% of those in
the nivolumab-plus-ipilimumab group, and 27.3% of those in the ipilimumab group.
CONCLUSIONS: Among previously untreated patients with metastatic melanoma,
nivolumab alone or combined with ipilimumab resulted in significantly longer
progression-free survival than ipilimumab alone. In patients with PD-L1-negative
tumors, the combination of PD-1 and CTLA-4 blockade was more effective than
either agent alone. (Funded by Bristol-Myers Squibb; CheckMate 067
ClinicalTrials.gov number, NCT01844505.). BACKGROUND: Patients with advanced squamous-cell non-small-cell lung cancer
(NSCLC) who have disease progression during or after first-line chemotherapy
have limited treatment options. This randomized, open-label, international,
phase 3 study evaluated the efficacy and safety of nivolumab, a fully human IgG4
programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, as compared with
docetaxel in this patient population.
METHODS: We randomly assigned 272 patients to receive nivolumab, at a dose of 3
mg per kilogram of body weight every 2 weeks, or docetaxel, at a dose of 75 mg
per square meter of body-surface area every 3 weeks. The primary end point was
overall survival.
RESULTS: The median overall survival was 9.2 months (95% confidence interval
[CI], 7.3 to 13.3) with nivolumab versus 6.0 months (95% CI, 5.1 to 7.3) with
docetaxel. The risk of death was 41% lower with nivolumab than with docetaxel
(hazard ratio, 0.59; 95% CI, 0.44 to 0.79; P<0.001). At 1 year, the overall
survival rate was 42% (95% CI, 34 to 50) with nivolumab versus 24% (95% CI, 17
to 31) with docetaxel. The response rate was 20% with nivolumab versus 9% with
docetaxel (P=0.008). The median progression-free survival was 3.5 months with
nivolumab versus 2.8 months with docetaxel (hazard ratio for death or disease
progression, 0.62; 95% CI, 0.47 to 0.81; P<0.001). The expression of the PD-1
ligand (PD-L1) was neither prognostic nor predictive of benefit.
Treatment-related adverse events of grade 3 or 4 were reported in 7% of the
patients in the nivolumab group as compared with 55% of those in the docetaxel
group.
CONCLUSIONS: Among patients with advanced, previously treated squamous-cell
NSCLC, overall survival, response rate, and progression-free survival were
significantly better with nivolumab than with docetaxel, regardless of PD-L1
expression level. (Funded by Bristol-Myers Squibb; CheckMate 017
ClinicalTrials.gov number, NCT01642004.). Nivolumab was developed as a monoclonal antibody against programmed death
receptor-1, an immune checkpoint inhibitor which negatively regulates T-cell
proliferation and activation. Intravenous administration of nivolumab was
approved for the treatment of unresectable maligt melanoma in 2014 in Japan.
When advanced melanoma patients were treated with nivolumab, median overall
survival became longer. Overall survival rate was significantly better in
nivolumab-treated melanoma patients than dacarbazine-treated melanoma patients.
Nivolumab had an acceptable long-term tolerability profile, with 22% of patients
experiencing grade 3 or 4 adverse events related to the drug. Therefore,
nivolumab can become an alternative therapy for advanced maligt melanoma. BACKGROUND: Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, was
associated with encouraging overall survival in uncontrolled studies involving
previously treated patients with advanced renal-cell carcinoma. This randomized,
open-label, phase 3 study compared nivolumab with everolimus in patients with
renal-cell carcinoma who had received previous treatment.
METHODS: A total of 821 patients with advanced clear-cell renal-cell carcinoma
for which they had received previous treatment with one or two regimens of
antiangiogenic therapy were randomly assigned (in a 1:1 ratio) to receive 3 mg
of nivolumab per kilogram of body weight intravenously every 2 weeks or a 10-mg
everolimus tablet orally once daily. The primary end point was overall survival.
The secondary end points included the objective response rate and safety.
RESULTS: The median overall survival was 25.0 months (95% confidence interval
[CI], 21.8 to not estimable) with nivolumab and 19.6 months (95% CI, 17.6 to
23.1) with everolimus. The hazard ratio for death with nivolumab versus
everolimus was 0.73 (98.5% CI, 0.57 to 0.93; P=0.002), which met the
prespecified criterion for superiority (P≤0.0148). The objective response rate
was greater with nivolumab than with everolimus (25% vs. 5%; odds ratio, 5.98
[95% CI, 3.68 to 9.72]; P<0.001). The median progression-free survival was 4.6
months (95% CI, 3.7 to 5.4) with nivolumab and 4.4 months (95% CI, 3.7 to 5.5)
with everolimus (hazard ratio, 0.88; 95% CI, 0.75 to 1.03; P=0.11). Grade 3 or 4
treatment-related adverse events occurred in 19% of the patients receiving
nivolumab and in 37% of the patients receiving everolimus; the most common event
with nivolumab was fatigue (in 2% of the patients), and the most common event
with everolimus was anemia (in 8%).
CONCLUSIONS: Among patients with previously treated advanced renal-cell
carcinoma, overall survival was longer and fewer grade 3 or 4 adverse events
occurred with nivolumab than with everolimus. (Funded by Bristol-Myers Squibb;
CheckMate 025 ClinicalTrials.gov number, NCT01668784.). |
The CXCR2 receptor is targeted in cancer. Name five antagonists. | There are numerous CXCR2 receptor antagonists, such as SB225002, G31P, SCH-527123, AZ10397767, SCH-479833. | Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has
implicated the N-terminal extracellular domain (N-ter) and regions vicinal to
the second extracellular loop (ECL2) in this activity. It was shown that
residues in the N-ter are necessary for binding of the physiologic ligands,
RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's
sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks
efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for
engagement by vMIP-II of CCR5 to its interaction with physiologic ligands.
RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated
partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants
of charged extracellular amino acids revealed that alteration of acidic residues
in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II.
Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of
RANTES and MIP-1 alpha and their ability to induce signaling, interaction with
vMIP-II was not altered by any mutation in the exoloops of the receptor.
Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine
binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with
the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an
affinity similar to that of the wild-type receptor. Engagement of CCR5 by
vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal
antibodies to the receptor, providing additional evidence for a distinct
mechanism for viral chemokine binding. Analysis of the coreceptor activity of
randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between
H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic
coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to
mutations in ECL2 provides a potential rationale to its inefficiency as an
antagonist of CCR5 coreceptor activity. These findings suggest that the
molecular anatomy of CCR5 binding plays a critical role in antagonism of
coreceptor activity. The brain is a target organ for recreational drugs and HIV-1. Epidemiological
data demonstrate that opioid abuse is a risk factor for HIV-1 infection and
progression to AIDS. Chemokines and their receptors have been implicated in the
neuropathogenesis of HIV-1 infections. However, little is known about the
effects of opioids on the expression of chemokines and their receptors (the
latter also are HIV-1 coreceptors) by cells of the CNS. Herein we describe the
effects of morphine on gene expression of the alpha- and beta-chemokines and
their receptors by the astrocytoma cell line U87 and by primary normal human
astrocyte (NHA) cultures. U87 cells treated with morphine showed significant
down-regulation of IL-8 gene expression, whereas expression of the IL-8 receptor
CXCR2 was reciprocally up-regulated as detected by RT-PCR. Treatment of NHAs
with morphine suppressed IL-8 and macrophage-inflammatory protein-1beta gene
expression, whereas expression of their receptor genes, CCR3 and CCR5, was
simultaneously enhanced. These morphine-induced effects on U87 and NHA cells
were reversed by the opioid mu receptor antagonist beta-funaltrexamine. Morphine
also enhanced the constitutive expression of the opioid mu receptor on
astroglial cells. Our results support the hypothesis that opioids play a
significant role in the susceptibility of the CNS to HIV-1 infection and
subsequent encephalopathy by inhibiting local production of HIV-1-protective
chemokines (IL-8 and macrophage-inflammatory protein-1beta) and enhancing
expression of HIV-1 entry coreceptor genes (CCR3, CCR5, and CXCR2) within the
CNS. These effects of opioids appear to be mediated through the opioid mu
receptor that we demonstrated on astroglial cells. To examine the significance of chemokine activation of CXCR2 in wound healing
after chemical burn, cutaneous injury was created by topical application of
nitrogen mustard on CXCR2 wild type (+/+), heterozygous (+/-), and knockout
(-/-) mice. Wounds were analyzed histologically for neutrophil and monocyte
infiltration and for reepithelialization at postwound days 4, 7, and 10.
Neutrophil recruitment to the wound site was reduced through postwound day 7 in
CXCR2 -/- mice as indicated by myeloperoxidase assay and by visual quantitation.
Because there is always concern that mice with targeted deletion of a specific
receptor may undergo developmental adaptations to offset the loss of the
receptor, we also accessed chemical wound repair in the presence of a small
molecule antagonist of CXCR2. Dietary supplementation with a CXCR2 antagonist
(SB-265610) during the wound repair process also markedly delayed healing
parameters in CXCR2 +/+ mice, even greater than treatment with glucocorticoids.
These parallel studies further establish that mice deficient in CXCR2 function
exhibit delayed cutaneous wound healing that may be primarily linked to impaired
neutrophil recruitment after chemical burn with nitrogen mustard. Thus, there
may be a potential therapeutic benefit of treating nitrogen mustard-induced skin
lesions with agonists of CXCR2 to facilitate the wound repair process. PURPOSE: This study aims to investigate the role of gastrin-17 (G17) on
angiogenesis features in gliomas both in vitro and in vivo.
EXPERIMENTAL DESIGN: The influences of G17 and G17 receptor antagonists were
characterized in vitro in terms of angiogenesis on human umbilical vein
endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with
respect to U373 orthotopic glioma xenografts. The influence of
phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB
inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis.
G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced
modifications in nuclear factor-kappaB DNA binding activity were characterized
by means of specific enzyme-linked immunosorbent assays. The influence of G17 on
E- and P-selectin expression was determined by means of computer-assisted
microscopy, whereas the influence of E- and P-selectin on HUVEC migration was
approached by means of antisense oligonucleotides. The chemotactic influence of
G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted
videomicroscopy with Dunn chambers.
RESULTS: Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors
were present in HUVECs and microvessels dissected from a human glioblastoma.
Whereas G17 significantly increased the levels of angiogenesis in vivo in the
U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor
antagonist L365,260 significantly counteracted the G17-mediated proangiogenic
effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor
alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these
endothelial cells. Gastrin significantly (but only transiently) decreased the
level of expression of E-selectin, but not P-selectin, whereas IL-8 increased
the expression of E-selectin. Specific antisense oligonucleotides against E- and
P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on
Matrigel.
CONCLUSIONS: The present study shows that gastrin has marked proangiogenic
effects in vivo on experimental gliomas and in vitro on HUVECs. This effect
depends in part on the level of E-selectin activation, but not on IL-8
expression/release by HUVECs. Substance P analogues, including [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP (SPA) are
broad-spectrum G protein-coupled receptor (GPCR) antagonists that have potential
antitumorigenic activities, although the mechanism(s) are not completely
understood. Here, we examined the effects of SPA in ductal pancreatic cancers
that express multiple GPCRs for mitogenic agonists and also produce
proangiogenic chemokines. Using HPAF-II, a well-differentiated pancreatic cancer
cell line as our model system, we showed that SPA inhibited multiple
neuropeptide-induced Ca(2+) mobilization, DNA synthesis, and
anchorage-independent growth in vitro. SPA also significantly attenuated the
growth of HPAF-II tumor xenografts in nude mice beyond the treatment period.
Interestingly, SPA markedly increased apoptosis but moderately decreased
proliferation marker, Ki-67 in the tumor xenografts implying additional
mechanism(s) for the significant growth inhibitory effect observed in vivo.
HPAF-II cells express ELR(+) CXC chemokines, including IL-8/CXCL8, which bind to
CXCR2 (a member of GPCR superfamily) and promote angiogenesis in multiple
cancers, including pancreatic cancer. SPA inhibited CXCR2-mediated Ca(2+)
mobilization and blocked specifically IL-8/CXCL8-induced angiogenesis in rat
corneal micropocket assay in vivo. A salient feature of the results presented
here is that SPA markedly reduced tumor-associated angiogenesis in the HPAF-II
xenografts in vivo. Our results show that SPA, a broad-spectrum GPCR antagonist
attenuates tumor growth in pancreatic cancer via a dual mechanism involving both
the antiproliferative and antiangiogenic properties. We conclude that this novel
dual-inhibitory property of SPA could be of significant therapeutic value in
pancreatic cancer, when used in combination with other antiproliferative and/or
antiangiogenic agents. Growth-related oncogene (GRO), a member of the CXC chemokine subfamily, plays a
major role in inflammation and wound healing. CXC chemokines have been found to
be associated with tumorigenesis, angiogenesis, and metastasis. Although
elevated expression of GRO has been reported in several human cancers, the
expression and role of GRO and its receptor, CXCR2, in esophageal cancer are
poorly understood. This study used real-time reverse transcription-PCR (RT-PCR)
and immunohistochemical approaches to show that GROalpha, GRObeta, and CXCR2 are
up-regulated in esophageal tumor tissue. Furthermore, GROalpha, GRObeta, and
CXCR2 are constitutively expressed in WHCO1, an esophageal cancer cell line that
was used as a model system here. GRObeta enhances transcription of EGR-1, via
the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be
blocked by a specific antagonist of CXCR2 (SB 225002) or specific antibody to
GRObeta. WHCO1 cells treated with SB 225002 exhibited a 40% reduction in cell
proliferation. A stable WHCO1 GROalpha RNA interference (RNAi) clone displayed a
43% reduction in GROalpha mRNA levels as determined by real-time RT-PCR, reduced
levels of GROalpha by fluorescence microscopy, and a 60% reduction in the levels
of phosphorylated ERK1/2. A stable clone expressing GRObeta RNAi displayed >95%
reduction in GRObeta mRNA levels, reduced levels of GRObeta by fluorescence
microscopy, and an 80% reduction in the levels of phosphorylated ERK1/2.
Moreover, these GROalpha RNAi- and GRObeta RNAi-expressing clones displayed a
20% and 50% decrease in cell proliferation, respectively. Our results suggest
that GROalpha-CXCR2 and GRObeta-CXCR2 signaling contributes significantly to
esophageal cancer cell proliferation and that this autocrine signaling pathway
may be involved in esophageal tumorigenesis. Although originally identified as mediators of inflammation, it is now apparent
that chemokines play a fundamental role in tissue development. In this study,
ELR(+)-CXC chemokine family members CXCL2 and CXCL7, along with their preferred
receptor CXCR2, were expressed at the earliest stages of metanephric development
in the rat, and signaling through this receptor was required for the survival
and maintece of the undifferentiated metanephric mesenchyme (MM). A specific
antagonist of the CXCR2 receptor SB225002 induced apoptosis in this population
but did not affect more mature structures or cells in the ureteric bud. CXCL7
treatment of isolated MM elicited an angiogenic response by upregulation of
matrix metalloprotease 9 and endothelial and mesangial markers
(platelet-endothelial cell adhesion molecule, Megsin, Thy-1, PDGF receptor
alpha, and vascular alpha-actin) and induced SB225002-sensitive cell invasion
through a matrix. Because Wilms' tumor cells may similarly depend on CXCR2
signaling for survival, primary tumor samples were analyzed, and 15 of 16 Wilms'
tumors were found to be CXCR2 positive, whereas grossly normal kidney tissues
from tumor patients or renal cell carcinomas were CXCR2 negative. Furthermore,
cell lines derived from Wilms' tumors but not those from renal cell carcinomas
were sensitive to SB225002-induced apoptosis. These data provide evidence for a
prosurvival and proangiogenic role of ELR(+)-CXC chemokines and their receptor
CXCR2 during metanephric development and suggest a novel mechanism for
chemotherapeutic intervention in Wilms' tumor. Benzo(a)pyrene (BP) is an environmental contamit known to favor airway
inflammation likely through up-regulation of pro-inflammatory cytokines. The
present study was designed to characterize its effects toward interleukin-8
(IL-8), a well-established pulmonary inflammatory cytokine. In primary human
macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion
levels in a dose-dependent manner. Such an up-regulation was likely linked to
aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was
reduced after AhR expression knock-down through RNA interference. Moreover,
electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation
experiments showed BP-triggered binding of AhR to a consensus xenobiotic
responsive element (XRE) found in the human IL-8 promoter. Finally, BP
administration to mice led to over-expression of keratinocyte chemoattractant
(KC), the murine functional homologue of IL-8, in lung. It also triggered the
recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was
however fully abolished in the presence of a chemical antagonist of the KC/IL-8
receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of
KC in BP-induced lung inflammation. Overall, these data highlight an
AhR-dependent regulation of IL-8 in response to BP that likely contributes to
the airway inflammatory effects of this environmental chemical. Constitutive activation of nuclear factor (NF)-kappaB is linked with the
intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic
chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-kappaB
whose expression is elevated in AIPC. This study sought to determine the
significance of CXCL8 signaling in regulating the response of AIPC cells to
oxaliplatin, a drug whose activity is reportedly sensitive to NF-kappaB
activity. Administration of oxaliplatin to PC3 and DU145 cells increased
NF-kappaB activity, promoting antiapoptotic gene transcription. In addition,
oxaliplatin increased the transcription and secretion of CXCL8 and the related
CXC-chemokine CXCL1 and increased the transcription and expression of
CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which
transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells
with CXCL8 potentiated NF-kappaB activation in AIPC cells, increasing the
transcription and expression of NF-kappaB-regulated antiapoptotic genes of the
Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist,
AZ10397767 (Bioorg Med Chem Lett 18:798-803, 2008), attenuated
oxaliplatin-induced NF-kappaB activation, increased oxaliplatin cytotoxicity,
and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological
inhibition of NF-kappaBorRNA interference-mediated suppression of Bcl-2 and
survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results
further support NF-kappaB activity as an important determit of cancer cell
sensitivity to oxaliplatin and identify the induction of autocrine CXCR2
signaling as a novel mode of resistance to this drug. Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of
prostate cancer cells to undergo apoptosis. In this study, we investigated how
endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis
pathway by determining the sensitivity of LNCaP and PC3 cells to administration
of the death receptor agonist tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3
cell viability, coincident with increased levels of apoptosis and the
potentiation of IL-8 secretion. Administration of recombit human IL-8 was
shown to increase the mRNA transcript levels and expression of c-FLIP(L) and
c-FLIP(S), two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with
the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP
mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear
factor-kappaB-mediated transcription attenuated IL-8-induced c-FLIP expression
in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown
to induce spontaneous apoptosis in both cell lines and to sensitize these
prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel.
Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to
the apoptosis-inducing effects of recombit TRAIL, most likely due to the
ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of
c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8
signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells
through direct transcriptional regulation of c-FLIP. Therefore, targeted
inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an
attractive therapeutic strategy to sensitize this stage of disease to
chemotherapy. PURPOSE: Melanoma, the most aggressive form of skin cancer, accounts for 75% of
all skin cancer-related deaths and current therapeutic strategies are not
effective in advanced disease. In the current study, we have investigated the
efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1.
EXPERIMENTAL DESIGN: Human A375SM melanoma cells were treated with SCH-479833 or
SCH-527123, and their effect on proliferation, motility, and invasion was
evaluated in vitro. We examined the downstream signaling events in the cells
following treatment with antagonists. For in vivo studies, A375SM cells were
implanted subcutaneously into athymic nude mice followed by administration of
SCH-479833, SCH-527123, or hydroxypropyl-beta-cyclodextrin (20%) orally for 21
days and their effect on tumor growth and angiogenesis was evaluated.
RESULTS: Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell
proliferation, chemotaxis, and invasive potential in vitro. Treatment of
melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth.
Histologic and histochemical analyses showed significant (P < 0.05) decreases in
tumor cell proliferation and microvessel density in tumors. Moreover, we
observed a significant increase in melanoma cell apoptosis in SCH-479833- or
SCH-527123-treated animals compared with controls.
CONCLUSION: Together, these studies show that selectively targeting CXCR2/CXCR1
with orally active small-molecule inhibitors is a promising therapeutic approach
for inhibiting melanoma growth and angiogenesis. The prostaglandin F(2alpha) (PGF(2alpha)) receptor (FP) is elevated in
endometrial adenocarcinoma. This study found that PGF(2alpha) signaling via FP
regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial
adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated
in cancer tissue compared with normal endometrium and localized to glandular
epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably
transfected with the FP receptor (FPS cells) with 100 nmol/L PGF(2alpha)
increased CXCL1 promoter activity, mRNA, and protein expression, and these
effects were abolished by cotreatment of cells with FP antagonist or chemical
inhibitors of Gq, epidermal growth factor receptor, and extracellular
signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L
PGF(2alpha) in endometrial adenocarcinoma explant tissue. CXCL1 is a potent
neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils
in endometrial adenocarcinoma and increased neutrophils were present in
endometrial adenocarcinoma compared with normal endometrium. Conditioned media
from PGF(2alpha)-treated FPS cells stimulated neutrophil chemotaxis, which could
be abolished by CXCL1 protein immunoneutralization of the conditioned media or
antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from
inoculation with FPS cells showed increased neutrophil infiltration compared
with tumors arising from wild-type cells or following treatment of mice bearing
FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a
novel PGF(2alpha)-FP pathway that may regulate the inflammatory microenvironment
in endometrial adenocarcinoma via neutrophil chemotaxis. BACKGROUND: We determined how CXC-chemokine signalling and necrosis
factor-kappaB (NF-kappaB) activity affected heat-shock protein 90 (Hsp90)
inhibitor (geldanamycin (GA) and 17-allylamino-demethoxygeldanamycin (17-AAG))
cytotoxicity in castrate-resistant prostate cancer (CRPC).
METHODS: Geldanamycin and 17-AAG toxicity, together with the CXCR2 antagonist
AZ10397767 or NF-kappaB inhibitor BAY11-7082, was assessed by 3-(4,
5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in two CRPC
lines, DU145 and PC3. Flow cytometry quantified apoptotic or necrosis profiles.
Necrosis factor-kappaB activity was determined by luciferase readouts or
indirectly by quantitative PCR and ELISA-based determination of CXCL8
expression.
RESULTS: Geldanamycin and 17-AAG reduced PC3 and DU145 cell viability, although
PC3 cells were less sensitive. Addition of AZ10397767 increased GA (e.g., PC3
IC(20): from 1.67+/-0.4 to 0.18+/-0.2 nM) and 17-AAG (PC3 IC(20): 43.7+/-7.8 to
0.64+/-1.8 nM) potency in PC3 but not DU145 cells. Similarly, BAY11-7082
increased the potency of 17-AAG in PC3 but not in DU145 cells, correlating with
the elevated constitutive NF-kappaB activity in PC3 cells. AZ10397767 increased
17-AAG-induced apoptosis and necrosis and decreased NF-kappaB activity/CXCL8
expression in 17-AAG-treated PC3 cells.
CONCLUSION: Ansamycin cytotoxicity is enhanced by inhibiting NF-kappaB activity
and/or CXC-chemokine signalling in CRPC cells. Detecting and/or inhibiting
NF-kappaB activity may aid the selection and treatment response of CRPC patients
to Hsp90 inhibitors. Increased expression of lymphangiogenesis factors VEGF-C/D and heparanase has
been correlated with the invasion of cancer. Furthermore, chemokines may modify
matrix to facilitate metastasis, and they are associated with VEGF-C and
heparanase. The chemokine CXCL7 binds heparin and the G-protein-linked receptor
CXCR2. We investigated the effect of CXCR2 blockade on the expression of
VEGF-C/D, heparanase, and on invasion. CXCL7 siRNA and a specific antagonist of
CXCR2 (SB225002) were used to treat CXCL7 stably transfected MCF10AT cells.
Matrigel invasion assays were performed. VEGF-C/D expression and secretion were
determined by real-time PCR and ELISA assay, and heparanase activity was
quantified by ELISA. SB225002 blocked VEGF-C/D expression and secretion (P <
.01). CXCL7 siRNA knockdown decreased heparanase (P < .01). Both SB225002 and
CXCL7 siRNA reduced the Matrigel invasion (P < .01). The MAP kinase signaling
pathway was not involved. The CXCL7/CXCR2 axis is important for cell invasion
and the expression of VEGF-C/D and heparanase, all linked to invasion. CXCR1 and CXCR2 are G-protein coupled receptors, that have been shown to play
important role in tumor growth and metastasis, and are prime targets for the
development of novel therapeutics. Here, we report that targeting CXCR2 and
CXCR1 activity using orally active small molecule antagonist (SCH-527123,
SCH-479833) inhibits human colon cancer liver metastasis mediated by decreased
neovascularization and enhanced maligt cell apoptosis. There were no
differences in primary tumor growth. These studies demonstrate the important
role of CXCR2/1 in colon cancer metastasis and that inhibition of CXCR2 and
CXCR1, small molecule antagonists provides a novel therapeutic strategy. This study evaluated the relevance of CXCR2 chemokine receptors in oral squamous
cell carcinoma, by means of in vitro and in vivo approaches. The in vitro
incubation of the selective and non-peptide CXCR2 receptor antagonist
N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) Urea (SB225002; 25 to 800 nM)
produced a time- and concentration-dependent inhibition of SCC158 (rat) and HN30
(human) cell lines viability. Conversely, this antagonist did not significantly
affect the viability of the immortalized keratinocyte lineage, HaCaT.
Additionally, the incubation of human IL-8 and rat CINC-1 CXCR2 agonists
produced a concentration-related increase on HN30 and SCC158 proliferation. The
submucosal injection of SCC158 cells (5 × 10(6) cells) into the tongue of
Fischer 344 rats induced tumor development, which displayed typical clinical
features. Immunohistochemical analysis of rat tongue biopsies revealed a marked
increase of CXCR2 receptor immunoreactivity, which was accompanied by augumented
expression of VEGF and caspase-3. Our data suggests an important role for CXCR2
receptors in oral squamous cell carcinoma. BACKGROUND: The current study was undertaken to characterize the effect of
anti-metabolites on inducing CXCL8 signaling and determining whether the
constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer
(CaP) cells modulates their sensitivity to this class of agent.
METHODS: The response of metastatic CaP cells to 5-Fluorouracil (5-FU),
Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and
PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to
determine effects of drugs or CXCL8 administration on target gene/protein
expression.
RESULTS: Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion
and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells.
Consistent with this, the inhibition of CXCL8 signaling using a CXCR2
antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001),
and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while
administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the
CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of
PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent
experiments confirmed that administration of recombit human CXCL8 increased
TS expression, a response mediated in part by the CXCR2 receptor. Moreover,
siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the
sensitivity of PC3 cells to 5-FU.
CONCLUSIONS: CXCL8 signaling provides a selective resistance of metastatic
prostate cancer cells to specific anti-metabolites by promoting a
target-associated resistance, in addition to underpinning an evasion of
treatment-induced apoptosis. Prostate cancer is the most common maligcy in Western countries. Chemokine
C-X-C motif receptor 1 (CXCR1) and CXCR2 play a key role in generation and
regulation of CXC chemokine signaling. CXCR1 is a receptor for interleukin 8
(IL8), a pro-inflammatory chemokine, and CXCR1/2 are crucially involved in the
prostate cancer development and progression. Thus, we generated a high-affinity
human CXCR1/CXCR2 inhibitor, CXCL8 (3-72) K11R/G31P, named G31P, which is a
synthetic derivative of the human cytokine, IL-8. In this study, we investigated
the effects of G31P on regulation of prostate cancer cell growth in vitro and in
nude mouse xenografts. Cell viability, adhesion, and wound healing assays were
used to assess the effects of G31P on growth, adhesion, and migration of PC-3
human prostate cancer cells in vitro, respectively. Nude mouse xenografts and
xenograft implantation assays were performed to determine the effect of G31P on
PC-3 cells in vivo. Immunohistochemistry was used to detect gene expression, and
fluorescence imaging was used to detect tumor volume and microvessel density in
tumor xenografts. The data showed that G31P treatment significantly reduced PC-3
cell viability, adhesion and migration capacity in a dose-dependent manner (up
to 100 ng/ml). Additionally, G31P treatment of nude mice suppressed the growth
of orthotopically transplanted tumor xenografts. G31P also inhibited tumor
tissue vascularization, which was associated with the decreased expression of
vascular endothelial growth factor and nuclear transcription factor (NF)-κB in
orthotopic xenograft tissues. This study provides evidence that G31P, a CXCR1/2
inhibitor, may effectively control prostate cancer. CXCR2 in non-small cell lung cancer (NSCLC) has been studied mainly in stromal
cells and is known to increase tumor inflammation and angiogenesis. Here, we
examined the prognostic importance of CXCR2 in NSCLC and the role of CXCR2 and
its ligands in lung cancer cells. The effect of CXCR2 expression on tumor cells
was studied using stable knockdown clones derived from a murine KRAS/p53-mutant
lung adenocarcinoma cell line with high metastatic potential and an orthotopic
syngeneic mouse model and in vitro using a CXCR2 small-molecule antagonist
(SB225002). CXCR2 protein expression was analyzed in tumor cells from 262 NSCLC.
Gene expression profiles for CXCR2 and its ligands (CXCR2 axis) were analyzed in
52 human NSCLC cell lines and 442 human lung adenocarcinomas. Methylation of
CXCR2 axis promoters was determined in 70 human NSCLC cell lines. Invasion and
metastasis were decreased in CXCR2 knockdown clones in vitro and in vivo.
SB225002 decreased invasion in vitro. In lung adenocarcinomas, CXCR2 expression
in tumor cells was associated with smoking and poor prognosis. CXCR2 axis gene
expression profiles in human NSCLC cell lines and lung adenocarcinomas defined a
cluster driven by CXCL5 and associated with smoking, poor prognosis, and RAS
pathway activation. Expression of CXCL5 was regulated by promoter methylation.
The CXCR2 axis may be an important target in smoking-related lung
adenocarcinoma. Recent evidence indicates that CXCR2 signaling is crucial for cancer
progression, and its antagonist SB225002 induces apoptosis in Wilms' tumor
cells. Here, we investigated the effect of SB225002 on cell cycle progression
and apoptosis induction in vitro, using CDDP-sensitive and -resistant OVCA cell
lines with different p53 status (wild type, mutant or null). Adenovirus
infection of wild-type p53 or transfection of p53 siRNA was used to over-express
or knock-down p53. Cell cycle and apoptosis were determined by flow cytometry or
Hoechst staining and observation of nuclear morphology. Our data demonstrated
that SB225002 induced apoptosis in both wild-type and p53-deficient ovarian
cancer (OVCA) cells through alternative mechanisms. SB225002 promoted mitotic
catastrophe, as evidenced by the accumulation of mitotic cells with spindle
abnormalities, chromosome mis-segregation, multi-polar cell division, multiple
nuclei, aneuploidy/polyploidy and subsequent extensive apoptosis.
SB225002-induced mitotic catastrophe appeared to be mediated by down-regulation
of checkpoint kinase Chk1 and Cdk1-cyclin B activation. In cells expressing
wild-type p53 (OV2008 and C13*), SB225002 increased total and phospho-Ser p53
levels, and p53 knock-down decreased SB225002-induced apoptosis, without
affecting premature mitosis. These results suggest that SB225002 induces
p53-dependent apoptosis, and provokes mitotic catastrophe in p53-independent
manner in p53 wild-type cells. Reconstitution with wild-type P53 in P53-null
SKOV3 cell attenuated SB225002-induced mitotic catastrophe, suggesting p53
prevented mitotic catastrophe induced by SB225002 in p53-deficient OVCA cells.
Finally, the effect of SB225002 could not be prevented by pretreatment with
CXCR2 ligand or its neutralizing antibody. The present studies demonstrate for
the first time that SB225002 has dual actions in OVCA cells, inducing classic
apoptosis through p53 activation and provoking mitotic catastrophe in both p53
wild-type and deficient cells by Chk1 inhibition and Cdk activation. These
findings raise the possibility of SB225002 as a new candidate molecule for OVCA
therapy independent of the p53 status. |
What is the genomic structure of the FAA (FANCA) gene? | The FAA (FANCA) gene contains 43 exons spanning approximately 80 kb. Exons range from 34 to 188 bp, whereas sequence analysis of the 5' region upstream of the putative transcription start site showed no obvious TATA and CAAT boxes, but did show a GC-rich region, typical of housekeeping genes. | |
What is Snord116? | SNORD116 is a small nucleolar (sno) RNA gene cluster (HBII-85) implicated as a major contributor the Prader-Willi phenotype.
SNORD116 genes appears to be responsible for the major features of PWS.
SNORD116 is a paternally expressed box C/D snoRNA gene cluster.
The mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs.
Snord116 expression in the medial hypothalamus, particularly within nuclei that are part of feeding circuitry. High expression of Snord116 was evident in the paraventricular (PVN) and ventromedial (VMH) nuclei, but particularly prevalent in the arcuate nucleus (ARC) according to in situ hybridization. Snord116 expression level in ventral hypothalamic dissections including ARC was significantly greater (by 2-fold) than that in cortex; and its expression level in dorsal hypothalamic dissections including PVN was double that in cortex. The enhanced expression pattern of Snord116 in hypothalamic nuclei was observed at weaning and young adult stages, but less obvious neonatally when expression was significantly more widespread. Therefore the expression of Snord116 likely is regulated developmentally.
Snord116del mice with paternally derived deletion lack expression of this snoRNA. They have early-onset postnatal growth deficiency, but normal fertility and lifespan. While pituitary structure and somatotrophs are normal, liver Igf1 mRNA is decreased. In cognitive and behavior tests, Snord116del mice are deficient in motor learning and have increased anxiety. Around three months of age, they develop hyperphagia, but stay lean on regular and high-fat diet. On reduced caloric intake, Snord116del mice maintain their weight better than wild-type littermates, excluding increased energy requirement as a cause of hyperphagia. Normal compensatory feeding after fasting, and ability to maintain body temperature in the cold indicate normal energy homeostasis regulation. Metabolic chamber studies reveal that Snord116del mice maintain energy homeostasis by altered fuel usage. Prolonged mealtime and increased circulating ghrelin indicate a defect in meal termination mechanism. Snord116del mice, the first snoRNA deletion animal model, reveal a novel role for a non-coding RNA in growth and feeding regulation. | Prader-Willi syndrome (PWS) is a neurobehavioral disorder manifested by
infantile hypotonia and feeding difficulties in infancy, followed by morbid
obesity secondary to hyperphagia. It is caused by deficiency of paternally
expressed transcript(s) within the human chromosome region 15q11.2. PWS patients
harboring balanced chromosomal translocations with breakpoints within small
nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence
for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA)
genes encoded downstream of SNRPN. In addition, recently published data provide
strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85)
in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic,
and molecular analyses including chromosome analysis, array comparative genomic
hybridization (array CGH), expression studies, and single-nucleotide
polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2
microdeletion on an 11-year-old child expressing the major components of the PWS
phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the
larger Prader-Willi/Angelman syndrome critical region that included the SNORD116
cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided
evidence in support of the deletion being on the paternal chromosome 15. This
child also met most of the major PWS diagnostic criteria including infantile
hypotonia, early-onset morbid obesity, and hypogonadism. Identification and
characterization of this case provide unequivocal evidence for a critical role
for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for
genomic copy-number changes in cases with complex phenotypes is proving to be
invaluable in detecting novel alterations and enabling better genotype-phenotype
correlations. The imprinted Snurf-Snrpn chromosomal domain contains two large arrays of
tandemly repeated, paternally expressed box C/D small-nucleolar RNA (snoRNA)
genes: the SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters believed
to play key roles in the fine-tuning of serotonin receptor (5-HT2C) pre-mRNA
processing and in the etiology of the Prader-Willi Syndrome (PWS), respectively.
SNORD115 and SNORD116 were recently proposed to undergo significant conversion
into shorter RNA species, the so-called psnoRNAs. Here, we provide evidence that
argues against the existence of abundant psnoRNAs in human or mouse brain.
Instead, we characterize a previously unsuspected low-abundance,
fibrillarin-associated SNORD115-derived smaller RNA species. Based on these
findings, we strongly recommend that PWS-encoded SNORD115 and SNORD116 be
considered as bona fide box C/D snoRNAs. The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of
imprinted genes expressed from the paternal chromosome only. Whereas loss of
function of the SNORD116 genes appears to be responsible for the major features
of PWS, the role of the other genes is less clear. One of these genes is
C15orf2, which has no orthologues in rodents, but appears to be under strong
positive selection in primates. C15orf2 encodes a 1156 amino acid protein with
six nuclear localisation sequences. By protein BLAST analysis and InterProScan
signature recognition search, we found sequence similarity of C15orf2 to the
nuclear pore complex (NPC) protein POM121. To determine whether C15orf2 is
located at nuclear pores, we generated a stable cell line that inducibly
expresses FLAG-tagged C15orf2 and performed immunocytochemical studies. We found
that C15orf2 is present at the nuclear periphery, where it colocalizes with NPCs
and nuclear lamins. At very high expression levels, we observed invaginations of
the nuclear envelope. Extending these observations to three-dimensional
structured illumination microscopy, which achieves an 8-fold improved volumetric
resolution over conventional imaging, we saw that C15orf2 is located at the
inner face of the nuclear envelope where it strongly associates with the NPC. In
nuclear envelope isolation and fractionation experiments, we detected C15orf2 in
the NPC and lamina fractions. These experiments for the first time demonstrate
that C15orf2 is part of the NPC or its associated molecular networks. Based on
our findings, we propose 'Nuclear pore associated protein 1' as the new name for
C15orf2. Genetic analyses were performed in a male patient with suspected Prader-Willi
syndrome who presented with hypogonadism, excessive eating, central obesity,
small hands and feet and cognition within the low normal range. However, he had
no neonatal hypotonia or feeding problems during infancy. Chromosome analysis
showed a normal male karyotype. Further analysis with array-CGH identified a
mosaic 847 kb deletion in 15q11-q13, including SNURF-SNRPN, the snoRNA gene
clusters SNORD116 (HBII-85), SNORD115, (HBII-52), SNORD109 A and B (HBII-438A
and B), SNORD64 (HBII-13), and NPAP1 (C15ORF2). MLPA confirmed the deletion and
the results were compatible with a paternal origin. Metaphase-FISH verified the
mosaicism with the deletion present in 58% of leukocytes analyzed. Three smaller
deletions in this region have previously been reported in patients with
Prader-Willi syndrome phenotype. All three deletions included SNORD116, but only
two encompassed parts of SNURF-SNRPN, implicating SNORD116 as the major
contributor to the Prader-Willi phenotype. Our case adds further information
about genotype-phenotype correlation and supports the hypothesis that SNORD116
plays a major role in the pathogenesis of Prader-Willi syndrome. Furthermore, it
examplifies diagnostic difficulties in atypical cases and illustrates the need
for additional testing methods when Prader-Willi syndrome is suspected. |
What is the functional role of the protein Drp1? | Drp1 is involved in the regulation of mitochondrial fission. | The endoplasmic reticulum (ER) can elicit proapoptotic signalling that results
in transmission of Ca(2+) to the mitochondria, which in turn stimulates
recruitment of the fission enzyme DRP1 to the surface of the organelle. Here, we
show that BH3-only BIK activates this pathway at the ER in intact cells,
resulting in mitochondrial fragmentation but little release of cytochrome c to
the cytosol. The BIK-induced transformations in mitochondria are dynamic in
nature and involve DRP1-dependent remodelling and opening of cristae, where the
major stores of cytochrome c reside. This novel function for DRP1 is distinct
from its recognized role in regulating mitochondrial fission. Selective
permeabilization of the outer membrane with digitonin confirmed that BIK
stimulation results in mobilization of intramitochondrial cytochrome c. Of note,
BIK can cooperate with a weak BH3-only protein that targets mitochondria, such
as NOXA, to activate BAX by a mechanism that is independent of DRP1 enzyme
activity. When expressed together, BIK and NOXA cause rapid release of mobilized
cytochrome c and activation of caspases. Dorsal root ganglia (DRG) neurons degenerate in diabetic neuropathy (DN) and
exhibit mitochondrial damage. We studied mitochondria of cultured DRG neurons
exposed to high glucose as an in vitro model of DN. High glucose sequentially
increases the expression, activation and localization of the pro-apoptotic
proteins Bim and Bax and the mitochondrial fission protein dynamin-regulated
protein 1 (Drp1). High glucose causes association of Drp1/Bax, similar to other
apoptotic stimuli. Collectively, these events promote mitochondrial
fragmentation and reduce mitochondrial number, suggestive of apoptotic
mitochondrial fission. Drp1 is also upregulated in DRG from experimentally
diabetic rats, suggesting a role for mitochondrial fission in DN. Insulin-like
growth factor-I (IGF-I) protects high glucose-treated DRG neurons by preventing
mitochondrial accumulation of Bim and Bax but does not modulate Drp1 expression
or localization. We propose that mitochondria are compromised by convergence of
Bim/Bax proteins with Drp1, which contributes to high glucose-induced injury in
DRG neurons. Mitochondria in cells comprise a tubulovesicular network shaped continuously by
complementary fission and fusion events. The mammalian Drp1 protein plays a key
role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in
the balance between these opposing processes can occur rapidly, indicating that
modifications to these proteins may regulate mitochondrial membrane dynamics. We
highlight posttranslational modifications of the mitochondrial fission protein
Drp1, for which these regulatory mechanisms are best characterized. This
dynamin-related GTPase undergoes a number of steps to mediate mitochondrial
fission, including translocation from cytoplasm to the mitochondrial outer
membrane, higher-order assembly into spirals, GTP hydrolysis associated with a
conformational change and membrane deformation, and ultimately disassembly. Many
of these steps may be influenced by covalent modification of Drp1. We discuss
the dynamic nature of Drp1 modifications and how they contribute not only to the
normal regulation of mitochondrial division, but also to neuropathologic
processes. In response to many apoptotic stimuli, oligomerization of Bax is essential for
mitochondrial outer membrane permeabilization and the ensuing release of
cytochrome c. These events are accompanied by mitochondrial fission that appears
to require Drp1, a large GTPase of the dynamin superfamily. Loss of Drp1 leads
to decreased cytochrome c release by a mechanism that is poorly understood. Here
we show that Drp1 stimulates tBid-induced Bax oligomerization and cytochrome c
release by promoting tethering and hemifusion of membranes in vitro. This
function of Drp1 is independent of its GTPase activity and relies on arginine
247 and the presence of cardiolipin in membranes. In cells, overexpression of
Drp1 R247A/E delays Bax oligomerization and cell death. Our findings uncover a
function of Drp1 and provide insight into the mechanism of Bax oligomerization. Bcl-2/adenovirus E1B 19-kDa interacting protein 1 (BNIP1), which is
predomitly localized to the endoplasmic reticulum (ER), is a pro-apoptotic
Bcl-2 homology domain 3 (BH3)-only protein. Here, we show that the expression of
BNIP1 induced not only a highly interconnected ER network but also mitochondrial
fragmentation in a BH3 domain-dependent manner. Functional analysis demonstrated
that BNIP1 expression increased dynamin-related protein 1 (Drp1) expression
followed by the mitochondrial translocation of Drp1 and subsequent mitochondrial
fission. Both BNIP1-induced mitochondrial fission and the translocation of Drp1
were abrogated by Bcl-2 overexpression. These results collectively indicate that
ER-specific BNIP1 plays an important role in mitochondrial dynamics by
modulating the mitochondrial fission protein Drp1 in a BH3 domain-dependent
fashion. The mechanoenzyme dynamin-related protein 1 (Drp1) hydrolyzes GTP to power
mitochondrial fission, a process required for organelle biogenesis, quality
control, transport, and apoptosis. The pleckstrin homology domain of dynamin is
essential for targeting to and severing of lipid tubules, but the function of
the corresponding variable domain (VD, or insert B) of Drp1 is unknown. We
replaced the VD of Drp1 with a panel of linker sequences of varying length and
secondary structure composition and found that the VD is dispensable for
mitochondrial recruitment, association with the Drp1-anchoring protein Mff
(mitochondrial fission factor), and basal and protonophore-induced mitochondrial
fragmentation. Indeed, several ΔVD mutants constitutively localized to the outer
mitochondrial membrane (OMM) and fragmented mitochondria more efficiently than
wild-type Drp1. Consistent with an autoinhibitory role of the VD, we identified
Arg-376 in the Drp1 stalk domain as necessary for Mff interaction, assembly into
spirals, and mitochondrial fission. Switching the length of N- and C-terminal
α-helical segments in the VD-replacing linker converted Drp1 from constitutively
active and OMM-localized to inactive and cytosolic. Other hypoactive ΔVD mutants
formed stable and characteristically shaped aggregates, including extended
filaments. Phosphorylation of a PKA site bordering the VD disassembled the
filamentous ΔVD mutant and accelerated cytosolic diffusion of full-length Drp1.
We propose a model for regulation of Drp1-dependent mitochondrial fission, in
which posttranslational modifications in or near the VD alter the conformation
of a membrane-proximal oligomerization interface to influence Drp1 assembly rate
and/or geometry. This in turn modulates Arg-376-dependent OMM targeting of Drp1
via multivalent interactions with Mff. We recently reported increased mitochondrial fission and decreased fusion,
increased amyloid beta (Aβ) interaction with the mitochondrial fission protein
Drp1, increased mitochondrial fragmentation, impaired axonal transport of
mitochondria and synaptic degeneration in neurons affected by AD. In the present
study, we extended our previous investigations to determine whether
phosphorylated tau interacts with Drp1 and to elucidate mitochondrial damage in
the progression of AD. We also investigated GTPase activity, which is critical
for mitochondrial fragmentation, in postmortem brain tissues from patients with
AD and brain tissues from APP, APP/PS1 and 3XTg.AD mice. Using
co-immunoprecipitation and immunofluorescence analyses, for the first time, we
demonstrated the physical interaction between phosphorylated tau and Drp1.
Mitochondrial fission-linked GTPase activity was significantly elevated in the
postmortem frontal cortex tissues from AD patients and cortical tissues from
APP, APP/PS1 and 3XTg.AD mice. On the basis of these findings, we conclude that
Drp1 interacts with Aβ and phosphorylated tau, likely leading to excessive
mitochondrial fragmentation, and mitochondrial and synaptic deficiencies,
ultimately possibly leading to neuronal damage and cognitive decline. Treatment
designed to reduce the expression of Drp1, Aβ and/or phosphorylated tau may
decrease the interaction between Drp1 and phosphorylated tau and the interaction
between Drp1 and Aβ, conferring protection to neurons from toxic insults of
excessive Drp1, Aβ and/or phosphorylated tau. AIMS: Glucocorticoids, such as dexamethasone, enhance hepatic energy metabolism
and gluconeogenesis partly through changes in mitochondrial function.
Mitochondrial function is influenced by the balance between mitochondrial fusion
and fission events. However, whether glucocorticoids modulate mitochondrial
function through the regulation of mitochondrial dynamics is currently unknown.
RESULTS: Here, we report that the effects of dexamethasone on mitochondrial
function and gluconeogenesis in hepatoma cells are dependent on the
mitochondrial fission protein dynamin-related protein 1 (Drp1). Dexamethasone
increased routine oxygen consumption, maximal respiratory capacity, superoxide
anion, proton leak, and gluconeogenesis in hepatoma cells. Under these
conditions, dexamethasone altered mitochondrial morphology, which was paralleled
by a large increase in Drp1 expression, and reduced mitofusin 1 (Mfn1) and Mfn2.
In vivo dexamethasone treatment also enhanced Drp1 expression in mouse liver. On
the basis of these observations, we analyzed the dependence on the Drp1 function
of dexamethasone effects on mitochondrial respiration and gluconeogenesis. We
show that the increase in mitochondrial respiration and gluconeogenesis induced
by dexamethasone are hampered by the inhibition of Drp1 function.
INNOVATION: Our findings provide the first evidence that the effects of
glucocorticoids on hepatic metabolism require the mitochondrial fission protein
Drp1.
CONCLUSION: In summary, we demonstrate that the mitochondrial effects of
dexamethasone both on mitochondrial respiration and on the gluconeogenic pathway
depend on Drp1. The mechano-enzyme dynamin-related protein 1 plays an important role in
mitochondrial fission and is implicated in cell physiology. Dysregulation of
Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage.
Drp1 shares structural and functional similarities with dynamin 1 with respect
to domain organization, ability to self-assemble into spiral-like oligomers and
GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1
have greatly improved the understanding of this prototypical member of the
dynamin superfamily. However, high-resolution structural information for
full-length human Drp1 covering the GTPase domain, the middle domain and the
GTPase effector domain (GED) is still lacking. In order to obtain mechanistic
insights into the catalytic activity, a nucleotide-free GTPase-GED fusion
protein of human Drp1 was expressed, purified and crystallized. Initial X-ray
diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped
crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a =
53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent
content of 42%. Expression of selenomethionine-labelled protein is currently in
progress. Here, the expression, purification, crystallization and X-ray
diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which
form a basis for more detailed structural and biophysical analysis. Mitochondria are highly dynamic and undergo constant fusion and fission that are
essential for maintaining physiological functions of cells. Although dysfunction
of mitochondria has been implicated in tumorigenesis, little is known about the
roles of mitochondrial dynamics in metastasis, the major cause of cancer death.
In the present study, we found a marked upregulation of mitochondrial fission
protein dynamin-related protein 1 (Drp1) expression in human invasive breast
carcinoma and metastases to lymph nodes. Compared with non-metastatic breast
cancer cells, mitochondria also were more fragmented in metastatic breast cancer
cells that express higher levels of total and active Drp1 and less mitochondrial
fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in
mitochondria elongation or clusters, respectively, and significantly suppressed
metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins
led to mitochondrial fragmentation and enhanced metastatic abilities of breast
cancer cells. Interestingly, these manipulations of mitochondrial dynamics
altered the subcellular distribution of mitochondria in breast cancer cells. For
example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia
formation, a key step for cancer metastasis, and suppressed
chemoattractant-induced recruitment of mitochondria to lamellipodial regions.
Conversely, silencing Mfn proteins resulted in more cell spreading and
lamellipodia formation, causing accumulation of more mitochondria in
lamellipodia regions. More importantly, treatment with a mitochondrial
uncoupling agent or adenosine triphosphate synthesis inhibitor reduced
lamellipodia formation and decreased breast cancer cell migration and invasion,
suggesting a functional importance of mitochondria in breast cancer metastasis.
Together, our findings show a new role and mechanism for regulation of cancer
cell migration and invasion by mitochondrial dynamics. Thus targeting
dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy
for suppressing breast cancer metastasis. RATIONALE: Closure of the ductus arteriosus (DA) is essential for the transition
from fetal to neonatal patterns of circulation. Initial PO2-dependent
vasoconstriction causes functional DA closure within minutes. Within days a
fibrogenic, proliferative mechanism causes anatomic closure. Though modulated by
endothelial-derived vasodilators and constrictors, O2 sensing is intrinsic to
ductal smooth muscle cells and oxygen-induced DA constriction persists in the
absence of endothelium, endothelin, and cyclooxygenase mediators. O2 increases
mitochondrial-derived H2O2, which constricts ductal smooth muscle cells by
raising intracellular calcium and activating rho kinase. However, the mechanism
by which oxygen changes mitochondrial function is unknown.
OBJECTIVE: The purpose of this study was to determine whether mitochondrial
fission is crucial for O2-induced DA constriction and closure.
METHODS AND RESULTS: Using DA harvested from 30 term infants during correction
of congenital heart disease, as well as DA from term rabbits, we demonstrate
that mitochondrial fission is crucial for O2-induced constriction and closure.
O2 rapidly (<5 minutes) causes mitochondrial fission by a cyclin-dependent
kinase- mediated phosphorylation of dynamin-related protein 1 (Drp1) at serine
616. Fission triggers a metabolic shift in the ductal smooth muscle cells that
activates pyruvate dehydrogenase and increases mitochondrial H2O2 production.
Subsequently, fission increases complex I activity. Mitochondrial-targeted
catalase overexpression eliminates PO2-induced increases in
mitochondrial-derived H2O2 and cytosolic calcium. The small molecule Drp1
inhibitor, Mdivi-1, and siDRP1 yield concordant results, inhibiting O2-induced
constriction (without altering the response to phenylephrine or KCl) and
preventing O2-induced increases in oxidative metabolism, cytosolic calcium, and
ductal smooth muscle cells proliferation. Prolonged Drp1 inhibition reduces DA
closure in a tissue culture model.
CONCLUSIONS: Mitochondrial fission is an obligatory, early step in mammalian O2
sensing and offers a promising target for modulating DA patency. Mitochondrial functions are altered in many human diseases including cancer.
Development of mitochondria-targeted therapies, either through restoring normal
mitochondrial function or promoting mitochondrial-induced cell death, is one of
the attractive strategies to improve the outcome of cancer treatment. Recent
advances have revealed the important functional involvement of mitochondrial
dynamics in cancer biology. Dynamin-related protein 1 (Drp1), a member of the
dynamin family of GTPases required for mitochondrial fission, has been found
upregulated in certain types of cancers, such as lung and breast cancers. In
addition, the roles of Drp1 in cell cycle progression, genome instability, cell
migration and apoptosis in cancer cells have also been recently uncovered. These
findings raise the possibility of targeting Drp1-mediated mitochondrial fission
as an effective therapy for treating cancer. This article explores the function
of Drp1 in cancer cells and discusses the theoretical basis for the development
of potential targeted therapy. Mitochondria are dynamic organelles forming a tubular network that is
continuously fusing and dividing to control their morphology and functions.
Recent literature has shed new light on a potential link between the dynamic
behavior of mitochondria and muscle development. In this study, we investigate
the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in
myogenic differentiation. We found that differentiation of C2C12 myoblasts
induced by serum starvation was accompanied by a gradual increase in Drp1
protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1
phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein
from the cytosol to mitochondria. During differentiation, treatment of myoblasts
with mitochondrial division inhibitor (mdivi-1), a specific inhibitor of Drp1
GTPase activity, caused extensive formation of elongated mitochondria, which
coincided with increased apoptosis evidenced by both enhanced caspase-3 activity
and increased number of terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated
myotubes (day 3 in differentiation media) showed a reduction in mitochondrial
DNA content, mitochondrial mass, and membrane potential in a dose-dependent
manner indicating defects in mitochondrial biogenesis during myogenic
differentiation. Most interestingly, mdivi-1 treatment significantly suppressed
myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable
overexpression of a domit negative mutant Drp1 (K38A) dramatically reduced
myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial
division is a necessary step for successful myogenic differentiation, and
perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations
during muscle development. Therefore, in the present study, we report a novel
physiological role of mitochondrial dynamics in myogenic differentiation. It is believed that mitochondrial dynamics is coordinated with endosomal traffic
rates during cytoskeletal remodeling, but the mechanisms involved are largely
unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling
by Src family kinases (SFK) to perturb cellular morphology, membrane traffic,
and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we
uncovered a functional connection between mitochondria-shaping proteins and the
small GTPase Rab11a, a key regulator of polarized transport via recycling
endosomes. We found that E4orf4 induced dramatic changes in the morphology of
mitochondria along with their mobilization at the vicinity of a polarized actin
network typifying E4orf4 action, in a manner controlled by SFK and Rab11a.
Mitochondrial remodeling was associated with increased proximity between Rab11a
and mitochondrial membranes, changes in fusion-fission dynamics, and
mitochondrial relocalization of the fission factor dynamin-related protein 1
(Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown
of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and
actin assembly, involving Rab11a-mediated mitochondrial dynamics in
E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich
structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and
contributed to the biogenesis of podosome rosettes. These findings suggest a
role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation
and unravel a novel functional interplay between Rab11a and mitochondria during
reshaping of the cell cytoskeleton, which would facilitate mitochondria
redistribution near energy-requiring actin-rich structures. BACKGROUND: Hypertension leads to cardiac hypertrophy as an adaptive response to
increased workload. While initial development of hypertrophy is compensatory
when contractile function is maintained, persistent stress on heart leads to
deteriorated cardiac function and onset of heart failure. Mitochondrial
dysfunction develops in the failing heart; however, whether it presents in
compensatory cardiac hypertrophy is controversial.
METHODS: Spontaneously hypertensive rats (SHRs) and age-matched normotensive
Wistar Kyoto rats were used in the study. Mitochondrial function and
remodeling-related mechanisms in the left ventricles were measured by enzyme
activity tests, Western blots, and reverse transcriptase polymerase chain
reaction.
RESULTS: Compensatory cardiac hypertrophy in SHR was indicated by higher
heart/weigh ratio, left ventricular systolic pressure and ±dp/dt(max) (P<.001,
P<.05, and P<.01, respectively). Enzyme activities of mitochondrial complex I
and II were significantly reduced (P<.05 and P<.01) in SHR in concert with
decreased expression of complex subunits (P<.01 for NDUFS3, P=.068 for SDHB, and
P<.05 for ATP5A1). Mitochondrial fission protein Drp1 was decreased (P<.05),
while fusion protein OPA1 was increased (P<.01). Parkin and SirT1/AMPK-PGC-1α
signaling, responsible for mitochondrial elimination and biogenesis
respectively, were decreased in SHR (P<.01 for Parkin, P<.001 for SirT1 and
p-AMPK).
CONCLUSION: Our results implicated that mitochondrial function and remodeling,
indicated by mitochondrial enzyme activities and remodeling-related molecules,
were compromised in compensatory hypertrophied myocardium of the SHR
hypertensive model.
SUMMARY: Mitochondrial function in compensatory hypertrophied myocardium is
controversial. Our present study found mitochondrial dysfunction in the left
ventricle of spontaneously hypertensive rats, which was possibly a result of
compromised mitochondrial remodeling including mitochondrial dynamics,
elimination, and biogenesis. Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is
required for mitochondrial fission in healthy and apoptotic cells. Drp1
activation is a complex process that involves translocation from the cytosol to
the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the
MOM, leading to membrane constriction/division. Similar to dynamins, Drp1
contains GTPase (G), bundle signaling element (BSE) and stalk domains. However,
instead of the lipid-interacting Pleckstrin Homology (PH) domain present in the
dynamins, Drp1 contains the so-called B insert or variable domain that has been
suggested to play an important role in Drp1 regulation. Different proteins have
been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1
acquires its fully functional status remains poorly understood. We found that
Drp1 can interact with pure lipid bilayers enriched in the
mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous
study, we now explore the specificity and functional consequences of this
interaction. We show that a four lysine module located within the B insert of
Drp1 interacts preferentially with CL over other anionic lipids. This
interaction dramatically enhances Drp1 oligomerization and assembly-stimulated
GTP hydrolysis. Our results add significantly to a growing body of evidence
indicating that CL is an important regulator of many essential mitochondrial
functions. It is well established that mitochondrial fragmentation plays a key role in the
pathogenesis of Alzheimer's disease (AD). Mitochondrial fission is mediated by
dynamin-related protein 1 (Drp1), which is highly expressed in nervous system
and regulated by various posttranslational modifications including
phosphorylation. We identified glycogen synthase kinase (GSK)3β-dependent Drp1
phosphorylation at Ser(40) and Ser(44), which increases Drp1 GTPase activity and
its mitochondrial distribution and could induce mitochondrial fragmentation.
Moreover, neurons transfected with Ser(40)Ser(44) phosphomimic Drp1 showed
increased mitochondria fragmentation and were more vulnerable to amyloid-β
(Aβ)-induced apoptosis. Therefore, blocking GSK3β-induced Drp1 phosphorylation
may be an effective way to protect neurons from Aβ toxicity. To address this, we
designed and synthesized an artificial polypeptide named TAT-Drp1-SpS, which
could specifically block GSK3β-induced Drp1 phosphorylation. Our results
demonstrated that TAT-Drp1-SpS treatment could significantly reduce Aβ-induced
neuronal apoptosis in cultured neurons. Notably, TAT-Drp1-SpS administration in
hippocampus Cornu Ammonis 1 (CA1) region significantly reduced Aβ burden and
rescued the memory deficits in AD transgenic mice. Although Aβ has multiple
targets to exert its neurotoxicity, our findings suggested that GSK3β-induced
mitochondrial fragmentation was, at least partially, mediated by Aβ toxicity and
contribute to the pathogenesis of AD. Taken together, GSK3β-induced Drp1
phosphorylation provides a novel mechanism for mitochondrial fragmentation in
AD, and our findings suggested a novel therapeutic strategy for AD. Drp1 is a dynamin-like GTPase that mediates mitochondrial and peroxisomal
division in a process dependent on self-assembly and coupled to GTP hydrolysis.
Despite the link between Drp1 malfunction and human disease, the molecular
details of its membrane activity remain poorly understood. Here we reconstituted
and directly visualized Drp1 activity in giant unilamellar vesicles. We
quantified the effect of lipid composition and GTP on membrane binding and
remodeling activity by fluorescence confocal microscopy and flow cytometry. In
contrast to other dynamin relatives, Drp1 bound to both curved and flat
membranes even in the absence of nucleotides. We also found that Drp1 induced
membrane tubulation that was stimulated by cardiolipin. Moreover, Drp1 promoted
membrane tethering dependent on the intrinsic curvature of the membrane lipids
and on GTP. Interestingly, Drp1 concentrated at membrane contact surfaces and,
in the presence of GTP, formed discrete clusters on the vesicles. Our findings
support a role of Drp1 not only in the formation of lipid tubes but also on the
stabilization of tightly apposed membranes, which are intermediate states in the
process of mitochondrial fission. RATIONALE: Both fusion and fission contribute to mitochondrial quality control.
How unopposed fusion affects survival of cardiomyocytes and left ventricular
function in the heart is poorly understood.
OBJECTIVE: We investigated the role of dynamin-related protein 1 (Drp1), a
GTPase that mediates mitochondrial fission, in mediating mitochondrial
autophagy, ventricular function, and stress resistance in the heart.
METHODS AND RESULTS: Drp1 downregulation induced mitochondrial elongation,
accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes
at baseline. Drp1 downregulation also suppressed autophagosome formation and
autophagic flux at baseline and in response to glucose deprivation in
cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted
Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy.
Mitochondrial elongation and accumulation of damaged mitochondria were also
observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1
downregulation, cardiac-specific Drp1 knockout mice developed left ventricular
dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks.
Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout
mice. Although left ventricular function in cardiac-specific Drp1 heterozygous
knockout mice was normal at 12 weeks of age, left ventricular function decreased
more severely after 48 hours of fasting, and the infarct size/area at risk after
ischemia/reperfusion was significantly greater in cardiac-specific Drp1
heterozygous knockout than in control mice.
CONCLUSIONS: Disruption of Drp1 induces mitochondrial elongation, inhibits
mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting
cardiac dysfunction and increased susceptibility to ischemia/reperfusion. Author information:
(1)Department of Protein Biochemistry, Institute of Life Science, Kurume
University, Kurume, Japan.
(2)Department of Protein Biochemistry, Institute of Life Science, Kurume
University, Kurume, Japan Department of Physiology and Cell Biology, Tokyo
Medical and Dental University, Tokyo, Japan.
(3)Department of Physiology and Cell Biology, Tokyo Medical and Dental
University, Tokyo, Japan.
(4)Department of Medicine, Division of Cardiovascular Medicine, Kurume
University School of Medicine, Kurume, Japan.
(5)Cardiovascular Research Institute, Kurume University, Kurume, Japan.
(6)Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki,
Japan.
(7)Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka,
Japan.
(8)Department of Physiology and Cell Biology, Tokyo Medical and Dental
University, Tokyo, Japan Department of Biochemistry and Molecular Biology,
Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.
(9)Department of Protein Biochemistry, Institute of Life Science, Kurume
University, Kurume, Japan Department of Molecular Biology, Graduate School of
Medical Science, Kyushu University, Fukuoka, Japan.
(10)Department of Protein Biochemistry, Institute of Life Science, Kurume
University, Kurume, Japan Department of Physiology and Cell Biology, Tokyo
Medical and Dental University, Tokyo, Japan [email protected]. RATIONALE: The role of Parkin in hearts is unclear. Germ-line Parkin knockout
mice have normal hearts, but Parkin is protective in cardiac ischemia.
Parkin-mediated mitophagy is reportedly either irrelevant, or a major factor, in
the lethal cardiomyopathy evoked by cardiac myocyte-specific interruption of
dynamin-related protein 1 (Drp1)-mediated mitochondrial fission.
OBJECTIVE: To understand the role of Parkin-mediated mitophagy in normal and
mitochondrial fission-defective adult mouse hearts.
METHODS AND RESULTS: Parkin mRNA and protein were present at low levels in
normal mouse hearts, but were upregulated after cardiac myocyte-directed Drp1
gene deletion in adult mice. Alone, forced cardiac myocyte Parkin overexpression
activated mitophagy without adverse effects. Likewise, cardiac myocyte-specific
Parkin deletion evoked no adult cardiac phenotype, revealing no essential
function for, and tolerance of, Parkin-mediated mitophagy in normal hearts.
Concomitant conditional Parkin deletion with Drp1 ablation in adult mouse hearts
prevented Parkin upregulation in mitochondria of fission-defective hearts, also
increasing 6-week survival, improving ventricular ejection performance,
mitigating adverse cardiac remodeling, and decreasing cardiac myocyte necrosis
and replacement fibrosis. Underlying the Parkin knockout rescue was suppression
of Drp1-induced hyper-mitophagy, assessed as ubiquitination of mitochondrial
proteins and mitochondrial association of autophagosomal p62/sequestosome 1
(SQSTM1) and processed microtubule-associated protein 1 light chain 3 (LC3-II).
Consequently, mitochondrial content of Drp1-deficient hearts was preserved.
Parkin deletion did not alter characteristic mitochondrial enlargement of
Drp1-deficient cardiac myocytes.
CONCLUSIONS: Parkin is rare in normal hearts and dispensable for constitutive
mitophagic quality control. Ablating Drp1 in adult mouse cardiac myocytes not
only interrupts mitochondrial fission, but also markedly upregulates Parkin,
thus provoking mitophagic mitochondrial depletion that contributes to the lethal
cardiomyopathy. |
Which is the genetic cause for the development of Fanconi anemia complementation group D1? | Fanconi anemia complementation group D1 (FANCD1) was shown to be induced by biallelic mutations in the BRCA2 breast-cancer-susceptibility gene. | Surprisingly, biallelic mutations in the BRCA2 breast-cancer-susceptibility gene
were found in Fanconi anemia (FA), a rare hereditary disorder characterized by
chromosomal instability, hypersensitivity to DNA cross-linking agents, and
cancer susceptibility. This suggests that a defect in the FA pathway might
predispose to familial breast cancer. A previously reported molecular
interaction between BRCA1 and the FA protein, FANCD2, supports the hypothesis
that both breast-cancer-susceptibility genes are components of the FA pathway,
functioning in DNA-damage response. However, an alternative hypothesis, that
group FA-D1 with mutated BRCA2 represents a FA-like syndrome that is involved in
a pathway distinct from the FA pathway, cannot be excluded. Similar syndromes
would also be expected when recombination genes, such as Rad51 and its paralogs,
are mutated. Fanconi anemia is an inherited disease characterized by bone marrow failure,
congenital malformations, and predisposition to cancer. The breast cancer
susceptibility gene BRCA2 was recently found to be associated with Fanconi
anemia complementation group D1 (FA-D1). We examined four kindreds afflicted
with Fanconi anemia for the presence of germline BRCA2 mutations. One kindred,
of Ashkenazi Jewish ancestry, had five members who were diagnosed with breast
cancer and two cousins who were BRCA2*6174delT/C3069X compound heterozygotes and
had Fanconi anemia and brain tumors. In another kindred of Ashkenazi Jewish and
Lithuanian Catholic ancestry, a child with Fanconi anemia and a medulloblastoma
was a BRCA2*6174delT/886delGT compound heterozygote. Two other kindreds each
contained a Fanconi anemia-afflicted child who developed medulloblastoma; one
child was of Latin American ancestry and a compound heterozygote for
BRCA2*I2490T/ 5301insA and the other was African American and a compound
heterozygote for BRCA2*Q3066X/E1308X. Median age of the Fanconi anemia-afflicted
children at brain tumor diagnosis was 3.5 years. The co-occurrence of brain
tumors, Fanconi anemia, and breast cancer observed in one of these kindreds
constitutes a new syndromic association. Individuals who carry a germline BRCA2
mutation and who plan to have children with a partner of Ashkenazi Jewish
descent should consider undergoing genetic counseling. Fanconi Anemia (FA) is an autosomal recessive syndrome characterized by
congenital abnormalities, progressive bone marrow failure, and susceptibility to
cancer. FA has eight known complementation groups and is caused by mutations in
at least seven genes. Biallelic BRCA2 mutations were shown recently to cause
FA-D1. Monoallelic (heterozygous) BRCA2 mutations confer a high risk of breast
cancer and are a major cause of familial breast cancer. To investigate whether
heterozygous variants in other FA genes are high penetrance breast cancer
susceptibility alleles, we screened germ-line DNA from 88 BRCA1/2-negative
families, each with at least three cases of breast cancer, for mutations in
FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG. Sixty-nine sequence variants were
identified of which 25 were exonic. None of the exonic variants resulted in
translational frameshifts or nonsense codons and 14 were polymorphisms
documented previously. Of the remaining 11 exonic variants, 2 resulted in
synonymous changes, and 7 were present in controls. Only 2 conservative missense
variants, 1 in FANCA and 1 in FANCE, were each found in a single family and were
not present in 300 controls. The results indicate that FA gene mutations, other
than in BRCA2, are unlikely to be a frequent cause of highly penetrant breast
cancer predisposition. Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by
congenital and developmental abnormalities, hypersensitivity to DNA
cross-linking agents such as mitomycin C (MMC), and strong predisposition to
acute myeloid leukemia (AML). In this article, we describe clinical and
molecular findings in a boy with a severe FA phenotype who developed AML by the
age of 2. Although he lacked a strong family history of cancer, he was
subsequently shown to carry biallelic mutations in the FANCD1/BRCA2 gene. These
included an IVS7 splice-site mutation, which is strongly associated with early
AML in homozygous or compound heterozygous carrier status in FA-D1 patients.
Myeloid leukemia cells from this patient have been maintained in culture for
more than 1 year and have been designated as the SB1690CB cell line. Growth of
SB1690CB is dependent on granulocyte macrophage colony stimulating factor or
interleukin-3. This cell line has retained its MMC sensitivity and has undergone
further spontaneous changes in the spectrum of cytogenetic aberrations compared
with the primary leukemia. This is the second AML cell line derived from an
FA-D1 patient and the first proof that maligt clones arising in FA patients
can retain inherited MMC sensitivity. As FA-derived maligcies are normally
not very responsive to treatment, this implies there are important mechanisms of
acquiring correction of the cellular FA phenotype that would explain the poor
chemoresponsiveness observed in FA-associated maligcies and might also play a
role in the initiation and progression of cancer in the general population. Fanconi anemia (FA) is an inherited cancer-susceptibility disorder,
characterized by genomic instability and hypersensitivity to DNA cross-linking
agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation
group allows for the first time to study the characteristics of primary
BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared
hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane
sulfonate (MMS), and like cells derived from other FA complementation groups,
not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells
were slightly sensitive to UV irradiation. Despite the observed lack of X-ray
sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was
observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient
fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells
displayed greatly enhanced levels of spontaneous as well as MMC-induced
chromosomal aberrations (CA), similar to cells deficient in homologous
recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent
cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels,
both spontaneous as well as after MMC treatment. Hence, these data indicate that
human cells with biallelic BRCA2 mutations display typical features of both FA-
and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the
integrated FA/BRCA DNA damage response pathway but also controls other functions
outside the FA pathway. |
Which diseases are caused by mutations in Calsequestrin 2 (CASQ2) gene? | CASQ2 mutations are associated with autosomal recessive catecholaminergic polymorphic ventricular tachycardia (CPVT) and familial hypertrophic cardiomyopathy. | Catecholamine-induced polymorphic ventricular tachycardia (CPVT), a rare disease
that occurs in subjects without obvious organic heart disease, is characterized
by episodes of syncope, seizures, or sudden death in response to physiologic or
emotional stress. This report reviews evidence that a missense mutation in the
CASQ2 gene is associated with autosomal-recessive CPVT. Mutations of two myocardial calcium signaling molecules, ryanodine receptor 2
(RYR2) and calsequestrin 2 (CASQ2), may cause catecholaminergic polymorphic
ventricular tachycardia (CPVT), a severe inherited arrhythmic disease
manifesting with salvoes of exercise-induced bidirectional and polymorphic
tachycardias. We screened 12 Finnish CPVT probands for mutations in these genes
and identified three novel RYR2 mutations (V2306I, P4902L, R4959Q), which were
absent in unaffected and control individuals. Although no obvious
disease-causing mutations were identified in the CASQ2 gene, the molecular
screening revealed two novel amino-acid polymorphisms (T66A and V76M). The
frequencies of these polymorphisms in 185 unrelated probands with long QT
syndrome and in 280 healthy blood donors were not significantly different. These
data, combined with our previous findings, show that RYR2 mutations are present
in at least 6/16 (38%) of the catecholaminergic polymorphic ventricular
tachycardia families, while CASQ2 mutations must be a rare cause of CPVT. Cardiac excitation-contraction coupling occurs by a calcium ion-mediated
mechanism in which the signal of action potential is converted into Ca2+ influx
into the cardiomyocytes through the sarcolemmal L-type calcium channels. This is
followed by Ca2+-induced release of additional Ca2+ ions from the lumen of the
sarcoplasmic reticulum into the cytosol via type 2 ryanodine receptors (RyR2).
RyR2 channels form large complexes with additional regulatory proteins,
including FKBP12.6 and calsequestrin 2 (CASQ2). Catecholamines, released into
the body fluids during emotional or physical stress, activate Ca2+-induced Ca2+
release by protein kinase A-mediated phosphorylation of RyR2. Catecholaminergic
polymorphic ventricular tachycardia (CPVT) is an insidious, early-onset and
highly maligt, inherited disorder characterized by effort-induced ventricular
arrhythmias in the absence of structural alterations of the heart. At least some
cases of sudden, unexplained death in young individuals may be ascribed to CPVT.
Mutations of the RyR2 gene cause autosomal domit CPVT, while mutations of the
CASQ2 gene may cause an autosomal recessive or domit form of CPVT. The steps
of the molecular pathogenesis of CPVT are not entirely clear, but inappropriate
"leakiness" of RyR2 channels is thought to play a role; the underlying
mechanisms may involve an increase in the basal activity of the RyR2 channel,
alterations in its phosphorylation status, a defective interaction of RyR2 with
other molecules or ions, such as FKBP12.6, CASQ2, or Mg2+, or its abnormal
activation by extra- or intraluminal Ca2+ ions. Beta-adrenergic antagonists have
proven to be of value in prevention of arrhythmias in CPVT patients, but
occasional treatment failures call for alternative measures. There is great
interest at present for the development of novel antiarrhythmic drugs for CPVT,
as the same approaches may be applied for treatment of more common forms of
life-threatening arrhythmias, such as those arising during ischemia and heart
failure. Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is a familial
disorder caused by cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2
(CASQ2) gene mutations. To define how CASQ2 mutations cause CPVT, we produced
and studied mice carrying a human D307H missense mutation (CASQ(307/307)) or a
CASQ2-null mutation (CASQ(DeltaE9/DeltaE9)). Both CASQ2 mutations caused
identical consequences. Young mutant mice had structurally normal hearts but
stress-induced ventricular arrhythmias; aging produced cardiac hypertrophy and
reduced contractile function. Mutant myocytes had reduced CASQ2 and increased
calreticulin and RyR2 (with normal phosphorylated proportions) but unchanged
calstabin levels, as well as reduced total sarcoplasmic reticulum (SR) Ca(2+),
prolonged Ca(2+) release, and delayed Ca(2+) reuptake. Stress further diminished
Ca(2+) transients, elevated cytosolic Ca(2+), and triggered frequent,
spontaneous SR Ca(2+) release. Treatment with Mg(2+), a RyR2 inhibitor,
normalized myocyte Ca(2+) cycling and decreased CPVT in mutant mice, indicating
RyR2 dysfunction was critical to mutant CASQ2 pathophysiology. We conclude that
CPVT-causing CASQ2 missense mutations function as null alleles. In the absence
of CASQ2, calreticulin, a fetal Ca(2+)-binding protein normally downregulated at
birth, remains a prominent SR component. Adaptive changes to CASQ2 deficiency
(increased posttranscriptional expression of calreticulin and RyR2) maintained
electrical-mechanical coupling, but increased RyR2 leakiness, a paradoxical
response further exacerbated by stress. The central role of RyR2 dysfunction in
CASQ2 deficiency unifies the pathophysiologic mechanism underlying CPVT due to
RyR2 or CASQ2 mutations and suggests a therapeutic approach for these inherited
cardiac arrhythmias. OBJECTIVE: To identify mutations and variants in CASQ2 gene in 27 CPVT
patients/family members.
METHODS: Mutational analysis was performed with direct DNA sequence analysis.
The frequency of an identified CASQ2 variant was determined using the Taqman
genotyping assay.
RESULTS: A novel heterozygous mutation, F189L, in CASQ2 gene was identified in
one family with CPVT. This mutation occurred at the evolutionarily, highly
conserved phenylalanine residue at codon 189, and was not present in 1400
control individuals. No other disease-causing mutations were identified in the
CASQ2 gene.
CONCLUSION: A novel mutation of F189L in the CASQ2 gene was identified. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an uncommon
heritable disease presenting with syncope or sudden cardiac death. Two genes
involved in calcium homeostasis, the ryanodine receptor gene and the
calsequestrin 2 (CASQ2) gene, have been implicated in this disease. We describe
a young man presenting with exercise-induced syncope, clinically diagnosed as
CPVT. Genetic analysis revealed two mutations, p.Y55C (c.164A>G) and p.P308L
(c.923C>T), in the CASQ2 gene. Subsequent familial analysis indicates a compound
heterozygous form of inheritance. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial
cardiac arrhythmia that is related to RYR2 or CASQ2 gene mutation. It occurs in
patients with structurally normal heart and causes exercise-emotion-triggered
syncope and sudden cardiac death. We experienced a case of CPVT in an 11
year-old female patient who was admitted for sudden cardiovascular collapse. The
initial electrocardiogram (ECG) on emergency department revealed ventricular
fibrillation. After multiple defibrillations, sinus rhythm was restored.
However, recurrent ventricular fibrillation occurred during insertion of
nasogastric tube without sedation in coronary care unit. On ECG monitoring,
bidirectional ventricular tachycardia occurred with sinus tachycardia and then
degenerated into ventricular fibrillation. To our knowledge, there has been no
previous case report of CPVT triggered by sinus tachycardia in Korea. Therefore,
we report the case as well as a review of the literature. RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is an inherited
disease that predisposes to cardiac arrest and sudden death. The disease is
associated with mutations in the genes encoding for the cardiac ryanodine
receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a
major loss of CASQ2 monomers, possibly because of enhanced degradation of the
mutant protein. The decrease of CASQ2 is associated with a reduction in the
levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2
and RyR2, a macromolecular complex devoted to control of calcium release from
the sarcoplasmic reticulum.
OBJECTIVE: We intended to evaluate whether viral gene transfer of wild-type
CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2.
METHODS AND RESULTS: We used an adeno-associated serotype 9 viral vector to
express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after
intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed
normalization of the levels of calsequestrin, triadin, and junctin, rescue of
electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation,
and lack of life-threatening arrhythmias.
CONCLUSIONS: We have proven the concept that induction of CASQ2 expression in
knockout mice reverts the molecular, structural, and electric abnormalities and
prevents life-threatening arrhythmias in CASQ2-defective catecholaminergic
polymorphic ventricular tachycardia mice. These data support the view that
development of CASQ2 viral gene transfer could have clinical application. Cardiac calsequestrin (Casq2) is the major Ca2+ binding protein in the
sarcoplasmic reticulum, which is the principle Ca2+ storage organelle of cardiac
muscle. During the last decade, experimental studies have provided new concepts
on the role of Casq2 in the regulation of cardiac muscle Ca2+ handling.
Furthermore, mutations in the gene encoding for cardiac calsequestrin, CASQ2,
cause a rare but severe form of catecholaminergic polymorphic ventricular
tachycardia (CPVT). Here, we review the physiology of Casq2 in cardiac Ca2+
handling and discuss pathophysiological mechanisms that lead to CPVT caused by
CASQ2 mutations. We also describe the clinical aspects of CPVT and provide an
update of its contemporary clinical management. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic disease so far related to mutations in the cardiac ryanodine
receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in
RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations
in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT
patients with no detected mutation in these two genes. Based on a candidate gene
approach, we focused our investigations on triadin and junctin, two proteins
that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin
(ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three
mutations in triadin which cosegregated with the disease on a recessive mode of
transmission in two families, but no mutation was found in junctin. Two TRDN
mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop
codons; the third mutation, a p.T59R missense mutation, was further studied.
Expression of the p.T59R mutant in COS-7 cells resulted in intracellular
retention and degradation of the mutant protein. This was confirmed after in
vivo expression of the mutant triadin in triadin knock-out mice by viral
transduction. In this work, we identified TRDN as a new gene responsible for an
autosomal recessive form of CPVT. The mutations identified in the two families
lead to the absence of the protein, thereby demonstrating the importance of
triadin for the normal function of the cardiac calcium release complex in
humans. Catecholaminergic polymorphic ventricular tachycardia is a familial cardiac
arrhythmia that is related to RYR2 or CASQ2 gene mutation. It occurs in patients
with structurally normal heart and causes exercise-emotion triggered syncope and
sudden cardiac death. We present a 13 year-old girl with recurrent episodes of
exercise-related syncope and prior history of sudden death in a first degree
relative. Regulation of calcium flux in the heart is a key process that affects cardiac
excitability and contractility. Degenerative diseases, such as coronary artery
disease, have long been recognized to alter the physiology of intracellular
calcium regulation, leading to contractile dysfunction or arrhythmias. Since the
discovery of the first gene mutation associated with catecholaminergic
polymorphic ventricular tachycardia (CPVT) in 2001, a new area of interest in
this field has emerged--the genetic abnormalities of key components of the
calcium regulatory system. Such anomalies cause a variety of genetic diseases
characterized by the development of life-threatening arrhythmias in young
individuals. In this Review, we provide an overview of the structural
organization and the function of calcium-handling proteins and describe the
mechanisms by which mutations determine the clinical phenotype. Firstly, we
discuss mutations in the genes encoding the ryanodine receptor 2 (RYR2) and
calsequestrin 2 (CASQ2). These proteins are pivotal to the regulation of calcium
release from the sarcoplasmic reticulum, and mutations can cause CPVT. Secondly,
we review defects in genes encoding proteins that form the voltage-dependent
L-type calcium channel, which regulates calcium entry into myocytes. Mutations
in these genes cause various phenotypes, including Timothy syndrome, Brugada
syndrome, and early repolarization syndrome. The identification of mutations
associated with 'calcium-handling diseases' has led to an improved understanding
of the role of calcium in cardiac physiology. BACKGROUND: The genetic background of catecholaminergic polymorphic ventricular
tachycardia (CPVT) has been extensively investigated for the last decade in
Western countries, but it remains unstudied in the Asian population.
METHODS AND RESULTS: In 50 Japanese probands from unrelated families who
satisfied clinical criteria for CPVT, genetic testing was conducted in all exons
on 3 CPVT-related genes: cardiac ryanodine receptor 2 (RYR2), calsequestrin 2
(CASQ2) and inward rectifier potassium channel 2 (KCNJ2), and the clinical
features between RYR2-genotyped and -non-genotyped patient groups were compared.
Genetic and clinical evaluation was also done in 46 family members. In the
genetic screening, 28 (18 novel) RYR2 (56.0%), 1 compound heterozygous CASQ2
(2.0%) and 1 KCNJ2 (2.0%) mutation carriers were identified. In the RYR2
mutation-positive group, the frequency of bidirectional ventricular tachycardia
and the use of β-blockers were significantly higher than in the
mutation-negative group. In contrast, there was no significant difference in
supraventricular arrhythmias between the 2 groups. With regard to disease
penetrance, the number of family members of RYR2-genotyped probands with a
clinical diagnosis of CPVT was high.
CONCLUSIONS: Thirty gene mutation carriers were found for 3 genes in 50 probands
clinically diagnosed as having CPVT. The penetrance of CPVT phenotype was
significantly higher in RYR2 mutation carriers, thus RYR2 gene screening in CPVT
patients would be indispensable to prevent unexpected cardiac sudden death of
young family members. BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a
lethal, rare hereditary disease with an estimated prevalence of 1:10 000. The
genetic variants that cause CPVT are usually highly penetrant. To date, about
189 variants in 5 genes (RYR2, CASQ2, CALM1, TRND, and KCNJ2) have been
associated with CPVT pathogenesis.
METHODS AND RESULTS: The Exome Sequencing Project database (ESP; n=6503) was
systematically searched for previously published missense and nonsense
CPVT-associated variants reported in several comprehensive reviews and in 2
databases: The Human Gene Mutation Database and The Inherited Arrhythmias
Database. We used 4 different prediction tools to assess all missense variants
previously associated with CPVT and compared the prediction of protein damage
between CPVT-associated variants identified in the ESP and those variants not
identified in the ESP. We identified 11% of the variants previously associated
with CPVT in the ESP population. In the literature, 57% of these variants were
reported as novel disease-causing variants absent in the healthy control
subjects. These putative CPVT variants were identified in 41 out of 6131
subjects in the ESP population, corresponding to a prevalence of CPVT of up to
1:150. Using an agreement of ≥3, in silico prediction tools showed a
significantly higher frequency of damaging variants among the CPVT-associated
variants not identified in the ESP database (83%) compared with those variants
identified in the ESP (50%; P=0.021).
CONCLUSIONS: We identified a substantial overrepresentation of CPVT-associated
variants in a large exome database, suggesting that these variants are not
necessarily the monogenic cause of CPVT. |
What are the most frequent non-canonical sequence motifs at the donor and acceptor splice sites in vertebrates? | There are two major exceptions to the canonical GT-AG dinucleotides at donor and acceptor sites: the GG-AG splice site pairs, recognized through the typical U2 splicing machinery, and the AT-AC splice pairs recognized by the U12 splicing machinery. | A set of 43 337 splice junction pairs was extracted from mammalian GenBank
annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them.
Of these, 98.71% contain canonical dinucleotides GT and AG for donor and
acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs;
and the remaining 0.73% occurs in a lot of small groups (with a maximum size of
0.05%). Studying these groups we observe that many of them contain splicing
dinucleotides shifted from the annotated splice junction by one position. After
close examination of such cases we present a new classification consisting of
only eight observed types of splice site pairs (out of 256 a priori possible
combinations). EST alignments allow us to verify the exonic part of the splice
sites, but many non-canonical cases may be due to intron sequencing errors. This
idea is given substantial support when we compare the sequences of human genes
having non-canonical splice sites deposited in GenBank by high throughput genome
sequencing projects (HTG). A high proportion (156 out of 171) of the human
non-canonical and EST-supported splice site sequences had a clear match in the
human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which
one was an error that corrected to GC-AG), 61 errors that were corrected to
GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected
to AT-AC), one case was produced from non-existent intron, seven cases were
found in HTG that were deposited to GenBank and finally there were only two
cases left of supported non-canonical splice sites. If we assume that
approximately the same situation is true for the whole set of annotated
mammalian non-canonical splice sites, then the 99.24% of splice site pairs
should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist
of other types of non-canonical splice sites. We analyze several characteristics
of EST-verified splice sites and build weight matrices for the major groups,
which can be incorporated into gene prediction programs. We also present a set
of EST-verified canonical splice sites larger by two orders of magnitude than
the current one (22 199 entries versus approximately 600) and finally, a set of
290 EST-supported non-canonical splice sites. Both sets should be significant
for future investigations of the splicing mechanism. U12-dependent introns are found in small numbers in most eukaryotic genomes, but
their scarcity makes accurate characterisation of their properties challenging.
A computational search for U12-dependent introns was performed using the draft
version of the human genome sequence. Human expressed sequences confirmed 404
U12-dependent introns within the human genome, a 6-fold increase over the total
number of non-redundant U12-dependent introns previously identified in all
genomes. Although most of these introns had AT-AC or GT-AG terminal
dinucleotides, small numbers of introns with a surprising diversity of termini
were found, suggesting that many of the non-canonical introns found in the human
genome may be variants of U12-dependent introns and, thus, spliced by the minor
spliceosome. Comparisons with U2-dependent introns revealed that the
U12-dependent intron set lacks the 'short intron' peak characteristic of
U2-dependent introns. Analysis of this U12-dependent intron set confirmed
reports of a biased distribution of U12-dependent introns in the genome and
allowed the identification of several alternative splicing events as well as a
surprising number of apparent splicing errors. This new larger reference set of
U12-dependent introns will serve as a resource for future studies of both the
properties and evolution of the U12 spliceosome. MOTIVATION: mRNA sequences and expressed sequence tags represent some of the
most abundant experimental data for identifying genes and alternatively spliced
products in metazoans. These transcript sequences are frequently studied by
aligning them to a genomic sequence template. For existing programs,
error-prone, polymorphic and cross-species data, as well as non-canonical splice
sites, still present significant barriers to producing accurate, complete
alignments.
RESULTS: We took a novel approach to spliced alignment that meaningfully
combined information from sequence similarity with that obtained from PSSM
splice site models. Scoring systems were chosen to maximize their power of
discrimination, and dynamic programming (DP) was employed to guarantee optimal
solutions would be found. The resultant program, EXALIN, performed better than
other popular tools tested under a wide range of conditions that included
detection of micro-exons and human-mouse cross-species comparisons. For improved
speed with only a marginal decrease in splice site prediction accuracy, EXALIN
could perform limited DP guided by a result from BLASTN.
AVAILABILITY: The source code, binaries, scripts, scoring matrices and splice
site models for human, mouse, rice and Caenorhabditis elegans utilized in this
study are posted at http://blast.wustl.edu/exalin. The software (scripts, source
code and binaries) is copyrighted but free for all to use. MOTIVATION: The vast majority of introns in protein-coding genes of higher
eukaryotes have a GT dinucleotide at their 5'-terminus and an AG dinucleotide at
their 3' end. About 1-2% of introns are non-canonical, with the most abundant
subtype of non-canonical introns being characterized by GC and AG dinucleotides
at their 5'- and 3'-termini, respectively. Most current gene prediction
software, whether based on ab initio or spliced alignment approaches, does not
include explicit models for non-canonical introns or may exclude their
prediction altogether. With present amounts of genome and transcript data, it is
now possible to apply statistical methodology to non-canonical splice site
prediction. We pursued one such approach and describe the training and
implementation of GC-donor splice site models for Arabidopsis and rice, with the
goal of exploring whether specific modeling of non-canonical introns can enhance
gene structure prediction accuracy.
RESULTS: Our results indicate that the incorporation of non-canonical splice
site models yields dramatic improvements in annotating genes containing GC-AG
and AT-AC non-canonical introns. Comparison of models shows differences between
monocot and dicot species, but also suggests GC intron-specific biases
independent of taxonomic clade. We also present evidence that GC-AG introns
occur preferentially in genes with atypically high exon counts.
AVAILABILITY: Source code for the updated versions of GeneSeqer and
SplicePredictor (distributed with the GeneSeqer code) isavailable at
http://bioinformatics.iastate.edu/bioinformatics2go/gs/download.html. Web
servers for Arabidopsis, rice and other plant species are accessible at
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/AtGDBgs.cgi,
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/OsGDBgs.cgi and
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/PlantGDBgs.cgi, respectively. A
SplicePredictor web server is available at
http://bioinformatics.iastate.edu/cgi-bin/sp.cgi. Software to generate training
data and parameterizations for Bayesian splice site models is available at
http://gremlin1.gdcb.iastate.edu/~volker/SB05B/BSSM4GSQ/ U12-type introns are spliced by the U12-dependent spliceosome and are present in
the genomes of many higher eukaryotic lineages including plants, chordates and
some invertebrates. However, due to their relatively recent discovery and a
systematic bias against recognition of non-canonical splice sites in general,
the introns defined by U12-type splice sites are under-represented in genome
annotations. Such under-representation compounds the already difficult problem
of determining gene structures. It also impedes attempts to study these introns
genome-wide or phylum-wide. The resource described here, the U12 Intron Database
(U12DB), aims to catalog the U12-type introns of completely sequenced eukaryotic
genomes in a framework that groups orthologous introns with each other. This
will aid further investigations into the evolution and mechanism of
U12-dependent splicing as well as assist ongoing genome annotation efforts.
Public access to the U12DB is available at
http://genome.imim.es/cgi-bin/u12db/u12db.cgi. |
What does isradipine do to L-type channels? | Isradipine antagonizes/blocks the L-type channels. | Previous studies have shown that 17 beta-estradiol (beta-E2) has a direct acute
inhibitory effect on vascular smooth muscle (VSM) contraction. To investigate
the mechanisms underlying this phenomenon, we utilized whole cell patch-clamping
techniques to study effects of beta-E2 on voltage-dependent Ca2+ channels in
cultured VSM cells (VSMC). T- and L-type Ca2+ currents were characterized with
ramp and pulse protocols in A7r5 cultured VSMC. T-type current, inactivated in <
100 ms, was reduced by Ba2+ and was comparatively little affected by isradipine.
L-type current required higher voltages to activate, inactivated slowly, was
greatly increased by Ba2+, and could be completely inhibited by 5 microM
isradipine. beta-E2 (10 microM) significantly reduced peak L-type Ba2+ current
and T-type Ca2+ current within 1-2 min, whereas alpha E2 (a hormonally inactive
isomer of estradiol) caused significantly less reduction in both types of
current. Vehicle (0.1% ethanol) had no significant effect on either current. The
inhibitory effect of beta-E2 on voltage-dependent Ca2+ currents may contribute
to previously demonstrated beta-E2 attenuation of VSM contraction. We have demonstrated that both high-K+ and electrical stimulation of the vagus
nerves release endogenous glutamate from the vascularly-perfused rat stomach in
a calcium-dependent manner. In the present study, we examined properties of
calcium channel subtypes mediating endogenous glutamate release from the
stomach. Application of 50 mM KCl elicited a release of glutamate, and this
release was abolished in calcium-free medium. The release of glutamate was
significantly inhibited by both omega-agatoxin IVA, a P/Q-type calcium channel
antagonist, and isradipine, an L type calcium channel antagonist.
Omega-conotoxin GVIA, an N type calcium channel antagonist and flunarizine, a
nonselective T-type calcium channel antagonist were without effect. In contrast
to this case of glutamate, omega-conotoxin GVIA induced a marked inhibition in
the release of gastric noradrenaline. The combined treatment with omega-agatoxin
IVA plus isradipine produced a marked synergistic inhibition of the glutamate
release. This inhibition was, however, much less than that by cadmium. The
present results suggest that P/Q and L type calcium channels coexist to regulate
the release of gastric glutamate. Furthermore, it is possible that unidentified
calcium channels other than P/Q and L type channels are also involved in the
release of glutamate in the stomach. Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate
into myofibroblast-like cells, which develop contractile properties and
contribute to increased resistance to blood flow. We investigated whether this
phenotypic activation includes changes in the expression of L-type
voltage-operated Ca2+ channels (VOCC), which mediate Ca2+ influx and regulate
cell contraction in vascular cell types. Rat HSCs were studied in the quiescent
phenotype and after their activation in vitro (cultured on plastic for 14 days)
and in vivo (isolated from rats with CCl(4)-induced cirrhosis). Patch-clamp
studies showed Ca2+ currents through L-type VOCC in HSCs activated both in vitro
and in vivo, whereas no currents were detected in quiescent HSCs. Moreover,
binding studies with (3)H-isradipine, a specific L-type VOCC antagonist, showed
a large number of binding sites in activated HSCs, while no specific binding was
found in quiescent HSCs. Finally, messenger RNA (mRNA) encoding L-type VOCC was
not detected in quiescent HSCs as assessed by reverse transcription-polymerase
chain reaction (RT-PCR) and Northern blot analysis, whereas it was present in
activated HSCs. Stimulation of L-type VOCC with KCl resulted in a marked
increase in [Ca2+](i) followed by cell contraction in HSCs activated both in
vitro and in vivo, whereas no effects were observed in quiescent HSCs. We
conclude that the activation of HSCs is associated with up-regulation of L-type
VOCC that mediate Ca2+ influx and cell contraction. These results may be
relevant to the pathogenesis of portal hypertension. In cochlea inner hair cells (IHCs), L-type Ca(2+) channels (LTCCs) formed by
alpha1D subunits (D-LTCCs) possess biophysical and pharmacological properties
distinct from those of alpha1C containing C-LTCCs. We investigated to which
extent these differences are determined by alpha1D itself by analyzing the
biophysical and pharmacological properties of cloned human alpha1D splice
variants in tsA-201 cells. Variant alpha1D(8A,) containing exon 8A sequence in
repeat I, yielded alpha1D protein and L-type currents, whereas no intact protein
and currents were observed after expression with exon 8B. In whole cell
patch-clamp recordings (charge carrier 15-20 mm Ba(2+)), alpha1D(8A) - mediated
currents activated at more negative voltages (activation threshold, -45.7 versus
-31.5 mV, p < 0.05) and more rapidly (tau(act) for maximal inward currents 0.8
versus 2.3 ms; p < 0.05) than currents mediated by rabbit alpha1C. Inactivation
during depolarizing pulses was slower than for alpha1C (current inactivation
after 5-s depolarizations by 90 versus 99%, p < 0.05) but faster than for LTCCs
in IHCs. The sensitivity for the dihydropyridine (DHP) L-type channel blocker
isradipine was 8.5-fold lower than for alpha1C. Radioligand binding experiments
revealed that this was not due to a lower affinity for the DHP binding pocket,
suggesting that differences in the voltage-dependence of DHP block account for
decreased sensitivity of D-LTCCs. Our experiments show that alpha1D(8A) subunits
can form slowly inactivating LTCCs activating at more negative voltages than
alpha1C. These properties should allow D-LTCCs to control physiological
processes, such as diastolic depolarization in sinoatrial node cells,
neurotransmitter release in IHCs and neuronal excitability. This study examines the molecular basis for the T-type and L-type Ca(2+)
currents in canine Purkinje cells. The I(CaT) in Purkinje cells was completely
suppressed by 200 nM kurtoxin, a specific blocker of both Ca(v)3.1 and Ca(v)3.2
channels. Since only Ca(v)3.2 mRNA is expressed at high levels in Purkinje
fibres, being approximately 100-fold more abundant than either Ca(v)3.1 or
Ca(v)3.3 mRNAs, it is concluded that the Ca(v)3.2 gene encodes the bulk of the
T-type Ca(2+) channels in canine Purkinje cells. This conclusion is consistent
with the sensitivity of the current to blockade by Ni(2+) ions (K(D) = 32
microM). For L-type channels, Ca(v)1.2 mRNA was most abundant in Purkinje fibres
but a significant level of Ca(v)1.3 mRNA expression was also found. A comparison
of the sensitivity to blockade by isradipine of the L-type currents in Purkinje
cells and ventricular epicardial myocytes, which only express Ca(v)1.2, suggests
that the Ca(v)1.3 channels make, at most, a minor contribution to the L-type
current in canine Purkinje cells. Activation of large conductance Ca(2+)-activated potassium (BK) channels hastens
action potential repolarisation and generates the fast afterhyperpolarisation in
hippocampal pyramidal neurons. A rapid coupling of Ca(2+) entry with BK channel
activation is necessary for this to occur, which might result from an identified
coupling of Ca(2+) entry through N-type Ca(2+) channels to BK channel
activation. This selective coupling was extremely rapid and resistant to
intracellular BAPTA, suggesting that the two channel types are close. Using
reciprocal co-immunoprecipitation, we found that N-type channels were more
abundantly associated with BK channels than L-type channels (Ca(V)1.2) in rat
brain. Expression of only the pore-forming alpha-subunits of the N-type
(Ca(V)2.2) and BK (Slo(27)) channels in a non-neuronal cell-line gave robust
macroscopic currents and reproduced the interaction. Co-expression of
Ca(V)2.2/Ca(V)beta(3) subunits with Slo(27) channels revealed rapid functional
coupling. By contrast, extremely rare examples of rapid functional coupling were
observed with co-expression of Ca(V)1.2/Ca(V)beta(3) and Slo(27) channels.
Action potential repolarisation in hippocampal pyramidal neurons was slowed by
the N-type channel blocker omega-conotoxin GVIA, but not by the L-type channel
blocker isradipine. These data showed that selective functional coupling between
N-type Ca(2+) and BK channels provided rapid activation of BK channels in
central neurons. Parkinson's disease is a common neurodegenerative disorder of unknown cause.
There is no cure or proven strategy for slowing the progression of the disease.
Although there are signs of pathology in many brain regions, the core symptoms
of Parkinson's disease are attributable to the selective degeneration of
dopaminergic neurons in the substantia nigra pars compacta. A potential clue to
the vulnerability of these neurons is their increasing reliance on Ca(2+)
channels to maintain autonomous activity with age. This reliance could pose a
sustained metabolic stress on mitochondria, accelerating cellular ageing and
death. The Ca(2+) channels underlying autonomous activity in dopaminergic
neurons are closely related to the L-type channels found in the heart and smooth
muscle. Systemic administration of isradipine, a dihydropyridine blocker of
L-type channels, forces dopaminergic neurons in rodents to revert to a juvenile,
Ca(2+)-independent mechanism to generate autonomous activity. More importantly,
reversion confers protection against toxins that produce experimental
parkinsonism, pointing to a potential neuroprotective strategy for Parkinson's
disease with a drug class that has been used safely in human beings for decades.
These studies also suggest that, although genetic and environmental factors can
hasten its onset, Parkinson's disease stems from a distinctive neuronal design
common to all human beings, making its appearance simply a matter of time. The motor symptoms of Parkinson's disease (PD) are due to the progressive loss
of dopamine (DA) neurons in substantia nigra pars compacta (SNc). Nothing is
known to slow the progression of the disease, making the identification of
potential neuroprotective agents of great clinical importance. Previous studies
using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have
shown that antagonism of L-type Ca2+ channels protects SNc DA neurons. However,
this was not true in a 6-hydroxydopamine (6-OHDA) model. One potential
explanation for this discrepancy is that protection in the 6-OHDA model requires
greater antagonism of Cav1.3 L-type Ca2+ channels thought to underlie
vulnerability and this was not achievable with the low affinity dihydropyridine
(DHP) antagonist used. To test this hypothesis, the DHP with the highest
affinity for Cav1.3L-type channels-isradipine-was systemically administered and
then the DA toxin 6-OHDA injected intrastriatally. Twenty-five days later,
neuroprotection and plasma concentration of isradipine were determined. This
analysis revealed that isradipine produced a dose-dependent sparing of DA fibers
and cell bodies at concentrations achievable in humans, suggesting that
isradipine is a potentially viable neuroprotective agent for PD. Alzheimer's disease is the most devastating neurodegenerative disorder in the
elderly, yet treatment options are severely limited. The drug development effort
to modify Alzheimer's disease pathology by intervention at beta amyloid
production sites has been largely ineffective or inconclusive. The greatest
challenge has been to identify and define downstream mechanisms reliably
predictive of clinical symptoms. Beta amyloid accumulation leads to
dysregulation of intracellular calcium by plasma membrane L-type calcium
channels located on neuronal somatodendrites and axons in the hippocampus and
cortex. Paradoxically, L-type calcium channel subtype Ca(v)1.2 also promotes
synaptic plasticity and spatial memory. Increased intracellular calcium
modulates amyloid precursor protein processing and affects multiple downstream
pathways including increased hyperphosphorylated tau and suppression of
autophagy. Isradipine is a Federal Drug Administration-approved dihydropyridine
calcium channel blocker that binds selectively to Ca(v)1.2 in the hippocampus.
Our studies have shown that isradipine in vitro attenuates beta amyloid oligomer
toxicity by suppressing calcium influx into cytoplasm and by suppressing
Ca(v)1.2 expression. We have previously shown that administration of isradipine
to triple transgenic animal model for Alzheimer's disease was well-tolerated.
Our results further suggest that isradipine became bioavailable, lowered tau
burden, and improved autophagy function in the brain. A better understanding of
brain pharmacokinetics of calcium channel blockers will be critical for
designing new experiments with appropriate drug doses in any future clinical
trials for Alzheimer's disease. This review highlights the importance of
Ca(v)1.2 channel overexpression, the accumulation of hyperphosphorylated tau and
suppression of autophagy in Alzheimer's disease and modulation of this pathway
by isradipine. |
Why does cranberry juice help combat urinary tract infections? | Cranberry products affect the surface properties, such as fimbriae and lipopolysaccharides, and adhesion of fimbriated and nonfimbriated E. coli. | Most research suggests that ingestion of cranberry juice may be useful in
preventing urinary tract infections. This pilot study examines the effect of
drinking moderate amounts of commercially available cranberry juice cocktail on
urinary pH in older, institutionalized adults. The results of the study have
implications for home care nurses who have similar patients in their case loads. CONTEXT: Cranberry juice has long been recognized in folk medicine as a
therapeutic agent, mainly in urinary tract infections. Its proposed mechanism of
action is antiadhesion of bacteria.
OBJECTIVE: Investigation of the potential antiadhesion effect of nondialyzed
material of cranberry (NDM) via its influence on secretion, gene expression, and
promoter activity of the fructosyltransferase (FTF), which is among the
extracellular enzymes associated with dental biofilm formation and pathogenesis
of oral bacteria.
MAIN OUTCOME MEASURES: Secretion of FTF from Streptococcus mutans, in the
presence of NDM, was measured by immunoblotting and confocal scanning laser
microscopy. Its influence on ftf gene expression was determined by reverse
transcription followed by real-time RT-PCR. The luciferase assay was used to
detect bioluminescence expressed by the ftf promoter activity of bacteria
exposed to NDM.
RESULTS: NDM at concentrations between 0.2/mL and 1mg/mL significantly (P<.05)
decreased secretion of extracellular FTF, as well as down-regulated ftf
expression in a dose-dependent manner. NDM also markedly reduced the luciferase
activity under the ftf promoter. Cranberry (Vaccinium macrocarpon) has been used for decades to prevent urinary
tract infections (UTIs) that are among the most common bacterial infections in
women. As to the traditional use of cranberry and its A-type proanthocyanidins'
ability to inhibit adherence of the bacterial P fimbriae in a dose-dependent
manner, clinical trials have been conducted on different subpopulations. A
Cochrane meta-analysis in 244 females with symptomatic UTI suggests that the
effect was more pronounced in women with recurrent UTIs than elderly males and
females or people requiring catheterization. A first head-to-head trial in older
females has been published comparing effectiveness of a low-dose antibiotic
versus cranberry in which investigators suggest that cranberry products may have
a role in older females with recurrent UTI. Still with regard to antibiotic
treatment in women, a recently published study investigated also the potential
cranberry juice interaction with beta-lactam antibiotics supporting the
hypothesis that cranberry juice in usual quantities as prophylaxis for UTI is
not likely to alter the pharmacokinetics of these oral antibiotics. In addition,
the effects of cranberry in pregt female patients have been investigated. A
first pilot trial has been published in which, while a possible protective
effect was shown, more than one third of the females withdrew mainly for
gastrointestinal upset. Proanthocyanidin is commonly used for inhibiting urinary tract infection (UTI)
of sensitive strains of Escherichia coli. The aim of this study was to
investigate the effect of proanthocyanidin on adherence of uropathogenic
multi-drug resistant E. coli to uroepithelial cells, which has not yet been
investigated so far. Extracts of the purified proanthocyanidin were prepared
from dried cranberry juice. Purity and structural assignment of proanthocyanidin
was assessed using high performance liquid chromatography and (13)C nuclear
magnetic resoce spectroscopy, respectively. Subsequently, its affect on
multi-drug resistant bacteria as well as quantification of anti-adherence
bioactivity on human vaginal and bladder epithelial cells was appraised.
Inhibition of adherence to an extent of about 70% with multi-drug resistant E.
coli strains was observed on uroepithelial cell. The anti-adherence bioactivity
of the proanthocyanidin was detected at concentrations of 10-50 µg/ml with
significant bacteriuria. Probable proanthocyanidin through A-type linkages
either combines to P-fimbriae of bacterial cells or modifies the structural
entity of P-fimbriae and inhibits bacterial adherence to uroepithelial cells.
The proanthocyanidin exhibited anti-adherence property with multi-drug resistant
strains of uropathogenic P-fimbriated E. coli with in vitro study. Hence
proanthocyanidin may be considered as an inhibitory agent for multi-drug
resistant strains of E. coli adherence to uroepithelial cells. Question Plusieurs enfants de ma clinique se rétablissent d’une infection des
voies urinaires (IVU). La mère de l’un d’eux m’a demandé si je recommandais le
jus de canneberge pour prévenir de futurs épisodes d’IVU. On lui avait
recommandé d’en boire lorsqu’elle a souffert d’une IVU il y a quelques mois.
Réponse Il a été démontré que le jus de canneberge était efficace pour prévenir
l’adhésion de bactéries comme l’Escherichia coli à l’épithélium de la vessie.
Les données scientifiques actuelles appuient l’utilisation du jus de canneberge
pour la prévention des IVU chez les femmes adultes, mais il n’en existe pas pour
le moment sur la prévention des IVU chez l’enfant. Si le jus de canneberge est
très sécuritaire pour la plupart des enfants, son acidité fait que son goût est
moins apprécié des enfants. Il reste aussi à déterminer la quantité de jus de
canneberge nécessaire pour prévenir les IVU chez les enfants. Flavonoids, present in high levels in cranberries, are potent bioactives known
for their health-promoting benefits, but cranberry beverages (CB) are not
typically recommended as part of a healthy diet. We examine the association
between CB consumption with macronutrient intake and weight status. Data for US
adults (≥19 years, n = 10,891) were taken from the National Health and Nutrition
Examination Survey (NHANES) Survey 2005-2008. Total CB consumption was measured
over two non-consecutive 24-h dietary recalls. Linear and logistic regression
models adjusting for important covariates were used to examine predicted
differences between CB consumers and non-consumers on macronutrient and
anthropometric outcomes. Results are weighted to be nationally representative.
CB consumers (n = 581) were older (>50 year) non-Hispanic black females. They
consumed an average 221 mL (7.5 oz) CB per day. In fully adjusted models CB
consumers (vs. non-consumers) had higher carbohydrates and total sugars and
lower percent energy from protein and total fat (all p < 0.001), but no
difference in total energy. A significantly higher proportion of CB consumers
were predicted to be normal weight (BMI < 25 kg/m2; p = 0.001) and had to have
lower waist circumferences (p = 0.001). Although there was not a significant
trend across level of CB intake, low and middle level CB consumers compared to
non-consumers were more likely to be normal weight (p < 0.001) and less likely
to be overweight/obese (BMI ≥ 25 kg/m2, p < 0.001). Despite having slightly
higher daily macronutrient intakes, CB consumers have more desirable
anthropometric measures compared to non-consumers. |