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Which are the musculoskeletal manifestations of Marfan syndrome? | Musculoskeletal manifestations of Marfan syndrome include scoliosis, dural ectasia, pectus excavatum and carinatum, arachnodactyly, otto pelvis (protrusio acetabuli), dolichostenomelia and ligamentous laxity. | Marfan's syndrome is a hereditary disorder of connective tissue, involving the
ophthalmic, cardiovascular and musculoskeletal systems. Two cases are described
in which protrusio acetabuli was a major problem. Otto pelvis should be
considered as one of the musculoskeletal manifestations of Marfan's syndrome. Marfan syndrome is an inherited multisystemic connective-tissue disease that is
caused by a mutation of the fibrillin-1 gene. The syndrome is characterized by a
wide range of clinical manifestations. Common cardiovascular manifestations,
most of which are substantial contributors to mortality, include annuloaortic
ectasia with or without aortic valve insufficiency, aortic dissection, aortic
aneurysm, pulmonary artery dilatation, and mitral valve prolapse. Scoliosis,
pectus excavatum and carinatum, arachnodactyly, and acetabular protrusion are
common musculoskeletal manifestations. Dural ectasia is a characteristic central
nervous system manifestation. In some patients with Marfan syndrome, there is
also pulmonary and ocular involvement. Early identification and treatment of
these conditions contribute to an improved quality of life and a life expectancy
close to the average for the general population in the United States.
Radiologists play a key role in the diagnosis of Marfan syndrome. Knowledge
about the various manifestations of Marfan syndrome and awareness of their
radiologic appearances permit a comprehensive diagnostic approach that allows
better patient care. Marfan syndrome is a variable autosomal domit disorder; most cases result
from mutations of fibrillin-1. Diagnosis is guided by the Ghent nosology. The
condition may manifest in the cardiovascular and ocular systems. Musculoskeletal
manifestations include scoliosis, dural ectasia, protrusio acetabuli, and
ligamentous laxity. Compared with patients with idiopathic scoliosis, patients
with Marfan syndrome tend to have scoliosis that progresses at a faster rate and
is more resistant to bracing; undergo scoliosis surgery complicated by greater
blood loss, pseudarthrosis, and additional curvature; and have more frequent
occurrences of dural ectasia, which may cause headaches, leg pain, or perineal
pain. Protrusio acetabuli may result in hip joint arthritis and may require
valgus osteotomy or total hip arthroplasty. BACKGROUND: Marfan syndrome (MS) is an autosomic domit condition of the
connective tissue that involves the ocular, cardiovascular and musculoskeletal
systems. MS is caused by mutations in the fibrillin-1 gene, leading to joint
ligaments flaccidity, joint hypermobility and an overgrowth of the long bones.
OBJECTIVES: The aim of the present study was to assess anthropometry,
musculoskeletal alterations and the prevalence of physical therapy treatments
among patients with MS.
METHODS: Twenty-six patients were included in this study [17 females (age:
13.23±2.77 years; body mass 51.5±24-68 Kg; height 1.70±1.40-1.81 m; arm span:
1.73±0.12 m) and 9 males (age: 14.44±2.18; body mass: 61.0±42-72 Kg; height:
1.83±1.66-1.97 m; arm span: 1.93±0.13 m)]. Anthropometric measurements and
musculoskeletal abnormalities were determined in a standardized fashion: pectus
and scoliosis were assessed through radiography and angulation (â) of the
scoliosis curve using the Cobb method; arachnodactyly was assessed through the
thumb sign and Walker-Murdoch test and dolichostenomelia was assessed by arm
span in relation to height. Patients also responded to a questionnaire
addressing participation in physical therapy.
RESULTS: In comparison to values estimated for the Brazilian population, mass
and height were greater among the patients with MS (females: p=0.001 e p<0.0005
e males p=0.019 e p=0.0001, respectively). The following musculoskeletal
abnormalities were found: pectus in 3 patients (11%), pectus and scoliosis in 19
(73%), dolichostenomelia in 11 (42%) and arachnodactyly in 21 (80%). Eleven
patients (42%) with MS had previously undergone physical therapy.
CONCLUSIONS: Patients with MS exhibit altered musculoskeleto and anthropometry
and have infrequent physical therapy treatment. |
What is the "Proteomic ruler"? | The MS signal of histones can be used as a "proteomic ruler" because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. | Absolute protein quantification using mass spectrometry (MS)-based proteomics
delivers protein concentrations or copy numbers per cell. Existing methodologies
typically require a combination of isotope-labeled spike-in references, cell
counting, and protein concentration measurements. Here we present a novel method
that delivers similar quantitative results directly from deep eukaryotic
proteome datasets without any additional experimental steps. We show that the MS
signal of histones can be used as a "proteomic ruler" because it is proportional
to the amount of DNA in the sample, which in turn depends on the number of
cells. As a result, our proteomic ruler approach adds an absolute scale to the
MS readout and allows estimation of the copy numbers of individual proteins per
cell. We compare our protein quantifications with values derived via the use of
stable isotope labeling by amino acids in cell culture and protein epitope
signature tags in a method that combines spike-in protein fragment standards
with precise isotope label quantification. The proteomic ruler approach yields
quantitative readouts that are in remarkably good agreement with results from
the precision method. We attribute this surprising result to the fact that the
proteomic ruler approach omits error-prone steps such as cell counting or
protein concentration measurements. The proteomic ruler approach is readily
applicable to any deep eukaryotic proteome dataset-even in retrospective
analysis-and we demonstrate its usefulness with a series of mouse organ
proteomes. |
What is the mechanism by which HIV-1-encoded Vif protein allows virus replication? | The HIV-1 Vif protein counteracts the antiviral activity of the APOBEC3 family by targeting the proteins for degradation through the ubiquitin-proteasome pathway. More specifically, Vif, serving as a substrate receptor, facilitates ubiquitination of APOBEC3 proteins by forming a Cullin5-based E3 ubiquitin ligase complex, which targets APOBEC3 proteins for rapid proteasomal degradation. | The viral infectivity factor (Vif) is essential for the productive infection and
dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1
target cells. Vif counteracts the packaging of two cellular cytidine deaminases
named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of
an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the
inhibition of A3G mRNA translation or by a direct competition mechanism. In
addition, Vif appears to be an active partner of the late steps of viral
replication by participating in virus assembly and Gag processing, thus
regulating the final stage of virion formation notably genomic RNA dimerization
and by inhibiting the initiation of reverse transcription. Vif is a small
pleiotropic protein with multiple domains, and recent studies highlighted the
importance of Vif conformation and flexibility in counteracting A3G and in
binding RNA. In this review, we will focus on the oligomerization and RNA
chaperone properties of Vif and show that the intrinsic disordered nature of
some Vif domains could play an important role in virus assembly and replication.
Experimental evidence demonstrating the RNA chaperone activity of Vif will be
presented. Several members of the APOBEC3 family of cellular restriction factors provide
intrinsic immunity to the host against viral infection. Specifically, APOBEC3DE,
APOBEC3F, APOBEC3G, and APOBEC3H haplotypes II, V, and VII provide protection
against HIV-1Δvif through hypermutation of the viral genome, inhibition of
reverse transcription, and inhibition of viral DNA integration into the host
genome. HIV-1 counteracts APOBEC3 proteins by encoding the viral protein Vif,
which contains distinct domains that specifically interact with these APOBEC3
proteins to ensure their proteasomal degradation, allowing virus replication to
proceed. Here, we review our current understanding of APOBEC3 structure, editing
and non-editing mechanisms of APOBEC3-mediated restriction, Vif-APOBEC3
interactions that trigger APOBEC3 degradation, and the contribution of APOBEC3
proteins to restriction and control of HIV-1 replication in infected patients. |
Which proteins are the different isoforms of the p38 MAP kinase? | The p38 Mitogen-Activated Protein (MAP) kinase, a serine/threonine kinase, is one of the best characterized kinases in the inflammatory process. There are four isoforms of the enzyme (p38alpha, p38beta, p38gamma and p38delta), which differ in tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates. Among the four identified p38 isoforms (p38α, p38β, p38γ, and p38δ), the α-form is the most fully studied. | The mitogen-activated protein kinase (MAPK) p38 is a Ser/Thr kinase, originally
isolated from lipopolysaccharide-stimulated monocytes. There are four isoforms
of the enzyme (p38alpha, p38beta, p38gamma and p38delta), which differ in tissue
distribution, regulation of kinase activation and subsequent phosphorylation of
downstream substrates. These enzymes also differ in sensitivity to p38 MAPK
inhibitors. The most thoroughly studied isoform is p38alpha, for which
activation has been observed in many hematopoietic and non-hematopoietic cell
types upon appropriate stimuli. p38alpha kinase is involved in the biosynthesis
of the cytokines tumor necrosis factor-alpha and interleukin-1beta at the
translational and transcriptional level. MAPK p38alpha represents a point of
convergence for multiple signaling processes that are activated during
inflammation, making it a key potential target for the modulation of cytokine
production. The discovery and publication of p38alpha and a
pyridinyl-imidazole-based p38alpha inhibitor initiated a huge effort by many
companies to develop p38alpha inhibitors as potential treatments for
inflammatory diseases. Herein, a brief overview is provided of the discovery and
development of AMG-548 (Amgen Inc), a selective and efficacious p38alpha
inhibitor, and its pharmacodynamic effects in a first-in-human study. Data from
a phase I multidose clinical trial are also included. In addition, other
p38alpha inhibitors that have advanced to clinical trials over the last three
years are discussed, such as BIRB-796 (Boehringer Ingelheim Pharmaceuticals
Inc), SCIO-469 and SCIO-323 (Scios Inc), and VX-702 (Vertex Pharmaceuticals
Inc/Kissei Pharmaceutical Co). The p38 family of kinases is a subgroup of the mitogen-activated protein kinase
family. It is composed of four isoforms and is involved in critical biological
processes as well as in inflammatory diseases. The exact unique role of each p38
isoform in these processes is not understood well. To approach this question we
have been developing intrinsically active variants of p38s. Recently we
described a series of mutants of the human p38alpha, which were spontaneously
active as recombit proteins purified from Escherichia coli cells. We show
here that some of these mutants are spontaneously active in several mammalian
cells in culture. The spontaneous activity of some mutants is higher than the
activity of the fully activated wild type counterpart. We further produced
mutants of the other p38 isoforms and found that p38beta(D176A),
p38gamma(D179A), p38delta(D176A), and p38delta(F324S) are spontaneously active
in vivo. The active mutants are also spontaneously phosphorylated. To test
whether the mutants actually fulfill downstream duties of p38 proteins, we
tested their effect on activating protein 1(AP-1)-mediated transcription. Active
mutants of p38alpha induced AP-1-driven reporter genes, as well as the c-jun and
c-fos promoters. An active variant of p38gamma suppressed AP-1-mediated
transcription. When active variants of p38alpha and p38gamma were co-expressed,
AP-1 activity was not induced, showing that p38gamma is domit over p38alpha
with respect to AP-1 activation. Thus, intrinsically active variants that are
spontaneously active in vivo have been obtained for all p38 isoforms. These
variants have disclosed different effects of each isoform on AP-1 activity. The p38 mitogen-activated protein kinases are activated in response to various
extracellular signals in eukaryotic cells and play a critical role in the
cellular responses to these signals. The four mammalian isoforms (p38alpha,
p38beta, p38gamma, and p38delta) are coexpressed and coactivated in the same
cells. The exact role of each p38 isoform has not been entirely identified, in
part due to the inability to activate each member individually. This could be
resolved by the use of intrinsically active mutants. Based on previous studies
on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001)
J Biol Chem276, 25351-2538] and human p38alpha[Diskin R, Askari N, Capone R,
Engelberg D & Livnah O (2004) J Biol Chem279, 47040-47049] we have generated
intrinsically active p38beta, p38gamma and p38delta mutants. In addition, we
have identified a new activating mutation site in p38alpha. Most of the
activating mutations are located in the L16 loop, in which conformational
changes were shown to induce activation. We show that these changes impose
substantial autophosphorylation activity, providing a mechanistic explanation
for the intrinsic activity of the mutants. The new active variants maintain
specificity towards substrates and inhibitors similar to that of the parental
wild-type proteins, and are phosphorylated by mitogen-activated protein kinase
kinase 6, their upstream activator. Thus, we have completed the development of a
series of intrinsically active mutants of all p38 isoforms. These active
variants could now become powerful tools for the elucidating the activation
mechanism and specific biological roles of each p38 isoform. PURPOSE: Increased expression of stromelysin-1 (matrix metalloproteinase
[MMP]-3) by the trabecular meshwork (TM) initiates extracellular matrix turnover
and increases aqueous humor outflow facility. Tumor necrosis factor (TNF)alpha
and interleukin (IL)-1alpha are efficacious inducers of MMP-3 in TM. To
facilitate understanding of the regulation of MMP-3, the authors investigated
the involvement of p38 MAP kinase pathway proteins in this process.
METHODS: Western immunoblots were used to determine the effects of these
cytokines and p38 MAP kinase pathway inhibitors on MMP-3 protein levels, p38 MAP
kinase isoforms, and phosphorylation levels in human and porcine TM cells. The
effects of a domit-negative p38 MAP kinase construct on MMP-3 expression were
evaluated. Morphologic changes in the cells were also examined.
RESULTS: Both cytokines increased MMP-3 levels. The p38 MAP kinase inhibitor
SB202190 diminished MMP-3 induction by TNFalpha at all times and at 24 hours by
IL-1alpha but potentiated the IL-1alpha-induced increase in MMP-3 at later
times. MMP-3 induction by both cytokines was blocked by domit-negative p38
MAP kinase constructs. Each cytokine increased phosphorylation of the p38 MAP
kinase pathway components and altered TM cell morphology. The p38 inhibitor
blocked only the morphologic changes produced by TNFalpha. Human and porcine TM
cells expressed p38 alpha, beta, delta, and gamma isoforms, which migrate
coincident with bands of specific phosphorylation.
CONCLUSIONS: The effects of p38 inhibitors and the domit-negative construct
on TNFalpha and IL-1alpha induction of MMP-3 demonstrate an essential role for
p38 in this signaling process. Differences between p38 inhibitor effects on
TNFalpha and IL-1alpha induction of MMP-3 suggest divergent p38 isoform use, as
do the morphologic responses. The anomalous p38 inhibitor effect on IL-1alpha
induction of MMP-3 and phosphorylation of p38 delta/gamma suggests complex
interactions between p38 MAP kinase isoforms and their differential uses by
TNFalpha and IL-1alpha in TM. The p38 MAP kinase signal transduction pathway is an important regulator of
proinflammatory cytokine production and inflammation. Defining the roles of the
various p38 family members, specifically p38alpha and p38beta, in these
processes has been difficult. Here we use a chemical genetics approach using
knock-in mice in which either p38alpha or p38beta kinase has been rendered
resistant to the effects of specific inhibitors along with p38beta knock-out
mice to dissect the biological function of these specific kinase isoforms. Mice
harboring a T106M mutation in p38alpha are resistant to pharmacological
inhibition of LPS-induced TNF production and collagen antibody-induced
arthritis, indicating that p38beta activity is not required for acute or chronic
inflammatory responses. LPS-induced TNF production, however, is still completely
sensitive to p38 inhibitors in mice with a T106M point mutation in p38beta.
Similarly, p38beta knock-out mice respond normally to inflammatory stimuli.
These results demonstrate conclusively that specific inhibition of the p38alpha
isoform is necessary and sufficient for anti-inflammatory efficacy in vivo. We and others previously showed that p38 mitogen-activated protein kinase is
indispensable for myogenic differentiation. However, it is less clear which of
the four p38 isoforms in the mouse genome participates in this process. Using
C2C12 myogenic cells as a model, we showed here that p38alpha, beta, and gamma
are expressed with distinct expression patterns during differentiation.
Knockdown of any of them by small interfering RNA inhibits myogenic
differentiation, which suggests that the functions of the three p38 isoforms are
not completely redundant. To further elucidate the unique role of each p38
isoform in myogenic differentiation, we individually knocked down one p38
isoform at a time in C2C12 cells, and we compared the whole-genome gene
expression profiles by microarrays. We found that some genes are coregulated by
all three p38 isoforms, whereas others are uniquely regulated by one particular
p38 isoform. Furthermore, several novel p38 target genes (i.e., E2F2, cyclin D3,
and WISP1) are found to be required for myogenin expression, which provides a
molecular basis to explain why different p38 isoforms are required for myogenic
differentiation. Adult skeletal muscle is a very stable tissue containing a small population of
myofiber-associated quiescent satellite cells compared with late
embryonic/neonatal skeletal muscle, which contains highly proliferating
myoblasts and small actively growing myofibers, suggesting that specific
regulatory pathways may control myogenesis at distinct developmental stages. The
p38 MAPK signaling pathway is central for myogenesis, based on studies using
immortalized and neonatal primary myoblasts in vitro. However, the contribution
of this pathway to adult myogenesis has never been investigated. Four p38
isoforms (p38alpha, p38beta, p38gamma and p38delta) exist in mammalian cells,
being p38alpha and p38gamma the most abundantly expressed isoforms in adult
skeletal muscle. Given the embryonic/neonatal lethality of p38alpha-deficient
mice, here we investigate the relative contribution of p38beta, p38gamma and
p38delta to adult myogenesis. Regeneration and myofiber growth of adult muscle
proceeds with similar efficiency in mice lacking p38beta, p38gamma and p38delta
as in wild-type control mice. In agreement with this, there is no difference in
adult primary myoblasts behavior in vitro among the different genotypes.
Importantly, the pattern of p38 activation (ascribed to p38alpha) remains
unperturbed during satellite cell-mediated myogenesis in vitro and adult muscle
regeneration in wild type and p38beta-, p38gamma- and p38delta-deficient mice,
rendering p38alpha as the essential p38 isoform sustaining adult myogenesis.
This study constitutes the first analysis addressing the functionality of
p38beta, p38gamma and p38delta in satellite cell-dependent adult muscle
regeneration and growth. The mammalian p38 mitogen-activated protein kinases (MAPKs) family is composed
of four members (p38α, p38β, p38γ, and p38δ), which are very similar in amino
acid sequence but differ in their expression patterns. This suggests that they
may have specific functions in different organs. In the last years most of the
effort has been centred on the study of the function of the p38α isoform, which
is widely referred to as p38 in the literature. However, the role that other p38
isoforms play in cellular functions and their implication in some of the
pathological conditions have not been precisely defined so far. In this paper we
highlight recent advances made in defining the functions of the two less studied
alternative p38MAPKs, p38γ and p38δ. We describe that these p38MAPKs show
similarities to the classical p38α isoform, although they may play central and
distinct role in certain physiological and pathological processes. Inflammation in the central nervous system (CNS) is a common feature of
age-related neurodegenerative diseases. Proinflammatory cytokines, such as IL-1β
and TNFα, are produced primarily by cells of the innate immune system, namely
microglia in the CNS, and are believed to contribute to the neuronal damage seen
in the disease. The p38 mitogen-activated protein kinase (MAPK) is one of the
kinase pathways that regulate the production of IL-1β and TNFα. Importantly,
small molecule inhibitors of the p38 MAPK family have been developed and show
efficacy in blocking the production of IL-1β and TNFα. The p38 family consists
of at least four isoforms (p38α, β, γ, δ) encoded by separate genes. Recent
studies have begun to demonstrate unique functions of the different isoforms,
with p38α being implicated as the key isoform involved in CNS inflammation.
Interestingly, there is also emerging evidence that two downstream substrates of
p38 may have opposing roles, with MK2 being pro-inflammatory and MSK1/2 being
antiinflammatory. This review discusses the properties, function and regulation
of the p38 MAPK family as it relates to cytokine production in the CNS. Oncogene-induced senescence is a stable proliferative arrest that serves as a
tumor-suppressing defense mechanism. p38 mitogen-activated protein kinase (MAPK)
has been implicated in oncogene-induced senescence and tumor suppression.
However, the specific role of each of the four p38 isoforms in oncogene-induced
senescence is not fully understood. Here, we demonstrate that p38δ mediates
oncogene-induced senescence through a p53- and p16(INK4A)-independent mechanism.
Instead, evidence suggests a link between p38δ and the DNA damage pathways.
Moreover, we have discovered a novel mechanism that enhances the expression of
p38δ during senescence. In this mechanism, oncogenic ras induces the
Raf-1-MEK-extracellular signal-regulated kinase (ERK) pathway, which, in turn,
activates the AP-1 and Ets transcription factors that are bound to the p38δ
promoter, leading to increased transcription of p38δ. These findings indicate
that induction of the prosenescent function of p38δ by oncogenic ras is achieved
through 2 mechanisms, transcriptional activation by the Raf-1-MEK-ERK-AP-1/Ets
pathway, which increases the cellular concentration of the p38δ protein, and
posttranslational modification by MKK3/6, which stimulates the enzymatic
activity of p38δ. In addition, these studies identify the AP-1 and Ets
transcription factors as novel signaling components in the senescence-inducing
pathway. |
Are pseudogenes enriched with housekeeping protein families? | Yes, housekeeping families tend to be enriched with a large number of pseudogenes. | Pseudofam (http://pseudofam.pseudogene.org) is a database of pseudogene families
based on the protein families from the Pfam database. It provides resources for
analyzing the family structure of pseudogenes including query tools, statistical
summaries and sequence alignments. The current version of Pseudofam contains
more than 125,000 pseudogenes identified from 10 eukaryotic genomes and aligned
within nearly 3000 families (approximately one-third of the total families in
PfamA). Pseudofam uses a large-scale parallelized homology search algorithm
(implemented as an extension of the PseudoPipe pipeline) to identify
pseudogenes. Each identified pseudogene is assigned to its parent protein family
and subsequently aligned to each other by transferring the parent domain
alignments from the Pfam family. Pseudogenes are also given additional
annotation based on an ontology, reflecting their mode of creation and
subsequent history. In particular, our annotation highlights the association of
pseudogene families with genomic features, such as segmental duplications. In
addition, pseudogene families are associated with key statistics, which identify
outlier families with an unusual degree of pseudogenization. The statistics also
show how the number of genes and pseudogenes in families correlates across
different species. Overall, they highlight the fact that housekeeping families
tend to be enriched with a large number of pseudogenes. |
What is the mechanism of cementogenesis in pulp regeneration? | The dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone. Dental follicle cells attach to Hertwig's epithelial root sheath (HERS), and pulp cells in the cementum promoting cementogenesis. The temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts. | Stem cell-mediated root regeneration offers opportunities to regenerate a
bio-root and its associated periodontal tissues to restore tooth loss.
Periodontal ligament (PDL) and cementum complex and dentin pulp complex have
been tissue engineered using human dental pulp stem cells and PDL stem cells,
respectively. The aim of this study was to explore whether dentin formation
could be induced using an inductive substrate and whether bioengineered dentin
could induce cementum and PDL formation. First, dentin was bioengineered from
tooth papillae of Sprague-Dawley (SD) rats with an inductive substrate, and its
phenotype was characterized; then primarily cultured human PDL cells were seeded
on the surface of dentin and transplanted under the skin of immunocompromised
mice. Histological, immunohistochemical, and scanning electronic microscopy
examinations results showed that bioengineered dentin could induce
cementogenesis and PDL formation, and condense PDL arranged perpendicularly on
the dentin surface via a layer of cementum-like tissue. The results indicated
that tissue-engineered dentin could be induced using an inductive substrate and
could be used as a further substrate for cementum and PDL tissue engineering. It is known that the dental follicle (DF) consists of progenitor cells that give
rise to the cementum, periodontal ligament, and alveolar bone; but little
information is available about the regulation of DF cell differentiation into
either cementogenic or osteogenic cell lineages for the regeneration of diseased
periodontal tissue. Here, we investigated the roles of DF, Hertwig's epithelial
root sheath (HERS), and pulp cells in the cementum and during alveolar bone
formation. We cultured these cells; transplanted them alone or in combination
into immunocompromised mice; and observed their effects at 6 and 12 weeks.
Histological and immunohistochemical results revealed that DF cells formed
cementum-like tissues with immunoreactivity to cementum-derived attached
protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In
addition, HERS cells played a role in the induction and maturation of
cementum-like tissues formed by DF cells. In contrast, implants of DF cells in
the presence of pulp cells led to the formation of bone-like tissues.
Interestingly, in the presence of both HERS and pulp cells, DF cells formed both
cementum-like and bone-like tissues. We demonstrated that while HERS cells are
able to induce DF cell differentiation into cementoblasts and promote cementum
formation, pulp cells could direct DF cell differentiation into osteoblasts and
enhance alveolar bone formation. These results suggest that the combined use of
DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts
and/or osteoblasts in vivo, thus providing a novel strategy for the successful
repair and regeneration of diseased periodontal tissue. INTRODUCTION: The loss of dental pulp may weaken teeth, rendering them
susceptible to reinfection, fracture, and subsequent tooth loss. Therefore,
regeneration of pulp is considered an ideal treatment to preserve teeth. The aim
of this study was to explore the capacity of dental pulp stem cells (DPSCs) and
platelet-rich plasma (PRP) to regenerate dental pulp in canine mature permanent
teeth.
METHODS: Pulpectomy with apical foramen enlarged to a #80 file was performed in
16 upper premolars of 4 beagle dogs. Four experimental groups were randomly
established: (1) the blood clot group, (2) the autologous DPSCs group, (3) the
PRP group, and (4) the DP + PRP group (a mixture of DPSCs and PRP). Four lower
premolars without any further treatment after pulpectomy were used as the
control group. All teeth were sealed with mineral trioxide aggregate and
composite. Twelve weeks after transplantation, the teeth were subjected to
radiographic and histologic examination.
RESULTS: Twenty-four of 32 experimental root canals gained newly formed tissues.
All canals with an introduction of a blood clot showed histologic evidence of
vital tissue formation. Cementum-like and periodontal ligament-like tissues
along the internal root canal walls were typical structures in most cases. There
is no significant difference between groups with or without autologous DPSC
transplantation (exact chi-square test, P < .05).
CONCLUSIONS: New vital tissues can be regenerated in permanent canine teeth
after pulpectomy and enlargement of the apical foramen. Histologically,
transplantation of DPSCs and/or PRP into root canals showed no enhancement in
new tissue formation compared with inducement of a blood clot into the root
canals alone. |
What are the reported adverse effects of gabapentin used in children? | Limited literature data, suggest that gabapentin may cause rash that is severe enough to necessitate discontinuation in a small percentage of children.
In a large survey of all age groups: The commonest adverse effects seen were somnolence, fainting, ataxia, nystagmus, tremor and headache, fatigue. However, their incidence was low and intensity mild. In a pediatric group only somnolence and dizziness were reported in 2 out of 33 patients.
Behavioural adverse effects are more common in children with intellectual disability and attention deficit, worse in <10yo : hyperactivity, defiance, irritability, agitation, aggression, explosive outbursts, oppositional behavior, often warranting discontinuation of the medication. | Pediatric experience with gabapentin (GBP), a new antiepileptic drug (AED), is
limited. We described 3 learning disabled children, 1 aged 7 and 2 aged 10
years, with intractable partial seizures who developed severe behavioral
problems while receiving modest doses of GBP. The children became hyperactive
and had explosive outburst consisting of aggressive and oppositional behavior.
The behavioral problems were sufficiently severe to require discontinuation of
GBP despite moderately improved seizure control. We report 7 children who received gabapentin (GBP) as adjunctive medic ation and
subsequently developed behavioral side effects. These behavioral changes
consisted of intensification of baseline behaviors as well as new behavioral
problems. Behaviors that parents considered most troublesome were tantrums,
aggression directed toward others, hyperactivity, and defiance. All behavioral
changes were reversible and were managed by dose reduction or discontinuation of
GBP. All children had baseline attention deficit hyperactivity disorder and
developmental delays. Efficacy and safety of gabapentin monotherapy were evaluated in 33 children with
newly diagnosed absence epilepsy in two identical, double-blind,
placebo-controlled trials in which a 2-week double-blind treatment phase was
followed by a 6-week open-label phase. Primary efficacy criterion was seizure
frequency change from baseline to end of double-blind treatment derived from
quantified electroencephalograms. Primary efficacy analyses compared treatment
differences in the 2-week double-blind phase. Gabapentin did not significantly
decrease or increase seizure frequency compared with placebo. Low dosages with
possibly subtherapeutic plasma levels may have contributed to the lack of
demonstrable efficacy. Somnolence and dizziness were the only adverse events
reported by at least two patients during gabapentin treatment. No clinically
important changes in laboratory assessments or other safety parameters were
observed. Gabapentin monotherapy at dosages ranging from 9.7 through 19.1
mg/kg/day is well tolerated in pediatric patients aged 4 through 12 years with
absence epilepsy. Twenty-six children with intellectual disability and six normal children, all
suffering from refractory partial seizures, received open-label gabapentin
(range = 10-50 mg kg(-1) day(-1); mean = 26.7 mg kg(-1) day(-1) as an add-on
medication to their antiepileptic drug regimen. Mean seizure frequency during
baseline was 9.5 seizures per week. Both groups had a significant reduction in
seizure frequency. Response scores and response ratios did not differ between
the intellectually disabled and normal groups (1.67+/-0.67 and 1.25+/-0.69, P =
0.697, and -0.400+/-0.089 and -0.283+/-0.159, P = 0.961, respectively).
Behavioural side-effects were more likely to occur in patients with intellectual
disability in comparison with the mentally normal group (P = 0.0107). In the
present patient population, patients younger than 10 years of age, all of whom
had intellectual disability, were more likely to have side-effects than those
older than 10 years of age. Observed adverse effects, which were generally mild,
occurred in patients with baseline intellectual disability, attention deficit
disorder and behavioural problems. Behavioural adverse effects warranted
discontinuation of the medication in only three patients. The severity of
intellectual disability (mild versus moderate or severe) did not affect the
extent of the response or the occurrence of side-effects. It is concluded that
gabapentin is equally effective as an add-on medication against partial seizures
in patients with or without intellectual disability. However, children with
intellectual disability who also are less than 10 years of age with baseline
attention deficit appear to be at a higher risk of behavioural side-effects. OBJECTIVE: To compare the tolerability of two different dose-initiation regimens
of gabapentin for the adjunctive treatment of partial seizures.
BACKGROUND: Patient compliance is a key feature of successful outpatient
pharmacologic therapy for epilepsy, and one aspect of compliance is simplicity
of initiation. By using a rapid titration rate, leading to a rapid therapeutic
gabapentin dose, perhaps there could be an improvement with compliance.
METHODS: Male or female patients, at least 12 years old, with a recent history
of partial seizures with or without secondary generalization, were randomized to
receive gabapentin (following a blinded placebo period of an undisclosed number
of days) as either a Slow initiation (300 mg day 1, 600 mg day 2, then 900
mg/day) or a Rapid initiation (900 mg/day immediately following the placebo
lead-in).
RESULTS: Starting gabapentin therapy at an initial therapeutic dosage of 900
mg/day is well tolerated by patients with epilepsy and is as safe as initiating
with a titration schedule over 3 days. Of the four most common adverse events
(somnolence, dizziness, ataxia, fatigue), only one, dizziness, occurred more
often in the nontitrated (Rapid initiation) group than in the titrated (Slow
initiation) group.
CONCLUSION: Initiation of gabapentin at 900 mg/day is as well tolerated as is a
3-day titration, except for a higher incidence of dizziness. The efficacy and safety of gabapentin as add-on therapy for refractory partial
seizures in 237 children, aged 3 to 12 years were evaluated over a 6-month
period. All children received gabapentin at 24 to 70 mg/kg/day. Efficacy
variables included the percent change in seizure frequency and the responder
rate (defined as those patients who showed >50% reduction in seizure frequency).
For all partial seizures, the median percent change in seizure frequency was
-34% and the overall responder rate was 34%. Simple partial seizures showed a
median reduction of -53%; complex partial seizures, -38%; and secondarily
generalized tonic-clonic seizures, -35%. Thirteen patients (5%) withdrew during
the 6-month period because of adverse events. Concurrent antiepileptic
medication remained unchanged in 185 patients (78%), was decreased in 27 (11%),
and increased in 25 (11%) patients. This 6-month follow-up study has
demonstrated that gabapentin was well tolerated and appeared to show a sustained
efficacy in a large population of children with refractory partial and
secondarily generalized tonic-clonic seizures. Gabapentin (Neurontin) is a gamma-aminobutyric acid analogue indicated in adults
for adjunctive treatment of partial seizures with or without secondary
generalization. Two studies were conducted to determine the single-dose
pharmacokinetics of gabapentin in healthy subjects age 1 month to 12 years and
to guide dose selection in safety and efficacy trials in pediatric patients.
Forty-eight subjects were given single oral doses of gabapentin (10 mg/kg) while
fasting. Enrollment was homogeneously distributed throughout the age range.
Plasma samples were drawn predose and then serially for 24 hours postdose.
Single doses of gabapentin were well tolerated by healthy pediatric subjects.
Plots of pharmacokinetic parameters versus age suggested significant differences
between younger (1 month to < 5 years) and older (> or =5 to 12 years) subjects.
Mean area under the plasma concentration-time curve from zero to infinity
(AUC(0-infinity)) was 25.6 microg x h/mL in younger subjects and 36.0 microg x
h/mL in older subjects (p < 0.001). Corresponding mean peak plasma
concentrations (Cmax) were 3.74 and 4.52 microg/ml (p < 0.05). Oral clearance
(normalized for body weight) was 7.40 and 4.41 mL/min/kg in younger subjects and
older subjects, respectively (p < 0.001). It was concluded that children between
1 month and < 5 years of age require approximately 30% higher daily doses of
gabapentin than those > or =5 to 12 years of age. PURPOSE: Our aim was to monitor the use of gabapentin (GBP) by patients
prescribed this drug by primary care physicians in England soon after it was
marketed in the United Kingdom.
METHODS: A noninterventional observational cohort study was conducted by using
the technique of prescription-event monitoring. Patients were identified from
dispensed National Health Service prescriptions. Outcome data were obtained from
questionnaires sent to the doctor approximately 6 months after the initial
prescription. These data included demographic information, events reported since
starting GBP, and reason for stopping the drug, if it was stopped. Incidence
rates were calculated for given periods for all events reported. Additional
information was requested for selected events of medical interest, including
pregcies. Standardised mortality ratio (SMR) was calculated.
RESULTS: The cohort comprised 3,100 patients, of whom 136 (4%) were children.
The median duration of treatment was 8.1 months. The most frequently reported
adverse events reported during the first month of treatment,
drowsiness/sedation, dizziness, and malaise/lassitude, also were the commonest
reasons for discontinuing GBP and reported as suspected adverse drug reactions
(ADRs). There were no congenital anomalies in the 11 babies born to women who
used GBP during the first trimester of pregcy. Crude mortality rate was 5
times that in general population but similar to that in other published studies.
CONCLUSIONS: Neurologic-related events were the most frequently reported adverse
events. They also were the commonest reasons for discontinuing treatment and
reported as suspected ADRs. No previously unrecognised adverse events were
detected in this large cohort of patients who were among the first treated with
gabapentin in England. OBJECTIVE: To describe a child who developed a skin reaction during gabapentin
therapy and discuss how we evaluated the probability of an adverse drug
reaction.
CASE SUMMARY: An 8-year-old boy with a neurodegenerative disease of unknown
origin and an epilepsy disorder developed an urticarial rash and irritability 10
and 4 days, respectively, after the start of gabapentin 20 mg/kg 3 times a day
for epilepsy control. Otherwise, the child was well; no changes in other
medication or diet had recently been made. After gabapentin discontinuation and
administration of one dose of methylprednisolone 10 mg/kg intravenously and
diphenhydramine 1 mg/kg every 4 hours via gastric tube, the rash disappeared
over 3 weeks.
DISCUSSION: In contrast to other antiepileptic drugs, skin reactions to
gabapentin are considered uncommon. In adults, reported prevalence of rash
possibly related to gabapentin range from 1% to 10%. A postmarketing
surveillance study reported gabapentin treatment failure as a consequence of
rash in 0.4% of 3000 patients. The product monograph does not mention rash in
children. In our patient, assessment using an objective causality scale revealed
that the rash was probably caused by gabapentin.
CONCLUSIONS: This case, and limited literature data, suggest that gabapentin may
cause rash that is severe enough to necessitate discontinuation in a small
percentage of children. Further research is needed to determine the actual
incidence and severity of gabapentin-related rash in this population. Insomnia is prevalent in pediatrics, particularly in those with
neurodevelopmental disorders. Gabapentin has shown promise in treating insomnia
in adults. The purpose of our study was to review our experience with using
gabapentin to treat insomnia in children. We identified 23 children, seen by the
authors in our Pediatric Sleep Clinic from January 2009 to March 2012. The mean
age was 7.2 years and 70% were male. The majority (87%) had been given diagnoses
of neurodevelopmental or neuropsychiatric disorders. All parents received
education in sleep behavioral interventions. The majority of children (70%) had
both sleep-onset and sleep maintece insomnia. The average starting dose of
gabapentin was 5 mg/kg every bedtime and the maximal dose was 15 mg/kg every
bedtime. At follow-up, improved sleep was noted in 78% of children. Adverse
effects were noted in 6 children. |
What is the effect of ranolazine in diastolic heart failure? | Data from in vitro and animal studies indicate that ranolazine improves diastolic function by inhibiting the late sodium current. Ranolazine is an innovative anti-ischemic and antianginal agent that reduces the Na-dependent Ca-overload, which improves diastolic tone and oxygen handling during myocardial ischemia. Furthermore, ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity. | To assess the effects of ranolazine, a new antiischemic drug, on regional
myocardium of the left ventricle, left ventricular (LV) hemodynamic and
angiographic data were obtained in 15 patients with previous transmural
myocardial infarction before and after intravenous infusion of ranolazine (200
or 500 micrograms/kg body weight). LV angiogram was analyzed by the area method
and was divided into six segments. Regional LV segments were classified as
normal (perfused by intact coronary vessels, n = 20), ischemic (perfused by
stenotic vessels but without ECG evidence suggesting myocardial necrosis, n =
25), or infarcted (total coronary occlusion and with the ECG evidence for
necrosis, n = 45). Regional area fractional shortening, peak filling rate, and
segmental wall motion during isovolumic relaxation period were analyzed. After
ranolazine, regional area fractional shortening was unchanged in all segments.
However, regional peak filling rate was decreased in the normal segments (1499
+/- 315 to 1368 +/- 303 mm2/sec, p < 0.05). In the ischemic segments, by
contrast, the administration of ranolazine significantly increased the regional
peak filling rate (1050 +/- 410 to 1133 +/- 439 mm/sec, p < 0.05) and regional
wall lengthening during the isovolumic relaxation period (0.9 +/- 4.1% to 2.8
+/- 5.7% of end-diastolic segmental area, p < 0.05), which indicates an
improvement of regional diastolic function. Infarct segments were little
affected by ranolazine. Thus, ranolazine improves diastolic function of the
noninfarcted myocardium under chronic ischemic conditions and also may exert a
mild negative lusitropic effect on the normal myocardium, although the former
beneficial effect appears to be more clinically important.(ABSTRACT TRUNCATED AT
250 WORDS) The goal of this study was to test the hypothesis that the novel anti-ischemic
drug ranolazine, which is known to inhibit late I(Na), could reduce
intracellular [Na(+)](i) and diastolic [Ca(2+)](i) overload and improve
diastolic function. Contractile dysfunction in human heart failure (HF) is
associated with increased [Na(+)](i) and elevated diastolic [Ca(2+)](i).
Increased Na(+) influx through voltage-gated Na(+) channels (late I(Na)) has
been suggested to contribute to elevated [Na(+)](i) in HF. In isometrically
contracting ventricular muscle strips from end-stage failing human hearts,
ranolazine (10 micromol/L) did not exert negative inotropic effects on twitch
force amplitude. However, ranolazine significantly reduced frequency-dependent
increase in diastolic tension (i.e., diastolic dysfunction) by approximately 30%
without significantly affecting sarcoplasmic reticulum (SR) Ca(2+) loading. To
investigate the mechanism of action of this beneficial effect of ranolazine on
diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to
increase intracellular Na(+) loading in ventricular rabbit myocytes. ATX-II
caused a significant rise in [Na(+)](i) typically seen in heart failure via
increased late I(Na). In parallel, ATX-II significantly increased diastolic
[Ca(2+)](i). In the presence of ranolazine the increases in late I(Na), as well
as [Na(+)](i) and diastolic [Ca(2+)](i) were significantly blunted at all
stimulation rates without significantly decreasing Ca(2+) transient amplitudes
or SR Ca(2+) content. In summary, ranolazine reduced the frequency-dependent
increase in diastolic tension without having negative inotropic effects on
contractility of muscles from end-stage failing human hearts. Moreover, in
rabbit myocytes the increases in late I(Na), [Na(+)](i) and [Ca(2+)](i) caused
by ATX-II, were significantly blunted by ranolazine. These results suggest that
ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction
due to elevated [Na(+)](i) and diastolic [Ca(2+)](i). Inhibition of the persistent or late Na current (INa) using ranolazine (Ranexa)
represents a novel mechanism of action that was approved in the United States in
2006 and only recently in the European Union for use in patients with stable
angina pectoris. In general, myocardial ischemia is associated with reduced
adenosine triphosphate fluxes and decreased energy supply, resulting in severe
disturbances of intracellular ion homeostasis in cardiac myocytes. In the recent
years, increased late INa was suggested to contribute to this phenomenon by
elevating intracellular Na concentration with subsequent rise in diastolic Ca
levels by means of the sarcolemmal Na-Ca exchange system. Ranolazine, a specific
inhibitor of late INa, reduces Na influx and hence ameliorates disturbed Na and
Ca homeostasis. This is associated with a symptomatic improvement of angina in
patients unlike other antianginal drugs without affecting heart rate or systemic
blood pressure as shown in placebo-controlled studies. Therefore, ranolazine is
a useful new option for patients with chronic stable angina not only as an
add-on therapy. New clinical and experimental studies even point to potential
antiarrhythmic effects, beneficial effects in diastolic heart failure, and under
hyperglycemic conditions. In the present article, the relevant
pathophysiological concepts for the role of late INa inhibition are reviewed and
the most recent data from basic studies and clinical trials are summarized. Palmitoyl-L-carnitine (PC), an ischemic metabolite, causes cellular Na(+) and
Ca(2+) overload and cardiac dysfunction. This study determined whether
ranolazine [(+/-)-1-piperazineacetamide,
N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-] attenuates
PC-induced Na(+) current and ventricular contractile dysfunction of the isolated
heart. PC (4 microM, 30 min) increased late Na(+) current by 1034 +/- 349% in
guinea pig isolated ventricular myocytes; ranolazine (10 microM) and
tetrodotoxin (TTX, 3 microM) significantly attenuated this effect of PC. PC
increased left ventricular end-diastolic pressure (LVEDP), coronary perfusion
pressure (CPP), wall stiffness, and cardiac lactate and adenosine release from
the isolated heart. Ranolazine (10 microM) significantly reduced the PC-induced
increase in LVEDP by 72 +/- 6% (n = 6, p < 0.001), reduced left ventricular wall
stiffness, and attenuated the PC-induced increase of CPP by 53 +/- 10% (n = 6-7,
p < 0.05). Ranolazine (10 microM) reduced the PC-induced increases of lactate
and adenosine release by 70 +/- 8 and 81 +/- 5%, respectively (n = 6, p <or=
0.05 for both). TTX (2 microM) significantly (p < 0.05) reduced PC-induced
increases of CPP and LVEDP. Pretreatment of isolated myocytes or hearts with the
free radical scavenger tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid, disodium
salt) (1 mM) significantly reduced the effects of PC to cause increases of late
Na(+) current and LVEDP, respectively, but unlike ranolazine or TTX, tiron did
not reverse increases of late Na(+) current and LVEDP caused by PC. In summary,
ranolazine and TTX, inhibitors of the late Na(+) current, attenuated the
PC-induced ventricular contractile dysfunction and increase of coronary
resistance in the guinea pig isolated heart. BACKGROUND: Ranolazine (Ran), an antianginal agent, inhibits late Na(+) current.
The purpose of this study was to determine whether there was an added benefit of
adding Ran to cardioplegia (CP) in a model of global ischemia/reperfusion.
METHODS AND RESULTS: Isolated rat hearts were Langendorff-perfused and exposed
to 40-minute normothermic, cardioplegic global ischemia and 30 minutes of
reperfusion. Before ischemia and during reperfusion, hearts were treated with no
drug (control) or with the late Na(+) current inhibitors Ran (5 micromol/L) or
tetrodotoxin (1 micromol/L). Ischemic cardioplegic arrest led to an increase of
left ventricular end-diastolic pressure (LVEDP) by > or =20 mm Hg (ie, cardiac
contracture). Ten out of 11 hearts treated with CP alone developed contracture,
whereas 6 out of 11 hearts treated with CP plus Ran developed contracture. Ran
added to CP reduced LVEDP at the end of ischemia from 41+/-5 mm Hg in CP alone
to 26+/-3 mm Hg in CP plus Ran (P=0.024). Area under the curve for LVEDP during
the entire ischemic period was also smaller in CP plus Ran versus CP alone. The
percent increase (from baseline) of LVEDP measured at the end of 30-minute
reperfusion was smaller for CP plus Ran (66+/-18%) versus CP alone (287+/-90%;
P=0.035). The area under the curve for LVEDP during reperfusion was smaller in
CP plus Ran versus CP alone. Tetrodotoxin (1 micromol/L) also reduced cardiac
contracture during ischemia/reperfusion, compared to CP alone.
CONCLUSIONS: Our results suggest that Ran may have therapeutic potential as an
adjunct to CP and further support a protective role of Na(+) current inhibition
during ischemia/reperfusion. PURPOSE: Ranolazine is a novel antianginal medication that acts by ameliorating
disturbed sodium and calcium homeostasis. By preventing myocyte sodium and
calcium overload, ranolazine also have potential beneficial effects on
myocardial function. Experimental models support this concept, as do 2 small
studies in human participants receiving ranolazine intravenously. We evaluated
changes in parameters of left ventricular function in stable angina patients
treated with oral ranolazine.
METHODS: Twenty-two participants were enrolled with Doppler echocardiography
performed at baseline and a mean of 2 months after initiation of treatment.
RESULTS: Global left ventricular function, as assessed by the myocardial
performance index, was significantly improved on drug therapy (P < .0001). This
was due to improvement in both diastolic and systolic parameters. Of 21
patients, 17 reported less angina and 8 patients reported an increase in
activity level.
CONCLUSIONS: We report improved parameters of left ventricular function in
response to ranolazine as used in the clinical setting. BACKGROUND: Heart failure with preserved ejection fraction (HFpEF), formerly
referred to as diastolic heart failure (DHF), accounts for >50% of all HF
patients. So far, there has been no specific treatment for impaired left
ventricular (LV) relaxation. Data from in vitro and animal studies indicate that
ranolazine improves diastolic function by inhibiting the late sodium current.
HYPOTHESIS: RAnoLazIne for the Treatment of Diastolic Heart Failure (RALI-DHF)
is a prospective, single-center, randomized, double-blind, placebo-controlled
proof-of-concept study to determine if ranolazine compared with placebo will be
more effective in improving diastolic function in patients with HFpEF.
METHODS: Twenty patients with HFpEF (EF ≥ 50% and ratio of transmitral Doppler
early filling velocity to tissue Doppler early diastolic mitral annular velocity
[E/E'] >15 or N-terminal pro-type brain natriuretic peptide >220 pg/mL) will be
randomized to receive ranolazine or placebo in a 1.5:1 ratio during their
catheterization if the LV end-diastolic pressure is ≥18 mm Hg and the time
constant of relaxation (τ) is ≥50 ms. Treatment will consist of intravenous
infusion of study drug (or placebo) for 24 hours, followed by oral treatment for
a total of 14 days.
ENDPOINTS: The study will include the following exploratory endpoints: (1)
change from baseline to 30 minutes from initiation of intravenous study drug
administration during cardiac catheterization hemodynamic parameters at both
resting and paced (120 beats per minute) conditions: τ, LV end-diastolic
pressure, and dP/dt(min) .; and (2) change from baseline to day 14 in E/E',
maximal oxygen consumption, and N-terminal pro-type brain natriuretic peptide.
CONCLUSIONS: The RALI-DHF study is designed as a translational study to bridge
the gap between basic science and therapeutics and to determine if ranolazine,
compared with placebo, will be more effective in improving diastolic function in
patients with HFpEF. RATIONALE: Previously, we demonstrated that a deoxycorticosterone acetate
(DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and
diastolic dysfunction with preserved systolic function. Oxidative stress has
been shown to increase late inward sodium current (I(Na)), reducing the net
cytosolic Ca(2+) efflux.
OBJECTIVE: Oxidative stress in the DOCA-salt model may increase late I(Na),
resulting in diastolic dysfunction amenable to treatment with ranolazine.
METHODS AND RESULTS: Echocardiography detected evidence of diastolic dysfunction
in hypertensive mice that improved after treatment with ranolazine (E/E':sham,
31.9 ± 2.8, sham+ranolazine, 30.2 ± 1.9, DOCA-salt, 41.8 ± 2.6, and
DOCA-salt+ranolazine, 31.9 ± 2.6; P=0.018). The end-diastolic pressure-volume
relationship slope was elevated in DOCA-salt mice, improving to sham levels with
treatment (sham, 0.16 ± 0.01 versus sham+ranolazine, 0.18 ± 0.01 versus
DOCA-salt, 0.23 ± 0.2 versus DOCA-salt+ranolazine, 0.17 ± 0.0 1 mm Hg/L;
P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with
ranolazine (DOCA-salt, 0.18 ± 0.02, DOCA-salt+ranolazine, 0.13 ± 0.01, sham,
0.11 ± 0.01, sham+ranolazine, 0.09 ± 0.02 seconds; P=0.0004). Neither late I(Na)
nor the Ca(2+) transients were different from sham myocytes. Detergent extracted
fiber bundles from DOCA-salt hearts demonstrated increased myofilament response
to Ca(2+) with glutathionylation of myosin binding protein C. Treatment with
ranolazine ameliorated the Ca(2+) response and cross-bridge kinetics.
CONCLUSIONS: Diastolic dysfunction could be reversed by ranolazine, probably
resulting from a direct effect on myofilaments, indicating that cardiac
oxidative stress may mediate diastolic dysfunction through altering the
contractile apparatus. Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3
encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from
gene mutations to the HCM phenotype remain incompletely understood, partially
because current mouse models of HCM do not faithfully reflect the human
situation and early hypertrophy confounds the interpretation of functional
alterations. The goal of this study was to evaluate whether myofilament Ca(2+)
sensitization and diastolic dysfunction are associated or precede the
development of left ventricular hypertrophy (LVH) in HCM. We evaluated the
function of skinned and intact cardiac myocytes, as well as the intact heart in
a recently developed Mybpc3-targeted knock-in mouse model carrying a point
mutation frequently associated with HCM. Compared to wild-type, 10-week old
homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in
skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster
Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional
shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and
Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast,
heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit
LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+)
sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These
data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction
are early phenotypic consequences of Mybpc3 mutations independent of LVH. The
accelerated Ca(2+) transients point to compensatory mechanisms directed towards
normalization of relaxation. We propose that HCM is a model for diastolic heart
failure and this mouse model could be valuable in studying mechanisms and
treatment modalities. Calpain is an intracellular Ca²⁺-activated protease that is involved in numerous
Ca²⁺ dependent regulation of protein function in many cell types. This paper
tests a hypothesis that calpains are involved in Ca²⁺-dependent increase of the
late sodium current (INaL) in failing heart. Chronic heart failure (HF) was
induced in 2 dogs by multiple coronary artery embolization. Using a conventional
patch-clamp technique, the whole-cell INaL was recorded in enzymatically
isolated ventricular cardiomyocytes (VCMs) in which INaL was activated by the
presence of a higher (1 μM) intracellular [Ca²⁺] in the patch pipette. Cell
suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1-2
h before INaL recordings. The numerical excitation-contraction coupling (ECC)
model was used to evaluate electrophysiological effects of calpain inhibition in
silico. MDL caused acceleration of INaL decay evaluated by the two-exponential
fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26
control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control
with vehicle, P<0.05 ANOVA). MDL significantly reduced INaL density recorded at
-30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in
vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage
relationships demonstrated that the INaL density was decreased by MDL in a wide
range of potentials, including that for the action potential plateau. At the
same time the membrane potential dependency of the steady-state activation and
inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted
that calpain inhibition greatly improves myocyte function by reducing the action
potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse
train.
CONCLUSIONS: Calpain inhibition reverses INaL changes in failing dog ventricular
cardiomyocytes in the presence of high intracellular Ca²⁺. Specifically it
decreases INaL density and accelerates INaL kinetics resulting in improvement of
myocyte electrical response and Ca²⁺ handling as predicted by our in silico
simulations. ACCORDING TO THE EJECTION FRACTION, PATIENTS WITH HEART FAILURE MAY BE DIVIDED
INTO TWO DIFFERENT GROUPS: heart failure with preserved or reduced ejection
fraction. In recent years, accumulating studies showed that increased mortality
and morbidity rates of these two groups are nearly equal. More importantly,
despite decline in mortality after treatment in regard to current guideline in
patients with heart failure with reduced ejection fraction, there are still no
trials resulting in improved outcome in patients with heart failure with
preserved ejection fraction so far. Thus, novel pathophysiological mechanisms
are under development, and other new viewpoints, such as multiple comorbidities
resulting in increased non-cardiac deaths in patients with heart failure and
preserved ejection fraction, were presented recently. In this review, we will
focus on the tested as well as the promising therapeutic options that are
currently studied in patients with heart failure with preserved ejection
fraction, along with a brief discussion of pathophysiological mechanisms and
diagnostic options that are helpful to increase our understanding of novel
therapeutic strategies. |
Elaborate on the potential efficacy of gemcitabine for the treatment of recurrent, platinum-resistant epithelial ovarian cancer. | Gemcitabine is a novel agent that has shown consistent activity as a single agent in the treatment of platinum-resistant ovarian cancer and a favorable toxicity profile. Because of its clinical and preclinical synergism with platinum analogs, gemcitabine has been combined with carboplatin as a convincing approach in the treatment of platinum-sensitive recurrent ovarian cancer patients. Gemcitabine and prolonged oral etoposide have shown reproducible single-agent activity in patients with platinum/paclitaxel-resistant ovarian cancer. The combination of carboplatin and gemcitabine resulted in significantly higher response rates and improved progression-free survival when compared with carboplatin alone. A biweekly schedule of gemcitabine combined with PLD is an active and safe chemotherapy regimen with acceptable and easily manageable toxicities in women with recurrent platinum-resistant ovarian cancer. Pertuzumab may add activity to gemcitabine for the treatment of platinum-resistant ovarian cancer. The regimen of gemcitabine combined with ifosfamide and anthracycline is feasible, tolerable and effective in patients with recurrent platinum resistant/refractory epithelial ovarian cancer. Gemcitabine plus endostar significantly improved the prognosis in patients with platinum-resistant recurrent ovarian cancer, especially in those with malignant effusion. Though the endostar cohort also improved median OS by 2.1 months, there was no statistically significant difference compared with gemcitabine alone cohort in this case. | The ovarian cancer presentations at the 2000 ASCO meeting did not yield any
major paradigm shifts in the treatment of women with epithelial ovarian cancer.
Emphasis at this year's meeting focused on the potential incorporation of drugs
such as topotecan, oxaliplatin, doxil, and gemcitabine into the initial
treatment strategies of women with advanced ovarian cancer. These studies
included the introduction of several active and tolerable regimens that are
potentially worthy of direct comparison to the carboplatin and paclitaxel
combination. In the woman with recurrent or persistent ovarian cancer there was
a greater focus on phase III studies directly comparing various chemotherapy
strategies in the treatment of women with recurrent disease. This included the
comparisons of single-versus two-drug salvage regimens, alternate salvage
schedules, and direct comparison of agents active in taxane- and
platinum-resistant disease. Finally, several early studies of novel
non-chemotherapeutic strategies were presented. Newer agents and combinations are needed in order to improve current results in
ovarian cancer treatment. Gemcitabine is a novel agent that has shown consistent
activity as a single agent in the treatment of platinum-resistant ovarian cancer
and a favorable toxicity profile. Because of its clinical and preclinical
synergism with platinum analogs, gemcitabine has been combined with carboplatin
as a convincing approach in the treatment of platinum-sensitive recurrent
ovarian cancer patients. Further combination of gemcitabine and other agents,
including paclitaxel, is also feasible and has been actively studied in order to
establish the role of gemcitabine in the management of treated and untreated
ovarian cancer patients. BACKGROUND: Current chemotherapy in platinum-resistant ovarian cancer patients
has demonstrated minimal to no improvements in survival. Despite the lack of
benefit, significant resources are utilized with such therapies. Therefore, the
objective in the current study was to assess the cost-effectiveness of salvage
chemotherapy for patients with platinum-resistant epithelial ovarian cancer
(EOC).
METHODS: A decision analysis model evaluated a hypothetical cohort of 4000
platinum-resistant patients with recurrent EOC. Several chemotherapy strategies
were analyzed: 1) best supportive care (BSC); 2) second-line
chemotherapy-monotherapy; 3) second-line chemotherapy-combination therapy; 4)
third-line chemotherapy after disease progression on second-line monotherapy;
and 5) third-line chemotherapy after disease progression on second-line
combination therapy. Sensitivity analyses were performed on all pertinent
uncertainties.
RESULTS: Using costs alone, BSC was the only definitive cost-effective treatment
for platinum-resistant recurrent ovarian cancer patients, and second-line
monotherapy was a reasonable cost-effective strategy with an incremental
cost-effectiveness ratio (ICER) of 64,104 dollars. The cost-effectiveness ranged
from 4,065 dollars per month of overall survival (OS) for BSC to 12,927 dollars
for third-line previous combination therapy. Compared with BSC, second-line
monotherapy gained an additional 3 months of OS, with a cost-effectiveness of
4,703 dollars per month of OS. Second-line combination therapy and third-line
therapies exhibited unfavorable ICER.
CONCLUSIONS: The current decision analysis was intended to be thought-provoking
and bring awareness to the high costs of subsequent chemotherapy with limited
effectiveness in patients with recurrent platinum-resistant EOC. Although actual
patients may receive multiple lines of chemotherapy, from the perspective of
costs alone this model using a hypothetical cohort demonstrated that best
supportive care was the only cost-effective strategy, with second-line
monotherapy appearing to be a reasonable cost-effective strategy given current
chemotherapeutic options. OBJECTIVE: To evaluate the anti-tumor effect and toxicity of gemcitabine
combined with platinum chemotherapy on recurrent epithelial ovarian cancer.
METHODS: Phase II study of gemcitabine combined with platinum chemotherapy was
carried out in 22 patients with recurrent epithelial ovarian cancer. Median age
of patients was 50.5 years old. Seven patients were platinum-sensitive and 15
patients were platinum-resistant or -refractory. All patients received
gemcitabine combined with carboplatin or oxaliplatin chemotherapy. Patients'
response rate (RR) and toxicity of gemcitabine combined with platinum
chemotherapy were evaluated.
RESULTS: A total of 98 gemcitabine-based chemotherapy cycles were performed.
Total RR was 36.4%, RR of platinum-sensitive patients was 4/7 and
platinum-resistant and -refractory patients was 4/15. The estimated median
survival time was 10.0 months (95% CI: 7.0-13.0) after initiation of gemcitabine
combined with platinum chemotherapy. There was no significant difference in
survival time between platinum-resistant/refractory group and platinum-sensitive
group (P = 0.061). Side effects of gemcitabine combined with platinum
chemotherapy were observed in 81.8% of patients. Grade II/III anemia (54.5%) and
grade III/IV neutropenia (54.5%) were most common toxicities. Ten (45.5%)
patients had to delay their chemotherapy cycles or reduce the dose of
chemotherapeutic drugs because of the severe side effects. Fourteen (63.6%)
patients received granulocyte colony-stimulating factor to relieve neutropenia,
and 8 (36.4%) patients received component blood transfusion to treat anemia or
thrombocytopenia. There was no treat-ment-associated death.
CONCLUSION: Gemcitabine combined with platinum chemotherapy appears to be an
effective and well-tolerant treatment for recurrent epithelial ovarian cancer,
including platinum-resistant or -refractory diseases. BACKGROUND: Treatment of patients with platinum resistant/refractory ovarian
cancer is a significant problem. In this study, we evaluated the efficacy and
tolerability of the combination of gemcitabine and pegylated liposomal
doxorubicin (PLD) in patients with platinum resistant/refractory ovarian cancer.
PATIENTS AND METHODS: We retrospectively evaluated the activity and toxicity of
gemcitabine and PLD combination in 35 patients with recurrent platinum
resistant/refractory ovarian cancer who had been treated and followed up in 7
centers in Turkey between December 2005 and June 2008. The patients received
gemcitabine 1.000 mg/m(2) on day 1 and 8, and PLD 25 mg/m(2) on day 1 every 28
days.
RESULTS: A total of 187 cycles (median, 6 cycles) were delivered. An objective
response rate of 28,6 % (1 complete, 9 partial response) was achieved.
Additionally, 16 patients (45.7 %) had disease stabilization. The median
time-to-progression was 6 months (95 % confidence interval, 4-8) and the median
overall survival was 17 months (95 % confidence interval, 12-22). Grade 3-4
hematologic toxicities were as follows: leucopenia (14.3%), neutropenia (8.6%),
and anemia (2.9%). One febrile neutropenic episode (2.9%) was observed.
Non-hematologic toxicity was well tolerated and easily managed and no grade 3-4
palmoplantar erytrodysestesia (PPE) was observed.
CONCLUSION: The combination of gemcitabine and PLD is an effective and tolerable
treatment option, with 74.3 % disease control rate for patients with platinum
resistant/refractory ovarian cancer. OBJECTIVES: The optimal treatment for women with recurrent epithelial ovarian
cancer is evolving. The objective of this review is to outline the transition
away from platinum doublets toward nonplatinum combinations and review emerging
data on antiangiogenesis therapy in this setting.
MATERIALS AND METHODS: Recently published and presented data as well as ongoing
clinical trials are discussed.
RESULTS: Current clinical practice largely harmonizes with a paradigm that
outlines a treatment algorithm for recurrent ovarian cancer based on the
duration of platinum-free exposure. In this model, patients whose penultimate
platinum compound exposure (platinum-free interval [PFI]) is longer than 6
months are generally offered a platinum agent or a platinum-containing doublet;
those with a shorter interval are usually treated with a single nonplatinum
agent. This is based on the simple contention that better clinical outcomes will
be realized with platinum in those deemed platinum sensitive (PFI >6 months).
However, it is becoming clear from various phase II and phase III clinical
studies that the performance of many nonplatinum chemotherapeutic agents is also
influenced by this parameter (PFI). Indeed, although definitive comparisons of
nonplatinum drugs to novel cytotoxic agents are lacking, the clinical activity
of these compounds might approach or exceed that of platinum agents. Although
recognized by clinicians, the dichotomy that determines therapy based on PFI has
not been formally accepted by the US Food and Drug Administration in all cases
of drug labeling. For instance, whereas the combination of gemcitabine and
carboplatin is now approved for patients with platinum-sensitive recurrent
ovarian cancer, traditionally used platinum-resistant (PFI <6 months) agents
such as topotecan and pegylated liposomal doxorubicin are also approved by the
US Food and Drug Administration as single agents in platinum-sensitive patients.
Furthermore, the nonplatinum doublet pegylated liposomal doxorubicin and
trabectedin has recently documented comparable activity to platinum combinations
among patients with a PFI of longer than 6 months. To that end, the most
prolific developmental therapeutics arena in ovarian cancer is biologically
targeted therapy, particularly angiogenesis inhibitors. Although it is unknown
if the clinical activity from these new agents will respect the
chemotherapy-sensitive dichotomy, it is clear that they have the potential to
augment efficacy, possibly in both traditionally chemosensitive and
chemoresistant phenotypes.
CONCLUSIONS: The term platinum sensitive should probably be replaced by
chemotherapy sensitive, particularly as new nonplatinum agents and combinations
are identified as active in this setting. Nonplatinum doublets are effective in
treating platinum-sensitive recurrent disease, and adding antiangiogenesis
agents to these combinations is a research priority. To evaluate the activity of gemcitabine and docetaxel in patients with recurrent
ovarian cancer.
METHODS: Patients with platinum-resistant disease and prior treatment with
paclitaxel received treatment with docetaxel on day 1 and gemcitabine on days 1
and 8, repeated every three weeks.
RESULTS: Twenty patients, with a platinum-free interval of three months, were
enrolled. Overall response rate was 25%. Treatment was associated with
significant myelosuppression.
CONCLUSIONS: In chemotherapy-resistant patients, this regimen exhibited
encouraging activity. Excessive myelosuppression led to early closure. This was
prevented by administering docetaxel on day 8 (instead of day 1) and
prophylactic use of G-CSF. Anti-angiogenic agents have played crucial roles in the treatment of ovarian
cancer in recent years, but potential benefits of endostatin have been largely
unexplored. The present retrospective study evaluated its efficacy and toxicity
with two cohorts of patients with platinum-resistant recurrent ovarian cancer.
One cohort received gemcitabine plus endostar (rh-endostatin), and the second
cohort received gemcitabine regimen alone, with totals of 31 and 27 patients,
respectively. The main endpoints were disease control rate (DCR), PFS, overall
survival (OS) and safety. There were statistically significant differences in
DCR (70.9% vs. 40.7%; P = 0.02) and PFS (6.3 months vs. 3.2 months, P = 0.001)
between the two cohorts. Though the endostar cohort also improved median OS by
2.1 months, there was no statistically significant difference compared with
gemcitabine alone cohort in this case (12.5 months vs. 10.4 months, P = 0.201).
Treatment was well tolerated for most patients, and toxicity of endostar was
negligible. Gemcitabine plus endostar significantly improved the prognosis in
patients with platinum-resistant recurrent ovarian cancer, especially in those
with maligt effusion. The endostar- containing regimen is recommended in this
setting. BACKGROUND: Currently, no clearly superior management strategy exists for
recurrent, platinum-resistant ovarian cancer. We tested the efficacy and safety
of gemcitabine combined with oxaliplatin (GEMOX) in a multicentre phase II
clinical trial.
METHODS: Forty one patients with recurrent, platinum-resistant ovarian cancer
were enrolled. Prior to study entry, all the participants had received at least
one platinum-based regimen. Gemcitabine was administered at 1000 mg/m2 as
protracted infusion (100 min) on day 1, and oxaliplatin at the dose of 100 mg/m2
on day 2 in a 2 hour infusion. Cycles were repeated every two weeks.
RESULTS: We observed an overall response rate of 37% [95% Confidence Interval
(CI), 22.3-51.7]. Objective responses plus disease stabilization (clinical
benefit) occurred in 78% of patients. Median progression-free survival was
6.8 months (95% CI, 5.8-7.8), and median overall survival was 16.5 months (95%
CI, 12.2-20.8). Median time to self-reported symptom relief, which was described
by 22 out of 27 symptomatic patients (81.5%), was 4 weeks (range, 2-8). Grade 4
neutropenia and febrile neutropenia were observed in 2 (5%) and 1 (2.5%)
patients, while grade 3 anemia was encountered in 2 (5%) patients, respectively.
The most common adverse effects of any grade were gastrointestinal symptoms,
fatigue and neutropenia. Nine patients (22%) experienced mild allergic reaction
to oxaliplatin, with no treatment discontinuation.
CONCLUSIONS: In our cohort of recurrent, platinum-resistant ovarian cancer
patients, GEMOX showed encouraging activity and manageable toxicity. Under
circumstances requiring a rapid disease control, this combination regimen may
offer a particularly viable option, particularly in heavily pretreated patients. The aim of this study was to evaluate progression-free survival, overall
survival (OS), response rate (RR), and clinical benefit in recurrent ovarian
cancer patients treated with gemcitabine and carboplatin and to compare the
outcome among platinum-resistant and platinum-sensitive patients. A
retrospective study using the medical records of patients diagnosed and treated
for recurrent epithelial ovarian cancer, fallopian tube carcinoma, or primary
peritoneal carcinoma with gemcitabine and carboplatin from 2005 through 2012 at
the Tel Aviv Sourasky Medical Center. The treatment regimen was carboplatin
(area under the curve=5) administered on day 1 and gemcitabine 850 mg/m
administered on days 1 and 8 in a 21-day cycle. Seventy patients with a median
age of 57 years (range: 38-86) were included in the study. Most patients (94.3%)
were initially diagnosed with stage III-IV disease and 44.3% had
platinum-sensitive disease. Median progression-free survival in
platinum-sensitive patients was 6.3 months [95% confidence interval (CI):
4.3-8.3] and 6.3 months (95% CI: 4.6-7.9) in platinum-resistant patients. Median
overall survival was 15.8 months (95% CI: 13.6-18.1) in the platinum-sensitive
patients and 18.4 months (95% CI: 10.0-27.8) in the platinum-resistant patients.
Platinum-sensitive patients had a RR of 43.2% and platinum-resistant patients
had a RR of 39.1%. The clinical benefit was 70.5% in platinum-sensitive patients
and 65.2% in platinum-resistant patients. Overall treatment had a favorable
safety profile. Gemcitabine and carboplatin demonstrate moderate toxicity with
similar efficacy in both platinum-sensitive and platinum-resistant epithelial
ovarian cancer, suggesting reversal of platinum resistance by gemcitabine. |
How does Foxa transcription factor exhibits its pioneering function? | The conceptional framework of the mechanism of action of the FoxA proteins is that these 'pioneer factors' that can engage chromatin before other transcription factors. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. FoxA induces local DNA demethylation, nucleosome destabilization and binds to mitotic chromosomes. When associated with mitotic chromatin, FoxA may "bookmark" active genes and ensure their reactivation in postmitotic cells (epigenetic memory). About one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. The "pioneer" features of FoxA factors involve various chromatin-binding parameters seen in linker histones and distinguish the factors with respect to their regulatory and mechanistic functions. | The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors has
been the subject of genetic and biochemical study for over 15 years. During this
time its three members, Foxa1, Foxa2 and Foxa3, have been found to play
important roles in multiple stages of mammalian life, beginning with early
development, continuing during organogenesis, and finally in metabolism and
homeostasis in the adult. Foxa2 is required for the formation of the node and
notochord, and in its absence severe defects in gastrulation, neural tube
patterning, and gut morphogenesis result in embryonic lethality. Foxa1 and Foxa2
cooperate to establish competence in foregut endoderm and are required for
normal development of endoderm-derived organs such as the liver, pancreas,
lungs, and prostate. In post-natal life, members of the Foxa family control
glucose metabolism through the regulation of multiple target genes in the liver,
pancreas, and adipose tissue. Insight into the unique molecular basis of Foxa
function has been obtained from recent genetic and genomic data, which identify
the Foxa proteins as 'pioneer factors' whose binding to promoters and enhancers
enable chromatin access for other tissue-specific transcription factors. FoxA proteins are pioneer transcription factors, among the first to bind
chromatin domains in development and enable gene activity. The Fox DNA-binding
domain structurally resembles linker histone and binds nucleosomes stably. Using
fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move
much more slowly in nuclei than other transcription factor types, including
c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates
with high nonspecific nucleosome binding, and point mutations that disrupt
nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear
mobility is consistent with its pioneer activity in chromatin. The genetic analysis of the Foxa genes in both total and conditional mutant mice
has clearly established that organogenesis of multiple systems is controlled by
this subfamily of winged helix transcription factors. These discoveries followed
the establishment of the conceptional framework of the mechanism of action of
the FoxA proteins as 'pioneer factors' that can engage chromatin before other
transcription factors. Recent molecular and genomic studies have also shown that
FoxA proteins can facilitate binding of several nuclear receptors to their
respective targets in a context-dependent manner, greatly increasing the range
and importance of FoxA factors in biology. Induced pluripotent stem cell (iPSC) technology is a promising approach for
converting one type of a differentiated cell into another type of differentiated
cell through a pluripotent state as an intermediate step. Recent studies,
however, indicate the possibility of directly converting one cell type to
another without going through a pluripotent state. This direct reprogramming
approach is dependent on a combination of highly potent transcription factors
for cell-type conversion, presumably skipping more physiological and multi-step
differentiation processes. A trial-and-error strategy is commonly used to screen
many candidate transcription factors to identify the correct combination of
factors. We speculate, however, that a better understanding of the functional
mechanisms of exemplary transcriptional activators will facilitate the
identification of novel factor combinations capable of direct reprogramming. The
purpose of this review is to critically examine the literature on three highly
potent transcriptional activators: the herpes virus protein, VP16; the master
regulator of skeletal muscle differentiation, MyoD and the "pioneer" factor for
hepatogenesis, FoxA. We discuss the roles of their functional protein domains,
interacting partners and chromatin remodeling mechanisms during gene activation
to understand how these factors open the chromatin of inactive genes and reset
the transcriptional pattern during cell type conversion. There exists a hierarchy by which transcription factors can engage their target
sites in chromatin, in that a subset of factors can bind transcriptionally
silent, nucleosomal DNA, whereas most factors cannot, and this hierarchy is
reflected, at least in part, in the developmental function of the factors. For
example, transcription factors possessing the Forkhead box (Fox) DNA-binding
domain contain an overall fold resembling that of linker histone and thus are
structured to bind DNA, site specifically, in a nucleosomal context. Where
tested, Fox factors bind early in the developmental or physiological activation
of target genes, thereby enabling the binding of other factors that cannot
engage chromatin on their own. To investigate the basis for early chromatin
binding, we have used fluorescence recovery after photobleaching (FRAP) to
analyze the mobility, in the live cell nucleus, of FoxA factors in comparison to
linker histone and other transcription factors. We have further analyzed the
factors for their ability to bind to chromatin in mitosis and thereby serve as
epigenetic marks. The results indicate that the "pioneer" features of FoxA
factors involve various chromatin-binding parameters seen in linker histones and
that distinguish the factors with respect to their regulatory and mechanistic
functions. Understanding how silent genes can be competent for activation provides insight
into development as well as cellular reprogramming and pathogenesis. We
performed genomic location analysis of the pioneer transcription factor FoxA in
the adult mouse liver and found that about one-third of the FoxA bound sites are
near silent genes, including genes without detectable RNA polymerase II.
Virtually all of the FoxA-bound silent sites are within conserved sequences,
suggesting possible function. Such sites are enriched in motifs for
transcriptional repressors, including for Rfx1 and type II nuclear hormone
receptors. We found one such target site at a cryptic "shadow" enhancer 7
kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional
activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory
properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically
induced in the early metaplastic condition of Barrett's esophagus, its
expression is not necessarily present in progressive Barrett's with dysplasia or
adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal
epithelium becomes gradually extinguished during progression to cancer, i.e,
expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We
propose that this decreased expression of Rfx1 could be an indicator of
progression from Barrett's esophagus to adenocarcinoma and that similar analyses
of other transcription factors bound to silent genes can reveal uticipated
regulatory insights into oncogenic progression and cellular reprogramming. EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is
indispensable for the maintece of the viral episomes in latently infected
cells. EBNA1 may induce genetic alterations by upregulating cellular
recombinases, production of reactive oxygen species (ROS) and affecting p53
levels and function. All these changes may contribute to tumorigenesis. In this
overview we focus, however, on the epigenetic alterations elicited by EBNA1 by
drawing a parallel between EBNA1 and the FoxA family of pioneer transcription
factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome
destabilization and bind to mitotic chromosomes. Local DNA demethylation and
nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1
and FoxA, when associated with mitotic chromatin may "bookmark" active genes and
ensure their reactivation in postmitotic cells (epigenetic memory). We speculate
that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular
genome. Such chromatin loops, sustained in mitotic chromatin similarly to the
long-distance interactions mediated by the insulator protein CTCF, may also
mediate the epigenetic inheritance of gene expression patterns. We suggest that
EBNA1 has the potential to induce patho-epigenetic alterations contributing to
tumorigenesis. |
Which are the 3 basic transcription factors that have been used for the direct reprogramming of fibroblasts into cardiomyocytes or cardiomyocyte like-cells? | Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We found that functional cardiomyocytes can be directly induced from fibroblasts by a combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5, in vitro and in vivo. | Recent advances in stem cell biology have established the feasibility of
reprogramming human and murine fibroblast cells into induced pluripotent stem
cells. Three master regulators have been demonstrated to be sufficient in the
management of cell status of 'pluripotent' versus 'differentiated'. The same
strategy has been used to directly convert one somatic cell type into another
cell type, such as the converting of exocrine pancreas cells into cells closely
resembling beta cells and the reprogramming of fibroblast cells into functional
neuron cells. Srivastava's group reported the first direct reprogramming of
mouse fibroblast cells into mesoderm lineage cells (cardiomyocytes) with the
enforced expression of three cardiac transcriptional factors: Gata4, Mef2c, and
Tbx5. The induced cardiomyocytes exhibit a global gene expression profile and
basic electrophysiological characteristics similar to those of postnatal
cardiomyocytes. This study made significant advances in cardiovascular and stem
cell fields and has important implications in understanding heart developmental
biology as well as in potential therapies of human cardiovascular diseases. The simultaneous overexpression of several transcription factors has emerged as
a successful strategy to convert fibroblasts into other cell types including
pluripotent cells, neurons, and cardiomyocytes. The selection and screening of
factors are critical, and have often involved testing a large pool of
transcription factors, followed by successive removal of single factors. Here,
to identify a cardiac transcription factor combination facilitating mouse
fibroblast reprogramming into cardiomyocytes, we directly screened all triplet
combinations of 10 candidate factors combined with a Q-PCR assay reporting
induction of multiple cardiac-specific genes. Through this screening method the
combination of Tbx5, Mef2c, and Myocd was identified to upregulate a broader
spectrum of cardiac genes compared to the combination of Tbx5, Mef2c, and Gata4
that was recently shown to induce reprogramming of fibroblasts into
cardiomyocytes. Cells cotransduced with Tbx5, Mef2c, Myocd expressed cardiac
contractile proteins, had cardiac-like potassium and sodium currents and action
potentials could be elicited. In summary the alternative screening approach that
is presented here avoided the elimination of transcription factors whose potency
is masked in complex transcription factor mixes. Furthermore, our results point
to the importance of verifying multiple lineage specific genes when assessing
reprogramming. RATIONALE: Direct reprogramming of fibroblasts into cardiomyocytes is a novel
strategy for cardiac regeneration. However, the key determits involved in
this process are unknown.
OBJECTIVE: To assess the efficiency of direct fibroblast reprogramming via viral
overexpression of GATA4, Mef2c, and Tbx5 (GMT).
METHODS AND RESULTS: We induced GMT overexpression in murine tail tip
fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of
transgenic mice carrying different cardiomyocyte lineage reporters. We found
that the induction of GMT overexpression in TTFs and CFs is inefficient at
inducing molecular and electrophysiological phenotypes of mature cardiomyocytes.
In addition, transplantation of GMT infected CFs into injured mouse hearts
resulted in decreased cell survival with minimal induction of cardiomyocyte
genes.
CONCLUSIONS: Significant challenges remain in our ability to convert fibroblasts
into cardiomyocyte-like cells and a greater understanding of cardiovascular
epigenetics is needed to increase the translational potential of this strategy. Loss of terminally differentiated cardiomyocytes due to heart disease is
irreversible and current therapeutic regimes are limited. Cell therapy using
stem cell-derived cardiomyocytes is an attractive option to repair injured
hearts. The discovery of direct reprogramming of fibroblasts into induced
pluripotent stem cells (iPSCs) and successful differentiation of iPSCs into
cardiomyocytes provided a revolutionary paradigm in heart regenerative research.
During the past decades, significant advances in stem cell culture,
differentiation and purification protocols, as well as in cell transplantation
methodologies, have been achieved. On the other hand, recent studies
demonstrated that a somatic cell could be converted into an alternative
differentiated cell type without first becoming a stem cell by overexpression of
lineage-specific factors. We found that functional cardiomyocytes can be
directly induced from fibroblasts by a combination of three cardiac
transcription factors, Gata4, Mef2c and Tbx5, in vitro and in vivo. I will
review the perspectives of heart regeneration using reprogramming technology. Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced
expression of cardiac transcription factors or microRNAs has recently been
demonstrated. The potential clinical applicability of these findings is based on
the minimal regenerative potential of the adult human heart and the limited
availability of human heart tissue. An initial but mandatory step toward
clinical application of this approach is to establish conditions for conversion
of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought
to determine the optimal combination of factors necessary and sufficient for
direct myocardial reprogramming of human fibroblasts. Here we show that four
human cardiac transcription factors, including GATA binding protein 4, Hand2,
T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac
marker expression in neonatal and adult human fibroblasts. After maintece in
culture for 4-11 wk, human fibroblasts reprogrammed with these proteins and
microRNAs displayed sarcomere-like structures and calcium transients, and a
small subset of such cells exhibited spontaneous contractility. These phenotypic
changes were accompanied by expression of a broad range of cardiac genes and
suppression of nonmyocyte genes. These findings indicate that human fibroblasts
can be reprogrammed to cardiac-like myocytes by forced expression of cardiac
transcription factors with muscle-specific microRNAs and represent a step toward
possible therapeutic application of this reprogramming approach. Heart disease remains a leading cause of death worldwide. Owing to the limited
regenerative capacity of heart tissue, cardiac regenerative therapy has emerged
as an attractive approach. Direct reprogramming of human cardiac fibroblasts
(HCFs) into cardiomyocytes may hold great potential for this purpose. We
reported previously that induced cardiomyocyte-like cells (iCMs) can be directly
generated from mouse cardiac fibroblasts in vitro and vivo by transduction of
three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In
the present study, we sought to determine whether human fibroblasts also could
be converted to iCMs by defined factors. Our initial finding that GMT was not
sufficient for cardiac induction in HCFs prompted us to screen for additional
factors to promote cardiac reprogramming by analyzing multiple cardiac-specific
gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT
up-regulated a broader spectrum of cardiac genes in HCFs more efficiently
compared with GMT alone. The HCFs and human dermal fibroblasts transduced with
GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape
to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins,
increased a broad range of cardiac genes and concomitantly suppressed fibroblast
genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells
matured to exhibit action potentials and contract synchronously in coculture
with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the
iCMs thus generated do not pass through a mitotic cell state. These findings
demonstrate that human fibroblasts can be directly converted to iCMs by defined
factors, which may facilitate future applications in regenerative medicine. |
What is the name of Bruton's tyrosine kinase inhibitor that can be used for treatment of chronic lymphocytic leukemia? | Ibrutinib is the covalent inhibitor of Bruton's tyrosine kinase that can be used for treatment of chronic lymphocytic leukemia (CLL). Ibrutinib has shown highly encouraging results in phase I/II trials in patients with treatment-naive, relapsed and refractory CLL even in the presence of high risk disease or poor prognostic markers. Ibrutinib demonstrated that Bruton's tyrosine kinase inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior. Ibrutinib has excellent activity in other B cell malignancies, including in particular mantle cell lymphoma and Waldenstrom macroglobulinemia. | INTRODUCTION: There has been a significant paradigm shift in the manner in which
lymphoid maligcies are treated and managed. Treatment has been moving away
from conventional chemotherapy and towards targeted therapy. The success of new
classes of agents such as monoclonal antibodies, proteasome inhibitors and
immunomodulatory derivatives has sparked further searches for novel pathways to
inhibit. The Bruton's tyrosine kinase (Btk) pathway is a downstream mediator of
the B-cell receptor (BCR) pathway, which is crucial in B-cell production and
maintece, and a potential therapeutic target.
AREAS COVERED: This review will summarize the current knowledge of the Btk
pathway and its role in lymphoid maligcies. It will also discuss the present
data about PCI-32765 in both the preclinical and clinical setting.
EXPERT OPINION: PCI-32765 is an oral irreversible Btk inhibitor with high
potency and both preclinical and clinical activity in chronic lymphocytic
leukemia (CLL) and non-Hodgkin's lymphoma (NHL). Phase I studies have
demonstrated that it is well tolerated and has an excellent safety profile.
Further studies are ongoing as a single agent and in combination with other
targeted and conventional therapies. PCI-32765 is a very promising targeted
therapy, and the data from these trials will ultimately decide its future role
and success. Protein kinases (PKs) and lipid kinases (LKs) are good choices for targets of
signal transduction therapy as these enzymes are involved in signaling pathways,
and are often related to the pathogenesis of lymphoid maligcies. The
attractiveness of PKs and LKs as drug able targets is enhanced by the fact that
they are enzymes whose biological activity can be turned off by drugs that block
their catalytic site. In the last few years small molecular kinase inhibitors
(KIs) have been synthesized and become available for preclinical studies and
clinical trials. The first KI, introduced into clinical practice in 1998, was
imatinib mesylate, which became the first choice drug in chronic myeloid
leukemia. More recently, several KIs have been developed to target the proximal
B-cell receptor (BCR) signaling pathway including spleen tyrosine kinase
inhibitor (Fostamatinib) and Bruton's tyrosine kinase inhibitors (Ibrutinib,
AVL-263). These agents are currently evaluated in early clinical trials in
chronic lymphocytic leukemia (CLL) and other diseases. Cyclin-dependent kinase
(Cdk) inhibitors, flavopiridol (alvocidib), BMS-387032 (SNS-032), sunitinib and
sorafenib are currently under evaluation in clinical trials for
relapsed/refractory CLL. Multi-tyrosine kinase inhibitors including vandetanib
(ZD6474) bosutinib (SKI-606), TKI258 (CHIR-258), pazopanib (GW786034) and
axitinib (AG013736) have been also developed for the treatment of lymphoid
maligcies. Phosphatidylinositol 3-kinases (PI3K ) are a family of lipid
kinases that mediate signals from cell surface receptors. CAL-101 (GS-1101) is
an oral PI3Kδ-specific inhibitor which has shown preclinical and clinical
activity against CLL. This article summarizes recent achievements in the
mechanism of action, pharmacological properties and clinical activity and
toxicity of PK and LK inhibitors in CLL. PURPOSE OF REVIEW: Despite ongoing efforts to decipher the cancer genome,
discoveries of new targetable genetic lesions within cancer cells are rare.
Therefore, alternative approaches are needed. Signals from the microenvironment
are increasingly recognized as drivers of disease progression in hematologic and
solid cancers. Consequently, there is growing interest in targeting the
tumor-microenvironment cross-talk. This review highlights recent therapeutic
advances in targeting the microenvironment in chronic lymphocytic leukemia
(CLL).
RECENT FINDINGS: CLL is the poster child for microenvironment-dependent
maligcies, because the clonal CLL B cells are highly dependent on external
signals for maintece and expansion. These pathways recapitulate those
responsible for normal B-cell expansion in germinal centers. The most prominent,
conserved mechanism is B-cell receptor (BCR) signaling, which promotes CLL cell
survival and expansion in lymphatic tissue areas designated proliferation
centers. BCR signaling now can be targeted by new targeted kinase inhibitors.
SUMMARY: Small molecule inhibitors of BCR signaling kinases, Bruton's tyrosine
kinase (Btk) inhibitor ibrutinib and the phosphoinositide 3'-kinase delta
(PI3Kδ) inhibitor GS-1101, are currently transforming the landscape of CLL
therapy. This development exemplifies that the microenvironment has become a
lively successful area of translational research. In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR)
play a major role in disease development and progression. In this light, new
therapies that specifically target signaling molecules downstream of the BCR
continue to be developed. While first studies on the selective small molecule
inhibitor of Bruton's tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated
that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory
behavior, these studies however did not address whether Btk-mediated signaling
is involved in the process of CLL leukemogenesis. To investigate the requirement
of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETμ
CLL mouse model, which is based on sporadic expression of the simian oncovirus
SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETμ mice on a
Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here
we show that Btk deficiency fully abrogates CLL formation in IgH.ETμ mice, and
that leukemias formed in Btk haplo-insufficient mice selectively expressed the
wild-type Btk allele on their active X chromosome. Conversely, Btk
overexpression accelerated CLL onset, increased mortality, and was associated
with selection of non-stereotypical BCRs into CLL clones. Taken together, these
data show that Btk expression represents an absolute prerequisite for CLL
development and that Btk mediated signaling enhances leukemogenesis in mice. We
therefore conclude that in CLL Btk expression levels set the threshold for
maligt transformation. 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. New treatment options are urgently needed for patients with relapsed chronic
lymphocytic leukemia (CLL) who fail to respond to currently available therapies
or cannot achieve a sustained response. Moreover, targeted agents with less
myelotoxicity are necessary to treat patients with multiple comorbidities who
would otherwise be unable to tolerate standard regimens. Ibrutinib, a Bruton's
tyrosine kinase inhibitor, has shown highly encouraging results in phase I/II
trials in patients with treatment-naive, relapsed and refractory CLL even in the
presence of high risk disease or poor prognostic markers. In phase I/II trials,
ibrutinib 420 mg or 840 mg - given continuously as single agent or at a dose of
420 mg daily in combination with a monoclonal antibody or chemoimmunotherapy -
has been associated with high response rates and durable clinical remissions.
Phase II and III trials are currently under way for treatment-naive patients,
relapsed/refractory patients, and for those patients harboring a 17p deletion. Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors
for proliferation and survival. In particular, the B-cell receptor (BCR) and
nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN)
microenvironment. Thus, model systems mimicking tumor-host interactions are
important tools to study CLL biology and pathogenesis. We investigated whether
the recently established NOD/scid/γc(null) (NSG) mouse xenograft model can
recapitulate the effects of the human microenvironment. We assessed, therefore,
tumor characteristics previously defined in LN-resident CLL cells, including
proliferation, and activation of the BCR and NF-κB pathways. We found that the
murine spleen (SP) microenvironment supported CLL cell proliferation and
activation to a similar degree than the human LN, including induction of BCR and
NF-κB signaling in the xenografted cells. Next, we used this model to study
ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development.
Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment,
decreased proliferation, induced apoptosis and reduced the tumor burden in vivo.
Thus, our data demonstrate that the SP of xenografted NSG mice can, in part,
recapitulate the role of the human LN for CLL cells. In addition, we show that
ibrutinib effectively disrupts tumor-host interactions essential for CLL cell
proliferation and survival in vivo. Recent clinical data suggest remarkable activity of ibrutinib, the
first-in-class covalent inhibitor of Bruton's tyrosine kinase (BTK), in chronic
lymphocytic leukemia (CLL), as well as excellent activity in other B cell
maligcies, including in particular mantle cell lymphoma and Waldenstrom
macroglobulinemia. This review evaluates the data from ongoing clinical and
correlative studies of ibrutinib in B cell maligcies with a particular focus
on CLL, and considers these data in the context of other B cell receptor pathway
inhibitors. BACKGROUND: The treatment of relapsed chronic lymphocytic leukemia (CLL) has
resulted in few durable remissions. Bruton's tyrosine kinase (BTK), an essential
component of B-cell-receptor signaling, mediates interactions with the tumor
microenvironment and promotes the survival and proliferation of CLL cells.
METHODS: We conducted a phase 1b-2 multicenter study to assess the safety,
efficacy, pharmacokinetics, and pharmacodynamics of ibrutinib (PCI-32765), a
first-in-class, oral covalent inhibitor of BTK designed for treatment of B-cell
cancers, in patients with relapsed or refractory CLL or small lymphocytic
lymphoma. A total of 85 patients, the majority of whom were considered to have
high-risk disease, received ibrutinib orally once daily; 51 received 420 mg, and
34 received 840 mg.
RESULTS: Toxic effects were predomitly grade 1 or 2 and included transient
diarrhea, fatigue, and upper respiratory tract infection; thus, patients could
receive extended treatment with minimal hematologic toxic effects. The overall
response rate was the same in the group that received 420 mg and the group that
received 840 mg (71%), and an additional 20% and 15% of patients in the
respective groups had a partial response with lymphocytosis. The response was
independent of clinical and genomic risk factors present before treatment,
including advanced-stage disease, the number of previous therapies, and the
17p13.1 deletion. At 26 months, the estimated progression-free survival rate was
75% and the rate of overall survival was 83%.
CONCLUSIONS: Ibrutinib was associated with a high frequency of durable
remissions in patients with relapsed or refractory CLL and small lymphocytic
lymphoma, including patients with high-risk genetic lesions. (Funded by
Pharmacyclics and others; ClinicalTrials.gov number, NCT01105247.). 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. Understanding the pathogenesis of CLL has uncovered a plethora of novel targets
for human application of monoclonal antibodies, engineered T cells, or
inhibitors of signal transduction pathways. The B-cell receptor signaling
pathway is being actively explored as a therapeutic target in CLL. Ibrutinib, an
inhibitor of Bruton's tyrosine kinase is showing impressive responses in heavily
pre-treated high-risk CLL, whether alone or in combination with MoAbs or
chemotherapy. Other key components of the BCR pathway, namely PI3K-δ, are also
being targeted with novel therapies with promising results as well. Future
trials would likely evaluate ibrutinib in the front-line setting. Moreover,
improvements in allogeneic HCT mostly by continuing to reduce associated
toxicity as well as incorporating cellular therapies such as autologous CLL
tumor vaccines, among others, will continue to expand. This is also the case for
the next generation of chimeric antigen receptor therapy for CLL once
genetically modified T cells are available at broad scale and with improved
efficacy. As our ability to further refine and integrate these therapies
continues to improve, and we gain further knowledge from gene sequencing, we
anticipate that treatment algorithms will continue to be revised to a more
personalized approach to treat this disease with improved efficacy and devoid of
unnecessary toxicity. Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation
of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of
the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive
activity in CLL and is the target of irreversible inhibition by ibrutinib, an
orally bioavailable kinase inhibitor that has shown outstanding activity in CLL.
Early clinical results in CLL with other reversible and irreversible BTK
inhibitors have been less promising, however, raising the question of whether
BTK kinase activity is an important target of ibrutinib and also in CLL. To
determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1)
transgenic mouse model of CLL, which results in spontaneous leukemia
development. Inhibition of BTK in primary human CLL cells by small interfering
RNA promotes apoptosis. Inhibition of BTK kinase activity through either
targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly
delays the development of CLL, demonstrating that BTK is a critical kinase for
CLL development and expansion and thus an important target of ibrutinib.
Collectively, our data confirm the importance of kinase-functional BTK in CLL. |
Is physical performance influenced by thyroid hormone metabolism? | Yes. | A 40-year-old woman was admitted for investigation of weakness and angina
pectoris. She had generalised weakness of muscles, cold intolerance and a
reduced physical performance. A previous neurological examination had already
revealed a carpal-tunnel syndrome of the right hand. This syndrome, combined
with a rough, cool skin and a periorbital edema, lead to the assumption of
hypothyroidism. The diagnosis was confirmed by a combination of very high
concentrations of TSH and decreased concentrations of the thyroid hormones.
Replacement therapy by oral administration of L-thyroxin resulted in a gradual
improvement of the patient's state. CONTEXT: Physiological changes in thyroid hormone concentrations might be
related to changes in the overall physical function in the elderly.
OBJECTIVE: We determined to what extent thyroid hormone concentrations are
related to physical function and mortality in elderly men.
DESIGN: A longitudinal population study (the Zoetermeer study) was conducted.
Mortality was registered in the subsequent 4 yr.
PARTICIPANTS: Four hundred three independently and ambulatory living men (aged
73-94 yr) participated.
MAIN OUTCOME MEASURES: The study examined the association between serum thyroid
hormones and parameters of physical function as well as the association with
mortality.
METHODS: TSH, free T4 (FT4) total T4, T3, rT3, and T4-binding globulin were
measured. Physical function was estimated by the number of problems in
activities of daily living, a measure of physical performance score (PPS), leg
extensor strength and grip strength, bone density, and body composition.
RESULTS: Serum rT3 increased significantly with age and the presence of disease.
Sixty-three men met the biochemical criteria for the low T3 syndrome (decreased
serum T3 and increased serum rT3). This was associated with a lower PPS,
independent of disease. Furthermore, higher serum FT4 (within the normal range
of healthy adults) and rT3 (above the normal range of healthy adults) were
related with a lower grip strength and PPS, independent of age and disease.
Isolated low T3 was associated with a better PPS and a higher lean body mass.
Low FT4 was related to a decreased risk of 4-yr mortality.
CONCLUSIONS: In a population of independently living elderly men, higher FT4 and
rT3 concentrations are associated with a lower physical function. High serum rT3
may result from a decreased peripheral metabolism of thyroid hormones due to the
aging process itself and/or disease and may reflect a catabolic state. Low serum
FT4 is associated with a better 4-yr survival; this may reflect an adaptive
mechanism to prevent excessive catabolism. Subclinical hyperthyroidism (SH) may be responsible for many cardiovascular
changes, including an impaired exercise performance. The aim of our study was to
evaluate the response to the treadmill cardiopulmonary test in patients with SH.
We studied 14 female patients from our endocrine clinic with exogenous SH, free
from cardiovascular diseases, with mean age of 38.6 +/- 10.2 years, body mass
index (BMI) of 24.4 +/- 4.0 kg/m(2), and disease duration of 4.9 +/- 4.9 years.
The mean serum thyrotropin (TSH) was 0.03 +/- 0.03 mU/L, serum free thyroxine
(FT(4)), 1.72 +/- 0.21 ng/dL, and serum triiodothyronine level, 137 +/- 32
ng/dL. The control group comprised 15 euthyroid, healthy women, with mean age of
35.4 +/- 7.4 years and BMI of 27.3 +/- 5.9 kg/m(2). Both groups had a sedentary
lifestyle and underwent the cardiopulmonary test using a treadmill with the
Balke protocol. Gas concentrations and the respiratory outflow were measured and
the electrocardiogram (ECG) was registered in real time. We calculated the
minute ventilation (V(E)), the oxygen consumption (peak VO(2)), the carbonic gas
exhalation (peak VCO(2)) and the anaerobic threshold (AT). The heart rate (HR)
at rest (90.9 +/- 15.7 versus 78.9 +/- 8.7 beats per minute; p = 0.03) was
higher in the patients from our clinic. There was no difference between groups
regarding age, BMI, fat percentage, blood pressure, peak HR, exercise duration,
mean treadmill peak inclination, V(E), peak VO(2), peak VCO(2), and AT. There
was no correlation between peak VO(2) and FT(4), TSH, or disease duration. Our
results show that exercise capacity in young and middle-aged female patients is
not significantly affected by exogenous SH. OBJECTIVE: Previous experimental studies have provided evidence showing that
changes in thyroid hormone signaling correspond to alterations in myocardial
function in animal models of heart failure. The present study further explores
whether thyroid hormone alterations are correlated with the functional status of
the myocardium in patients with heart failure.
METHODS: In this study, 37 patients with mean ejection fraction (EF%) of 26.2
(8.2) were included. Myocardial performance was assessed by echocardiography and
cardiopulmonary exercise testing. Total tri-iodothyronine (T3), thyroxine, and
TSH levels were measured in plasma.
RESULTS: Total T3 was strongly correlated with VO2max (r = 0.78, P = 2 x
10(-8)). Furthermore, multivariate analysis revealed that total T3 was an
independent predictor of VO2max (P = 0.000 005). A weaker but significant
correlation was also found between total T3 and EF% (r = 0.56, P = 0.0004),
systolic (r = 0.43, P = 0.009) and diastolic (r = 0.46, P = 0.004) blood
pressure.
CONCLUSIONS: changes in thyroid hormone were closely correlated to myocardial
functional status in patients with heart failure. These data probably indicate a
possible role of thyroid hormone in the pathophysiology of heart failure and
confirm previous experimental reports. OBJECTIVE: We report a very rare case of Hoffmann's syndrome with
musclehypertrophy complicating hypothyroidism.
CLINICAL PRESENTATION: A 24-year-old man presented with a 2-year history of
forgetfulness, swelling in his face, shoulder and calf, and motor weakness in
his lower extremities. His calf and shoulder muscles were hypertrophic.
Neurological examination revealed hoarseness of the voice, proximal muscle
weakness, reduced deep tendon reflexes and a mildly ataxic gait. Laboratory
tests indicated markedly elevated serum muscle enzymes and lipids, a high
thyroid-stimulating hormone level and low free triiodothyronine and free
thyroxine levels. Electromyographic evaluation showed myopathy.
INTERVENTION: Oral L-thyroxine treatment was started and at a 1-month follow-up
examination, mental status and physical performance were improved.
CONCLUSION: This report shows that in the differential diagnosis of myopathy
with pseudohypertrophy, Hoffmann's syndrome should be considered. Although muscle metabolism and exercise capacity seem to be affected in patients
with subclinical hypothyroidism, there is little evidence indicating improvement
of the exercise tolerance due to levothyroxine (L-T(4)) replacement. The aim of
the present study was to verify possible cardiopulmonary changes during exercise
in patients with subclinical hypothyroidism on L-T(4) replacement with a normal
serum TSH for six months. Twenty-three patients with subclinical hypothyroidism
were randomized into treated (no.=11) and untreated (no.=12) patients. A
cardiopulmonary test was performed with a treadmill, using the modified Balke
protocol. Heart rate, oxygen uptake, minute ventilation and other
cardiopulmonary parameters were assessed at the 5th minute of exercise. FT4
levels increased while TSH normalized after hormone replacement. Oxygen uptake
decreased significantly after hormone replacement (24.1+/-6.3 vs 17.1+/-4.2 ml x
kg x min(-1); p=0.03).Minute ventilation also showed an enhanced performance in
treated patients (28.0+/-8.1 vs 23.5+/-5.6 l x min(-1); p=0.03), as did the
heart rate (128+/-17 vs 121+/-17 bpm; p=0.03). There were no changes in the
untreated group. The results demonstrate that submaximal cardiopulmonary
exercise performance improved after six months of TSH normalization and this
improvement can help enhance the ability to carry out daily life activities in
patients with subclinical hypothyroidism. |
Do plant genomes contain CpG islands? | In plant genomes, there exist discrete regions rich in CpG dinucleotides, namely CpG clusters. In rice, most of these CpG clusters are associated with genes. Rice genes are grouped into one of the five classes according to the position of an associated CpG cluster. Among them, class 1 genes, which harbor a CpG cluster at the 5 -terminus, share similarities with human genes having CpG islands | We screened plant genome sequences, primarily from rice and Arabidopsis
thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides
within these sequences. These CpG-rich clusters appeared in the analysed
sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in
rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the
CpG-rich clusters were associated with genes, which suggests that these clusters
are useful landmarks in genome sequences for identifying genes in plants with
small genomes. In contrast, in plants with larger genomes, only a few of the
clusters were associated with genes. These plant CpG-rich clusters satisfied the
criteria used for identifying human CpG islands, which suggests that these CpG
clusters may be regarded as plant CpG islands. The position of each island
relative to the 5'-end of its associated gene varied considerably. Genes in the
analysed sequences were grouped into five classes according to the position of
the CpG islands within their associated genes. A large proportion of the genes
belonged to one of two classes, in which a CpG island occurred near the 5'-end
of the gene or covered the whole gene region. The position of a plant CpG island
within its associated gene appeared to be related to the extent of
tissue-specific expression of the gene; the CpG islands of most of the widely
expressed rice genes occurred near the 5'-end of the genes. The identification of promoters and their regulatory elements is one of the
major challenges in bioinformatics and integrates comparative, structural, and
functional genomics. Many different approaches have been developed to detect
conserved motifs in a set of genes that are either coregulated or orthologous.
However, although recent approaches seem promising, in general, unambiguous
identification of regulatory elements is not straightforward. The delineation of
promoters is even harder, due to its complex nature, and in silico promoter
prediction is still in its infancy. Here, we review the different approaches
that have been developed for identifying promoters and their regulatory
elements. We discuss the detection of cis-acting regulatory elements using
word-counting or probabilistic methods (so-called "search by signal" methods)
and the delineation of promoters by considering both sequence content and
structural features ("search by content" methods). As an example of search by
content, we explored in greater detail the association of promoters with CpG
islands. However, due to differences in sequence content, the parameters used to
detect CpG islands in humans and other vertebrates cannot be used for plants.
Therefore, a preliminary attempt was made to define parameters that could
possibly define CpG and CpNpG islands in Arabidopsis, by exploring the
compositional landscape around the transcriptional start site. To this end, a
data set of more than 5,000 gene sequences was built, including the promoter
region, the 5'-untranslated region, and the first introns and coding exons.
Preliminary analysis shows that promoter location based on the detection of
potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward.
Nevertheless, because the landscape of CpG/CpNpG islands differs considerably
between promoters and introns on the one side and exons (whether coding or not)
on the other, more sophisticated approaches can probably be developed for the
successful detection of "putative" CpG and CpNpG islands in plants. BACKGROUND: A GC-compositional strand bias or GC-skew (=(C-G)/(C+G)), where C
and G denote the numbers of cytosine and guanine residues, was recently reported
near the transcription start sites (TSS) of Arabidopsis genes. However, it is
unclear whether other eukaryotic species have equally prominent GC-skews, and
the biological meaning of this trait remains unknown.
RESULTS: Our study confirmed a significant GC-skew (C > G) in the TSS of Oryza
sativa (rice) genes. The full-length cDNAs and genomic sequences from
Arabidopsis and rice were compared using statistical analyses. Despite marked
differences in the G+C content around the TSS in the two plants, the degrees of
bias were almost identical. Although slight GC-skew peaks, including opposite
skews (C < G), were detected around the TSS of genes in human and Drosophila,
they were qualitatively and quantitatively different from those identified in
plants. However, plant-like GC-skew in regions upstream of the translation
initiation sites (TIS) in some fungi was identified following analyses of the
expressed sequence tags and/or genomic sequences from other species. On the
basis of our dataset, we estimated that > 70 and 68% of Arabidopsis and rice
genes, respectively, had a strong GC-skew (> 0.33) in a 100-bp window (that is,
the number of C residues was more than double the number of G residues in a
+/-100-bp window around the TSS). The mean GC-skew value in the TSS of
highly-expressed genes in Arabidopsis was significantly greater than that of
genes with low expression levels. Many of the GC-skew peaks were preferentially
located near the TSS, so we examined the potential value of GC-skew as an index
for TSS identification. Our results confirm that the GC-skew can be used to
assist the TSS prediction in plant genomes.
CONCLUSION: The GC-skew (C > G) around the TSS is strictly conserved between
monocot and eudicot plants (ie. angiosperms in general), and a similar skew has
been observed in some fungi. Highly-expressed Arabidopsis genes had overall a
more marked GC-skew in the TSS compared to genes with low expression levels. We
therefore propose that the GC-skew around the TSS in some plants and fungi is
related to transcription. It might be caused by mutations during transcription
initiation or the frequent use of transcription factor-biding sites having a
strand preference. In addition, GC-skew is a good candidate index for TSS
prediction in plant genomes, where there is a lack of correlation among CpG
islands and genes. In plant genomes, there exist discrete regions rich in CpG dinucleotides, namely
CpG clusters. In rice, most of these CpG clusters are associated with genes.
Rice genes are grouped into one of the five classes according to the position of
an associated CpG cluster. Among them, class 1 genes, which harbor a CpG cluster
at the 5'-terminus, share similarities with human genes having CpG islands. In
the present study, by analyzing plant genome sequence data, primarily from rice,
we investigated the chromosomal distribution of genes of each class, mainly
class 1 genes. Class 1 genes were not uniformly distributed across the rice
genome, but were clustered into discrete chromosomal segments. EST-based
analysis of the distribution of expressed genes indicates that this segmental
distribution of class 1 genes caused a preferential distribution of expressed
genes within class 1 gene-rich segments. We then compared the methylation status
of genes of each class to examine the possibility that differential DNA
methylation, if any, is relevant to the observed differential expression level
of genes inside and outside the class 1 segments. The difference in the
methylation level between these genes was revealed to be fairly small, which
does not support the above-mentioned possibility. The genomes of many higher plant species are the most highly methylated among
eukaryotes. We report here that in spite of their heavy methylation, genomic
DNAs from four plant species contain a fraction that is very rich in
non-methylated sites. The fraction was characterized in maize where it
represents about 2.5% of the total nuclear genome. In order to establish the
genomic origin of the fraction, three maize genes containing clustered CpG were
tested for methylation and were found to be non-methylated in the CpG-rich
regions. By contrast, tested CpGs were methylated in a gene whose sequence
showed no clustering of CpG. These observations suggest that the CpG-rich
fraction of plants is at least partially derived from non-methylated regions
that are associated with genes. A similar phenomenon has been described in
vertebrate genomes. We discuss the evolution of CpG islands in both groups of
organisms, and their possible uses in mapping and gene isolation in plants. BACKGROUND: Due to its overarching role in genome function, sequence-dependent
DNA curvature continues to attract great attention. The DNA double helix is not
a rigid cylinder, but presents both curvature and flexibility in different
regions, depending on the sequence. More in depth knowledge of the various
orders of complexity of genomic DNA structure has allowed the design of
sophisticated bioinformatics tools for its analysis and manipulation, which, in
turn, have yielded a better understanding of the genome itself. Curved DNA is
involved in many biologically important processes, such as transcription
initiation and termination, recombination, DNA replication, and nucleosome
positioning. CpG islands and tandem repeats also play significant roles in the
dynamics and evolution of genomes.
RESULTS: In this study, we analyzed the relationship between these three
structural features within rice (Oryza sativa) and Arabidopsis (Arabidopsis
thaliana) genomes. A genome-scale prediction of curvature distribution in rice
and Arabidopsis indicated that most of the chromosomes of both genomes have
maximal chromosomal DNA curvature adjacent to the centromeric region. By
analyzing tandem repeats across the genome, we found that frequencies of repeats
are higher in regions adjacent to those with high curvature value. Further
analysis of CpG islands shows a clear interdependence between curvature value,
repeat frequencies and CpG islands. Each CpG island appears in a local minimal
curvature region, and CpG islands usually do not appear in the centromere or
regions with high repeat frequency. A statistical evaluation demonstrates the
significance and non-randomness of these features.
CONCLUSIONS: This study represents the first systematic genome-scale analysis of
DNA curvature, CpG islands and tandem repeats at the DNA sequence level in plant
genomes, and finds that not all of the chromosomes in plants follow the same
rules common to other eukaryote organisms, suggesting that some of these genomic
properties might be considered as specific to plants. |
Describe the isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads, as applied for the case of the essential hematopoietic transcription factor GATA-1. | Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes. | GATA-1 is essential for the generation of the erythroid, megakaryocytic,
eosinophilic and mast cell lineages. It acts as an activator and repressor of
different target genes, for example, in erythroid cells it represses cell
proliferation and early hematopoietic genes while activating erythroid genes,
yet it is not clear how both of these functions are mediated. Using a
biotinylation tagging/proteomics approach in erythroid cells, we describe
distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the
repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in
addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we
show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus
providing an explanation for the overlapping functions of these two factors in
erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in
vivo by distinct complexes, thus linking specific GATA-1 partners to distinct
aspects of its functions. Based on these findings, we suggest a model for the
different roles of GATA-1 in erythroid differentiation. We have described the application of a simple biotinylation tagging approach for
the direct purification of tagged transcription factor complexes, based on the
use of artificial short peptide tags that are specifically and efficiently
biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells
with the tagged factor. We used this approach to initially characterize
complexes formed by the hematopoietic transcription factor GATA-1 in erythroid
cells. GATA-1 is essential for the erythroid differentiation, its functions
encompassing upregulation of erythroid genes, repression of alternative
transcription programs, and suppression of cell proliferation. However, it was
not clear how all of these GATA-1 functions are mediated. Our work describes,
for the first time, distinct GATA-1 interactions with the essential
hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin
remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and
GATA-1/TAL-1 complexes. We also provide evidence that distinct GATA-1 complexes
are associated with specific GATA-1 functions in erythroid differentiation, for
example, GATA-1/Gfi-1b with the suppression of cell proliferation and
GATA-1/FOG-1/MeCP1 with the repression of other hematopoietic transcription
programs. We next applied the biotinylation tag to Ldb-1, a known partner of
GATA-1, and characterized a number of novel interaction partners that are
essential in erythroid development, in particular, Eto-2, Lmo4, and CdK9. Last,
we are in the process of applying the same technology to characterize the
factors that are bound to the suppressed gamma-globin promoter in vivo. BACKGROUND: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays
(Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide
identification of binding sites of chromatin associated proteins. However, the
highly variable quality of antibodies and the availability of epitopes in
crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have
often been used as more reliable alternatives. In addition, we have employed
protein in vivo biotinylation tagging as a very high affinity alternative to
antibodies. In this paper we describe the optimization of biotinylation tagging
for ChIP and its coupling to a known epitope tag in providing a reliable and
efficient alternative to antibodies.
RESULTS: Using the biotin tagged erythroid transcription factor GATA-1 as
example, we describe several optimization steps for the application of the high
affinity biotin streptavidin system in ChIP. We find that the omission of SDS
during sonication, the use of fish skin gelatin as blocking agent and choice of
streptavidin beads can lead to significantly improved ChIP enrichments and lower
background compared to antibodies. We also show that the V5 epitope tag performs
equally well under the conditions worked out for streptavidin ChIP and that it
may suffer less from the effects of formaldehyde crosslinking.
CONCLUSION: The combined use of the very high affinity biotin tag with the less
sensitive to crosslinking V5 tag provides for a flexible ChIP platform with
potential implications in ChIP sequencing outcomes. |
Are high-flow nasal cannulae effective for treatment of preterm infants? | Yes. The use of high-flow nasal cannulae is an increasingly popular alternative to nasal continuous positive airway pressure for noninvasive respiratory support of preterm infants after extubation. However, the use of high-flow nasal cannulae in preterm infants was shown to be associated with a higher rate of reintubation, increased exposure to oxygen and longer duration of respiratory support. High-flow nasal cannulae are also effective for treatment of apnea of prematurity. | Apnea of prematurity (AOP) is frequently managed with nasal continuous positive
airway pressure (NCPAP). Nasal cannula (NC) are used at low flows (<0.5 L/min)
to deliver supplemental oxygen to neonates. A number of centers use high-flow
nasal cannula (HFNC) in the management of AOP without measuring the positive
distending pressure (PDP) generated. Objective. To determine the NC flow
required to generate PDP equal to that provided by NCPAP at 6 cm H(2)O and to
assess the effectiveness of HFNC as compared NCPAP in the management of AOP.
Method. Forty premature infants, gestation 28.7 +/- 0.4 weeks (mean +/- standard
error of mean), postconceptual age at study 30.3 +/- 0.6 weeks, birth weight
1256 +/- 66 g, study weight 1260 +/- 63 g who were being managed with
conventional NCPAP for at least 24 hours for clinically significant apnea of
prematurity, were enrolled in a trial of ventilator-generated conventional NCPAP
versus infant NC at flows of up to 2.5 L/min. End expiratory esophageal pressure
was measured on NCPAP and on NC, and the gas flow on NC was adjusted to generate
an end expiratory esophageal pressure equal to that measured on NCPAP. Two
6-hour periods were continuously recorded and the data were stored on computer.
Results. The flow required to generate a comparable PDP with NC varied with the
infant's weight and was represented by the equation: flow (L/min) = 0.92 +
0.68x, x = weight in kg, R = 0.72. There was no difference in the frequency and
duration of apnea, bradycardia or desaturation per recording between the 2
systems. Conclusion. NC at flows of 1 to 2.5 L/min can deliver PDP in premature
neonates. HFNC is as effective as NCPAP in the management of AOP. OBJECTIVE: The aim of this study was to measure pharyngeal pressures in preterm
infants receiving high-flow nasal cannulae.
STUDY DESIGN: A total of 18 infants were studied (median gestational age 34
weeks, weight 1.619 kg). A catheter-tip pressure transducer was introduced into
the nasopharynx. Flow was sequentially increased to a maximum of 8 l min(-1) and
decreased to a minimum of 2 l min(-1).
RESULT: There was a strong association between pharyngeal pressure and both flow
rate and infant weight (P<0.001, r (2)=0.61), but not mouth closure. This
relationship could be expressed as pharyngeal pressure (cm H(2)O)=0.7+1.1 F
(F=flow per kg in l min(-1) kg(-1)).
CONCLUSION: High-flow nasal cannulae at flow rates of 2 to 8 l min(-1) can lead
to clinically significant elevations in pharyngeal pressure in preterm infants.
Flow rate and weight but not mouth closure are important determits of the
pressure transmitted. OBJECTIVE: To determine the better approach for weaning preterm infants from
nasal continuous positive airway pressure (NCPAP) with or without transitioning
to nasal cannula (NC).
DESIGN/METHODS: This is a randomized, open label, controlled trial. Preterm
infants born at ≥28 weeks gestation who were clinically stable on NCPAP of 5 cm
H(2)O with FiO(2)<0.30 for at least 24 h were randomly assigned to one of 2
groups. The no-NC group were kept on NCPAP until they were on FiO(2)=0.21 for 24
h, and then were weaned off NCPAP completely without any exposure to NC. If they
met failing criteria, NCPAP was re-instituted. The NC-group was weaned off NCPAP
when FiO(2) was ≤0.30 to NC (2 L/min) followed by gradual weaning from oxygen.
Infants who failed NC were supported back with NCPAP for 24 h before making a
second attempt of NC.
RESULTS: Sixty neonates were enrolled; 30 in each group. The two groups were
similar in birthweight, gestational age, sex, antenatal steroids, mode of
delivery, use of surfactant and xanthines, and duration of mechanical
ventilation. After randomization, the no-NC group had fewer days on oxygen
[median (interquartile range): 5 (1-8) vs 14 (7.5-19.25) days, p<0.001] and
shorter duration of respiratory support [10.5 (4-21) vs 18 (11.5-29) days,
p=0.03]. There were no differences between groups regarding success of weaning
from NCPAP.
CONCLUSIONS: Weaning preterm infants from NCPAP to NC is associated with
increased exposure to oxygen and longer duration of respiratory support. BACKGROUND: High flow nasal cannulae (HFNC) are small, thin, tapered cannulae
used to deliver oxygen or blended oxygen and air at flow rates of > 1 L/min.
HFNC can be used to provide high concentrations of oxygen and may deliver
positive end-expiratory pressure.
OBJECTIVES: To compare the safety and efficacy of HFNC with other forms of
non-invasive respiratory support in preterm infants.
SEARCH STRATEGY: The strategy included searches of the Cochrane Central Register
of Controlled Trials (CENTRAL) (The Cochrane Library 2010), MEDLINE, CINAHL,
EMBASE and abstracts from conference proceedings.
SELECTION CRITERIA: Randomised or quasi-randomised trials comparing HFNC with
other non-invasive forms of respiratory support in preterm infants immediately
after birth or following extubation.
DATA COLLECTION AND ANALYSIS: Data were extracted and analysed by the authors.
Relative risk, risk difference and number needed to treat were calculated.
MAIN RESULTS: Four studies were identified for inclusion in the review. The
studies differed in the interventions compared (nasal continuous positive airway
pressure (CPAP), humidified HFNC, non-humidified HFNC), the flow rates provided
and the indications for respiratory support. Meta-analysis and subgroup analysis
were not possible. When used as primary respiratory support after birth, one
trial found similar rates of treatment failure in infants treated with HFNC and
nasal CPAP. Following extubation, one trial found that infants treated with HFNC
had a significantly higher rate of reintubation than those treated with nasal
CPAP. Another trial found similar rates of reintubation for humidified and
non-humidified HFNC, and the fourth trial found no difference between two
different models of equipment used to deliver humidified HFNC.
AUTHORS' CONCLUSIONS: There is insufficient evidence to establish the safety or
effectiveness of HFNC as a form of respiratory support in preterm infants. When
used following extubation, HFNC may be associated with a higher rate of
reintubation than nasal CPAP. Further adequately powered randomised controlled
trials should be undertaken in preterm infants comparing HFNC with nasal CPAP
and with other means of respiratory support; or of support following extubation.
These trials should measure clinically important outcomes. BACKGROUND: Limited data are available to describe the CPAP effects that can be
expected when using high flow with a traditional nasal cannula.
OBJECTIVE: To describe the relationship between the pressure generated at the
airway opening and flow through a nasal cannula using a simulated infant model.
We hypothesized that positive pressure generated by a standard cannula at flows
> 2 L/min would be minimal and clinically unimportant.
METHODS: Nares were simulated with holes drilled in a plastic fixture. A nares
template for CPAP prongs served as a sizing template for the holes. Small,
medium, and large nares fixtures were constructed and connected to a lung
simulator that simulated spontaneous breathing. Respiratory muscle pressure was
simulated by setting a waveform and adjusting the amplitude to deliver a range
of tidal volumes (V(T)) from 3 mL to 12 mL. Lung compliance and resistance were
set at 0.5 mL/cm H(2)O and 125 cm H(2)O/L/s, respectively. Nasal cannulas were
inserted in the model nares. We assured that the prong occlusion of the nares
did not exceed 50%. Cannula flow was adjusted from 2-6 L/min in 1-L/min
increments. Data were averaged over 20 breaths. Mean airway pressure and percent
change in V(T) were recorded.
RESULTS: The greatest effect on V(T) (mean ± SD 0.16 ± 0.10 mL) and pressure
change (mean ± SD 0.7 ± 0.5 cm H(2)O) occurred with the premature cannula. The
least effect on pressure (mean ± SD 0.3 ± 0.22 cm H(2)O) and V(T) change (mean ±
SD 0.01 ± 0.02 mL) occurred with the infant cannula.
CONCLUSIONS: Clinically important pressures were not generated by high flows
with a standard nasal cannula. The differences in spontaneous V(T) across all
flows were negligible. Continuous positive airway pressure (CPAP) is widely used in neonatal units both
as a primary mode of respiratory support and following extubation from
mechanical ventilation. In this review, the evidence for CPAP use particularly
in prematurely born infants is considered. Studies comparing methods of CPAP
generation have yielded conflicting results, but meta-analysis of randomised
trials has demonstrated that delivering CPAP via short nasal prongs is most
effective in preventing re-intubation. At present, there is insufficient
evidence to establish the safety or efficacy of high flow nasal cannulae for
prematurely born infants. Observational studies highlighted that early CPAP use
rather than intubation and ventilation was associated with a lower incidence of
bronchopulmonary dysplasia (BPD), but this has not been confirmed in three large
randomised trials. Meta-analysis of the results of randomised trials has
demonstrated that use of CPAP reduces extubation failure, particularly if a CPAP
level of 5 cm H2O or more is used. Nasal injury can occur and is related to the
length of time CPAP is used; weaning CPAP by pressure rather than by
"time-cycling" reduces the weaning time and may reduce BPD. In conclusion,
further studies are required to identify the optimum mode of CPAP generation and
it is important that prematurely born infants are weaned from CPAP as soon as
possible. Respiratory failure in the premature infants remains a difficult challenge. An
alternative to the use of nasal continuous positive airway pressure (NCPAP) as a
non-invasive modality to support respiratory distress in premature infants has
been the recent introduction of high flow nasal cannula (HFNC) devices in many
neonatal units. There has been increased use of HFNC presumably because of
anecdotal reports and experience that it is easy to use, and well tolerated by
the infants, while experiencing decreased nasal septumerosion. The paucity of
evidence regarding its efficacy and safety, would support a caution approach to
the use of HFNC. Particular concern has focused on the imprecise regulation and
generation of pressure that may occur at higher flows especially in the smallest
of infants. BACKGROUND: High-flow nasal cannulae (HFNC) are gaining in popularity as a form
of non-invasive respiratory support for preterm infants in neonatal intensive
care units around the world. They are proposed as an alternative to nasal
continuous positive airway pressure (NCPAP) in a variety of clinical situations,
including post-extubation support, primary therapy from birth and 'weaning' from
NCPAP.
OBJECTIVES: To present and discuss the available evidence for the use of HFNC in
the preterm population.
METHODS: An internet-based literature search for relevant, original research
articles (both randomised studies and not) on the use of HFNC in preterm infants
was undertaken.
RESULTS: A total of 19 studies were included in the review. Distending pressure
generated by HFNC in preterm infants increases with increasing flow rate and
decreasing infant size and varies according to the amount of leak around the
prongs. HFNC may be as effective as NCPAP at improving respiratory parameters
such as tidal volume and work of breathing in preterm infants, but probably only
at flow rates >2 litres/min. The efficacy and safety of HFNC in preterm infants
remain to be determined.
CONCLUSIONS: There is growing evidence of the feasibility of HFNC as an
alternative to other forms of non-invasive ventilation in preterm infants.
However, there remains uncertainty about the efficacy and safety of HFNC in this
population. Until the results of larger randomised trials are known, widespread
use of HFNC to treat preterm infants cannot be recommended. BACKGROUND: Heated, humidified, high-flow nasal cannula oxygen therapy (HHHFNC)
has been used to improve ventilation in preterm infants. There are no data on
airway pressures generated and efficacy in bronchiolitis.
OBJECTIVE: The objective of this study was to determine nasopharyngeal (NP)
pressures generated with HHHFNC therapy in bronchiolitis.
METHODS: We conducted a prospective, observational study to measure NP pressures
at varying flow rates of HHHFNC therapy in moderate to severe bronchiolitis.
Vital signs, bronchiolitis severity scores, and oxygen saturation were also
noted.
RESULTS: Twenty-five patients were enrolled (mean, 78.1 [SD, 30.9] days; weight,
5.3 [SD, 1.1] kg). Nasopharyngeal pressures increased linearly with flow rates
up to 6 L/min. Beyond 6 L/min, pressure increase was linear but less
accelerated. On average, NP pressure increased by 0.45 cm H2O for each 1-L/min
increase in flow rate. There were significant differences between pressures in
open- and closed-mouth states for flow rates up to 6 L/min. At 6 L/min, the
pressure in open-mouth state was 2.47 cm H2O and that in closed-mouth state was
2.74 cm H2O (P < 0.001). Linear regression analysis revealed that only flow (not
weight or gender) had an effect on generated pressure. Bronchiolitis severity
scores improved significantly with HHHFNC therapy (pre: 14.5 [SD, 1.4], post:
10.4 [SD, 1.2]; P < 0.001).
CONCLUSIONS: Increasing flow rates of HHHFNC therapy are associated with linear
increases in NP pressures in bronchiolitis patients. Larger studies are needed
to assess the clinical efficacy of HHHFNC therapy in bronchiolitis. BACKGROUND: High flow nasal cannula (HFNC) systems utilize higher gas flow rates
than standard nasal cannulae. The use of HFNC as a respiratory support modality
is increasing in the infant, pediatric, and adult populations as an alternative
to non-invasive positive pressure ventilation.
OBJECTIVES: This critical review aims to: (1) appraise available evidence with
regard to the utility of HFNC in neonatal, pediatric, and adult patients; (2)
review the physiology of HFNC; (3) describe available HFNC systems (online
supplement); and (4) review ongoing and planned trials studying the utility of
HFNC in various clinical settings.
RESULTS: Clinical neonatal studies are limited to premature infants. Only a few
pediatric studies have examined the use of HFNC, with most focusing on this
modality for viral bronchiolitis. In critically ill adults, most studies have
focused on acute respiratory parameters and short-term physiologic outcomes with
limited investigations focusing on clinical outcomes such as duration of therapy
and need for escalation of ventilatory support. Current evidence demonstrates
that HFNC generates positive airway pressure in most circumstances; however, the
predomit mechanism of action in relieving respiratory distress is not well
established.
CONCLUSION: Current evidence suggests that HFNC is well tolerated and may be
feasible in a subset of patients who require ventilatory support with
non-invasive ventilation. However, HFNC has not been demonstrated to be
equivalent or superior to non-invasive positive pressure ventilation, and
further studies are needed to identify clinical indications for HFNC in patients
with moderate to severe respiratory distress. OBJECTIVE: To determine whether postextubation respiratory support via heated,
humidified, high-flow nasal cannulae (HHHFNC) results in a greater proportion of
infants younger than 32 weeks' gestation being successfully extubated after a
period of endotracheal positive pressure ventilation compared with conventional
nasal continuous positive airway pressure (NCPAP).
STUDY DESIGN: We randomly assigned preterm ventilated infants to Vapotherm
HHHFNC or NCPAP after extubation. The primary outcome, extubation failure, was
defined by prespecified failure criteria in the 7 days after extubation.
RESULTS: A total of 132 ventilated infants younger than 32 weeks' gestation were
randomized to receive either HHHFNC (n = 67) or NCPAP (n = 65). Extubation
failure occurred in 15 (22%) of the HHHFNC group compared with 22 (34%) of the
NCPAP group. There was no difference in the number of infants reintubated in the
first week. Treatment with HHHFNC reduced the nasal trauma score 3.1 (SD 7.2)
versus NCPAP 11.8 (SD 10.7), P < .001.
CONCLUSIONS: HHHFNC and NCPAP produced similar rates of extubation failure. AIMS: This study aims to determine if there is a difference in the pharyngeal
pressure, measured as a surrogate for continuous positive distending airway
pressure, delivered to premature infants between two commonly used heated,
humidified high-flow nasal cannulae (HHHFNC) devices: Fisher & Paykel Healthcare
HHHFNC and Vapotherm 2000i.
METHODS: Pharyngeal pressure measurements were taken from stable premature
infants receiving HHHFNC for respiratory support. Flow rates of 2-8 L/min were
studied.
RESULTS: Nine infants had pharyngeal pressure measurements recorded with both
HHHFNC devices at flow rates of 2-8 L/min. There was no difference in pharyngeal
pressures recorded between devices at flow rates of 2-6 L/min; measured pressure
was linearly associated with flow (R(2) = 0.9). At flow rates of 7 L/min,
Vapotherm delivered a mean (standard deviation) pharyngeal pressure of 4.7 (2.2)
cmH2 O compared with 4.23 (2.2) cmH2 O by the Fisher & Paykel device (P = 0.04).
At a flow of 8 L/min, the mean pharyngeal pressure via Vapotherm was 4.9 (2.2)
cmH2 O compared with 4.1 (2.3) cmH2 O with the Fisher & Paykel device (P =
0.05).
CONCLUSIONS: Both HHHFNC delivered similar pharyngeal pressures at flow rates of
2-6 L/min. The pressure limiter valve of the Fisher & Paykel device attenuated
the pharyngeal pressures at flows of 7 and 8 L/min. Vapotherm trended towards
higher delivered pharyngeal pressure at flow rates 7 and 8 L/min, but the
clinical significance of the difference remains unclear. BACKGROUND: High-flow nasal cannula (HFNC) is a safe, well-tolerated, and
noninvasive method of respiratory support that has seen increasing use in the
care of children with respiratory distress. High-flow nasal cannula may be able
to prevent intubations in infants and children with respiratory distress.
OBJECTIVE: The objective of this study was to determine the clinical and patient
characteristics that predict success or failure of HFNC therapy in children
presenting to the pediatric emergency department (PED) with respiratory
distress.
DESIGN/METHODS: A retrospective cohort review was conducted of all children
younger than 2 years evaluated in 2 PEDs between June 2011 and September 2012
who received HFNC therapy within 24 hours of initial triage. Data extraction
included clinical variables, demographic variables, and patient outcomes.
Therapy failure was defined as the clinical decision to intubate a patient after
an antecedent trial of HFNC. Multivariable logistic regression was performed to
identify factors associated with intubation following HFNC.
RESULTS: Four hundred ninety-eight cases meeting criteria for inclusion were
identified. The most common final diagnosis was acute bronchiolitis (n = 231,
46%), followed by pneumonia (n = 138, 28%) and asthma (n = 38, 8%). Of the 498
patients, 42 (8%) of patients failed therapy and required intubation following
HFNC trial. Risk factors associated with HFNC failure were triage respiratory
rate greater than 90th percentile for age (odds ratio [OR], 2.11; 95% confidence
interval [CI], 1.01-4.43), initial venous PCO2 greater than 50 mm Hg (OR, 2.51;
95% CI, 1.06-5.98), and initial venous pH less than 7.30 (OR, 2.53; 95% CI,
1.12-5.74). A final diagnosis of bronchiolitis was observed to be protective
with respect to intubation (OR, 0.40; 95% CI, 0.17-0.96).
CONCLUSIONS: In infants with all-cause respiratory distress presenting in the
PED, triage respiratory rate greater than 90th percentile for age, initial
venous PCO2 greater than 50 mm Hg, and initial venous pH less than 7.30 were
associated with failure of HFNC therapy. A diagnosis of acute bronchiolitis was
protective with respect to intubation following HFNC. This finding may help
guide clinicians who use HFNC by identifying a patient population at higher risk
of failing therapy. |
What are the molecular characteristics of the FAA (FANCA) cDNA? | The 5.5-kb cDNA of the FAA (FANCA) gene has an open reading frame of 4,368 nucleotides, whereas the FAA protein is predicted to have a molecular weight of approximately 163 kDa. | |
What are the skeletal muscle satellite cells? | Skeletal muscle satellite cells (SCs) are Pax7(+) myogenic stem cells that reside between the basal lamina and the plasmalemma of the myofiber. In mature muscles, SCs are typically quiescent, but can be activated in response to muscle injury. Depending on the magnitude of tissue trauma, SCs may divide minimally to repair subtle damage within individual myofibers or produce a larger progeny pool that forms new myofibers in cases of overt muscle injury | Skeletal muscle satellite cells are quiescent mononucleated myogenic cells,
located between the sarcolemma and basement membrane of
terminally-differentiated muscle fibres. These are normally quiescent in adult
muscle, but act as a reserve population of cells, able to proliferate in
response to injury and give rise to regenerated muscle and to more satellite
cells. The recent discovery of a number of markers expressed by satellite cells
has provided evidence that satellite cells, which had long been presumed to be a
homogeneous population of muscle stem cells, may not be equivalent. It is
possible that a sub-population of satellite cells may be derived from a more
primitive stem cell. Satellite cell-derived muscle precursor cells may be used
to repair and regenerate damaged or myopathic skeletal muscle, or to act as
vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to
3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5)
satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells
per person. (2) Main functions: repair and maintece of skeletal muscle. (3)
Turnover rate: close to zero in non-traumatic conditions-high in disease or
severe trauma. Skeletal muscle satellite cells play key roles in postnatal muscle growth and
regeneration. To study molecular regulation of satellite cells, we directly
prepared satellite cells from 8- to 12-week-old C57BL/6 mice and performed
genome-wide gene expression analysis. Compared with activated/cycling satellite
cells, 507 genes were highly upregulated in quiescent satellite cells. These
included negative regulators of cell cycle and myogenic inhibitors. Gene set
enrichment analysis revealed that quiescent satellite cells preferentially
express the genes involved in cell-cell adhesion, regulation of cell growth,
formation of extracellular matrix, copper and iron homeostasis, and lipid
transportation. Furthermore, reverse transcription-polymerase chain reaction on
differentially expressed genes confirmed that calcitonin receptor (CTR) was
exclusively expressed in dormant satellite cells but not in activated satellite
cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore,
we next examined the expression of CTR in vivo. CTR was specifically expressed
on quiescent satellite cells, but the expression was not found on
activated/proliferating satellite cells during muscle regeneration. CTR-positive
cells reappeared at the rim of regenerating myofibers in later stages of muscle
regeneration. Calcitonin stimulation delayed the activation of quiescent
satellite cells. Our data provide roles of CTR in quiescent satellite cells and
a solid scaffold to further dissect molecular regulation of satellite cells.
Disclosure of potential conflicts of interest is found at the end of this
article. Research focusing on the canonical adult myogenic progenitor, the skeletal
muscle satellite cell, is still an ever-growing field 46 years from their
initial description. Recent publications revealed numerous new aspects of
satellite cell biology, starting from their developmental life to their role as
the principal self-renewing myogenic stem cell in adult skeletal muscle and
finally their loss during aging. The myogenic potential of satellite cells is
under the molecular control of specific paired-box and bHLH transcription
factors whose tightly orchestrated balance accounts for an effective skeletal
muscle regeneration. New reports also demonstrate satellite cells relationships
with blood vessels and the high myogenic potential of stem cell subsets related
to both lineages. Skeletal muscle satellite cells are adult muscle-derived stem cells receiving
increasing attention. Sheep satellite cells have a greater similarity to human
satellite cells with regard to metabolism, life span, proliferation and
differentiation, than satellite cells of the rat and mouse. We have used 2-step
enzymatic digestion and differential adhesion methods to isolate and purify
sheep skeletal muscle satellite cells, identified the cells and induced
differentiation to examine their pluripotency. The most efficient method for the
isolation of sheep skeletal muscle satellite cells was the type I collagenase
and trypsin 2-step digestion method, with the best conditions for in vitro
culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence
staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin,
MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed,
as indicated by the expression of MyoG and fast muscle myosin. After osteogenic
induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline
phosphatase) staining results both being positive. After adipogenic induction,
cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear
lipid droplets were present around the cells, with Oil Red-O staining giving a
positive result. In summary, a successful system has been established for the
isolation, purification and identification of sheep skeletal muscle satellite
cells. The outstanding regenerative capacity of skeletal muscle is attributed to the
resident muscle stem cell termed satellite cell. Satellite cells are essential
for skeletal muscle regeneration as they ultimately provide the myogenic
precursors that rebuild damaged muscle tissue. Satellite cells
characteristically are a heterogeneous population of stem cells and committed
progenitor cells. Delineation of cellular hierarchy and understanding how
lineage fate choices are determined within the satellite cell population will be
invaluable for the advancement of muscle regenerative therapies. Satellite cells (SC) are quiescent adult muscle stem cells critical for
postnatal development. Children with cerebral palsy have impaired muscular
growth and develop contractures. While flow cytometry previously demonstrated a
reduced SC population, extracellular matrix abnormalities may influence the cell
isolation methods used, systematically isolating fewer cells from CP muscle and
creating a biased result. Consequently, the purpose of this study was to use
immunohistochemistry on serial muscle sections to quantify SC in situ. Serial
cross-sections from human gracilis muscle biopsies (n = 11) were labeled with
fluorescent antibodies for Pax7 (SC transcriptional marker), laminin (basal
lamina), and 4',6-diamidino-2-phenylindole (nuclei). Fluorescence microscopy
under high magnification was used to identify SC based on labeling and location.
Mean SC/100 myofibers was reduced by ∼70% (p < 0.001) in children with CP
(2.89 ± 0.39) compared to TD children (8.77 ± 0.79). Furthermore, SC
distribution across fields was different (p < 0.05) with increased percentage of
SC in fields being solitary cells (p < 0.01) in children with CP. Quantification
of SC number in situ, without any other tissue manipulation confirms children
with spastic CP have a reduced number. This stem cell loss may, in part, explain
impaired muscle growth and apparent decreased responsiveness of CP muscle to
exercise. Skeletal muscle mass, function, and repair capacity all progressively decline
with aging, restricting mobility, voluntary function, and quality of life.
Skeletal muscle repair is facilitated by a population of dedicated muscle stem
cells (MuSCs), also known as satellite cells, that reside in anatomically
defined niches within muscle tissues. In adult tissues, MuSCs are retained in a
quiescent state until they are primed to regenerate damaged muscle through
cycles of self-renewal divisions. With aging, muscle tissue homeostasis is
progressively disrupted and the ability of MuSCs to repair injured muscle
markedly declines. Until recently, this decline has been largely attributed to
extrinsic age-related alterations in the microenvironment to which MuSCs are
exposed. However, as highlighted in this Perspective, recent reports show that
MuSCs also progressively undergo cell-intrinsic alterations that profoundly
affect stem cell regenerative function with aging. A more comprehensive
understanding of the interplay of stem cell-intrinsic and extrinsic factors will
set the stage for improving cell therapies capable of restoring tissue
homeostasis and enhancing muscle repair in the aged. |
What is the role of neurogranin in Alzheimer's disease patients? | Dendritic protein neurogranin is markedly increased in cerebrospinal fluid in Alzheimer's disease patients. Neurogranin might reflect the neurodegenerative processes within the brain, indicating a role for neurogranin as a potential novel clinical biomarker for synaptic degeneration in AD.
Neurogranin is important for synaptic plasticity and memory. | Synaptic pathology occurs early in Alzheimer's disease (AD) development, and
cerebrospinal fluid biomarkers for synaptic damage may be altered early in the
disease process. In the present study we examined cerebrospinal fluid levels of
the postsynaptic protein neurogranin in patients with mild cognitive impairment
(MCI) or AD and controls. The low neurogranin level in cerebrospinal fluid
required enrichment by immunoprecipitation prior to mass spectrometric
identification and semi-quantitative immunoblot analysis. Relative
quantification revealed a significant increase of neurogranin in the AD group
compared with controls, while the MCI group was not statistically different from
either controls or the AD group. The concentrations of the AD biomarkers T-tau,
P-tau(181) and Aβ(42) were significantly changed in the control and MCI groups
compared with the AD group, but no significant differences were found between
the MCI group and controls for the three biomarkers. Nevertheless, a trend
towards increasing levels of neurogranin, T-tau and P-tau(181) was found in
cerebrospinal fluid from MCI patients compared with controls. The elevated
neurogranin levels in the MCI and AD groups might reflect synaptic degeneration.
These results together suggest that cerebrospinal fluid neurogranin might be
valuable together with the established AD biomarkers in the early diagnosis of
AD and warrants further studies to determine the diagnostic value of
neurogranin. The multifactorial causes impacting the risk of developing sporadic forms of
Alzheimer's disease (AD) remain to date poorly understood. Epidemiologic studies
in humans and research in rodents have suggested that hypothyroidism could
participate in the etiology of AD. Recently, we reported that adult-onset
hypothyroidism in rats favors β-amyloid peptide production in the hippocampus.
Here, using the same hypothyroidism model with the antithyroid molecule
propythiouracyl (PTU), we further explored AD-related features, dysfunctional
cell-signaling mechanisms and hippocampal-dependent learning and memory. In vivo
MRI revealed a progressive decrease in cerebral volume of PTU-treated rats. In
the hippocampus, hypothyroidism resulted in tau hyperphosphorylation and
increases in several proinflammatory cytokines. These modifications were
associated with impaired spatial memory and reduced hippocampal expression of
signaling molecules important for synaptic plasticity and memory, including
neurogranin, CaMKII, ERK, GSK3β, CREB, and expression of the transcription
factor EGR1/Zif268. These data strengthen the idea that hypothyroidism
represents an important factor influencing the risk of developing sporadic forms
of AD. INTRODUCTION: Synaptic dysfunction and degeneration are central events in
Alzheimer's disease (AD) pathophysiology that are thought to occur early in
disease progression. Synaptic pathology may be studied by examining protein
biomarkers specific for different synaptic elements. We recently showed that the
dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76
(Ng48-76), is markedly increased in cerebrospinal fluid (CSF) in AD and that
Ng48-76 is the domit peptide in human brain tissue. The aim of this study was
to characterize Ng in plasma and CSF using mass spectrometry and to investigate
the performance of plasma Ng as an AD biomarker.
METHODS: Paired plasma and CSF samples from patients with AD (n = 25) and
healthy controls (n = 20) were analyzed in parallel using an immunoassay
developed in-house on the Meso Scale Discovery platform and hybrid
immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients
with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS.
High-resolution mass spectrometry was used for identification of endogenous
plasma Ng peptides.
RESULTS: Ng in human plasma is present as several endogenous peptides. Of the 16
endogenous Ng peptides identified, seven were unique for plasma and not
detectable in CSF. However, Ng48-76 was not present in plasma. CSF Ng was
significantly increased in AD compared with controls (P < 0.0001), whereas the
plasma Ng levels were similar between the groups in both studies. Plasma and CSF
Ng levels showed no correlation. CSF Ng was stable during storage at -20°C for
up to 2 days, and no de novo generation of peptides were detected.
CONCLUSIONS: For the first time, to our knowledge, we have identified several
endogenous Ng peptides in human plasma. In agreement with previous studies, we
show that CSF Ng is significantly increased in AD as compared with healthy
controls. The origin of Ng in plasma and its possible use as a biomarker need to
be further investigated. The results suggest that CSF Ng, in particular Ng48-76,
might reflect the neurodegenerative processes within the brain, indicating a
role for Ng as a potential novel clinical biomarker for synaptic function in AD. |
Does triiodothyronine play a regulatory role in insulin secretion from pancreas? | YES | Using medium with a low ionic strength, a low concentration of Ca2+ and Mg2+ and
devoid of K+, we have measured Ca(2+)-ATPase activity in the homogenates of rat
islets preincubated for 3 min with several hormones in the presence of 3.3 mmol
glucose/l. Insulin secretion was also measured in islets incubated for 5 min
under identical experimental conditions. Islets preincubated with glucose (3.3
mmol/l) and glucagon (1.4 mumol/l) plus theophylline (10 mmol/l), ACTH (0.11
nmol/l), bovine GH (0.46 mumol/l), prolactin (0.2 mumol/l) or tri-iodothyronine
(1.0 nmol/l) have significantly lower Ca(2+)-ATPase activity than those
preincubated with only 3.3 mmol glucose/l. All these hormones increased the
release of insulin significantly. Dexamethasone (0.1 mumol/l) and somatostatin
(1.2 mumol/l) enhanced the Ca(2+)-ATPase activity while adrenaline (10 mumol/l)
did not produce any significant effect on the activity of the enzyme. These
hormones decreased the release of insulin significantly. These results
demonstrated that islet Ca(2+)-ATPase activity was modulated by the hormones
tested. Their inhibitory or enhancing effect seemed to be related to their
effect on insulin secretion; i.e. those which stimulated the secretion of
insulin inhibited the activity of the enzyme and vice versa. Hence, their effect
on insulin secretion may be due, in part, to their effect on enzyme activity and
consequently on the concentration of cytosolic Ca2+. These results reinforce the
assumption that Ca(2+)-ATPase activity participates in the physiological
regulation of insulin secretion, being one of the cellular targets for several
agents which affect this process. To elucidate the mechanism of thyroid hormone-induced hyperinsulinemia, the
acute and direct effect of thyroid hormone administration on insulin secretion
was investigated in rats in vivo and in vitro. In the perfused rat pancreas, the
addition of thyroxine (10 micrograms/dL) or 3,5,3'-triiodothyronine (150 ng/dL)
to the perfusing medium did not affect insulin secretion. The administration of
thyroxine (40 micrograms/kg, s.c.) in vivo increased the plasma insulin level
from 11 +/- 2 microUnits/mL (mean +/- SD) to 30 +/- 7 microUnits/mL, while blood
glucose and plasma glucagon were unchanged. This phenomenon was inhibited
completely by the preadministration of oxprenolol hydrochloride (2 mg/kg, s.c.),
and inhibited partly by the preadministration of metoprolol tartrate (35 mg/kg,
s.c.). These results suggest that thyroid hormone induces hyperinsulinemia via
beta-adrenergic stimulation in the rat. Thyroxine dehalogenation by rat pancreatic islets was studied incubating
isolated islets with labelled T4. [125I]T4 added to the incubation medium was
deiodinated by the islets with the consequent production of T3, rT3 and iodide.
This deiodination process showed a clear glucose-dependence, being significantly
increased in the presence of 16.6 mmol/l glucose. The existence of high and low
affinity binding sites for T3 was also demonstrated incubating [125I]T3 with
islets under different experimental conditions. The properties of these binding
sites were greatly influenced by the extracellular concentration of glucose.
Addition of T3 to the incubation medium, significantly modified the insulin
release, but its effect varied according to the glucose concentration in the
medium, i.e. it enhanced the insulin release at a glucose concentration between
2 to 8 mmol/l; it has no effect at 12 mmol/glucose, and significantly inhibited
the secretion of insulin in the presence of 16.6 mmol/l glucose. Our results
suggest that thyroid hormones might play a direct regulatory effect on insulin
secretion, probably mediated by its deiodination and interaction with specific
receptors in the islet cell. Thyroid hormones modulate the immune system and metabolism, influence insulin
secretion, and cause decreased glucose tolerance. Thyroid hormones have been
described to change the incidence of spontaneous autoimmune thyroiditis in
Bio-Breeding/Worcester (BB) rats but it is unknown how these hormones affect the
development of type 1 diabetes mellitus (T1DM). The aim was to investigate the
influence of changes in thyroid function during postnatal development on the
prevalence of T1DM in BB rats and the influence of T3 on the beta cell mass in
non-diabetic Wistar rats. BB rats were treated with sodium iodine (NaI) or
thyroid stimulating hormone (TSH) neonatally or with tri-iodo-thyronine (T3)
during adolescence. At the age of 19 weeks the incidence of T1DM and the degree
of insulitis were evaluated. The influence of T3 treatment on the beta cell mass
was evaluated in Wistar rats by unbiased stereological methods. The incidence of
T1DM in control BB rats was 68% at the age of 19 weeks. NaI and T3 reduced the
incidence, whereas TSH had no effect. In Wistar rats T3 treatment increased the
beta cell mass per bodyweight. The modulation of thyroid function during
postnatal development may thus affect the precipitation of T1DM in genetically
susceptible individuals. Thyroid hormones have crucial developmental effect during fetal life. This study
investigates the effects of maternal hypothyroidism on the carbohydrate
metabolism and insulin secretion capacity of islets of the adult male offspring
of rats. One group of pregt mothers (fetal hypothyroid) of Wistar rats drank
water containing 0.02% of 6-propyl-2-thiouracil during pregcy, while the
control group consumed only tap water. After delivery, survival and weight of
the neonates from both groups were followed. In adult male offspring, the
intravenous glucose tolerance test was performed and 5-6 weeks later,
glucose-stimulated insulin secretion of isolated islets was assessed. Plasma
glucose concentration of the fetal hypothyroid group during intravenous glucose
tolerance test was significantly higher (p=0.003) at 5-20 min as compared to the
control group, whereas plasma insulin concentration was significantly lower
(p=0.012) at 5-20 min. Insulin secretion of the isolated islets stimulated with
16 mM glucose of the offspring in the fetal hypothyroid group (376.2 ± 57.1
pmol/islet/60 min) was significantly lower (p=0.02) as compared to the control
group (618.1 ± 85.2). Although adult offspring born from hypothyroid mothers
were euthyroid, their glucose tolerance and glucose stimulated insulin secretion
of islets were altered, which may eventually contribute to the development of
diabetes. 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. |
How many disulfide bridges has the protein hepcidin got? | Hepcidin contains eight cysteine residues that form four disulfide bridges. | We report the isolation of a novel antimicrobial peptide, bass hepcidin, from
the gill of hybrid striped bass, white bass (Morone chrysops) x striped bass (M.
saxatilis). After the intraperitoneal injection of Micrococcus luteus and
Escherichia coli, the peptide was purified from HPLC fractions with
antimicrobial activity against Escherichia coli. Sequencing by Edman degradation
revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative
cysteines. Molecular mass measurements of the native peptide and the reduced and
alkylated peptide confirmed the sequence with four intramolecular disulfide
bridges. Peptide sequence homology to human hepcidin and other predicted
hepcidins, indicated that the peptide is a new member of the hepcidin family.
Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A
predicted prepropeptide (85 amino acids) consists of three domains: a signal
peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21
amino acids). The gene has two introns and three exons. A TATA box and several
consensus-binding motifs for transcription factors including C/EBP, nuclear
factor-kappaB, and hepatocyte nuclear factor were found in the region upstream
of the transcriptional start site. In white bass liver, hepcidin gene expression
was induced 4500-fold following challenge with the fish pathogen, Streptococcus
iniae, while expression levels remained low in all other tissues tested. A novel
antimicrobial peptide from the gill, bass hepcidin, is predomitly expressed
in the liver and highly inducible by bacterial exposure. Hepcidin, a 25 amino acid peptide hormone containing a complex network of four
disulfide bonds is the hormone regulator of iron homeostasis. Three bridges
synthetic peptide analogs have been prepared following two synthetic strategies
and two oxidation procedures: i) a microwave-assisted solid phase synthesis
followed by air oxidation of the six free cysteines ii) a manual solid phase
synthesis followed by stepwise deprotection and oxidation of cysteine pairs. All
the peptides with different connectivities have been characterized by MALDI ToF
spectrometry, and tested for their ability to degrade the cellular iron
exporter, ferroportin. While linear peptides are inactive, the one-bridge and
two-bridge peptides retaining protected cysteines by bulky substituents are
active. Similarly, the three-bridge peptides are active irrespective of their
disulfide connectivities. Hepcidin was first identified as an antimicrobial peptide present in human serum
and urine. It was later demonstrated that hepcidin is the long sought hormone
that regulates iron homeostasis in mammals. The native peptide of 25 amino acids
(Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The
aim of the present study was to assess whether the intramolecular disulfide
bridges are necessary for Hepc25 antimicrobial activity. We show that a
synthetic peptide corresponding to human Hepc25, and which contains the four
disulfide bridges, has an antibacterial activity against several strains of
Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide
where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable
activity against the same strains of bacteria. In a further step, the mode of
action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to
assess bacterial membrane integrity. No permeabilization of the membrane was
observed with Hepc25, indicating that this peptide does not kill bacteria by
destroying their membranes. Gel retardation assay showed that the Hepc25 binds
to DNA with high efficiency, and that this binding ability is dependent on the
presence of the intramolecular disulfide bridges. Reduction of Hepc25 or
replacement of the eight cysteines by alanine residues led to peptides that were
no longer able to bind DNA in the in vitro assay. Altogether, these results
demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by
the intramolecular disulfide bridges in order to have antibacterial activity. Hepcidin, a cysteine-rich cationic antibacterial peptide, plays an important
role in human defense against pathogen infection. However, its role in reptile
immune response and whether it is involved in antibacterial immune have not yet
been proven. In order to study the antibacterial activity of Crocodylus
siamensis hepcidin (Cshepc), a common reptile which lives in topic region of
Southeast Asia, a cDNA sequence of Cshepc was cloned, which included an open
reading frame (ORF) of 300 bp encoding a 99 amino acid preprohepcidin. Cshepc
has eight cysteines formed four conserved disulfide bridges, similarly to that
of human's. Sequence analysis showed that Cshepc mature peptide was more
conserved than that of preprohepcidin. Tissue expression analysis indicated that
Cshepc transcripts were highly expressed in the liver, muscle and heart of C.
siamensis. Recombit expressed hepcidin could significantly inhibit the growth
of the Gram-negative bacteria Escherichia coli and Aeromonas sobria as well as
the Gram-positive bacterium Staphylococcus aureus, and Bacillus subtilis in
vitro, suggesting that Cshepc, like human hepcidin could play a role in the
antibacterial function in hosts innate immune response. Particularly in the field of middle- and top-down peptide and protein analysis,
disulfide bridges can severely hinder fragmentation and thus impede sequence
analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS
approach, which was applied to the analysis of the primary structure of
oxytocin, containing one disulfide bridge, and of hepcidin, containing four
disulfide bridges. The presented workflow provided up to 80% (on-line)
conversion of disulfide bonds in both peptides. With minimal sample preparation,
such reduction resulted in a higher number of peptide backbone cleavages upon
CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle
times, including electrode recovery, were rapid and, therefore, might very well
be coupled with liquid chromatography for protein or peptide separation, which
has great potential for high-throughput analysis. |
What is the association between GERD and gluten ? | GERD symptoms are common in classically symptomatic untreated CD patients. The GFD is associated with a rapid and persistent improvement in reflux symptoms that resembles the healthy population.
Food intolerance is a common complaint amongst patients with functional gastrointestinal (GI) disorders (FGIDs), including those with irritable bowel syndrome (IBS), functional dyspepsia, as well as gastroesophageal reflux disease. There is a great interest in the role of a major dietary protein, gluten, in the production of symptoms...several published studies have consistently shown the efficacy of a gluten-free diet in rapidly controlling esophageal symptoms and in preventing their recurrence | BACKGROUND & AIMS: Celiac disease (CD) patients often complain of symptoms
consistent with gastroesophageal reflux disease (GERD). We aimed to assess the
prevalence of GERD symptoms at diagnosis and to determine the impact of the
gluten-free diet (GFD).
METHODS: We evaluated 133 adult CD patients at diagnosis and 70 healthy
controls. Fifty-three patients completed questionnaires every 3 months during
the first year and more than 4 years after diagnosis. GERD symptoms were
evaluated using a subdimension of the Gastrointestinal Symptoms Rating Scale for
heartburn and regurgitation domains.
RESULTS: At diagnosis, celiac patients had a significantly higher reflux symptom
mean score than healthy controls (P < .001). At baseline, 30.1% of CD patients
had moderate to severe GERD (score >3) compared with 5.7% of controls (P < .01).
Moderate to severe symptoms were significantly associated with the classical
clinical presentation of CD (35.0%) compared with atypical/silent cases (15.2%;
P < .03). A rapid improvement was evidenced at 3 months after initial treatment
with a GFD (P < .0001) with reflux scores comparable to healthy controls from
this time point onward.
CONCLUSIONS: GERD symptoms are common in classically symptomatic untreated CD
patients. The GFD is associated with a rapid and persistent improvement in
reflux symptoms that resembles the healthy population. Celiac disease (CD) may often be associated with various motor disorders
affecting the different segments of the digestive tract, including the
esophagus. Although it has not been universally reported, some available
evidences indicate that pediatric and adult celiac patients could manifest a
higher frequency of esophagitis and gastroesophageal reflux disease-related
symptoms compared to nonceliac patients. In addition, several published studies
have consistently shown the efficacy of a gluten-free diet in rapidly
controlling esophageal symptoms and in preventing their recurrence. Since the
participation of gluten in the esophageal symptoms of CD seems clear, its
intimate mechanisms have yet to be elucidated, and several hypothesis have been
proposed, including the specific immune alterations characterizing CD, the
reduction in nutrient absorption determining the arrival of intact gluten to
distal gastrointestinal segments, and various dysregulations in the function of
gastrointestinal hormones and peptides. Recent studies have suggested the
existence of a possible relationship between CD and eosinophilic esophagitis,
which should be more deeply investigated. |
Which are the major intramolecular phosphorylation sites of human Chk2 involved in cell cycle control? | The major phosphorylation sites of human Chk2 involved in cell cycle control are T68, S19, and S33/35. | A comparative biochemical analysis was performed using recombit human protein
kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells.
Dephosphorylated, inactive, recombit human Chk2 could be reactivated in a
concentration-dependent manner. Despite distinct time-dependent
autophosphorylation kinetics by monitoring the phosphorylation of amino acid
residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D
and Q69E) they gave identical specific activities. However, upon gel filtration
of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to
the formation of a 'pure' dimer; dephosphorylated wildtype Chk2 eluted as a
monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the
absence or presence of DNA damage showed significant T68 phosphorylation already
in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of
additional Chk2 sites was observed (S19, S33/35). A comparison of ATM+/+ and
ATM-/- cells with respect to phosphorylation of residues T68, S19, S33/35 in the
absence and presence of DNA damage showed in all cases phosphorylation of T68,
although signal intensity was increased ca. three-fold after DNA damage. Mass
spectrometric analyses of human recombit Chk2 isolated from bacteria and
insect cells showed distinct differences. The number of phosphorylated residues
in human recombit Chk2 isolated from bacteria was 16, whereas in the case of
the recombit human Chk2 from insect cells it was 8. Except for phosphorylated
amino acid T378 which was not found in the Chk2 isolated from bacteria, all
other phosphorylated residues identified in human Chk2 from insect cells were
present also in Chk2 from bacteria. |
Is the Histidine-Rich Calcium Binding protein (HRC) related to arrhythmias and cardiac disease? | Histidine-rich calcium binding protein (HRC) is a high capacity, low affinity Ca(2+) binding protein with a potential role in heart failure and arrhythmogenesis due to its activity as regulator of SR Ca(2+) uptake and Ca(2+) release.In addition, HRC null mice displayed a significantly exaggerated response to the induction of cardiac hypertrophy by isoproterenol compared to their wild-type littermates. A human genetic variant (Ser96Ala) in HRC has been linked to ventricular arrhythmia and sudden death in dilated cardiomyopathy. | AIMS: To investigate whether genetic variants of the histidine-rich calcium
(HRC)-binding protein are associated with idiopathic dilated cardiomyopathy
(DCM) and its progression.
METHODS AND RESULTS: We screened 123 idiopathic DCM patients and 96 healthy
individuals by single-strand conformation polymorphism analysis and direct
sequencing for genetic variants in HRC. Six polymorphisms were detected:
Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (-/GAG),
Asp261del (GAT/-), and an in-frame insertion of 51 amino acids at His321. The
analysis of their frequencies did not reveal any significant correlation with
DCM development. However, the Ser96Ala polymorphism exhibited a statistically
significant correlation with the occurrence of life-threatening ventricular
arrhythmias. During a follow-up of 4.02 +/- 2.4 years, the risk for ventricular
arrhythmias was higher (HR, 9.620; 95% CI, 2.183-42.394; P = 0.003) in the
Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable
Cox regression analysis, the Ser96Ala polymorphism was the only significant
genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI,
0.838-20.967; P = 0.018).
CONCLUSION: The Ser96Ala genetic variant of HRC is associated with
life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an
independent predictor of susceptibility to arrhythmogenesis in the setting of
DCM. Contractile dysfunction and ventricular arrhythmias associated with heart
failure have been attributed to aberrant sarcoplasmic reticulum (SR) Ca(2+)
cycling. The study of junctin (JCN) and histidine-rich Ca(2+) binding protein
(HRC) becomes of particular importance since these proteins have been shown to
be critical regulators of Ca(2+) cycling. Specifically, JCN is a SR membrane
protein, which is part of the SR Ca(2+) release quaternary structure that also
includes the ryanodine receptor, triadin and calsequestrin. Functionally, JCN
serves as a bridge between calsequestrin and the Ca(2+) release channel,
ryanodine receptor. HRC is a SR luminal Ca(2+) binding protein known to
associate with both triadin and the sarcoplasmic reticulum Ca(2+)-ATPase, and
may thus mediate the crosstalk between SR Ca(2+) uptake and release. Indeed,
evidence from genetic models of JCN and HRC indicate that they are important in
cardiophysiology as alterations in these proteins affect SR Ca(2+) handling and
cardiac function. In addition, downregulation of JCN and HRC may contribute to
Ca(2+) cycling perturbations manifest in the failing heart, where their protein
levels are significantly reduced. This review examines the roles of JCN and HRC
in SR Ca(2+) cycling and their potential significance in heart failure. Histidine-rich calcium binding protein (HRC) is a high capacity, low affinity
Ca(2+) binding protein, specifically expressed in striated muscles of mammals.
In rabbit skeletal and cardiac muscles, HRC binds to sarcoplasmic reticulum (SR)
membranes via triadin, a junctional SR protein. Recently, a potential role in
heart failure and arrhythmogenesis has been assigned to HRC due to its activity
as regulator of SR Ca(2+) uptake and Ca(2+) release. HRC might play a
particularly relevant role in the equine heart, given its slower resting heart
rate (20-35 beats/min) and longer action potential duration (APD) (0.6-1.0 s)
than are found in other mammals. The results from this study showed for the
first time direct evidence that HRC protein in equine cardiac muscle was
expressed in association with the SR membranes and that HRC transcriptional
activity was three times higher in the ventricles compared to the atria. The
predomice of HRC mRNA up-regulation in ventricular myocardium was specific to
the horse heart, since a more even distribution between atria and ventricles was
found in animals of similar body size or species, such as cattle or domestic
donkeys. BACKGROUND: Histidine-rich calcium binding protein (HRC) is located in the lumen
of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA
affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may
disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of
cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under
basal condition, but exhibited a significantly increased susceptibility to
isoproterenol-induced cardiac hypertrophy. In the present study, we
characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of
cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated
virus (AAV)-mediated HRC knock-down (KD) systems, respectively.
METHODOLOGY/PRINCIPAL FINDINGS: AAV-mediated HRC-KD system was used with or
without C57BL/6 mouse model of transverse aortic constriction-induced failing
heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in
failing heart (FH). Initially we expected that HRC-KD could elicit cardiac
functional recovery in failing heart (FH), since predesigned siRNA-mediated
HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2
without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and
HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with
decreased fractional shortening and increased cardiac fibrosis compared with
control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and
phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly
increased level of cleaved caspase-3, a cardiac cell death marker was also
found, consistent with the result of TUNEL assay.
CONCLUSIONS/SIGNIFICANCE: Increased Ca(2+) leak and cytosolic Ca(2+)
concentration due to a partial KD of HRC could enhance activity of CaMKII and
phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in
TAC-FH. Our results present evidence that down-regulation of HRC could
deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+)
cycling. BACKGROUND: A human genetic variant (Ser96Ala) in the sarcoplasmic reticulum
(SR) histidine-rich Ca(2+)-binding (HRC) protein has been linked to ventricular
arrhythmia and sudden death in dilated cardiomyopathy. However, the precise
mechanisms affecting SR function and leading to arrhythmias remain elusive.
METHODS AND RESULTS: We generated transgenic mice with cardiac-specific
expression of human Ala96 HRC or Ser96 HRC in the null background to assess
function in absence of endogenous protein. Ala96 HRC decreased (25% to 30%)
cardiomyocyte contractility and Ca2+ kinetics compared with Ser96 HRC in the
absence of any structural or histological abnormalities. Furthermore, the
frequency of Ca2+ waves was significantly higher (10-fold), although SR Ca2+
load was reduced (by 27%) in Ala96 HRC cells. The underlying mechanisms involved
diminished interaction of Ala96 HRC with triadin, affecting ryanodine receptor
(RyR) stability. Indeed, the open probability of RyR, assessed by use of
ryanodine binding, was significantly increased. Accordingly, stress conditions
(5 Hz plus isoproterenol) induced aftercontractions (65% in Ala96 versus 12% in
Ser96) and delayed afterdepolarizations (70% in Ala96 versus 20% in Ser96). The
increased SR Ca2+ leak was accompanied by hyperphosphorylation (1.6-fold) of RyR
at Ser2814 by calmodulin-dependent protein kinase II. Accordingly, inclusion of
the calmodulin-dependent protein kinase II inhibitor KN93 prevented Ser2814
phosphorylation and partially reversed the increases in Ca2+ spark frequency and
wave production. Parallel in vivo studies revealed ventricular ectopy on
short-term isoproterenol challenge and increased (4-fold) propensity to
arrhythmias, including nonsustained ventricular tachycardia, after myocardial
infarction in Ala96 HRC mice.
CONCLUSIONS: These findings suggest that aberrant SR Ca2+ release and increased
susceptibility to delayed afterdepolarizations underlie triggered arrhythmic
activity in human Ala96 HRC carriers. |
Which are the methods for in silico prediction of the origin of replication (ori) among bacteria? | Several in silico methods have been applied for prediction of the origin of replication (ori). DNA base composition asymmetry, such as GC skew, is the basis of numerous in silico methods used to detect the ori in prokaryotes. The Z curve analysis is also used for ori identification. Comparative genomics, by BLAST analyses of the intergenic sequences compared to related species have been applied in ori prediction. The finding of the dnaA gene and its binding sites, DnaA boxes, as well as the finding of the binding sites of other proteins, such as CtrA and IHF, are fundamental characteristics used for in silico prediction of the ori. Also, the localization of boundary genes, such as cell division cycle (cdc6) gene, and consensus origin recognition box (ORB) sequences have been employed for ori detection. The study of the gene order around the origin sequence and the distribution of the genes encoded in the leading versus the lagging strand are also used for in silico detection of the ori. | Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model
for studying respiratory mycoplasmosis. The complete genome of this bacterium,
which belongs to the class Mollicutes, has recently been sequenced, but studying
the role of specific genes requires improved genetic tools. In silico
comparative analysis of sequenced mollicute genomes indicated the lack of
conservation of gene order in the region containing the predicted origin of
replication (oriC) and the existence, in most of the mollicute genomes examined,
of putative DnaA boxes lying upstream and downstream from the dnaA gene. The
predicted M. pulmonis oriC region was shown to be functional after cloning it
into an artificial plasmid and after transformation of the mycoplasma, which was
obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid
DNA. However, after a few in vitro passages, this plasmid integrated into the
chromosomal oriC region. Reduction of this oriC region by subcloning experiments
to the region either upstream or downstream from dnaA resulted in plasmids that
failed to replicate in M. pulmonis, except when these two intergenic regions
were cloned with the tetM determit as a spacer in between them. An internal
fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC
plasmid, and the resulting construct was used to transform M. pulmonis. Targeted
integration of this genetic element into the chromosomal hlyA by a single
crossing over, which results in the disruption of the gene, could be documented.
These mycoplasmal oriC plasmids may therefore become valuable tools for
investigating the roles of specific genes, including those potentially
implicated in pathogenesis. BACKGROUND: Chromosomal DNA replication in bacteria starts at the origin (ori)
and the two replicores propagate in opposite directions up to the terminus (ter)
region. We hypothesize that the two replicores need to reach ter at the same
time to maintain a physical balance; DNA insertion would disrupt such a balance,
requiring chromosomal rearrangements to restore the balance. To test this
hypothesis, we needed to demonstrate that ori and ter are in a physical balance
in bacterial chromosomes. Using wavelet analysis, we documented GC skew, AT
skew, purine excess and keto excess on the published bacterial genomic sequences
to locate the turning (minimum and maximum) points on the curves. Previously,
the minimum point had been supposed to correlate with ori and the maximum to
correlate with ter.
RESULTS: We observed a strong tendency of the bacterial chromosomes towards a
physical balance, with the minima and maxima corresponding to the known or
putative ori and ter and being about half chromosome separated in most of the
bacteria studied. A nonparametric method based on wavelet transformation was
employed to perform significance tests for the predicted loci.
CONCLUSIONS: The wavelet approach can reliably predict the ori and ter regions
and the bacterial chromosomes have a strong tendency towards a physical balance
between ori and ter. Bacterial chromosomes are highly polarized in their nucleotide composition
through mutational selection related to replication. Using compositional skews
such as the GC skew, replication origin and terminus can be predicted in silico
by observing the shift points. However, the genome sequence is affected by
myriad functional requirements and selection on numerous subgenomic features,
and elimination of this "noise" should lead to better predictions. Here, we
present a noise-reduction approach that uses low-pass filtering through Fast
Fourier transform coupled with cumulative skew graphs. It increases the
prediction accuracy of the replication termini compared with previously
documented methods based on genomic base composition. BACKGROUND: The annotated genomes of two closely related strains of the
intracellular bacterium Wolbachia pipientis have been reported without the
identifications of the putative origin of replication (ori). Identifying the ori
of these bacteria and related alpha-Proteobacteria as well as their patterns of
sequence evolution will aid studies of cell replication and cell density, as
well as the potential genetic manipulation of these widespread intracellular
bacteria.
RESULTS: Using features that have been previously experimentally verified in the
alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication
(ori) regions were identified in silico for Wolbachia strains and eleven other
related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These
features include DnaA-, CtrA- and IHF-binding sites as well as the flanking
genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE
and COG1253 protein (CBS domain protein). Comparisons of the putative ori region
among related Wolbachia strains showed higher conservation of bases within
binding sites.
CONCLUSION: The sequences of the ori regions described here are only similar
among closely related bacteria while fundamental characteristics like presence
of DnaA and IHF binding sites as well as the boundary genes are more widely
conserved. The relative paucity of CtrA binding sites in the ori regions, as
well as the absence of key enzymes associated with DNA replication in the
respective genomes, suggest that several of these obligate intracellular
bacteria may have altered replication mechanisms. Based on these analyses,
criteria are set forth for identifying the ori region in genome sequencing
projects. BACKGROUND: Chromosomal replication is the central event in the bacterial cell
cycle. Identification of replication origins (oriCs) is necessary for almost all
newly sequenced bacterial genomes. Given the increasing pace of genome
sequencing, the current available software for predicting oriCs, however, still
leaves much to be desired. Therefore, the increasing availability of genome
sequences calls for improved software to identify oriCs in newly sequenced and
unotated bacterial genomes.
RESULTS: We have developed Ori-Finder, an online system for finding oriCs in
bacterial genomes based on an integrated method comprising the analysis of base
composition asymmetry using the Z-curve method, distribution of DnaA boxes, and
the occurrence of genes frequently close to oriCs. The program can also deal
with unotated genome sequences by integrating the gene-finding program ZCURVE
1.02. Output of the predicted results is exported to an HTML report, which
offers convenient views on the results in both graphical and tabular formats.
CONCLUSION: A web-based system to predict replication origins of bacterial
genomes has been presented here. Based on this system, oriC regions have been
predicted for the bacterial genomes available in GenBank currently. It is hoped
that Ori-Finder will become a useful tool for the identification and analysis of
oriCs in both bacterial and archaeal genomes. Caulobacter crescentus (CB15) initiates chromosome replication only in stalked
cells and not in swarmers. To better understand this dimorphic control of
chromosome replication, we isolated replication origins (oris) from freshwater
Caulobacter (FWC) and marine Caulobacter (MCS) species. Previous studies
implicated integration host factor (IHF) and CcrM DNA methylation sites in
replication control. However, ori IHF and CcrM sites identified in the model FWC
CB15 were only conserved among closely related FWCs. DnaA boxes and CtrA binding
sites are established CB15 ori components. CtrA is a two-component regulator
that blocks chromosome replication selectively in CB15 swarmers. DnaA boxes and
CtrA sites were found in five FWC and three MCS oris. Usually, a DnaA box and a
CtrA site were paired, suggesting that CtrA binding regulates DnaA activity. We
tested this hypothesis by site-directed mutagenesis of an MCS10 ori which
contains only one CtrA binding site overlapping a critical DnaA box. This
overlapping site is unique in the whole MCS10 genome. Selective DnaA box
mutations decreased replication, while selective CtrA binding site mutations
increased replication of MCS10 ori plasmids. Therefore, both FWC and MCS oris
use CtrA to repress replication. Despite this similarity, phylogenetic analysis
unexpectedly shows that CtrA usage evolved separately among these Caulobacter
oris. We discuss consensus oris and convergent ori evolution in differentiating
bacteria. Genome replication is a crucial and essential process for the continuity of
life.In all organisms it starts at a specific region of the genome known as
origin of replication (Ori) site. The number of Ori sites varies in prokaryotes
and eukaryotes. Replication starts at a single Ori site in bacteria, but in
eukaryotes multiple Ori sites are used for fast copying across all chromosomes.
The situation becomes complex in archaea, where some groups have single and
others have multiple origins of replication. Themococcales, are a
hyperthermophilic order of archaea. They are anaerobes and heterotrophs-peptide
fermenters, sulphate reducers, methanogens being some of the examples of
metabolic types. In this paper we have applied a combination of multiple in
silico approaches - Z curve, the cell division cycle (cdc6) gene location and
location of consensus origin recognition box (ORB) sequences for location of
origin of replication in Thermococcus onnurineus, Thermococcus gammatolerans and
other Themococcales and compared the results to that of the well-documented case
of Pyrococcus abyssi. The motivation behind this study is to find the number of
Ori sites based on the data available for members of this order. Results from
this in silico analysis show that the Themococcales have a single origin of
replication. Computational prediction of the origin of replication is a challenging problem
and of immense interest to biologists. Several methods have been proposed for
identifying the replicon site for various classes of organisms. However, these
methods have limited applicability since the replication mechanism is different
in different organisms. We propose a correlation measure and show that it is
correctly able to predict the origin of replication in most of the bacterial
genomes. When applied to Methanocaldococcus jannaschii, Plasmodium falciparum
apicoplast and Nicotiana tabacum plastid, this correlation based method is able
to correctly predict the origin of replication whereas the generally used GC
skew measure fails. Thus, this correlation based measure is a novel and
promising tool for predicting the origin of replication in a wide class of
organisms. This could have important implications in not only gaining a deeper
understanding of the replication machinery in higher organisms, but also for
drug discovery. Replication of chromosomes is one of the central events in the cell cycle.
Chromosome replication begins at specific sites, called origins of replication
(oriCs), for all three domains of life. However, the origins of replication
still remain unknown in a considerably large number of bacterial and archaeal
genomes completely sequenced so far. The availability of increasing complete
bacterial and archaeal genomes has created challenges and opportunities for
identification of their oriCs in silico, as well as in vivo. Based on the
Z-curve theory, we have developed a web-based system Ori-Finder to predict oriCs
in bacterial genomes with high accuracy and reliability by taking advantage of
comparative genomics, and the predicted oriC regions have been organized into an
online database DoriC, which is publicly available at
http://tubic.tju.edu.cn/doric/ since 2007. Five years after we constructed
DoriC, the database has significant advances over the number of bacterial
genomes, increasing about 4-fold. Additionally, oriC regions in archaeal genomes
identified by in vivo experiments, as well as in silico analyses, have also been
added to the database. Consequently, the latest release of DoriC contains oriCs
for >1500 bacterial genomes and 81 archaeal genomes, respectively. DNA replication initiation, which starts at specific chromosomal site (known as
replication origins), is the key regulatory stage of chromosome replication.
Archaea, the third domain of life, use a single or multiple origin(s) to
initiate replication of their circular chromosomes. The basic structure of
replication origins is conserved among archaea, typically including an AT-rich
unwinding region flanked by several conserved repeats (origin recognition box,
ORB) that are located adjacent to a replication initiator gene. Both the ORB
sequence and the adjacent initiator gene are considerably diverse among
different replication origins, while in silico and genetic analyses have
indicated the specificity between the initiator genes and their cognate origins.
These replicator-initiator pairings are reminiscent of the oriC-dnaA system in
bacteria, and a model for the negative regulation of origin activity by a
downstream cluster of ORB elements has been recently proposed in haloarchaea.
Moreover, comparative genomic analyses have revealed that the mosaics of
replicator-initiator pairings in archaeal chromosomes originated from the
integration of extrachromosomal elements. This review summarizes the research
progress in understanding of archaeal replication origins with particular focus
on the utilization, control and evolution of multiple replication origins in
haloarchaea. Precise DNA replication is critical for the maintece of genetic integrity in
all organisms. In all three domains of life, DNA replication starts at a
specialized locus, termed as the replication origin, oriC or ORI, and its
identification is vital to understanding the complex replication process. In
bacteria and eukaryotes, replication initiates from single and multiple origins,
respectively, while archaea can adopt either of the two modes. The Z-curve
method has been successfully used to identify replication origins in genomes of
various species, including multiple oriCs in some archaea. Based on the Z-curve
method and comparative genomics analysis, we have developed a web-based system,
Ori-Finder, for finding oriCs in bacterial genomes with high accuracy. Predicted
oriC regions in bacterial genomes are organized into an online database, DoriC.
Recently, archaeal oriC regions identified by both in vivo and in silico methods
have also been included in the database. Here, we summarize the recent advances
of in silico prediction of oriCs in bacterial and archaeal genomes using the
Z-curve based method. DNA replication is one of the most basic processes in all three domains of
cellular life. With the advent of the post-genomic era, the increasing number of
complete archaeal genomes has created an opportunity for exploration of the
molecular mechanisms for initiating cellular DNA replication by in vivo
experiments as well as in silico analysis. However, the location of replication
origins (oriCs) in many sequenced archaeal genomes remains unknown. We present a
web-based tool Ori-Finder 2 to predict oriCs in the archaeal genomes
automatically, based on the integrated method comprising the analysis of base
composition asymmetry using the Z-curve method, the distribution of origin
recognition boxes identified by FIMO tool, and the occurrence of genes
frequently close to oriCs. The web server is also able to analyze the
unotated genome sequences by integrating with gene prediction pipelines and
BLAST software for gene identification and function annotation. The result of
the predicted oriCs is displayed as an HTML table, which offers an intuitive way
to browse the result in graphical and tabular form. The software presented here
is accurate for the genomes with single oriC, but it does not necessarily find
all the origins of replication for the genomes with multiple oriCs. Ori-Finder 2
aims to become a useful platform for the identification and analysis of oriCs in
the archaeal genomes, which would provide insight into the replication
mechanisms in archaea. The web server is freely available at
http://tubic.tju.edu.cn/Ori-Finder2/. |
What is the use of emulsion PCR in Next Generation Sequencing? | Prior to Next Generation Sequencing reactions, DNA libraries are constructed, amplified with emulsion PCR, and enriched with the use of enrichment beads. The library samples are then loaded to a sequencing chip and analyzed on an NGS platform. | The motivation of this research is to establish a system of target genomic DNA
capture and enrichment, which could be used in deep sequencing of target regions
with next-generation sequencing. To design the 120 bp capture probes (baits) and
prepare the SureSelect reagents, 2,414,977 bp human genomic sequence of 11,824
exons in 1250 genes were submitted to the Agilent eArray platform and
manufactured by Agilent. Two human genomic DNA samples were used and conducted
the successive experiments for sequencing library construction: shearing
fragmentation by sonication, blunt-ending and phosphorylation, adaptor ligation,
150-200 bp fragments size selection, followed by hybridization with the baits,
hybrid selection with magnetic beads, and PCR amplification. Prior to SOLiD
sequencing reaction, the libraries were amplified with emulsion PCR and enriched
with the P2 enrichment beads. The library samples were loaded to sequencing Chip
for Work Flow Analysis (WFA) or sequencing running with default parameters. The
results displayed that 46 509 baits were designed and synthesized for 11,147
gene regions, and SureSelect capture probe regent was prepared. Real-time PCR
showed the target enrichment efficiency up to 2(9) times with the SureSelect
system. WFA revealed that the libraries were suitable for SOLiD Sequencing. The
sequencing data revealed that 70% of the unique mapped sequence tags matched the
target regions, and the average coverage of the target regions were above
200-fold. All these demonstrated the feasibility of the established system of
target genome sequence capture for next generation DNA sequencing. The 454 Genome Sequencer (GS) FLX System is one of the next-generation
sequencing systems featured by long reads, high accuracy, and ultra-high
throughput. Based on the mechanism of emulsion PCR, a unique DNA template would
only generate a unique sequence read after being amplified and sequenced on GS
FLX. However, biased amplification of DNA templates might occur in the process
of emulsion PCR, which results in production of artificial duplicate reads.
Under the condition that each DNA template is unique to another, 3.49%-18.14% of
total reads in GS FLX-sequencing data were found to be artificial duplicate
reads. These duplicate reads may lead to misunderstanding of sequencing data and
special attention should be paid to the potential biases they introduced to the
data. Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent
sequencing are based on amplification of library fragments on the surface of
beads prior to sequencing. Commonly, libraries are barcoded and pooled, to
maximise the sequence output of each sequence run. Here, we describe a novel
approach for normalization of multiplex next generation sequencing libraries
after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are
prepared by fluorescent tagging of complementary sequences and then resolved by
high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol
is simple and provides an even sequence distribution of multiplex libraries when
sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion
PCR beads are removed, the approach gives rise to a substantial increase in
sequence quality and mean read length, as compared to that obtained by standard
enrichment protocols. BACKGROUND: Aortopathies are a group of disorders characterized by aneurysms,
dilation, and tortuosity of the aorta. Because of the phenotypic overlap and
genetic heterogeneity of diseases featuring aortopathy, molecular testing is
often required for timely and correct diagnosis of affected individuals. In this
setting next generation sequencing (NGS) offers several advantages over
traditional molecular techniques.
METHODS: The purpose of our study was to compare NGS enrichment methods for a
clinical assay targeting the nine genes known to be associated with aortopathy.
RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment
methods were directly compared by enriching DNA from eight samples. Enriched
samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform.
Depth of coverage, consistency of coverage across samples, and the overlap of
variants identified were assessed. This data was also compared to whole-exome
sequencing data from ten individuals.
RESULTS: Read depth was greater and less variable among samples that had been
enriched using the RNA-bait hybridization capture enrichment method. In
addition, samples enriched by hybridization capture had fewer exons with mean
coverage less than 10, reducing the need for followup Sanger sequencing.
Variants sets produced were 77% concordant, with both techniques yielding
similar numbers of discordant variants.
CONCLUSIONS: When comparing the design flexibility, performance, and cost of the
targeted enrichment methods to whole-exome sequencing, the RNA-bait
hybridization capture enrichment gene panel offers the better solution for
interrogating the aortopathy genes in a clinical laboratory setting. The human leukocyte antigen (HLA) class I and class II loci are the most
polymorphic genes in the human genome; distinguishing the thousands of HLA
alleles is challenging. Next generation sequencing of exonic amplicons with the
454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides
high resolution, high throughput HLA genotyping for eight class I and class II
loci. HLA typing of potential donors for unrelated bone marrow donor registries
typically uses a subset of these loci at high sample throughput and low cost per
sample. The Fluidigm Access Array System enables the incorporation of 48
different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples
with up to 48 different primer pairs in a microfluidic device generating 2304
parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are
used. During genomic PCR, in this 4-primer system, the outer set of primers
containing the MID and the 454 adaptor sequences are incorporated into an
amplicon generated by the inner HLA target-specific primers each containing a
common sequence tag at the 5' end of the forward and reverse primers. Pools of
the resulting amplicons are used for emulsion PCR and clonal sequencing on the
454 Life Sciences GS FLX System, followed by genotyping with Conexio software.
We have genotyped 192 samples with 100% concordance to known genotypes using 8
primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1,
3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per
amplicon was obtained. We have also genotyped 96 samples at high resolution (14
primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1,
3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173
sequence reads per amplicon. Next-generation sequencing technology can now provide population biologists and
phylogeographers with information at the genomic scale; however, many pertinent
questions in population genetics and phylogeography can be answered effectively
with modest levels of genomic information. For the past two decades, most
population-level studies have lacked nuclear DNA (nDNA) sequence data due to the
complications and cost of amplifying and sequencing diploid loci. However,
pyrosequencing of emulsion PCR reactions, amplifying from only one molecule at a
time, can generate megabases of clonally amplified loci at high coverage,
thereby greatly simplifying allelic sequence determination. Here, we present a
step-by-step methodology for utilizing the 454 GS FLX Titanium pyrosequencing
platform to simultaneously sequence 16 populations (at 20 individuals per
population) at 10 different nDNA loci (3,200 loci in total) in one plate of
sequencing for less than the cost of traditional Sanger sequencing. Sanger sequencing of multigenic disorders can be technically challenging, time
consuming, and prohibitively expensive. High-throughput next-generation
sequencing (NGS) can provide a cost-effective method for sequencing targeted
genes associated with multigenic disorders. We have developed a NGS clinical
targeted gene assay for the mitochondrial genome and for 108 selected nuclear
genes associated with mitochondrial disorders. Mitochondrial disorders have a
reported incidence of 1 in 5000 live births, encompass a broad range of
phenotypes, and are attributed to mutations in the mitochondrial and nuclear
genomes. Approximately 20% of mitochondrial disorders result from mutations in
mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or
mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is
enriched by long-range PCR and the 108 nuclear genes (which represent 1301
amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is
performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis
is performed using commercial and in-house developed bioinformatics pipelines. A
total of 16 validation and 13 clinical samples were examined. All previously
reported variants associated with mitochondrial disorders were found in
validation samples, and 5 of the 13 clinical samples were found to have
mutations associated with mitochondrial disorders in either the mitochondrial
genome or the 108 nuclear genes. All variants were confirmed by Sanger
sequencing. OBJECTIVE: To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion
Torrent Personal Genome Machine (PGM) and to validate the result of targeted
next-generation semiconductor sequencing for the diagnosis of genetic disorders.
METHODS: Peripheral blood samples were collected from three MFS patients and a
normal control with informed consent. Genomic DNA was isolated by standard
method and then subjected to targeted sequencing using an Ion Ampliseq(TM)
Inherited Disease Panel. Three multiplex PCR reactions were carried out to
amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA
fragments from different samples were ligated with barcoded sequencing adaptors.
Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were
carried out using an Ion One Touch system. The ion sphere particles were
sequenced on a 318 chip using the PGM platform. Data from the PGM runs were
processed using an Ion Torrent Suite 3.2 software to generate sequence reads.
After sequence alignment and extraction of SNPs and indels, all the variants
were filtered against dbSNP137. DNA sequences were visualized with an Integrated
Genomics Viewer. The most likely disease-causing variants were analyzed by
Sanger sequencing.
RESULTS: The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 ×
median sequencing depth and a coverage of > 98% for the targeted regions in all
the four samples. After data analysis and database filtering, one known missense
mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and
p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All
mutations were verified by conventional Sanger sequencing.
CONCLUSION: Pathogenic FBN1 mutations have been identified in all patients with
MFS, indicating that the targeted next-generation sequencing on the PGM
sequencers can be applied for accurate and high-throughput testing of genetic
disorders. Due to the difficulties in deep sequencing, high-throughput sequencing of
ancient DNA has been limited to exceptionally well-preserved ancient materials.
The primary factor is microbial attack popularly observed in the buried
materials, and it causes drastic increase in relative ratio of microbial DNA in
the extracted DNA. We present a unified strategy in which emulsion PCR is
coupled with target enrichment followed by next-generation sequencing. The
method made it possible to obtain efficiently non-duplicated reads mapped to
target sequences of interest, and this can achieve deep and reliable sequencing
of ancient DNA from typical materials, even though poorly preserved. |
Can adult humans be induced to produce fetal hemoglobin? | Fetal hemoglobin, or foetal haemoglobin, is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old. Functionally, fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream.
Unusually high levels of fetal haemoglobin production can ameliorate sickle cell disease and β thalassaemia. Although efforts directed at the pharmacological stimulation of fetal haemoglobin as an approach to managing these conditions have met with limited success, there is wide variation in individual responses.
Based on results, adults humans could be induced to produce fetal hemoglobin. | Patients who achieved bone marrow engraftment of cord blood-derived progenitor
cells provided an opportunity to examine the expression of fetal Hb by neonatal
hematopoietic progenitors in a postneonatal host. Cord blood cells from
histocompatible siblings were successfully transplanted in two children with the
Fanconi anemia syndrome. One of the transplant donors had heterocellular
hereditary persistence of fetal Hb, apparently due to gamma-globin gene
triplication; the other donor was hematologically normal. The G gamma/A gamma
ratio of the patient who received his transplant from the donor with hereditary
persistence of fetal Hb was markedly elevated, similar to that of the transplant
donor's cord blood, and this ratio remained elevated in subsequent months. In
the other child, the G gamma/A gamma ratio immediately after her transplant was
typical of the normal newborn, and over the next several months it reverted to
the adult pattern. Globin synthesis studies performed shortly after engraftment
demonstrated ratios of fetal Hb/adult Hb synthesis in both patients that were
typical of those of normal newborns. Over the next several months, both patients
converted to the adult pattern. Fetal Hb to adult Hb switching in these patients
seemed to follow a temporal sequence intrinsic to the transplanted neonatal
progenitor cells, without discernible influence of postneonatal environmental
factors. The program for Hb switching seems to be an inherent feature of
neonatal hematopoietic progenitor cells. Globin gene switching may be mediated by proteins expressed during different
stages of development. Their identification may clarify the mechanisms of the
conversion from fetal to adult globin production and lead to new approaches to
reversing or retarding the gamma- to beta-globin gene switch. To explore this
hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25,
2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and
72 h. Erythroid differentiation was verified by benzidine staining and by
measuring the activity of a transduced A gamma-globin gene promoter linked to a
luciferase reporter gene. Using differential display polymerase chain reaction
(PCR), total mRNA extracted from induced cells at each time point of induction
was reverse transcribed in the presence of A, G, and C anchored primers and 16
arbitrary primers, calculated to amplify approximately 50% of expressed genes.
Amplified mRNAs from induced and uninduced cells were separated in
polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to
represent either up- or downregulated mRNA species in induced K562 cells were
identified. Sixty-four of these fragments had more than 95% homology to known
GenBank sequences. Seventeen fragments with characteristics of transcription
factors were cloned. These include differentiation-related gene-1 (drg-1), PAX
3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger
protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC
transcriptional coactivator, LIM domain protein, and trophoblast hypoxia
regulating factor. Differential expression of all 17 fragments over 72 h was
confirmed by reverse Northern dot blot analysis, semiquantitative PCR using
nested primers, and Northern analysis. Erythroid maturation in induced K562
cells is associated with differential expression of numerous genes. Some encode
transcription factors that could effect the initiation of HbF synthesis. Almost
half of the differentially expressed clones contained cDNAs of unidentified open
reading frames and these are the object of continued study. The persistence of fetal hemoglobin in many patients with deletion type beta
thalassemias and the expression patterns of human globin genes in transgenic
mice suggest that gamma- to beta-globin gene switching results primarily from
competition of gamma- and beta-globin genes for interaction with the beta-globin
locus control region (LCR). To define regulatory sequences that are essential
for the competitive advantage of the gamma gene at early developmental stages,
stable transgenic mouse lines were produced with LCR gamma-beta constructs
containing deletions of gamma 5'-flanking DNA. All constructs contained the full
22 kb LCR, a 4.1 kb beta-globin gene and a gamma-globin gene with 1348, 383,
202, 130, 72 or 52 bp of 5'-flanking sequence. Primer extension analysis of yolk
sac, fetal liver and blood RNA from these lines demonstrated that a region
between -202 and -130 of the human gamma-globin gene promoter was required to
suppress beta-globin gene expression at early developmental stages. Four
transcription factor binding sites within this region [GATA(p), Oct1, GATA(d)
and CACCC] were mutated independently in LCR gamma-beta constructs and
transgenic mouse lines were produced. Only the gamma CACCC box mutation resulted
in high levels of beta-globin gene expression in early embryos. These results
demonstrate that the CACCC box of the human gamma-globin gene plays a critical
role in human beta-globin gene developmental specificity. The data also suggest
that gamma CACCC box binding factors mediate LCR-gamma interactions which
normally enhance gamma-globin and suppress beta-globin gene expression in fetal
erythroid cells. The expression of epsilon- and gamma-globin mRNA and protein has been determined
in three Old World monkey species (Macaca mulatta, Macaca nemestrina, and
Cercopithecus aethiops). Using RT-PCR with primers for epsilon- and
gamma-globin, both mRNAs were detected in early fetal stages, whereas at 128
days (85% of full term), only gamma was expressed. High-performance liquid
chromatography was used for separation and quantitation, and matrix-assisted
laser desorption/ionization mass spectrometry was used for identification of
globin polypeptides. An alpha-globin polymorphism was observed in all of the
species examined. During fetal life, gamma-globin was the predomit expressed
beta-type globin. The red blood cells of infants still contained substantial
amounts of gamma-globin, which declined to negligible levels in 14 weeks as
beta-globin expression reached adult values. The ratio of gamma1- to
gamma2-globins (equivalent to Ggamma/Agamma in humans) was approximately 2.5,
similar to the Ggamma/Agamma ratio observed in humans. Thus, gamma-globin gene
expression in these Old World monkeys species has three features in common with
human expression: expression of both duplicated gamma genes, the relative
preponderance of gamma1 over gamma2 expression, and the delay of the switch from
gamma- to beta-globin until the perinatal period. Thus, the catarrhines seem to
share a common pattern of developmental switching in the beta-globin gene
cluster, which is distinct from the timing of expression in either prosimians or
the New World monkeys. Our results indicate that an Old World monkey, such as
Rhesus, could serve as a model organism (resembling humans) for experimentally
investigating globin gene expression patterns during the embryonic, fetal, and
postnatal stages. The design and evaluation of therapies for the sickle cell and beta-thalassemia
syndromes rely on our understanding of hemoglobin accumulation during human
erythropoiesis. Here we report direct measurements of hemoglobin composition and
messenger RNA (mRNA) levels in cultured CD34(+) cells and correlate those
measurements with studies of freshly obtained bone marrow samples. Hemoglobin
levels in differentiating cells were also compared with morphologic,
immunophenotypic, and cell cycle assessments. A population of large
preproerythroblasts was first identified within 24 hours and became the domit
population by day 5. The transition from proerythroblast to basophilic
normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1%
+/- 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts
were the domit and final cell type by day 13. High-performance liquid
chromatography-based quantitation of fetal (HbF) and adult (HbA) hemoglobin and
real-time polymerase chain reaction globin mRNA quantitation demonstrated a
coordinate rise in the accumulation of both proteins and mRNA among these
developmentally staged populations. Quantitative analyses on freshly sorted bone
marrow populations demonstrated a similar rising pattern with beta-globin and
HbA as the domit species at both early and late stages of differentiation. We
found no evidence for HbF domit populations or switching during
differentiation in adult cells. Instead, rapid increases in both HbF
(heterocellular) and HbA (pancellular) content were observed, which coincided
with the apex in cell cycling and the proerythroblast-basophilic normoblast
transition. Based on these measurements, we conclude that HbF and HbA content
are regulated with the rate of proliferation during adult erythropoiesis. Treatment of adult blood-derived stem cells with transforming growth factor
(TGF-beta) during the first 3-4 days in culture increases the proportions and
absolute numbers of erythroid cells subsequently expressing fetal hemoglobin (F+
cells). The change in F+ cell proportions may be due to globin switching or to
selective effects on the expansion of stem cell subpopulations with different
globin expression programs. To distinguish between the two mechanisms, we
compared the effects of TGF-beta on proliferation and globin expression with the
effects of well-researched agents known to increase fetal hemoglobin (HbF) in
sickle cell patients. Hydroxyurea suppressed F+ and F- erythroid cells equally
and thus did not affect the F+ proportions. Aza-cytidine and sodium butyrate,
known reactivators of gamma-globin expression, suppressed F+ and F- cells
differentially and increased F+ cell proportions with a dependence on treatment
timing similar to that of TGF-beta. In contrast to TGF-beta, these agents had no
superimposed stimulatory effect. The data suggest that TGF-beta reactivates
gamma-globin expression, combined with a sequential stimulation and suppression
of erythropoiesis. The similarities between the actions of TGF-beta and
therapeutic reactivators of fetal hemoglobin make it conceivable that TGF-beta
may have the potential to increase HbF in patients with beta-hemoglobin
disorders. Interruption of the normal fetal-to-adult transition of hemoglobin expression
should largely ameliorate sickle cell and beta-thalassemia syndromes.
Achievement of this clinical goal requires a robust understanding of
gamma-globin gene and protein silencing during human development. For this
purpose, age-related changes in globin phenotypes of circulating human erythroid
cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors.
Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes
expressed HbA and the adult beta-globin gene, as well as HbF and the
gamma-globin genes. The distribution of hemoglobin and globin gene expression
then changed abruptly due to the expansion of cells lacking HbF or gamma-globin
mRNA (silenced cells). In adult reticulocytes, less than 5% expressed
gamma-globin mRNA. These data are consistent with a "switching" model in humans
that initially results largely from gamma- and beta-globin gene coexpression and
competition during fetal development. In contrast, early postnatal life is
marked by the rapid accumulation of cells that possess undetectable gamma-globin
mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular
replacement. This novel silencing pattern may be important for the development
of HbF-enhancing therapies. We studied the effects of rapamycin on cultures of erythroid progenitors derived
from the peripheral blood of 10 beta-thalassaemia patients differing widely with
respect to their potential to produce foetal haemoglobin (HbF). For this, we
employed the two-phase liquid culture procedure for growing erythroid
progenitors, high performance liquid chromatography for analysis of HbF
production and reverse transcription polymerase chain reaction for
quantification of the accumulation of globin mRNAs. The results demonstrated
that rapamycin induced an increase of HbF in cultures from all the
beta-thalassaemia patients studied and an increase of their overall Hb
content/cell. The inducing effect of rapamycin was restricted to gamma-globin
mRNA accumulation, being only minor for beta-globin and none for alpha-globin
mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA
content and production of HbF in erythroid precursor cells from
beta-thalassaemia patients is of great importance as this agent (also known as
sirolimus or rapamune) is already in clinical use as an anti-rejection agent
following kidney transplantation. These data suggest that rapamycin warrants
further evaluation as a potential therapeutic drug in beta-thalassaemia and
sickle cell anaemia. Beta-thalassemia is a genetic, red blood cell disorder affecting the beta-globin
chain of the adult hemoglobin gene. This results in excess accumulation of
unpaired alpha-chain gene products leading to reduced red blood cell life span
and the development of severe anemia. Current treatment of this disease involves
regular blood transfusion and adjunct chelation therapy to lower blood
transfusion-induced iron overload. Fetal hemoglobin switching agents have been
proposed to treat genetic blood disorders, such as sickle cell anemia and
beta-thalassemia, in an effort to compensate for the dysfunctional form of the
beta-globin chain in adult hemoglobin. The rationale behind this approach is to
pair the excess normal alpha-globin chain with the alternative fetal gamma-chain
to promote red blood cell survival and ameliorate the anemia. Reprogramming of
differentiation in intact, mature, adult white blood cells in response to
inclusion of monoclonal antibody CR3/43 has been described. This form of
retrograde development has been termed "retrodifferentiation", with the ability
to re-express a variety of stem cell markers in a heterogeneous population of
white blood cells. This form of reprogramming, or reontogeny, to a more
pluripotent stem cell state ought to recapitulate early hematopoiesis and
facilitate expression of a fetal and/or adult program of hemoglobin synthesis or
regeneration on infusion and subsequent redifferentiation. Herein, the outcome
of infusion of autologous retrodifferentiated stem cells (RSC) into 21 patients
with beta-thalassemia is described. Over 6 months, Infusion of 3-h autologous
RSC subjected to hematopoietic-conducive conditions into patients with
beta-thalassemia reduced mean blood transfusion requirement, increased mean
fetal hemoglobin synthesis, and significantly lowered mean serum ferritin. This
was always accompanied by an increase in mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration
(MCHC) in such patients. No adverse side effects in response to the infusion of
autologous RSC were noted. This novel clinical procedure may profoundly modify
the devastating course of many genetic disorders in an autologous setting, thus
paving the way to harnessing pluripotency from differentiated cells to
regenerate transiently an otherwise genetically degenerate tissue such as
thalassemic blood. The mechanisms underlying the human fetal-to-adult beta-globin gene switch
remain to be determined. While there is substantial experimental evidence to
suggest that promoter DNA methylation is involved in this process, most data
come from studies in nonhuman systems. We have evaluated human gamma- and
beta-globin promoter methylation in primary human fetal liver (FL) and adult
bone marrow (ABM) erythroid cells. Our results show that, in general, promoter
methylation and gene expression are inversely related. However, CpGs at -162 of
the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and
FL, respectively. We also studied gamma-globin promoter methylation during in
vitro differentiation of erythroid cells. The gamma promoters are initially
hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become
hypomethylated during the preerythroid phase of differentiation and are then
remethylated later, during erythropoiesis. The period of promoter
hypomethylation correlates with transient gamma-globin gene expression and may
explain the previously observed fetal hemoglobin production that occurs during
early adult erythropoiesis. These results provide the first comprehensive survey
of developmental changes in human gamma- and beta-globin promoter methylation
and support the hypothesis that promoter methylation plays a role in human
beta-globin locus gene switching. beta-Thalassemia (thal) is the most common recessive inherited disorder in
Mediterranean populations. It is estimated that the frequency of this disease in
the Moroccan population is between 1.5 and 3.0%. Severe forms of homozygous
thalassemia cases require expensive and technically demanding curative (bone
marrow transplantation) or palliative (chronic transfusion/chelation) therapies.
The -158 (C-->T) polymorphism of the (G)gamma-globin gene (XmnI polymorphism) is
known to ameliorate the severity of the disease because of it strong association
with an increased production of fetal hemoglobin (Hb F). Among the many known
mutations in Morocco, six are common [codon 39 (C-->T), frameshift codon (FSC) 8
(-AA), IVS-II-745 (CG), FSC 6 (-A), -29 (A-->G) and IVS-I-1 (G-->A)]. In this
study, we have investigated, in 82 Moroccan beta-thalassemic chromosomes, the
correlation between the six common mutations and the XmnI polymorphism using the
Fisher exact test. The XmnI polymorphism was divided into two categories, (XmnI
[+] and XmnI [-]) and the six common Moroccan mutations into two groups (group I
with FSC 8 and group II without FSC 8). Correlation was carried out between the
XmnI [+] category and the six common mutations individually that showed that 68%
of chromosomes in the XmnI [+] category had the FSC 8 (-AA) mutation. The
results reported here show that there is a positive correlation between the XmnI
polymorphism and FSC 8 mutation in linkage with haplotype IV [- + - + + - +] (p
<10(-5)). In conclusion, molecular determination of genetic markers in early
childhood will help to identify candidates for pharmacological Hb F switching by
hydroxyurea (HU). In the Moroccan population, a good response to HU treatment
should be suspected in cases with the -158 (C-->T) polymorphism in linkage with
haplotype IV and internal beta-globin gene framework 3. Fetal hemoglobin (HbF), the predomit hemoglobin in the fetus, is a mixture of
two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ
only at position 136 reflecting the products of two nonallelic gamma-globin
genes. At the time of birth, HbF accounts for approximately 70% of the total Hb.
The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas
in the trace amounts of HbF that is found in the adult it reverses to 40:60
because of a gamma- to beta-globin gene switch. Alterations of these ratios are
indicative of a molecular defect at the level of the HbF synthesis. Qualitative
hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and
quantitative hemoglobinopathies affecting the synthesis of HbF such as
gamma-thalassemias, duplications, triplications, and even sextuplications of the
gamma-globin genes, which may be detected in newborn blood lysates, have been
described. Moreover, several pathological and nonpathological conditions
affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell
disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are
characterized by the continued synthesis of gamma-globin chains in the adult
life. Studies of these natural mutants associated with increased synthesis of
HbF in adult life have provided considerable insight into the understanding of
the control of globin gene expression and Hb switching. The study of haemoglobin switching has represented a focus in haematology due in
large part to the clinical relevance of the fetal to adult haemoglobin switch
for developing targeted approaches to ameliorate the severity of the
beta-haemoglobinopathies. Additionally, the process by which this switch occurs
represents an important paradigm for developmental gene regulation. In this
review, we provide an overview of both the embryonic primitive to definitive
switch in haemoglobin expression, as well as the fetal to adult switch that is
unique to humans and old world monkeys. We discuss the nature of these switches
and models of their regulation. The factors that have been suggested to regulate
this process are then discussed. With the increased understanding and discovery
of molecular regulators of haemoglobin switching, such as BCL11A, new avenues of
research may lead ultimately to novel therapeutic, mechanism-based approaches to
fetal haemoglobin reactivation in patients. Sickle cell disease (SCD) and ß-thalassemia represent the most common
hemoglobinopathies caused, respectively, by the alteration of structural
features or deficient production of the ß-chain of the Hb molecule. Other
hemoglobinopathies are characterized by different mutations in the α- or
ß-globin genes and are associated with anemia and might require periodic or
chronic blood transfusions. Therefore, ß-thalassemia, SCD and other
hemoglobinopathies are excellent candidates for genetic approaches since they
are monogenic disorders and, potentially, could be cured by introducing or
correcting a single gene into the hematopoietic compartment or a single stem
cell. Initial attempts at gene transfer of these hemoglobinopathies have proved
unsuccessful due to limitations of available gene transfer vectors. With the
advent of lentiviral vectors many of the initial limitations have been overcame.
New approaches have also focused on targeting the specific mutation in the
ß-globin genes, correcting the DNA sequence or manipulating the fate of RNA
translation and splicing to restore ß-globin chain synthesis. These techniques
have the potential to correct the defect into hematopoietic stem cells or be
utilized to modify stem cells generated from patients affected by these
disorders. This review discusses gene therapy strategies for the
hemoglobinopathies, including the use of lentiviral vectors, generation of
induced pluripotent stem cells (iPS) cells, gene targeting, splice-switching and
stop codon readthrough. Gene therapy might fall short in achieving a complete reversion of the
β-thalassemic phenotype due to current limitations in vector design and
myeloablative regimen. Following gene transfer, all or a large proportion of
erythroid cells might express suboptimal levels of β-globin, impairing the
therapeutic potential of the treatment. Our aim was to evaluate whether, in
absence of complete reversion of the β-globin phenotype upon gene transfer, it
is possible to use fetal hemoglobin induction to eliminate the residual α-globin
aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines
and erythroid precursor cells from β(0)39-thalassemia patients were employed.
Gene therapy was performed with the lentiviral vector T9W. Induction of fetal
hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were
determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on
K562 transgenic cell lines harboring different copies of a lentiviral vector
carrying the human β-globin gene, showing that γ-globin mRNA expression and HbF
production can be induced in the presence of high levels of β-globin gene
expression and HbA accumulation. We then treated erythroid progenitor cells from
β-thalassemic patients with T9W, which expresses the human β-globin gene and
mithramycin separately or in combination. When transduction with our lentiviral
vector is insufficient to completely eliminate the unpaired α-globin chains,
combination of β-globin gene transfer therapy together with fetal hemoglobin
induction might be very efficacious to remove the excess of α-globin proteins in
thalassemic erythroid progenitor cells. OBJECTIVE(S): Erythropoiesis is regulated by some extrinsic and intrinsic
factors as microRNAs (miRNAs). miRNAs are endogenously small non-coding
regulatory RNAs which play vital roles in the variety of cellular fate, critical
processes; growth, apoptosis, metabolism, survival of the cells and specially
differentiation. Several miRNAs such as miR-16 and miR-451 have been shown to be
correlated with erythroid differentiation. Taking into account the importance of
miRNAs in cellular differentiation, the goal of the present study was to examine
the role of miRNAs in hematopoietic stem cells (HSC) differentiation into the
erythroid cells in the absence of growth factors and stimulatory cytokines.
MATERIALS AND METHODS: CD133+ stem cells were infected with lentiviruses
containing miR-451/miR-16 precursor sequence, erythroid differentiation was
evaluated using RT-PCR for hemoglobin chains and surface antigens, also by
banzidine staining.
RESULTS: MiR-451up-regulation, but not miR-16, could induce α, β and γ-globin
expression in CD133+ cells and have strong correlation with appearance of CD71
and CD235a markers in these cells. Moreover, miR-451 up-regulation increases the
banzidine positive cells to ~ %40.
CONCLUSION: Our results provide strong evidence that miR-451 up-regulation
strongly induces erythroid differentiation and maturation of CD133+ stem cells.
Hence, this method may provide a useful technique for the production of
artificial blood RBC and be used as a new strategy for gene therapy of
hemoglobinopathies, such as β-thalassemias and sickle cell anemia. |
What is the role of thyroid hormone in Stem cell differentiation? | Thyroid hormone treatment of Human-induced pluripotent stem cell-derived cardiomyocytes attenuates the fetal gene expression and induces differentiation. Liganded T3 receptor (TR) regulates cell autonomous formation of adult intestinal progenitor cells and that T3 action in the connective tissue is important for the establishment of the stem cell niche. In the intestinal epithelium, TRα1 and TRβ2 are expressed at the level of stem/progenitor cell populations where they induce cell proliferation and differentiation, respectively. Thyroid hormone is implicated in neural stem cell function and differentiation and acts as a neurogenic switch in the adult neural stem cell niche. Furthermore, thyroid hormone enhances maturation of oligodendrocyte precursor cells. Thyroid hormones also induce hemopoietic pluripotent stem cell differentiation toward erythropoiesis and c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. Finally, thyroid hormone modulates late differentiation stages of mesenchymal stem cells chondrogenesis via BMP signaling. | We have previously reported that E pluripoietins are produced in mice after a
single 20-mg injection of cytosine arabinoside (Ara-C) and that they are able to
initiate the determination of hemopoietic pluripotent stem cells (CFU-S) toward
the erythrocytic lineage. However, the mechanism of E pluripoietin release is
still unclear. Since the stimulating effect of thyroid hormone on erythropoiesis
is well known, we postulated a link between this hormone and the E
pluripoietins. In previous papers we demonstrated that L-triiodothyronine (LT3)
exhibits the capacity of inducing CFU-S differentiation toward erythropoiesis in
vitro. Two series of data presented here suggest that LT3 acts indirectly on
CFU-S determination by promoting the release of E pluripoietin-like factors.
First, the Ara-C injection which induces the production of E pluripoietins in
mice also promotes an increase in the LT3 plasma level. Second, medium
conditioned with bone marrow cells exposed in vitro for 90 min to LT3 (even
though this medium does not contain LT3) has E pluripoietin-like effects,
inducing CFU-S differentiation toward the erythrocytic lineage. Thyroid hormone (T3) and retinoic acid (RA) are essential for normal vertebrate
development and are known to coregulate several genes. Early development is
predomitly retinoic acid sensitive, yet thyroid hormone receptor-alpha (T3R
alpha) is expressed along with retinoic acid receptors (RAR)-alpha, -beta, and
-gamma. To determine the role of unliganded T3R alpha in early development and
on RA-stimulated neural development, we used homologous recombination techniques
to inactivate both T3R alpha gene alleles in mouse embryonic stem (ES) cells.
Loss of both T3R alpha alleles resulted in an increase in basal and RA-induced
expression of the endogenous RA-responsive genes, RAR beta and alkaline
phosphatase, which demonstrates that T3R alpha has an inhibitory effect on the
RA response. A similar magnitude of T3R inhibition of the RA response was seen
in transient transfection assays of RA response elements in both ES and assays
of RA response elements in both ES and JEG cells. Cotransfection experiments
were used to demonstrate that inhibition of the RA response could be mediated by
T3R alpha 1. The addition of T3R alpha 1, but not the T3R alpha variant c-erbA
alpha 2, to T3R alpha-null ES cells restored the inhibitory effect on RA-induced
gene expression. RA-stimulated neural differentiation was seen in the wild-type,
but not in T3R alpha-null ES, cells, consistent with reports of abnormal neural
development as a consequence of premature RA stimulation. Our results
demonstrate that the early expression of unliganded T3R alpha functions to
modulate the RA response and RA-stimulated neural differentiation. Nuclear receptors are important regulators of erythroid cell development. Here
we investigated the impact of retinoid X receptor (RXR), retinoic acid receptor
(RAR), and of the c-erbA/thyroid hormone (T3) receptor (c-erbA/TR) on growth and
differentiation of erythroid cells using an in vitro culture system of stem cell
factor-dependent erythroid progenitors. RXR, RAR, and c-erbA/TR-specific ligands
were found to induce erythroid-specific gene expression and to accelerate
erythroid differentiation in culture, with T3 being most effective. Furthermore,
while ligand-activated c-erbA/TR accelerated differentiation, unliganded
c-erbA/TR effectively blocked differentiation and supported sustained progenitor
growth in culture. Thus, c-erbA/TR appears to act as a binary switch affecting
erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated
c-erbA/TR induces differentiation. Additionally, to determine the impact of RXR
for erythroid cell development, domit interfering mutant RXRs, lacking the
transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire
DNA-binding domain, were introduced into erythroid progenitor cells via
recombit retrovirus vectors and analyzed for RXR-specific effects. It was
found that expression of wild-type RXR and of the RXR mutants devoid of AF-1
and/or AF-2 supported a transient outgrowth of erythroid cells. In marked
contrast, expression of the domit interfering deltaDNA-binding domain RXR,
containing a deletion of the entire DNA-binding domain, was incompatible with
erythroid cell growth in vitro, suggesting a pivotal role of RXR for erythroid
cell development. Thyroid hormone and retinoic acid (RA) are essential for normal neural
development in vivo, yet all in vitro differentiation strategies of embryonic
stem (ES) cells use only RA. We developed a novel differentiation strategy of
mouse ES cells using T(3). A domit-negative knock-in point mutation (P398H)
was introduced into the thyroid hormone receptor alpha gene to determine the
influence of T(3) on ES cell differentiation. Differentiation promoted by T(3)
(1 nM), RA (1 microM), or combined T(3)/RA was assessed in wild-type (wt) and
mutant (m) ES cells on the basis of neuronal-specific gene expression and cell
cycle. T(3) alone stimulated neural differentiation in a similar fashion as that
seen with RA in both wtES and mES cells. Expression of neurogranin and
Ca(2+)/calmodulin-dependent kinase IV mRNA (identified in vivo as T(3)-regulated
genes), however, was markedly reduced in mES, compared with wtES cells. RA
treatment enhanced apoptosis, significantly greater than that seen with T(3)
stimulation. T(3) treatment given with RA significantly reduced the apoptotic
effects of RA, an effect not seen in mES cells. T(3)-induced ES cell neural
differentiation of thyroid hormone alpha mutant and wtES cells provides an in
vitro model to study T(3)-dependent gene regulation in neural development. This
system could also be used to identify novel T(3)-regulated genes. The modulation
of the apoptotic effects of RA by T(3) may have implications for stem cell
therapy. Outgrowth, long-term self-renewal, and terminal maturation of human erythroid
progenitors derived from umbilical cord blood in serum-free medium can be
modulated by steroid hormones. Homogeneous erythroid cultures, as characterized
by flow cytometry and dependence on a specific mixture of physiologic
proliferation factors, were obtained within 8 days from a starting population of
mature and immature mononuclear cells. Due to previous results in mouse and
chicken erythroblasts, the proliferation-promoting effect of glucocorticoids was
not unexpected. Surprisingly, however, androgen had a positive effect on the
sustained expansion of human female but not male erythroid progenitors. Under
optimal conditions, sustained proliferation of erythroid progenitors resulted in
a more than 10(9)-fold expansion within 60 days. Terminal erythroid maturation
was significantly improved by adding human serum and thyroid hormone
(3,5,3'-triiodothyronine [T3]) to the differentiation medium. This resulted in
highly synchronous differentiation of the cells toward enucleated erythrocytes
within 6 days, accompanied by massive size decrease and hemoglobin accumulation
to levels comparable to those in peripheral blood erythrocytes. Thus, obviously,
different ligand-activated nuclear hormone receptors massively influence the
decision between self-renewal and terminal maturation in the human erythroid
compartment. T3 is required for normal early development, but relatively few T3-responsive
target genes have been identified. In general, in vitro stem cell
differentiation techniques stimulate a wide range of developmental programs,
including thyroid hormone receptor (TR) pathways. We developed several in vitro
stem cell models to more specifically identify TR-mediated gene expression in
early development. We found that embryonic carcinoma (EC) cells have reduced T3
nuclear binding capacity and only modestly express the known T3 target genes,
neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in
response to T3. Full T3 induction in transient transfection of EC cells was
restored with cotransfection of a TR expression vector. We, therefore, performed
gene expression profiles in wild-type embryonic stem (ES) cells compared with
expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES
cells), to identify T3 target genes. T3 stimulation of wild-type ES cells
altered mRNA expression of 610 known genes (26% of those studied), although only
approximately 60 genes (1%) met criteria for direct T3 stimulation based on the
magnitude of induction and requirement for the presence of TR. We selected five
candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding
protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane
antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness
of these genes was evaluated in both in vitro endogenous gene expression and in
vivo mouse model systems. These genes identified in a novel stem cell system,
including those induced and repressed in response to T3, may mediate thyroid
hormone actions in early development. Thyroid hormones (TH) are essential for brain development. However, information
on if and how this key endocrine factor affects adult neurogenesis is
fragmentary. We thus investigated the effects of TH on proliferation and
apoptosis of stem cells in the subventricular zone (SVZ), as well as on
migration of transgene-tagged neuroblasts out of the stem cell niche.
Hypothyroidism significantly reduced all three of these processes, inhibiting
generation of new cells. To determine the mechanisms relaying TH action in the
SVZ, we analyzed which receptor was implicated and whether the effects were
played out directly at the level of the stem cell population. The alpha TH
receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive
progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha
to be required to maintain full proliferative activity. Finally, a direct TH
transcriptional effect, not mediated through other cell populations, was
revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly
modulated transcription from the c-myc promoter reporter construct containing a
functional TH response element containing TRE but not from a mutated TRE
sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the
adult mammalian brain. Here we demonstrate that physiological concentrations of the thyroid hormones T3
and T4 enhance the KERATIN 15 promoter activity and expression in epithelial
stem cells of adult human scalp hair follicles in situ and in vitro.
Additionally, T3 and T4 stimulate expression of the immuno-inhibitory surface
molecule CD200. Subsequently, T3 and T4 induce apoptosis and differentiation and
inhibit clonal growth of these progenitor cells in vitro. These data suggest
that human hair follicle bulge-derived epithelial stem cells underlie profound,
previously unknown hormonal regulation by thyroid hormones, and show that
primary human keratin 15-GFP+ progenitor cells can be exploited to further
elucidate fundamental endocrine controls of human epithelial stem cells. Embryonic stem cells (ESCs) can differentiate into functional cardiomyocytes and
thus represent a promising cell source for cardiac regenerative therapy.
Nevertheless, the therapeutic application of ESC-derived cardiomyocytes is
limited by the low efficacy of the current protocol for cardiac differentiation
and their immature phenotypes. Although thyroid hormone is essential for normal
cardiac development and function, its role in the cardiac differentiation of
ESCs, as well as the maturation of ESC-derived cardiomyocytes, remains unclear.
In this study, we examined the cardiac differentiation of murine ESCs in the
presence of T(3) for 7 d using flow cytometry, RT-PCR, cellular
electrophysiology study, and confocal calcium imaging. Compared with control
conditions, T(3) supplementation increased the number of ESC-derived
cardiomyocytes and was accompanied by up-regulation of a panel of cardiac
markers, including Nkx2.5, myosin light chain-2V, as well as alpha- and
beta-myosin heavy chain. More importantly, electrophysiological study revealed
that ESC-derived cardiomyocytes exhibited more adult-like phenotypes after T(3)
supplementation based on action potential characteristics. They also exhibited
more adult-like calcium homeostasis properties. These phenotypic changes were
associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase-2a
and ryanodine receptor-2 expression. In addition, the classical (genomic)
pathway was shown to be involved in T(3)-induced cardiac differentiation of
ESCs. Our results show that T(3) supplementation promotes cardiac
differentiation of ESCs and enhances maturation of electrophysiological, as well
as calcium homeostasis, properties of ESC-derived cardiomyocytes. Adult organ-specific stem cells are essential for organ homeostasis and repair
in adult vertebrates. The intestine is one of the best-studied organs in this
regard. The intestinal epithelium undergoes constant self-renewal throughout
adult life across vertebrates through the proliferation and subsequent
differentiation of the adult stem cells. This self-renewal system is established
late during development, around birth, in mammals when endogenous thyroid
hormone (T3) levels are high. Amphibian metamorphosis resembles mammalian
postembryonic development around birth and is totally dependent upon the
presence of high levels of T3. During this process, the tadpole intestine,
predomitly a monolayer of larval epithelial cells, undergoes drastic
transformation. The larval epithelial cells undergo apoptosis and concurrently,
adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and
then differentiate to establish a trough-crest axis of the epithelial fold,
resembling the crypt-villus axis in the adult mammalian intestine. We and others
have studied the T3-dependent remodeling of the intestine in Xenopus laevis.
Here we will highlight some of the recent findings on the origin of the adult
intestinal stem cells. We will discuss observations suggesting that liganded T3
receptor (TR) regulates cell autonomous formation of adult intestinal progenitor
cells and that T3 action in the connective tissue is important for the
establishment of the stem cell niche. We will further review evidence suggesting
similar T3-dependent formation of adult intestinal stem cells in other
vertebrates. A deficiency of maternal thyroid hormones (THs) during pregcy may have severe
impacts on fetal brain development. However, the cellular targets of THs and
their underlying mechanisms are still unclear. In this study, we found that
maternal hypothyroidism during pregcy in mice inhibited neurogenesis in the
embryonic telencephalon and caused learning and memory impairment in the
offspring. To explore the underlying mechanisms, we treated cultured mouse
embryonic neural stem cells (eNSCs) with a physiological level of 3, 5,
3'-triiodo-L-thyronine (T3). We found that T3 promoted the neuronal
differentiation of eNSCs, while inhibiting astrocytic differentiation. In
addition, the proliferation and maintece of eNSCs were inhibited by T3.
Furthermore, the TH receptor alpha 1 (TRα1) was detected in the eNSCs both in
vivo and in vitro. Silencing TRα1 protein expression with specific siRNA
eliminated the effects of T3 on eNSCs. We also found that T3 decreased STAT3
phosphorylation and STAT3-DNA binding activity through TRα1. The over expression
of STAT3 attenuated the promotive effects of T3 on neuronal differentiation of
eNSCs. Taken together, these results suggest that T3 promotes the neuronal
differentiation of eNSCs by inhibiting STAT3 signaling activity through TRα1 and
contributes to early neurogenesis in the embryonic telencephalon. Our studies
reveal the physiological effects of TH in regulating eNSCs differentiation and
suggest that eNSCs are one of the major cellular targets in the central nervous
system by which TH influences early brain development. These findings also
provide new insights into the mechanisms of neurological deficits caused by TH
deficiency during embryogenesis. Disrupted thyroid hormone function evokes severe physiological consequences in
the immature brain. In adulthood, although clinical reports document an effect
of thyroid hormone status on mood and cognition, the molecular and cellular
changes underlying these behavioural effects are poorly understood. More
recently, the subtle effects of thyroid hormone on structural plasticity in the
mature brain, in particular on adult hippocampal neurogenesis, have come to be
appreciated. However, the specific stages of adult hippocampal progenitor
development that are sensitive to thyroid hormone are not defined. Using
nestin-green fluorescent protein reporter mice, we demonstrate that thyroid
hormone mediates its effects on hippocampal neurogenesis by influencing Type 2b
and Type 3 progenitors, although it does not alter proliferation of either the
Type 1 quiescent progenitor or the Type 2a amplifying neural progenitor. Thyroid
hormone increases the number of doublecortin (DCX)-positive Type 3 progenitors,
and accelerates neuronal differentiation into both DCX-positive immature
neurones and neuronal nuclei-positive granule cell neurones. Furthermore, we
show that this increase in neuronal differentiation is accompanied by a
significant induction of specific transcription factors involved in hippocampal
progenitor differentiation. In vitro studies using the neurosphere assay support
a direct effect of thyroid hormone on progenitor development because
neurospheres treated with thyroid hormone are shifted to a more differentiated
state. Taken together, our results indicate that thyroid hormone mediates its
neurogenic effects via targeting Type 2b and Type 3 hippocampal progenitors, and
suggests a role for proneural transcription factors in contributing to the
effects of thyroid hormone on neuronal differentiation of adult hippocampal
progenitors. The subventricular zone (SVZ) neural stem cell niche contains mixed populations
of stem cells, transit-amplifying cells, and migrating neuroblasts. Deciphering
how endogenous signals, such as hormones, affect the balance between these cell
types is essential for understanding the physiology of niche plasticity and
homeostasis. We show that Thyroid Hormone (T(3)) and its receptor, TRα1, are
directly involved in maintaining this balance. TRα1 is expressed in amplifying
and migrating cells. In vivo gain- and loss-of-function experiments demonstrate
first, that T(3)/TRα1 directly repress Sox2 expression, and second, that TRα1
overexpression in the niche favors the appearance of DCX+ migrating neuroblasts.
Lack of TRα increases numbers of SOX2+ cells in the SVZ. Hypothyroidism
increases proportions of cells in interphase. Thus, in the adult SVZ, T(3)/TRα1
together favor neural stem cell commitment and progression toward a migrating
neuroblast phenotype; this transition correlates with T(3)/TRα1-dependent
transcriptional repression of Sox2. BACKGROUND: Thyroid hormones are involved in developmental and homeostatic
processes in several tissues. Their action results in different outcomes
depending on the developmental stage, tissue and/or cellular context.
Interestingly, their pleiotropic roles are conserved across vertebrates. It is
largely documented that thyroid hormones act via nuclear receptors, the TRs,
which are transcription factors and whose activity can be modulated by the local
availability of the hormone T3. In the "classical view", the T3-induced
physiological response depends on the expression of specific TR isoforms and the
iodothyronine deiodinase selenoenzymes that control the local level of T3, thus
TR activity.
SCOPE OF THE REVIEW: Recent data have clearly established that the functionality
of TRs is coordinated and integrated with other signaling pathways, specifically
at the level of stem/progenitor cell populations. Here, we summarize these data
and propose a new and intriguing role for thyroid hormones in two selected
examples.
MAJOR CONCLUSIONS: In the intestinal epithelium and the retina, TRα1 and TRβ2
are expressed at the level of the precursors where they induce cell
proliferation and differentiation, respectively. Moreover, these different
functions result from the integration of the hormone signal with other intrinsic
pathways, which play a fundamental role in progenitor/stem cell physiology.
GENERAL SIGNIFICANCE: Taken together, the interaction of TRs with other
signaling pathways, specifically in stem/progenitor cells, is a new concept that
may have biological relevance in therapeutic approaches aimed to target stem
cells such as tissue engineering and cancer. This article is part of a Special
Issue entitled Thyroid hormone signalling. Thyroid hormone (TH) is essential for vertebrate brain development. Most
research on TH and neuronal development focuses on late development, mainly the
perinatal period in mammals. However, in human infants neuromotor development
correlates best with maternal TH levels in the first trimester of pregcy,
suggesting that TH signaling could affect early brain development. Studying TH
signaling in early embryogenesis in mammals is experimentally challenging. In
contrast, free-living embryos, such as Xenopus laevis, permit physiological
experimentation independent of maternal factors. We detailed key elements of TH
signaling: ligands, receptors (TR), and deiodinases during early X. laevis
development, before embryonic thyroid gland formation. Dynamic profiles for all
components were found. Between developmental stages 37 and 41 (~48 h after
hatching, coincident with a phase of continuing neurogenesis) significant
increases in T(3) levels as well as in mRNA encoding deiodinases and TR
occurred. Exposure of embryos at this developmental stage for 24 h to either a
TH antagonist, NH-3, or to tetrabromobisphenol A, a flame retardant and known TH
disruptor, differentially modulated the expression of a number of TH target
genes implicated in neural stem cell function or neural differentiation.
Moreover, 24-h exposure to either NH-3 or tetrabromobisphenol A diminished cell
proliferation in the brain. Thus, these data show first, that TH signaling
exerts regulatory roles in early X. laevis neurogenesis and second, that this
period represents a potential window for endocrine disruption. Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of
hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes
ossification, this is a concern for the application of MSCs in articular
cartilage tissue engineering. To identify mechanisms that elicit this
phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for
differential gene expression analysis and functional experiments with the focus
on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in
chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and
dexamethasone withdrawal and addition of triiodothyronine. Differential gene
expression analysis of BMPs and their receptors was performed. Based on these
results, the in vitro hypertrophy model was used to investigate the effect of
recombit BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy
could be shown clearly by an increased cell size, alkaline phosphatase activity,
and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP
receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in
the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone
induced hypertrophy by upregulation of BMP4 and this induced enhancement of
hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an
important modulator of the late differentiation stages in MSC chondrogenesis and
the thyroid hormone induces this pathway. As cartilage tissue engineering
constructs will be exposed to this factor in vivo, this study provides important
insight into the biology of MSC-based cartilage. Furthermore, the possibility to
engineer hypertrophic cartilage may be helpful for critical bone defect repair. INTRODUCTION: Thyroid hormones play important roles in the development of neural
cells in the central nervous system. Even minor changes to normal thyroid
hormone levels affect dendritic and axonal outgrowth, sprouting and myelination
and might even lead to irreversible damages such as cretinism. Despite our
knowledge of the influence on the mammalian CNS, the role of thyroid hormones in
the development of the enteric nervous system (ENS) still needs to be
elucidated. In this study we have analyzed for the first time the influence of
3,5,3'-triiodothyronine (T3) on ENS progenitor cells using cell biological
assays and a microarray technique.
RESULTS: In our in vitro model, T3 inhibited cell proliferation and stimulated
neurite outgrowth of differentiating ENS progenitor cells. Microarray analysis
revealed a group of 338 genes that were regulated by T3 in differentiating
enterospheres. 67 of these genes are involved in function and development of the
nervous system. 14 of them belong to genes that are involved in axonal guidance
or neurite outgrowth. Interestingly, T3 regulated the expression of netrin G1
and endothelin 3, two guidance molecules that are involved in human enteric
dysganglionoses.
CONCLUSION: The results of our study give first insights how T3 may affect the
enteric nervous system. T3 is involved in proliferation and differentiation
processes in enterospheres. Microarray analysis revealed several interesting
gene candidates that might be involved in the observed effects on enterosphere
differentiation. Future studies need to be conducted to better understand the
gene to gene interactions. |
Which receptors can be evaluated with the [18F]altanserin? | 5-HT2A (5-hydroxytryptamine type 2a) receptor can be evaluated with the [18F]altanserin. | Changes in serotonin-2 receptors have been demonstrated in brain autopsy
material from patients with various neurodegenerative and affective disorders.
It would be desirable to locate a ligand for the study of these receptors in
vivo with positron emission tomography (PET). Altanserin is a
4-benzoylpiperidine derivative with a high affinity and selectivity for S2
receptors in vitro. Dynamic PET studies were carried out in nine normal
volunteers with high-specific activity (376-1,680 mCi/mumol) [18F]altanserin.
Arterial blood samples were obtained and the plasma time-activity curves were
corrected for the presence of labeled metabolites. Thirty minutes after
injection, selective retention of the radioligand was observed in cortical
areas, while the cerebellum, caudate, and thalamus had low radioactivity levels.
Specific binding reached a plateau between 30 and 65 min postinjection at 1.8%
of the injected dose/L of brain and then decreased, indicating the reversibility
of the binding. The total/nonspecific binding ratio reached 2.6 for times
between 50 and 70 min postinjection. The graphical analysis proposed by Logan et
al. allowed us to estimate the binding potential (Bmax/KD). Pretreatment with
ketanserin was given to three volunteers and brain activity remained uniformly
low. An additional study in one volunteer showed that [18F]altanserin can be
displaced from the receptors by large doses of ketanserin. At the end of the
study, unchanged altanserin was 57% of the total plasma activity. These results
suggest that [18F]altanserin is selective for S2 receptors in vivo as it is in
vitro. They indicate that [18F]altanserin is suitable for imaging and
quantifying S2 receptors with PET in humans. To further validate its use in positron emission tomography (PET), we studied
the binding of [18F]altanserin, a specific 5HT2 radioligand, in the rat brain
using in vivo autoradiography. Distribution of [18F]altanserin binding was
comparable to the in vitro mapping of 5HT2 receptors reported in the literature.
Selective displacers were used to test the reversibility and the selectivity of
this radioligand. Specific binding of [18F]altanserin in the rat frontal cortex
was quantified by direct counting with an electronic imaging system and by
quantification on digitalized autoradiograms. Close results of about 30 pmol/g
were obtained with both methods. Our data confirmed that [18F]altanserin is a
valid tracer for 5HT2 receptors binding studies. [18F]altanserin has been used to label serotonin 5-HT2A receptors, which are
believed to be important in the pathophysiology of schizophrenia and depression.
The purpose of this study was to test the feasibility of a constant infusion
paradigm for equilibrium modeling of [18F]altanserin with PET. Kinetic modeling
with [18F]altanserin may be hampered by the presence of lipophilic
radiometabolites observed in plasma after intravenous administration.
METHODS: Eight healthy volunteers were injected with [18F]altanserin as a bolus
(208+/-9 MBq [5.62+/-0.25 mCi]) plus constant infusion (65+/-3 MBq/h
[1.76+/-0.08 mCi/h]) ranging from 555 to 626 min (615+/-24 min) after injection.
PET acquisitions (10-20 min) and venous blood sampling were performed every
30-60 min throughout the infusion period.
RESULTS: Linear regression analysis revealed that time-activity curves for both
brain activity and plasma [18F]altanserin and metabolite concentrations
stabilized after about 6 h. This permitted equilibrium modeling and estimation
of V3' (ratio of specific uptake [cortical-cerebellar] to total plasma parent
concentration after 6 h). Values of V3' ranged from 1.57+/-0.38 for anterior
cingulate cortex to 1.02+/-0.39 for frontal cortex. The binding potential V3
(ratio of specific uptake to free plasma parent concentration after 6 h, using
group mean f1) was also calculated and ranged from 169+/-41 for anterior
cingulate cortex to 110+/-42 for frontal cortex. From 6 h onward, the rate of
change for V3' and V3 was only 1.11+/-1.69 %/h.
CONCLUSION: These results demonstrate the feasibility of equilibrium imaging
with [18F]altanserin over more than 5 radioactive half-lives and suggest a
method to overcome difficulties associated with lipophilic radiolabeled
metabolites. The stability in V3 and V3' once equilibrium is achieved suggests
that a single PET acquisition obtained at 6 h may provide a reasonable measure
of 5-HT2A receptor density. AIM: The study presented here firstly compares the distribution of the binding
potential of the serotonin-5HT2A receptor as measured in vivo with data of
receptor density taken from literature. Secondly, the sensitivity of the method
to detect gradual differences in receptor densities is evaluated.
METHODS: Positron emission tomography (PET) studies were carried out in 6
healthy volunteers using the selective serotonin-5HT2A ligand 18F-altanserin.
The binding potential was quantified in 12 regions using Logan's graphical
method and the equilibrium method. These data were compared to the distribution
of receptor density as taken from literature.
RESULTS: The binding data in vivo correlated to autoradiography data (post
mortem) with r = 0.83 (Pearson regression coefficient; p < 0.0001). A difference
in the receptor density between two regions could be detected with p < 0.05 when
it amounted at least to 18%.
CONCLUSION: This study demonstrates a good agreement between in vivo data
obtained with 18F-altanserin and PET in healthy volunteers and the true
autoradiographically determined distribution of 5HT2A receptors in human brains.
The in vivo method seems to be sensitive enough to detect changes in receptor
density of more than 18%. The feasibility of in vivo serotonin 5HT(2) receptor binding measurement using
[18F]altanserin as a radioligand has been well established. In this study, the
postsynaptic receptor binding potential of this ligand was examined as a
possible indicator of synaptic serotonin content after pharmacological
challenge. Studies were performed in 11 subjects with a history of recurrent
major depression. Six of them received serotonergic antidepressive treatment at
the time of the experiment, the other five patients were untreated. Two PET
measurements were carried out in each subject within 2 or 3 days. Before one of
the measurements, 25 mg of the serotonin re-uptake inhibitor clomipramine were
given intravenously, the other measurement was done without pharmacological
challenge. The data were analyzed using non-linear least-square regression and
Logan's graphical method. In the whole group of subjects, binding potential and
distribution volume of altanserin decreased following clomipramine challenge.
The decrease was between 14 (P=0.03) and 23% (P=0.004). This effect was mainly
seen in subjects not on antidepressive medication. Clomipramine challenge
probably increased the synaptic serotonin level, which competed with altanserin
leading to the lowered binding potential. The paradigm might, thus, be useful to
estimate serotonin release in vivo. Pretreatment with serotonergic
antidepressants reduces the effect of clomipramine. The aim of the present study is to describe and validate a method for accurate
quantification of 5-hydroxytryptamine (5-HT)(2A) receptors using
[18F]altanserin-positron emission tomography (PET) and the bolus/infusion
approach. A bolus/infusion ratio of 1.75 h aimed at attaining rapid steady state
in blood and brain was predicted from previous bolus studies performed in our
laboratory. The infusion schedule was tested in normal subjects (n = 10) using
dynamic PET and frequent plasma sampling for 6 h. Steady state was attained in
brain and plasma within 2 h, and time-activity curves remained constant for
another 3 h. To represent free and nonspecifically bound [18F]altanserin and its
radiolabeled metabolites only, cerebellum must show no displacement in 5-HT(2A)
displacement studies. To validate this, saturating doses of cold ketanserin were
administered and it was found that specific binding of [18F]altanserin decreased
uniformly to the level of the cerebellum and no change in the cerebellar
time-activity curve was found after ketanserin administration. A shorter
experimental setup was tested in a second group (n = 20) including patients with
neuropsychiatric disorders. Dynamic PET (five frames of 8 minutes each) and
venous blood sampling at midscan time started 2 h after [18F]altanserin
administration. The mean percentage rate of change per hour in the outcome
parameter, DV(3)', was low (mean -0.3% h-1; range -7.3-7.2% h-1) and no
correlation of DV(3)' versus time was demonstrated. It is concluded that
5-HT(2A) receptor studies can be conducted within 2 h of [18F]altanserin
infusion, yielding reliable results. The aim of the present study was to develop an experimental paradigm for the
study of serotonergic neurotransmission in humans using positron emission
tomography and the 5-HT2A selective radioligand [18F]altanserin. [18F]altanserin
studies were conducted in seven subjects using the bolus/infusion approach
designed for attaining steady state in blood and brain 2 hours after the initial
[18F]altanserin administration. Three hours after commencement of radiotracer
administration, 0.25 mg/kg of the selective serotonin reuptake inhibitor,
citalopram (Lundbeck, Valby, Denmark), was administered to all subjects as a
constant infusion for 20 minutes. To reduce 5-HT1A-mediated autoinhibition of
cortical 5-HT release, four of the seven subjects were pretreated with the
partial 5-HT1A agonist pindolol for 3 days at an increasing oral dose (25 mg on
the day of scanning). In each subject, the baseline condition (120 to 180
minutes) was compared with the stimulated condition (195 to 300 minutes).
Despite a pronounced increase in plasma prolactin and two subjects reporting hot
flushes compatible with an 5-HT-induced adverse effect, cortical [18F]altanserin
binding was insensitive to the citalopram challenge, even after pindolol
pretreatment. The biochemical and cellular events possibly affecting the
unsuccessful translation of the citalopram/pindolol challenge into a change in
5-HT2A receptor binding of [18F]altanserin are discussed. To study the 5-HT(2A) receptors in the living human brain, using positron
emission tomography (PET), two selective radiotracers are currently in use:
[(18)F]altanserin and [(11)C]MDL 100907. It is, however, currently unknown to
what extent data obtained with either tracer are directly comparable. The aim of
this study was to compare binding characteristics of these two radiotracers in
rat brain with respect to affinity (K(d)), receptor binding density (B(max)),
binding potential (BP), and nonspecific binding. Further, binding kinetics,
sensitivity towards competition with the endogenous transmitter serotonin, and
the competitive/noncompetitive interaction between the two radioligands were
evaluated. In addition, the selectivity of [(18)F]altanserin for the 5-HT(2A)
receptor was assessed. The K(d) value of [(18)F]altanserin and [(3)H]MDL 100907
was in the order of 0.3 nM. B(max) in frontal cortex was 523 and 527 fmol/mg
protein, respectively. The binding of [(18)F]altanserin was not influenced by
blocking either the 5-HT(2B/2C) or the alpha(1)-adrenergic receptors. At 37
degrees C the association t(1/2) was 2.8 and 2.7 min and the dissociation t(1/2)
was 11 and 13.5 min for [(18)F]altanserin and [(3)H]MDL 100907, respectively.
Both radioligands were displaced by 5-HT, only at high concentrations; the K(i)
value of 5-HT ranging between 650 and 3,300 nM. This indicates that binding of
both radioligands in PET studies is not directly influenced by changes in
endogenous 5-HT.Overall, the binding of [(18)F]altanserin and [(3)H]MDL 100907
to the 5-HT(2A) receptor was very comparable, showing selective high affinity
binding in the subomolar range. It has been suggested that impulsive behavior is caused by dysfunctional
serotonergic 5-HT neurotransmission in the central nervous system (CNS). Brain
neuroimaging studies have shown that behavioral inhibition is linked to the
activation of cortex sites such as the ventral frontal cortex. Positron emission
tomography (PET) imaging with [(18)F]altanserin to characterize 5-HT(2A)
receptor binding revealed a reduction in 5-HT(2A) binding in the ventral frontal
cortex in women who had recovered from impulsive diseases. These clinical,
neuroimaging, and pharmacological studies appear to support the hypothesis that
functional alteration of neurotransmission due to genetic polymorphisms of the
5-HT receptors may be involved in impulsive behavior modulation. Following
evaluation by a self-reporting measure, it was proposed that a polymorphism in
the promoter of the 5-HT(2A) receptor gene is the underlying cause of impulsive
behavior; however, this hypothesis is not convincing. We examined whether the
polymorphism in the 5-HT(2A) receptor gene promoter is involved in impulsive
aggression by evaluating a behavioral task (Go/No-go task) in normal volunteers.
The polymorphism of the 5-HT(2A) receptor gene promoter in lymphocytes from 71
volunteers was analyzed by using PCR. Impulsivity was defined as the number of
commission errors (responding when one should not) recorded during a Go/No-go
task; a larger number of commission errors indicate greater difficulty in
inhibiting impulsive behavior. The subjects of the A-1438A allele group for the
5-HT(2A) receptor gene made more commission errors under the punishment-reward
(PR)condition in a Go/No-go task than those in the G-1438G group. In the present
review, we discuss and suggest the possible involvement of the A-1438A
polymorphism of the 5HT2A receptor gene promoter in impulsive behavior. This
hypothesis was evaluated by using a behavioral task measure that could directly
reveal impulsive behavioral traits in humans. PURPOSE: To determine the reproducibility of measurements of brain 5-HT2A
receptors with an [18F]altanserin PET bolus/infusion approach. Further, to
estimate the sample size needed to detect regional differences between two
groups and, finally, to evaluate how partial volume correction affects
reproducibility and the required sample size.
METHODS: For assessment of the variability, six subjects were investigated with
[18F]altanserin PET twice, at an interval of less than 2 weeks. The sample size
required to detect a 20% difference was estimated from [18F]altanserin PET
studies in 84 healthy subjects. Regions of interest were automatically
delineated on co-registered MR and PET images.
RESULTS: In cortical brain regions with a high density of 5-HT2A receptors, the
outcome parameter (binding potential, BP1) showed high reproducibility, with a
median difference between the two group measurements of 6% (range 5-12%),
whereas in regions with a low receptor density, BP1 reproducibility was lower,
with a median difference of 17% (range 11-39%). Partial volume correction
reduced the variability in the sample considerably. The sample size required to
detect a 20% difference in brain regions with high receptor density is
approximately 27, whereas for low receptor binding regions the required sample
size is substantially higher.
CONCLUSION: This study demonstrates that [18F]altanserin PET with a
bolus/infusion design has very low variability, particularly in larger brain
regions with high 5-HT2A receptor density. Moreover, partial volume correction
considerably reduces the sample size required to detect regional changes between
groups. BACKGROUND: Many studies have found disturbances of serotonin (5-HT) activity in
anorexia nervosa (AN). Because little is known about 5-HT receptor function in
AN, positron emission tomography (PET) imaging with 5-HT receptor-specific
radioligands was used to characterize 5-HT1A and 5-HT2A receptors.
METHODS: Fifteen women ill with AN (ILL AN) were compared with 29 healthy
control women (CW); PET and [11C]WAY100635 were used to assess binding potential
(BP) of the 5-HT1A receptor, and [18F]altanserin was used to assess postsynaptic
5-HT2A receptor BP. [15O] water and PET were used to assess cerebral blood flow.
RESULTS: The ILL AN women had a highly significant (30%-70%) increase in
[11C]WAY100635 BP in prefrontal and lateral orbital frontal regions, mesial and
lateral temporal lobes, parietal cortex, and dorsal raphe nuclei compared with
CW. The [18F]altanserin BP was normal in ILL AN but was positively and
significantly related to harm avoidance in suprapragenual cingulate, frontal,
and parietal regions. Cerebral blood flow was normal in ILL AN women.
CONCLUSIONS: Increased activity of 5-HT1A receptor activity may help explain
poor response to 5-HT medication in ILL AN. This study extends data suggesting
that 5-HT function, and, specifically, the 5-HT2A receptor, is related to
anxiety in AN. PURPOSE: The serotonin 2A (5-HT(2A)) receptor is of interest in several
psychiatric and neurological diseases. In the present study we investigated the
longitudinal stability of 5-HT(2A) receptors and the stability of the
quantification procedure in the elderly in order to be able to study elderly
patients with neuropsychiatric diseases on a longitudinal basis.
METHODS: [(18)F]-Altanserin PET was used to quantify 5-HT(2A) receptors in 12
healthy elderly individuals at baseline and at 2 years in six volumes of
interest. A bolus/infusion protocol was used to achieve the binding potential,
BP(P). The reproducibility as assessed in terms of variability and the
reliability as assessed in terms of intraclass correlation coefficient (ICC)
were used to compare inter- and intraobserver stability and to evaluate the
effects of increasing complexity of partial volume (PV) corrections. We also
compared the stability of our measurements over 2 years with the stability of
data from an earlier study with 2-week test-retest measurements.
RESULTS: BP(P) was unaltered at follow-up without the use of PV correction and
when applying two-tissue PV correction, test-retest reproducibility was 12-15%
and reliability 0.45-0.67 in the large bilateral regions such as the parietal,
temporal, occipital and frontal cortices, while orbitofrontal and anterior
cingulate cortical regions were less stable. The use of PV correction decreased
the variability but also decreased the between-subject variation, thereby
worsening the reliability.
CONCLUSION: In healthy elderly individuals, brain 5-HT(2A) receptor binding
remains stable over 2 years, and acceptable reproducibility and reliability in
larger regions and high intra- and interobserver stability allow the use of
[(18)F]-altanserin in longitudinal studies of patients with neuropsychiatric
disorders. There is a large body of evidence that serotonin [5-hydroxytryptamine (5-HT)]
plays an important role in the transmission and regulation of pain. Here we used
positron emission tomography (PET) to study the relationship between baseline
5-HT(2A) binding in the brain and responses to noxious heat stimulation in a
group of young healthy volunteers. Twenty-one healthy subjects underwent PET
scanning with the 5-HT(2A) antagonist, [(18)F]altanserin. In addition,
participants underwent a battery of pain tests using noxious heat stimulation to
assess pain threshold, pain tolerance and response to short-lasting phasic and
long-lasting (7-minute) tonic painful stimulation. Significant positive
correlations were found between tonic pain ratings and [(18)F]altanserin binding
in orbitofrontal (r=0.66; p=0.005), medial inferior frontal (r=0.60; p=0.014),
primary sensory-motor (r=0.61; p=0.012) and posterior cingulate (r=0.63;
p=0.009) cortices. In contrast, measures of regional [(18)F]altanserin binding
did not correlate with pain threshold, pain tolerance, or suprathreshold phasic
pain responses. These data suggest that cortical 5-HT(2A) receptor availability
co-varies with responses to tonic pain. The correlation between
[(18)F]altanserin binding in prefrontal cortex and tonic pain suggests a
possible role of this brain region in the modulation and/or cognitive-evaluative
appreciation of pain. Neurobiological evidence implicates the amygdala as well as serotonergic
(serotonin, 5-HT) signaling via postsynaptic 5-HT(2A) receptors as essential
substrates of anxiety behaviors. Assuming a functional interdependence of these
substrates, we hypothesized that a low-fear behavioral phenotype due to
bilateral lesion of the amygdala would be associated with significant 5-HT(2A)
receptor changes. Thus, we used [(18)F]altanserin positron emission tomography
(PET) referenced to radioligand plasma levels and corrected for partial volume
effects to quantify the spatial distribution of 5-HT(2A) receptor binding
potential (BP(P)) in a rare patient with Urbach-Wiethe disease and selective
bilateral amygdala calcification damage relative to 10 healthy control subjects.
Consistent with our a priori hypothesis, we observed a 70% global decrease in
5-HT(2A) receptor BP(P) in the Urbach-Wiethe patient relative to controls. Thus,
brain abnormalities in this patient are not restricted to the amygdala, but
extend to overall 5-HT neurotransmission via 5-HT(2A) receptors. Our findings
provide important insights into the molecular architecture of human anxiety
behaviors and suggest the 5-HT(2A) receptor as a promising pharmacological
target to control pathological anxiety. [(18)F]altanserin is the preferred radiotracer for in-vivo labeling of serotonin
2A receptors by positron emission tomography (PET). We report a modified
synthesis procedure suited for reliable production of multi-GBq amounts of
[(18)F]altanserin useful for application in humans. We introduced thermal
heating for drying of [(18)F]fluoride as well as for the reaction instead of
microwave heating. We furthermore describe solid phase extraction and HPLC
procedures for quantitative determination of [(18)F]altanserin and metabolites
in plasma. The time course of arterial plasma activity with and without
metabolite correction was determined. 90 min after bolus injection, 38.4% of
total plasma activity derived from unchanged [(18)F]altanserin. Statistical
comparison of kinetic profiles of [(18)F]altanserin metabolism in plasma samples
collected in the course of two ongoing studies employing placebo, the serotonin
releaser dexfenfluramine and the hallucinogen psilocybin, revealed the same
tracer metabolism. We conclude that metabolite analysis for correction of
individual plasma input functions used in tracer modeling is not necessary for
[(18)F]altanserin studies involving psilocybin or dexfenfluramine treatment. A dysfunction of multiple neurotransmitter systems is assumed as a neurochemical
basis of the akinetic-rigid syndrome of progressive supranuclear palsy (PSP). In
vitro studies have produced conflicting results on the serotoninergic system in
PSP. We, therefore, studied the binding potential of the serotonin 2A (5-HT(2A))
receptor ligand [18F]altanserin in 8 patients with clinically probable PSP and
13 healthy controls using positron emission tomography. We found an
up-regulation of 5-HT(2A) receptors in the substantia nigra and, to a lower
degree, in the striatum, while neocortical 5- HT(2A) receptor densities showed
no changes upon partial-volume correction. Nigral and striatal receptor changes
were significantly correlated with patients' scores of motor dysfunction (UPDRS
III, PSP-rating scale) pointing to a functional relevance of the described
findings. The vulnerability to mood disorders, impulsive-aggression, eating disorders, and
suicidal behavior varies greatly with gender, and may reflect gender differences
in central serotonergic function. We investigated the relationships of gender,
mood, impulsivity, aggression and temperament to 5HT(2A) receptor binding in 21
healthy subjects using [18F]altanserin and PET neuroimaging. Binding potentials
in pre-defined regions-of-interest (ROI) were calculated using the Logan
graphical method, corrected for partial volume effects, and compared by gender
with age co-varied. SPM analysis was used for voxel level comparisons.
Altanserin binding (BP(P)) was greater in male than female subjects in the
following nine ROIs: hippocampus (HIP) and Lt. HIP, lateral orbital frontal
cortex (LOF) and Lt. LOF, left medial frontal cortex (Lt. MFC), left medial
temporal cortex (Lt. MTC), left occipital cortex (Lt. OCC), thalamus (THL) and
Lt. THL. Differences in Lt. HIP and Lt. MTL remained significant after
Bonferroni correction. Gender differences were noted in the co-variation of
psychological traits with BP(P) values in specific ROIs. Among males alone,
aggression was negatively correlated with BP(P) values in Lt. LOF and Lt. MFC,
and Suspiciousness positively correlated in LOF, Lt. LOF and Lt. MFC. Among
female subjects alone, Negativism was positively correlated with BP(P) values in
HIP, and Verbal Hostility in Lt. HIP. Altanserin binding in Lt. MTC was
positively correlated with Persistence, with no significant gender effect.
Gender differences in 5HT(2A) receptor function in specific ROIs may mediate
expression of psychological characteristics such as aggression, suspiciousness
and negativism. Future studies of 5HT(2A) receptor function and its relationship
to behavior should control for gender. Serotonin (5-hydroxytryptamine, 5-HT) has diverse physiologic roles as a
neurotransmitter in the central nervous system (1). It is involved in regulation
and modulation of sleep, affective and personality behaviors, and pain. It also
is a regulator of smooth muscle function and platelet aggregation. The brain
cortical 5-HT system has been implicated in several neuropsychiatric disorders,
including major depression, anxiety, obsessive-compulsive disorder, and
schizophrenia (2, 3). The effects of 5-HT are mediated by as many as seven
classes of receptor populations (5-HT1 to 5-HT7), many of which include several
subtypes (4). There are three receptor subtypes within the G protein-coupled
5-HT2 receptor family: 5-HT2A, 5-HT2B, and 5-HT2C. 5-HT2A receptors are
abundantly present in the cerebral cortex, basal forebrain, hippocampus,
amygdala, dorsal thalamus, hypothalamus, superior colliculus, substantia nigra,
pedunculopontine nucleus, legmental area, and myelencephalon (5). 5-HT2A
receptors are involved in mediation of normal and psychotic states, working
memory, regulation of GABAergic and cholinergic neuronal cells, sleep,
peripheral pain, and cardiovascular functions. 5-HT2B receptors are found mainly
in several peripheral tissues, such as the stomach, intestine, and pulmonary
smooth muscle, and in the myocardium. In the brain, 5-HT2B receptors are found
in discrete nuclei of the cerebellum, lateral septum, dorsal hypothalamus,
dorsal raphe, and amygdala. 5-HT2C receptors are found in the choroid plexus,
substantia nigra, globus pallidus, and ventromedial thalamus. 5-HT2A receptors
are implicated in several psychiatric disorders, such as schizophrenia,
depression, and obsessive-compulsive disorder. Thus, there is a need for
selective ligands to investigate the pharmacologic role of 5-HT2A receptors.
There have been several studies to develop specific 5-HT2A radioligands, such as
[11C]ketanserin (6), [18F]spiperone (7), [11C]methylspiperone ([11C]NMSP), and
[18F]setoperone [PubMed], for positron emission tomography (PET) imaging.
However, none has proven specific for 5-HT2A receptors because these compounds
also bind to other receptors, such as dopamine receptors and 5-HT1 receptor
subtypes. Altanserin, a fluorobenzoyl derivative related to ketanserin, was
reported to be a potent inhibitor of 5-HT2A receptors with >100-fold selectivity
over D2/3 receptors, 5-HT1A, 5-HT6, and 5-HT7 (8, 9). This led to the
development of
3-[2-[4-(4-[18F]fluorobenzoyl)-1-piperidyl]ethyl]-2-sulfanyl-3H-quinazolin-4-one
([18F]altanserin) as a useful tool for 5-HT2A receptor PET imaging in vivo (10). A dysfunctional glutamatergic system is thought to be central to the negative
symptoms and cognitive deficits recognized as determit to the poor quality of
life of people with schizophrenia. Modulating glutamate uptake has, thus, been
suggested as a novel target for antipsychotics. Alstonine is an indole alkaloid
sharing with atypical antipsychotics the profile in animal models relevant to
schizophrenia, though divergent in its mechanism of action. The aim of this
study was to evaluate the effects of alstonine on glutamate uptake.
Additionally, the effects on glutathione content and extracellular S100B levels
were assessed. Acute hippocampal slices were incubated with haloperidol (10μM),
clozapine (10 and 100μM) or alstonine (1-100μM), alone or in combination with
apomorphine (100μM), and 5-HT(2) receptor antagonists (0.01μM altanserin and
0.1μM SB 242084). A reduction in glutamate uptake was observed with alstonine
and clozapine, but not haloperidol. Apomorphine abolished the effect of
clozapine, whereas 5-HT(2A) and 5-HT(2C) antagonists abolished the effects of
alstonine. Increased levels of glutathione were observed only with alstonine,
also the only compound that failed to decrease the release of S100B. This study
shows that alstonine decreases glutamate uptake, which may be beneficial to the
glutamatergic deficit observed in schizophrenia. Noteworthily, the decrease in
glutamate uptake is compatible with the reversal of MK-801-induced social
interaction and working memory deficits. An additional potential benefit of
alstonine as an antipsychotic is its ability to increase glutathione, a key
cellular antioxidant reported to be decreased in the brain of patients with
schizophrenia. Adding to the characterization of the novel mechanism of action
of alstonine, the lack of effect of apomorphine in alstonine-induced changes in
glutamate uptake reinforces that D(2) receptors are not primarily implicated.
Though clearly mediated by 5-HT(2A) and 5-HT(2C) serotonin receptors, the
precise mechanisms that result in the effects of alstonine on glutamate uptake
warrant elucidation. Numerous studies indicate that the serotonergic (5-HT) transmitter system is
involved in the regulation of impulsive aggression and there is from
post-mortem, in vivo imaging and genetic studies evidence that the 5-HT2A
receptor may be involved. We investigated 94 healthy individuals (60 men, mean
age 47.0±18.7, range 23-86) to determine if trait aggression and trait
impulsivity were related to frontal cortex 5-HT2A receptor binding (5-HT2AR) as
measured with [18F]-altanserin PET imaging. Trait aggression and trait
impulsivity were assessed with the Buss-Perry Aggression Questionnaire (AQ) and
the Barratt Impulsiveness Scale 11 (BIS-11). Statistical analyses were conducted
using a multiple linear regression model and internal consistency reliability of
the AQ and BIS-11 was evaluated by Cronbach's alpha. Contrary to our hypothesis,
results revealed no significant associations between 5-HT2AR and the AQ or
BIS-11 total scores. Also, there was no significant interaction between gender
and frontal cortex 5-HT2AR in predicting trait aggression and trait impulsivity.
This is the first study to examine how 5-HT2AR relates to trait aggression and
trait impulsivity in a large sample of healthy individuals. Our findings are not
supportive of a selective role for 5-HT2AR in mediating the 5-HT related effects
on aggression and impulsivity in psychiatrically healthy individuals. Imaging the cerebral serotonin 2A (5-HT2A ) receptors with positron emission
tomography (PET) has been carried out in humans with [(11) C]MDL 100907 and
[(18) F]altanserin. Recently, the MDL 100907 analogue [(18) F]MH.MZ was
developed combining the selectivity profile of MDL 100907 and the favourable
radiophysical properties of fluorine-18. Here, we present a direct comparison of
[(18) F]altanserin and [(18) F]MH.MZ. 5-HT2A receptor binding in pig cortex and
cerebellum was investigated by autoradiography with [(3) H]MDL 100907, [(18)
F]MH.MZ, and [(18) F]altanserin. [(18) F]MH.MZ and [(18) F]altanserin were
investigated in Danish Landrace pigs by brain PET scanning at baseline and after
i.v. administration of blocking doses of ketanserin. Full arterial input
function and high performance liquid chromatography (HPLC) analysis allowed for
tissue-compartment kinetic modeling of PET data. In vitro autoradiography showed
high binding in cortical regions with both [(18) F]MH.MZ and [(18) F]altanserin.
Significant 5-HT2A receptor binding was also found in the pig cerebellum, thus
making this region unsuitable as a reference region for in vivo data analysis in
this species. The cortical binding of [(18) F]MH.MZ and [(18) F]altanserin was
blocked by ketanserin supporting that both radioligands bind to 5-HT2A receptors
in the pig brain. In the HPLC analysis of pig plasma, [(18) F]MH.MZ displayed a
fast and reproducible metabolism resulting in hydrophilic radiometabolites only
whereas the metabolic profile of [(18) F]altanserin as expected showed
lipophilic radiometabolites. Due to the slow kinetics of [(18) F]MH.MZ in
high-binding regions in vivo, we suggest that [(18) F]MH.MZ will be an
appropriate tracer for low binding regions where kinetics will be faster,
whereas [(18) F]altanserin is a suitable tracer for high-binding regions. INTRODUCTION: The selective 5-hydroxytryptamine type 2a receptor (5-HT(2A)R)
radiotracer [(18)F]altanserin is a promising ligand for in vivo brain imaging in
rodents. However, [(18)F]altanserin is a substrate of P-glycoprotein (P-gp) in
rats. Its applicability might therefore be constrained by both a differential
expression of P-gp under pathological conditions, e.g. epilepsy, and its
relatively low cerebral uptake. The aim of the present study was therefore
twofold: (i) to investigate whether inhibition of multidrug transporters (MDT)
is suitable to enhance the cerebral uptake of [(18)F]altanserin in vivo and (ii)
to test different pharmacokinetic, particularly reference tissue-based models
for exact quantification of 5-HT(2A)R densities in the rat brain.
METHODS: Eighteen Sprague-Dawley rats, either treated with the MDT inhibitor
cyclosporine A (CsA, 50 mg/kg, n=8) or vehicle (n=10) underwent 180-min PET
scans with arterial blood sampling. Kinetic analyses of tissue time-activity
curves (TACs) were performed to validate invasive and non-invasive
pharmacokinetic models.
RESULTS: CsA application lead to a two- to threefold increase of
[(18)F]altanserin uptake in different brain regions and showed a trend toward
higher binding potentials (BP(ND)) of the radioligand.
CONCLUSIONS: MDT inhibition led to an increased cerebral uptake of
[(18)F]altanserin but did not improve the reliability of BP(ND) as a
non-invasive estimate of 5-HT(2A)R. This finding is most probable caused by the
heterogeneous distribution of P-gp in the rat brain and its incomplete blockade
in the reference region (cerebellum). Differential MDT expressions in
experimental animal models or pathological conditions are therefore likely to
influence the applicability of imaging protocols and have to be carefully
evaluated. |
Which diseases have been associated with the PTPN22 620W allele? | The functional polymorphism 620W in the intracellular tyrosine phosphatase PTPN22 gene has been shown to confer susceptibility to the development of type 1 diabetes, seropositive rheumatoid arthritis, systemic lupus erythematosus, Hashimoto thyroiditis, and Wegener's granulomatosis (granulomatosis with polyangiitis). | Rheumatoid arthritis (RA), like other autoimmune diseases, has a complex genetic
basis. Rapid technical advances in high-throughput genotyping and analysis have
now reached a point where genes of low-to-moderate risk can be identified using
a variety of study designs, including whole genome association studies. The
availability of large, well-characterized populations of cases and controls are
critical to the success of these efforts. A functional variant (R620W) of the
intracellular protein tyrosine phosphatase N22 (PTPN22) has now been
conclusively shown to confer approximately two-fold risk for seropositive RA as
well as several other autoimmune disorders. PTPN22 appears to act primarily by
setting thresholds for T-cell receptor signaling, and the current data suggest
that the PTPN22 620W allele is likely to be a general risk factor for the
development of humoral autoimmunity. PTPN22 is expressed widely in hematopoietic
cells, but other than in T cells, its role is unknown. These results provide
strong evidence for the longstanding hypothesis that common genes underlie
different autoimmune phenotypes and emphasize that finding genes of only
moderate risk can provide important insights into disease pathogenesis. The protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene encodes for
lymphoid tyrosine phosphatase LYP, involved in the negative regulation of early
T-cell activation. An association has recently been reported between the
PTPN22-620W functional allele and rheumatoid factor-positive (RF+) rheumatoid
arthritis (RA), among other autoimmune diseases. Expected linkage proof for
consistency cannot be definitely produced by an affected sib-pair (ASP)
analysis. Our aim was therefore to search for linkage evidence with the
transmission disequilibrium test. DNA from the French Caucasian population was
available for two samples of 100 families with one RA patient and both parents,
and for 88 RA index cases from RA ASP families. Genotyping was carried out by
PCR-restriction fragment length polymorphism. The analysis was performed using
the transmission disequilibrium test, genotype relative risk and ASP-based
analysis. The transmission disequilibrium test of the PTPN22-620W allele
revealed linkage and association for RF+ RA (61% of transmission, P = 0.037).
The genotype relative risk showed the risk allele in 34% of RF+ RA patients and
in 24% of controls derived from nontransmitted parental chromosomes (P = 0.047,
odds ratio = 1.69, 95% confidence interval = 1.03-2.78). The ASP investigation
showed no enriched risk allele in RA multiplex families, resulting in a lack of
power of ASP analysis, explaining the published negative results. This study is
the first to show linkage of PTPN22 to RF+ RA, consistent with PTPN22 as a new
RA gene. OBJECTIVE: Analyses of families with multiple autoimmune disorders have revealed
a functional polymorphism, 620W, in the intracellular tyrosine phosphatase gene
PTPN22 as a predisposing factor for type 1 diabetes, seropositive rheumatoid
arthritis, systemic lupus erythematosus, and Hashimoto thyroiditis, and the
presence of the PTPN22 protein appears to herald the development of
autoantibodies in these disorders. This study therefore examined whether the
functionally relevant PTPN22 polymorphism is associated with Wegener's
granulomatosis (WG).
METHODS: A population-based study was performed for the PTPN22 polymorphism in
199 patients with WG and in 399 healthy individuals. The R620W variation was
investigated by simple restriction fragment-length polymorphism analysis.
RESULTS: The PTPN22 620W allele frequency was significantly increased in
antineutrophil cytoplasmic antibody (ANCA)-positive WG patients compared with
healthy controls (P < 0.001). The association was particularly striking in
patients with kidney, lung, eye, and peripheral nervous system involvement
(i.e., those with generalized WG).
CONCLUSION: The PTPN22 620W allele appears to be involved in the pathogenesis of
WG, and ANCA positivity seems to be the hallmark. The 620W allele of PTPN22 has been associated with susceptibility to several
different forms of chronic inflammatory disease, including Type 1 diabetes
(T1D), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and
autoimmune thyroiditis (AIT). We set out to explore its possible role in two
other inflammatory diseases: multiple sclerosis (MS) and Crohn's disease (CD).
In our cohort of 496 MS trios from the United Kingdom, we observed reduced
transmission of the PTPN22 620W allele. The CD sample consisted of 169 trios as
well as 249 cases of CD with their 207 matched control subjects collected in the
province of Québec, Canada; there was also no evidence of association between
the PTPN22 620W allele and susceptibility for CD. Pooled analyses combining our
data with published data assessed a total of 1496 cases of MS and 1019 cases of
CD but demonstrated no evidence of association with either disease. Given the
modest odds ratios of known risk alleles for inflammatory diseases, these
analyses do not exclude a role for the PTPN22 allele in susceptibility to CD or
MS, but they do suggest that such a putative role would probably be more modest
than that reported so far in T1D, RA, SLE, and AIT. The minor allele of the R620W missense single-nucleotide polymorphism (SNP;
rs2476601) in the PTPN22 (protein tyrosine phosphatase non-receptor 22) gene has
been reported to be associated with multiple autoimmune diseases, including type
1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, juvenile
idiopathic arthritis, autoimmune thyroiditis and vitiligo. Systemic sclerosis
(SSc) is a connective tissue disease with some autoimmune abnormalities. The aim
of our study was to test for association of the PTPN22*620W allele with SSc in a
French Caucasian cohort with a case-control study of 121 patients with SSc and
103 controls. All patients and controls were genotyped for the PTPN22*R620W SNP.
No association was found between the PTPN22*620W allele and SSc (7% v 9.2%, p =
0.39). The frequency of genotypes carrying at least one 620W allele was similar
in both groups (13% v 17%, p = 0.38). The PTPN22*620W allele was also not
associated with autoantibody patterns. Thus, the PTPN22*R620W polymorphism
cannot be regarded as a genetic susceptibility factor for SSc in the French
Caucasian population. OBJECTIVES: PTPN22 is involved in T-cell activation and its R620W
single-nucleotide polymorphism (SNP) has been shown to predispose to different
autoimmune diseases. The aims of this study were to investigate the role of the
PTPN22 R620W SNP in conferring susceptibility to the ANCA-associated
vasculitides (AAVs), and to explore potential associations between the PTPN22
genotype and the disease manifestations.
METHODS: PTPN22 R620W SNP was genotyped in a cohort of 344 AAV patients [143
with granulomatosis with polyangiitis (Wegener's) (GPA), 102 with microscopic
polyangiitis (MPA) and 99 with Churg-Strauss syndrome (CSS)] and in 945 healthy
controls.
RESULTS: The frequency of the minor allele (620W) was significantly higher in
GPA patients than in controls [P = 0.005, χ(2 )= 7.858, odds ratio (OR) = 1.91],
while no statistically significant association was found with MPA or CSS. Among
GPA patients, the 620W allele was particularly enriched in ANCA-positive
patients as compared with controls (P = 0.00012, χ(2 )= 14.73, OR = 2.31); a
particularly marked association was also found with ENT involvement (P = 0.0071,
χ(2 )= 7.258, OR = 1.98), lung involvement (P = 0.0060, χ(2 )= 7.541, OR = 2.07)
and skin manifestations of all kinds (P = 0.000047, χ(2 )= 16.567, OR = 3.73).
CONCLUSION: The PTPN22 620W allele confers susceptibility to the development of
GPA (but not of MPA or CSS), and particularly of its ANCA-positive subset. |
Which extra thyroid tissues have thyrotropin (TSH) receptors? | TSH receptors are expressed also in extrathyroid tissues. TSH receptors seem to be functional. Extrathyroid tissues include fibrobasts of the orbit and adipose tissue
The principal tissues with TSH receptors are:
adippose tissue
orbital fibrotic tissue | CONTEXT: Graves' disease (GD) is a systemic autoimmune syndrome comprising
manifestations in thyroid and orbital connective tissue. The link between these
two tissues in GD eludes our understanding. Patients with GD have increased
frequency of circulating monocyte lineage cells known as fibrocytes. These
fibrocytes infiltrate orbital connective tissues in thyroid-associated
ophthalmopathy and express functional TSH receptor (TSHR).
OBJECTIVE: The aim of the study was to identify and characterize CD34(+)
fibrocytes in thyroid tissue.
DESIGN/SETTING/PARTICIPANTS: Patients undergoing surgical thyroidectomy at two
academic medical centers were recruited to the study.
MAIN OUTCOME MEASURES: We performed immunohistochemistry, flow cytometry,
real-time PCR, cytokine-specific ELISA, and cell differentiation.
RESULTS: CD34(+)ColI(+)CXCR4(+)TSHR(+) cells can be identified in situ in
thyroid tissue from donors with GD, Hashimoto's thyroiditis, or in
normal-appearing tissue. Thyroid fibroblasts cultivated from these glands
express a CD34(-)ColI(+)CXCR4(+)TSHR(+) phenotype. TSHR levels are higher than
those in orbital fibroblasts. When treated with TSH, thyroid fibroblasts
generate IL-6 and IL-8. The induction of IL-6 can be blocked by dexamethasone, a
chemical inhibitor of Akt/Pkb, and by knocking down Akt with a specific small
interfering RNA. When treated with TGF-β or rosiglitazone, thyroid fibroblasts
differentiate into myofibrocytes or adipocytes, respectively.
CONCLUSIONS: ColI(+)CXCR4(+)TSHR(+) thyroid fibroblasts resemble orbital
fibroblasts and circulating fibrocytes. CD34(+) fibrocytes appear to infiltrate
both tissues in GD. Thyroid fibroblasts lose CD34 display in culture, unlike
orbital fibroblasts and circulating fibrocytes. Fibrocytes and their fibroblast
derivatives may participate in the pathogenesis of thyroid autoimmunity after
TSHR activation. They could represent a therapeutic target for these diseases. BACKGROUND: Obesity is known to be associated with higher risks of
cardiovascular disease, metabolic syndrome, and diabetes mellitus.
Thyroid-stimulating hormone (TSHR) is the receptor for thyroid-stimulating
hormone (TSH, or thyrotropin), the key regulator of thyroid functions. The
expression of TSHR, once considered to be limited to thyrocytes, has been so far
detected in many extrathyroidal tissues including liver and fat. Previous
studies have shown that TSHR expression is upregulated when preadipocytes
differentiate into mature adipocytes, suggestive of a possible role of TSHR in
adipogenesis. However, it remains unclear whether TSHR expression in adipocytes
is implicated in the pathogenesis of obesity.
METHODS: In the present study, TSHR expression in adipose tissues from both mice
and human was analyzed, and its association with obesity was evaluated.
RESULTS: We here showed that TSHR expression was increased at both mRNA and
protein levels when 3T3-L1 preadipocytes were induced to differentiate.
Knockdown of TSHR blocked the adipocyte differentiation of 3T3-L1 preadipocytes
as evaluated by Oil-red-O staining for lipid accumulation and by RT-PCR analyses
of PPAR-γ and ALBP mRNA expression. We generated obesity mice (C57/BL6) by
high-fat diet feeding and found that the TSHR protein expression in visceral
adipose tissues from obesity mice was significantly higher in comparison with
the non-obesity control mice (P < 0.05). Finally, the TSHR expression in adipose
tissues was determined in 120 patients. The results showed that TSHR expression
in subcutaneous adipose tissue is correlated with BMI (body mass index).
CONCLUSION: Taken together, these results suggested that TSHR is an important
regulator of adipocyte differentiation. Dysregulated expression of TSHR in
adipose tissues is associated with obesity, which may involve a mechanism of
excess adipogenesis. To determine the relative importance of TSH in white adipose tissue, we compared
the adipose phenotypes of two distinct mouse models of hypothyroidism. These
models differed in that the normal reciprocal relationship between thyroid
hormone and TSH was intact in one and disrupted in the other. One model,
thyroidectomized (THYx) mice, had a 100-fold increase in TSH and a normal TSH
receptor (TSHR); in contrast, the other model, hyt/hyt mice, had a 120-fold
elevation of TSH but a nonfunctional TSHR. Although both THYx and hyt/hyt mice
were in a severe hypothyroid state, the epididymal fat (mg)/body wt (g) (F/B)
ratio of THYx mice was much smaller than that of hyt/hyt mice (8.2 ± 0.43 vs.
14.4 ± 0.40, respectively, P < 0.001). The fat cell diameter in THYx mice was
also smaller than that in hyt/hyt mice (79 ± 2.8 vs. 105 ± 2.2 μm, respectively,
P < 0.001), suggesting that TSH induced lipolysis in adipose tissues. When we
transferred a functional mouse TSHR gene and a control plasmid into opposite
sides of epididymal fat of hyt/hyt mice by plasmid injection combined with
electroporation, fat weight of the TSHR side was decreased to 60% of that of the
control side. Messenger RNA levels of hormone-sensitive lipase in epididymal fat
containing the transferred TSHR gene were twofold higher than those in tissue
from the control side. These results indicated that TSH worked as a lipolytic
factor in white adipose tissues, especially in mice in a hypothyroid state. |
Which histone modifications distinguish between promoters and enhancers? | H3K27ac is a marker of active enhancers. An enhancer chromatin state signature associated with active developmental enhancers may be defined by high levels of H3K27ac marking, nucleosome displacement, hypersensitivity to sonication, and strong depletion of H3K27me3. | The regulation of gene expression during thymocyte development provides an ideal
experimental system to study lineage-commitment processes. In particular,
expression of the CD4, CD8A and CD8B genes seems to correlate well with the
cell-fate decisions that are taken by thymocytes, and elucidating the molecular
mechanisms that underlie the differential expression of these genes could reveal
key events in differentiation processes. Here, we review examples of how gene
cis elements (such as promoters, enhancers and locus control regions) and trans
elements (such as transcription factors, chromatin-remodelling complexes and
histone-modification enzymes) come together to orchestrate a finely tuned
sequence of events that results in the complex pattern of CD4, CD8A and CD8B
gene expression that is observed during thymocyte development. The identification of transcriptional regulatory modules within mammalian
genomes is a prerequisite to understanding the mechanisms controlling regulated
gene expression. While high-throughput microarray- and sequencing-based
approaches have been used to map the genomic locations of sites of nuclease
hypersensitivity or target DNA sequences bound by specific protein factors, the
identification of regulatory elements using functional assays, which would
provide important complementary data, has been relatively rare. Here we present
a method that permits the functional identification of active transcriptional
regulatory modules using a simple procedure for the isolation and analysis of
DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome
that contains these elements. The more than 100 new active regulatory DNAs
identified in this manner from F9 cells correspond to both promoter-proximal and
distal elements, and display several features predicted for endogenous
transcriptional regulators, including localization within DNase-accessible
chromatin and CpG islands, and proximity to expressed genes. Furthermore,
comparison with published ChIP-seq data of ES-cell chromatin shows that the
functional elements we identified correspond with genomic regions enriched for
H3K4me3, a histone modification associated with active transcriptional
regulatory elements, and that the correspondence of H3K4me3 with our
promoter-distal elements is largely ES-cell specific. The majority of the distal
elements exhibit enhancer activity. Importantly, these functional DNA fragments
are an average 149 bp in length, greatly facilitating future applications to
identify transcription factor binding sites mediating their activity. Thus, this
approach provides a tool for the high-resolution identification of the
functional components of active promoters and enhancers. BACKGROUND: Recent genomic scale survey of epigenetic states in the mammalian
genomes has shown that promoters and enhancers are correlated with distinct
chromatin signatures, providing a pragmatic way for systematic mapping of these
regulatory elements in the genome. With rapid accumulation of chromatin
modification profiles in the genome of various organisms and cell types, this
chromatin based approach promises to uncover many new regulatory elements, but
computational methods to effectively extract information from these datasets are
still limited.
RESULTS: We present here a supervised learning method to predict promoters and
enhancers based on their unique chromatin modification signatures. We trained
Hidden Markov models (HMMs) on the histone modification data for known promoters
and enhancers, and then used the trained HMMs to identify promoter or enhancer
like sequences in the human genome. Using a simulated annealing (SA) procedure,
we searched for the most informative combination and the optimal window size of
histone marks.
CONCLUSION: Compared with the previous methods, the HMM method can capture the
complex patterns of histone modifications particularly from the weak signals.
Cross validation and scanning the ENCODE regions showed that our method
outperforms the previous profile-based method in mapping promoters and
enhancers. We also showed that including more histone marks can further boost
the performance of our method. This observation suggests that the HMM is robust
and is capable of integrating information from multiple histone marks. To
further demonstrate the usefulness of our method, we applied it to analyzing
genome wide ChIP-Seq data in three mouse cell lines and correctly predicted
active and inactive promoters with positive predictive values of more than 80%.
The software is available at http://http:/nash.ucsd.edu/chromatin.tar.gz. The human body is composed of diverse cell types with distinct functions.
Although it is known that lineage specification depends on cell-specific gene
expression, which in turn is driven by promoters, enhancers, insulators and
other cis-regulatory DNA sequences for each gene, the relative roles of these
regulatory elements in this process are not clear. We have previously developed
a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate
promoters, enhancers and insulators in the human genome. Here we use the same
approach to identify these elements in multiple cell types and investigate their
roles in cell-type-specific gene expression. We observed that the chromatin
state at promoters and CTCF-binding at insulators is largely invariant across
diverse cell types. In contrast, enhancers are marked with highly
cell-type-specific histone modification patterns, strongly correlate to
cell-type-specific gene expression programs on a global scale, and are
functionally active in a cell-type-specific manner. Our results define over
55,000 potential transcriptional enhancers in the human genome, significantly
expanding the current catalogue of human enhancers and highlighting the role of
these elements in cell-type-specific gene expression. We reported previously that well-characterized enhancers but not promoters for
typical tissue-specific genes, including the classic Alb1 gene, contain
unmethylated CpG dinucleotides and evidence of pioneer factor interactions in
embryonic stem (ES) cells. These properties, which are distinct from the
bivalent histone modification domains that characterize the promoters of genes
involved in developmental decisions, raise the possibility that genes expressed
only in differentiated cells may need to be marked at the pluripotent stage.
Here, we demonstrate that the forkhead family member FoxD3 is essential for the
unmethylated mark observed at the Alb1 enhancer in ES cells, with FoxA1
replacing FoxD3 following differentiation into endoderm. Up-regulation of FoxD3
and loss of CpG methylation at the Alb1 enhancer accompanied the reprogramming
of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells.
Studies of two genes expressed in specific hematopoietic lineages revealed that
the establishment of enhancer marks in ES cells and iPS cells can be regulated
both positively and negatively. Furthermore, the absence of a pre-established
mark consistently resulted in resistance to transcriptional activation in the
repressive chromatin environment that characterizes differentiated cells. These
results support the hypothesis that pluripotency and successful reprogramming
may be critically dependent on the marking of enhancers for many or all
tissue-specific genes. Developmental programs are controlled by transcription factors and chromatin
regulators, which maintain specific gene expression programs through epigenetic
modification of the genome. These regulatory events at enhancers contribute to
the specific gene expression programs that determine cell state and the
potential for differentiation into new cell types. Although enhancer elements
are known to be associated with certain histone modifications and transcription
factors, the relationship of these modifications to gene expression and
developmental state has not been clearly defined. Here we interrogate the
epigenetic landscape of enhancer elements in embryonic stem cells and several
adult tissues in the mouse. We find that histone H3K27ac distinguishes active
enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This
indicates that the amount of actively used enhancers is lower than previously
anticipated. Furthermore, poised enhancer networks provide clues to unrealized
developmental programs. Finally, we show that enhancers are reset during nuclear
reprogramming. Post-translational histone modifications, acting alone or in a context-dependent
manner, influence numerous cellular processes via their regulation of gene
expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly
understood histone modification. Some reports describe depletion of K27Me1 at
promoters and transcription start sites (TSS), implying its depletion at TSS is
necessary for active transcription, while others have associated enrichment of
H3K27me1 at TSS with increased levels of mRNA expression. Tissue- and
gene-specific patterns of H3K27me1 enrichment and their correlation with gene
expression were determined via chromatin immunoprecipitation on chip microarray
(ChIP-chip) and human mRNA expression array analyses. Results from erythroid
cells were compared with those in neural and muscle cells. H3K27me1 enrichment
varied depending on levels of cell-type specific gene expression, with highest
enrichment over transcriptionally active genes. Over individual genes, the
highest levels of H3K27me1 enrichment were found over the gene bodies of highly
expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of
actively transcribing genes, H3K27me1 was selectively depleted at the TSS of
actively transcribed genes. There was markedly decreased to no H3K27me1
enrichment in genes with low expression. At some locations, H3K27
monomethylation was also found to be associated with chromatin signatures of
gene enhancers. The regulatory elements that direct tissue-specific gene expression in the
developing mammalian embryo remain largely unknown. Although chromatin profiling
has proven to be a powerful method for mapping regulatory sequences in cultured
cells, chromatin states characteristic of active developmental enhancers have
not been directly identified in embryonic tissues. Here we use
whole-transcriptome analysis coupled with genome-wide profiling of H3K27ac and
H3K27me3 to map chromatin states and enhancers in mouse embryonic forelimb and
hindlimb. We show that gene-expression differences between forelimb and
hindlimb, and between limb and other embryonic cell types, are correlated with
tissue-specific H3K27ac signatures at promoters and distal sites. Using H3K27ac
profiles, we identified 28,377 putative enhancers, many of which are likely to
be limb specific based on strong enrichment near genes highly expressed in the
limb and comparisons with tissue-specific EP300 sites and known enhancers. We
describe a chromatin state signature associated with active developmental
enhancers, defined by high levels of H3K27ac marking, nucleosome displacement,
hypersensitivity to sonication, and strong depletion of H3K27me3. We also find
that some developmental enhancers exhibit components of this signature,
including hypersensitivity, H3K27ac enrichment, and H3K27me3 depletion, at lower
levels in tissues in which they are not active. Our results establish histone
modification profiling as a tool for developmental enhancer discovery, and
suggest that enhancers maintain an open chromatin state in multiple embryonic
tissues independent of their activity level. Histone modifications are now well-established mediators of transcriptional
programs that distinguish cell states. However, the kinetics of histone
modification and their role in mediating rapid, signal-responsive gene
expression changes has been little studied on a genome-wide scale. Vascular
endothelial growth factor A (VEGFA), a major regulator of angiogenesis, triggers
changes in transcriptional activity of human umbilical vein endothelial cells
(HUVECs). Here, we used chromatin immunoprecipitation followed by
high-throughput sequencing (ChIP-seq) to measure genome-wide changes in histone
H3 acetylation at lysine 27 (H3K27ac), a marker of active enhancers, in
unstimulated HUVECs and HUVECs stimulated with VEGFA for 1, 4, and 12 h. We show
that sites with the greatest H3K27ac change upon stimulation were associated
tightly with EP300, a histone acetyltransferase. Using the variation of H3K27ac
as a novel epigenetic signature, we identified transcriptional regulatory
elements that are functionally linked to angiogenesis, participate in rapid
VEGFA-stimulated changes in chromatin conformation, and mediate VEGFA-induced
transcriptional responses. Dynamic H3K27ac deposition and associated changes in
chromatin conformation required EP300 activity instead of altered nucleosome
occupancy or changes in DNase I hypersensitivity. EP300 activity was also
required for a subset of dynamic H3K27ac sites to loop into proximity of
promoters. Our study identified thousands of endothelial, VEGFA-responsive
enhancers, demonstrating that an epigenetic signature based on the variation of
a chromatin feature is a productive approach to define signal-responsive genomic
elements. Further, our study implicates global epigenetic modifications in
rapid, signal-responsive transcriptional regulation. Enhancers play a pivotal role in regulating the transcription of distal genes.
Although certain chromatin features, such as the histone acetyltransferase P300
and the histone modification H3K4me1, indicate the presence of enhancers, only a
fraction of enhancers are functionally active. Individual chromatin marks, such
as H3K27ac and H3K27me3, have been identified to distinguish active from
inactive enhancers. However, the systematic identification of the most
informative single modification, or combination thereof, is still lacking.
Furthermore, the discovery of enhancer RNAs (eRNAs) provides an alternative
approach to directly predicting enhancer activity. However, it remains
challenging to link chromatin modifications to eRNA transcription. Herein, we
develop a logistic regression model to unravel the relationship between
chromatin modifications and eRNA synthesis. We perform a systematic assessment
of 24 chromatin modifications in fetal lung fibroblast and demonstrate that a
combination of four modifications is sufficient to accurately predict eRNA
transcription. Furthermore, we compare the ability of eRNAs and H3K27ac to
discriminate enhancer activity. We demonstrate that eRNA is more indicative of
enhancer activity. Finally, we apply our fibroblast trained model to six other
cell-types and successfully predict eRNA synthesis. Thus, we demonstrate the
learned relationships are general and independent of cell-type. We provided a
powerful tool to identify active enhancers and reveal the relationship between
chromatin modifications, eRNA production and enhancer activity. |
Where is the metaxin complex localized? | The metaxin complex is localized to the outer mitochondrial membrane. | Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin
3 genes in the mouse, is essential for survival of the postimplantation mouse
embryo. In this study, the subcellular location, domain structure, and
biochemical function of metaxin were investigated. Anti-recombit metaxin
antibodies recognized 35- and 70-kDa proteins in mitochondria from various
tissues; the 35-kDa protein is consistent in size with the predicted translation
product of metaxin cDNA. When metaxin cDNA was transfected into COS cells,
immunofluorescence staining demonstrated that the protein is located in
mitochondria. Metaxin contains a putative mitochondrial outer membrane signal
anchor domain at its C terminus, and a truncated form of metaxin lacking this
signal anchor domain had a reduced association with mitochondria. In addition,
metaxin was highly susceptible to proteases in intact mitochondria. We therefore
conclude that metaxin is a mitochondrial protein that extends into the cytosol
while anchored into the outer membrane at its C terminus. In its N-terminal
region, metaxin shows significant sequence identity to Tom37, a component of the
outer membrane portion of the mitochondrial preprotein translocation apparatus
in Saccharomyces cerevisiae, but important structural differences, including
apparently different mechanisms of targeting to membranes, also exist between
the two proteins. Given the similar subcellular locations of metaxin and Tom37,
the possible role of metaxin in mitochondrial preprotein import was
investigated. Antibodies against metaxin, when preincubated with mitochondria,
partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria.
Metaxin is therefore the second mammalian component of the protein translocation
apparatus of the mitochondrial outer membrane to be characterized at the
molecular level and the first for which an inherited mutation has been
described. The early embryonic lethal phenotype of mice lacking metaxin
demonstrates that efficient import of proteins into mitochondria is crucial for
cellular survival. The characterization of metaxin provides an opportunity to
elucidate similarities and possible differences in the mechanisms of protein
import between fungi and mammals and in the phenotypes of fungi and mammals
lacking mitochondrial import receptors. Thrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose regulation
of expression and function are not well understood. Mouse Thbs3 is located just
downstream from the divergently transcribed metaxin gene (Mtx), which encodes an
outer mitochondrial membrane import protein. Although Thbs3 and Mtx share a
common promoter region, previous studies showed that Mtx is regulated by
proximal elements that had little effect on Thbs3 expression. In this study,
transient transfection of rat chondrosarcoma cells and NIH-3T3 fibroblasts
demonstrated that Thbs3 is regulated in a cell type-specific manner by a
position- and orientation-independent far upstream enhancer located within
intron 6 of Mtx. Despite its greater proximity to the transcription start site
of Mtx, the Thbs3 enhancer did not have a significant effect on Mtx expression.
Two DNA-protein complexes, which were both required for activity, were
identified when nuclear extracts were assayed with a probe containing the
enhancer sequence. The protein in one of these complexes was identified as Sp1,
while the other DNA-protein complex remains uncharacterized. A 6-kilobase pair
promoter containing the enhancer was able to direct specific expression of the
E. coli lacZ gene in transgenic mice, whereas a 2-kilobase pair promoter that
lacked the enhancer was inactive. Thus, despite their close proximity, the genes
of the Mtx/Thbs3 gene cluster are regulated independently. A recently described protein, metaxin 1, serves as a component of a preprotein
import complex in the outer membrane of the mammalian mitochondrion. A yeast
two-hybrid screen with metaxin 1 as bait has now identified a novel protein,
which we have termed metaxin 2, as a metaxin 1-binding protein. Metaxin 2 shares
29% identity with metaxin 1 at the amino acid level, but metaxin 2, unlike
metaxin 1, lacks a C-terminal mitochondrial outer membrane signal-anchor domain.
Two C. elegans hypothetical proteins, CelZC97.1 and CelF39B2.i, share high
sequence similarity with metaxin 2 and metaxin 1, respectively, and likely
represent the C. elegans orthologs. Affinity-purified antibodies against metaxin
2 were prepared against the recombit protein produced in E. coli and were
used to analyze the subcellular distribution of metaxin 2. In subcellular
fractions of mouse liver, a 29 kD immunoreactive protein, consistent in size
with the predicted translation product of metaxin 2 cDNA, was found solely in
mitochondria. Alkali extraction of mitochondria indicated that metaxin 2 is
peripherally associated with mitochondrial membranes. Metaxin 2 in intact
mitochondria was susceptible to digestion with proteinase K, indicating that
metaxin 2 is located on the cytosolic face of the mitochondrial outer membrane.
Finally, baculoviruses encoding a His6-tagged metaxin 2 and an untagged metaxin
1 lacking its C-terminal transmembrane domain were produced and used separately
or in combination to infect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate
affinity column only in the presence of metaxin 2, indicating that metaxin 1 and
metaxin 2 interact when overexpressed in insect cells. These results suggest
that metaxin 2 is bound to the cytosolic face of the mitochondrial outer
membrane by means of its interaction with membrane-bound metaxin 1, and that
this complex may play a role in protein import into mammalian mitochondria. Metaxin is an outer membrane protein of mammalian mitochondria which is
suggested to be involved in protein import into the organelle. RNA blot analysis
showed that distribution of metaxin mRNA in human tissues differs from that of
mRNA for the translocase component Tom20. Effect of overexpression of human
metaxin on mitochondrial preprotein import and processing in COS-7 cells was
studied. Overexpression of metaxin resulted in impaired mitochondrial import of
natural and chimeric preproteins and in their accumulation. We previously
reported that overexpression of Tom20 in cultured cells causes inhibition of
import of mitochondrial preprotein. Coexpression of metaxin with Tom20 had no
further effect on the preprotein import. Overexpression of the cytosolic domain
of metaxin also caused inhibition of preprotein import, although less strongly
than the full-length metaxin. In blue native PAGE, Tom40, Tom22, and a portion
of Tom20 migrated as a complex of approximately 400 kDa, and the other portion
of Tom20 migrated in smaller forms of approximately 100 and approximately 40
kDa. On the other hand, metaxin migrated at a position of approximately 50 kDa.
These results confirm earlier in vitro results that metaxin participates in
preprotein import into mammalian mitochondria, and indicates that it does not
associate with the Tom complex. Voltage-dependent anion-selective channel (VDAC) is a beta-barrel protein in the
outer mitochondrial membrane that is necessary for metabolite exchange with the
cytosol and is proposed to be involved in certain forms of apoptosis. We studied
the biogenesis of VDAC in human mitochondria by depleting the components of the
mitochondrial import machinery by using RNA interference. Here, we show the
importance of the translocase of the outer mitochondrial membrane (TOM) complex
in the import of the VDAC precursor. The deletion of Sam50, the central
component of the sorting and assembly machinery (SAM), led to both a strong
defect in the assembly of VDAC and a reduction in the steady-state level of
VDAC. Metaxin 2-depleted mitochondria had reduced levels of metaxin 1 and were
deficient in import and assembly of VDAC and Tom40, but not of three
matrix-targeted precursors. We also observed a reduction in the levels of
metaxin 1 and metaxin 2 in Sam50-depleted mitochondria, implying a connection
between these three proteins, although Sam50 and metaxins seemed to be in
different complexes. We conclude that the pathway of VDAC biogenesis in human
mitochondria involves the TOM complex, Sam50 and metaxins, and that it is
evolutionarily conserved. A monoclonal antibody (mAb) has been produced which reacts with human mitofilin,
a mitochondrial inner membrane protein. This mAb immunocaptures its target
protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3,
CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins,
CHCHD3 has been assigned to the matrix space, and the other two proteins have
not been described in mitochondria previously. The functional role of this new
complex is uncertain. However, a role in protein import related to maintece
of mitochondrial structure is suggested as mitofilin helps regulate
mitochondrial morphology and at least four of the associated proteins (metaxins
1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11
is a chaperone-like protein that may have a similar role. The role of plant mitochondrial outer membrane proteins in the process of
preprotein import was investigated, as some of the principal components
characterized in yeast have been shown to be absent or evolutionarily distinct
in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria
were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN,
and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional
Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit
translocase of the outer membrane complex. Simultaneous inactivation of two of
the three TOM20 genes changed the rate of import for some precursor proteins,
revealing limited isoform subfunctionalization. Inactivation of all three TOM20
genes resulted in severely reduced rates of import for some but not all
precursor proteins. The outer membrane protein METAXIN was characterized to play
a role in the import of mitochondrial precursor proteins and likely plays a role
in the assembly of beta-barrel proteins into the outer membrane. An outer
mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity
to a chloroplast import receptor was shown to interact with a variety of
precursor proteins. All three proteins have domains exposed to the cytosol and
interacted with a variety of precursor proteins, as determined by pull-down and
yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted
in protein abundance changes in the others, suggesting functional redundancy.
Thus, it is proposed that all three components directly interact with precursor
proteins to participate in early stages of mitochondrial protein import. Translocator protein (18 kDa, TSPO), previously known as the peripheral-type
benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein
necessary for cholesterol import and steroid production. We reconstituted the
mitochondrial targeting and insertion of TSPO into the OMM to analyze the
signals and mechanisms required for this process. Initial studies indicated the
formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis.
The formation of this complex was found to be dependent on the presence of ATP
and the cytosolic chaperone Hsp90. Through mutational analysis we identified two
areas necessary for TSPO targeting, import, and function: amino acids 103-108
(Schellman motif), which provide the necessary structural orientation for
import, and the cholesterol-binding C-terminus required for insertion. Although
the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22
and Tom40 were present in the OMM, the TOM complex did not interact with TSPO.
In search of proteins involved in TSPO import, we analyzed complexes known to
interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was
found to be dependent on an identified protein, Metaxin 1, for formation and
TSPO import. The level of import of TSPO into steroidogenic cell mitochondria
was increased following treatment of the cells with cAMP. These findings suggest
that the initial targeting of TSPO to mitochondria is dependent upon the
presence of cytosolic chaperones interacting with the import receptor Tom70. The
C-terminus plays an important role in targeting TSPO to mitochondria, whereas
its import into the OMM is dependent upon the presence of the Schellman motif.
Final integration of TSPO into the OMM occurs via its interaction with Metaxin
1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP. |
What are the major classes of retrotransposons active in the human genome? | LINE-1 (L1), Alu, SVA | L1 (LINE-1), a long interspersed repetitive DNA family of mammalian genomes, is
thought to be a sequence family derived from a retrotransposon-like element(s),
but its actively transposable unit(s) has not been identified yet. We developed
a novel method for selective isolation of the human L1 sequences which
transposed in a relatively recent past and may have still retained a feature of
the 'active L1' unit. From the inspection of the nucleotide sequences, we
conjectured that the 'active L1' or 'nearly active L1' units should have a high
content of the CpG dinucleotide sequence, a mutation hot spot sequence, and
contain several sites for rare cutters such as BssH II and Nar I at their 5'
terminal regions. Using these rare cutter sites as selection markers, the L1
sequences were isolated, which had the high content of CpG at the 5' terminal
regions and over 90% homology to L1 transcripts found in a human teratocarcinoma
cell line. These L1s were shown to be 'relatively new L1' units which had
integrated into chromosomes within these several million years during evolution.
From the sequence data of these L1s and L1 cDNA, a consensus sequence of the 5'
terminal region of high CpG L1s were constructed. A region of the consensus
sequence showed about 69% homology to the 5' terminal region of Drosophila
jockey element. Several distinct families of endogenous retrovirus-like elements (ERVs) exist in
the genomes of primates. Despite the important evolutionary consequences that
carrying these intragenomic parasites may have for their hosts, our knowledge
about their evolution is still scarce. A matter of particular interest is
whether evolution of ERVs occurs via a master lineage or through several
lineages coexisting over long periods of time. In this work, the paleogenomic
approach has been applied to the study of the evolution of ERV9, one of the
human endogenous retrovirus families mobilized during primate evolution. By
searching the GenBank database with the first 676 bp of the ERV9 long terminal
repeat, we identified 156 different element insertions into the human genome.
These elements were grouped into 14 subfamilies based on several characteristic
nucleotide differences. The age of each subfamily was roughly estimated based on
the average sequence divergence of its members from the subfamily consensus
sequence. Determination of the sequential order of diagnostic substitutions led
to the identification of four distinct lineages, which retained their capacity
of transposition over extended periods of evolution. Strong evidence for mosaic
evolution of some of these lineages is presented. Taken altogether, the
available data indicate that the possibility of ERV9 still being active in the
human lineage can not be discarded. All autonomous non-long terminal repeat (non-LTR) retrotransposons reported to
date in vertebrates encode an apurinic/apyrimidinic endonuclease-like enzyme
necessary for target sequence cleavage and subsequent target-primed reverse
transcription. We describe here vertebrate non-LTR retrotransposons encoding
another type of endonuclease more related to type IIS restriction enzymes. Such
retrotransposons have been detected until now only in trypanosomes, nematodes,
and arthropods. The retrotransposon Rex6 was identified in the genome of several
teleost fish including Xiphophorus maculatus (platyfish), Oryzias latipes
(medakafish), Oreochromis niloticus (Nile tilapia), and Fugu rubripes (Japanese
pufferfish). Rex6 encodes a reverse transcriptase and a putative restriction
enzyme-like endonuclease and is a member of the R4 family of non-LTR
retrotransposons containing the Dong and R4 elements found in nematodes and
insects. Rex6 was active in many species during teleost evolution and underwent
several bursts of retrotransposition (some of them being relatively recent)
leading to a high copy number of Rex6 in the genome of numerous fish. Extremely
truncated Rex6-related sequences were detected by database screening in
reptiles, including the snake Trimeresus flavoviridis and the lizard Anolis
carolinensis, but not in sequences from the human genome project, suggesting
that this element might have been lost from certain vertebrate lineages. Long and short interspersed elements (LINEs and SINEs) are retroelements that
make up almost half of the human genome. L1 and Alu represent the most prolific
human LINE and SINE families, respectively. Only a few Alu elements are able to
retropose, and the factors determining their retroposition capacity are poorly
understood. The data presented in this paper indicate that the length of Alu
"A-tails" is one of the principal factors in determining the retropositional
capability of an Alu element. The A stretches of the Alu subfamilies analyzed,
both old (Alu S and J) and young (Ya5), had a Poisson distribution of A-tail
lengths with a mean size of 21 and 26, respectively. In contrast, the A-tails of
very recent Alu insertions (disease causing) were all between 40 and 97 bp in
length. The L1 elements analyzed displayed a similar tendency, in which the
"disease"-associated elements have much longer A-tails (mean of 77) than do the
elements even from the young Ta subfamily (mean of 41). Analysis of the draft
sequence of the human genome showed that only about 1000 of the over one million
Alu elements have tails of 40 or more adenosine residues in length. The presence
of these long A stretches shows a strong bias toward the actively amplifying
subfamilies, consistent with their playing a major role in the amplification
process. Evaluation of the 19 Alu elements retrieved from the draft sequence of
the human genome that are identical to the Alu Ya5a2 insert in the NF1 gene
showed that only five have tails with 40 or more adenosine residues. Sequence
analysis of the loci with the Alu elements containing the longest A-tails (7 of
the 19) from the genomes of the NF1 patient and the father revealed that there
are at least two loci with A-tails long enough to serve as source elements
within our model. Analysis of the A-tail lengths of 12 Ya5a2 elements in diverse
human population groups showed substantial variability in both the Alu A-tail
length and sequence homogeneity. On the basis of these observations, a model is
presented for the role of A-tail length in determining which Alu elements are
active. Alu elements are the most successful SINEs (Short INterspersed Elements) in
primate genomes and have reached more than 1,000,000 copies in the human genome.
The amplification of most Alu elements is thought to occur through a limited
number of hyperactive "master" genes that produce a high number of copies during
long evolutionary periods of time. However, the existence of long-lived,
low-activity Alu lineages in the human genome suggests a more complex
propagation mechanism. Using both computational and wet-bench approaches, we
reconstructed the evolutionary history of the AluYb lineage, one of the most
active Alu lineages in the human genome. We show that the major AluYb lineage
expansion in humans is a species-specific event, as nonhuman primates possess
only a handful of AluYb elements. However, the oldest existing AluYb element
resided in an orthologous position in all hominoid primate genomes examined,
demonstrating that the AluYb lineage originated 18-25 million years ago. Thus,
the history of the AluYb lineage is characterized by approximately 20 million
years of retrotranspositional quiescence preceding a major expansion in the
human genome within the past few million years. We suggest that the evolutionary
success of the Alu family may be driven at least in part by "stealth-driver"
elements that maintain low retrotranspositional activity over extended periods
of time and occasionally produce short-lived hyperactive copies responsible for
the formation and remarkable expansion of Alu elements within the genome. HERV-H family is the most abundant HERV families in the human genome with more
than 1000 copies including full-length, truncated form, and solitary LTRs. We
investigated envelope (env) gene fragments of HERV-H family in various human
tissues and cancer cells. The env fragments were detected in mRNA of several
human tissues (placenta, skeletal muscle, spleen, and thymus) and cancer cells
(RT4, BT-474, HCT-116, TE-1, UO-31, Jurkat, HepG2, A549, MCF7, OVCAR-3,
MIA-PaCa-2, PC3, LOX-IMVI, AZ521, 2F7, U-937, and C-33A) by RT-PCR approach. The
RT-PCR products were cloned and sequenced. New 12 clones from human tissues and
48 clones from cancer cells of env gene sequences belonging to the HERV-H family
showed 84.3-98.1% sequence similarity to that of HERV-H (AF108843). Deduced
amino acid sequences of 60 clones from human tissues and cancer cell lines
showed multiple frameshifts and termination codons caused by deletion/insertion
or point mutation with the exception of eight clones as following: HHE9-1,
HHE9-5 (skeletal muscle), HHE10-5 (spleen), CHE10-9 (MCF7), CHE12-4, CHE12-5
(MIA-PaCa-2), and CHE18-1, CHE18-3 (C-33A) to that of HERV-H (AF108843). A
phylogenetic tree of the HERV-H family was constructed to understand their
relationship, indicating that they were divided into three groups, one major
(group I) and two minor (group II and III), through sequence divergence. The
HERV-H families in group I has been proliferated on human genome during hominoid
evolution. These active HERV-H elements are worthy of further investigations as
potential pathogenic effects to various human diseases including cancers. A potential relationship between transposon-derived repeats (TDR) and human
germline methylation is of biological importance since many genes are flanked by
TDR and methylation could affect the expression of nearby genes. Furthermore,
DNA methylation has been suggested as a global defense mechanism against genome
instability threatened by TDR. We studied the correlation between the density of
HapMap methyl-associated SNPs (mSNPs), a marker of germline methylation, and
proportion of TDR. After correcting for confounding variables, we found a
negative correlation between proportion of Alu repeats and mSNP density for
125-1000 kb windows. Similar results were found for the most active subgroup of
repeats. In contrast, a negative correlation between proportion of L1 repeats
and mSNP density was found only in the larger 1000 kb windows. Using methylation
data on germ cells (sperm) from the Human Epigenome Project, we found a lower
proportion of Alu repeats adjacent (3-15 kb) to hypermethylated amplicons. On
the contrary, there was a higher proportion of L1 repeats in the 3-5 kb of
sequence flanking hypermethylated amplicons but not in the 10-15 kb flanks. Our
data indicate a differential response to the major repeat families and that DNA
methylation is unlikely to be a uniform global defense system against all TDR.
It appears to play a role for the L1 subgroup, with sequences adjacent to L1
repeats methylated in response to their proximity. In contrast, sequences
adjacent to Alu repeats appear to be hypomethylated, arguing against a role of
methylation in germline defense against those elements. BACKGROUND: Cancer is a significant and growing problem worldwide. While this
increase may, in part, be attributed to increasing longevity, improved case
notifications and risk-enhancing lifestyle (such as smoking, diet and obesity),
hygiene-related factors resulting in immuno-regulatory failure may also play a
major role and call for a revision of vaccination strategies to protect against
a range of cancers in addition to infections.
DISCUSSION: Human endogenous retroviruses (HERVs) are a significant component of
a wider family of retroelements that constitutes part of the human genome. They
were originated by the integration of exogenous retroviruses into the human
genome millions of years ago. HERVs are estimated to comprise about 8% of human
DNA and are ubiquitous in somatic and germinal tissues.Physiologic and
pathologic processes are influenced by some biologically active HERV families.
HERV antigens are only expressed at low levels by the host, but in circumstances
of inappropriate control their genes may initiate or maintain pathological
processes. Although the precise mechanism leading to abnormal HERVs gene
expression has yet to be clearly elucidated, environmental factors seem to be
involved by influencing the human immune system.HERV-K expression has been
detected in different types of tumors.Among the various human endogenous
retroviral families, the K series was the latest acquired by the human species.
Probably because of its relatively recent origin, the HERV-K is the most
complete and biologically active family.The abnormal expression of HERV-K
seemingly triggers pathological processes leading to melanoma onset, but also
contributes to the morphological and functional cellular modifications
implicated in melanoma maintece and progression.The HERV-K-MEL antigen is
encoded by a pseudo-gene incorporated in the HERV-K env-gene. HERV-K-MEL is
significantly expressed in the majority of dysplastic and normal naevi, as well
as other tumors like sarcoma, lymphoma, bladder and breast cancer. An amino acid
sequence similar to HERV-K-MEL, recognized to cause a significant protective
effect against melanoma, is shared by the antigenic determits expressed by
some vaccines such as BCG, vaccinia virus and the yellow fever virus.HERV-K are
also reactivated in the majority of human breast cancers. Monoclonal and
single-chain antibodies against the HERV-K Env protein recently proved capable
of blocking the proliferation of human breast cancer cells in vitro, inhibiting
tumor growth in mice bearing xenograft tumors.
SUMMARY: A recent epidemiological study provided provisional evidence of how
melanoma risk could possibly be reduced if the yellow fever virus vaccine (YFV)
were received at least 10 years before, possibly preventing tumor initiation
rather than culling melanoma cells already compromised. Further research is
recommended to confirm the temporal pattern of this protection and
eliminate/attenuate the potential role of relevant confounders as socio-economic
status and other vaccinations.It appears also appropriate to examine the
potential protective effect of YFV against other maligcies expressing high
levels of HERV-K antigens, namely breast cancer, sarcoma, lymphoma and bladder
cancer.Tumor immune-therapy, as described for the monoclonal antibodies against
breast cancer, is indeed considered more complex and less advantageous than
immune-prevention. Cellular immunity possibly triggered by vaccines as for YFV
might also be involved in anti-cancer response, in addition to humoral immunity. |
Which are the clinical characteristics of Diamond-Blackfan anemia? | Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplastic anemia that usually presents early in infancy and is characterized by red cell aplasia, congenital anomalies, and a predisposition to cancer. | Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplastic anemia
that usually presents early in infancy and is inherited in up to 45% of cases.
It is characterized by red cell aplasia, congenital anomalies, and a
predisposition to cancer. Corticosteroids and red blood cell transfusions are
the mainstays of therapy. We describe a case of 3-month-old infant who presented
with severe anemia, elevated levels of HbF and adenosine deaminase and bilateral
hydronephrosis, who was later confirmed as DBA by mutation analysis using the
direct sequencing method. Direct sequencing analysis of RPS19 gene was performed
with both cDNA and genomic DNA extracted from peripheral blood and a c.3G>A
point mutation of exon 2 resulting in p.Met1Ile was identified in this patient.
The patient showed an inadequate response to steroid therapy and a partial
response to RBC transfusion with a follow-up Hb level of 8.3 g/dL on her last
visit to the outpatient clinic. DBA is a genetically and phenotypically
heterogeneous disease, and we have reviewed the clinical characteristics of 25
Korean patients thus far reported in the literature. To our knowledge, this is
the first case of DBA confirmed by mutation analysis in Korea, and mutation
identification using molecular method is recommended for confirmation of this
genetically and phenotypically heterogeneous disease. In order to explore the diagnosis and therapy of Diamond Blackfan anemia (DBA),
the clinical data of 45 cases of DBA admitted in our hospital from February 1994
to July 2011 were analyzed retrospectively. The clinical characteristics,
results of laboratory examination, treatment reaction and outcome of disease
were investigated. The results indicated that out of 45 children diagnosed as
DBA, 14 cases (31.1%) had short stature and physical malformation. All patients
had anemia with reticulocytopenia. Thirty-four patients (75.6%) had mean
corpuscular volume. Eleven patients (24.4%) had macrocytic anemia. Bone marrow
examination showed a marked erythroid hypoplasia in all patients. Out of 29
cases tested for fetal hemoglobin (HbF), 13 cases (44.8%) had high level of HbF.
Erythroid colony-forming unit of bone marrow was tested in 25 patients, among
them 12 patients (48%) showed normal plasia, 13 (52%) showed hypoplasia. The
erythropoietin (EPO) levels of 17 patients were elevated. Karyotypes were
examined in 28 patients, and showed all normal. The treatment was based on
corticosteroids and Cyclosporine A. Thirty patients had good response to
corticosteroid therapy, and 10 of them obtained a sustained
corticosteroid-induced remission. Twenty cases discontinued corticosteroid
therapy after remission, as a result, 15 cases (75%) relapsed, moreover all the
relapsed cases still had good response to corticosteroid. Two relapsed patients
suffered from aplastic anemia, one of them died of therapy failure. Six patients
were unresponsive to corticosteroid, 1 of which achieved remission with
cyclosporine A and the others continued to receive regular transfusions. 3
patients received iron chelation therapy. It is concluded that the clinical
characteristics, complete blood count, bone marrow smear, HbF level and EPO
level are useful to make a diagnosis of DBA. Most patients have a good response
to corticosteroid therapy, but relapse rate is high when drug was discontinued.
Patients unresponsive to corticosteroid should receive regular transfusions and
chelation therapy. Diamond Blackfan Anemia (DBA) is a rare hypoplastic anemia that presents in
infancy with macrocytic anemia and reticulocytopenia. It is a ribosomopathy with
autosomal domit inheritance. In our series of 10 patients with DBA,
congenital malformations were observed in 50% of the cases. Age at symptom onset
ranged from 0-12 months. Age at diagnosis ranged from 4 months to 96 months.
Male: female ratio was 9:1. Response to prednisolone was observed in 4 out of
the 10 patients (either during initial treatment or during re-challenge).
Response to cyclosporine was found to be poor. Bone marrow transplantation was
successful in attaining remission in one patient. Maligcies were not reported
in any patient possibly due to a short follow up period. Diamond-Blackfan anemia is a rare, inherited disease that characteristically
presents as a chronic, normochromic macrocytosis due to red cell lineage bone
marrow failure. Although studies are elaborating on the genetic basis for its
associated comorbidities, little has been published comparing this anemia to
other chronic anemias that have similar laboratory results in children. This
article offers a global perspective of the disease and compares it with anemia
due to vitamin B12 and folate deficiency in children. |
How can the expression of SerH3 immobilization antigen be regulated? | The expression of Tetrahymena surface proteins serotype H3 (SerH3) is under temperature regulation. SerH3 is expressed when cells are incubated between the temperatures of 20 and 35 degrees C. | In ciliates, only one of the alternative forms of the immunodomit membrane
glycoprotein usually coats the external surface of the cell. Such mutual
exclusion is regulated at the pretranslational level by mechanisms that result
in the expression of a single protein gene. In the holotrich Tetrahymena
thermophila five alternative cell surface immobilization proteins (i-antigens)
are expressed under different conditions of temperature (L, H, T) and culture
media (I, S). Using polyclonal and monoclonal antibodies to these proteins and a
cDNA probe derived from the SerH3 gene, we have reinvestigated expression of
i-antigens in media supplemented with 0.2 M NaCl. We find that in addition to S,
the H and L antigens are also present on the cell surface. While all three
i-antigens may be simultaneously present on the cell surface, the combinations
S/L and S/H are more frequent. Compared to cells expressing H and L singly, the
level of H3 mRNA is diminished, and a subset of the L family of polypeptides is
variably expressed. The expression of S begins within 30 min after transfer to
NaCl-supplemented medium, while the expression of L begins three days to several
weeks after transfer. When cells are transferred out of NaCl-supplemented
medium, S is turned off within 24 h, and L is expressed for at least 1 wk prior
to the return of full H expression. Although these differences in kinetics
suggest differences in control mechanism(s), the absence of I and T on the
surface of NaCl-grown cells suggests that there is also a common regulatory link
among H, S and L.(ABSTRACT TRUNCATED AT 250 WORDS) The DNA sequences of a cDNA clone and the macronuclear genomic fragment
corresponding to the functional copy of the SerH3 surface antigen gene of
Tetrahymena thermophila were determined. Primer extension and nuclease
protection assays show that the SerH3 transcription unit is 1,425 nucleotides
long and contains no introns. The predicted polypeptide encoded by the SerH3
gene has a molecular mass of 44,415 daltons; one-third of its 439 residues are
either cysteine, serine, or threonine. The central half of the polypeptide
consists of three homologous domains in tandem array; within these domains, the
cysteine, proline, and tryptophan residues occur in highly regular patterns. The expression of Tetrahymena surface proteins serotype H3 (SerH3) and serotype
T (SerT) is under environmental regulation. SerH3 is expressed when cells are
incubated between the temperatures of 20 and 35 degrees C, while SerT is
expressed when cells are grown at temperatures above 35 degrees C. Using a SerH3
cDNA clone as a hybridization probe, we determined that (i) the SerH3 gene is a
member of a multigene family; (ii) most members of this multigene family are
variably rearranged during macronuclear development; and (iii) the gene which
produces the SerH3 mRNA is reproducibly rearranged during macronuclear
development. Synthesis of the serotype H3 (SerH3) surface antigen is temperature dependent
and responds within 1 h to a change in incubation conditions (G.A. Bannon, R.
Perkins-Dameron, and A. Allen-Nash, Mol. Cell. Biol. 6:3240-3245, 1986).
Recently, a Tetrahymena thermophila cDNA clone (pC6; D.W. Martindale and P.J.
Bruns, Mol. Cell. Biol. 3:1857-1865, 1983) has been shown to be homologous to a
portion of the SerH3 mRNA (F.P. Doerder and R.L. Hallberg, personal
communication), and it was shown that the cellular levels of this RNA rapidly
decreased when cells were shifted from 30 to 41 degrees C (R.L. Hallberg, K.W.
Kraus, and R.C. Findly, Mol. Cell. Biol. 4:2170-2179, 1984). These observations
indicate that synthesis of the SerH3 protein is highly regulated in response to
temperature and led us to initiate studies to determine the mechanism(s) by
which SerH3 gene expression is controlled. Using pC6 as a hybridization probe
for the SerH3 mRNA, we have determined that (i) the level of SerH3 protein
synthesis is directly correlated with the amount of SerH3 message available for
translation; (ii) there is, at most, a twofold difference between the relative
transcription rates of SerH3 genes at 30 and 40 degrees C; (iii) the SerH3 mRNA
half-life in cells incubated at 30 degrees C is greater than 1 h, whereas the
half-life in cells incubated at 40 degrees C is only approximately 3 min. These
results demonstrate that Tetrahymena SerH3 surface protein expression is
regulated by mRNA abundance. Furthermore, the major mechanism controlling mRNA
abundance is a dramatic temperature-dependent change in SerH3 mRNA stability. In Tetrahymena thermophila, the expression of a temperature-specific surface
protein known as SerH3 is primarily controlled by a temperature-dependent change
in the stability of the mRNA that encodes this protein. At 30 degrees C the
SerH3 mRNA displays a half-life of 60 minutes while at 40 degrees C the
half-life decreases to only 3 minutes. We used a Tetrahymena mutant cell line
(rseB) defective in expression of SerH3 at 30 degrees C to explore the
mechanisms involved in temperature-dependent mRNA stability. The results of in
vitro nuclear run-off assays and Northern and slot blot analysis of cytoplasmic
and nuclear RNAs show that the rseB locus encodes a temperature-sensitive
product that has no effect on SerH3 gene transcription or the steady-state
levels of SerH3 nuclear RNA. However, the product of this locus does have a
dramatic effect on cytoplasmic levels of the SerH3 mRNA at 30 degrees C,
indicating that SerH3 gene expression is affected post-transcriptionally within
the cytoplasm. To explore the possibility that the rseB locus controls SerH3
mRNA stability we developed an in vitro mRNA decay assay. This assay
successfully duplicates the differential decay of the SerH3 mRNA observed in
wild-type cells grown at different temperatures. The apparent half-life of the
SerH3 mRNA in cytoplasmic extracts derived from cells grown at 30 degrees C is
approximately 45 minutes while in cytoplasmic extracts derived from cells grown
at 40 degrees C it is only 6 minutes. When similar experiments are performed
using extracts prepared from the Tetrahymena rseB cell line, we find that the
SerH3 mRNA is only stable in extract prepared from cells grown under conditions
in which the mRNA accumulates to detectable levels in the cytoplasm. These
results indicate that the product of the rseB locus is a trans-acting
cytoplasmic factor that exerts its effect on SerH3 gene expression by regulating
SerH3 mRNA stability. The SerH locus of Tetrahymena thermophila is one of several paralogous loci with
genes encoding variants of the major cell surface protein known as the
immobilization antigen (i-ag). The locus is highly polymorphic, raising
questions concerning functional equivalency and selective forces acting on its
multiple alleles. Here, we compare the sequences and expression of SerH1, SerH3,
SerH4, SerH5, and SerH6. The precursor i-ags are highly similar. They are rich
in alanine, serine, threonine, and cysteine and they share nearly identical ER
translocation and GPI addition signals. The locations of the 39 cysteines are
highly conserved, particularly in the 3.5 central, imperfect tandem repeats in
which 8 periodic cysteines punctuate alternating short and long stretches of
amino acids. Hydrophobicity patterns are also conserved. Nevertheless, amino
acid sequence identity is low, ranging from 60.7 to 82.9%. At the nucleotide
level, from 9.7 to 26.7% of nucleotide sites are polymorphic in pairwise
comparisons. Expression of each allele is regulated by temperature-sensitive
mRNA stability. H mRNAs are stable at <36 degrees but are unstable at >36
degrees. The H5 mRNA, which is less affected by temperature, has a different
arrangement of the putative mRNA destabilization motif AUUUA. Statistical
analysis of SerH genes indicates that the multiple alleles are neutral.
Significantly low ratios of the rates of nonsynonymous to synonymous amino acid
substitutions suggest that the multiple alleles are subject to purifying
(negative) selection enforcing constraints on structure. |
Which factors play a role in promoter proximal pausing of RNA polymerase II? | NELF (negative elongator factor) and DSIF (DRB Sensitivity Inducing Factor) | NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in
extracts from human cells. A multifaceted approach was taken to investigate the
potential role of these factors in promoter proximal pausing on the hsp70 gene
in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced
the level of polymerase that paused in the promoter proximal region of hsp70.
Depletion of one NELF subunit in salivary glands using RNA interference also
reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed
that NELF and DSIF associate with the promoter region before heat shock.
Immunofluorescence analysis of polytene chromosomes corroborated the
cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa
colocalize at the hsp70 genes, small heat shock genes, and many other
chromosomal locations. Finally, following heat shock induction, DSIF and
polymerase but not NELF were strongly recruited to chromosomal puffs harboring
the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the
promoter proximal region of hsp70. The transcriptional activator, HSF, might
cause NELF to dissociate from the elongation complex. DSIF continues to
associate with the elongation complex and could serve a positive role in
elongation. The transcription elongation factor
5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing
factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in
concert with negative elongation factor (NELF), promoter-proximal pausing of
RNAPII. DSIF is also reportedly involved in transcriptional activation. However,
the role of DSIF in transcriptional activation by DNA-binding activators is
unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator,
Gal4-VP16, to promote transcriptional activation. In the absence of DSIF,
Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during
elongation in vitro. The presence of DSIF reduced pausing, thereby supporting
Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects
within a short time-frame from initiation to elongation, and that NELF does not
affect the positive regulatory function of DSIF. Knockdown of the gene encoding
the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced
Gal4-VP16-mediated activation of a reporter gene, but had no effect on
expression in the absence of activator. Together, these results provide evidence
that higher-level transcription has a stronger requirement for DSIF, and that
DSIF contributes to efficient transcriptional activation by preventing RNAPII
pausing during transcription elongation. The eukaryotic transcription elongation factor
5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing
factor (DSIF), is involved in regulating the processivity of RNA polymerase II.
DSIF plays also a role in transcriptional activation, and in concert with the
negative elongation factor NELF causes promoter proximal pausing of RNA
polymerase II. Furthermore, DSIF has also been implicated in regulating the
transcription of the human immunodeficiency virus proviral DNA. Human DSIF is
composed of the two subunits, hSpt4 (p14) and hSpt5 (p160), corresponding to the
yeast homologs Spt4 and Spt5. Here we show the purification and characterization
of the small subunit, hSpt4. We were able to purify the protein in a soluble
form separately from the larger hSpt5 subunit. CD and NMR spectroscopy show that
the purified protein hSpt4 exhibits an alpha/beta topology with a well defined
tertiary structure. Furthermore metal analysis by ICP-OES indicates that the
protein contains a functional 4-Cys Zn-finger. The eukaryotic transcription elongation factor DSIF [DRB
(5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor]
is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast
factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA
polymerase II and plays an essential role in transcriptional activation of
eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative
elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In
the present paper we describe the crystal structure of hSpt4 in complex with the
dimerization region of hSpt5 (amino acids 176-273) at a resolution of 1.55 A (1
A=0.1 nm). The heterodimer shows high structural similarity to its homologue
from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to
the NTD (N-terminal domain) of the bacterial transcription factor NusG. A
homologue for hSpt4 has not yet been found in bacteria. However, the archaeal
transcription factor RpoE" appears to be distantly related. Although a
comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity
of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4
could not be observed by NMR titration experiments. A conserved glutamate
residue, which was shown to be crucial for dimerization in yeast, is also
involved in the human heterodimer, but is substituted for a glutamine residue in
Escherichia coli NusG. However, exchanging the glutamine for glutamate proved
not to be sufficient to induce hSpt4 binding. Negative elongation factor (NELF) and
5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor
(DSIF) are involved in pausing RNA Polymerase II (Pol II) in the
promoter-proximal region of the hsp70 gene in Drosophila, before heat shock
induction. Such blocks in elongation are widespread in the Drosophila genome.
However, the mechanism by which DSIF and NELF participate in setting up the
paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF,
and a reconstituted Drosophila Pol II elongation complex to gain insight into
the mechanism of pausing. Our results show that DSIF and NELF require a nascent
transcript longer than 18 nt to stably associate with the Pol II elongation
complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of
DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex.
Taken together, these results provide a possible model by which DSIF binds the
elongation complex via association with the nascent transcript and subsequently
recruits NELF. Although DSIF and NELF were both required for inhibition of
transcription, we did not detect a NELF-RNA contact when the nascent transcript
was between 22 and 31 nt long, which encompasses the region where
promoter-proximal pausing occurs on many genes in Drosophila. This raises the
possibility that RNA binding by NELF is not necessary in promoter-proximal
pausing. Divergent transcription occurs at the majority of RNA polymerase II (RNAPII)
promoters in mouse embryonic stem cells (mESCs), and this activity correlates
with CpG islands. Here we report the characterization of upstream antisense
transcription in regions encoding transcription start site associated RNAs
(TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and
Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5'
termini and heterogeneous nonpolyadenylated 3' ends. uaRNAs are short-lived with
average half-lives of 18 minutes and are present at 1-4 copies per cell,
approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs.
These uaRNAs are probably initiation products because their capped termini
correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are
associated with these antisense polymerases and their sense partners. Knockdown
of either NELF or DSIF results in an increase in the levels of uaRNAs.
Consistent with P-TEFb controlling release from pausing, treatment with its
inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with
similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in
transcriptional activity in sense and antisense directions. Together these data
show divergent polymerases are regulated after P-TEFb recruitment with uaRNA
levels controlled by the exosome. The expression of many metazoan genes is regulated through controlled release of
RNA polymerase II (Pol II) that has paused during early transcription
elongation. Pausing is highly enriched at genes in stimulus-responsive pathways,
where it has been proposed to poise downstream targets for rapid gene
activation. However, whether this represents the major function of pausing in
these pathways remains to be determined. To address this question, we analyzed
pausing within several stimulus-responsive networks in Drosophila and discovered
that paused Pol II is much more prevalent at genes encoding components and
regulators of signal transduction cascades than at inducible downstream targets.
Within immune-responsive pathways, we found that pausing maintains basal
expression of critical network hubs, including the key NF-κB transcription
factor that triggers gene activation. Accordingly, loss of pausing through
knockdown of the pause-inducing factor NELF leads to broadly attenuated immune
gene activation. Investigation of murine embryonic stem cells revealed that
pausing is similarly widespread at genes encoding signaling components that
regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude
that the role of pausing goes well beyond poising-inducible genes for activation
and propose that the primary function of paused Pol II is to establish basal
activity of signal-responsive networks. Elongation is becoming increasingly recognized as a critical step in eukaryotic
transcriptional regulation. Although traditional genetic and biochemical studies
have identified major players of transcriptional elongation, our understanding
of the importance and roles of these factors is evolving rapidly through the
recent advances in genome-wide and single-molecule technologies. Here, we focus
on how elongation can modulate the transcriptional outcome through the
rate-liming step of RNA polymerase II (Pol II) pausing near promoters and how
the participating factors were identified. Among the factors we describe are the
pausing factors--NELF (negative elongation factor) and DSIF (DRB
sensitivity-inducing factor)--and P-TEFb (positive elongation factor b), which
is the key player in pause release. We also describe the high-resolution view of
Pol II pausing and propose nonexclusive models for how pausing is achieved. We
then discuss Pol II elongation through the bodies of genes and the roles of FACT
and SPT6, factors that allow Pol II to move through nucleosomes. |
Which are the main clinical features of Fanconi anemia? | Fanconi anaemia (FA) is an autosomal recessive disease characterised by congenital abnormalities, defective haemopoiesis, and increased risk of malignancies. | Features of chromosomal aberrations, hypersensitivity to DNA crosslinking
agents, and predisposition to maligcy have suggested a fundamental anomaly of
DNA repair in Fanconi anemia. The function of the recently isolated FACC
(Fanconi anemia group C complementing) gene for a subset of this disorder is not
yet known. The notion that FACC plays a direct role in DNA repair would predict
that the polypeptide should reside in the nucleus. In this study, a polyclonal
antiserum raised against FACC was used to determine the subcellular location of
the polypeptide. Immunofluorescence and subcellular fractionation studies of
human cell lines as well as COS-7 cells transiently expressing human FACC showed
that the protein was localized primarily to the cytoplasm under steady-state
conditions, transit through the cell cycle, and exposure to crosslinking or
cytotoxic agents. However, placement of a nuclear localization signal from the
simian virus 40 large tumor antigen at the amino terminus of FACC directed the
hybrid protein to the nuclei of transfected COS-7 cells. These observations
suggest an indirect role for FACC in regulating DNA repair in this group of
Fanconi anemia. Fanconi anemia (FA) consists of a group of at least five autosomal recessive
disorders that share both clinical (e.g., birth defects and hematopoietic
failure) and cellular (e.g., sensitivity to cross-linking agents and
predisposition to apoptosis) features with each other. However, a common
pathogenetic link among these groups has not been established. To identify
genetic pathways that are altered in FA and characterize shared molecular
defects, we used mRNA differential display to isolate genes that have altered
expression patterns in FA cells. Here, we report that the expression of an
interferon-inducible gene, MxA, is highly upregulated in cells of FA
complementation groups A, B, C, and D, but it is suppressed in FA group C cells
complemented with wild-type FAC cDNA as well as in non-FA cells. A
posttranscriptional mechanism rather than transcriptional induction appears to
account for MxA overexpression. Forced expression of MxA in Hep3B cells enhances
their sensitivity to mitomycin C and induces apoptosis, similar to the FA
phenotype. Thus, MxA is a downstream target of FAC and is the first genetic
marker to be identified among multiple FA complementation groups. These data
suggest that FA subtypes converge onto a final common pathway, which is
intimately related to the interferon signaling mechanism. Constitutive activity
of this pathway may explain a number of the phenotypic features of FA,
particularly the pathogenesis of bone marrow failure. Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer
predisposition. Bone marrow failure resulting in pancytopenia is the main cause
of death of FA patients. Diagnosis of FA is based on their cellular
hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic
complementation experiments suggest the involvement of at least eight genes in
FA. The gene for complementation group A (FANCA) is defective in the majority of
FA patients. We show here that mice deficient of FANCA: are viable and have no
detectable developmental abnormalities. The hematological parameters showed a
slightly decreased platelet count and a slightly increased erythrocyte mean cell
volume in mice at young age, but this did not progress to anemia. Consistent
with the clinical phenotype of FA patients, both male and female mice showed
hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the
knock-out mice exhibited spontaneous chromosomal instability and were
hyper-responsive to the clastogenic effect of the crosslinker mitomycin C. Fanconi anaemia (FA) is an autosomal recessive disease characterised by
congenital abnormalities, defective haemopoiesis, and a high risk of developing
acute myeloid leukaemia and certain solid tumours. Chromosomal instability,
especially on exposure to alkylating agents, may be shown in affected subjects
and is the basis for a diagnostic test. FA can be caused by mutations in at
least seven different genes. Interaction pathways have been established, both
between the FA proteins and other proteins involved in DNA damage repair, such
as ATM, BRCA1 and BRCA2, thereby providing a link with other disorders in which
defective DNA damage repair is a feature. This review summarises the clinical
features of FA and the natural history of the disease, discusses diagnosis and
management, and puts the recent molecular advances into the context of the
cellular and clinical FA phenotype. Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked
inheritance, features diverse clinical symptoms, such as progressive bone marrow
failure, hypersensitivity to DNA cross-linking agents, chromosomal instability
and susceptibility to cancer. At least 12 genetic subtypes have been described
(FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked
to a distinct gene. Most FA proteins form a complex that activates the FANCD2
protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream
of this step. The FA proteins typically lack functional domains, except for
FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an
E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking
agents, the FA proteins are thought to function in the repair of DNA interstrand
cross-links, which block the progression of DNA replication forks. Here we
present a hypothetical model, which not only describes the assembly of the FA
pathway, but also positions this pathway in the broader context of DNA
cross-link repair. Finally, the possible role for the FA pathway, in particular
FANCF and FANCB, in the origin of sporadic cancer is discussed. 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. Fanconi anaemia (FA) is a rare, autosomal recessive, genetically complex, DNA
repair deficiency syndrome in man. Patients with FA exhibit a heterogeneous
spectrum of clinical features. The most significant and consistent phenotypic
characteristics are stem cell loss, causing progressive bone marrow failure and
sterility, diverse developmental abnormalities and a profound predisposition to
neoplasia. To date, 15 genes have been identified, biallelic disruption of any
one of which results in this clinically defined syndrome. It is now apparent
that all 15 gene products act in a common process to maintain genome stability.
At the molecular level, a fundamental defect in DNA repair underlies this
complex phenotype. Cells derived from FA patients spontaneously accumulate
broken chromosomes and exhibit a marked sensitivity to DNA-damaging
chemotherapeutic agents. Despite complementation analysis defining many
components of the FA DNA repair pathway, no direct link to DNA metabolism was
established until recently. First, it is now evident that the FA pathway is
required to make incisions at the site of damaged DNA. Second, a specific
component of the FA pathway has been identified that regulates nucleases
previously implicated in DNA interstrand crosslink repair. Taken together, these
data provide genetic and biochemical evidence that the FA pathway is a bona fide
DNA repair pathway that directly mediates DNA repair transactions, thereby
elucidating the specific molecular defect in human Fanconi anaemia. Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia,
a disorder characterized by sensitivity to DNA interstrand crosslinking agents.
The clinical features of cytopenia, developmental defects, and tumor
predisposition are similar in each group, suggesting that the gene products
participate in a common pathway. The Fanconi anemia DNA repair pathway consists
of an anchor complex that recognizes damage caused by interstrand crosslinks, a
multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several
downstream repair proteins including nucleases and homologous recombination
enzymes. We review progress in the use of structural and biochemical approaches
to understanding how each FANC protein functions in this pathway. |
Which is the receptor for the immunosuppressive drug cyclosporin A (CsA)? | Cyclophilin is the intracellular receptor protein for cyclosporin A (CsA). | Cyclosporine mediates its immunosuppressive effect by preventing the synthesis
of lymphokine mRNA during the process of T lymphocyte activation. Although the
detailed molecular mechanism by which CsA achieves this effect is unknown, two
proteins have been identified as putative intracellular CsA-receptor proteins.
One of these, calmodulin, is an important Ca++-binding protein and enzyme
cofactor and the other, cyclophilin, is a novel protein that is reported to have
protein kinase activity. In this study the CsA-binding capacity of both these
proteins has been assessed using CsA-coated ELISA plates and CsA-affinity gel
matrices. CsA binding was shown by cyclophilin whereas no CsA-calmodulin binding
could be detected under identical conditions. However, it was not possible to
demonstrate any cyclophilin-associated protein kinase activity. Jurkat cells
were probed for the presence of CsA-binding proteins using the CsA-affinity gel
matrix; a 17 KD protein, most probably cyclophilin, was identified as the major
CsA-binding protein. In addition, a previously unidentified CsA-binding 45 KD
phosphoprotein was precipitated from 32P-labeled Jurkat cells. These results
would support cyclophilin as the major, if not only, intracellular receptor
protein for CsA. However, the relationship between binding of CsA to cyclophilin
and/or the 45 KD phosphoprotein and the immunosuppressive effects of CsA is
still unknown. BACKGROUND: Since its isolation in 1970, and discovery of its potent inhibitory
activity on T-cell proliferation, cyclosporin A (CsA) has been shown to play a
significant role in diverse fields of biology. Furthermore, chemical
modification of CsA has led to analogs with distinct biological activities
associated with its protein receptor family, cyclophilins.
SCOPE OF REVIEW: This review systematically collates the synthetic chemistry
performed at each of the eleven amino acids, and provides examples of the
utility of such transformations. The various modifications of CsA are traced
from early, modest chemistry performed at the unique Bmt residue, through the
remarkable use of a polyanion enolate that can be stereoselectively manipulated,
and onto application of more recently developed olefin metathesis chemistry to
prepare new CsA derivatives with unexpected biological activity.
MAJOR CONCLUSIONS: The myriad biological activities of CsA and its synthetic
derivatives have inspired the development of new approaches to modify the CsA
ring. In turn, these new CsA derivatives have served as tools in the discovery
of new roles for cyclophilins.
GENERAL SIGNIFICANCE: This review provides information on the types of
cyclosporin derivatives that are available to the many biologists working in
this field, and should be of value to the medicinal chemist trying to discover
drugs based on CsA. This article is part of a Special Issue entitled
Proline-directed foldases: Cell signaling catalysts and drug targets. |
Which drugs may interfere thyroxine absorption? | bile acid sequestrants, ferrous sulphate, sucralfate, calcium carbonate, aluminium-containing antacids, phosphate binders, raloxifene and proton-pump inhibitors, have also been shown to interfere with the absorption of levothyroxine
sevelamer hydrochloride or chromium picolinate should be advised to separate the time of ingestion of these drugs from their thyroid hormone preparation by several hours | A patient with hypothyroidism who was euthyroid on a fixed-dosage, long-term
maintece regimen of levothyroxine sodium developed persistently elevated
serum thyrotropin levels while receiving an aluminum hydroxide-containing
antacid. The thyrotropin levels returned to normal shortly after cessation of
the antacid therapy. These observations indicate that aluminum hydroxide may
interfere with the bioavailability of thyroxine. The thyroid function of
patients who are receiving replacement or suppressive thyroxine therapy should
be monitored following the commencement of concurrent treatment with medications
containing aluminum hydroxide. CONTEXT: The effect of calcium carbonate on the absorption of levothyroxine has
not been studied systematically. Such a potential drug interaction merits
investigation because concurrent treatment with both drugs is common,
particularly in postmenopausal women.
OBJECTIVE: To investigate the potential interference of calcium carbonate in the
absorption of levothyroxine.
DESIGN: Prospective cohort study conducted from November 1998 to June 1999,
supplemented with an in vitro study of thyroxine (T(4)) binding to calcium
carbonate.
SETTING: Veterans Affairs Medical Center in West Los Angeles, Calif.
PATIENTS: Twenty patients (age range, 27-78 years; n=11 men) with hypothyroidism
who were taking a stable long-term regimen of levothyroxine were included in the
study. All patients had serum free T(4) and thyrotropin values in the normal
range before beginning the study.
INTERVENTION: Subjects were instructed to take 1200 mg/d of elemental calcium as
calcium carbonate, ingested with their levothyroxine, for 3 months.
MAIN OUTCOME MEASURES: Levels of free T(4), total T(4), total triiodothyronine
(T(3)), and thyrotropin, measured in all subjects at baseline (while taking
levothyroxine alone), at 2 and 3 months (while taking calcium carbonate and
levothyroxine), and 2 months after calcium carbonate discontinuation (while
continuing to take levothyroxine).
RESULTS: Mean free T(4) and total T(4) levels were significantly reduced during
the calcium period and increased after calcium discontinuation. Mean free T(4)
levels were 17 pmol/L (1.3 ng/dL) at baseline, 15 pmol/L (1.2 ng/dL) during the
calcium period, and 18 pmol/L (1.4 ng/dL) after calcium discontinuation (overall
P<.001); mean total T(4) levels were 118 nmol/L (9.2 microg/dL) at baseline, 111
nmol/L (8.6 microg/dL) during the calcium period, and 120 nmol/L (9.3 microg/dL)
after calcium discontinuation (overall P=.03). Mean thyrotropin levels increased
significantly, from 1.6 mIU/L at baseline to 2.7 mIU/L during the calcium
period, and decreased to 1. 4 mIU/L after calcium discontinuation (P=.008).
Twenty percent of patients had serum thyrotropin levels higher than the normal
range during the calcium period; the highest observed level was 7.8 mIU/L. Mean
T(3) levels did not change during the calcium period. The in vitro study of T(4)
binding to calcium showed that adsorption of T(4) to calcium carbonate occurs at
acidic pH levels.
CONCLUSIONS: This study of 20 patients receiving long-term levothyroxine
replacement therapy indicates that calcium carbonate reduces T(4) absorption and
increases serum thyrotropin levels. Levothyroxine adsorbs to calcium carbonate
in an acidic environment, which may reduce its bioavailability. JAMA.
2000;283:2822-2825 The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on
triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured
cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C
in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM).
The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6);
[125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4
uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of
[125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with
1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The
relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM
verapamil or nifedipine was less than after 15 min or 1 h, indicating that the
major inhibitory effect of the Ca2+ channel blockers occurred at the level of
the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM
verapamil or nifedipine was proportional to the reduction of cellular [125I]T3
uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake
but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested.
Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium
affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel
blockers on T3 uptake in cardiomyocytes are not secondary to their effects on
Ca2+ influx but, rather, reflect interference with the putative T3 carrier in
the plasma membrane. To our knowledge, raloxifene hydrochloride, a selective estrogen receptor
modulator, has never been reported to interfere with absorption of
levothyroxine. We describe a 79-year-old woman with chronic, treated primary
hypothyroidism, presenting with increasing levothyroxine requirement while
taking raloxifene at the same time as levothyroxine. For two 6- to 8-week
periods, we separated the ingestion of raloxifene and levothyroxine by about 12
hours. In addition, we tested the absorption of 1.0 mg of levothyroxine sodium
with and without the coadministration of 60 mg of raloxifene hydrochloride on 2
separate occasions by collecting serial blood samples for 6 hours.
Hypothyroidism occurred in a reproducible fashion whenever levothyroxine and
raloxifene were administered together and improved whenever they were taken
separately. Combined administration of levothyroxine and raloxifene resulted in
lower levels of serum thyroxine compared with administration of levothyroxine
alone. By a yet unknown mechanism, raloxifene caused malabsorption of
levothyroxine in our patient when coadministered. The comparative bioavailability of oral doses of levothyroxine (LT(4))
formulation taken as tablets, after being crushed, or chewed before swallowing
has not been well studied. Three patients with hypothyroidism who showed
persistent elevation of serum thyrotropin (TSH) despite taking 200, 150, and 125
microg of LT(4) tablets per day are presented. They did not show signs and
symptoms of gastrointestinal illness that could interfere with the absorption of
LT(4) nor history of such a condition. They did not concurrently take
medications known to affect the absorption of LT(4) from the gut. Their serum
TSH levels normalized when the tablets were taken after being pulverized. The
difference appeared attributable to the slow dissolution of the tablets in the
gut of these patients. Studies in hypothyroid rats show that, when infused with a combination of
thyroxine (T4) plus triiodothyronine (T3) to normalize thyrotropin (TSH),
euthyroidism in all organs is only ensured when T(4) and T(3) are administered
in a ratio as normally secreted by the rat thyroid. As substitution with
T(4)-only results in an abnormal serum T(4)/T(3) ratio, it is also possible that
in humans, euthyroidism does not exist at the tissue level in many organs,
considering that iodothyronine metabolism in the human and the rat share many
similar mechanisms. Recent reports in which cognitive function and well-being
are compared in patients with primary hypothyroidism substituted with T(4)-only
versus substitution with T(4) plus T(3) result in controversial findings in that
either positive or no effects were found. In all these studies T(3) was used in
the plain form that results in nonphysiologic serum T(3) peaks. In these studies
it is suggested that substitution with T(3 )should preferably be performed with
a preparation that slowly releases T(3) to avoid these peaks. In the study
reported here we show that treatment of hypothyroid subjects with a combination
of T(4) plus slow-release T(3) leads to a considerable improvement of serum T(4)
and T(3) values, the T(4)/T(3) ratio and serum TSH as compared to treatment with
T(4)- only. Serum T(3) administration with slow-release T(3) did not show serum
peaks, in contrast to plain T(3). Thyroid hormones (THs) are involved in the occurrence of anxiety and affective
disorders; however, the effects following an anxiolytic benzodiazepine
treatment, such as diazepam administration, on the mechanism of action of
thyroid hormones has not yet been investigated. The effect of diazepam on the in
vitro nuclear T3 binding, on the relative expression of the TH receptors (TRs)
and on the synaptosomal TH availability were examined in adult rat cerebral
hemispheres 24 h after a single intraperitoneal dose (5 mg/kg BW) of this
tranquillizer. Although, diazepam did not affect the availability of TH either
in blood circulation or in the synaptosomal fraction, it decreased (33%) the
nuclear T3 maximal binding density (B(max)). No differences were observed in the
equilibrium dissociation constant (K(d)). The TRalpha2 variant (non-T3-binding)
mRNA levels were increased by 33%, whereas no changes in the relative expression
of the T3-binding isoforms of TRs (TRalpha1, TRbeta1) were observed. This study
shows that a single intraperitoneal injection of diazepam affects within 24 h,
the density of the nuclear TRs and their expression pattern. The latest effect
occurs in an isoform-specific manner involving specifically the TRalpha2 mRNA
levels in adult rat brain. BACKGROUND: Oral iodized poppy seed oil is an appropriate measure for
controlling iodine deficiency in areas where iodized salt is not yet available.
However, a more effective and cheaper iodized oil preparation is needed.
OBJECTIVE: The aim of this study was to compare the efficacy of iodized peanut
oil with that of iodized poppy seed oil.
DESIGN: Schoolchildren aged 8-10 y were supplemented with a single oral dose of
iodized peanut oil (P200, P400, or P800 mg I), iodized poppy seed oil (PS400 mg
I), or peanut oil (placebo). The concentration of urinary iodine (UI) was
measured at 0, 4, 12, 25, and 50 wk, whereas thyroid volume and serum
thyrotropin and free thyroxine concentrations were measured at 0, 25, and 50 wk.
RESULTS: UI was higher in all treatment groups than in the placebo group, except
at baseline. UI in the P200 group was not significantly different from that in
the PS400 group at all times of measurement. In a comparison of preparations
supplying 400 mg I conducted by using a mathematical model, iodine retention
from the peanut oil preparation was 3 times that from the poppy seed oil, and
the protection period for peanut oil was twice as long as that for the poppy
seed oil (P < 0.001 for both). The reduction in thyroid volume was greater in
the treatment groups than in the placebo group (P < 0.001). No significant
differences in serum hormone concentrations were observed between groups before
or after treatment.
CONCLUSION: Iodized peanut oil is more efficacious in controlling iodine
deficiency than is iodized poppy seed oil containing the same amount of iodine. INTRODUCTION: Thyroxine supplementation of patients with hypothyroidism is
usually simple. A few patients, however, continue to present elevated TSH levels
despite large doses of L-thyroxine.
CASE: We report the case of a 71-year-old women who had had a thyroidectomy 10
years earlier and had since been hospitalized repeatedly for profound
hypothyroidism. Despite her consistent claims of good adherence to her treatment
regimen, we considered the diagnosis of L-thyroxine pseudomalabsorption and
confirmed it by thyroid hormone absorption tests.
DISCUSSION: L-thyroxine pseudomalabsorption due to concealed poor treatment
adherence should be considered after ruling out drug or dietary interference and
true organic malabsorption. Diagnosis of this factitious disease can be
confirmed by L-thyroxine absorption tests. BACKGROUND: Dosage of T(4) in central hypothyroidism is primarily guided by the
free serum T(4) level (fT4). However, the optimum fT4 range is ill defined, and
subtle hypothyroidism might be missed using this approach.
OBJECTIVES: Our aim was to investigate the effects of a body weight (bw)-adapted
T(4) treatment, alone or in combination with T(3), on metabolism, well-being,
and cognitive function in comparison to a regimen leading to normal fT4.
DESIGN: This was a placebo-controlled trial (double-blind, crossover).
PATIENTS: A total of 29 patients (age 52 +/- 2 yr; females/males, 8/21) with
hypopituitarism, including TSH deficiency, participated in the study.
INTERVENTIONS: Three regimens were compared (5 wk each): "EMPIRICAL-T4,"
empirical T(4) dosage (1 +/- 0.05 microg/kg bw) leading to normal fT4;
BW-ADAPTED-T4 (1.6 microg/kg bw T(4)); and "BW-ADAPTED-T3T4," bw-adapted
combination of T(3) and T(4) (ratio of 1:10).
RESULTS: BW-ADAPTED-T4 administration increased mean fT4 concentrations to the
upper limit of the normal range (peak levels). Compared with EMPIRICAL-T4,
BW-ADAPTED-T4 treatment resulted in a lower body mass index (BMI) (29.0 +/- 0.7
vs. 29.5 +/- 0.7 kg/m(2); P < 0.03), lower total cholesterol (198 +/- 9 vs. 226
+/- 7 mg/dl; P < 0.01), and lower low-density lipoprotein (LDL) cholesterol (116
+/- 5 vs. 135 +/- 7 mg/dl; P < 0.01). BW-ADAPTED-T3T4 treatment was associated
with additional beneficial effects on ankle reflex time and working memory but
resulted in supraphysiological free serum T(3) (fT(3)) levels.
LIMITATIONS: Long-term side effects may have been missed.
CONCLUSIONS: Using a dose of 1.6 microg/kg bw improved markers commonly
associated with central hypothyroidism. This suggests that T(4) dosage based on
bw and aiming at fT4 in the upper reference range is superior to titration of
T(4) aiming at middle normal fT4 concentrations in those patients. OBJECTIVE: Medications may sometimes interfere with the intestinal absorption of
levothyroxine, primarily by forming an insoluble complex with the thyroid
hormone in the intestinal lumen. The goal of this study was to examine the acute
effects of three previously unstudied medications on levothyroxine absorption.
DESIGN: We studied the effects of three medications on thyroxine absorption in
seven normal volunteers. On each study day, the subjects ingested 1 mg
levothyroxine sodium, either taken separately or co-administered with sevelamer
hydrochloride (Renagel, a phosphate-binding medication used in the treatment of
hyperphosphatemia), chromium picolinate (an over-the-counter nutritional
supplement), or ezetimibe (Zetia, a drug used in the treatment of
hypercholesterolemia). Serum thyroxine was measured at intervals over a 6-hour
period following drug ingestion.
MAIN OUTCOME: Sevelamer hydrochloride and chromium picolinate each significantly
(p < 0.05) decreased the area under the serum thyroxine concentration curve,
while ezetimibe had no effect.
CONCLUSION: Hypothyroid patients taking sevelamer hydrochloride or chromium
picolinate should be advised to separate the time of ingestion of these drugs
from their thyroid hormone preparation by several hours. Absorbed thyroid dose and effective half-life were determined in 46 hyperthyroid
cats after treatment with a low dose (mean 111MBq) of radioiodine intravenously.
Thirteen of these cats had received iohexol for glomerular filtration rate (GFR)
measurement within 24h before treatment with radioiodine in view of another
ongoing study at our institution. Pre-therapy values were obtained for total
thyroxine (TT(4)) and for the thyroid to salivary gland ratio with sodium
pertechnetate gamma-camera imaging. All cats underwent post-therapy scans at 24,
48 and 120 h for evaluation of radioactive iodine uptake (RAIU) and the
effective half-life of radioiodine. The absorbed dose was calculated from the
cumulative activity with Olinda software. Both groups were comparable in age,
TT(4) and the ratio of thyroid activity to salivary gland activity. Statistical
analysis revealed a significant decreased absorbed dose in the thyroid in the
iohexol group. This decreased uptake was not accompanied by an decreased
effective half-life of the radioiodine. The variation of inter-individual RAIU
decreased in this group and more homogenous absorbed doses were obtained. No
significant difference in outcome could be demonstrated. However, a tendency
towards a higher number of residual hyperthyroidism in the iohexol group was
noted (15 versus 6% in control group). This study demonstrates that iohexol
interferes with the uptake of radioiodine in the hyperthyroid cat but does not
provoke increased turnover. In this study, albeit including a small number of
cats, outcome did not seem to be significantly affected. BACKGROUND: FDA Guidance for pharmacokinetic (PK) testing of levothyroxine
(L-T(4)) for interbrand bioequivalence has evolved recently. Concerns remain
about efficacy and safety of the current protocol, based on PK analysis
following supraphysiological L-T(4) dosing in euthyroid volunteers, and recent
recalls due to intrabrand manufacturing problems also suggest need for further
refinement. We examine these interrelated issues quantitatively, using simulated
what-if scenarios testing efficacy of a TSH-based protocol and tablet stability
and absorption, to enhance precision of L-T(4) bioequivalence methods.
METHODS: We use an updated simulation model of human thyroid hormone regulation
quantified and validated from data that span a wide range of normal and abnormal
thyroid system function. Bioequivalence: We explored a TSH-based protocol, using
normal replacement dosing in simulated thyroidectomized patients, switching
brands after 8 weeks of full replacement dosing. We simulated effects of tablet
potency differences and intestinal absorption differences on predicted plasma
TSH, T(4), and triiodothyronine (T(3)) dynamics. Stability: We simulated effects
of potency decay and lot-by-lot differences in realistic scenarios, using actual
tablet potency data spanning 2 years, comparing the recently reduced 95-105%
FDA-approved potency range with the original 90-110% range.
RESULTS: A simulated decrease as small as 10-15% in L-T(4) or its absorption
generated TSH concentrations outside the bioequivalence target range (0.5-2.5
mU/L TSH), whereas T(3) and T(4) plasma levels were maintained normal. For a 25%
reduction, steady-state TSH changed 300% (from 1.5 to 6 mU/L) compared with <25%
for both T(4) and T(3) (both within their reference ranges). Stability: TSH,
T(4), and T(3) remained within normal ranges for most potency decay scenarios,
but tablets of the same dose strength and brand were not bioequivalent between
lots and between fresh and near-expired tablets.
CONCLUSIONS: A pharmacodynamic TSH-measurement bioequivalence protocol, using
normal L-T(4) replacement dosing in athyreotic volunteers, is likely to be more
sensitive and safer than current FDA Guidance based on T(4) PK. The tightened
95-105% allowable potency range for L-T(4) tablets is a significant improvement,
but otherwise acceptable potency differences (whether due to potency decay or
lot-by-lot inconsistencies) may be problematic for some patients, for example,
those undergoing high-dose L-T(4) therapy for cancer. Persistent elevation of TSH levels in patients under treatment for
hypothyroidism is a relatively common clinical problem in endocrinology
practice. The most common cause for this phenomenon is poor patient compliance
with their thyroid hormone tablets. In the compliant patient, however, multiple
aetiologies are possible and a methodological and stepwise approach to the
patient's problem will uniformly identify a cause, or at least a resolution. To analyse the influence of thyroid status on the effect of aluminium (Al) upon
intestinal calcium (Ca) absorption, adult male Wistar rats with experimentally
altered thyroid hormones circulating levels, were orally treated (o.g.) with 0
(control), or 50 mg elemental Al (as chloride)/kg body weight (b.w.) per day,
for a 14 d period. Hyper- and hypo-thyroid conditions were respectively achieved
by means of administration of either sodium levothyroxine (50 microg/kg b.w. per
day, o.g.) or methimazole, a thyroxine synthesis inhibitor (1mg/kg b.w. per day,
o.g.). In duodenum-jejunum segments, in vitro mucosa-to-serosa (45)Ca flux
(JCa(ms)) and kinetics of (45)Ca uptake in isolated enterocytes, were
determined. In serum, concentrations of thyroxine (T4) and triiodothyronine (T3)
were measured by chemiluminescent enzyme immunoassay. Unlike non-Al-treated
rats, JCa(ms) of Al-exposed rats decreased as serum levels of T4 and T3
increased, showing a significant inverse correlation in both cases (T4:
r(2)=0.414, P=0.024; T3: r(2)=0.443, P=0.018). Enterocytes isolated from rats
treated with Al plus thyroxine showed a reduction of both maximum Ca uptake
(4.86+/-0.44 vs. 6.85+/-1.04 nmol Ca/mg protein, P<0.05) and K(m) (0.84+/-0.18
vs. 1.05+/-0.36 mM, P<0.05) when compared to control. The observed variability
in the Al effect on Ca transport with thyroid status of rats could be reflecting
a negative interaction of Al with thyroid hormone action mechanisms on
intestinal Ca absorption, which would take place mainly at Ca entry into
enterocyte from lumen. Food, dietary fibre and espresso coffee interfere with the absorption of
levothyroxine. Malabsorptive disorders reported to affect the absorption of
levothyroxine include coeliac disease, inflammatory bowel disease, lactose
intolerance as well as Helicobacter pylori (H. pylori) infection and atrophic
gastritis. Many commonly used drugs, such as bile acid sequestrants, ferrous
sulphate, sucralfate, calcium carbonate, aluminium-containing antacids,
phosphate binders, raloxifene and proton-pump inhibitors, have also been shown
to interfere with the absorption of levothyroxine. Forty bitches in anoestrus for more than six months from the last heat, with a
serum progesterone level less than 1 ng/ml were subjected to oestrus induction
trials using anti-prolactin drugs and levothyroxine, once daily orally for 20
consecutive days. The mean serum progesterone level among them was found to be
0.57 +/- 0.03 ng/ml. Out of 10 animals treated in each group, five (50%) in
Group I (bromocriptine @ 50 microg/kg body weight), nine (90%) in Group II
(cabergoline @ 5 microg/kg body weight), eight (80%) in Group III (thyroxine @10
microg/kg body weight) and seven (70%) in Group IV (thyroxine @ 5 microg/kg body
weight) responded by evincing proestrual bleeding. The mean (+/-SEM) time taken
from initiation of treatment to onset of proestrual bleeding in Groups I, II,
III and IV was 28 +/- 3.39, 13.44 +/- 3.12 (P < 0.05), 24.50 +/- 3.18 and 33 +/-
2.21 days respectively. The mean (+/-SEM) duration of proestrus and oestrus in
the treatment groups was 9.80 +/- 0.86, 10.11 +/- 0.68, 11.25 +/- 0.88 and 10.71
+/- 0.68 days and 7.60 +/- 0.24, 8 +/- 0.29, 8.5 +/- 0.63 and 7.85 +/- 0.46 days
respectively. The conception rate in relation to the number of animals
responding to oestrus induction in the treatment groups was 80%, 78%, 63% and
57%, respectively. The mean (+/-SEM) gestation length calculated from the last
breeding date and litter size in the treatment groups varied from 60.50 +/- 1.55
to 64.00 +/- 0.82 days and 5.14 +/- 0.34 to 6.40 +/- 0.40 respectively. BACKGROUND: Helicobacter pylori infection is a most frequent cause of chronic
gastritis. H. pylori may decrease absorption of oral thyroxine by decreasing
gastric acid secretion in the stomach. In this study, we aimed to investigate
the change in thyroid function tests of the cases after H. pylori eradication
who were not responding to high doses of thyroxine treatment before H. pylori
eradication.
METHODS: Hypothyroid cases who were not responding to high doses of thyroxine
among the ones presented to Endocrinology and Gastroenterohepatology Clinics of
Sisli Etfal Training and Research Hospital between 2009 and 2010 were included
in the study. Thyroid function tests were performed two times in all cases
before and after H. pylori eradication. Duodenal, antral and corporal biopsies,
and jejunal aspirates and biopsies were taken during upper gastrointestinal
system endoscopies performed in all patients. Cases without intestinal pathology
were included in the study.
RESULTS: Serum thyrotropin (TSH), free T3, and free T4 values before H. pylori
eradication were 30.5 ± 28.8 IU/mL, 2.64 ± 0.56 pg/mL, and 0.92 ± 0.32 ng/mL,
respectively, and after eradication were found to be 4.2 ± 10.6 IU/mL, 3.02 ±
0.61 pg/mL, and 1.3 ± 0.34 ng/mL, respectively (p values <.001, .002, and <.001,
respectively). After H. pylori eradication treatment, TSH decreased in all of
the cases, factitious thyrotoxicosis developed in % 21 of these cases.
CONCLUSION: In hypothyroid cases, H. pylori gastritis may be responsible for an
inadequate response to the treatment. H. pylori eradication in the cases
receiving high doses of thyroxine has a risk for thyrotoxicosis. OBJECTIVE: To assess the pharmacokinetic equivalence of a new soft capsule
formulation of levothyroxine versus a marketed reference product and to assess
the soft capsule formulated with stricter potency guidelines versus the capsule
before the implementation of the new potency rule.
METHOD: Two single-dose randomized two-way crossover pharmacokinetic equivalence
studies and one dosage form proportionality single-dose study comparing low,
medium, and high strengths of the new formulation. All three studies were
performed in a clinical setting. Participants were healthy male and female adult
subjects with normal levothyroxine levels. A total of 90 subjects participated
in the three studies.
RESULTS: Pharmacokinetic parameters were calculated on baseline- adjusted
concentrations. The first pharmacokinetic equivalence study compared the
levothyroxine sodium soft capsule formulation (Tirosint) with the reference
Synthroid tablets and the two products were considered bioequivalent. The dosage
form proportionality study compared the 50-, 100-, and 150-μg test capsules
strengths dosed at the same level (600 μg) and all three strengths were
considered equivalent when given at the same dosage. The last study compared the
test capsule used in the first two studies with a new capsule formulation
following the new potency guideline (±5%) set forward by the Food and Drug
Administration and the two capsules were considered bioequivalent. Doses were
well tolerated by subjects in all three studies with no serious adverse events
reported.
CONCLUSIONS: The levothyroxine soft capsule formulated with the stricter new
potency guideline set forward by the Food and Drug Administration met
equivalence criteria in terms of rate and extent of exposure under fasting
conditions to the reference tablet formulation. Clinical doses of the capsule
formulation can be given using any combination of the commercialized strengths. BACKGROUND: Roux-en-Y gastric bypass (RYGB) modifies the anatomical structure of
the upper intestine tract, reduces gastric acid secretion, and may impair LT4
absorption. The aim of this study was to evaluate the LT4 absorption in morbidly
obese patients before and after RYGB.
METHODS: Thirty morbidly obese patients were divided in two groups: The NS group
included 15 patients before RYGB surgery (BMI = 43.1 ± 4 kg/m(2)), and the S
group included 15 patients after surgery (BMI = 37.3 ± 4 kg/m(2)). Two baseline
samples were collected, and 600 μg of oral LT4 tablets were administered. Blood
samples were collected at 30, 60, 120, 180, 240, 300, and 1440 min. Serum-free
T4 (FT4), total T4 (TT4), and TSH were measured at each time point. The increase
in TT4, FT4, and TSH (ΔTT4, ΔFT4, and ΔTSH) was calculated, subtracting from the
baseline mean value.
RESULTS: The pharmacokinetics parameters regarding LT4 absorption, maximum ΔTT4,
and area under the curve(AUC) of both ΔTT4 and ΔFT4 were significantly higher in
the S group compared with the NS group (p < 0.05). It was observed, however,
that there was a significant delay in the absorption of LT4 in the S group.
Basal serum TSH and leptin levels were higher in the NS group (p = 0.016 and
0.026, respectively), whereas basal serum TT4, FT4, ΔTSH, and the AUC of ΔTSH
were similar between groups.
CONCLUSIONS: In this study, we have demonstrated that Roux-en-Y bypass surgery
does not diminish LT4 absorption. A small but significant delayed absorption of
LT4, however, was observed in patients after surgery. A model of thyroidectomized sheep intravenously supplemented with thyroid
hormone (TH) was developed to mimic endogenous TH exposure and to analyze the
impact on plasma TH homeostasis of xenobiotic interference with TH binding to
plasma proteins. TH was displaced from plasma protein binding sites by using
phenylbutazone (PBZ) as a test xenobiotic, to compare the effect of PBZ on
steady state free and total plasma TH concentrations between the in vivo
situation and an in vitro system. While PBZ increased free TH in vitro, PBZ
administration in vivo produced an immediate reduction in both total and free
plasma TH. The decrease in the total TH was consistent with a PBZ-induced
displacement of TH from its plasma binding proteins, leading to an increase in
total TH plasma clearance. However, this reduction in total TH was not expected
to be accompanied by a parallel decrease in free plasma TH since the free TH is
determined by the clearance of the free plasma TH. This suggested that PBZ may
also have interfered with the clearance mechanisms of free TH. It can be
concluded that our thyroidectomized sheep model enables a dual action of a
xenobiotic on plasma TH to be distinguished, namely a displacement of TH from
its binding proteins leading to a decrease in the total plasma concentration,
which is not relevant to thyroid function versus an interference with the
intrinsic TH clearance leading to a change in the free plasma TH, which has a
major impact in terms of thyroid disruption. The effects of aluminium (Al) on thyroid function were evaluated in adult Wistar
rats intraperitoneally (i.p) injected with 7 mg Al (as lactate)/kg body weight
(b.w) per day during a six week period. The time-course kinetics of Na(125)I (3
μCi per 100 g b.w, i.p) was analysed by measuring gamma-radioactivity of
thyroid, serum, serum protein precipitate and bile, at times ranging from 2 to
96 h post-dosing. In Al-treated group the (125)I(-) thyroid uptake at 24 h
(15,840 ± 570 vs. 18,030 ± 630 dpm/mg, P<0.05) as well as the rate of (125)I(-)
release from the gland, calculated as the slope of the plot between 24 and 96 h
(84 ± 8 vs. 129 ± 11 dpm/mg/h, P<0.05) were significantly reduced as compared to
control. The biliary (125)I(-) excretion was not modified at all studied times.
The Al content and lipid peroxidation (69.1 ± 8.5 vs. 53.2 ± 7.0 nmol MDA/g wet
weight, P<0.05) of thyroid tissue were increased in Al-treated rats. The serum
concentrations of total thyroxine (T4, 3.78 ± 0.14 vs. 4.68 ± 0.12 μg/dL,
P<0.05) and total triiodothyronine (T3, 47 ± 4 vs. 66 ± 5 ng/dL, P<0.05) were
decreased by effect of Al, but free-T4 (1.05 ± 0.05 vs. 1.04 ± 0.04 ng/dL, NS)
and thyrotropin (TSH, 2.7 ± 0.4 vs. 2.6 ± 0.5 ng/ml, NS) remain unchanged. In
spite of the Al could indirectly affect thyroid iodide uptake and hormones
secretion by a mechanism involving the induction of an oxidative stress state,
however, these changes could be managed by the hypothalamus-pituitary-thyroid
endocrine axis. We can conclude that in adult rats the Al would not act as a
thyroid disruptor. OBJECTIVE: Replacement T4 dose in hypothyroid patients bearing both chronic
autoimmune thyroiditis and atypical celiac disease (CD) has been analyzed.
DESIGN: Replacement T4 dose has been analyzed in 35 hypothyroid patients with
Hashimoto's thyroiditis (HT) and atypical CD, as defined by the American
Gastroenterological Association. We have evaluated the ability of the same dose
of T4 to reach target TSH in 21 patients before and during gluten-free diet
(GFD). In the remaining 14 patients, noncompliant with GFD, we analyzed
replacement T4 dose and compared it with that in a similar group consisting of
68 patients with hypothyroid HT but no evidence of celiac sprue or other
conditions interfering with T4 absorption.
RESULTS: In patients with isolated HT, the desired serum TSH (median=1.02
mU/liter) was reached in all patients after 5±2 months of treatment at a median
T4 dose of 1.31 μg/kg·d. After a similar period and dose of T4, higher levels of
TSH (median=4.20 mU/liter) were observed in patients with HT and CD. In 21 CD
patients, target TSH (median TSH=1.25 mU/liter) has been attained after 11±3
months of GFD without increasing T4 dose (1.32 μg/kg·d). In the remaining 14
patients, who were noncompliant with GFD, target TSH has also been achieved but
at a higher T4 dose (median=1.96 μg/kg·d; +49%; P=0.0002) than in hypothyroid
patients without CD.
CONCLUSIONS: Atypical CD increases the need for T4. The effect was reversed by
GFD or by increasing T4 dose. Malabsorption of T4 may provide the opportunity to
detect CD that was overlooked until the patients were put under T4 therapy. To better understand the pharmacokinetics and potential advantages of a
levothyroxine oral solution vs. tablets and soft gel capsules.4 randomized,
2-treatment, single-dose (600 mcg levothyroxine), 2-way crossover bioequivalence
studies in 84 healthy subjects were analyzed. Samples were collected before
dosing and until 48-72 h post-dose to calculate noncompartmental
baseline-adjusted pharmacokinetic parameters: maximum concentration, time to
maximum concentration, and area-under-the-concentration-time-curve from 0 to
48 h and from 0 to 2 h.Mean pharmacokinetic parameters (±standard deviation) for
tablets, capsules and solution, respectively, were:
area-under-the-concentration-time-curve from 0 to 2 h (ng*h/mL)=68.4±32.8,
64.4±24.4, 99.1±22.7; area-under-the-concentration-time-curve from 0 to 48 h
(ng*h/mL)=1 632±424, 1 752±445, 1 862±439; maximum concentration
(ng/mL)=67.6±20.9, 68.0±15.9, 71.4±16.0; time of maximum concentration
(hours)=2.25±0.99, 2.38±1.58, 1.96±1.07. Overall rate and extent of exposure
were not statistically different between formulations, but a faster onset of
absorption for the solution was suggested (greater
area-under-the-concentration-time-curve from 0 to 2 h and faster time to maximum
concentration by an average of 30 min).Levothyroxine rate and extent of exposure
are similar between tested formulations. The solution appears however to reach
systemic circulation quicker as dissolution is not needed before absorption
starts. The solution's greater early exposure and a faster time to maximal
concentration of around 30 min may be of benefit to minimize drug-food
interactions and deserves further investigations. CONTEXT: In the United States, generic substitution of levothyroxine (L-T(4)) by
pharmacists is permitted if the formulations are deemed to be bioequivalent by
the Federal Drug Administration, but there is widespread concern that the
pharmacokinetic standard used is too insensitive.
OBJECTIVE: We aimed to evaluate the bioequivalence of a brand-name L-T(4)
(Synthroid) and an AB-rated generic formulation (Sandoz, Princeton, NJ) in
children with severe hypothyroidism.
DESIGN: This was a prospective randomized crossover study in which patients
received 8 weeks of one L-T(4) formulation followed by 8 weeks of the other.
SETTING: The setting was an academic medical center.
PATIENTS: Of 31 children with an initial serum TSH concentration >100 mU/L, 20
had congenital hypothyroidism (CH), and 11 had autoimmune thyroiditis.
MAIN OUTCOME MEASURES: The primary endpoint was the serum TSH concentration.
Secondary endpoints were the free T(4) and total T(3) concentrations.
RESULTS: The serum TSH concentration was significantly lower after 8 weeks of
Synthroid than after generic drug (P = .002), but thyroid hormone levels did not
differ significantly. Subgroup analysis revealed that the difference in TSH was
restricted to patients with CH (P = .0005). Patients with CH required a higher
L-T(4) dose (P < .0004) and were younger (P = .003) but were not resistant to
thyroid hormone; 15 of 16 CH patients had severe thyroid dysgenesis or agenesis
on imaging. The response to generic vs brand-name preparation remained
significant when adjusted for age.
CONCLUSIONS: Synthroid and an AB-rated generic L-T(4) are not bioequivalent for
patients with severe hypothyroidism due to CH, probably because of diminished
thyroid reserve. It would therefore seem prudent not to substitute L-T(4)
formulations in patients with severe CH, particularly in those <3 yr of age. Our
results may have important implications for other severely hypothyroid patients
in whom precise titration of L-T(4) is necessary. |
Is Turcot syndrome associated with glioblastoma? | Yes, Turcot syndrome is associated with glioblastoma. Turcot syndrome is an autosomal recessive disorder clinically characterized by the occurrence of primary glial tumors of the central nervous system, including glioblastoma, and adenomatous colonic polyps during the first or second decades of life, with a spectrum of clinical features such as "café-au-lait" spots, axillary freckling, and hyperpigmented spots. | The authors describe two patients with the association of polyposis-coli and
central nervous system tumor (Turcot's syndrome). Clinical, genetic and
therapeutic aspects are discussed. We report a case of Turcot's syndrome in a 20-year old man with multiple
adenomatous polyps of the colon and glioblastoma multiforme. Detailed
histopathological study of all 25 polyps removed from his colon confirmed the
distinct morphological and numerical features of the colonic polyposis in
Turcot's syndrome. Moreover, 45% of the total polyps and all polyps with a
diameter exceeding 2 cm showed maligt transformation, indicating the
precancerous nature of these polyps. These findings are discussed together with
data obtained from a literature review of 32 histopathologically confirmed cases
of Turcot's syndrome with reference to the possible heterogeneous nature of the
syndrome at the present time. The current views on the relationship of Turcot's
syndrome to other polyposis coli syndromes are presented. The authors analyzed a family in which three descendants presented with
adenocarcinoma of the colon. In two of them the presence of colonic adenomatosis
was observed. Another family member, a 13-year-old girl, presented with Turcot
syndrome, that is, brain tumor associated with colonic adenomatosis. The nature
of the hereditary transmission of Turcot syndrome is hence analyzed, discussing
whether it happens through an autosomal recessive or a domit gene.
Undoubtedly the family has colonic adenomatosis, a disease considered of
autosomal domit transmission. Based on the clinical observation, the authors
suggest that Turcot syndrome may be determined by an autosomal gene with a
pleiotropic effect and variable expressivity. Another unusual autopsy case of the Turcot syndrome is reported in a 23-year-old
woman with polyposis coli, who developed primary carcinoma of the jejunum and
glioblastoma multiforme of the left frontal lobe. Previously documented cases
are reviewed. Discussion focuses on the occurrence of other extracolonic
abnormalities observed with the Turcot syndrome. The Turcot syndrome (TS) is a rare, probably autosomal recessive, disorder
characterized by development of primary neuroepithelial tumors of the central
nervous system (CNS) and numerous adenomatous colorectal polyps. To examine the
possible involvement of mutations of the APC gene, which is responsible for
familial adenomatous polyposis (FAP), in Turcot syndrome, we examined DNAs from
TS patients for alterations in this gene by means of ribonuclease protection
analysis. Germ-line APC mutations were detected in each of three unrelated cases
of TS, and additional (somatic) mutations were observed in colonic adenomas that
had developed in one of these patients. However, no somatic mutations in APC
were found among 91 neuroepithelial tumors (medulloblastoma, glioblastoma,
astrocytoma, and oligodendroglioma), whether sporadic or associated with TS.
These results suggest that the APC gene is associated with pathogenesis of one
feature of TS, but that at least one other gene is responsible for the genesis
of neuroepithelial tumors in the CNS. Tumors of the central nervous system (CNS) are common causes of morbidity and
mortality. These tumors can occur sporadically or in individuals with genetic
disorders predisposing to cancer development. Such syndromes include
neurofibromatosis type 2, neurofibromatosis type 1, Li-Fraumeni syndrome, as
well as von Hippel-Lindau disease, tuberous sclerosis, and Turcot syndrome.
There may also be familial syndromes resulting in glioma or meningioma alone,
but these are not well understood. Development of sporadic gliomas is
accompanied by a number of molecular genetic alterations, including activation
of domit oncogenes and inactivation of tumor suppressor genes. Some of these
alterations may be associated with progression of gliomas to their most
maligt form, glioblastoma multiforme. However, at this time molecular genetic
analysis of gliomas does not provide better prognosis than histopathological
staging. Recently, experimental treatments of gliomas in rodents, using gene
therapy, have been reported. Results of these studies have been promising, and
these techniques may represent a future direction for therapy in humans. We report on a 12-year-old patient with Turcot Syndrome (Glioma polyposis). This
patient's case deals with the association between a glioblastoma, anaplastic
glioma (WHO Grade III) and colonic adenocarcinoma based on familial polyposis
coli. Possible etiology and neurosurgical, clinically important characteristics
of this rare syndrome, such as the young age of the patient and the relatively
long survival time, will be discussed. In maligt gliomas, the characteristically heterogeneous features and frequent
diffuse spread within the brain have raised the question of whether maligt
gliomas arise monoclonally from a single precursor cell or polyclonally from
multiple transformed cells forming confluent clones. Although monoclonality has
been shown in surgically resected tissues, these may not include the full
spectrum of patterns seen on autopsy material. Little is known about the
clonality of low-grade gliomas from which maligt gliomas may sometimes arise.
We sought to investigate the clonality of low-grade and maligt gliomas by
using and comparing surgical and autopsy material with a Polymerase chain
reaction (PCR)-based assay for nonrandom X chromosome inactivation. For that,
purpose, archival surgical and autopsy material from 15 female patients (group
A) (age 4 to 73 years; median, 45) with maligt gliomas (12 glioblastomas, one
gliosarcoma, one anaplastic oligoastrocytoma, one gliomatosis cerebri), surgical
material only from 21 female patients (group S) (age 6 to 78 years; median, 60)
with low-grade and maligt gliomas (four low-grade astrocytomas, three
oligoastrocytomas, two anaplastic astrocytomas, one gemistocytic astrocytoma,
four oligodendrogliomas, seven glioblastomas) were analyzed. In group A,
representative areas (mean = 5/patient; median = 7) were microdissected from
tissue sections and assayed by PCR amplification of a highly polymorphic
microsatellite marker locus of the human androgen receptor gene (HUMARA) in the
presence of alpha32P with and without predigestion with a methylation-sensitive
restriction enzyme (HhaI). Products were resolved by denaturing gel
electrophoresis and autoradiographed. In group S, selected tumor areas were used
for the assay. Each patient's normal brain tissue was used for control. The band
intensity of alleles were measured by densitometric scanning. In group A, 13 of
15 cases were informative (heterozygous). The same pattern of nonrandom X
chromosome inactivation was present in all areas of solid dense and moderate
tumor infiltration in eight including all components of the gliosarcoma. Two of
eight also showed focal loss of heterozygosity (LOH). One of 13 presented global
LOH. Two of 13 showed microsatellite instability, one of which in a patient with
Turcot syndrome, the other in gliomatosis cerebri. Opposite skewing patterns
were seen in distant areas of gliomatosis cerebri consistent with oligoclonal
derivation. Clonality remained indeterminate in one glioblastoma and in the
anaplastic oligoastrocytoma because of skewed lyonization in the normal control.
In group S, 19 of 21 cases were informative. Fifteen of 19 were monoclonal (four
low-grade astrocytomas, one anaplastic astrocytoma, one gemistocytic
astrocytoma, two oligodendrogliomas, one oligoastrocytoma, six glioblastomas).
Four of 19 were indeterminate. We conclude that (1) Low-grade and maligt
gliomas are usually monoclonal tumors, and extensively infiltrating tumors must
result from migration of tumor cells (2) Gliomatosis cerebri may initiate as an
oligoclonal process or result from collision gliomas (3) Biphasic gliomas likely
arise from a single precursor cell. (4) LOH at the HUMARA locus is probably
related to partial or complete deletion of an X-chromosome, which occurs in
maligt gliomas during clonal evolution. The Turcot syndrome has been defined as the simultaneous presence of multiple
polyposis of the colon and a maligt brain tumor. This association is supposed
to be genetically transmitted, even though we still do not exactly know whether
this occurs in a domit or recessive way. The case of a 47-year-old man
submitted to a right hemicolectomy for cancer and polyposis, following a series
of endoscopic polypectomies and, finally, removal of left temporal glioma is
here presented. BACKGROUND: Turcot syndrome (TS) or the glioma-polyposis syndrome, is a rare,
heritable disorder thought by some authors to be a variant of familial
adenomatous polyposis (FAP). It is characterized by central nervous system (CNS)
neoplasms and gastrointestinal polyposis.
METHODS: We present a case report of a patient who developed a medulloblastoma
at age 5 years. Ten years later, she developed adenocarcinoma of the colon.
Seven months after resection of this Dukes' C2 adenocarcinoma, she presented
with a second primary CNS tumor, a glioblastoma multiforme. The patient's
colonic adenocarcinoma and glioblastoma were evaluated histologically and
cytogenetically.
RESULTS: Cytogenetic analysis revealed the presence of chromosomal instability
in both tumors. This unusual case of two primary CNS neoplasms in a patient with
TS is presented with a review of the literature.
CONCLUSIONS: The implications of the cytogenetic analysis are discussed in
conjunction with the present knowledge of the molecular biology of TS. Turcot syndrome is the association of colorectal polyposis with primary
neuroepithelial tumors of the central nervous system such as glioblastoma and
medulloblastoma. Including putative patients, more than 150 familial or sporadic
cases of the syndrome have been reported in literature. Since early reports,
there is considerable controversy regarding the modality of genetic transmission
and the distinction from other syndromes like familial adenomatous
polyposis(FAP). Recent molecular evidence suggests that Turcot syndrome could be
divided into the following two entities based on the distinct genetic
backgrounds. (1) True Turcot syndrome(autosomal recessive): Intestinal polyps
are less in number(< 100), large in size and apt to transform to the maligt
tumor. Brain tumor is mainly diagnosed as glioblastoma or astrocytoma and
mismatch repair genes might be involved. (2) FAP-associated type(autosomal
domit): Predisposing to medulloblastoma. Genetic testing for colon cancer: joint statement of the American College of
Medical Genetics and American Society of Human Genetics. Joint Test and
Technology Transfer Committee Working Group. Turcot's syndrome, clinically characterized by the coincident occurrence of
primary tumors of the colon and the central nervous system, can genetically be
divided into two syndromes: familial adenomatous polyposis (FAP) and hereditary
nonpolyposis colon carcinoma (HNPCC). In the present case, a 60-year-old patient
with glioblastoma multiforme and a history of hereditary malignomas is described
as an example of a HNPCC-associated Turcot's syndrome. New molecular biological
methods and results give deeper insight into clinical syndromes, and the better
understanding improves diagnostics, therapy, and outcome estimations, even in
rare diseases. In the present case, a new germinal mutation could be identified. Heterozygous mutations in one of the DNA mismatch repair genes cause hereditary
nonpolyposis colorectal cancer (MIM114500). Turcot syndrome (MIM276300) has been
described as the association of central nervous system maligt tumors and
familial colorectal cancer and has been reported to be both a domit and
recessive disorder. Homozygous and compound heterozygous mutations in APC, MLH1,
MSH2, and PMS2 genes have been reported in five families. Here we describe a
nonconsanguineous Pakistani family, including a son with lymphoma and colorectal
cancer diagnosed at ages 5 and 8, respectively, and an 8-year-old daughter with
glioblastoma multiforme. Both children had features of neurofibromatosis type 1
including atypical café au lait spots and axillary freckling without a family
history consistent with neurofibromatosis type 1, familial adenomatous
polyposis, or hereditary nonpolyposis colorectal cancer. Mutational analysis was
done for MLH1, MSH2, and MSH6 using denaturing high-performance liquid
chromatography and sequencing of a blood sample from the daughter. A novel
homozygous single base insertion mutation was identified (3634insT) resulting in
a premature stop at codon 1,223 in exon 7 of the MSH6 gene. Both parents were
found to be heterozygous for the 3634insT mutation. Microsatellite instability
testing showed instability in the glioblastoma sample. We report here the first
identification of a homozygous mutation in MSH6 in a family with childhood-onset
brain tumor, lymphoma, colorectal cancer, and neurofibromatosis type 1
phenotype. Our findings support a role for MSH6 in Turcot syndrome and are
consistent with an autosomal recessive mode of inheritance. We report the clinicopathological, genetic, and immunohistochemical
characterization of an atypical Turcot syndrome (TS) family with small bowel
cancer. The tumor family history of a patient with cafè-au-lait spots (CALS) and
early onset adenomas, duodenal cancer, and glioblastoma was positive for colonic
adenoma (mother), jejunal (maternal grandfather), lung (father), and colorectal
(paternal uncle) cancers. PMS2 genetic testing identified the nonsense 1951C>T
(Q643X) and the missense 161C>T (S46I) mutations. PMS2 expression was absent in
the proband's duodenal cancer with high microsatellite instability. The normal
cells also displayed no PMS2 expression and some degree of instability. Our
findings point out the association between PMS2 and TS, and support the
hypothesis that patients with a few polyps, small bowel tumors with a very early
onset, glioblastoma, and CALS should be considered as a variant of hereditary
nonpolyposis colorectal cancer. A recessive model of inheritance caused by
compound heterozygous mutations was consistent with the observed severe clinical
phenotype and has important implications for predicting cancer risk in both the
proband and his relatives. Familial glioblastoma multiforme is a rather uncommon entity, being in most
cases associated to known genetic disorders (as Turcot syndrome, Li-Fraumeni
syndrome, neurofibromatosis, etc.). However, familial gliomas have also been
described, although less frequently, independently of these genetic syndromes
showing some special features regarding its etiology and clinical
manifestations. Less than 10% of gliomas may be considered as true multicentric
tumours either synchronous or metachronous in clinical presentation.
Metachronous glioblastomas have been associated to better prognosis in some
studies, with genetic studies having found clear differences among the tumors
within same patients. Familial glioblastoma with metachronous presentation is an
exceptional disorder. These tumors show special therapeutic implications due to
the limitations of radiotherapy once the patient has already irradiated. A
variety of non-specific mutations have been found in these patients but true
characterization of this disorder remains unclear and will be based on further
genetic studies. We present a clinical report on a patient harbouring a familial
and metachronous glioblastoma. The main aspects of this entity are reviewed. Microsatellite instability (MSI) is present in hereditary conditions due to
mismatch repair (MMR) gene mutations. Following MSI analysis, tumor samples are
classified into MSS (stable), MSI-L (low instability), and MSI-H (high
instability) based on the fraction of unstable loci. Another MSI-based
classification takes into account the size difference between mutant alleles in
tumor DNA compared to wild-type alleles; two types of MSI, A and B, are
recognized using this approach, type A being characterized by smaller, more
subtle allelic shifts compared to type B. Biallelic mutations of MMR genes are
associated with pediatric cancers, including glial tumors, in Turcot syndrome
type 1 (TS1). However, most TS1-associated gliomas so far analyzed did not
display MSI. We investigated the frequency of MSI in a series of 34 pediatric
gliomas of different grade using a panel of five mononucleotide quasimonomorphic
markers. Subtle qualitative changes were observed for the majority of markers in
two glioblastomas (5.9% of the total series and 33.3% of glioblastomas). In both
cases, family histories were compatible with TS1, and mutations of the PMS2 and
MLH1 genes were identified. In one family, the MSI patterns were compared
between the glioblastoma and a colon cancer from an affected relative, showing a
clear qualitative difference, with the former displaying type A and the latter
type B instability, respectively. These results were confirmed using additional
microsatellite markers, indicating that knowledge of the association between
TS1-related glial tumors and subtle type A MSI is important for full
ascertainment of TS1 patients and appropriate counselling. Familial adenomatous polyposis (FAP) is characterized by the development of many
tens to thousands of adenomas in the rectum and colon during the second decade
of life. FAP has an incidence at birth of about 1/8,300, it manifests equally in
both sexes, and accounts for less than 1% of colorectal cancer (CRC) cases. In
the European Union, prevalence has been estimated at 1/11,300-37,600. Most
patients are asymptomatic for years until the adenomas are large and numerous,
and cause rectal bleeding or even anemia, or cancer develops. Generally, cancers
start to develop a decade after the appearance of the polyps. Nonspecific
symptoms may include constipation or diarrhea, abdominal pain, palpable
abdominal masses and weight loss. FAP may present with some extraintestinal
manifestations such as osteomas, dental abnormalities (unerupted teeth,
congenital absence of one or more teeth, supernumerary teeth, dentigerous cysts
and odontomas), congenital hypertrophy of the retinal pigment epithelium
(CHRPE), desmoid tumors, and extracolonic cancers (thyroid, liver, bile ducts
and central nervous system). A less aggressive variant of FAP, attenuated FAP
(AFAP), is characterized by fewer colorectal adenomatous polyps (usually 10 to
100), later age of adenoma appearance and a lower cancer risk. Some lesions
(skull and mandible osteomas, dental abnormalities, and fibromas on the scalp,
shoulders, arms and back) are indicative of the Gardner variant of FAP. Classic
FAP is inherited in an autosomal domit manner and results from a germline
mutation in the adenomatous polyposis (APC) gene. Most patients (~70%) have a
family history of colorectal polyps and cancer. In a subset of individuals, a
MUTYH mutation causes a recessively inherited polyposis condition,
MUTYH-associated polyposis (MAP), which is characterized by a slightly increased
risk of developing CRC and polyps/adenomas in both the upper and lower
gastrointestinal tract. Diagnosis is based on a suggestive family history,
clinical findings, and large bowel endoscopy or full colonoscopy. Whenever
possible, the clinical diagnosis should be confirmed by genetic testing. When
the APC mutation in the family has been identified, genetic testing of all
first-degree relatives should be performed. Presymptomatic and prenatal
(amniocentesis and chorionic villous sampling), and even preimplantation genetic
testing is possible. Referral to a geneticist or genetic counselor is mandatory.
Differential diagnoses include other disorders causing multiple polyps (such as
Peutz-Jeghers syndrome, familial juvenile polyps or hyperplastic polyposis,
hereditary mixed polyposis syndromes, and Lynch syndrome). Cancer prevention and
maintaining a good quality of life are the main goals of management and regular
and systematic follow-up and supportive care should be offered to all patients.
By the late teens or early twenties, colorectal cancer prophylactic surgery is
advocated. The recommended alternatives are total proctocolectomy and ileoanal
pouch or ileorectal anastomosis for AFAP. Duodenal cancer and desmoids are the
two main causes of mortality after total colectomy, they need to be identified
early and treated. Upper endoscopy is necessary for surveillance to reduce the
risk of ampullary and duodenal cancer. Patients with progressive tumors and
unresectable disease may respond or stabilize with a combination of cytotoxic
chemotherapy and surgery (when possible to perform). Adjunctive therapy with
celecoxib has been approved by the US Food and Drug Administration and the
European Medicines Agency in patients with FAP. Individuals with FAP carry a
100% risk of CRC; however, this risk is reduced significantly when patients
enter a screening-treatment program. BACKGROUND: Turcot syndrome (TS) is a rare genetic disorder of DNA mismatch
repair predisposing to glioblastoma (GBM) in the type 1 variant.
OBJECTIVE: We report the clinicopathological and genetic features of 3 gliomas
in TS type 1 patients.
METHODS: Three cases were reviewed from our clinical and pathology files at
Washington University with the diagnosis of TS 1 and GBM over the past 14 years.
All 3 had classic features of GBM, but also demonstrated bizarre multinucleated
giant cells and remarkably high mitotic indices. Sarcomatous regions were found
in 2. Despite these features, the patients had prolonged survival times of 44,
55, and >29 months (ie, currently alive). Demographic and clinical courses were
abstracted from retrospective chart review. Histopathology was reviewed from all
cases and reticulin histochemistry was added to identify possible foci of
sarcomatous differentiation.
RESULTS: All 3 had classic features of GBM, and Ki-67 labeling indices ranged
from 18 to 45%. All 3 also showed strong nuclear p53 positivity. Two cases were
negative for the isocitrate dehydrogenase 1 (IDH1) mutation, and O-Methylguanine
methyltransferase promoter methylation was seen in one. Fluorescence in situ
hybridization was done using 1p/1q, 19p/19q, centromere 7/epithelial growth
factor receptor (EGFR), and PTEN/DMBT1 probes. Focal EGFR amplification was seen
in one case, although other common alterations of either primary GBMs or gliomas
with prolonged survival (1p/19q codeletion) were lacking.
CONCLUSION: We conclude that 1) the giant cell variant of GBM is overrepresented
in TS; 2) gliosarcomas may also be encountered; and 3) survival is often
favorable, despite histological anaplasia and exuberant proliferation. A 15-year-old boy was admitted with the diagnosis of colonic polyposis, and
during a 2-year follow-up, he underwent operation for right parieto-occipital
anaplastic astrocytoma, left-side colonic non-Hodgkin lymphoma (NHL) and
cerebella glioblastoma which were all confirmed by histology. Although cases of
Turcot's syndrome (TS) (colonic polyposis and primary brain tumour occurring in
the same patient) have been previously described, association with
haematological maligcy is rare. We hereby report such a case with TS. Turcot syndrome (TS) is a rare hereditary disorder clinically characterized by
the occurrence of primary tumors of the colon and the central nervous system
(CNS). Here we present the case of an 11-year-old boy with a synchronous
clinical presentation of both glioblastoma multiforme (GBM) and colonic
adenocarcinoma. A molecular genetic study revealed microsatellite instability in
the DNA mismatch repair (MMR) gene. This patient ultimately survived for 13
months after clinical presentation. Based on this case study, the synchronous
presentation of glioblastoma multiforme and adenocarcinoma of the colon might
suggest a shorter survival rate for patients with Turcot syndrome. A literature
review complements this paper. A 13-year-old child presented with three simultaneous maligcies: glioblastoma
multiforme, Burkitt lymphoma, and colonic adenocarcinoma. She was treated for
her diseases without success and died 8 months after presentation. Genetic
analysis revealed a homozygous mutation in the PMS2 gene, consistent with
constitutional mismatch repair deficiency. Her siblings and parents were
screened: three of four siblings and both parents were heterozygous for this
mutation; the fourth sibling did not have the mutation. Lynch syndrome, familial adenomatous polyposis, and Mut Y homolog
(MYH)-associated polyposis are three major known types of inherited colorectal
cancer, which accounts for up to 5% of all colon cancer cases. Lynch syndrome is
most frequently caused by mutations in the mismatch repair genes MLH1, MSH2,
MSH6, and PMS2 and is inherited in an autosomal domit manner. Familial
adenomatous polyposis is manifested as colonic polyposis caused by mutations in
the APC gene and is also inherited in an autosomal domit manner. Finally,
MYH-associated polyposis is caused by mutations in the MUTYH gene and is
inherited in an autosomal recessive manner but may or may not be associated with
polyps. There are variants of both familial adenomatous polyposis (Gardner
syndrome--with extracolonic features--and Turcot syndrome, which features
medulloblastoma) and Lynch syndrome (Muir-Torre syndrome features sebaceous skin
carcinomas, and Turcot syndrome features glioblastomas). Although a clinical
diagnosis of familial adenomatous polyposis can be made using colonoscopy,
genetic testing is needed to inform at-risk relatives. Because of the
overlapping phenotypes between attenuated familial adenomatous polyposis,
MYH-associated polyposis, and Lynch syndrome, genetic testing is needed to
distinguish among these conditions. This distinction is important, especially
for women with Lynch syndrome, who are at increased risk for gynecological
cancers. Clinical testing for these genes has progressed rapidly in the past few
years with advances in technologies and the lower cost of reagents, especially
for sequencing. To assist clinical laboratories in developing and validating
testing for this group of inherited colorectal cancers, the American College of
Medical Genetics and Genomics has developed the following technical standards
and guidelines. An algorithm for testing is also proposed. |
Which is the gene most commonly mutated in Tay-Sachs disease? | HEXA gene, encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A | Tay-Sachs disease displays a variety of forms on the clinical and biochemical
level. On the molecular level it has been shown, that poly (A)+ RNA preparations
from fibroblasts of patients with classical Tay-Sachs disease lack detectable
alpha-chain message when analyzed by Northern blotting with complementary DNA
encoding the alpha-chain of human beta-hexosaminidase A. In this report the p
beta H alpha-5 clone was used to investigate whether patients with two different
variants of Tay-Sachs disease also lack the alpha-chain message. On the basis of
RNA hybridization analyses, we could show that our patients which synthesize an
altered alpha-chain, as judged by testing enzyme activity and substrate
specificity, have the 2.1 kb mRNA which is also seen in healthy control
patients. Tay-Sachs disease is an autosomal recessive genetic disorder resulting from
mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme,
beta-N-acetylhexosaminidase A (ref. 1). A relatively high frequency of carriers
(1/27) of a lethal, infantile form of the disease is found in the Ashkenazi
Jewish population, but it is not yet evident whether this has resulted from a
founder effect and random genetic drift or from a selective advantage of
heterozygotes. We have identified a single-base mutation in a cloned fragment of
the HEXA gene from an Ashkenazi Jewish patient. This change, the substitution of
a C for G in the first nucleotide of intron 12 is expected to result in
defective splicing of the messenger RNA. A test for the mutant allele based on
amplification of DNA by the 'polymerase chain rection and cleavage of a DdeI
restriction site generated by the mutation revealed that this case and two other
cases of the Ashkenazi, infantile form of Tay-Sachs disease are heterozygous for
two different mutations. The occurrence of multiple mutant alleles warrants
further examination of the selective advantage hypothesis. GM2-gangliosidosis is a group of neurological disorders resulting from
genetically defective catabolism, and consequent abnormal accumulation, of
GM2-ganglioside. Three major types are distinguished: the B variant (Tay-Sachs
disease), the O variant (Sandhoff disease), and the AB variant, caused by
genetic abnormalities in the genes coding for the beta-hexosaminidase alpha- or
beta-subunit, or the GM2-activator protein, respectively. A number of gene
abnormalities responsible for Tay-Sachs disease have already been identified and
the correlation between the beta-hexosaminidase alpha gene abnormality and the
clinical phenotype has been explained in many cases. In the severest phenotype
of Tay-Sachs disease (infantile form), mRNA of beta-hexosaminidase alpha subunit
is not produced or is unstable such as in French Canadian patients or in Jewish
patients with infantile Tay-Sachs disease, or the polypeptide does not have any
catalytic activities because of the alteration of glycosylation such as the
mutation of Glu482-to-Lys found in a Italian patient or the altered structure of
polypeptide. The mutation identified in a large majority of the Japanese
infantile Tay-Sachs disease patients, which is a G-to-T substitution at 3'-end
of intron 5, generates a short mRNA with complete skipping of exon 6 and a
polypeptide lacking 34 amino acids is generated but catalytically inactive. On
the other hand, some active alpha beta dimers must be generated in patients with
milder phenotypes of Tay-Sachs disease such as Gly269-to-Ser mutation in an
adult form. Some of the mutations appear in high frequency among certain ethnic
groups such as Ashkenazi Jewish patients and French Canadians.(ABSTRACT
TRUNCATED AT 250 WORDS) We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in
a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of
HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=),
located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G
(p.Asn399Asp) in exon 11, has been previously characterized as a common
polymorphism in African-Americans. Hex A activity measured in TSD Glial cells,
transfected with HEXA cDNA constructs bearing these mutations, was unaltered
from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR
products revealed three aberrant transcripts in the patient, one where exon 8
was absent, one where exon 11 was absent and a third lacking both exons 10 and
11. All three novel transcripts contain frameshifts resulting in premature
termination codons (PTCs). Transfection of mini-gene constructs carrying the
c.814G>A and c.1305C>T mutations proved that the two mutations result in exon
skipping. mRNAs that harbor a PTC are detected and degraded by the
nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal
proteins. However, although NMD is functional in the patient's fibroblasts,
aberrant transcripts are still present. We suggest that the level of correctly
spliced transcripts as well as the efficiency in which NMD degrade the
PTC-containing transcripts, apparently plays an important role in the phenotype
severity of the unique patient and thus should be considered as a potential
target for drug therapy. Tay-Sachs disease (TSD) is a recessively inherited disorder caused by the
deficient activity of hexosaminidase A due to mutations in the HEXA gene. Up to
date there is no information regarding the molecular genetics of TSD in
Argentinean patients. In the present study we have studied 17 Argentinean
families affected by TSD, including 20 patients with the acute infantile form
and 3 with the sub-acute form. Overall, we identified 14 different mutations
accounting for 100% of the studied alleles. Eight mutations were novel: 5 were
single base changes leading to drastic residue changes or truncated proteins, 2
were small deletions and one was an intronic mutation that may cause a splicing
defect. Although the spectrum of mutations was highly heterogeneous, a high
frequency of the c.459+5G>A mutation, previously described in different
populations was found among the studied cohort. Haplotype analysis suggested
that in these families the c.459+5G>A mutation might have arisen by a single
mutational event. Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase
A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay
Sachs disease in India are yet unknown. We aimed to determine mutations leading
to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion
criteria included neuroregression, seizures, exaggerated startle reflex,
macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging
criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the
brain. Biochemical criteria included deficiency of hexosaminidase A (less than
2% of total hexosaminidase activity for infantile patients). Total leukocyte
hexosaminidase activity was assayed by
4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A
activity was assayed by heat inactivation method and
4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons
and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using
an automated sequencer. Mutations were confirmed in parents and looked up in
public databases. In silico analysis for mutations was carried out using SIFT,
Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen
families were included in the study. We identified six novel missense mutations,
c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G
(p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously
reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation
p.E462V was found in six unrelated families from Gujarat indicating a founder
effect. A previously known splice site mutation c.805+1 G>C and another intronic
mutation c.672+30 T>G of unknown significance were also identified. Mutations
could not be identified in one family. We conclude that TSD patients from
Gujarat should be screened for the common mutation p.E462V. |
In which types of DNA repair is the UvrAB complex involved? | UvrB and the lesion-recognition factor UvrA form the UvrAB complex, which plays a key role in bacterial nucleotide excision repair (NER). In transcription-coupled repair (TCR), the transcription repair coupling factor Mfd recruits uvrA, and the assembled UvrAB complex initiates repair. UvrAB complex also suppresses illegitimate recombination. | Ultraviolet light induced pyrimidine dimers in DNA are recognized and repaired
by a number of unique cellular surveillance systems. At the highest level of
complexity Escherichia coli (E. coli) has a uvr DNA repair system comprising the
UvrA, UvrB and UvrC proteins responsible for incision. There are several
preincision steps governed by this pathway which includes an ATP-dependent UvrA
dimerization reaction required for UvrAB nucleoprotein formation. This complex
formation driven by ATP binding, is associated with localized topological
unwinding of DNA. This protein complex can catalyze an ATP-dependent 5'----3'
directed strand displacement of D-loop DNA or short single strands annealed to a
single stranded circular or linear DNA. This putative translocational process is
arrested when damaged sites are encountered. The complex is now primed for dual
incision catalyzed by UvrC. The remainder of the repair process involves UvrD
(helicase II) and DNA polymerase I for a coordinately controlled "excision
resynthesis" step accompanied by UvrABC turnover. Furthermore, it is proposed
that levels of repair proteins can be regulated by proteolysis. UvrB is
converted to truncated UvrB* by a stress induced protease which also acts at
similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to
DNA it cannot participate in helicase or incision reactions. It is also a
DNA-dependent ATPase. Transcription when coupled to nucleotide excision repair specifies the location
in active genes where preferential DNA repair is to take place. During DNA
damage-induced recruitment of RNA polymerase (RNAP), there is a physical
association of the beta subunit of Escherichia coli RNAP and the UvrA component
of the repair apparatus (G. C. Lin and L. Grossman, submitted for publication).
This molecular affinity is reflected in the ability of the RNAP to increase, in
a promoter-dependent manner, DNA supercoiling by the UvrAB complex. In the
presence of the RNAP, the UvrAB complex is able to bind to promoter regions and
to translocate in a 5' to 3' direction along the non-transcribed strand. As a
consequence of this helicase-catalyzed translocation, preferential incision of
DNA damaged sites occurs downstream on the transcribed strand. Because of the
helicase directionality, the initial binding of the UvrAB complex to the
transcribed strand would inevitably lead to its collision with the RNAP. These
results imply that the RNAP-induced DNA structure in the vicinity of the
transcription start site signals a landing or entry site for the UvrAB complex
on DNA. The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide
excision repair of a large variety of DNA damages. The damage recognition and
incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent
DNA helix-tracking activity associated with the UvrA2B1 complex. The latter
activity leads to the generation of highly positively supercoiled DNA in the
presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled
DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is
resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and
relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can
contribute to the above reaction by abolishing the accumulation of highly
positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision.
Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in
restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to
incise highly positively supercoiled uvDNA results from the failure of the
formation of UvrAB-dependent obligatory intermediates associated with the DNA
conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV
endonuclease efficiently incises uvDNA regardless of its topological state. The
in vitro topodynamic system proposed in this study may provide a simple model
for studying a topological aspect of nucleotide excision repair and its
interaction with other DNA topology-related processes in E. coli. To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision
repair proteins during the formation of the pre-incision complex at a damaged
DNA site, we used substrates with modifications around a single
2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing
oligonucleotides from a single-stranded DNA circle, we conclude that during
interaction with our substrates UvrAB introduces changes in DNA which are
localized at the lesion and are limited to 1-3 bp. Since these changes might
include a denaturation of DNA at the lesion site and, consequently, a bubble
structure might be present in a pre-incision complex, we studied incision
activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an
AAF adduct. Opening more than one base on either or both sides of the lesion
caused a significant decrease in the incision activity of UvrABC, but did not
change the position of the incision sites. We conclude that the UvrAB action
leading to a pre-incision complex does not include the formation of a bubble
intermediate generated by extensive denaturation of base pairs. The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a
variety of lesions with diverse chemical structures and the presence of helicase
activity in the UvrAB complex which can displace short oligonucleotides annealed
to single-stranded DNA led to a model in which this activity moves UvrAB along
undamaged DNA to damaged sites where the lesion blocks further translocation and
the protein-DNA pre-incision complex is formed. To evaluate this mechanism for
damage recognition, we constructed substrates with oligonucleotides of different
lengths annealed to single-stranded DNA circles and placed a single
2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the
circle. For the substrates with no lesion, the UvrAB complex effectively
displaced a 22-mer but not a 27-mer or longer fragments. The presence of AAF on
the oligonucleotide significantly increased the release of the 27-mer but
oligomers of 30 or longer were not separated. Placing the lesion on the circular
strand did not block the release of the fragments. Instead, the releasing
activity of UvrAB was stimulated and also depended on the length of the annealed
oligonucleotide. These observations do not agree with the predictions of a
damage recognition mechanism that depends on helicase-driven translocation. Most
likely, the strand-separating activity of UvrAB is a consequence of local
changes occurring during the formation of a DNA-protein pre-incision complex at
the damaged site and is not due to translocation of the protein along undamaged
DNA to locate a lesion. Illegitimate recombination is a major cause of genetic instability in
prokaryotes as well as in eukaryotes. This recombination usually occurs at a low
frequency, but it is greatly enhanced by UV irradiation or other environmental
stresses. DNA damages produced by these environmental stresses are thought to
induce DNA double-strand breaks, leading to illegitimate recombination. In this
paper we show that UV-induced illegitimate recombination is enhanced by
mutations of nucleotide excision repair genes, uvrA or uvrB, and partially by
uvrC mutation, but not by uvrD mutation. Unexpectedly, the recombination was
enhanced by the uvrA uvrB double mutation even without UV irradiation, but the
uvrB uvrC double mutation has not shown this effect, suggesting that
illegitimate recombination is mostly suppressed by UvrA and UvrB. Moreover,
illegitimate recombination was synergistically enhanced by the recQ uvrA double
mutation. In addition, overproduction of the UvrA protein suppressed the
hyperrecombination phenotype of the recQ or uvrB mutant, but it did not affect
the UV-sensitive phenotype of the uvrB mutant. We concluded that the UvrAB
complex suppresses illegitimate recombination in a pathway shared with RecQ
helicase. In addition, UvrA protein alone can suppress illegitimate
recombination in the pathway, in which RecQ helicase and UvrAB complex work.
Possible functions of the proteins involved in these pathways are also
discussed. Escherichia coli nucleotide excision repair (NER) is responsible for removing
bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the
activity of the UvrAB complex which can induce DNA conformational change is
employed in NER, the involvement of DNA topology and DNA topoisomerases remains
unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo
system. The repair analysis of intracellular plasmid revealed that the DNA
damage on positive supercoils generated by gyrase inhibition remained
unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants.
These results suggest that DNA topology affects the NER process and the removal
of positive supercoils by gyrase is vital for the efficiency of the E. coli NER
system. The dual-incision nature of the reaction of UV-irradiated DNA catalyzed by the
UvrABC complex potentially leads to excision of a damaged fragment. However,
neither fragment release under nondenaturing conditions nor the UvrBC proteins
are turned over. The addition of the UvrD protein to the incised DNA-UvrBC
complex results in excision of the incised damaged strand and in the turnover of
the UvrC protein. In an effort to better understand the involvement of UvrD in
the excision step, immunoprecipitation was used to detect interacting proteins
with UvrD in the DNA repair. In this communication, it is shown that UvrA and
UvrB are precipitated with UvrD in solution but the UvrAB complex is not. In the
incision complex, UvrB could be precipitated and the preincubation of UvrD with
UvrB revealed an inhibitory effect on the turnover of the incision complex.
These data imply that UvrB in the incision complex seems to recruit UvrD to the
3' incised site of the incised strand by protein-protein interaction and to
allow initiation of unwinding by UvrD from the resulting nick in a 3' to 5'
direction. The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of
nucleotide excision repair. The uvrC gene product is involved in at least one of
the dual incisions produced by the UvrABC complex. Using single-stranded (ss)
DNA affinity chromatography, we have separated two forms of UvrC from both
wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and
UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and
UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same
fashion; i.e., they incise six to eight nucleotides 5' to and three to five
nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce
different incisions, however, at a CC-1065-N3-adenine adduct in the sequence
5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises
at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises
only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both
UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are
proportional to the amount of UvrCI and UvrCII. DNase I footprints of the
MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct
at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct
site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding
differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI
binding. Interestingly, while the presence of unirradiated DNA enhances the
efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance
UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These
results show that two different forms of UvrC differ in DNA binding properties
as well as incision modes at some kinds of DNA damage. It is generally accepted that the damage recognition complex of nucleotide
excision repair in Escherichia coli consists of two UvrA and one UvrB molecule,
and that in the preincision complex UvrB binds to the damage as a monomer. Using
scanning force microscopy, we show here that the damage recognition complex
consists of two UvrA and two UvrB subunits, with the DNA wrapped around one of
the UvrB monomers. Upon binding the damage and release of the UvrA subunits,
UvrB remains a dimer in the preincision complex. After association with the UvrC
protein, one of the UvrB monomers is released. We propose a model in which the
presence of two UvrB subunits ensures damage recognition in both DNA strands.
Upon binding of the UvrA(2)B(2) complex to a putative damaged site, the DNA
wraps around one of the UvrB monomers, which will subsequently probe one of the
DNA strands for the presence of a lesion. When no damage is found, the DNA will
wrap around the second UvrB subunit, which will check the other strand for
aberrations. UvrB plays a key role in bacterial nucleotide excision repair. It is the
ultimate damage-binding protein that interacts with both UvrA and UvrC. The
oligomeric state of UvrB and the UvrAB complex have been subject of debate for a
long time. Using fluorescence resoce energy transfer (FRET) between GFP and
YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show
that in solution two UvrB subunits bind to UvrA, most likely as part of a
UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the
ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA
promotes dimerization of the two C-terminal domain 4 regions of UvrB. The
presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when
the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When
the complex is bound to a damaged site, FRET between the two UvrB subunits could
still be detected, but only as long as UvrA remains associated. Dissociation of
UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of
the FRET signal, indicating that the domain 4 regions no longer interact. We
propose that the UvrA-induced dimerization of the domain 4 regions serves to
shield these domains from premature UvrC binding. Only after specific binding of
the UvrB dimer to a damaged site and subsequent release of UvrA is the contact
between the domain 4 regions broken, allowing recruitment of UvrC and subsequent
incisions. How DNA repair proteins sort through a genome for damage is one of the
fundamental uswered questions in this field. To address this problem, we
uniquely labeled bacterial UvrA and UvrB with differently colored quantum dots
and visualized how they interacted with DNA individually or together using
oblique-angle fluorescence microscopy. UvrA was observed to utilize a
three-dimensional search mechanism, binding transiently to the DNA for short
periods (7 s). UvrA also was observed jumping from one DNA molecule to another
over approximately 1 microm distances. Two UvrBs can bind to a UvrA dimer and
collapse the search dimensionality of UvrA from three to one dimension by
inducing a substantial number of UvrAB complexes to slide along the DNA. Three
types of sliding motion were characterized: random diffusion, paused motion, and
directed motion. This UvrB-induced change in mode of searching permits more
rapid and efficient scanning of the genome for damage. Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair
(NER) that acts specifically on lesions in the transcribed strand of expressed
genes. First reported in mammalian cells, TCR was then documented in Escherichia
coli. In this organism, an RNA polymerase arrested at a lesion is displaced by
the transcription repair coupling factor, Mfd. This protein recruits the NER
lesion-recognition factor UvrA, and then dissociates from the DNA. UvrA binds
UvrB, and the assembled UvrAB* complex initiates repair. In mutants lacking
active Mfd, TCR is absent. A gene transcribed by the bacteriophage T7 RNA
polymerase in E. coli also requires Mfd for TCR. The CSB protein (missing or
defective in cells of patients with Cockayne syndrome, complementation group B)
is essential for TCR in humans. CSB and its homologs in higher eukaryotes are
likely functional equivalents of Mfd. |
What is the localization of the protein encoded by the gene DNAJC11? | mitochondrial inner membrane | A monoclonal antibody (mAb) has been produced which reacts with human mitofilin,
a mitochondrial inner membrane protein. This mAb immunocaptures its target
protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3,
CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins,
CHCHD3 has been assigned to the matrix space, and the other two proteins have
not been described in mitochondria previously. The functional role of this new
complex is uncertain. However, a role in protein import related to maintece
of mitochondrial structure is suggested as mitofilin helps regulate
mitochondrial morphology and at least four of the associated proteins (metaxins
1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11
is a chaperone-like protein that may have a similar role. |
Is Calcium/Calmodulin dependent protein kinase II (CaMKII) involved in cardiac arrhythmias and heart failure? | Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase expressed abundantly in the heart. CaMKII targets numerous proteins involved in excitation-contraction coupling and excitability, and its activation may simultaneously contribute to heart failure and cardiac arrhythmias. | In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is
increased. Altered Na(+) channel gating is linked to and may promote ventricular
tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part
perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and
Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na))
in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch
clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage
dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was
Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed
recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related
inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent
(late) inward I(Na) and intracellular Na(+) concentration (as measured by the
Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was
prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C)
overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+)
channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS
duration and repolarization (QT intervals), decreased effective refractory
periods, and increased the propensity to develop VT. We conclude that CaMKII
associates with and phosphorylates cardiac Na(+) channels. This alters I(Na)
gating to reduce availability at high heart rate, while enhancing late I(Na)
(which could prolong action potential duration). In mice, enhanced
CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of
Na(+) channel function may contribute to arrhythmogenesis in HF. Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter
Na(+) channel gating and recapitulate a human Na(+) channel genetic mutation
that causes an unusual combined arrhythmogenic phenotype in patients:
simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in
heart failure where arrhythmias are common, and CaMKII inhibition can reduce
arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to
acquired arrhythmic disease. We developed a Markovian Na(+) channel model
including CaMKII-dependent changes, and incorporated it into a comprehensive
myocyte action potential (AP) model with Na(+) and Ca(2+) transport. CaMKII
shifts Na(+) current (I(Na)) availability to more negative voltage, enhances
intermediate inactivation, and slows recovery from inactivation (all
loss-of-function effects), but also enhances late noninactivating I(Na) (gain of
function). At slow heart rates, with long diastolic time for I(Na) recovery,
late I(Na) is the predomit effect, leading to AP prolongation (long QT
syndrome). At fast heart rates, where recovery time is limited and APs are
shorter, there is little effect on AP duration, but reduced availability
decreases I(Na), AP upstroke velocity, and conduction (Brugada syndrome). CaMKII
also increases cardiac Ca(2+) and K(+) currents (I(Ca) and I(to)), complicating
CaMKII-dependent AP changes. Incorporating I(Ca) and I(to) effects individually
prolongs and shortens AP duration. Combining I(Na), I(Ca), and I(to) effects
results in shortening of AP duration with CaMKII. With transmural heterogeneity
of I(to) and I(to) downregulation in heart failure, CaMKII may accentuate
dispersion of repolarization. This provides a useful initial framework to
consider pathways by which CaMKII may contribute to arrhythmogenesis. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)
regulates a rich variety of downstream targets in heart. Ca(2+) homeostatic
proteins are important CaMKII targets that support myocardial
excitation-contraction coupling. Under stress conditions, excessive CaMKII
activity promotes heart failure and arrhythmias, in part through actions at
Ca(2+) homeostatic proteins. Here, we briefly review the molecular and cellular
physiology of CaMKII in myocardium. Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional
serine/threonine kinase expressed abundantly in the heart. CaMKII targets
numerous proteins involved in excitation-contraction coupling and excitability,
and its activation may simultaneously contribute to heart failure and cardiac
arrhythmias. In this review, we summarize the modulatory effects of CaMKII on
cardiac ion channel function and expression and illustrate potential
implications in the onset of arrhythmias via a computer model. Phosphorylation of the cardiac ryanodine receptor (RyR2) is a key mechanism
regulating sarcoplasmic reticulum (SR) Ca2+ release. Differences in opinion have
arisen over the importance assigned to specific phosphorylation sites on RyR2,
over the kinase (s) suggested to directly phosphorylate RyR2 and surrounding the
possibility that altered phosphorylation of RyR2 is associated with contractile
dysfunction observed in heart failure. Ca2+/calmodulin dependent protein kinase
II (CaMKII) can phosphorylate RyR2 and modulate its activity. This
phosphorylation positively modulates cardiac inotropic function but in extreme
situations such as heart failure, elevated CaMKII activity can adversely
increase Ca2+ release from the SR and lead to arrhythmogenesis. Although other
kinases can phosphorylate RyR2, most notably cAMP-dependent protein kinase
(PKA), evidence for a key role of CaMKII in mediating RyR2-dependent Ca2+
release is emerging. Future challenges include (i) fully identifying mechanisms
of CaMKII interaction with the RyR2 complex and (ii) given the ubiquitous
expression of CaMKII, developing selective strategies to modulate RyR2-targeted
CaMKII activity and allow improved understanding of its role in normal and
diseased heart. Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and
heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing
disordered cell membrane excitability and myocardial dysfunction remain
uncertain. Failing human cardiomyocytes exhibit increased CaMKII and
voltage-gated Ca(2+) channel (Ca(V)1.2) activity, and enhanced expression of a
specific Ca(V)1.2 beta-subunit protein isoform (beta(2a)). We recently
identified Ca(V)1.2 beta(2a) residues critical for CaMKII phosphorylation (Thr
498) and binding (Leu 493), suggesting the hypothesis that these amino acids are
crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT
beta(2a) expression causes cellular Ca(2+) overload, arrhythmia-triggering cell
membrane potential oscillations called early afterdepolarizations (EADs), and
premature death in paced adult rabbit ventricular myocytes. Prevention of
intracellular Ca(2+) release by ryanodine or global cellular CaMKII inhibition
reduced EADs and improved cell survival to control levels in WT
beta(2a)-expressing ventricular myocytes. In contrast, expression of beta(2a)
T498A or L493A mutants mimicked the protective effects of ryanodine or global
cellular CaMKII inhibition by reducing Ca(2+) entry through Ca(V)1.2 and
inhibiting EADs. Furthermore, Ca(V)1.2 currents recorded from cells
overexpressing CaMKII phosphorylation- or binding-incompetent beta(2a) subunits
were incapable of entering a CaMKII-dependent high-activity gating mode (mode
2), indicating that beta(2a) Thr 498 and Leu 493 are required for Ca(V)1.2
activation by CaMKII in native cells. These data show that CaMKII binding and
phosphorylation sites on beta(2a) are concise but pivotal components of a
molecular and biophysical and mechanism for EADs and impaired survival in adult
cardiomyocytes. Transgenic (TG) Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) δ(C) mice
develop systolic heart failure (HF). CaMKII regulates intracellular Ca(2+)
handling proteins as well as sarcolemmal Na(+) channels. We hypothesized that
CaMKII also contributes to diastolic dysfunction and arrhythmias via
augmentation of the late Na(+) current (late I(Na)) in early HF (8-week-old TG
mice). Echocardiography revealed severe diastolic dysfunction in addition to
decreased systolic ejection fraction. Premature arrhythmogenic contractions
(PACs) in isolated isometrically twitching papillary muscles only occurred in TG
preparations (5 vs. 0, P < 0.05) which could be completely terminated when
treated with the late I(Na) inhibitor ranolazine (Ran, 5 μmol/L).
Force-frequency relationships revealed significantly reduced twitch force
amplitudes in TG papillary muscles. Most importantly, diastolic tension
increased with raising frequencies to a greater extent in TG papillary muscles
compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm²; P < 0.05).
Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm² (at 10 Hz; P
< 0.05) without negative inotropic effects. Mechanistically, the late I(Na) was
markedly elevated in myocytes isolated from TG mice and could be completely
reversed by Ran. In conclusion, our results show for the first time that TG
CaMKIIδ(C) overexpression induces diastolic dysfunction and arrhythmogenic
triggers possibly via an enhanced late I(Na). Inhibition of elevated late I(Na)
had beneficial effects on arrhythmias as well as diastolic function in papillary
muscles from CaMKIIδ(C) TG mice. Thus, late I(Na) inhibition appears to be a
promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is
found to be increased. Many signals have risen and fallen in the tide of investigation into mechanisms
of myocardial hypertrophy and heart failure (HF). In our opinion, the
multifunctional Ca and calmodulin-dependent protein kinase II (CaMKII) has
emerged as a molecule to watch, in part because a solid body of accumulated data
essentially satisfy Koch's postulates, showing that the CaMKII pathway is a core
mechanism for promoting myocardial hypertrophy and heart failure. Multiple
groups have now confirmed the following: (1) that CaMKII activity is increased
in hypertrophied and failing myocardium from animal models and patients; (2)
CaMKII overexpression causes myocardial hypertrophy and HF and (3) CaMKII
inhibition (by drugs, inhibitory peptides and gene deletion) improves myocardial
hypertrophy and HF. Patients with myocardial disease die in equal proportion
from HF and arrhythmias, and a major therapeutic obstacle is that drugs designed
to enhance myocardial contraction promote arrhythmias. In contrast, inhibiting
the CaMKII pathway appears to reduce arrhythmias and improve myocardial
responses to pathological stimuli. This brief paper will introduce the molecular
physiology of CaMKII and discuss the impact of CaMKII on ion channels, Ca
handling proteins and transcription in myocardium. This article is part of a
special issue entitled "Key Signaling Molecules in Hypertrophy and Heart
Failure". Aim of this review is to give an overview and discuss recent findings on the
role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the heart.
Special attention is drawn to excitation-contraction coupling (ECC) and
excitation-transcription coupling (ETC). Because CaMKII expression and activity
are increased in cardiac hypertrophy, heart failure, and during arrhythmias both
in animal models as well as in the human heart a clinical significance of CaMKII
is implied. Ryanodine receptor (RyR2) dysfunction, which may result from a variety of
mechanisms, has been implicated in the pathogenesis of cardiac arrhythmias and
heart failure. In this review, we discuss the important role of
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the regulation of
RyR2-mediated Ca(2+) release. In particular, we examine how pathological
activation of CaMKII can lead to an increased risk of sudden arrhythmic death.
Finally, we discuss how reduction of CaMKII-mediated RyR2 hyperactivity might
reduce the risk of arrhythmias and may serve as a rationale for future
pharmacotherapeutic approaches. Treating arrhythmias is a challenge for clinicians because pharmacological
therapies are often ineffective or have severe side effects. Patients with heart
failure frequently present with supreventricular and ventricular arrhythmias.
New antiarrhythmic therapies are needed that modulate the specific
pathomechanisms underlying the development of cardiac arrhythmias and may have a
better safety-profile. The Ca-calmodulin dependent kinase II (CaMKII) seems to
be involved in the development of heart failure and arrhythmias and may
therefore be a promising target for the development of antiarrhythmic therapies.
The current review aims at discussing some novel as well as known cytosolic and
sarcolemmal mechanisms involved in CaMKII-dependent arrhythmias without being
able to cover all aspects known in the field. BACKGROUND: CaMKII activation is proarrhythmic in heart failure where myocardium
is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle
has not been well understood.
OBJECTIVE: We tested abnormal impulse inducibility by stretch current in
myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where
CaMKII activity is reduced by ≈ 62%.
METHODS AND RESULTS: Action potentials were recorded by whole-cell patch clamp,
and abnormal impulses were induced in LV myocytes by a simulation of
stretch-activated channel (SAC) current. SAC activation failed to induce
abnormal impulses in wild type (WT) myocytes but steadily produced early
after-depolarizations and automaticity in KO myocytes in which an increase in
L-type calcium channel (LTCC) current (I(Ca)) and a reduction of sarcoplasmic
reticulum Ca(2+) leak and action potential duration (APD) were observed. The
abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low
concentration of nifedipine eliminated abnormal impulses without shortening APD,
implicating I(Ca) in promoting stretch-induced abnormal impulses. In addition,
APD prolongation by LTCC opener S(-)Bay K 8644 or isoproterenol facilitated
abnormal impulse induction in WT ventricular myocytes even in the presence of
CaMKII inhibitor AIP, whereas APD prolongation by K(+) channel blocker
4-aminopyridine promoted abnormal impulses in KO myocytes but not in WT
myocytes.
CONCLUSION: I(Ca) activation plays a central role in stretch-induced abnormal
impulses and APD prolongation is arrhythmogenic only when I(Ca) is highly
activated. At increased I(Ca) activation, CaMKII inhibition cannot suppress
abnormal impulse induction. Arrhythmias can develop in various cardiac diseases, such as ischemic heart
disease, cardiomyopathy and congenital heart disease. It can also contribute to
the aggravation of heart failure and sudden cardiac death. Redox stress and
Ca(2+) overload are thought to be the important triggering factors in the
generation of arrhythmias in failing myocardium. From recent studies, it appears
evident that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a
central role in the arrhythmogenic processes in heart failure by sensing
intracellular Ca(2+) and redox stress, affecting individual ion channels and
thereby leading to electrical instability in the heart. CaMKII, a
multifunctional serine/threonine kinase, is an abundant molecule in the neuron
and the heart. It has a specific property as "a memory molecule" such that the
binding of calcified calmodulin (Ca(2+)/CaM) to the regulatory domain on CaMKII
initially activates this enzyme. Further, it allows autophosphorylation of T287
or oxidation of M281/282 in the regulatory domain, resulting in sustained
activation of CaMKII even after the dissociation of Ca(2+)/CaM. This review
provides the understanding of both the structural and functional properties of
CaMKII, the experimental findings of the interactions between CaMKII, redox
stress and individual ion channels, and the evidences proving the potential
participation of CaMKII and oxidative stress in the diverse arrhythmogenic
processes in a diseased heart. Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the
treatment of cardiac arrhythmia, cardiac inflammation, and heart failure.
Amiodarone is a noncompetitive inhibitor of the α - and β -adrenergic receptors
and prevents calcium influx in the slow-response cells of the sinoatrial and
atrioventricular nodes. Overexpression of Ca(2+)/calmodulin-dependent protein
kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias.
We hypothesised that administration of WXKL and amiodarone can reduce the
incidence of arrhythmias by regulating CaMKII signal transduction. A total of
100 healthy Sprague Dawley rats were used in the study. The rats were randomly
divided into four groups (a sham group, a myocardial infarction (MI) group, a
WXKL-treated group, and an amiodarone-treated group). A myocardial infarction
model was established in these rats by ligating the left anterior descending
coronary artery for 4 weeks. Western blotting was used to assess CaMKII,
p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6
(FKBP12.6) levels. The Ca(2+) content in the sarcoplasmic reticulum (SR) and the
calcium transient amplitude were studied by confocal imaging using the
fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and
cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar
to amiodarone. INTRODUCTION: Calcium-calmodulin-dependent protein kinase II (CaMKII) has
emerged as a central mediator of cardiac stress responses which may serve
several critical roles in the regulation of cardiac rhythm, cardiac
contractility and growth. Sustained and excessive activation of CaMKII during
cardiac disease has, however, been linked to arrhythmias, and maladaptive
cardiac remodeling, eventually leading to heart failure (HF) and sudden cardiac
death.
AREAS COVERED: In the current review, the authors describe the unique structural
and biochemical properties of CaMKII and focus on its physiological effects in
cardiomyocytes. Furthermore, they provide evidence for a role of CaMKII in
cardiac pathologies, including arrhythmogenesis, myocardial ischemia and HF
development. The authors conclude by discussing the potential for CaMKII as a
target for inhibition in heart disease.
EXPERT OPINION: CaMKII provides a promising nodal point for intervention that
may allow simultaneous prevention of HF progression and development of
arrhythmias. For future studies and drug development there is a strong rationale
for the development of more specific CaMKII inhibitors. In addition, an improved
understanding of the differential roles of CaMKII subtypes is required. In the recent years, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was
suggested to be associated with cardiac hypertrophy and heart failure but also
with arrhythmias both in animal models as well as in the human heart. This
article focuses on the role of CaMKII for excitation-contraction coupling but
more explicitly it highlights major CaMKIIdependent proarrhythmogenic mechanisms
including SR Ca(2+) leak and late Na(+) current. Because a clinical significance
of CaMKII is implied for both mechanisms, CaMKII inhibition is suggested to be a
therapeutical approach in the near future. BACKGROUND: Sarcoplasmic reticulum (SR) Ca(2+) leak through ryanodine receptor
type 2 (RyR2) dysfunction is of major pathophysiological relevance in human
heart failure (HF); however, mechanisms underlying progressive RyR2
dysregulation from cardiac hypertrophy to HF are still controversial.
METHODS AND RESULTS: We investigated healthy control myocardium (n=5) and
myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In
hypertrophy, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein
kinase A (PKA) both phosphorylated RyR2 at levels that were not different from
healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR
Ca(2+) leak. In HF, however, the SR Ca(2+) leak was nearly doubled compared with
hypertrophy, which led to reduced systolic Ca(2+) transients, a depletion of SR
Ca(2+) storage and elevated diastolic Ca(2+) levels. This was accompanied by a
significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast,
PKA-dependent RyR2 phosphorylation was not increased in HF and was independent
of previous β-blocker treatment. In HF, CaMKII inhibition but not inhibition of
PKA yielded a reduction of the SR Ca(2+) leak. Moreover, PKA inhibition further
reduced SR Ca(2+) load and systolic Ca(2+) transients.
CONCLUSIONS: In human hypertrophy, both CaMKII and PKA functionally regulate
RyR2 and may induce SR Ca(2+) leak. In the transition from hypertrophy to HF,
the diastolic Ca(2+) leak increases and disturbed Ca(2+) cycling occurs. This is
associated with an increase in CaMKII- but not PKA-dependent RyR2
phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic
target for the treatment of arrhythmias and contractile dysfunction. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with
important regulatory functions in the heart and brain, and its chronic
activation can be pathological. CaMKII activation is seen in heart failure, and
can directly induce pathological changes in ion channels, Ca(2+) handling and
gene transcription. Here, in human, rat and mouse, we identify a novel mechanism
linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a
key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia
causes covalent modification of CaMKII by O-linked N-acetylglucosamine
(O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII
autonomously, creating molecular memory even after Ca(2+) concentration
declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of
diabetic humans and rats. In cardiomyocytes, increased glucose concentration
significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic
reticulum Ca(2+) release events that can contribute to cardiac mechanical
dysfunction and arrhythmias. These effects were prevented by pharmacological
inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact
perfused hearts, arrhythmias were aggravated by increased glucose concentration
through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute
blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of
CaMKII is a novel signalling event in pathways that may contribute critically to
cardiac and neuronal pathophysiology in diabetes and other diseases. |
What is the lay name of the treatment for CCSVI (chronic cerebro-spinal venous insufficiency) in multiple sclerosis. | The so-called "LIberation therapy" is in fact Endovascular Treatment and consists of PTA (Percutaneous Transluminal Angioplasty), which is dilatation of the internal jugular and/or azygous veins by a catheter venography. Stent placement is optional but has been strongly advised against as being dangerous. | OBJECTIVE: Chronic cerebrospinal venous insufficiency (CCSVI) is characterized
by combined stenoses of the principal pathways of extracranial venous drainage,
including the internal jugular veins (IJVs) and the azygous (AZY) vein, with
development of collateral circles and insufficient drainage shown by increased
mean transit time in cerebral magnetic resoce (MR) perfusion studies. CCSVI
is strongly associated with multiple sclerosis (MS). This study evaluated the
safety of CCSVI endovascular treatment and its influence on the clinical outcome
of the associated MS.
METHODS: Sixty-five consecutive patients with CCSVI, subdivided by MS clinical
course into 35 with relapsing remitting (RR), 20 with secondary progressive
(SP), and 10 with primary progressive (PP) MS, underwent percutaneous
transluminal angioplasty (PTA). Mean follow-up was 18 months. Vascular outcome
measures were postoperative complications, venous pressure, and patency rate.
Neurologic outcome measures were cognitive and motor function assessment, rate
of MS relapse, rate of MR active positive-enhanced gadolinium MS lesions (Gad+),
and quality of life (QOL) MS questionnaire.
RESULTS: Outpatient endovascular treatment of CCSVI was feasible, with a minor
and negligible complication rate. Postoperative venous pressure was
significantly lower in the IJVs and AZY (P < .001). The risk of restenosis was
higher in the IJVs compared with the AZY (patency rate: IJV, 53%; AZY, 96%; odds
ratio, 16; 95% confidence interval, 3.5-72.5; P < .0001). CCSVI endovascular
treatment significantly improved MS clinical outcome measures, especially in the
RR group: the rate of relapse-free patients changed from 27% to 50%
postoperatively (P < .001) and of MR Gad+ lesions from 50% to 12% (P < .0001).
The Multiple Sclerosis Functional Composite at 1 year improved significantly in
RR patients (P < .008) but not in PP or SP. Physical QOL improved significantly
in RR (P < .01) and in PP patients (P < .03), with a positive trend in SP (P <
.08). Mental QOL showed significant improvement in RR (P < .003) and in PP (P <
.01), but not in SP.
CONCLUSIONS: PTA of venous strictures in patients with CCSVI is safe, and
especially in patients with RR, the clinical course positively influenced
clinical and QOL parameters of the associated MS compared with the preoperative
assessment. Restenosis rates are elevated in the IJVs but very promising in the
AZY, suggesting the need to improve endovascular techniques in the former. The
results of this pilot study warrant a subsequent randomized control study. The pyramidal pathway is frequently affected early on in multiple sclerosis (MS)
and impaired motor performance is a major cause of disability. Pyramidal tract
function can be assessed using transcranial magnetic stimulation (TMS). TMS
supports the diagnosis of MS, detecting corticospinal tract involvement and
monitoring its course with or without treatment. It has been never investigated
whether any relationship exists between the TMS outcome measure and minimally
invasive treatment of multiple severe extracranial stenosis, affecting the
principal ce rebrospinal venous segments in MS patients. We report the clinical
and transcranial magnetic stimulation follow-up of a patient during a relapse in
relapsing-remitting MS. She underwent percutaneous balloon angioplasty of the
associated chronic cerebrospinal venous insufficiency (CCSVI), due to membranous
obstruction of the proximal azygous vein, with severe stenosis of the left
internal jugular vein. Treatment of the associated CCSVI made a parallel
improvement in both clinical and neurophysiological parameters, allowing us to
avoid high dose steroid therapy. The relationship between the clinical and
neurophysiological course on the one hand, and haemodynamic correction of the
associated CCSVI on the other, calls for further exploration on a wider number
of patients. The impact of CCSVI on the different neuro-physiological parameters
has not been fully estimated, but the intriguing case here reported suggests
that it may be greater than previously assumed. The demonstration of a
modification of the cerebrovenous function with both clinical manifestation and
via TMS suggests that the hampered cerebral venous return may contribute to the
clinical course of MS. A chronic state of impaired venous drainage from the central nervous system,
termed chronic cerebrospinal venous insufficiency (CCSVI), is claimed to be a
pathologic phenomenon exclusively seen in multiple sclerosis (MS). This has
invigorated the causal debate of MS and generated immense interest in the
patient and scientific communities. A potential shift in the treatment paradigm
of MS involving endovascular balloon angioplasty or venous stent placement has
been proposed as well as conducted in small patient series. In some cases, it
may have resulted in serious injury. In this Point of View, we discuss the
recent investigations that led to the description of CCSVI as well as the
conceptual and technical shortcomings that challenge the potential relationship
of this phenomenon to MS. The need for conducting carefully designed and
rigorously controlled studies to investigate CCVSI has been recognized by the
scientific bodies engaged in MS research. Several scientific endeavors examining
the presence of CCSVI in MS are being undertaken. At present, invasive and
potentially dangerous endovascular procedures as therapy for patients with MS
should be discouraged until such studies have been completed, analyzed, and
debated in the scientific arena. OBJECTIVES: The aim of this report is to assess the safety of endovascular
treatment for chronic cerebrospinal venous insufficiency (CCSVI). Although
balloon angioplasty and stenting seem to be safe procedures, there are currently
no data on the treatment of a large group of patients with this vascular
pathology.
METHODS: A total of 564 endovascular procedures (balloon angioplasty or, if this
procedure failed, stenting) were performed during 344 interventions in 331 CCSVI
patients with associated multiple sclerosis.
RESULTS: Balloon angioplasty alone was performed in 192 cases (55.8%), whereas
the stenting of at least one vein was required in the remaining 152 cases
(44.2%). There were no major complications (severe bleeding, venous thrombosis,
stent migration or injury to the nerves) related to the procedure, except for
thrombotic occlusion of the stent in two cases (1.2% of stenting procedures) and
surgical opening of femoral vein to remove angioplastic balloon in one case
(0.3% of procedures). Minor complications included occasional technical problems
(2.4% of procedures): difficulty removing the angioplastic balloon or problems
with proper placement of stent, and other medical events (2.1% of procedures):
local bleeding from the groin, minor gastrointestinal bleeding or cardiac
arrhythmia.
CONCLUSIONS: The procedures appeared to be safe and well tolerated by the
patients, regardless of the actual impact of the endovascular treatments for
venous pathology on the clinical course of multiple sclerosis, which warrants
long-term follow-up. Chronic cerebrospinal venous insufficiency (CCSVI) is a putative new theory that
has been suggested by some to have a direct causative relation with the
symptomatology associated with multiple sclerosis (MS) [1]. The core foundation
of this theory is that there is abnormal venous drainage from the brain due to
outflow obstruction in the draining jugular vein and/or azygos veins. This
abnormal venous drainage, which is characterised by special ultrasound criteria,
called the "venous hemodynamic insufficiency severity score" (VHISS), is said to
cause intracerebral flow disturbance or outflow problems that lead to
periventricular deposits [2]. In the CCSVI theory, these deposits have a great
similarity to the iron deposits seen around the veins in the legs in patients
with chronic deep vein thrombosis. Zamboni, who first described this new theory,
has promoted balloon dilatation to treat the outflow problems, thereby curing
CCSVI and by the same token alleviating MS complaints. However, this theory does
not fit into the existing bulk of scientific data concerning the pathophysiology
of MS. In contrast, there is increasing worldwide acceptance of CCSVI and the
associated balloon dilatation treatment, even though there is no supporting
scientific evidence. Furthermore, most of the information we have comes from one
source only. The treatment is called "liberation treatment," and the results of
the treatment can be watched on YouTube. There are well-documented testimonies
by MS patients who have gained improvement in their personal quality of life
(QOL) after treatment. However, there are no data available from patients who
underwent unsuccessful treatments with which to obtain a more balanced view. The
current forum for the reporting of success in treating CCSVI and thus MS seems
to be the Internet. At the CIRCE office and the MS Centre in Amsterdam, we
receive approximately 10 to 20 inquiries a month about this treatment. In
addition, many interventional radiologists, who are directly approached by MS
patients, contact the Cardiovascular and Interventional Radiological Society of
Europe (CIRSE) for advice. Worldwide, several centres are actively promoting and
performing balloon dilatation, with or without stenting, for CCSVI. Thus far, no
trial data are available, and there is currently no randomized controlled trial
(RCT) in progress Therefore, the basis for this new treatment rests on anecdotal
evidence and successful testimonies by patients on the Internet. CIRSE believes
that this is not a sound basis on which to offer a new treatment, which could
have possible procedure-related complications, to an often desperate patient
population. PURPOSE: To evaluate the safety of endovascular treatment of chronic
cerebrovascular insufficiency (CCSVI) in patients with multiple sclerosis (MS).
METHODS: In a 1-year period, 461 MS patients (261 women; mean age 45.4 years,
range 21-79) with CCSVI underwent endovascular treatment of 1012 venous lesions
during 495 procedures [34 (6.9%) reinterventions]. While balloon angioplasty was
preferred, 98 stents were implanted in 76 patients for lesion recoil,
restenosis, or suboptimal dilation. The procedures were analyzed for incidences
of major adverse events (death, major bleeding, or clinical deterioration of
MS), access site complications, procedure-related complications, and procedural
safety-related variables (fluoroscopy and contrast times). The complication
rates were compared to published data for similar endovascular methods.
RESULTS: There were no deaths, major bleeding events, or clinical deterioration
of MS. Access site complications included limited groin hematoma (5, 1.0%);
there were no arteriovenous fistulas or puncture site infections. Systemic
complications included only rare cardiac arrhythmias (6, 1.2%).
Procedure-related complications included vein rupture (2, 0.4%), vein dissection
(15, 3.0%), acute in-stent/in-segment thrombosis (8, 1.6%), and acute recoil (1,
0.2%); there was no stent migration or fracture or distal embolization. Mean
fluoroscopy time was 22.7 minutes, and mean contrast volume was 136.3 mL.
CONCLUSION: Endovascular therapy appears to be a safe and reliable method for
treating CCSVI. Innovations such as purpose-specific materials and devices are
needed, as are case-controlled and randomized data to establish efficacy in
ameliorating MS symptoms. The Canadian Institutes of Health Research and the Multiple Sclerosis (MS)
Society of Canada recently convened an Invitational Panel to consider the
scientific evidence linking chronic cerebrospinal venous insufficiency (CCSVI)
and MS. The panel supported studies to determine whether CCSVI causes MS, but
felt that there is currently so much uncertainty about the relationship between
CCSVI and MS that a clinical trial is not indicated at this time. This
commentary argues that the decision about whether a clinical trial is warranted
must be informed by science, but should be addressed from a broader societal
perspective. We suggest that members of the public should be more actively
involved in scientifically based, but patient-relevant and emotionally charged
issues considered by organizations that fund health research. Chronic cerebrospinal venous insufficiency (CCSVI) is a hypothesis through which
cerebral venous drainage abnormalities contribute towards the pathogenesis of
multiple sclerosis. CCSVI venoplasty is already practised worldwide. We report
the case of a 33-year-old lady with multiple sclerosis who underwent left
internal jugular venoplasty resulting in iatrogenic jugular thrombosis requiring
open thrombectomy for symptom relief. This occurred without insertion of a stent
and while fully anticoagulated. Clinicians should be aware that endovenous
treatment of CCSVI could cause paradoxical deterioration of cerebral venous
drainage. Patients with complications post venoplasty are now presenting to
geographically distant vascular units. Recent reports have emerged suggesting that multiple sclerosis (MS) may be due
to abnormal venous outflow from the central nervous system, termed chronic
cerebrospinal venous insufficiency (CCSVI). These reports have generated strong
interest and controversy over the prospect of a treatable cause of this chronic
debilitating disease. This review aims to describe the proposed association
between CCSVI and MS, summarize the current data, and discuss the role of
endovascular therapy and the need for rigorous randomized clinical trials to
evaluate this association and treatment. Multiple sclerosis (MS) is a chronic disease of the central nervous system with
not yet completely understood pathogenesis. The so called "chronic cerebrospinal
venous insufficiency (CCSVI) theory" has recently emerged, supporting the
concept of a cerebrospinal venous drainage impairment as the cause of MS. Since
the first publication on this topic with a claimed 100% specificity and
sensitivity of the condition for MS diagnosis, CCSVI theory has generated a
scientific and mass media debate with a great hope for the miracle of a new
possible endovascular treatment of MS ("liberation procedure"). We critically
summarize the available evidence on CCSVI discussing inconsistent and incomplete
replication of the original results by different groups, methodological
limitations and potential therapeutic implications. We conclude that the
available data are insufficient to establish conclusively a clear relationship
between MS and CCSVI and do not support the role of CCSVI as the primary cause
of MS. Until credible scientific evidence replicates the original results, any
proposed invasive treatments of CCSVI should be discouraged. OBJECTIVES: Chronic cerebrospinal venous insufficiency (CCSVI) is associated
with multiple sclerosis (MS). The objective of the study was to see if
percutaneous transluminal angioplasty (PTA) of duplex-detected lesions, of the
internal jugular and/or azygous veins, was safe, burdened by a significant
restenosis rate, and whether there was any evidence that treatment reduced MS
disease activity.
DESIGN: This was a case-control study.
MATERIALS: We studied 15 patients with relapsing-remitting MS and
duplex-detected CCSVI.
METHODS: Eight patients had PTA in addition to medical therapy (immediate
treatment group (ITG)), whereas seven had treatment with PTA after 6 months of
medical therapy alone (delayed treatment group (DTG)).
RESULTS: No adverse events occurred. At 1 year, there was a restenosis rate of
27%. Overall, PTA was followed by a significant improvement in functional score
compared with baseline (p < 0.02). The annualised relapse rate was 0.12% in the
ITG compared with 0.66% in the DTG (p = NS). Magnetic resoce imaging (MRI)
blindly demonstrates a trend for fewer T2 lesions in the ITG (p = 0.081),
corresponding to a 10% decrease in the ITG compared with a 23% increase in the
DTG over the first 6 months of the study.
CONCLUSIONS: This study further confirms the safety of PTA treatment in patients
with CCSVI associated with MS. The results, despite the significant rate of
restenosis, are encouraging and warrant a larger multicentre double-blinded,
randomised study. PURPOSE: To evaluate the safety of outpatient endovascular treatment in patients
with multiple sclerosis (MS) and chronic cerebrospinal venous insufficiency
(CCSVI).
MATERIALS AND METHODS: A retrospective analysis was performed to assess
complications occurring within 30 days of endovascular treatment of CCSVI. The
study population comprised 240 patients; 257 procedures were performed over 8
months. The indication for treatment in all patients was symptomatic MS. Of the
procedures, 49.0% (126 of 257) were performed in a hospital, and 51.0% (131 of
257) were performed in the office. Primary procedures accounted for 93.0% (239
of 257) of procedures, and repeat interventions accounted for 7% (18 of 257).
For patients treated primarily, 87% (208 of 239) had angioplasty, and 11% (26 of
239) had stent placement; 5 patients were not treated. Of patients with
restenosis, 50% (9 of 18) had angioplasty, and 50% (9 of 18) had stent
placement.
RESULTS: After the procedure, all but three patients were discharged within 3
hours. Headache after the procedure was reported in 8.2% (21 of 257) of
patients; headache persisted > 30 days in 1 patient. Neck pain was reported in
15.6% (40 of 257); 52.5% (21 of 40) of these patients underwent stent placement.
Three patients experienced venous thrombosis requiring retreatment within 30
days. Sustained intraprocedural arrhythmias were observed in three patients, and
two required hospital admission. One of these patients, who was being retreated
for stent thrombosis, was hospitalized because of a stress-induced
cardiomyopathy.
CONCLUSIONS: Endovascular treatment of CCSVI is a safe procedure; there is a
1.6% risk of major complications. Cardiac monitoring is essential to detect
intraprocedural arrhythmias. Ultrasonography after the procedure is recommended
to confirm venous patency and to identify patients experiencing acute venous
thrombosis. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central
nervous system (CNS) by an unknown pathogenesis. MR venography and postmortem
studies have demonstrated a topographic correspondence between multiple
sclerosis (MS) plaques and the cerebral venous system pathology. In recent
observational studies performed on patients from distinctive gene pools, the
prevalence of chronic cerebrospinal venous insufficiency (CCSVI) in MS ranged
from 56% to 100%. Endovascular treatment (percutaneous transluminal angioplasty
(PTA) with or without stenting) of CCSVI was reported to be feasible with a
minor complication rate. In 4 patients with different forms of multiple
sclerosis venography was performed that revealed stenosis of the proximal region
of the jugular vein (right or left). Percutaneous transluminal balloon
angioplasty (PTA) was performed in all patients. There were no complications and
mean stenosis was reduced after PTA from 59.75% to 36.75%. Follow-up included
clinical observations and magnetic resoce imaging (MRI). In all the cases we
observed positive remission of the disease, the first ever documented case of
MRI index improvement. PTA seems to be an effective treatment for patients with
CCVI and multiple sclerosis, However, randomized studies are warranted to
establish the efficacy of this new treatment for MS. An open study was conducted with the aim of reporting long-term clinical outcome
of endovascular treatment for chronic cerebrospinal venous insufficiency (CCSVI)
in patients with multiple sclerosis (MS). Twenty-nine patients with clinically
definite relapsing-remitting MS underwent percutaneous transluminal angioplasty
for CCSVI, outside a clinical relapse. All the patients were regularly observed
over at least two years before the first endovascular treatment and for at least
two years after it (mean post-procedure follow up 30.6±6.1 months). The
following clinical outcome measures were used: annual relapse rate and Expanded
Disability Status Scale (EDSS) score. All the patients were observed intensively
(mean 6 hours) on the day of the endovascular treatment to monitor for possible
complications (bleeding, shock, heart attack, death). We compared the annual
relapse rate before and after treatment (in the two years before and the two
years after the first endovascular treatment) and the EDSS score recorded two
years before versus two years after the treatment. Overall, 44 endovascular
procedures were performed in the 29 patients, without complications. Thirteen of
the 29 patients (45%) underwent more than one treatment session because of
venous re-stenosis: 11 and two patients underwent two and three endovascular
treatments respectively. The annual relapse rate of MS was significantly lower
post-procedure (0.45±0.62 vs 0.76±0.99; p=0.021), although it increased in four
patients. The EDSS score two years after treatment was significantly lower
compared to the EDSS score recorded at the examination two years before
treatment (1.98±0.92 vs 2.27±0.93; p=0.037), although it was higher in four
patients. Endovascular treatment of concurrent CCSVI seems to be safe and
repeatable and may reduce annual relapse rates and cumulative disability in
patients with relapsing-remitting MS. Randomized controlled studies are needed
to further assess the clinical effects of endovascular treatment of CCSVI in MS. PURPOSE: This study proposed to prospectively evaluate safety and clinical
changes in outpatient endovascular treatment in patients with multiple sclerosis
(MS) and chronic cerebrospinal venous insufficiency (CCSVI).
MATERIALS AND METHODS: Two hundred fifty-nine patients with MS were followed
with the Multiple Sclerosis Impact Scale (MSIS-29) before and for 1 and 6 months
after treatment of extracranial internal jugular vein and azygos vein stenoses
and occlusions using venous angioplasty, as well as stent placement in 2.5% of
patients. Before treatment, the patients were tested with magnetic resoce
(MR) venography and flow quantification.
RESULTS: We found statistically significant improvements in the MSIS-29 scores
(P < .01) at both 1 and 6 months. At 1 and 6 months, 67.9% and 53.6% were
improved on the physical scale, respectively, and 53.0% and 44.4% were improved
on the psychological scale, respectively. Women showed greater improvement than
did men on the physical scale at 6 months (P = .01). Patients with primary
progressive MS (PPMS) showed less improvement than did those with
relapsing-remitting MS (RRMS) on the psychological scale at 1 month, and
venoplasty treatment of more vein sites versus fewer vein sites showed greater
improvement on the physical scale at both 1 and 6 months. Fifteen patients
(6.3%) reported recurrent symptoms after clinical improvement and were treated
again. There was one serious adverse event, a deep venous thrombosis at the
catheter insertion site, which resolved with treatment.
CONCLUSIONS: Endovascular treatment of CCSVI in patients with MS appears to be a
safe procedure resulting in significant clinical improvement. OBJECTIVE: Chronic cerebrospinal venous insufficiency (CCSVI) was recently
described in patients with multiple sclerosis (MS). The hypothesis of the
vascular aetiology provides a new approach in the investigation and treatment of
MS.
METHODS: Our open-label study included 94 MS patients who fulfilled ultrasound
sonographic criteria required for CCSVI. The internal jugular and/or azygous
veins by a catheter venography were dilated.
RESULTS: In 34.8% of the patients unilateral, in 65.2% bilateral venous
abnormalities and in 2.1% no luminal obstructions were demonstrated. The patient
group with the higher disability score had a significantly higher number of
venous lesions (P < 0.005). Significant improvement of clinical disability in
relapsing-remitting patients was (P < 0.001) achieved. In our study no stents
were used. Re-stenosis occurred in 21.7% of the patients.
CONCLUSION: The number of venous narrowings is higher in more disabled patients.
A significant improvement in clinical disability in the relapsing-remitting
group was observed. Although it is still debated whether chronic cerebro-spinal venous insufficiency
(CCSVI) plays a role in multiple sclerosis (MS) development, many patients
underwent endovascular treatment (ET) of CCSVI. The objective of the study is to
evaluate the outcome and safety of ET in Italian MS patients. Italian MS centers
that are part of the Italian MS Study Group were all invited to participate to
this retrospective study. A structured questionnaire was used to collect
detailed clinical data before and after the ET. Data from 462 patients were
collected in 33 centers. ET consisted of balloon dilatation (93 % of cases) or
stent application. The mean follow-up duration after ET was 31 weeks. Mean EDSS
remained unchanged after ET (5.2 vs. 4.9), 144 relapses occurred in 98/462 cases
(21 %), mainly in RR-MS patients. Fifteen severe adverse events were recorded in
3.2 % of cases. Given the risk of severe adverse events and the lack of
objective beneficial effects, our findings confirm that at present ET should not
be recommended to patients with MS. Although it is debated whether chronic cerebro-spinal venous insufficiency
(CCSVI) plays a role in multiple sclerosis (MS) development, many patients
undergo endovascular treatment (ET) of CCSVI. A study is ongoing in Italy to
evaluate the clinical outcome of ET. Severe adverse events (AEs) occurred in
15/462 subjects at a variable interval after ET: jugular thrombosis in seven
patients, tetraventricular hydrocephalus, stroke, paroxysmal atrial
fibrillation, status epilepticus, aspiration pneumonia, hypertension with
tachicardia, or bleeding of bedsore in the remaining seven cases. One patient
died because of myocardial infarction 10 weeks after ET. The risk of severe AEs
related to ET for CCSVI must be carefully considered. BACKGROUND: In 2009, Dr. Paolo Zamboni proposed chronic cerebrospinal venous
insufficiency (CCSVI) as a possible cause of multiple sclerosis (MS). Although
his theory and the associated treatment ("liberation therapy") received little
more than passing interest in the international scientific and medical
communities, his ideas became the source of tremendous public and political
tension in Canada. The story moved rapidly from mainstream media to social
networking sites. CCSVI and liberation therapy swiftly garnered support among
patients and triggered remarkable and relentless advocacy efforts. Policy makers
have responded in a variety of ways to the public's call for action.
DISCUSSION: We present three different perspectives on this evolving story, that
of a health journalist who played a key role in the media coverage of this
issue, that of a health law and policy scholar who has closely observed the
unfolding public policy developments across the country, and that of a medical
ethicist who sits on an expert panel convened by the MS Society of Canada and
the Canadian Institutes of Health Research to assess the evidence as it emerges.
SUMMARY: This story raises important questions about resource allocation and
priority setting in scientific research and science policy. The growing power of
social media represents a new level of citizen engagement and advocacy, and
emphasizes the importance of open debate about the basis on which such policy
choices are made. It also highlights the different ways evidence may be
understood, valued and utilized by various stakeholders and further emphasizes
calls to improve science communication so as to support balanced and informed
decision-making. PURPOSE: To investigate characteristics of cine phase contrast-calculated
cerebrospinal fluid (CSF) flow and velocity measures in patients with
relapsing-remitting (RR) multiple sclerosis (MS) receiving standard medical
treatment who had been diagnosed with chronic cerebrospinal venous insufficiency
(CCSVI) and underwent percutaneous transluminal angioplasty (PTA).
MATERIALS AND METHODS: This case-controlled, magnetic resoce (MR)
imaging-blinded study included 15 patients with RR MS who presented with
significant stenoses (≥50% lumen reduction on catheter venography) in the
azygous or internal jugular veins. Eight patients underwent PTA in addition to
medical therapy immediately following baseline assessments (case group) and
seven had delayed PTA after 6 months of medical therapy alone (control group).
CSF flow and velocity measures were quantified over 32 phases of the cardiac
cycle by a semiautomated method. Outcomes were compared between groups at
baseline and at 6 and 12 months of the study by mixed-effect model analysis.
RESULTS: At baseline, no significant differences in CSF flow or velocity
measures were detected between groups. At month 6, significant improvement in
flow (P<.001) and velocity (P = .013) outcomes were detected in the immediate
versus the delayed group, and persisted to month 12 (P = .001 and P = .021,
respectively). Within-group flow comparisons from baseline to follow-up showed a
significant increase in the immediate group (P = .033) but a decrease in the
delayed group (P = .024). Altered CSF flow and velocity measures were associated
with worsening of clinical and MR outcomes in the delayed group.
CONCLUSIONS: PTA in patients with MS with CCSVI increased CSF flow and decreased
CSF velocity, which are indicative of improved venous parenchyma drainage. |
Which hormone abnormalities are characteristic to Pendred syndrome? | Thyroid hormone abnormalities are characteristic to Pendred syndrome. Hypothyroidism is the most common thyroid hormone abnormality in Pendred syndrome. Pendred syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter and a partial defect in iodide organification. | Four families, 29 members, with Pendred's syndrome were studied to clarify
hearing loss and hormonal status. The ages ranged from 3 to 50 years. Complete
Pendred's syndrome was found in 9 patients. They had bilateral profound hearing
loss with residual hearing at low frequencies. Goiter was diagnosed at the age
of 1 to 14 years with a positive perchlorate discharge test. Twelve of the
patients' relatives showed partial Pendred's syndrome. Mild sensorineural
hearing losses occurred in the low- and medium-range frequencies with normal
perchlorate discharge test results in 6 cases. The other 6 had a slight drop in
the perchlorate discharge test results with normal hearing. Five subjects were
normal and 3 had normal hormonal and normal perchlorate discharge test results,
but were not tested audiologically. This paper shows that patients with
Pendred's syndrome may have goiter at birth or develop it between 8 and 14
years, that their deafness is bilateral and profound, and that their perchlorate
discharge tests are positive. Relatives of Pendred's syndrome patients showed
mild low-frequency sensorineural hearing loss without goiter and normal
perchlorate discharge test results in half the cases, and a slight drop in the
perchlorate discharge test results with normal hearing and without goiter in the
other half. A correlation between these findings and genetic studies needs
further investigation. OBJECTIVE: Pendred's syndrome is an association between congenital neurosensory
deafness and goitre with abnormal discharge of iodide following perchlorate
challenge, indicating a defect of iodide organification. Although Pendred's
syndrome may cause up to 7.5% of all cases of congenital deafness, the molecular
basis of the association between the hearing loss and the thyroid organification
defect remains unknown. We chose to investigate the role of the thyroid
peroxidase (TPO) gene as the genetic defect in Pendred's syndrome.
DESIGN: A highly informative variable number tandem repeat (VNTR), located 1.5
kb downstream of exon 10 of the TPO gene, was used to search for genetic linkage
in multiple sibships affected by Pendred's syndrome.
PATIENTS: Seven kindreds were recruited from the UK, each with at least two
affected members. We have also examined a large inbred Israeli family with two
affected offspring and five unaffected children.
MEASUREMENTS: Individuals were assigned affected status based on the
characteristic clinical features of Pendred's syndrome, namely the presence of
congenital sensorineural hearing loss and the appearance in early life of a
goitre. Additionally, at least one affected member from each sibship had a
characteristic positive perchlorate discharge test (Morgans & Trotter, 1958).
PCR amplification of genomic DNA at the TPO VNTR allowed assignment of genotypes
to each individual and the calculation of a two-point LOD score.
RESULTS: In six of the nine sibships analysed we found obligatory recombination
between TPO and Pendred's syndrome. Non-complementation observed in affected
parents with an affected offspring excluded TPO in an affected sibship with
genotype sharing and supports a hypothesis of genetic homogeneity for Pendred's
syndrome. In two sibships, mutation of the TPO gene as the cause of Pendred's
syndrome could not be excluded.
CONCLUSIONS: These data suggest that defects at the thyroid peroxidase locus on
chromosome 2 are not the major cause of Pendred's syndrome. Pendred syndrome is an autosomal recessive disorder characterized by the
association between sensorineural hearing loss and thyroid swelling or goitre
and is likely to be the most common form of syndromic deafness. Within the
thyroid gland of affected individuals, iodide is incompletely organified with
variable effects upon thyroid hormone biosynthesis, whilst the molecular basis
of the hearing loss is unknown. The PDS gene has been identified by positional
cloning of chromosome 7q31, within the Pendred syndrome critical linkage
interval and encodes for a putative ion transporter called pendrin. We have
investigated a cohort of 56 kindreds, all with features suggestive of a
diagnosis of Pendred syndrome. Molecular analysis of the PDS gene identified 47
of the 60 (78%) mutant alleles in 31 families (includes three homozygous
consanguineous kindreds and one extended family segregating three mutant
alleles). Moreover, four recurrent mutations accounted for 35 (74%) of PDS
disease chromosomes detected and haplotype analysis would favour common founders
rather than mutational hotspots within the PDS gene. Whilst these findings
demonstrate molecular heterogeneity for PDS mutations associated with Pendred
syndrome, this study would support the use of molecular analysis of the PDS gene
in the assessment of families with congenital hearing loss. Pendred syndrome is a recessive inherited disorder that consists of
developmental abnormalities of the cochlea, sensorineural hearing loss, and
diffuse thyroid enlargement (goiter). This disorder may account for up to 10% of
cases of hereditary deafness. The disease gene (PDS) has been mapped to
chromosome 7q22-q31, and encodes a chloride-iodide transport protein. We
performed mutation analysis of individual exons of the PDS gene in one Spanish
family that shows intrafamilial variability of the deafness phenotype (two
patients with profound and one with moderate-severe deafness). We identified a
new splice-site mutation affecting intron 4 of the PDS gene, at nucleotide
position 639+7. RNA analysis from lymphocytes of the affected patients showed
that mutation 639+7A-->G generates a new donor splice site, leading to an mRNA
with an insertion of six nucleotides from intron 4 of PDS. Since the newly
created donor splice site is likely to compete with the normal one, variations
of the levels of normal and aberrant transcripts of the PDS gene in the cochlea
may explain the variability in the deafness presentation. Primary congenital hypothyroidism is characterized by low levels of circulating
thyroid hormones and raised levels of thyrotropin at birth. It can be either
permanent or transitory. Most permanent cases (80-85%) result from alterations
in the formation of the thyroid gland during embryogenesis (thyroid dysgenesis),
and several were shown recently to be produced by mutations in genes responsible
for the development of thyroid follicular cells (TITF1, TITF2, PAX8 and TSHR).
Less frequently, congenital hypothyroidism is determined by defects in thyroid
hormone synthesis (hormonogenesis defects). The latter are usually associated
with goiter. Recently, the molecular mechanisms of two forms of hormonogenesis
defects (iodine transport defects and Pendred syndrome) were elucidated. Pendred syndrome is an autosomal recessive disorder characterized by congenital
deafness and thyroid goiter. The thyroid disease typically develops around
puberty and is associated with a mild organification defect, characterized by an
inappropriate discharge of iodide upon perchlorate stimulation (a positive
perchlorate discharge test). The gene (PDS) mutated in Pendred syndrome is
expressed in thyroid and encodes a 780-amino acid protein (pendrin) that has
recently been shown to function as an iodide/chloride transporter. We sought to
establish the location of pendrin in the thyroid and to examine the regulatory
network controlling its synthesis. Using peptide-specific antibodies for
immunolocalization studies, pendrin was detected in a limited subset of cells
within the thyroid follicles, exclusively at the apical membrane of the
follicular epithelium. Interestingly, significantly greater amounts of pendrin
were encountered in thyroid tissue from patients with Graves' disease. Using a
cultured rat thyroid cell line (FRTL-5), PDS expression was found to be
significantly induced by low concentrations of thyroglobulin (TG), but not by
TSH, sodium iodide, or insulin. This is different from the established effect of
TG, more typically a potent suppressor of thyroid-specific gene expression.
Together, these results suggest that pendrin is an apical porter of iodide in
the thyroid and that the expression and function of both the apical and basal
iodide porters are coordinately regulated by follicular TG. Although the textbook view of Pendred syndrome is that of an autosomal recessive
condition characterized by deafness and goitre, it is increasingly clear that
not all such patients present this classical clinical picture. Malformations of
the inner ear, specifically enlargement of the vestibular aqueduct, are common
in Pendred syndrome and mutations in the PDS (Pendred Syndrome) gene have been
recorded in patients presenting with deafness and vestibular aqueduct dilatation
only, without other features of Pendred syndrome. Since this is the most common
radiological malformation of the cochlea in deaf patients, we investigated what
proportion of such cases were due to mutation of the PDS gene. We assessed 57
patients referred with radiological evidence of vestibular aqueduct enlargement,
by history, clinical examination, perchlorate discharge test and molecular
analysis of the PDS locus. Forty-one patients (72%) had unequivocal evidence of
Pendred syndrome. The finding of a single heterozygous mutation at the PDS gene
in a further eight was strongly suggestive of a critical role for pendrin, the
protein product of the PDS gene, in the generation of enlarged vestibular
aqueducts in at least 86% (49/57 cases) of patients with this radiological
malformation. Securing the diagnosis of Pendred syndrome may be difficult,
especially in the single case. Goitre is an inconstant finding, and the
perchlorate discharge test, although helpful, is of diagnostic value only if
abnormal. Enlargement of the vestibular aqueduct should be considered as the
most likely presentation of Pendred syndrome and should prompt specific
investigation of that diagnostic possibility. Pendred syndrome might henceforth
be recharacterized as deafness with enlargement of the vestibular aqueduct,
which is sometimes associated with goitre. Pendred syndrome and the enlarged vestibular aqueduct (EVA) are considered
phenotypic variations of the same entity due to mutations in the SLC26A4
(pendrin) gene. Pendred syndrome consists in sensorineural deafness, goiter and
impaired thyroid hormone synthesis while in EVA thyroid function seems to be
preserved. The aim of this study was to evaluate thyroid function and morphology
and to look for mutations in the SLC26A4 gene in patients presented with EVA.
Among 57 consecutive patients with sensorineural deafness 15 with EVA, as
assessed by magnetic resoce imaging (MRI), were identified and studied. A
complete evaluation of thyroid function including thyroid echography and
perchlorate discharge test was carried out in all patients with EVA; all exons
of the SLC26A4 gene were amplified from peripheral leukocytes and directly
sequenced, using specific intronic primers. Out of 15 patients with EVA, goiter
was present in 8 (53%), hypothyroidism in 7 (47%), increased serum thyroglobulin
levels in 8 (53%) and a positive perchlorate discharge test in 10 (67%). Nine
alleles of the SLC26A4 gene were mutated: 2 novel mutations (L465W and G497R)
and 4 already known mutations (T410M, R409H, T505N and IVS1001+1G>A) were found.
Four subjects were compound heterozygous and 1 heterozygous (G497R/wt). All
patients harbouring mutations in the SLC26A4 gene had goiter and a positive
perchlorate discharge test: 3 were slightly hypothyroid and 2 euthyroid. The
remaining 10 patients had no mutations in the SLC26A4 gene: 4 of them were
hypothyroid, 2 with goiter and positive perchlorate discharge test, 2 without
goiter and with negative perchlorate discharge test. Two patients without
mutations were euthyroid with positive perchlorate discharge test. Patients with
mutations in the SLC26A4 gene had larger thyroid volume (p<0.002), higher serum
thyroglobulin (Tg) levels (p<0.002) and greater radioiodine discharge after
perchlorate (p=0.09) than patients without mutations. The results of the present
study lend support to the concept that all patients with mutated SLC26A4 gene
have abnormalities of thyroid function tests. Sensorineural hearing defect and goiter are common features of Pendred's
syndrome. The clinical diagnosis of Pendred's syndrome remains difficult because
of the lack of sensitivity and specificity of the thyroid signs. The
identification of PDS as the causative gene allowed molecular screening and
enabled a re-evaluation of the syndrome to identify potential diagnostic
characteristics. This report presents the clinical and genotypic findings of 30
French families, for whom a diagnosis of Pendred's syndrome had been made.
Twenty-seven families had at least one mutated allele. Twenty-eight different
mutations were identified, 11 of which had never been previously reported. The
main clinical characteristics were: early hearing loss, fluctuation in terms of
during deafness evolution, and the presence of an enlarged vestibular aqueduct. OBJECTIVE: To determine the proportion of the various types of thyroid disorders
among newborns detected by the neonatal TSH screening programme, with a normally
located thyroid gland. Patients and methods Of the 882 575 infants screened in
our centre between 1981 and 2002, 85 infants with a normally located gland had
persistent elevation of serum TSH values (an incidence of 1/10 383). Six of
these 85 patients were lost to follow-up and were therefore excluded from the
study. During follow-up, patients were classified as having permanent or
transient hypothyroidism.
RESULTS: Among the 79 patients included in the study, transient (n = 30, 38% of
cases) and permanent (n = 49, 62% of cases) congenital hypothyroidism (CH) was
demonstrated during the follow-up at the age of 0.7 +/- 0.6 years and 2.6 +/-
1.8 years (P < 0.0001), respectively. The proportion of premature births was
significantly higher in the group with transient CH (57%) than in the group with
permanent CH (2%) (P < 0.0001). A history of iatrogenic iodine overload was
identified during the neonatal period in 69% of transient cases. Among permanent
CH cases (n = 49), patients were classified as having a goitre (n = 27, 55% of
cases), a normal sized and shaped thyroid gland (n = 14, 29% of cases) or a
hypoplastic gland (n = 8, 16% of cases). The latter patients demonstrated global
thyroid hypoplasia (n = 3), a right hemithyroid (n = 2), hypoplasia of the left
lobe (n = 2), or asymmetry in the location of the two lobes (n = 1). Patients
with a normal sized and shaped thyroid gland showed a significantly less severe
form of hypothyroidism than those with a goitre or a hypoplastic thyroid gland
(P < 0.0002). Among permanent CH cases, those with a goitre (n = 27) had an
iodine organification defect (n = 10), Pendred syndrome (n = 1), a defect of
thyroglobulin synthesis (n = 8), or a defect of sodium iodine symporter (n = 1),
and in seven patients no aetiology could be determined. Among permanent cases
with a normal sized and shaped thyroid gland (n = 14), a specific aetiology was
found in only one patient (pseudohypoparathyroidism) and two patients had Down's
syndrome. Among those with a globally hypoplastic gland, a TSH receptor gene
mutation was found in two patients.
CONCLUSIONS: A precise description of the phenotype can enhance our
understanding of various forms of neonatal hypothyroidism as well as their
prevalence and management. It also helps to identify cases of congenital
hypothyroidism of unknown aetiology, which will need to be investigated in
collaboration with molecular biologists. Congenital hypothyroidism is the most common neonatal metabolic disorder and
results in severe neurodevelopmental impairment and infertility if untreated.
Congenital hypothyroidism is usually sporadic but up to 2% of thyroid dysgenesis
is familial, and congenital hypothyroidism caused by organification defects is
often recessively inherited. The candidate genes associated with this
genetically heterogeneous disorder form two main groups: those causing thyroid
gland dysgenesis and those causing dyshormonogenesis. Genes associated with
thyroid gland dysgenesis include the TSH receptor in non-syndromic congenital
hypothyroidism, and Gsalpha and the thyroid transcription factors (TTF-1, TTF-2,
and Pax-8), associated with different complex syndromes that include congenital
hypothyroidism. Among those causing dyshormonogenesis, the thyroid peroxidase
and thyroglobulin genes were initially described, and more recently PDS (Pendred
syndrome), NIS (sodium iodide symporter), and THOX2 (thyroid oxidase 2) gene
defects. There is also early evidence for a third group of congenital
hypothyroid conditions associated with iodothyronine transporter defects
associated with severe neurological sequelae. This review focuses on the genetic
aspects of primary congenital hypothyroidism. Perchlorate has been detected in groundwater in many parts of the United States,
and recent detection in vegetable and dairy food products indicates that
contamination by perchlorate is more widespread than previously thought.
Perchlorate is a competitive inhibitor of the sodium iodide symporter, the
thyroid cell-surface protein responsible for transporting iodide from the plasma
into the thyroid. An estimated 4.3% of the U.S. population is subclinically
hypothyroid, and 6.9% of pregt women may have low iodine intake. Congenital
hypothyroidism affects 1 in 3,000 to 1 in 4,000 infants, and 15% of these cases
have been attributed to genetic defects. Our objective in this review is to
identify genetic biomarkers that would help define subpopulations sensitive to
environmental perchlorate exposure. We review the literature to identify genetic
defects involved in the iodination process of the thyroid hormone synthesis,
particularly defects in iodide transport from circulation into the thyroid cell,
defects in iodide transport from the thyroid cell to the follicular lumen
(Pendred syndrome), and defects of iodide organification. Furthermore, we
summarize relevant studies of perchlorate in humans. Because of perchlorate
inhibition of iodide uptake, it is biologically plausible that chronic ingestion
of perchlorate through contaminated sources may cause some degree of iodine
discharge in populations that are genetically susceptible to defects in the
iodination process of the thyroid hormone synthesis, thus deteriorating their
conditions. We conclude that future studies linking human disease and
environmental perchlorate exposure should consider the genetic makeup of the
participants, actual perchlorate exposure levels, and individual iodine
intake/excretion levels. For over 100 years after the first description of the disorder, the molecular
pathology underlying the deafness and thyroid pathology in Pendred syndrome (PS)
remained unknown. In 1997, early progress towards understanding the molecular
basis of the disorder was made when we identified the PS gene and found it to
belong to the SLC26 family of anion transporters. The realization that an anion
transporter was responsible for these clinical features soon highlighted a
potential role for pendrin in thyroid hormone biosynthesis. The role of pendrin
in deafness, however, remained unclear. Our determination of its expression
pattern in the inner ear along with the development of a mouse with a targeted
disruption of the Slc26a4 gene has revealed that Slc26a4 is expressed in areas
of the endolymphatic compartment known to play a role in endolymph reabsorption
and that absence of this protein leads to a profound prenatal endolymphatic
hydrops and destruction of many of the epithelial cells surrounding the scala
media. The precise mechanisms underlying endolymph reabsorption in the inner ear
are not yet known; these studies, however, provide some of the groundwork for
allowing the future delineation of these processes. Inherited as an autosomal recessive trait, Pendred syndrome is a disease that
shows congenital sensorineural hearing loss and goiter, with a positive finding
in the perchlorate discharge test. Pendred syndrome results from various
mutations in the PDS/SLC26A4 gene that cause production of an abnormal pendrin
protein. More than 90 mutations in the PDS/SLC26A4 gene have been reported
throughout the world. A recent study of 26 Korean patients with a relatively
high frequency (65%) of a mutated PDS/SLC26A4 gene exhibited nonsyndromic
deafness and an enlarged vestibular aqueduct. We report two patients with
characteristics of typical Pendred syndrome, a 26-yr-old female and a 61-yr-old
male, who were both homozygous for a previously reported missense mutation,
H723R (Histidine 723Arginine) in the PDS/SLC26A4 gene. Pendred syndrome, defined as the constellation of goiter, sensori-neural hearing
loss, and positive perchlorate discharge test, is the most frequent cause of
congenital deafness. Newly introduced diagnostic approaches to the disease are
rather expensive and complicated, therefore we evaluated the value of MRI as the
sole, or adjunctive diagnostic approach, and compared it with the traditional
ones. Presuming the classic triad as the gold standard, we compared MRI findings
in six such defined patients with six cases having goiter, hearing loss, and
normal perchlorate discharge test. Our results indicated that MRI was 83.6%
sensitive and 66.7% specific in patients fulfilling all three criteria
(complete), while in the 'partial' group the sensitivity and specificity were
66.7% and 100% respectively. In conclusion, MRI, although impressive as an
adjunctive diagnostic tool, may not replace the holistic approach, and the
latter may be more convenient, cheaper, and still more accurate. However in
'partial' cases with equivocal findings, and in relatives of the patients, MRI
may be a valuable diagnostic adjunct. Pendred syndrome (PS) is an autosomal recessive disease that is characterized by
congenital sensorineural hearing loss, goiter, and a partial iodine
organification defect. In this study, we characterized the thyroid status and
identified mutations in the SLC26A4 gene in Chinese subjects with PS. We
evaluated 7 unrelated Chinese subjects who had PS. Biochemical analysis, formal
audiogram, ultrasonography of the thyroid gland, perchlorate discharge test,
computerized tomography scan of the vestibular aqueducts, and DNA sequence
analysis of SLC26A4 were performed. Levels of thyroid hormones were essentially
normal in all patients: 2 patients had goiters and/or elevated serum
thyroglobulin levels, whereas 2 other patients had positive thyroid antibodies
and a positive perchlorate discharge test. We identified SLC26A4 gene mutations
in 6 of 7 probands and their affected relatives. The affected subjects in family
I was compound heterozygous for 2 missense mutations: a mutation in exon 9
(1079C>T) that resulted in the replacement of alanine by valine at codon 360
(A360V) and a mutation in exon 19 (2168A>G) that resulted in the replacement of
histidine by arginine at codon 723 (H723R). The affected subjects in families II
and III all were homozygous for a mutation in intron 7. The probands IV and V
were compound heterozygotes for the mutation in intron 7 and in exon 19, and the
proband VI was compound heterozygous for the intron 7 mutation and a missense
mutation in exon 12 (1343C>T) that resulted in the replacement of serine by
leucine at codon 448 (S448L). One novel mutation was identified (A360V). We
identified biallelic mutations in the SLC26A4 gene in 6 of 7 probands with PS in
Taiwan, including a novel missense mutation. The mild thyroid dysfunction in
these patients suggests that PS should be considered in all patients with
congenital or early-onset hearing impairment. Pendred syndrome is a recessive autosomal disorder characterized by thyroid
goiter and sensorineural hearing loss. The Pendred syndrome gene (SLC26A4)
encodes a new anion exchanger named pendrin which mediates iodide transport by
thyrocytes and regulates ion and fluid transport by the endolymphatic sac
epithelium. Pendrin defects result in inner ear malformations, with enlargement
of the endolymphatic sac and duct in association with a large vestibular
aqueduct. Furthermore, patients may develop endolymphatic hydrops requiring
diuretic therapy, mainly in the form of thiazides. Pendrin could also account
for apical Cl(-)/ HCO3(-) exchange at level of intercalated cells of the
cortical collecting duct in the kidneys, however, humans with Pendred syndrome
have no symptoms attributable to renal pendrin abnormalities in basal
conditions. We report the case of a child with Pendred syndrome and intercurrent
endolymphatic hydrops, who developed profound hypokalemia and severe
hypochloremic metabolic alkalosis (potassium 1.7, chloride 70, sodium 129, HCO3
43.8, base excess +17.8 mmol/l, pH 7.52) following thiazide therapy. In subjects
with Pendred syndrome thiazide therapy seems to provoke more severe Cl(-) and
extracellular volume depletion. A possible explanation could be the defective
action of the disrupted pendrin, which exacerbates the effects of the inhibition
of C1(-) reabsorption mediated by the thiazide-sensitive NaCl cotransporter
(SLC12A3). Pendred syndrome is an autosomal recessive disorder characterized by
sensorineural hearing impairment, presence of goiter, and a partial defect in
iodide organification, which may be associated with insufficient thyroid hormone
synthesis. Goiter development and development of hypothyroidism are variable and
depend on nutritional iodide intake. Pendred syndrome is caused by biallelic
mutations in the SLC26A4 gene, which encodes pendrin, a transporter of chloride,
bicarbonate and iodide. This review discusses the controversies surrounding the
potential role of pendrin in mediating apical iodide efflux into the lumen of
thyroid follicles, and discusses its functional role in the kidney and the inner
ear. We report a young woman with genetically confirmed Pendred syndrome and discuss
the current therapeutic strategies of dyshormonogenetic goitre. A small diffuse
thyroid enlargement developed during infancy and although substitution therapy
with L-thyroxine was adequate, it progressed and underwent multinodular
transformation. Cervical ultrasound at the age of 22 years demonstrated three
solid nodules and fine-needle aspiration biopsy showed a finding typical of
follicular adenoma. It is known that dyshormonogenetic goitres have a tendency
to grow despite appropriate treatment with L-thyroxine. Management of a patient
with Pendred syndrome requires careful follow-up and regular imaging of the
thyroid. Although the therapeutic approach to dyshormonogenetic goitres is still
controversial, in our patient we chose total thyroidectomy as the most
advantageous method to prevent the development of maligcies that may arise
more frequently from dyshormonogenetic goitres than from goitres of other
aetiologies. Thyroid hormones are essential for normal development and metabolism. Thyroid
hormone biosynthesis requires iodide uptake into the thyrocytes and efflux into
the follicular lumen, where it is organified on selected tyrosyls of
thyroglobulin. Uptake of iodide into the thyrocytes is mediated by an intrinsic
membrane glycoprotein, the sodium-iodide symporter (NIS), which actively
cotransports two sodium cations per each iodide anion. NIS-mediated transport of
iodide is driven by the electrochemical sodium gradient generated by the
Na(+)/K(+)-ATPase. NIS is expressed in the thyroid, the salivary glands, gastric
mucosa, and the lactating mammary gland. TSH and iodide regulate iodide
accumulation by modulating NIS activity via transcriptional and
posttranscriptional mechanisms. Biallelic mutations in the NIS gene lead to a
congenital iodide transport defect, an autosomal recessive condition
characterized by hypothyroidism, goiter, low thyroid iodide uptake, and a low
saliva/plasma iodide ratio. Pendrin is an anion transporter that is
predomitly expressed in the inner ear, the thyroid, and the kidney. Biallelic
mutations in the SLC26A4 gene lead to Pendred syndrome, an autosomal recessive
disorder characterized by sensorineural deafness, goiter, and impaired iodide
organification. In thyroid follicular cells, pendrin is expressed at the apical
membrane. Functional in vitro data and the impaired iodide organification
observed in patients with Pendred syndrome support a role of pendrin as an
apical iodide transporter. INTRODUCTION: The Pendred syndrome (PS) is an autosomally recessively inherited
disease. Its diagnosis requires identification of the classical triad of
symptoms, including hypoacusis, thyroid goitre and iodine organification defect
in the thyroid, which may lead to thyroid functional disorders of
hypothyroidism. SP is accompanied by anatomical anomalies. The objective is the
hearing and balance system evaluation and the analysis of the inner ear
structure and also the assessment of the function and structure of thyroid
gland.
MATERIAL AND METHODS: For the research four families were qualified, 7 persons
with PS, 12 persons altogether. In all the patients the anamnesis in the form of
a questionnaire and laryngological examination were performed. It was followed
by pure tone, speech and impedance audiometry and brainstem response testing as
well. ENG was also conducted. Patients with hearing loss were subjected to
magnetic resoce of temporal bone. For the whole group thyroid hormones levels
and iodine organification in the thyroid identified in a test with potassium
perchlorate were measured and also USG and scyntography were conducted.
RESULTS: In audiological examination in 3 cases deafness, in 2 cases profound
hypoacusis and in 2 mild hypoacusis were recognised. In the group in 2 patients
the hypoacusis was of a mixed type. In radiological assessment the labirynth
showed anatomical anomalies in the form of enlargement of the vestibular
aqueduct and the endolyphatic sac, yet in 3 patients the anomalies also
concerned the structure of cochlear and semicircular canals. Endocrine
examination showed hypothyroidism in 5, its subclinical form in 1, diffuse
thyroid goitre in 4 and nodular thyroid goiter in 2 cases.
CONCLUSIONS: A complex clinical evaluation: endocrine and audiological, together
with radiological diagnostic imaging, supported by molecular studies of SLC26A4
gene, are the procedures, necessary for complete and accurate diagnosis of PS
and EVAS. CONTEXT: Goiter and deafness can be associated in some genetic syndromes, e.g.
Pendred syndrome (PS) and resistance to thyroid hormone (RTH). PS is an
autosomal recessive disorder characterized by goiter and sensorineural hearing
impairment with an enlarged vestibular aqueduct bilaterally. RTH is an autosomal
domit condition of reduced tissue sensitivity to thyroid hormone in which
goiter is very frequent and hearing loss occurs in about 20% of patients.
OBJECTIVE, PATIENTS, AND DESIGN: The objective of this study was to identify the
cause of goiter and deafness in two sisters born to healthy unrelated parents.
We present their history, clinical presentation, and follow-up and report the
results of molecular genetic investigations.
RESULTS: The elder sister had an elevated TSH level at newborn screening
followed by subclinical hypothyroidism, childhood-onset goiter, and bilateral
progressive sensorineural hearing impairment with enlarged vestibular aqueducts,
consistent with a diagnosis of PS. Her younger sister had congenital goiter,
elevated free T3 and free T4 concentrations with unsuppressed TSH, sinus
tachycardia, and bilateral progressive sensorineural hearing impairment with
enlarged vestibular aqueducts. This clinical presentation was consistent with a
diagnosis of RTH, in which, however, inner ear malformations are uncommon.
Interestingly, molecular genetic testing showed that, whereas the elder sister
is affected by PS, the younger sister has both PS (due to compound heterozygous
SLC26A4 mutations) and RTH (due to a novel de novo heterozygous THRB mutation).
CONCLUSIONS: This is the first report of the cooccurrence, in the same
individual, of PS and RTH, two genetic syndromes both associated with goiter and
hearing impairment. Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic
deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an
anion exchanger located in the cochlea, thyroid, and kidney. The goal of the
present study was to determine whether developmental delays, possibly mediated
by systemic or local hypothyroidism, contribute to the failure to develop
hearing in mice lacking Slc26a4 (Slc26a4(-/-)). We evaluated thyroid function by
voltage and pH measurements, by array-assisted gene expression analysis, and by
determination of plasma thyroxine levels. Cochlear development was evaluated for
signs of hypothyroidism by microscopy, in situ hybridization, and quantitative
RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4(-/-) and
sex-matched Slc26a4(+/-) littermates between postnatal day 5 (P5) and P90. In
adult Slc26a4(-/-) mice, the transepithelial potential and the pH of thyroid
follicles were reduced. No differences in the expression of genes that
participate in thyroid hormone synthesis or ion transport were observed at P15,
when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold
enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to
generate a cochlear thyroid hormone peak at P7. Cochlear development, including
tunnel opening, arrival of efferent innervation at outer hair cells,
endochondral and intramembraneous ossification, and developmental changes in the
expression of Dio2, Dio3, and Tectb were delayed by 1-4 days. These data suggest
that pendrin functions as a HCO3- transporter in the thyroid, that Slc26a4(-/-)
mice are systemically euthyroid, and that delays in cochlear development,
possibly due to local hypothyroidism, lead to the failure to develop hearing. CONTEXT: Pendred syndrome is caused by mutations in the gene coding for pendrin,
an apical Cl-/I- exchanger.
OBJECTIVE: To analyze intrathyroidal compensatory mechanisms when pendrin is
lacking, we investigated the thyroid of a patient with Pendred syndrome. The
expression of proteins involved in thyroid hormone synthesis, markers of
oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes
were analyzed.
RESULTS: Three morphological zones were identified: nearly normal follicles with
iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without
iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone
2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were
localized at the apical pole, OS and cell apoptosis were absent, but ClC-5
expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly
present and increased in the cytosol and associated with high OS, apoptosis,
cell proliferation, and increased expression of peroxiredoxin-5, catalase, and
dehalogenase-1 but moderate ClC-5 expression.
CONCLUSION: In conclusion, the absence of pendrin is accompanied by increased
ClC-5 expression that may transiently compensate for apical iodide efflux. In
more affected follicles, Duox and TPO are relocated in the cytosol, leading to
abnormal intracellular thyroid hormone synthesis, which results in cell
destruction presumably because intracellular OS cannot be buffered by
antioxidant defenses. Biallelic mutations of SLC26A4 (encoding pendrin) cause Pendred syndrome (PS),
an autosomal recessive genetic disorder with deafness and goiter. The mechanism
underlying the development of the goiter is unknown. Here, we report clinical
and molecular findings of a patient with PS. This 27-year-old woman was born to
nonconsanguineous healthy parents. She was seen at our hospital due to hearing
loss at age 3 years, and subsequently developed goiter at age 10 years. From age
15 years, her thyroid gland showed progressive enlargement accompanied by
elevation of serum thyroglobulin reaching 10-fold the normal amount. Thyroidal
iodine uptake was also increased during goiter progression ((123)I uptake at 24
hr: 20.2% at age 17 years; 69.4% at age 24 years; reference, 8-40), while serum
thyrotropin (TSH) levels and iodine organification (examined by the perchrolate
or thiocyanate discharge test) remained normal. We sequenced SLC26A4 using
standard PCR-based technique, and found one novel (p.T537P) and one recurrent
(p.H723R) mutations in a compound heterozygous state. Expression experiments
using COS-7 cells showed that the two mutants were entrapped in the endoplasmic
reticulum and were poorly localized at the plasma membrane. In summary, a
molecularly confirmed PS patient showed goiter progression accompanied by
elevated serum thyroglobulin and increased thyroidal iodine uptake, but normal
serum TSH levels and normal iodine organification. This implies that some
pendrin mutations may involve direct stimulation of thyroid cell proliferation
with no TSH hyperstimulation and no iodine organification defect. Pendred syndrome is an autosomal recessive disorder defined by sensorineural
deafness, goiter and a partial defect in the organification of iodide. It is
caused by biallelic mutations in the SLC26A4 gene, which encodes pendrin, a
multifunctional anion exchanger. At the level of the inner ear, pendrin is
important for the creation of a normal endolymph composition and the maintece
of the endocochlear potential. In the thyroid, pendrin is expressed at the
apical membrane of thyroid follicular cells and it appears to be involved in
mediating iodide efflux into the lumen and/or maintece of the follicular pH.
Goiter development and hypothyroidism vary among affected individuals and seem
to be partially dependent on nutritional iodide intake. In the kidney, pendrin
functions as a chloride/bicarbonate exchanger. Elucidation of the molecular
basis of Pendred syndrome and the function of pendrin has provided unexpected
novel insights into the pathophysiology of the inner ear, thyroid hormone
synthesis, and chloride/bicarbonate exchange in the kidney. Thyroid hormones are essential for normal development and metabolism. Their
synthesis requires transport of iodide into thyroid follicles. The mechanisms
involving the apical efflux of iodide into the follicular lumen are poorly
elucidated. The discovery of mutations in the SLC26A4 gene in patients with
Pendred syndrome (congenital deafness, goiter, and defective iodide
organification) suggested a possible role for the encoded protein, pendrin, as
an apical iodide transporter. We determined whether TSH regulates pendrin
abundance at the plasma membrane and whether this influences iodide efflux.
Results of immunoblot and immunofluorescence experiments reveal that TSH and
forskolin rapidly increase pendrin abundance at the plasma membrane through the
protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin
membrane abundance correlates with a decrease in intracellular iodide as
determined by measuring intracellular (125)iodide and can be inhibited by
specific blocking of pendrin. Elimination of the putative protein kinase A
phosphorylation site T717A results in a diminished translocation to the membrane
in response to forskolin. These results demonstrate that pendrin translocates to
the membrane in response to TSH and suggest that it may have a physiological
role in apical iodide transport and thyroid hormone synthesis. BACKGROUND/AIMS: Pendrin (SLC26A4), a transporter accomplishing anion exchange,
is expressed in inner ear, thyroid gland, kidneys, lung, liver and heart. Loss
or reduction of function mutations of SLC26A4 underlie Pendred syndrome, a
disorder invariably leading to hearing loss with enlarged vestibular aqueducts
and in some patients to hypothyroidism and goiter. Renal pendrin expression is
up-regulated by mineralocorticoids such as aldosterone or deoxycorticosterone
(DOCA). Little is known about the impact of mineralocorticoids on pendrin
expression in extrarenal tissues.
METHODS: The present study utilized RT-qPCR and Western blotting to quantify the
transcript levels and protein abundance of Slc26a4 in murine kidney, thyroid,
heart and lung prior to and following subcutaneous administration of 100 mg/kg
DOCA.
RESULTS: Slc26a4 transcript levels as compared to Gapdh transcript levels were
significantly increased by DOCA treatment in kidney, heart, lung and thyroid.
Accordingly pendrin protein expression was again significantly increased by DOCA
treatment in kidney, heart, lung and thyroid.
CONCLUSION: The observations reveal mineralocorticoid sensitivity of pendrin
expression in kidney, heart, thyroid and lung. |
What is the mode of inheritance of long QT Jervell and Lange-Nielsen syndrome? | Jervell and Lange-Nielsen long QT syndrome (JLNS) is characterized by autosomal recessive mode of inheritance | Jervell and Lange-Nielsen syndrome is an autosomal recessive hereditary
condition that presents with cardiac abnormalities characterized by a prolonged
Q-T electrocardiographic pattern and congenital severe-to-profound auditory
deficits. This paper describes the auditory history of twin boys born out of
consanguinity and diagnosed with this syndrome. Both infants were products of
the neonatal intensive care unit (NICU) and failed initial ABR screening.
Diagnostic evaluation demonstrated profound hearing loss and developmental
delays for each infant. Because sudden death is a consequence, audiologists are
advised to recognize signs and symptoms associated with this syndrome. Deafness and electrocardiographic changes (prolongation of the Q-T interval and
inversion of the T wave) with a clinical picture of syncopal attacks and sudden
death, were described as a distinct syndrome by Jervell and Lange-Nielsen in
1957. The syndrome is inherited as an autosomal recessive trait. In this study,
all the cases reported since 1957 and their proposed prevalence are reviewed.
The authors describe the 4 cases they have studied, all of which presented
congenital sensorineural hearing loss and electrocardiographic changes
characteristic of the syndrome. The relatively high number of cases they have
encountered casts doubt on literature that states that the syndrome occurs more
frequently in Northern-European populations. Consequently, it is advisable to
perform an electrocardiogram in all children affected by congenital deafness. Jervell Lange-Nielsen syndrome (JLNS) is a recessive disorder with congenital
deafness and long-QT syndrome (LQTS 1). Mutations in the potassium-channel gene
KVLQT1 (LQTS 1) have been identified in JLNS and in autosomal-domit LQTS as
well. We performed haplotype analysis with microsatellite markers in a Lebanese
family with JLNS, but failed to detect linkage at LQTS 1. Moreover, using this
approach, we excluded two other ion-channel genes involved in autosomal-domit
LQTS, HERG (LQTS 2) and SCN5A (LQTS 3). Our findings indicate that JLNS is
genetically heterogeneous and that, in this family, an unknown LQTS gene causes
the disease. The LQT1 locus (KCNQ1) has been correlated with the most common form of
inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and
high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which
together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K(+) channels.
Mutant KvLQT1 subunits may be associated either with an autosomal domit form
of inherited LQT, Romano-Ward syndrome, or an autosomal recessive form, Jervell
and Lange-Nielsen syndrome (JLNS). We have identified a small domain between
residues 589 and 620 in the KvLQT1 C-terminus, which may function as an assembly
domain for KvLQT1 subunits. KvLQT1 C-termini do not assemble and KvLQT1 subunits
do not express functional K(+) channels without this domain. We showed that a
JLN deletion-insertion mutation at KvLQT1 residue 544 eliminates important parts
of the C-terminal assembly domain. Therefore, JLN mutants may be defective in
KvLQT1 subunit assembly. The results provide a molecular basis for the clinical
observation that heterozygous JLN carriers show slight cardiac dysfunctions and
that the severe JLNS phenotype is characterized by the absence of KvLQT1
channel. Jervell and Lange-Nielsen syndrome (MIM 220400; JLNS), is a rare form of
profound congenital deafness combined with syncopal attacks and sudden death due
to prolonged QTc; it is an autosomal recessive trait. After its first
description in Norway in 1957, later reports from many other countries have
confirmed its occurrence. Nowhere is the prevalence so high as in Norway, where
we estimate a prevalence of at least 1:200,000. The KCNQ1 and KCNE1 proteins
coassemble in a potassium channel, and mutations in either the KCNQ1 gene or the
KCNE1 gene disrupt endolymph production in the stria vascularis in the cochlea,
causing deafness. KCNQ1 seems to be the major gene in JLNS. Long QT syndrome
(LQTS) is a separate disorder of either autosomal domit or recessive
inheritance caused by mutations in four different ion channel genes; KCNQ1 is
the one most frequently involved. Some heterozygous carriers of JLNS mutations
in either gene may suffer from prolonged QTc and be symptomatic LQTS patients
with a need for appropriate medical treatment to prevent life-threatening
cardiac arrhythmia. In general, frameshift/stop mutations cause JLNS, and
missense/splice site mutations cause LQTS, but a precise genotype-phenotype
correlation in LQTS and JLNS is not established, which complicates both genetic
counseling and clinical risk evaluation in carriers. We review JLNS from a
Norwegian perspective because of the unusually high prevalence, the genetic
homogeneity associated with considerable mutational heterogeneity, and some
evidence for recurrent mutational events as well as one founder mutation. We
outline the clinical implications for investigation of deaf children and cases
of sudden infant death syndrome as well as careful electrocardiographic
monitoring of identified mutation carriers to prevent sudden death. Am. J. Med.
Genet. (Semin. Med. Genet.) 89:137-146, 1999. In contrast to the Romano-Ward (R-W) syndrome, the Jervell and Lange-Nielsen
(J-LN) syndrome is an autosomal recessive inherited disease characterized by
QT-prolongation in the electrocardiogram (ECG) and recurrent syncopal attacks
which are also typical for the R-W syndrome, but also by congenital deafness.
Recently, defect alleles in the genes for KCNQ1 and KCNE1 have been identified
in patients with the J-LN syndrome. These genes may be causative for the R-W
syndrome as well but in J-LN patients, they are only present in the homozygote
or compound heterozygote form. In the present paper, we review the clinical and
genetic similarities and differences of the J-LN and the R-W syndrome as well as
the diagnostic and therapeutic management of these patients and their family
members. 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. BACKGROUND- Homozygous or compound heterozygous mutations in KCNQ1 cause Jervell
and Lange-Nielsen syndrome, a rare, autosomal-recessive form of long-QT syndrome
characterized by deafness, marked QT prolongation, and a high risk of sudden
death. However, it is not understood why some individuals with mutations on both
KCNQ1 alleles present without deafness. In this study, we sought to determine
the prevalence and genetic determits of this phenomenon in a large referral
population of patients with long-QT syndrome. METHODS AND RESULTS- A
retrospective analysis of all patients with long-QT syndrome evaluated from July
1998 to April 2012 was used to identify those with ≥1 KCNQ1 mutation. Of the 249
KCNQ1-positive patients identified, 15 (6.0%) harbored a rare putative
pathogenic mutation on both KCNQ1 alleles. Surprisingly, 11 of these patients
(73%) presented without the sensorineural deafness associated with Jervell and
Lange-Nielsen syndrome. The degree of QT-interval prolongation and the number of
breakthrough cardiac events were similar between patients with and without
deafness. Interestingly, truncating mutations were more prevalent in patients
with Jervell and Lange-Nielsen syndrome (79%) than in nondeaf patients (36%;
P<0.001) derived from this study and those in the literature. CONCLUSIONS- In
this study, we provide evidence that the recessive inheritance of a severe
long-QT syndrome type 1 phenotype in the absence of an auditory phenotype may
represent a more common pattern of long-QT syndrome inheritance than previously
anticipated and that these cases should be treated as a higher-risk long-QT
syndrome subset similar to their Jervell and Lange-Nielsen syndrome
counterparts. Furthermore, mutation type may serve as a genetic determit of
deafness, but not cardiac expressivity, in individuals harboring ≥1 KCNQ1
mutation on each allele. Long QT syndrome is one of the most common cardiac ion channel diseases, but its
morbidity and mortality rate can be lessened with an early diagnosis and proper
treatment. This cardiac ventricular repolarization abnormality is characterized
by a prolonged QT interval and a propensity for ventricular tachycardia (VT) of
the torsades de pointes type. The long QT syndrome represents a high risk for
presyncope, syncope, cardiac arrest, and sudden death. Jervell and Lange-Nielsen
syndrome (JLNS) is a recessively inherited form of long QT syndrome
characterized by profound sensorineural deafness and prolongation of the QT
interval. Findings have shown that JLNS occurs due to homozygous and compound
heterozygous pathogenic variants in KCNQ1 or KCNE1. A 3.5-year-old girl
presented to the hospital with recurrent syncope, seizures, and congenital
sensorineural deafness. Her electrocardiogram showed a markedly prolonged QT
interval, and she had a diagnosis of JLNS. The sequence analysis of the proband
showed the presence of a pathogenic homozygous missense variant (c.728G>A,
p.Arg243His). Heterozygous mutations of KCNQ1 were identified in her mother,
father, and sister, demonstrating true homozygosity. Even with high-dose
beta-blocker therapy, the patient had two VT attacks, so an implantable
cardioverter defibrillator was fitted. The authors suggest early genetic
diagnosis for proper management of the disease in the proband and genetic
counseling for both the proband and the girl's extended family. |
List programs suitable for protein docking | Macromolecular docking is the computational modelling of the quaternary structure of complexes formed by two or more interacting biological macromolecules. Protein–protein complexes are the most commonly attempted targets of such modelling, followed by protein–nucleic acid complexes.
The ultimate goal of docking is the prediction of the three-dimensional structure of the macromolecular complex of interest as it would occur in a living organism. Docking itself only produces plausible candidate structures. These candidates must be ranked using methods such as scoring functions to identify structures that are most likely to occur in nature.
Nowadays there a lot of programs suitable for proteins docking such as CSBB-ConeExclusion, HADDOCK, ZDOCK, GalaxyDock, PHASE, DockRank, HotLig, SOL, AutodockVina, DockoMatic, DockoMatic, DockTrina, CAVITY, LiGenDock and DOCK. | Predicting protein-protein and protein-ligand docking remains one of the
challenging topics of structural biology. The main problems are (i) to reliably
estimate the binding free energies of docked states, (ii) to enumerate possible
docking orientations at a high resolution, and (iii) to consider mobility of the
docking surfaces and structural rearrangements upon interaction. Here we present
a novel algorithm, TreeDock, that addresses the enumeration problem in a
rigid-body docking search. By representing molecules as multidimensional binary
search trees and by exploring a sufficient number of docking orientations such
that two chosen atoms, one from each molecule, are always in contact, TreeDock
is able to explore all clash-free orientations at very fine resolution in a
reasonable amount of time. Due to the speed of the program, many contact pairs
can be examined to search partial or complete surface areas. The deterministic
systematic search of TreeDock is in contrast to most other docking programs that
use stochastic searches such as Monte Carlo or simulated annealing methods. At
this point, we have used the Lennard-Jones potential as the only scoring
function and show that this can predict the correct docked conformation for a
number of protein-protein and protein-ligand complexes. The program is most
powerful if some information is known about the location of binding faces from
NMR chemical-shift perturbation studies, orientation information from residual
dipolar coupling, or mutational screening. The approach has the potential to
include docking-site mobility by performing molecular dynamics or other
randomization methods of the docking site and docking families to families of
structures. The performance of the algorithm is demonstrated by docking three
complexes of immunoglobulin superfamily domains, CD2 to CD58, the V(alpha)
domain of a T-cell receptor to its V(beta) domain, and a T-cell receptor to a
pMHC complex as well as a small molecule inhibitor to a phosphatase. ClusPro (http://nrc.bu.edu/cluster) represents the first fully automated,
web-based program for the computational docking of protein structures. Users may
upload the coordinate files of two protein structures through ClusPro's web
interface, or enter the PDB codes of the respective structures, which ClusPro
will then download from the PDB server (http://www.rcsb.org/pdb/). The docking
algorithms evaluate billions of putative complexes, retaining a preset number
with favorable surface complementarities. A filtering method is then applied to
this set of structures, selecting those with good electrostatic and desolvation
free energies for further clustering. The program output is a short list of
putative complexes ranked according to their clustering properties, which is
automatically sent back to the user via email. The Fast Fourier Transform (FFT) correlation approach to protein-protein docking
can evaluate the energies of billions of docked conformations on a grid if the
energy is described in the form of a correlation function. Here, this
restriction is removed, and the approach is efficiently used with pairwise
interaction potentials that substantially improve the docking results. The basic
idea is approximating the interaction matrix by its eigenvectors corresponding
to the few domit eigenvalues, resulting in an energy expression written as
the sum of a few correlation functions, and solving the problem by repeated FFT
calculations. In addition to describing how the method is implemented, we
present a novel class of structure-based pairwise intermolecular potentials. The
DARS (Decoys As the Reference State) potentials are extracted from structures of
protein-protein complexes and use large sets of docked conformations as decoys
to derive atom pair distributions in the reference state. The current version of
the DARS potential works well for enzyme-inhibitor complexes. With the new
FFT-based program, DARS provides much better docking results than the earlier
approaches, in many cases generating 50% more near-native docked conformations.
Although the potential is far from optimal for antibody-antigen pairs, the
results are still slightly better than those given by an earlier FFT method. The
docking program PIPER is freely available for noncommercial applications. The accurate scoring of rigid-body docking orientations represents one of the
major difficulties in protein-protein docking prediction. Other challenges are
the development of faster and more efficient sampling methods and the
introduction of receptor and ligand flexibility during simulations. Overall,
good discrimination of near-native docking poses from the very early stages of
rigid-body protein docking is essential step before applying more costly
interface refinement to the correct docking solutions. Here we explore a simple
approach to scoring of rigid-body docking poses, which has been implemented in a
program called pyDock. The scheme is based on Coulombic electrostatics with
distance dependent dielectric constant, and implicit desolvation energy with
atomic solvation parameters previously adjusted for rigid-body protein-protein
docking. This scoring function is not highly dependent on specific geometry of
the docking poses and therefore can be used in rigid-body docking sets generated
by a variety of methods. We have tested the procedure in a large benchmark set
of 80 unbound docking cases. The method is able to detect a near-native solution
from 12,000 docking poses and place it within the 100 lowest-energy docking
solutions in 56% of the cases, in a completely unrestricted manner and without
any other additional information. More specifically, a near-native solution will
lie within the top 20 solutions in 37% of the cases. The simplicity of the
approach allows for a better understanding of the physical principles behind
protein-protein association, and provides a fast tool for the evaluation of
large sets of rigid-body docking poses in search of the near-native orientation. Structure-based virtual screening relies on scoring the predicted binding modes
of compounds docked into the target. Because the accuracy of this scoring relies
on the accuracy of the docking, methods that increase docking accuracy are
valuable. Here, we present a relatively straightforward method for improving the
probability of identifying accurately docked poses. The method is similar in
concept to consensus scoring schemes, which have been shown to increase ranking
power and thus hit rates, but combines information about predicted binding modes
rather than predicted binding affinities. The pose prediction success rate of
each docking program alone was found in this trial to be 55% for Autodock, 58%
for DOCK, and 64% for Vina. By using more than one docking program to predict
the binding pose, correct poses were identified in 82% or more of cases, a
significant improvement. In a virtual screen, these more reliably posed
compounds can be preferentially advanced to subsequent scoring stages to improve
hit rates. Consensus docking can be easily introduced into established
structure-based virtual screening methodologies. Scoring, the process of selecting the biologically relevant solution from a pool
of generated conformations, is one of the major challenges in the field of
biomolecular docking. A prominent way to cope with this challenge is to
incorporate information-based terms into the scoring function. Within this
context, low-resolution shape data obtained from either ion-mobility mass
spectrometry (IM-MS) or SAXS experiments have been integrated into the
conventional scoring function of the information-driven docking program HADDOCK.
Here, the strengths and weaknesses of IM-MS-based and SAXS-based scoring, either
in isolation or in combination with the HADDOCK score, are systematically
assessed. The results of an analysis of a large docking decoy set composed of
dimers generated by running HADDOCK in ab initio mode reveal that the content of
the IM-MS data is of too low resolution for selecting correct models, while
scoring with SAXS data leads to a significant improvement in performance.
However, the effectiveness of SAXS scoring depends on the shape and the
arrangement of the complex, with prolate and oblate systems showing the best
performance. It is observed that the highest accuracy is achieved when SAXS
scoring is combined with the energy-based HADDOCK score. The influenza virus hemagglutinin is a potential drug target for antivirus
treatment. A variety of membrane fusion inhibitors targeting hemagglutinin have
been discovered, but the binding sites and modes, important for understanding
membrane fusion and rational drug design, have not yet been elucidated. In this
article, we investigated the possible hemagglutinin binding sites for the
current membrane fusion inhibitors. Four possible binding pockets (Pocket A, B,
C, and D) at the stalk region of hemagglutinin were detected and defined using
the CAVITY program. Most of the current membrane fusion inhibitors were reported
to bind to Pocket C by amino acid mutation experiments and molecular modeling
simulation. However, our binding site prediction suggested that Pocket A is the
best ligand binding site other than Pocket C. Using a specific computational
protocol combining molecular docking, three-dimensional QSAR, and receptor
mimicking, we further found that Pocket A is the putative binding site for a
series of membrane fusion inhibitors (1-phenyl-cycloalkane carbamides). This is
further proven by the antiviral spectrum of the inhibitors. This protocol for
the identification of ligand binding sites in influenza hemagglutinin is also
applicable for the analysis of other protein targets with no explicit binding
information. In spite of the abundance of oligomeric proteins within a cell, the structural
characterization of protein-protein interactions is still a challenging task. In
particular, many of these interactions involve heteromeric complexes, which are
relatively difficult to determine experimentally. Hence there is growing
interest in using computational techniques to model such complexes. However,
assembling large heteromeric complexes computationally is a highly combinatorial
problem. Nonetheless the problem can be simplified greatly by considering
interactions between protein trimers. After dimers and monomers, triangular
trimers (i.e. trimers with pair-wise contacts between all three pairs of
proteins) are the most frequently observed quaternary structural motifs
according to the three-dimensional (3D) complex database. This article presents
DockTrina, a novel protein docking method for modeling the 3D structures of
nonsymmetrical triangular trimers. The method takes as input pair-wise contact
predictions from a rigid body docking program. It then scans and scores all
possible combinations of pairs of monomers using a very fast root mean square
deviation test. Finally, it ranks the predictions using a scoring function which
combines triples of pair-wise contact terms and a geometric clash penalty term.
The overall approach takes less than 2 min per complex on a modern desktop
computer. The method is tested and validated using a benchmark set of 220 bound
and seven unbound protein trimer structures. DockTrina will be made available at
http://o-d.inrialpes.fr/software/docktrina. DockoMatic is a free and open source application that unifies a suite of
software programs within a user-friendly graphical user interface (GUI) to
facilitate molecular docking experiments. Here we describe the release of
DockoMatic 2.0; significant software advances include the ability to (1) conduct
high throughput inverse virtual screening (IVS); (2) construct 3D homology
models; and (3) customize the user interface. Users can now efficiently setup,
start, and manage IVS experiments through the DockoMatic GUI by specifying
receptor(s), ligand(s), grid parameter file(s), and docking engine (either
AutoDock or AutoDock Vina). DockoMatic automatically generates the needed
experiment input files and output directories and allows the user to manage and
monitor job progress. Upon job completion, a summary of results is generated by
Dockomatic to facilitate interpretation by the user. DockoMatic functionality
has also been expanded to facilitate the construction of 3D protein homology
models using the Timely Integrated Modeler (TIM) wizard. The wizard TIM provides
an interface that accesses the basic local alignment search tool (BLAST) and
MODELER programs and guides the user through the necessary steps to easily and
efficiently create 3D homology models for biomacromolecular structures. The
DockoMatic GUI can be customized by the user, and the software design makes it
relatively easy to integrate additional docking engines, scoring functions, or
third party programs. DockoMatic is a free comprehensive molecular docking
software program for all levels of scientists in both research and education. Twelve homology models of the human M2 muscarinic receptor using different sets
of templates have been designed using the Prime program or the modeller program
and compared to crystallographic structure (PDB:3UON). The best models were
obtained using single template of the closest published structure, the M3
muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led
to worse models. Data document a key role of the template in homology modeling.
The models differ substantially. The quality checks built into the programs do
not correlate with the RMSDs to the crystallographic structure and cannot be
used to select the best model. Re-docking of the antagonists present in
crystallographic structure and relative binding energy estimation by calculating
MM/GBSA in Prime and the binding energy function in YASARA suggested it could be
possible to evaluate the quality of the orthosteric binding site based on the
prediction of relative binding energies. Although estimation of relative binding
energies distinguishes between relatively good and bad models it does not
indicate the best one. On the other hand, visual inspection of the models for
known features and knowledge-based analysis of the intramolecular interactions
allows an experimenter to select overall best models manually. The program VinaMPI has been developed to enable massively large virtual drug
screens on leadership-class computing resources, using a large number of cores
to decrease the time-to-completion of the screen. VinaMPI is a massively
parallel Message Passing Interface (MPI) program based on the multithreaded
virtual docking program AutodockVina, and is used to distribute tasks while
multithreading is used to speed-up individual docking tasks. VinaMPI uses a
distribution scheme in which tasks are evenly distributed to the workers based
on the complexity of each task, as defined by the number of rotatable bonds in
each chemical compound investigated. VinaMPI efficiently handles multiple
proteins in a ligand screen, allowing for high-throughput inverse docking that
presents new opportunities for improving the efficiency of the drug discovery
pipeline. VinaMPI successfully ran on 84,672 cores with a continual decrease in
job completion time with increasing core count. The ratio of the number of tasks
in a screening to the number of workers should be at least around 100 in order
to have a good load balance and an optimal job completion time. The code is
freely available and downloadable. Instructions for downloading and using the
code are provided in the Supporting Information. 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. Selecting near-native conformations from the immense number of conformations
generated by docking programs remains a major challenge in molecular docking. We
introduce DockRank, a novel approach to scoring docked conformations based on
the degree to which the interface residues of the docked conformation match a
set of predicted interface residues. DockRank uses interface residues predicted
by partner-specific sequence homology-based protein-protein interface predictor
(PS-HomPPI), which predicts the interface residues of a query protein with a
specific interaction partner. We compared the performance of DockRank with
several state-of-the-art docking scoring functions using Success Rate (the
percentage of cases that have at least one near-native conformation among the
top m conformations) and Hit Rate (the percentage of near-native conformations
that are included among the top m conformations). In cases where it is possible
to obtain partner-specific (PS) interface predictions from PS-HomPPI, DockRank
consistently outperforms both (i) ZRank and IRAD, two state-of-the-art
energy-based scoring functions (improving Success Rate by up to 4-fold); and
(ii) Variants of DockRank that use predicted interface residues obtained from
several protein interface predictors that do not take into account the binding
partner in making interface predictions (improving success rate by up to
39-fold). The latter result underscores the importance of using partner-specific
interface residues in scoring docked conformations. We show that DockRank, when
used to re-rank the conformations returned by ClusPro, improves upon the
original ClusPro rankings in terms of both Success Rate and Hit Rate. DockRank
is available as a server at http://einstein.cs.iastate.edu/DockRank/. In the present study, receptor induced 3D-QSAR model was developed for a set of
46 thieno[2,3-b]pyridine-5- carbonitrile PKC-θ inhibitors, to explore the
structural requirements of the molecules necessary for PKC-θ inhibition. Since
the chemical nature of the studied molecules was different from the crystal
ligand of the selected protein, induced fit docking (IFD) protocol was employed
to induce the conformational changes in the active site of the selected protein.
Thereafter, all molecules were docked into the newly generated active site
environment of the selected protein using glide docking program, and the 3D-QSAR
analysis was performed in PHASE program utilizing the docking based alignment of
the molecules. The best 3D-QSAR model was selected on the basis of the highest
value of Q(2)test (0.600), and the selected model also showed high values of
R(2)train , 0.915, Pearson-r, 0.801 and low value of SD, 0.241. The contour maps
corresponding to the selected 3D-QSAR model, in combination with docking
analysis, helped to explore the essential amino acid residues involved in
binding, and structural requirements of the ligand molecules necessary for
complementary fit with the active site of the protein. Therefore, the
information revealed from the generated model can further be explored as a novel
tool for the designing of new congener molecules that can serve as potential
therapeutics for the treatment of various disease conditions associated with
abnormal PKC-θ signalling. S100B is a calcium sensing protein belonging to the S100 protein family with
intracellular and extracellular roles. It is one of the EF hand homodimeric
proteins, which is known to interact with various protein targets to regulate
varied biological functions. Extracellular S100B has been recently reported to
interact with FGF2 in a RAGE-independent manner. However, the recognition
mechanism of S100B-FGF2 interaction at the molecular level remains unclear. In
this study, the critical residues on S100B-FGF2 interface were mapped by
combined information derived from NMR spectroscopy and site directed mutagenesis
experiments. Utilizing NMR titration data, we generated the structural models of
S100B-FGF2 complex from the computational docking program, HADDOCK which were
further proved stable during 15ns unrestrained molecular dynamics (MD)
simulations. Isothermal titration calorimetry studies indicated S100B
interaction with FGF2 is an entropically favored process implying domit role
of hydrophobic contacts at the protein-protein interface. Residue level
information of S100B interaction with FGF2 was useful to understand the varied
target recognition ability of S100B and further explained its role in effecting
extracellular signaling diversity. Mechanistic insights into the S100B-FGF2
complex interface and cell-based assay studies involving mutants led us to
conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation. We report the performance of our approaches for protein-protein docking and
interface analysis in CAPRI rounds 20-26. At the core of our pipeline was the
ZDOCK program for rigid-body protein-protein docking. We then reranked the ZDOCK
predictions using the ZRANK or IRAD scoring functions, pruned and analyzed
energy landscapes using clustering, and analyzed the docking results using our
interface prediction approach RCF. When possible, we used biological information
from the literature to apply constraints to the search space during or after the
ZDOCK runs. For approximately half of the standard docking challenges we made at
least one prediction that was acceptable or better. For the scoring challenges
we made acceptable or better predictions for all but one target. This indicates
that our scoring functions are generally able to select the correct binding
mode. We have determined the structure of the human integrin α1I domain bound to a
triple-helical collagen peptide. The structure of the α1I-peptide complex was
investigated using data from NMR, small angle x-ray scattering, and size
exclusion chromatography that were used to generate and validate a model of the
complex using the data-driven docking program, HADDOCK (High Ambiguity Driven
Biomolecular Docking). The structure revealed that the α1I domain undergoes a
major conformational change upon binding of the collagen peptide. This involves
a large movement in the C-terminal helix of the αI domain that has been
suggested to be the mechanism by which signals are propagated in the intact
integrin receptor. The structure suggests a basis for the different binding
selectivity observed for the α1I and α2I domains. Mutational data identify
residues that contribute to the conformational change observed. Furthermore,
small angle x-ray scattering data suggest that at low collagen peptide
concentrations the complex exists in equilibrium between a 1:1 and 2:1
α1I-peptide complex. Combinatorial chemical libraries produced on solid support offer fast and
cost-effective access to a large number of unique compounds. If such libraries
are screened directly on-bead, the speed at which chemical space can be explored
by chemists is much greater than that addressable using solution based synthesis
and screening methods. Solution based screening has a large supporting body of
software such as structure-based virtual screening tools which enable the
prediction of protein-ligand complexes. Use of these techniques to predict the
protein bound complexes of compounds synthesized on solid support neglects to
take into account the conjugation site on the small molecule ligand. This may
invalidate predicted binding modes, the linker may be clashing with protein
atoms. We present CSBB-ConeExclusion, a methodology and computer program which
provides a measure of the applicability of solution dockings to solid support.
Output is given in the form of statistics for each docking pose, a unique 2D
visualization method which can be used to determine applicability at a glance,
and automatically generated PyMol scripts allowing visualization of protein atom
incursion into a defined exclusion volume. CSBB-ConeExclusion is then
exemplarically used to determine the optimum attachment point for a purine
library targeting cyclin-dependent kinase 2 CDK2. |
Which genes have been found mutated in Gray platelet syndrome patients? | The genetic defects responsible for gray platelet syndrome are mutations in the genes NBEAL2, GATA1 and GFI1B. | We identified a family with gray platelet syndrome (GPS) segregating as a
sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia,
and large agranular platelets characteristic of GPS, while obligate carrier
females were asymptomatic but had dimorphic platelets on peripheral smear.
Associated findings included mild erythrocyte abnormalities in affected males.
Linkage analysis revealed a 63 cM region on the X chromosome between markers
G10578 and DXS6797, which segregated with the platelet phenotype and included
the GATA1 gene. Sequencing of GATA1 revealed a G-to-A mutation at position 759
corresponding to amino acid change Arg216Gln. This mutation was previously
described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but
not of gray platelet syndrome. Our findings suggest that XLTT is within a
spectrum of disorders constituting the gray platelet syndrome, and we propose
that GATA1 is an upstream regulator of the genes required for platelet
alpha-granule biogenesis. Gray platelet syndrome (GPS) is a predomitly recessive platelet disorder that
is characterized by mild thrombocytopenia with large platelets and a paucity of
α-granules; these abnormalities cause mostly moderate but in rare cases severe
bleeding. We sequenced the exomes of four unrelated individuals and identified
NBEAL2 as the causative gene; it has no previously known function but is a
member of a gene family that is involved in granule development. Silencing of
nbeal2 in zebrafish abrogated thrombocyte formation. Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is
characterized by large platelets that lack α-granules. Here we show that
mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain
protein, cause GPS and that megakaryocytes and platelets from individuals with
GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of
sucrose-gradient subcellular fractions of platelets indicated that NBEAL2
localizes to the dense tubular system (endoplasmic reticulum) in platelets. Next-generation RNA sequence analysis of platelets from an individual with
autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal
transcript reads, including intron retention, mapping to NBEAL2 (encoding
neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as
the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain
that is predicted to be involved in vesicular trafficking and may be critical
for the development of platelet α-granules. The gray platelet syndrome is a rare inherited bleeding disorder characterized
by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The
genetic defect responsible for gray platelet syndrome was recently identified in
biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with
inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All
of them underwent NBEAL2 DNA sequencing and evaluation of the platelet
phenotype, including a systematic assessment of the α-granule content by
immunofluorescence analysis for α-granule secretory proteins. We identified 9
novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included
missense, nonsense and frameshift mutations, as well as nucleotide substitutions
that altered the splicing mechanisms as determined at the RNA level. All the
individuals with NBEAL2 biallelic mutations showed almost complete absence of
platelet α-granules. Interestingly, the 13 individuals assumed to be
asymptomatic because carriers of a mutated allele had platelet macrocytosis and
significant reduction of the α-granule content. However, they were not
thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2
alterations, suggesting that other genetic defect(s) are responsible for their
platelet phenotype. Of note, these patients were characterized by a lower
severity of the α-granule deficiency than individuals with two NBEAL2 mutated
alleles. Our data extend the spectrum of mutations responsible for gray platelet
syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is
a genetic heterogeneous trait. In terms of practical applications, the screening
of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe
reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated
allele have mild laboratory abnormalities, suggesting that even
haploinsufficiency has an effect on platelet phenotype. Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with
macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to
loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here
a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit
splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and
their cargo, including von Willebrand factor (VWF), thrombospondin-1, and
platelet factor 4. The platelet α-granule membrane protein P-selectin is
expressed at 48% of wild-type levels and externalized upon platelet activation.
The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-)
platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later
stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown
by flow cytometry, platelet aggregometry, bleeding assays, and intravital
imaging of laser-induced arterial thrombus formation. Microscopic analysis
detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which
when cultured showed delayed maturation, decreased survival, decreased ploidy,
and developmental abnormalities, including abnormal extracellular distribution
of VWF. Our results confirm that α-granule secretion plays a significant role in
platelet function, and they also indicate that abnormal α-granule formation in
Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development,
and platelet production. The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding
disorder caused by a deficiency of alpha granules in platelets. We detected a
nonsense mutation in the gene encoding the transcription factor GFI1B (growth
factor independent 1B) that causes autosomal domit gray platelet syndrome.
Both gray platelets and megakaryocytes had abnormal marker expression. In
addition, the megakaryocytes had dysplastic features, and they were abnormally
distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant
GFI1B transcriptional activity in a domit-negative manner. Our studies show
that GFI1B, in addition to being causally related to the gray platelet syndrome,
is key to megakaryocyte and platelet development. |
Does dasatinib promote or inhibit T-cell proliferation? | Dasatinib inhibits T-cell proliferation | Dasatinib is an oral small molecule inhibitor of Abl and Src family tyrosine
kinases (SFK), including p56(Lck) (Lck). Given the central importance of Lck in
transmitting signals from the T-cell receptor (TCR) signaling complex and the
potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent
could provide a novel route of immunomodulation via targeted inhibition of
antigen-induced signaling. Herein, we show that dasatinib inhibits TCR-mediated
signal transduction, cellular proliferation, cytokine production, and in vivo
T-cell responses. However, dasatinib-mediated inhibition does not induce
apoptosis because the effect is reversible or may be overcome by signals
bypassing the TCR, such as phorbol ester. Signal transduction and proliferative
responses via IL-2 remain essentially unperturbed, suggesting that dasatinib
displays specificity for TCR signaling. In addition, dasatinib combined with
cyclosporine A or rapamycin led to a much more potent inhibition of T-cell
activation, suggesting that targeted inhibition of Lck could be a useful adjunct
for enhanced immunomodulation. In combination with currently available
immunomodulatory agents, SFK inhibition could potentially increase
immunomodulatory efficacy while minimizing toxicity of individual agents. Dasatinib (BMS-354825) is a Src/ABL tyrosine kinase inhibitor currently approved
for the treatment of chronic myeloid leukemia. Dasatinib has increased potency
against ABL compared to the current therapy imatinib, and is effective in many
cases where disease is resistant to imatinib. Dasatinib also inhibits many
Src-family tyrosine kinases. We have demonstrated in this study that dasatinib
is able to block the function of normal human T-lymphocytes in vitro at
clinically relevant concentrations. T-cell functions including proliferation,
activation and cytokine production were all uniformly inhibited in the presence
of dasatinib. We also demonstrated inhibition of TCR signalling through
Src-family kinase LCK, and predicted that inhibition of LCK and other kinases
involved in T-cell signalling by dasatinib is responsible for the suppression of
T-cell function. These findings raise the concern about potential T-cell
inhibition in patients taking dasatinib, and suggest a possible application for
the treatment of T-cell mediated immune disorders. PURPOSE: The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for
the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia.
Because SRC kinases are known to play an important role in physiologic T-cell
activation, we analyzed the immunobiological effects of dasatinib on T-cell
function. The effect of dasatinib on multiple T-cell effector functions was
examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine
kinase inhibitor staurosporine was used as a comparator.
EXPERIMENTAL DESIGN: Purified human CD3+ cells and virus-specific CD8+ T cells
from healthy blood donors were studied directly ex vivo; antigen-specific
effects were confirmed in defined T-cell clones. Functional outcomes included
cytokine production (interleukin-2, IFN gamma, and tumor necrosis factor alpha),
degranulation (CD107a/b mobilization), activation (CD69 up-regulation),
proliferation (carboxyfluorescein diacetate succinimidyl ester dilution),
apoptosis/necrosis induction, and signal transduction.
RESULTS: Both dasatinib and staurosporine inhibited T-cell activation,
proliferation, cytokine production, and degranulation in a dose-dependent
manner. Mechanistically, this was mediated by the blockade of early signal
transduction events and was not due to loss of T-cell viability. Overall, CD4+ T
cells seemed to be more sensitive to these effects than CD8+ T cells, and naïve
T cells more sensitive than memory T-cell subsets. The inhibitory effects of
dasatinib were so profound that all T-cell effector functions were shut down at
therapeutically relevant concentrations.
CONCLUSION: These findings indicate that caution is warranted with use of this
drug in the clinical setting and provide a rationale to explore the potential of
dasatinib as an immunosuppressant in the fields of transplantation and
T-cell-driven autoimmune diseases. OBJECTIVE: To investigate the inhibitory effects of dasatinib on proliferation,
function, and signaling events on CD8+T cells.
MATERIALS AND METHODS: Carboxyfluorescein diacetate succinimidyl ester and
5-bromo-2-deoxyuridine were used to detect proliferation and cell cycle of CD8+T
cells treated with dasatinib, respectively. Frequency and function of viral and
leukemia-antigen-specific CD8+T cells from healthy donors were measured by
tetramer staining and ELISPOT assay. Western blotting analysis was performed to
detect T-cell receptor (TCR), nuclear factor kappa B (NF-kappaB) and Src
signaling events in T cells treated with dasatinib or imatinib.
RESULTS: Dasatinib inhibited proliferation of CD8+T cells in a dose-dependent
manner, which was associated with lower secretion of interferon-gamma and
granzyme B, as well as with arrest of CD8+T cells in the G0/G1 phase of cell
cycle. Inhibition of CD8+T cells was proven for blood samples from a patient
under dasatinib medication when compared with their T-cell status without
dasatinib. Western blotting confirmed that these effects were mediated through
downregulation of the phosphorylation level of molecules from the TCR and the
NF-kappaB signaling transduction cascade. Dasatinib proved to be more potent
than imatinib on Src and TCR signaling events in Jurkat T cells.
CONCLUSION: Our study demonstrated that dasatinib impaired proliferation and
function of CD8+T cells via TCR and NF-kappaB signaling events without inducing
apoptosis. Therefore, dasatinib might alter the graft-vs-leukemia effect and the
graft-vs-host disease after allogeneic stem cell transplantation sustained by
CD8+T cells. Dasatinib might also be used as a novel immunosuppressant agent. CD4+CD25+ regulatory T cells (Tregs) can influence various immune responses.
Little is known about the effects of the Abl/Src kinase inhibitor dasatinib on
Tregs which regulate anti-tumor/leukaemia immune responses. The present study
demonstrated that dasatinib inhibited proliferation of Tregs and CD4+CD25- T
cells in a dose-dependent manner, which was associated with the decreased
production of corresponding cytokines. Treatment of Tregs with dasatinib
inhibited the suppressive capacity of Tregs. The mechanisms of this inhibition
included arrest of cells in the G0/G1 phase of cell cycle, down-regulation of
the transcription factor forkhead box P3, glucocorticoid-induced tumour necrosis
factor receptor and the cytotoxic T lymphocyte associated protein 4 as well as
inhibition of signaling events through Src and nuclear factor kappaB. Dasatinib
showed an inhibitory effect on the proliferation and function of both Tregs and
CD4+CD25- T cells at therapeutically relevant concentrations of the drug.
Clinical administration of dasatinib might influence not only the
graft-versus-leukaemia effect but also the graft-versus-host-disease in patients
receiving dasatinib after allogeneic stem cell transplantation and/or donor
lymphocytes infusion as the function of both Tregs and effector T cells are
hampered in a similar way by dasatinib. OBJECTIVE: Dasatinib (BMS-354825) is a small molecule Src/Abl tyrosine kinase
inhibitor approved for the treatment of chronic myeloid leukemia and
Philadelphia chromosome-positive acute lymphoblastic leukemia. Members of the
Src family of kinases are involved in the induction of innate and adaptive
immunity. The purpose of this study was to evaluate the inhibitory action of
dasatinib on antigen-specific CD8(+) and CD4(+) T-cell function, as well as
natural killer (NK) cell cytotoxicity.
MATERIALS AND METHODS: To assess dasatinib-mediated inhibition of
antigen-specific T-cell proliferation, transgenic CD4(+) and CD8(+) T cells
specific for ovalbumin were utilized. Endogenous CD4(+) and CD8(+) T-cell
responses were determined following immunization of dasatinib-treated or control
mice with a nonreplicating recombit virus. Clearance of the RMA-S cells, a
major histocompatibility complex (MHC) class I-deficient thymoma sensitive to
NK-cell lysis, was analyzed in mice undergoing dasatinib treatment.
RESULTS: Dasatinib inhibited antigen-specific proliferation of murine CD4(+) and
CD8(+) transgenic T cells in vitro and in vivo. Endogenous antigen-specific
helper T-cell recall responses and induction of T-cell-mediated cytotoxicity
following immunization with a nonreplicating recombit virus were also
inhibited. So to was the ability of NK cells to eliminate MHC class I-deficient
cells in vivo.
CONCLUSIONS: These findings suggest that dasatinib has the potential to modulate
the host immune response at clinical doses and highlights scope for off target
applications, e.g., therapeutic immunosuppression in the context of autoimmune
pathogenesis and allogeneic tissue transplantation. Dasatinib, a dual tyrosine kinase inhibitor, is known to modulate or suppress
T-cell activation and proliferation. We report a series of 8 patients who
developed chronic peripheral lymphocytosis, identified as natural killer cells
or natural killer/T-cells based on their large granular lymphocyte morphologies
and CD16(+), CD56(+), CD3(-) or CD3(+) immunophenotypic profiles, out of 18
patients receiving dasatinib therapy. All cases that developed large granular
lymphocyte lymphocytosis achieved optimal molecular response (8/8 in large
granular lymphocyte(+) patients vs. 3/10 in large granular lymphocyte(-)
patients, p=0.002). A (51)Cr release assay demonstrated that natural killer cell
cytotoxicity has been enhanced in a case of large granular lymphocyte
lymphocytosis compared to normal healthy donors, and that natural killer cell
cytotoxicity in dasatinib-responders was superior to that in non-responders. In
summary, the present study suggests that natural killer or natural killer/T cell
lineage large granular lymphocyte lymphocytosis develops in association with
dasatinib therapy and that large granular lymphocyte might have a therapeutic
effect on Ph(+) leukemic cells. |
Does SCRIB deregulation promote cancer? | Yes, deregulation of scribble promotes cancer. | Drosophila Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl) act
in concert as regulators of epithelial polarity, and human homologs of
Drosophila dlg, scrib, and lgl are cancer-associated genes. LLGL1, LLGL2, and
LLGL3/STXBP5 genes, encoding LGL1, LGL2, and LGL3/Tomosyn, respectively, are
human homologs of Drosophila lgl gene. Here, we identified and characterized
LLGL4 (also known as STXBP5L) gene encoding LGL4 protein, by using
bioinformatics. Uncharacterized human KIAA1006 cDNA (AB023223) was derived from
human LLGL4 gene. LLGL4 mRNA was expressed in kidney, brain hippocampus, and
also in lung carcinoid, and germ cell tumors. LLGL4 gene, consisting of 28
exons, was mapped to human chromosome 3q13.33. Mouse A830015P08Rik cDNA
(NM_172440.1) was a 3'-truncated partial Llgl4 cDNA. Nucleotide sequence of
full-length mouse Llgl4 cDNA was determined in silico by assembling
A830015P08Rik cDNA, BU609516 EST and last two exons of Llgl4 gene within mouse
genome clone RP24-174G4 (AC118742.3). Human LGL4 showed 95.8% total-amino-acid
identity with mouse Lgl4, and 68.4% total-amino-acid identity with human LGL3.
LGLH1 domain (codon 1-11 of LGL4), LGLH2 domain (codon 52-98) and LGLH3 domain
(codon 994-1054) were identified as novel conserved regions among LGL family
members. LGL1 and LGL2 consist of LGLH1, LGLH2, LGLH3 domains and five WD40
repeats, while LGL3 and LGL4 consist of LGLH1, LGLH2, LGLH3 domains, five WD40
repeats and the C-terminal Syntaxin-binding SNARE domain. This is the first
report on identification and characterization of human LLGL4 and mouse Llgl4
genes. Activating mutations in genes of the Ras-mitogen-activated protein kinase (MAPK)
pathway occur in approximately 30% of all human cancers; however, mutation of
Ras alone is rarely sufficient to induce tumour development. Scribble is a
polarity regulator recently isolated from a Drosophila screen for events that
cooperate with Ras mutation to promote tumour progression and cell invasion. In
mammals, Scribble regulates directed cell migration and wound healing in vivo;
however, no role has been identified for mammalian Scribble in oncogenic
transformation. Here we show that in human epithelial cells expressing oncogenic
Ras or Raf, loss of Scribble promotes invasion of cells through extracellular
matrix in an organotypic culture system. Further, we show that the mechanism by
which this occurs is in the regulation of MAPK signalling by Scribble. The
suppression of MAPK signalling is a highly conserved function of Scribble as it
also prevents Raf-mediated defects in Drosophila wing development. Our data
identify Scribble as an important mediator of MAPK signalling and provide a
molecular basis for the observation that Scribble expression is decreased in
many invasive human cancers. Loss of cell polarity proteins such as Scribble induces neoplasia in Drosophila
by promoting uncontrolled proliferation. In mammals, the role that polarity
proteins play during tumorigenesis is not well understood. Here, we demonstrate
that depletion of Scribble in mammary epithelia disrupts cell polarity, blocks
three-dimensional morphogenesis, inhibits apoptosis, and induces dysplasia in
vivo that progress to tumors after long latency. Loss of Scribble cooperates
with oncogenes such as c-myc to transform epithelial cells and induce tumors in
vivo by blocking activation of an apoptosis pathway. Like depletion,
mislocalization of Scribble from cell-cell junction was sufficient to promote
cell transformation. Interestingly, spontaneous mammary tumors in mice and
humans possess both downregulated and mislocalized Scribble. Thus, we
demonstrate that scribble inhibits breast cancer formation and that deregulation
of polarity pathways promotes dysplastic and neoplastic growth in mammals by
disrupting morphogenesis and inhibiting cell death. Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but
its potential role in human cancer is controversial. Herein, we show that Scrib
is nearly universally overexpressed in cultured tumor cell lines and genetically
disparate cancer patient series compared with matched normal tissues in vivo.
Instead of a membrane association seen in normal epithelia, tumor-associated
Scrib is mislocalized and found predomitly in the cytosol. Small-interfering
RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell
migration in wound-healing assays, suppressed tumor cell invasion across
Matrigel-coated inserts, and down-regulated the expression of cell motility
markers and mediators of epithelial-mesenchymal transition. These data uncover a
previously unrecognized exploitation of Scrib for aberrant tumor cell motility
and invasion, thus potentially contributing to disease progression in humans. The expression of small, non-coding RNA or microRNAs (miR), is frequently
deregulated in human cancer, but how these pathways affect disease progression
is still largely elusive. Here, we report on a miR, miR-296, which is
progressively lost during tumor progression and correlates with metastatic
disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts
cancers. Functionally, miR-296 controls a global cell motility gene signature in
epithelial cells by transcriptionally repressing the cell polarity-cell
plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly
increased and mislocalized Scrib in human tumors, resulting in exaggerated
random cell migration and tumor cell invasiveness. Re-expression of miR-296 in
MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels
predict tumor relapse in hepatocellular carcinoma patients. These data identify
miR-296 as a global repressor of tumorigenicity and uncover a previously
unexplored exploitation of Scrib in tumor progression in humans. Loss of cellular polarity is a hallmark of epithelial cancers, raising the
possibility that regulators of polarity have a role in suppressing
tumorigenesis. The Scribble complex is one of at least three interacting protein
complexes that have a critical role in establishing and maintaining epithelial
polarity. In human colorectal, breast, and endometrial cancers, expression of
the Scribble complex member SCRIB is often mislocalized and deregulated. Here,
we report that Scrib is indispensable for prostate homeostasis in mice. Scrib
heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib
loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically,
Scrib was shown to negatively regulate the MAPK cascade to suppress
tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in
mice combined with expression of an oncogenic Kras mutation promoted the
progression of prostate cancer that recapitulated the human disease. The
clinical significance of the work in mice was highlighted by our observation
that SCRIB deregulation strongly correlated with poor survival in human prostate
cancer. These data suggest that the polarity network could provide a new avenue
for therapeutic intervention. After years of extensive scientific discovery much has been learned about the
networks that regulate epithelial homeostasis. Loss of expression or functional
activity of cell adhesion and cell polarity proteins (including the PAR, crumbs
(CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages
of tumour progression and invasiveness. But the key roles of these proteins in
crosstalk with the Hippo and liver kinase B1 (LKB1)-AMPK pathways and in
epithelial function and proliferation indicate that they may also be associated
with the early stages of tumorigenesis. For example, deregulation of adhesion
and polarity proteins can cause misoriented cell divisions and increased
self-renewal of adult epithelial stem cells. In this Review, we highlight some
advances in the understanding of how loss of epithelial cell polarity
contributes to tumorigenesis. The γ subunit of the major histocompatibility complex (MHC) class II complex,
CD74, is overexpressed in a significant proportion of metastatic breast tumors,
but the mechanistic foundation and biologic significance of this phenomenon are
not fully understood. Here, we show that when CD74 is overexpressed in human
cancer and noncancerous epithelial cells, it interacts and interferes with the
function of Scribble, a product of a well-known tumor suppressor gene.
Furthermore, using epithelial cell lines expressing CD74 under the control of
tetracycline-inducible promoter and quantitative high-resolution mass
spectrometry, we demonstrate that, as a result of CD74 overexpression, the
phosphorylation pattern of the C-terminal part of Scribble undergoes specific
changes. This is accompanied with a translocation of the protein from the sites
of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is
likely to effectively enhance the motility and invasiveness of the cancer cells. Author information:
(1)Cell Cycle and Cancer Genetics, Research Division, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia.
(2)1] Cell Cycle and Cancer Genetics, Research Division, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia [2] The Sir Peter MacCallum Department of
Oncology, Melbourne, Victoria, Australia.
(3)Cell Signaling and Cell Death Division, The Walter and Eliza Hall Institute
of Medical Research, Parkville, Victoria, Australia.
(4)1] The Sir Peter MacCallum Department of Oncology, Melbourne, Victoria,
Australia [2] Translational Research Laboratory, Cancer Therapeutics Program,
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
(5)1] Cell Cycle and Cancer Genetics, Research Division, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia [2] The Sir Peter MacCallum Department of
Oncology, Melbourne, Victoria, Australia [3] Department of Pathology, University
of Melbourne, Parkville, Victoria, Australia [4] Department of Molecular Biology
and Biochemistry, University of Melbourne, Parkville, Victoria, Australia. |
Is cardiac magnetic resonance imaging indicated in the pre-participation screening of athletes? | Currently cardiac magnetic resonance imaging is not indicated in the pre-participation screening of athletes. However the potential of this imaging technique to provide new information on cardiac function morphology and myocardial composition, in particular with regard to myocardial fibrosis, gets it potentially suitable to be applied in the pre-participation screening of athletes | To evaluate left ventricular myocardial mass and function as well as ostial
coronary artery cross-sectional area in endurance athletes, an athlete group of
12 highly trained rowers and a control group of 12 sedentary healthy subjects
underwent MR examination. An ECG-gated breath-hold cine gradient-echo sequence
was used to calculate myocardial mass, end-diastolic and end-systolic volumes,
stroke volume, and cardiac output, all related to body surface area, as well as
ejection fraction. A 3D fat-saturated ECG- and respiratory-triggered navigator
echo sequence was used to evaluate coronary arteries: left main (LM), left
anterior descending (LAD), left circumflex (LCx), and right coronary artery
(RCA). Cross-sectional area was calculated and divided for body surface area.
Myocardial mass was found significantly larger in athlete group than in control
group (p = 0.0078), the same being for end-diastolic volume (p = 0.0078), stroke
volume (p = 0.0055), LM (p = 0.0066) and LAD (p = 0.0129). No significant
difference was found for all the remaining parameters. Significant correlation
with myocardial mass was found for LM (p < 0.001) and LAD (p = 0.0340), not for
LCx and RCA. Magnetic resoce imaging is a useful tool in evaluating the
myocardial hypertrophy and function of athlete's heart. Magnetic resoce
angiography is a valuable noninvasive method to visualize the correlated
cross-sectional area increase of the left coronary artery system. Many cardiac conditions found in athletes are amenable to athletic participation
if well-managed. As always, a respect for the causes of sudden cardiac death in
athletes is paramount. Although rare, sudden cardiac death in athletes is
frequently preventable by careful pre-participation screening. The authors hope
that more athletes will undergo screening, and that basic investigations,
including but not limited to electrocardiography, will become more widespread. A
high degree of surveillance for cardiac pathology in athletes is the duty of any
sports medicine practitioner. AIMS: Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in
young athletes, and substantial interest persists in strategies for timely
identification. We assessed the diagnostic efficacy of Italian pre-participation
screening programme with 12-lead ECG (in addition to history and physical
examination) for identification of HCM.
METHODS AND RESULTS: Four thousand four hundred and fifty members of the Italian
national teams, initially judged eligible for competition as a result of
systematic pre-participation screening across Italy, subsequently underwent
clinical and echocardiographic examination at the Institute of Sports Medicine
and Science (Rome) to assess the presence of previously undetected HCM. None of
the 4450 athletes showed clinical evidence of HCM. Other cardiac abnormalities
were detected in only 12 athletes, including myocarditis (n=4), mitral valve
prolapse (n=3), Marfan's syndrome (n=2), aortic regurgitation with bicuspid
valve (n=2), and arrhythmogenic right ventricular cardiomyopathy (n=1). In
addition, echocardiography identified four athletes with borderline left
ventricular wall thickness (i.e. 13 mm) in the 'grey zone' of overlap between
HCM and athlete's heart. In two of these athletes, subsequent genetic analysis
or clinical changes over an average 8-year follow-up resulted, respectively, in
a definitive or possible diagnosis of HCM.
CONCLUSION: The Italian national pre-participation screening programme including
12-lead ECG appears to be efficient in identifying young athletes with HCM,
leading to their timely disqualification from competitive sports. These data
also suggest that routine echocardiography is not an obligatory component of
broad-based screening programmes designed to identify young athletes with HCM. The cardiovascular pre-participation screening proposal for young competitive
athletes has the potential to save young lives. This study aimed to identify
individuals at risk for potentially lethal cardiovascular diseases in athletes
before competition. Between June 2005 and July 2005, 351 (170 male and 181
female) elite Chinese athletes from 21 sports were profiled. The 12-lead
electrocardiogram and echocardiography were employed to evaluate cardiovascular
diseases. The vast majority had no definitive evidence of cardiovascular
disease. However, abnormal ECGs were identified in 16 athletes (4.5%), including
4 with distinctly abnormal and 12 with mildly abnormal patterns. Only 13
athletes (3.7%) had echocardiographic evidence of relatively mild valve
regurgitation that had not been previously suspected. In three athletes with
relatively mild ventricular septal hypertrophy (13-14 mm), it was not possible
to discern with absolute certainty whether the wall thickening was a
manifestation of hypertrophic cardiomyopathy or secondary to athletic
conditioning ("athlete heart"). This screening protocol identified no athletes
with definite evidence of hypertrophic cardiomyopathy, Marfan's syndrome or
other cardiovascular diseases that convey a significant potential risk for
sudden death or disease progression during athletic activity. This is largely
due to the relative low prevalence of conditions resulting in sudden cardiac
death in young athletes and high false positive/negative rates in the tests used
as part of the screening process (due to a large overlap between cardiovascular
changes due to pathology and those due to intense training). In 1982 a nationwide program of pre-participation screening including 12-lead
electrocardiography (ECG) was launched in Italy. The aim of this article is to
examine whether this 25-year screening program should be considered a valid and
advisable public health strategy. The analysis of data coming from the
long-running Italian experience indicates that ECG screening has provided
adequate sensitivity and specificity for detection of potentially lethal
cardiomyopathy or arrhythmias and has led to substantial reduction of mortality
of young competitive athletes by approximately 90%. Screening was feasible
thanks to the Italian Health System, which is developed in terms of health care
and prevention services, and because of the limited costs of cardiovascular
evaluation in the setting of a mass program. On the basis of current scientific
evidence the implementation of a mass-screening program aimed to prevent
athletic-field sudden cardiac death should be at least carefully considered by
public health administrators worldwide. Advanced cardiac imaging, using cardiac magnetic resoce imaging (MRI) and
multidetector computed tomography (CT), is increasingly used in the work-up of
athletes with suspected abnormalities on screening. Both imaging modalities
produce highly accurate and reproducible structural and functional cardiac
information. Cardiac MRI has the advantage of imaging without radiation exposure
or the use of iodine-containing contrast agents, but is sometimes not possible
due to claustrophobia or other contraindications. Although cardiac MRI can rule
out coronary artery anomalies, multidetector CT is superior to cardiac MRI for
visualising the full extent of the coronary arteries and atherosclerotic
coronary artery disease. For patients less than 35 years of age, cardiac MRI is
the first option after initial echocardiography for further assessment of
cardiomyopathies, myocarditis and coronary anomalies, which are major causes of
sudden cardiac death in young athletes. For athletes over 35 years of age the
most common cause of sudden cardiac death is coronary artery disease, whereby
cardiovascular screening requires further diagnostic modalities and may include
multidetector CT. Although several investigations have demonstrated that prolonged aerobic
exercise results in decreased left ventricular (LV) function, few have examined
the impact of an acute bout of high-intensity exercise on right ventricular (RV)
and LV systolic and diastolic function. Cardiac magnetic resoce imaging with
tagging was used to study the impact of high-intensity interval exercise on
biventricular function in 9 endurance-trained (ET; Vo(2)max 69 +/- 7 ml/kg/min)
and 9 normally active (NA; Vo(2)max 44 +/- 9 ml/kg/min) men. Subjects underwent
baseline cardiac magnetic resoce imaging assessments (pre) and then performed
an average of 14 1-minute intervals at 97 +/- 11% (NA) and 99 +/- 6% (ET) of
peak power output, separated by 2 minutes of recovery at 21 +/- 6% (NA) and 21
+/- 9% (ET) of peak power output. After exercise, 2 cardiac magnetic resoce
imaging assessments (post 1 at 6.2 +/- 2.6 minutes and post 2 at 38.4 +/- 3.8
minutes) were completed. RV and LV ejection fractions, twist, basal and apical
rotation rates, rate of untwisting, circumferential strain, and timings were
examined. No significant change in RV and LV ejection fractions, twist,
untwisting rate, or strain after exercise occurred in the NA group. In the ET
group, RV ejection fraction (pre 56 +/- 4%, post 1 54 +/- 4%, post 2 54 +/- 3%)
and LV ejection fraction (pre 62 +/- 4%, post 1 59 +/- 4%, post 2 58 +/- 4%)
were decreased at post 1 and post 2, while untwisting rate, apical rotation
rate, and circumferential strain were decreased at post 2 (all p values <0.05).
In conclusion, biventricular systolic and diastolic dysfunction occurred after
14 minutes of high-intensity exercise in ET athletes, a phenomenon not observed
in NA subjects. Sudden cardiac death in competing athletes is usually caused by unsuspected
heart disease, and pre-participation screening may reduce the incidence of this
tragic event. Although the cost-effectiveness of screening programs is unclear,
international sports associations are currently implementing mandatory screening
of elite athletes. During the first year of screening in the top Danish soccer
league, all athletes were found to be eligible for continued participation in
the game, suggesting that concern about false positive screening results may be
exaggerated. Sudden cardiac arrest is most often the first clinical manifestation of an
underlying cardiovascular disease and usually occurs in previously asymptomatic
athletes. The risk benefit ratio of physical exercise differs between young
competitive athletes and middle-age/senior individuals engaged in leisure-time
sports activity. Competitive sports are associated with an increase in the risk
of sudden cardiovascular death (SCD) in susceptible adolescents and young adults
with underlying cardiovascular disorders. In middle-age/older individuals,
physical activity can be regarded as a 'two-edged sword': vigorous exertion
increases the incidence of acute coronary events in those who did not exercise
regularly, whereas habitual physical activity reduces the overall risk of
myocardial infarction and SCD. Although cardiovascular pre-participation
evaluation offers the potential to identify athletes with life-threatening
cardiovascular abnormalities before onset of symptoms and may reduce their risk
of SCD, there is a significant debate among cardiologists about efficacy, impact
of false-positive results and cost-effectiveness of routine screening. This
review presents an appraisal of the available data and criticisms concerning
screening programmes aimed to prevent SCD of either young competitive athletes
or older individuals engaged in leisure-time sports activity. Sudden cardiac death of a young athlete is the most tragic event in sports and
devastates the family, the sports medicine team, and the local community. Such a
fatality represents the first manifestation of cardiac disease in up to 80% of
young athletes who remain asymptomatic before sudden cardiac arrest occurs; this
explains the limited power of screening modalities based solely on history and
physical examination. The long-running Italian experience showed that
electrocardiogram (ECG) screening definitively improves the sensitivity of
pre-participation evaluation for heart diseases and substantially reduces the
risk of death in the athletic field (primary prevention). However, some cardiac
conditions, such as coronary artery diseases, present no abnormalities on
12-lead ECG. Moreover, cardiac arrest due to non-penetrating chest injury
(commotio cordis) cannot be prevented by screening. This justifies the efforts
for implementing programmes of early external defibrillation of unpredictable
arrhythmic cardiac arrest. This article reviews the epidemiology of sudden
cardiac arrest in the athlete in terms of incidence, sport-related risk,
underlying causes, and the currently available prevention programmes such as
pre-participation screening and early external defibrillation by using automated
external defibrillators. The best strategy is to combine synergistically primary
prevention of sudden cardiac death by pre-participation identification of
athletes affected by at-risk cardiomyopathies and secondary prevention with
back-up defibrillation of unpredictable sudden cardiac arrest on the athletic
field. BACKGROUND: The diagnosis of subtle structural heart disease in competitive
athletes with ventricular arrhythmias (VAs) and an apparently normal heart is
challenging. Three-dimensional electroanatomic mapping (EAM) has been
demonstrated to reliably identify low-voltage areas that correspond to different
cardiomyopathic substrates.
OBJECTIVE: The purpose of this study was to test whether EAM may help in the
diagnosis of concealed cardiomyopathies in athletes with VAs and an apparently
normal heart.
METHODS: We studied 13 consecutive competitive athletes (12 males, age 30 ± 13
years) who had documentation of VAs within the previous 6 months on 12-lead
electrocardiogram (ECG), 24-hour Holter ECG, or ECG exercise testing and who
were judged as having a structurally normal heart after a thorough noninvasive
evaluation, including signal-averaged ECG, transthoracic echocardiogram, and
cardiac magnetic resoce imaging. Depending on the presumed site of VA origin
according to 12-lead ECG criteria, patients underwent right or left ventricular
EAM and EAM-guided endomyocardial biopsy.
RESULTS: Presenting arrhythmias included sustained ventricular tachycardia (n =
3), multiple episodes of nonsustained ventricular tachycardia (n = 7), and
frequent ventricular ectopic beats (>1,000 during 24 hours; n = 3). Three
patients had a history of syncope. Twelve (92%) patients had at least one
low-voltage region at EAM, which corresponded at EAM-guided endomyocardial
biopsy to the histological diagnosis of active myocarditis in seven patients and
of arrhythmogenic right ventricular cardiomyopathy in five. In one patient the
histological evidence of contraction band necrosis allowed the unmasking of
caffeine and ephedrine abuse.
CONCLUSIONS: Electroanatomical substrate mapping may help diagnose concealed
myocardial diseases in competitive athletes presenting with recent-onset VAs and
an apparently normal heart. Further studies are warranted to assess the
prognostic implications of such subtle myocardial abnormalities. PURPOSE: To evaluate the prevalence of structural cardiac lesions using
echocardiography in apparently healthy boys referred for pre-participation
screening (PPS).
SUBJECTS AND METHODS: 3100 male soccer players were evaluated by
echocardiography in addition to the standard PPS.
RESULTS: In 56 subjects (1.8%), a structural cardiac lesion with potential
future complication was detected. Specifically, hypertrophic cardiomyopathy
(HCM) was found in two boys; bicuspid aortic valve (BAV) in 24; mitral valve
prolapse in 10 and atrial septal defects (ASDs) in 20. Resting physical
examination (PE) failed to identify any abnormalities in the majority of the
subjects. All the boys presented an uncomplicated echocardiography, except two
boys with HCM, one with BAV associated with aortic dilatation and one with a
large ASD.
CONCLUSION: Asymptomatic young athletes may have a structural cardiac alteration
with the potential of present or future haemodynamic and arrhythmic
consequences. A majority of mild cardiac lesions are difficult to diagnose or
suspect by the current screening based on medical history, PE and ECG.
Transthoracic echocardiography significantly improves the diagnostic power of
screening in the detection of both mild and serious cardiac conditions in the
athletic population. Sudden death in athletes occurs because of the existence of hidden
cardiovascular disorders which, during effort, may jeopardize the electrical
stability of the heart, triggering ventricular tachycardia and/or fibrillation.
Apart from rare conditions of ion channel diseases in the setting of a
structurally normal heart, in which the disorder may be easily diagnosed on
basal or stress test ECG, cardiac abnormalities at risk of causing sudden death
may affect the aorta (Marfan syndrome), the coronary arteries (congenital
coronary artery anomalies, premature coronary atherosclerosis), the myocardium
(hypertrophic and arrhythmogenic cardiomyopathy), the valves (bicuspid aortic
valve, mitral valve prolapse) and the conduction system (pre-excitation
syndromes). These structural heart disorders may be detected by ECG and/or echo.
The employment of these tools at pre-participation screening can help to
identify concealed anomalies, which may play a major role in early diagnosis,
risk stratification, and prevention of sudden death. The sudden cardiac death (SCD) of an athlete is always a dramatic event, and one
wonders if it could have been prevented by pre-participation cardiovascular
screening. For years now, a pro/con debate has been taking place on the
pre-participation screening of athletes: the method, who is responsible,
cost-effectiveness, obligatory or voluntary screening. In this pro-article,
which agrees with the "sudden cardiac death can be prevented by routinely
pre-participation cardiovascular screening"-standpoint, the unique Italian
experience is the best argument for the support of screening. This study clearly
demonstrated a reduction in SCD in those athletes who were screened in
accordance with Italian law. "Athlete's heart" is characterized by an increase in ventricular chamber sizes
and myocardial mass (MM), and is mainly observed in endurance athletes. At
present, it remains unclear whether cardiac adaptations in long-distance runners
differ from those in triathletes. Twenty male triathletes (mean age 38.7 ± 6.2
years) and 20 male marathon runners (mean age 44.1 ± 7.9) underwent cardiac
magnetic resoce imaging to calculate left and right ventricular end-diastolic
volume (EDV), end-systolic volume (ESV), stroke volume (SV), ejection fraction
(EF), and MM. Late-enhancement (LE) imaging was used to exclude structural
alterations or myocardial scarring. EDV, ESV, SV, and EF for the left and right
ventricles, as well as MM, did not differ between long-distance runners and
triathletes, although the weekly training volume was significantly higher in
triathletes (17.05 vs 9.95 h/week, P < 0.0001). There was a significant
correlation between weekly training volume and right and left EDV, right and
left ESV as well as MM within the study group. Myocardial LE was absent in all
athletes. Highly trained male long-distance runners and triathletes have
comparable cardiac parameters. However, the extent of physical training seems to
be associated with the degree of cardiac adaptation in endurance athletes. The
absence of LE supports the idea that athlete's heart is a nonpathological
adaptation of the cardiovascular system. Long-term high-intensity physical activity is associated with morphological
changes, termed as the 'athlete's heart'. The differentiation of physiological
cardiac adaptive changes in response to high-level exercise from pathological
changes consistent with an inherited cardiomyopathy is imperative.
Cardiovascular magnetic resoce (CMR) imaging allows definition of abnormal
processes occurring at the tissue level, including, importantly, myocardial
fibrosis. It is therefore vital in accurately making this differentiation. In
this review, we will review the role of CMR imaging of fibrosis, and detail CMR
characterisation of myocardial fibrosis in various cardiomyopathies, and the
implications of fibrosis. Additionally, we will outline advances in imaging
fibrosis, in particular T1 mapping. Finally we will address the role of CMR in
pre-participation screening. PURPOSE: Sudden cardiac death [SCD] in competitive athletes is caused by a
diverse set of cardiovascular diseases such as hypertrophic and dilated
cardiomyopathy [HCM/DCM], myocarditis, coronary anomalies or even coronary
artery disease. In order to identify potential risk factors responsible for SCD,
elite athletes underwent cardiac magnetic resoce [CMR] imaging.
MATERIALS AND METHODS: 73 male [M] and 22 female [F] athletes (mean age
35.2 ± 11.4 years) underwent CMR imaging. ECG-gated breath-hold cine SSFP
sequences were used for the evaluation of wall motion abnormalities and
myocardial hypertrophy as well as for quantitative analysis (left and right
ventricular [LV, RV] end-diastolic and end-systolic volume [EDV, ESV], stroke
volume [SV], ejection fraction [EF] and myocardial mass [MM]). Furthermore, left
and right atrial sizes were assessed by planimetry and delayed enhancement
imaging was performed 10 minutes after the application of contrast agent.
Coronary arteries were depicted using free-breathing Flash-3 D MR angiography.
RESULTS: The quantitative analyses showed eccentric hypertrophy of the left
ventricle (remodeling index [MM/LV-EDV]: M 0.75, F 0.665), enlargement of the RV
volumes (RV-EDV: M 122.6 ± 19.0 ml/m², F 99.9 ± 7.2 ml/m²) and an increased SV
(LV-SV: M 64.7 ± 10.0 ml/m², F 56.5 ± 5.7 ml/m²; RV-SV; M 66.7 ± 10.4 ml/m², F
54.2 ± 7.1 ml/m²). Abnormal findings were detected in 6 athletes (6.3 %)
including one benign variant of coronary anomaly and abnormal late gadolinium
enhancement in 2 cases. None of the athletes showed wall motion abnormalities or
signs of myocardial ischemia.
CONCLUSION: CMR imaging of endurance athletes revealed abnormal findings in more
than 5 % of the athletes. However, the prognostic significance remains unclear.
Thus, cardiac MRI cannot be recommended as a routine examination in the care of
athletes. Sudden cardiac death in young athletes is rare but tragic. The cardiology
community is faced with the challenge of providing a sensible strategy for the
prevention of SCD while simultaneously reaffirming that the benefits of regular
exercise far outweigh potential risks. At present, there is a broad range of
screening recommendations dependent upon country, sporting discipline, and
competition level. While much recent debate has focused on the efficacy of
screening with electrocardiography, a number of sporting bodies also mandate the
inclusion of exercise testing and echocardiography in screening protocols.
Cardiac magnetic resoce imaging, coronary calcium scoring and computed
tomography coronary angiography have also been promoted as potentially valuable
screening tools for competitive athletes. This review will examine the
controversial topic of utilizing cardiac imaging for athlete pre-participation
screening. Specifically, the limitations of screening for relatively rare
disorders using imaging tools with uncertain or imperfect accuracy will be
addressed. Current evidence suggests that the accuracy of all cardiac imaging
modalities is insufficient to justify their use as primary screening modalities
in athletes. Atypical findings such as marked cardiac dilation, reduced
deformation, or small patches of delayed gadolinium enhancement may be commonly
encountered in well-trained athletes, but, at present, the prognostic
significance of such findings is unknown. Resulting uncertainty for the
clinician and athlete has the potential for psychological stress, further
testing, and unnecessary exclusions from competition. However, these concerns
must not be confused with the extremely useful applications of cardiac imaging
for the assessment of athletes with symptoms, an abnormal electrocardiogram or a
positive family history. As modern imaging further enhances our understanding of
the spectrum of athlete's heart, its role may expand from the assessment of
athletes with suspected disease to being part of comprehensive pre-participation
screening in apparently healthy athletes. |
Is imatinib an antidepressant drug? | No. Imatinib is a tyrosine-kinase inhibitor used in the treatment of multiple cancers, most notably Chronic myelogenous leukemia (CML) and Gastrointestinal stromal tumor (GIST). | Imatinib mesylate (STI571), a specific Bcr-Abl inhibitor, has shown a potent
antileukemic activity in clinical studies of chronic myeloid leukemia (CML)
patients. Early prediction of response to imatinib cannot be anticipated. We
used a standardized quantitative reverse-transcriptase polymerase chain reaction
(QRT-PCR) for BCR-ABL transcripts on 191 out of 200 late-chronic phase CML
patients enrolled in a phase II clinical trial with imatinib 400 mg/day. Bone
marrow samples were collected before treatment, after 12, 20 and at the end of
study treatment (52 weeks) while peripheral blood samples were obtained after 2,
3, 6, 10, 14, 20 and 52 weeks of therapy. The amount of BCR-ABL transcript was
expressed as the ratio of BCR-ABL to beta2-microglobulin (beta2M). We show that,
following initiation of imatinib, the early BCR-ABL level trends in both bone
marrow and peripheral blood samples made it possible to predict the subsequent
cytogenetic outcome and response. We propose this method as the method of choice
for monitoring patients on imatinib therapy. QRT-PCR studies may be able to
identify degrees of molecular response that predict both complete cytogenetic
response and long term stability, as well as patterns of response that provide
an early indication of relapse and imatinib resistance. OBJECTIVES: The aim of the study was the analysis of women with gastrointestinal
stromal tumors (GIST) of small intestine treated and followed-up in Cancer
Center-Institute in Warsaw, who were primary operated in gynecological
departments due to suspicion of gynecological neoplasm.
MATERIALS AND METHODS: In the database of Clinical GIST Registry from 2001 to
2004 we identified 44 women with the diagnosis of CD117(+) GIST of small
intestine, what corresponds to 34% (44/130) all female GIST patients. Sixteen of
them (36.4%, 16/44) were primary operated on in gynecological departments due to
the tentative diagnosis of gynecological neoplasm.
RESULTS: The only indication for operation in 29 women was undiagnosed
microscopically tumor of the pelvis. Sixteen of them (55.2%, 16/29) were
operated on schedule in gynecological departments. The others 15 patients were
operated due to: ileus and perforation of digestive tract (8), gastrointestinal
bleeding (3), abdominal pain (2) and others (2). In analyzed group of patients
20 women (45.5%) after GIST excision remain without evidence of disease with
median follow-up time of 9 months, and in 24 patients (54.5%) GIST recurred in
median time of 18.5 months. In this latter group 23 patients were treated with
imatinib due to inoperable/metastatic lesions. Estimated 2-year overall survival
(calculated form the date of imatinib introduction) was 75%.
CONCLUSIONS: GISTs, especially of small intestine, may simulate in women
gynecological tumor, particularly of the ovary. Radical surgery remains the most
effective method of GIST treatment. In inoperable/metastatic lesion the
treatment of choice is tyrosinase kinase inhibitor--imatinib. Second-line treatments recommended by the National Cancer Center Network to
manage advanced-stage gastrointestinal stromal tumours (GIST) were evaluated to
determine the cost and cost-effectiveness of each intervention in the Mexican
insurance system, the Instituto Mexicano del Seguro Social (IMSS). Treatments
examined over a 5-year temporal horizon to estimate long-term costs included 800
mg day(-1) of imatinib mesylate, 50 mg day(-1) of sunitinib malate (administered
in a 4 week on/2 week rest schedule), and palliative care. The mean cost (MC),
cost-effectiveness, and benefit of each intervention were compared to determine
the best GIST treatment from the institutional perspective of the IMSS. As
sunitinib was not reimbursed at the time of the study, a Markov model and
sensitivity analysis were conducted to predict the MC and likelihood of
reimbursement. Patients taking 800 mg day(-1) of imatinib had the highest MC
(+/-s.d.) of treatment at $35,225.61 USD (+/-1253.65 USD); while sunitinib
incurred a median MC of $17,805.87 USD (+/-694.83 USD); and palliative care had
the least MC over treatment duration as the cost was $2071.86 USD (+/-472.88
USD). In comparison to palliative care, sunitinib is cost-effective for 38.9% of
patients; however, sunitinib delivered the greatest survival benefit as 5.64
progression-free months (PFM) and 1.4 life-years gained (LYG) were obtained in
the economic model. Conversely, patients on imatinib and palliative care saw a
lower PFM of 5.28 months and 2.58 months and also fewer LYG (only 1.31 and 1.08
years, respectively). Therefore, economic modeling predicts that reimbursing
sunitinib over high dose imatinib in the second-line GIST indication would
deliver cost savings to the IMSS and greater survival benefits to patients. BACKGROUND: Imatinib mesylate, an orally administered kinase inhibitor that
targets the Kit (CD117) protein, currently has 10 approved indications including
treatment of chronic myelogenous leukemia and metastatic gastrointestinal
stromal tumors (GIST). Treatment with adjuvant imatinib following surgical
resection of localized Kit-positive GIST, the most recent FDA-approved
indication (December 2008), has been shown to significantly improve
recurrence-free survival (RFS) compared with surgical resection alone. Although
adjuvant imatinib has proven effective in clinical trials, it is important to
consider the economic impact to health plans of introducing imatinib in
accordance with its new labeled indication.
OBJECTIVE: To evaluate the budgetary impact over a 3-year time horizon of
treating patients with localized Kit-positive GIST with 1 year of adjuvant
imatinib following surgical resection.
METHODS: A Markov model was developed to predict patients' transitions across
health states defined by initial treatment (surgical resection followed by
adjuvant imatinib 400 milligrams [mg] daily versus surgical resection alone),
recurrence, and progression. Treatments for a first recurrence were (a) imatinib
400 mg daily for recurrences following resection only or after completion of 1
year of treatment with imatinib 400 mg daily and (b) imatinib 800 mg daily for
recurrence during active treatment with imatinib 400 mg daily. Treatments for
further progression were imatinib 800 mg daily, sunitinib, or best supportive
care (BSC) following imatinib 400 mg per day, and sunitinib or BSC following
imatinib 800 mg daily. Recurrence rates were derived from the American College
of Surgeons Oncology Group (ACOSOG) Z9001 clinical trial, which compared 1 year
of adjuvant imatinib following surgical resection with surgical resection only.
The total number of patients with localized and surgically resected GIST
(incidence rate of 0.36 per 100,000) was estimated from epidemiologic studies of
GIST. Uptake of treatment with imatinib was estimated from unpublished data from
qualitative market research funded by the study sponsor. The uptake rate
assumptions reflected both (a) the percentage of patients with Kitpositive
disease and (b) the percentage of clinically eligible patients who would use
imatinib. Costs were estimated by combining unit costs from published sources
with expected resource utilization based on the clinical trial publication and
National Comprehensive Cancer Network guidelines on the treatment of patients
with GIST. To obtain estimates of the budgetary impact, we compared estimated
health care costs with versus without adjuvant imatinib, where health care costs
with imatinib reflected the costs of treatment minus cost offsets associated
with delayed or avoided recurrence or progression. All "with" scenarios assumed
no additional uses other than surgically resected localized Kit-positive GIST
(i.e., no change in off-label use of imatinib). The budgetary impact was
estimated for the first 3 years after the introduction of adjuvant imatinib in
accordance with its new labeled indication in a hypothetical plan population of
10 million persons. Results were calculated both as total budgetary impact and
as per member per month (PMPM) cost in 2009 dollars. Sensitivity analyses were
performed to test the robustness of model results to changes in parameter
estimates.
RESULTS: The model predicted 36 incident resected GIST cases per year in a
health plan of 10 million members. The estimated counts of cases treated with
adjuvant imatinib were 10.8, 16.2, and 21.6 in the first, second, and third
years after introduction, respectively, with the annual increases attributable
to changes in the proportion of patients with resected GIST assumed to use
imatinib (30% in year 1, rising to 45% in year 2 and 60% in year 3). The model
predicted that treatment of these cases with imatinib will increase pharmacy
costs by an additional $505,144 in the first year, $757,717 in the second year,
and $1,010,289 in the third year. Increased resource use associated with
monitoring patients during and after treatment with adjuvant imatinib would cost
an additional $21,564, $38,145, and $56,605 in the first, second, and third
years, respectively. Recurrence would be avoided or delayed in 7 patients over
the 3-year period. Avoided or delayed recurrences would result in cost offsets
of $61,583 in the first year, $156,702 in the second year, and $233,849 in the
third year. The net budgetary impact was estimated to be $465,126 in the first
year (less than $0.01 PMPM), $639,159 in the second year ($0.01 PMPM), and
$833,044 in the third year ($0.01 PMPM). Results of sensitivity analyses
indicated that the budgetary impact in the third year is most sensitive to
changes in the price of adjuvant imatinib and recurrence rates.
CONCLUSIONS: The model predicted that the introduction of adjuvant imatinib for
treatment of surgically resected, localized, Kit-positive GIST will lead to a
net budgetary impact of $0.01 PMPM in the third year after introduction assuming
change in use only in accordance with the new labeled indication. Approximately
11.7%-21.9% of the cost of adjuvant imatinib is offset by the reduction in costs
associated with GIST recurrence. We prospectively evaluated the efficacy and safety of imatinib plus hydroxyurea
in patients with progressive/recurrent meningioma. A total of 21 patients with
progressive/recurrent meningioma were enrolled in this dual center, single-arm,
phase II trial. All patients received 500 mg of hydroxyurea twice a day.
Imatinib was administered at 400 mg/day for patients not on CYP3A enzyme
inducing anti-epileptic drugs (EIAEDs) and at 500 mg twice a day for patients on
EIAEDs. The primary endpoint was progression-free survival at 6 months (PFS-6)
and secondary endpoints were safety, radiographic response rate, and overall
survival (OS). Best radiographic response was stable disease and was observed in
14 patients (67%). PFS-6 for all patients, those with grade I tumors (n = 8) and
those with grade II or III tumors (n = 13) was 61.9, 87.5 and 46.2%,
respectively. Patients with grade II or III tumors had poorer PFS and OS than
those with grade I tumors, (P = 0.025 and P = 0.018) respectively. The only
grade 3 or greater adverse event occurring in ≥ 10% of patients was anemia
(10%). Imatinib plus hydroxyurea is well tolerated among patients with
meningioma but has modest anti-tumor activity for this indication. Allogeneic hematopoietic stem cell transplantation (HSCT) is well-established as
a potentially curative treatment for patients who have chronic myeloid leukemia.
The success of imatinib and other tyrosine kinase inhibitors (TKI) as initial
therapy has changed the treatment paradigm for this disease. Allogeneic
hematopoietic transplants are now reserved for patients whose disease does not
respond optimally to TKI treatment. Patients whose disease does not have an
optimal response to imatinib may respond to a second-generation TKI, dasatinib
or nilotinib, and many achieve major or complete molecular and cytogenetic
responses. The indication for allogeneic HSCT versus continued second-line
therapy is not well-defined and is the subject of ongoing study. There has been
continued progress in reducing the toxicity and risks of HSCT with development
of reduced-intensity regimens; transplants can be routinely performed in
patients up to the age of 75 years who are in fair general medical condition.
Transplantation results from unrelated donors have improved, with survival rates
similar that achieved with matched siblings. Results with haploidentical and
cord blood transplants have markedly improved, and should be considered for
patients lacking a matched donor. Allogeneic hematopoietic transplants have the
best chance to be curative in patients with chronic phase that is under
hematologic control with 80% disease-free survival; patients progressing to the
accelerated phase or blast crisis have a much poorer prognosis. Thus, HSCT
should be considered for patients with imatinib failure. Patients receiving
second-line TKI therapy must be closely monitored and referred for
transplantation if a complete cytogenetic response and major molecular response
is not achieved. HSCT should be performed if feasible in patients without a
continued response to TKI treatment. In recent years, the panel of known molecular mutations in acute lymphoblastic
leukemia (ALL) has been continuously increased. In Philadelphia-positive ALL,
deletions of the IKZF1 gene were identified as prognostically adverse factors.
These improved insights in the molecular background and the clinical
heterogeneity of distinct cytogenetic subgroups may allow most differentiated
therapeutic decisions, for example, with respect to the indication to allogeneic
HSCT within genetically defined ALL subtypes. Quantitative real-time PCR allows
highly sensitive monitoring of the minimal residual disease (MRD) load, either
based on reciprocal gene fusions or immune gene rearrangements. Molecular
diagnostics provided the basis for targeted therapy concepts, for example,
combining the tyrosine kinase inhibitor imatinib with chemotherapy in patients
with Philadelphia-positive ALL. Screening for BCR-ABL1 mutations in
Philadelphia-positive ALL allows to identify patients who may benefit from
second-generation tyrosine kinase inhibitors or from novel compounds targeting
the T315I mutation. Considering the central role of the molecular techniques for
the management of patients with ALL, efforts should be made to facilitate and
harmonize immunophenotyping, cytogenetics, and molecular mutation screening.
Furthermore, the potential of high-throughput sequencing should be evaluated for
diagnosis and follow-up of patients with B-lineage ALL. Imatinib mesylate is the sole BCR-ABL tyrosine kinase inhibitor approved as
first-line treatment of accelerated-phase (AP) chronic myeloid leukemia (CML).
Indication was based on the STI571 0109 study, in which imatinib favorably
compared to historical treatments in patients failing prior therapies. The
relevance of these results to currently newly diagnosed AP-CML patients remains
unknown. We evaluated the benefit of imatinib in 42 newly diagnosed AP-CML
patients. In all, 16 patients had hematological acceleration without chromosomal
abnormalities in addition to the Philadelphia chromosome (ACAs; HEM-AP), 16
solely had ACAs (ACA-AP) and 10 had hematological acceleration plus ACAs (HEM-AP
+ ACA). Major cytogenetic responses were achieved in 93.7% of HEM-AP patients,
75% of patients with ACA-AP (P=NS) and 40% of patients with HEM-AP + ACA
(P=0.0053). The 24-month failure-free survival rate was 87.5% in HEM-AP
patients, 43.8% in ACA-AP patients and 15% in HEM-AP + ACA patients (P=0.022).
The 24-month estimate of progression-free survival was 100% in HEM-AP patients,
92.8% in ACA-AP patients and 58.3% in HEM-AP + ACA patients (P=0.0052). In
conclusion, frontline imatinib allows favorable outcomes in HEM-AP and ACA-AP
patients but appears insufficient for patients with HEM-AP + ACA. Broader-target
and/or more potent BCR-ABL tyrosine kinase inhibitors alone or in combination
may be considered in this setting. Hydroxyurea (HU) is an antimetabolic agent commonly used in myeloproliferative
disorders and hematological diseases as well as in severe psoriasis. Despite of
usually be well tolerated, sometimes it can induce immunosuppression and
mucocutaneous adverse effects associated with discomfort or pain. Nevertheless,
oral mucosal adverse reactions are extremely uncommon and present as ulcers,
tongue depapilation and dyschromia. Complete remission of adverse effects is
usually observed after withdrawal of the medication. The aim of this paper is to
report two patients with oral lesions related to HU treatment. T0 he patients
were adequately managed by changing hydroxyurea with imatinib mesilate. Oral
lesions are rare complications of long-term hydroxyurea treatment and may be an
indication of stopping therapy and substitution with imatinib mesilate. AIM: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor
of the gastrointestinal tract. Surgery remains the elective treatment. We
retrospectively compared two group of patients, who underwent surgery for GIST
before and after Imatinib advent in order to analyze the recurrence and survival
rate.
METHODS: Two patient groups who underwent surgery for GIST, from January 1997 to
December 2002 (Pre-Imatinib group) and from January 2003 to December 2008
(Post-Imatinib group) were compared. Patients were evaluated on the base of
gender, age, clinical manifestations, primary location and metastasis
positivity, tumor size, mitotic index, immunoreactivity for CD117 and the
outcome, including date of death.
RESULTS: In the Pre-IM group only one patient died for prostate cancer, 12
months after operation, the other died because of GIST with a 24.6 months of
median survival rate (range 15-51). In the remaining 12 patients the median
follow up period was 55 months (range 6-152 months). In the Post-IM group the
mean follow up was 50.7 months (range 26-74) and they are still being assessed
for oncological as well as surgical treatment.
CONCLUSION: Early diagnosis and radical resection remain the standard of cure
for GISTs. To date, the use of Imatinib lead to its utilization as adjuvant and
neo-adjuvant therapy in adults. Our experience suggests that there is a
correlation between the mutational status of KIT and clinical outcome. These
aspects should be explored for targeted therapy that can effectively combine
biological therapy to surgery. The outcome and quality of life of chronic myeloid leukemia (CML) patients has
remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs).
Currently, hematopoietic stem cell transplantation (HSCT) is considered mainly
as a third line salvage therapy in cases of TKIs resistance or intolerance. Here
we describe a patient with chronic phase CML who developed both resistance and
late occurrence of s severe thrombo-cytopenia on first and second generation
TKIs and eventually underwent HSCT. Although the mechanism of the
myelosuppression is not fully understood, we showed for the first time the
development of dose dependent platelet antibodies in the presence of TKIs,
suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes
that late development of severe myelosuppression during imatinib treatment is
probably an important indication for consideration of early HSCT. Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the alimentary
tract rarely observed in children. The treatment of choice for GIST is surgical
resection. Although the prognosis of GISTs with low maligt potential is
excellent, high-maligt-potential GISTs have a high rate of recurrence.
Prognostic factors such as tumor size, mitotic rate and presence of metastases
may provide an indication for adjuvant imatinib mesylate (IM) treatment. Here we
present a young patient with a large GIST with high-risk features who is in
complete remission after surgical excision and adjuvant IM treatment. This
patient is the only colon-located CD117-positive case where IM was administered.
The exact indications as well as the optimal dose and duration of IM need to be
clarified with the contribution of new cases and the growing experience of this
rare disease. BACKGROUND: The management of primary gastrointestinal stromal tumours (GISTs)
has evolved with the introduction of adjuvant therapy. Recently reported results
of the SSG XVIII/AIO trial by the Scandinavian Sarcoma Group (SSG) and the
German Working Group on Medical Oncology (AIO) represent a significant change in
the evidence for adjuvant therapy duration. The objectives of this European
Expert Panel meeting were to describe the optimal management and best practice
for the systemic adjuvant treatment of patients with primary GISTs.
MATERIALS AND METHODS: A panel of medical oncology experts from European sarcoma
research groups were invited to a 1-day workshop. Several questions and
discussion points were selected by the organising committee prior to the
conference. The experts reviewed the current literature of all clinical trials
available on adjuvant therapy for primary GISTs, considered the quality evidence
and formulated recommendations for each discussion point.
RESULTS: Clinical issues were identified and provisional clinical opinions were
formulated for adjuvant treatment patient selection, imatinib dose, duration and
patient recall, mutational analysis and follow-up of primary GIST patients.
Adjuvant imatinib 400 mg/day for 3 years duration is a standard treatment in all
patients with significant risk of recurrence following resection of primary
GISTs. Patient selection for adjuvant therapy should be based on any of the
three commonly used patient risk stratification schemes. R1 surgery (versus R0)
alone is not an indication for adjuvant imatinib in low-risk GIST. Recall and
imatinib restart could be proposed in patients who discontinued 1-year adjuvant
imatinib within the previous 3 months and may be considered on a case-by-case
basis in patients who discontinued within the previous year. Mutational analysis
is recommended in all cases of GISTs using centralised laboratories with good
quality control. Treatment is not recommended in an imatinib-insensitive
D842V-mutated GIST. During adjuvant treatment, patients are recommended to be
clinically assessed at 1- to 3-month intervals. Upon discontinuation, computed
tomography scan (CT) scans are recommended every 3 to 4 months for 2 years when
the risk of relapse is highest, followed by every 6 months until year 5 and
annually until year 10 after treatment discontinuation.
CONCLUSIONS: Key points in systemic adjuvant treatment and clinical management
of primary GISTs as well as open questions were identified during this European
Expert Panel meeting on GIST management. The use of complementary and alternative medicines (CAM) by cancer patients is
increasing. Concomitant use of CAM and anticancer drugs could lead to serious
safety issues in patients. CAM have the potential to cause pharmacokinetic
interactions with anticancer drugs, leading to either increased or decreased
plasma levels of anticancer drugs. This could result in unexpected toxicities or
a reduced efficacy. Significant pharmacokinetic interactions have already been
shown between St. John's Wort (SJW) and the anticancer drugs imatinib and
irinotecan. Most pharmacokinetic CAM-drug interactions, involve drug
metabolizing cytochrome P450 (CYP) enzymes, in particular CYP3A4. The effect of
CAM on CYP3A4 activity and expression can be assessed in vitro. However, no data
have been reported yet regarding the relevance of these in vitro data for the
prediction of CAM-anticancer drug interactions in clinical practice. To address
this issue, a literature research was performed to evaluate the relevance of in
vitro data to predict clinical effects of CAM frequently used by cancer
patients: SJW, milk thistle, garlic and Panax ginseng (P. ginseng). Furthermore,
in clinical studies the sensitive CYP3A4 substrate probe midazolam is often used
to determine pharmacokinetic interactions. Results of these clinical studies
with midazolam are used to predict pharmacokinetic interactions with other drugs
metabolized by CYP3A4. Therefore, this review also explored whether clinical
trials with midazolam are useful to predict clinical pharmacokinetic
CAM-anticancer drug interactions. In vitro data of SJW have shown CYP3A4
inhibition after short-term exposure and induction after long-term exposure. In
clinical studies using midazolam or anticancer drugs (irinotecan and imatinib)
as known CYP3A4 substrates in combination with SJW, decreased plasma levels of
these drugs were observed, which was expected as a consequence of CYP3A4
induction. For garlic, no effect on CYP3A4 has been shown in vitro and also in
clinical studies garlic did not affect the pharmacokinetics of both midazolam
and docetaxel. Milk thistle and P. ginseng predomitly showed CYP3A4
inhibition in vitro. However, in clinical studies these CAM did not cause
significant pharmacokinetic interactions with midazolam, irinotecan, docetaxel
and imatinib. Most likely, factors as poor pharmaceutical availability,
solubility and bioavailability contribute to the lack of significant clinical
interactions. In conclusion, in vitro data are useful as a first indication for
potential pharmacokinetic drug interactions with CAM. However, the discrepancies
between in vitro and clinical results for milk thistle and P. ginseng show that
clinical studies are required for confirmation of potential interactions. At
last, midazolam as a model substrate for CYP3A4, has convincingly shown to
correctly predict clinical interactions between CAM and anticancer drugs. |
What is the function of the MTH1 enzyme in cancer cells? | The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. | 8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in
their precursors due to the actions of oxygen radicals produced through a normal
cellular metabolism. Since oxidized guanine can pair with both cytosine and
adenine, it causes alterations in the phenotypic expression when it is present
in RNA. To prevent such an outcome, organisms must have some mechanism for
eliminating such oxidized guanine nucleotides from RNA and its precursors. In
mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to
8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins
functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has
been suggested to be a good candidate for such a role. The human PNP protein has
an ability to bind specifically to RNA containing 8-oxoGua. When human cells are
exposed to agents that induce oxidative stress, such as hydrogen peroxide and
menadion, the amounts of PNP protein decrease rapidly while amounts of other
proteins in the cells do not change after such treatments. No specific decrease
in the PNP protein level is observed when cells are treated with ACNU and
cycloheximide at doses sufficient to provide the same degree of growth
suppression. These results imply that the PNP protein might thus play a role in
excluding oxidized forms of RNA from the translation mechanism. The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides
including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as
an important defense system against mutagenesis, carcinogenesis, and cell death
induced by oxidized purine nucleotides. We previously reported that the
functional groups at the 2- and 6-positions of the purine ring affect the
recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are
substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this
study, three nucleotide analogs containing this motif were examined. A synthetic
purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino
function (dJTP) was hydrolyzed to the monophosphate with high efficiency by
MTH1. On the other hand, two analogs that lack the two-ring system of their
bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates.
FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less
efficiently than 8-hydroxy-dGTP. These results clarify the effects of the
2-amino group and the two-ring system of the purine base on the recognition by
the human MTH1 protein. A number of environmental factors, such as tobacco and alcohol, have been
implicated, through oxidative DNA damage, in the development of squamous cell
carcinomas of the head and neck (SCCHN). Several pathways are involved in the
repair of DNA lesions caused by oxidative stress, such as the base excision
repair system (BER), which repairs mutation involving 8-oxoguanine and comprises
the MUTYH, OGG1 and MTH1 genes. We analysed 29 patients, assessing germline
polymorphisms or mutations in these genes by complete genomic sequencing of
exons and adjacent intronic regions. Thirty healthy blood donors served as
controls. No pathogenic germline mutations were identified. We found common and
rare new variants in the coding and adjacent intronic regions. In summary, our
data do not support a major role for MUTYH, OGG1 and MTH1 variants in the
etiology of sporadic squamous oral/oropharyngeal carcinomas. This does not
exclude the involvement of the three BER genes in the tumorigenesis of SCCHN
through other mechanisms such as promotor hypermethylation, genomic
rearrangements or mutations involving regulatory sequences. To assess the functions of the three human MutT-type enzymes, MTH1, MTH2, and
NUDT5, mutation induction by an oxidized form of dGTP,
8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP;
7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), was examined using human
293T cells treated with their specific siRNAs. Shuttle plasmid DNA containing
the supF gene was first transfected into the cells, and then 8-OH-dGTP was
introduced by means of osmotic pressure. Escherichia coli cells were transformed
with the DNAs replicated in the treated cells. The knockdown of the MTH1, MTH2,
and NUDT5 proteins increased the A:T --> C:G substitution mutations induced by
8-OH-dGTP. In addition, the increase in the induced mutation frequency was more
evident in the triple-knockdown cells. These results indicate that all three of
the human MTH1, MTH2, and NUDT5 proteins act as a defense against the
mutagenesis induced by oxidized dGTP. MutT-related proteins, including Escherichia coli MutT and the human MTH1
(NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to
8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of
8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to
cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade
8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are
interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors
during DNA replication. The two activities associated with NUDT5 exhibit
different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9,
while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two
types of reactions indicated that ADP ribose is a better substrate for NUDT5
compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was
competitively inhibited by ADP ribose and its reaction product, AMP, and in
reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results
imply that the two types of substrates may share the same binding site for
catalysis. MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from
being incorporated into DNA. We here present the structures of human MTH1 (1.9Å)
and its complex with the product 8-oxo-dGMP (1.8Å). Unexpectedly MTH1 binds the
nucleotide in the anti conformation with no direct interaction between the 8-oxo
group and the protein. We suggest that the specificity depends on the
stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the
product induces no major structural changes. The structures reveal the mode of
nucleotide binding in MTH1 and provide the structural basis for inhibitor
design. Most of the proteins carrying the 23-residue MutT-related sequence are capable
of hydrolyzing compounds with a general structure of nucleoside diphosphate
linked to another moiety X and are called the Nudix hydrolases. Among the 22
human Nudix proteins (identified by the sequence signature), some remain
uncharacterized as enzymes without a defined substrate. Here, we reveal that the
NUDT18 protein, whose substrate was unknown, can degrade
8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the
monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2
(NUDT15), we propose that it should be named MTH3. Although these three human
proteins resemble each other in their sequences, their substrate specificities
differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2
can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme
activity of MTH2 is considerably lower than that of MTH1. On the other hand,
MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP.
Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and
8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the
MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can
degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the
finding that MTH1 and MTH2 show a limited activity against the ribonucleotide
counterpart, 8-oxo-GTP. These three enzymes may function together to help
maintain the high fidelity of DNA replication and transcription under oxidative
stress. Oxidative damage can be induced by many environmental stressors.
8-Hydroxydeoxyguanosine (8-OHdG) has been used as a biomarker of oxidative DNA
damage in both in vitro and in vivo studies. In the present study, Wistar rats
were exposed to radon gas at a concentration of 100,000Bq/m(3) for 12 h/d for
30, 60, and 120 d, equivalent to cumulative doses of 60, 120, and 240 working
level months (WLM), respectively. Changes in levels of 8-OHdG, reactive oxygen
species (ROS), and total antioxidant (T-AOC), as well as expressions of some DNA
repair enzymes such as 8-oxoguanine DNA glycosylase (OGG1) and MutT homolog 1
(oxidized purine nucleoside triphosphatase, MTH1), were determined in rat urine,
peripheral blood lymphocytes, and lung after exposure to radon. The results
revealed an increase in 8-OHdG and ROS levels, a decrease in T-AOC levels, and
reduced OGG1 and MTH1 expression levels. The elevated amount of 8-OHdG in urine
or lymphocytes was positively correlated with the cumulative exposure dose,
whereas OGG1 and MHT1 expression levels in lung were inversely correlated with
cumulative exposure dose. These findings indicate that oxidative damage induced
by radon may be involved in radon-induced carcinogenesis. Glucose uptake, the first, rate-limiting step of its utilization, is facilitated
by glucose transporters. Expression of several glucose transporter (HXT) genes
in yeast is repressed by the Rgt1 repressor, which recruits the
glucose-responsive transcription factor Mth1 and the general corepressor complex
Ssn6-Tup1 in the absence of glucose; however, it is derepressed when Mth1 is
inactivated by glucose. Here we show that Ssn6-Tup1 interferes with the
DNA-binding ability of Rgt1 in the absence of Mth1 and that the Rgt1 function
abrogated by Ssn6 overexpression is restored by co-overexpression of Mth1. Thus
Mth1 likely regulates Rgt1 function not by modulating its DNA-binding activity
directly but by functionally antagonizing Ssn6-Tup1. Mth1 does so by acting as a
scaffold-like protein to recruit Ssn6-Tup1 to Rgt1. Supporting evidence shows
that Mth1 blocks the protein kinase A-dependent phosphorylation of Rgt1 that
impairs the ability of Rgt1 to interact with Ssn6-Tup1. Of note, Rgt1 can bind
DNA in the absence of Ssn6-Tup1 but does not inhibit transcription, suggesting
that dissociation of Rgt1 from Ssn6-Tup1, but not from DNA, is necessary and
sufficient for the expression of its target genes. Taken together, these
findings show that Mth1 is a transcriptional corepressor that facilitates the
recruitment of Ssn6-Tup1 by Rgt1. The reversible nature of protein phosphorylation dictates that any protein
kinase activity must be counteracted by protein phosphatase activity. How
phosphatases target specific phosphoprotein substrates and reverse the action of
kinases, however, is poorly understood in a biological context. We address this
question by elucidating a novel function of the conserved PP4 family phosphatase
Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the
glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets
the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain
of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity
is required for the timely dephosphorylation of the downstream transcriptional
repressor Rgt1 upon glucose withdrawal, a critical event in the repression of
HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1,
an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites
associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase
antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein
kinase activated in response to glucose, in vitro and regulates Mth1 function
via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2
phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene
expression by glucose. |
Are ultraconserved elements depleted among copy number variants (CNVs)? | Yes. Interestingly, human ultraconserved elements (UCEs) have been reported to be strongly depleted among segmental duplications and benign copy number variants (CNVs). These elements may be interpreted as hallmarks for dose-sensitive genes, particularly for those genes whose gain or loss may be directly implied in neurodevelopmental disorders. Therefore, their presence in genomic imbalances of unknown effect might be suggestive of a clinically relevant condition | An earlier search in the human, mouse and rat genomes for sequences that are
100% conserved in orthologous segments and > or = 200 bp in length identified
481 distinct sequences. These human-mouse-rat sequences, which represent
ultraconserved elements (UCEs), are believed to be important for functions
involving DNA binding, RNA processing and the regulation of transcription and
development. In vivo and additional computational studies of UCEs and other
highly conserved sequences are consistent with these functional associations,
with some observations indicating enhancer-like activity for these elements.
Here, we show that UCEs are significantly depleted among segmental duplications
and copy number variants. Notably, of the UCEs that are found in segmental
duplications or copy number variants, the majority overlap exons, indicating,
along with other findings presented, that UCEs overlapping exons represent a
distinct subset. Ultraconserved elements (UCEs) are segments of >200 bp length showing absolute
sequence identity between orthologous regions of human, rat and mouse genomes.
The selection factors acting on these UCEs are still unknown. Recent studies
have shown that UCEs function as long-range enhancers of flanking genes or are
involved in splicing when overlapping with exons. The depletion of UCEs among
copy number variation as well as the significant under-representation of
single-nucleotide polymorphisms (SNPs) within UCEs have also revealed their
evolutional and functional importance indicating their potential impact on
disease, such as cancer. In the present study, we investigated the influence of
six SNPs within UCEs on familial breast cancer risk. Two out of six SNPs showed
an association with familial breast cancer risk. Whereas rs9572903 showed only a
borderline significant association, the frequency of the rare [G] allele of
rs2056116 was higher in cases than in controls indicating an increased familial
breast cancer risk ([G] versus [A]: odds ratio (OR) = 1.18, 95% confidence
interval (CI) 1.06-1.30, P = 0.0020; [GG] versus [AA]: OR = 1.41, 95% CI
1.15-1.74, P = 0.0011). Interestingly, comparing with the older age group, the
ORs were increased in woman younger than 50 years of age ([G] versus [A]: OR =
1.27, 95% CI 1.11-1.45, P = 0.0005; [GG] versus [AA]: OR = 1.60, 95% CI
1.22-2.10, P = 0.0007) pointing to an age- or hormone-related effect. This is
the first study indicating that SNPs in UCEs might be associated with cancer
risk. Ultraconserved elements (UCEs) are sequences that are identical between
reference genomes of distantly related species. As they are under negative
selection and enriched near or in specific classes of genes, one explanation for
their ultraconservation may be their involvement in important functions. Indeed,
many UCEs can drive tissue-specific gene expression. We have demonstrated that
nonexonic UCEs are depleted among segmental duplications (SDs) and copy number
variants (CNVs) and proposed that their ultraconservation may reflect a
mechanism of copy counting via comparison. Here, we report that nonexonic UCEs
are also depleted among 10 of 11 recent genomewide data sets of human CNVs,
including 3 obtained with strategies permitting greater precision in determining
the extents of CNVs. We further present observations suggesting that nonexonic
UCEs per se may contribute to this depletion and that their apparent dosage
sensitivity was in effect when they became fixed in the last common ancestor of
mammals, birds, and reptiles, consistent with dosage sensitivity contributing to
ultraconservation. Finally, in searching for the mechanism(s) underlying the
function of nonexonic UCEs, we have found that they are enriched in TAATTA,
which is also the recognition sequence for the homeodomain DNA-binding module,
and bounded by a change in A + T frequency. Ultraconserved elements (UCEs) are strongly depleted from segmental duplications
and copy number variations (CNVs) in the human genome, suggesting that deletion
or duplication of a UCE can be deleterious to the mammalian cell. Here we
address the process by which CNVs become depleted of UCEs. We begin by showing
that depletion for UCEs characterizes the most recent large-scale human CNV
datasets and then find that even newly formed de novo CNVs, which have passed
through meiosis at most once, are significantly depleted for UCEs. In striking
contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted
for UCEs and can even become significantly enriched. This observation raises the
possibility that CNVs that arise somatically and are relatively newly formed are
less likely to have established a CNV profile that is depleted for UCEs.
Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the
diseased state. In support of this latter explanation, somatic CNVs that are not
associated with disease are depleted for UCEs. Finally, we show that it is
possible to observe the CNVs of induced pluripotent stem (iPS) cells become
depleted of UCEs over time, suggesting that depletion may be established through
selection against UCE-disrupting CNVs without the requirement for meiotic
divisions. |
Is the microRNA 132 (miR-132) involved in brain pathologies? | Yes. MicroRNA 132 (miR-132), is involved in brain pathologies. | Micro-RNAs constitute a family of small noncoding ribonucleic acids that are
posttranscriptional regulators of messenger RNA activity. Although micro-RNAs
are known to be dynamically regulated during neural development, the role of
micro-RNAs in brain aging and neurodegeneration is not known. This study
examined micro-RNA abundance in the hippocampal region of fetal, adult and
Alzheimer's disease brain. The data indicate that micro-RNAs encoding miR-9,
miR-124a, miR-125b, miR-128, miR-132 and miR-219 are abundantly represented in
fetal hippocampus, are differentially regulated in aged brain, and an alteration
in specific micro-RNA complexity occurs in Alzheimer hippocampus. These data are
consistent with the idea that altered micro-RNA-mediated processing of messenger
RNA populations may contribute to atypical mRNA abundance and neural dysfunction
in Alzheimer's disease brain. Huntington's disease (HD) is an incurable, fatal neurodegenerative disorder that
is caused by a polyglutamine expansion in the huntingtin (Htt) protein. Neuronal
death in the striatum-the most obvious manifestation of the disease-is likely to
result from widespread dysregulation of gene expression in various brain
regions. To date, several potential mechanisms for this have been discovered,
including one involving REST (RE1-Silencing Transcription Factor), a master
regulator of neuronal genes. Recently, independent studies have demonstrated
that post-transcriptional gene regulation by microRNAs is also disrupted in HD.
Expression of key neuronal microRNAs-including mir-9/9*, mir-124 and mir-132-is
repressed in the brains of human HD patients and mouse models. These changes
occur downstream of REST, and are likely to result in major disruption of mRNA
regulation and neuronal function. In this study we will discuss these findings
and their implications for our understanding of HD. Using updated bioinformatic
analysis, we predict 21 new candidate microRNAs in HD. We propose future
strategies for unifying large-scale transcriptional and microRNA datasets with
the aim of explaining HD aetiology. By way of example, we show how available
genomic datasets can be integrated to provide independent, analytical validation
for dysregulation of REST and microRNA mir-124 in HD. As a consequence, gene
ontology analysis indicates that HD is characterised by a broad-based depression
of neural genes in the caudate and motor cortex. Thus, we propose that a
combination of REST, microRNAs and possibly other non-coding RNAs profoundly
affect the neuronal transcriptome in HD. Activity-dependent changes in gene-expression are believed to underlie the
molecular representation of memory. In this study, we report that in vivo
activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To
determine if production of miR-132 is regulated by neuronal activity its
expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR).
Pilocarpine-induced seizures led to a robust, rapid, and transient increase in
the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in
mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory
bulb, and striatum by contextual fear conditioning, odor-exposure, and
cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics
of pri-miR-132 were monitored and found to parallel those of immediate early
genes, peaking at 45 min and returning to basal levels within 2 h of
stimulation. Expression levels of primary and mature-miR-132 increased
significantly between postnatal Days 10 and 24. We conclude that miR-132 is an
activity-dependent microRNA in vivo, and may contribute to the long-lasting
proteomic changes required for experience-dependent neuronal plasticity. Preconditioning describes the ischemic stimulus that triggers an endogenous,
neuroprotective response that protects the brain during a subsequent severe
ischemic injury, a phenomenon known as 'tolerance'. Ischemic tolerance requires
new protein synthesis, leads to genomic reprogramming of the brain's response to
subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate
posttranscriptional gene expression by exerting direct effects on messenger RNA
(mRNA) translation. We examined miRNA expression in mouse cortex in response to
preconditioning, ischemic injury, and tolerance. The results of our microarray
analysis revealed that miRNA expression is consistently altered within each
group, but that preconditioning was the foremost regulator of miRNAs. Our
bioinformatic analysis results predicted that preconditioning-regulated miRNAs
most prominently target mRNAs that encode transcriptional regulators; methyl-CpG
binding protein 2 (MeCP2) was the most prominent target. No studies have linked
MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2
expression, is decreased in preconditioned cortex. Downregulation of miR-132 is
consistent with our finding that preconditioning ischemia induces a rapid
increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal
that ischemic preconditioning regulates expression of miRNAs and their predicted
targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could
serve as effectors of ischemic preconditioning-induced tolerance. Huntington's disease (HD) is a genetic neurodegenerative disease caused by
abnormal CAG expansion. MicroRNAs (miRNAs) are short RNA molecules regulating
gene expression, and are implicated in a variety of diseases including HD.
However, the profiles and regulation of miRNAs in HD are not fully understood.
Here, we analyzed the miRNA expression and miRNA regulators in two transgenic
models of HD, YAC128 and R6/2 mice, and in a 3-nitropropionic acid (3NP)-induced
striatal degeneration rat model. After characterizing the phenotypes by
behavioral tests and histological analyses, we profiled striatal miRNAs using a
miRNA microarray and we measured the key molecules involved in miRNA biogenesis
and function. YAC128 mice showed upregulation-domit miRNA expressions at 5
months and downregulation-domit expressions at 12 months. Concomitantly, the
expressions of Drosha-DGCR8, Exportin-5, and Dcp1 were increased at 5months, and
the expression of Dicer was decreased at 12 months. In 10-week-old R6/2 mice,
downregulation was domit in the miRNA expressions and the level of Drosha
decreased concomitantly. Nine miRNAs (miR-22, miR-29c, miR-128, miR-132,
miR-138, miR-218, miR-222, miR-344, and miR-674*) were commonly down-regulated
in both the 12-month-old YAC128 and 10-week-old R6/2 mice. Meanwhile, 3NP rats
showed dynamic changes in the miRNA profiles during disease development and a
few miRNAs with altered expression. Our results show that transgenic HD mice
have abnormal miRNA biogenesis. This information should aid in future studies on
therapeutic application of miRNAs in HD. The α-synuclein has been implicated in the pathophysiology of Parkinson's
disease (PD), because mutations in the alpha-synuclein gene cause
autosomal-domit hereditary PD and fibrillary aggregates of alpha-synuclein
are the major component of Lewy bodies. Since presynaptic accumulation of
α-synuclein aggregates may trigger synaptic dysfunction and degeneration, we
have analyzed alterations in synaptosomal proteins in early symptomatic
α-synuclein(A30P)-transgenic mice by two-dimensional differential gel
electrophoresis. Moreover, we carried out microRNA expression profiling using
microfluidic chips, as microRNA have recently been shown to regulate synaptic
plasticity in rodents and to modulate polyglutamine-induced protein aggregation
and neurodegeneration in flies. Differentially expressed proteins in
α-synuclein(A30P)-transgenic mice point to alterations in mitochondrial
function, actin dynamics, iron transport, and vesicle exocytosis, thus partially
resembling findings in PD patients. Oxygen consumption of isolated brain
mitochondria, however, was not reduced in mutant mice. Levels of several
microRNA (miR-10a, -10b, -212, -132, -495) were significantly altered. One of
them (miR-132) has been reported to be highly inducible by growth factors and to
be a key regulator of neurite outgrowth. Moreover, miR-132-recognition sequences
were detected in the mRNA transcripts of two differentially expressed proteins.
MicroRNA may thus represent novel biomarkers for neuronal malfunction and
potential therapeutic targets for human neurodegenerative diseases. Tauopathies represent a large class of neurological and movement disorders
characterized by abnormal intracellular deposits of the microtubule-associated
protein tau. It is now well established that mis-splicing of tau exon 10,
causing an imbalance between three-repeat (3R) and four-repeat (4R) tau
isoforms, can cause disease; however, the underlying mechanisms affecting tau
splicing in neurons remain poorly understood. The small noncoding microRNAs
(miRNAs), known for their critical role in posttranscriptional gene expression
regulation, are increasingly acknowledged as important regulators of alternative
splicing. Here, we identified a number of brain miRNAs, including miR-124,
miR-9, miR-132 and miR-137, which regulate 4R:3R-tau ratios in neuronal cells.
Analysis of miRNA expression profiles from sporadic progressive supranuclear
palsy (PSP) patients, a major 4R-tau tauopathy, showed that miR-132 is
specifically down-regulated in disease. We demonstrate that miR-132 directly
targets the neuronal splicing factor polypyrimidine tract-binding protein 2
(PTBP2), which protein levels were increased in PSP patients. miR-132
overexpression or PTBP2 knockdown similarly affected endogenous 4R:3R-tau ratios
in neuronal cells. Finally, we provide evidence that miR-132 is inversely
correlated with PTBP2 during post-natal brain development at the time when
4R-tau becomes expressed. Taken together, these results suggest that changes in
the miR-132/PTBP2 pathway could contribute to the abnormal splicing of tau exon
10 in the brain, and sheds light into the potential role played by miRNAs in a
subset of tauopathies. Recent reports of microRNA (miR) modulators of both neuronal and immune
processes (here termed NeurimmiRs) predict therapeutic potential for
manipulating NeurimmiR levels in diseases affecting both the immune system and
higher brain functions, such as Alzheimer's disease (AD), Parkinson's disease
(PD), multiple sclerosis (MS) and anxiety-related disorders. In our opinion,
NeurimmiRs that function within both the nervous and the immune systems, such as
miR-132 and miR-124, may act as 'negotiators' between these two interacting
compartments. We suggest that NeurimmiRs primarily target transcriptional or
other regulatory genes, which enables modulation of both immune and cognitive
processes through direct or indirect alterations of neuron-glia and/or
brain-to-body signaling. Thus, manipulating NeurimmiR control over the immune
contributions to cognitive pathways may offer new therapeutic targets. When an otherwise harmful insult to the brain is preceded by a brief,
noninjurious stimulus, the brain becomes tolerant, and the resulting damage is
reduced. Epileptic tolerance develops when brief seizures precede an episode of
prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding
RNAs that function as post-transcriptional regulators of gene expression. We
investigated how prior seizure preconditioning affects the miRNA response to
status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was
extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status
epilepticus in animals that had previously received either seizure
preconditioning (tolerance) or no preconditioning (injury), and mature miRNA
levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs
was increased, relative to control, after status epilepticus alone, and
expression of 12 miRNAs was decreased. Increased miR-132 levels were matched
with increased binding to Argonaute-2, a constituent of the RNA-induced
silencing complex. In tolerant animals, expression responses of >40% of the
injury-group-detected miRNAs differed, being either unchanged relative to
control or down-regulated, and this included miR-132. In vivo microinjection of
locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132
depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death.
Thus, our data strongly suggest that miRNAs are important regulators of
seizure-induced neuronal death. Schizophrenia is characterized by affective, cognitive, neuromorphological, and
molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs
(miRNAs) are small noncoding RNA sequences critical to neurodevelopment and
adult neuronal processes by coordinating the activity of multiple genes within
biological networks. We examined the expression of 854 miRNAs in prefrontal
cortical tissue from 100 control, schizophrenic, and bipolar subjects. The
cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was
significantly down-regulated in both the schizophrenic discovery cohort and a
second, independent set of schizophrenic subjects. Analysis of miR-132 target
gene expression in schizophrenia gene-expression microarrays identified 26 genes
up-regulated in schizophrenia subjects. Consistent with NMDA-mediated
hypofunction observed in schizophrenic subjects, administration of an NMDA
antagonist to adult mice results in miR-132 down-regulation in the prefrontal
cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits
significant developmental regulation and overlaps with critical
neurodevelopmental processes during adolescence. Adult prefrontal expression of
miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor
signaling during a brief postnatal period. Several key genes, including DNMT3A,
GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression
either during normal neurodevelopment or in tissue from adult schizophrenic
subjects. Our data suggest miR-132 dysregulation and subsequent abnormal
expression of miR-132 target genes contribute to the neurodevelopmental and
neuromorphological pathologies present in schizophrenia. Early stages of many neurodegenerative diseases, such as Alzheimer's disease,
vascular and frontotemporal dementia, and Parkinson's disease, are frequently
associated with Mild Cognitive Impairment (MCI). A minimally invasive screening
test for early detection of MCI may be used to select optimal patient groups in
clinical trials, to monitor disease progression and response to treatment, and
to better plan patient clinical care. Here, we examined the feasibility of using
pairs of brain-enriched plasma microRNA (miRNA), at least one of which is
enriched in synapses and neurites, as biomarkers that could differentiate
patients with MCI from age-matched controls. The identified biomarker pairs fall
into two sets: the "miR-132 family" (miR-128/miR-491-5p, miR-132/miR-491-5p and
mir-874/miR-491-5p) and the "miR-134 family" (miR-134/miR-370,
miR-323-3p/miR-370 and miR-382/miR-370). The area under the Receiver-Operating
Characteristic curve for the differentiation of MCI from controls using these
biomarker pairs is 0.91-0.95, with sensitivity and specificity at 79%-100%
(miR-132 family) and 79%-95% (miR-134 family), and p〈0.001. In a separate
longitudinal study, the identified miRNA biomarker pairs successfully detected
MCI in majority of patients at asymptomatic stage 1-5 years prior to clinical
diagnosis. The reported biomarker pairs also appear useful for detecting
age-related brain changes. Further testing in a larger study is necessary for
validation of these results. Prion diseases typically have long pre-clinical incubation periods during which
time the infectious prion particle and infectivity steadily propagate in the
brain. Abnormal neuritic sprouting and synaptic deficits are apparent during
pre-clinical disease, however, gross neuronal loss is not detected until the
onset of the clinical phase. The molecular events that accompany early neuronal
damage and ultimately conclude with neuronal death remain obscure. In this
study, we used laser capture microdissection to isolate hippocampal CA1 neurons
and determined their pre-clinical transcriptional response during infection. We
found that gene expression within these neurons is dynamic and characterized by
distinct phases of activity. We found that a major cluster of genes is altered
during pre-clinical disease after which expression either returns to basal
levels, or alternatively undergoes a direct reversal during clinical disease.
Strikingly, we show that this cluster contains a signature highly reminiscent of
synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation
of neuroprotective pathways. Additionally, genes involved in neuronal projection
and dendrite development were also altered throughout the disease, culminating
in a general decline of gene expression for synaptic proteins. Similarly,
deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p,
miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the
first in depth genomic study describing the pre-clinical response of hippocampal
neurons to early prion replication. Our findings suggest that prion replication
results in the persistent stimulation of a programmed response that is mediated,
at least in part, by synaptic NMDA receptor activity that initially promotes
cell survival and neurite remodelling. However, this response is terminated
prior to the onset of clinical symptoms in the infected hippocampus, seemingly
pointing to a critical juncture in the disease. Manipulation of these early
neuroprotective pathways may redress the balance between degeneration and
survival, providing a potential inroad for treatment. |
What is an approximate number of CTCF binding sites in the human genome? | The number of CTCF binding sites in the human genome lies between 31,000 and 50,000. | The molecular basis for commitment of progenitors to the eosinophil lineage and
mechanisms by which eosinophil-specific genes are expressed and regulated during
differentiation is unknown. Expression of eosinophil peroxidase (EPO) is
restricted to the eosinophil lineage. To understand the mechanisms involved in
transcriptional regulation of EPO gene expression, we clone the region of the
EPO gene upstream of the transcriptional start site and analyzed the cis-acting
elements required for EPO promoter activity in an eosinophil-inducible leukemic
cell line, HL-60-C15. The 5'-flanking region of the EPO gene containing 1.5
kilobases of sequence upstream of the transcriptional start site was subcloned
into the promoterless pXP2-luciferase vector. The EPO-pXP2 construct and 5'
deletion mutants were electroporated into HL-60-C15 cells and luciferase
reporter activity assessed. The -1.5-kilobase EPO-pXP2 promoter construct
reproducibly expressed > 120-fold more luciferase activity than did promoterless
pXP2, and a 12-fold (90%) decrease in promoter activity was obtained when
sequences between -122 and -45 base pairs (bp) were deleted. The specificity of
the EPO promoter for the eosinophil lineage was analyzed by transfecting the
EPO-pXP2 constructs and deletion mutants into HL-60-C15 cells and the parental
HL-60 line; EPO promoter activity was 8-10-fold less in the HL-60 parental line,
suggesting lineage specific elements in the -122 to -45 bp region. To further
characterize regulatory sequences important for promoter activity, we performed
linker-scanning analysis on the -122 to -45 bp region and identified a number of
positively and negatively acting elements in the promoter. DNase I footprinting
was performed with HL-60-C15, HL-60, and HeLa nuclear extracts to identify
nuclear proteins that may bind to the functional elements; these experiments
identified three protected regions of the EPO promoter which correspond to the
functional segments defined by linker-scanning analysis and which contain
consensus, potential binding sites for Egr-1, H4TF-1, PuF, CTCF, UBP-1, and
GaEII transcription factors. Further study of EPO promoter regulation should
elucidate unique transcriptional features of eosinophil gene regulation in
granulocyte development. The evolution of genomic imprinting in mammals occurred more than 100 million
years ago, and resulted in the formation of genes that are functionally haploid
because of parent-of-origin-dependent expression. Despite ample evidence from
studies in a number of species suggesting the presence of imprinted genes on
human chromosome 14, their identity has remained elusive. Here we report the
identification of two reciprocally imprinted genes, GTL2 and DLK1, which
together define a novel imprinting cluster on human chromosome 14q32. The
maternally expressed GTL2 (gene trap locus 2) gene encodes for a nontranslated
RNA. DLK1 (delta, Drosophila, homolog-like 1) is a paternally expressed gene
that encodes for a transmembrane protein containing six epidermal growth factor
(EGF) repeat motifs closely related to those present in the delta/notch/serrate
family of signaling molecules. The paternal expression, chromosomal
localization, and biological function of DLK1 also make it a likely candidate
gene for the callipyge phenotype in sheep. Many of the predicted structural and
regulatory features of the DLK1/GTL2 domain are highly analogous to those
implicated in IGF2/H19 imprint regulation, including two hemimethylated
consensus binding sites for the vertebrate enhancer blocking protein, CTCF.
These results provide evidence that a common mechanism and domain organization
may be used for juxtapositioned, reciprocally imprinted genes. Genomic imprinting, the differential expression of autosomal genes based on
their parent of origin, is observed in all eutherian mammals that have been
examined. In most instances the genes that are imprinted in one species are
imprinted in others as well, suggesting that imprinting predated eutherian
radiation. For example, the RNA-coding H19 gene is repressed upon paternal
inheritance in all species examined to date. Thus, it is surprising that there
is remarkably little sequence conservation among the cis-acting DNA regulatory
elements that are required for imprinting of H19 and the tightly linked Igf2
gene. The most conserved characteristic in the imprinting control region (ICR)
is the presence of multiple binding sites for the zinc finger protein CTCF,
raising the possibility that CTCF binding might be sufficient for the reciprocal
imprinting of H19 and Igf2. To investigate whether a human H19 transgene,
harboring seven CTCF sites, is correctly recognized and imprinted in the mouse,
a 100 kb transgene containing the human H19 gene was introduced into the mouse
germline. The human transgene was specifically methylated after passage through
the male germline in a copy number-dependent manner, but the methylation was
unstable, undergoing progressive loss during development. Consequently, the
transgene was highly expressed upon both maternal and paternal inheritance.
These results argue that the signals for both the acquisition and maintece of
methylation imprinting are diverging rapidly. The serotonin transporter (5-HTT) gene contains a variable number tandem repeat
(VNTR) domain within intron 2 that is often associated with a number of
neurological conditions, including affective disorders. The implications of this
polymorphism are not yet understood, however, we have previously demonstrated
that the 5-HTT VNTR is a transcriptional regulatory domain, and the allelic
variation supports differential reporter gene expression in vivo and in vitro.
The aim of this study was to identify transcription factors responsible for the
regulation of this VNTR. Using a yeast one-hybrid screen, we found the
transcription factor Y box binding protein 1 (YB-1) interacts with the 5-HTT
VNTR. Consistent with this, we demonstrate in a reporter gene assay that the
polymorphic VNTR domains differentially respond to exogenous YB-1 and that YB-1
will bind to the VNTR in vitro in a sequence-specific manner. Interestingly, the
transcription factor CCTC-binding factor (CTCF), previously shown to interact
with YB-1, interferes with the ability of the VNTR to support YB-1-directed
reporter gene expression. In addition, CTCF blocks the binding of YB-1 to its
DNA recognition sequences in vitro, thus providing a possible mechanism of
regulation of YB-1 activation of the VNTR by CTCF. Therefore, we have identified
YB-1 and CTCF as transcription factors responsible, at least in part, for
modulation of VNTR function as a transcriptional regulatory domain. Our data
suggest a novel mechanism that explains, in part, the ability of the distinct
VNTR copy numbers to support differential reporter gene expression based on YB-1
binding sites. Immune responses to invading pathogens are mediated largely through a family of
transmembrane Toll-like receptors and modulated by a number of downstream
effectors. In particular, a family of four interleukin 1 receptor-associated
kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological
targeting of particular IRAK components may be beneficial for treatment of
bacterial infections. Here, we studied transcriptional regulation of the human
IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites
for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and
AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2
promoter activity in naive and endotoxin-treated mononuclear cells, in dormant
and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found
that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility
shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a
single high-affinity binding site for the transcriptional regulator and a
chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site
within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2
promoter was confirmed by chromatin immunoprecipitation assay. Specific residues
that interacted with the CTCF protein, were identified by methylation
interference assay. In all cell lines analyzed, including cells of lung, renal,
monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing
an intact CTCF-binding site, showed strong promoter activity. However, IRAK2
promoter activity was decreased dramatically for the constructs with a mutated
CTCF-binding site. BACKGROUND: CTCF is a highly conserved and essential zinc finger protein
expressed in virtually all cell types. In conjunction with cohesin, it organizes
chromatin into loops, thereby regulating gene expression and epigenetic events.
The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less
clear.
RESULTS: Using immunohistochemistry on testis sections and fluorescence-based
microscopy on intact live seminiferous tubules, we show that CTCFL is only
transiently present during spermatogenesis, prior to the onset of meiosis, when
the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL
distribution overlaps completely with that of Stra8, a retinoic acid-inducible
protein essential for the propagation of meiosis. We find that absence of CTCFL
in mice causes sub-fertility because of a partially penetrant testicular
atrophy. CTCFL deficiency affects the expression of a number of testis-specific
genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL
has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES
cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are
downregulated in CTCFL-deficient testis are upregulated in ES cells. These data
indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide
DNA-binding analysis shows that CTCFL binds a consensus sequence that is very
similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and
~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled
nucleosomes, whereas CTCF favors consensus sites surrounded by phased
nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is
functionally different from CTCF.
CONCLUSIONS: Our data suggest that nucleosome composition specifies the
genome-wide binding of CTCFL and CTCF. We propose that the transient expression
of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a
subset of promoters and maintain the expression of male germ cell genes. Chromatin insulators are DNA elements that regulate the level of gene expression
either by preventing gene silencing through the maintece of heterochromatin
boundaries or by preventing gene activation by blocking interactions between
enhancers and promoters. CCCTC-binding factor (CTCF), a ubiquitously expressed
11-zinc-finger DNA-binding protein, is the only protein implicated in the
establishment of insulators in vertebrates. While CTCF has been implicated in
diverse regulatory functions, CTCF has only been studied in a limited number of
cell types across human genome. Thus, it is not clear whether the identified
cell type-specific differences in CTCF-binding sites are functionally
significant. Here, we identify and characterize cell type-specific and
ubiquitous CTCF-binding sites in the human genome across 38 cell types
designated by the Encyclopedia of DNA Elements (ENCODE) consortium. These cell
type-specific and ubiquitous CTCF-binding sites show uniquely versatile
transcriptional functions and characteristic chromatin features. In addition, we
confirm the insulator barrier function of CTCF-binding and explore the novel
function of CTCF in DNA replication. These results represent a critical step
toward the comprehensive and systematic understanding of CTCF-dependent
insulators and their versatile roles in the human genome. CTCF is a ubiquitously expressed regulator of fundamental genomic processes
including transcription, intra- and interchromosomal interactions, and chromatin
structure. Because of its critical role in genome function, CTCF binding
patterns have long been assumed to be largely invariant across different
cellular environments. Here we analyze genome-wide occupancy patterns of CTCF by
ChIP-seq in 19 diverse human cell types, including normal primary cells and
immortal lines. We observed highly reproducible yet surprisingly plastic genomic
binding landscapes, indicative of strong cell-selective regulation of CTCF
occupancy. Comparison with massively parallel bisulfite sequencing data
indicates that 41% of variable CTCF binding is linked to differential DNA
methylation, concentrated at two critical positions within the CTCF recognition
sequence. Unexpectedly, CTCF binding patterns were markedly different in normal
versus immortal cells, with the latter showing widespread disruption of CTCF
binding associated with increased methylation. Strikingly, this disruption is
accompanied by up-regulation of CTCF expression, with the result that both
normal and immortal cells maintain the same average number of CTCF occupancy
sites genome-wide. These results reveal a tight linkage between DNA methylation
and the global occupancy patterns of a major sequence-specific regulatory
factor. |
Does cucumber lower blood sugar in diabetics? | Yes. Based on several scientific reports, ethanolic extract of cucumber and some other Cucurbitaceae plants are associated with a significant reduction of elevated blood glucose level, suggesting that cucumber could have antidiabetic activity. | In the first part of this study, the effect of four isocaloric mixed breakfast
meals on the blood glucose and urinary glucose losses was tested in nine adult
diabetics and in three healthy subjects, ages 60 to 75. Three of the test meals
consisted of a base diet supplemented with applesauce sweetened with sucrose,
fructose, or sorbitol. In the fourth test meal, the starch was increased
together with saccharine. In the second part of the study, analyses for free
glucose and sucrose in several timed food preparations, ordinary as well as food
preparations specially designed for diabetics, were performed. The amount of
sucrose equivalents (S(eg)) in one ordinary serving of the various products was
estimated. No significant differences among sucrose, fructose, and
sorbitol-containing meals with respect to the effect on the blood glucose level
or on glucosuria were found. The saccharine-containing meal gave a significantly
greater blood glucose increase at 60 min only. The amount of sucrose in ordinary
marinated foods, such as herring, cucumber, and common beet was negligible.
Water-packed fruits supplied one half of the amount of S(eq) or less, compared
with fruits packed in sorbitol-sweetened syrup. The amount of S(eq) in the
latter products as well as in fruits packed in unsweetened juice equalled that
of the fleshy substance of ordinary sucrose-sweetened products. It was concluded
that fructose or sorbitol has no advantages over sucrose, as regards the effect
on blood glucose in well-regulated adult diabetics, and that it seems
unnecessary to have specially sweetened foods designed for diabetics. The anti-hyperglycemic effect of 12 edible plants was studied on 27 healthy
rabbits, submitted weekly to subcutaneous glucose tolerance tests after gastric
administration of water, tolbutamide or a traditional preparation of the plant.
Tolbutamide, Cucurbita ficifolia, Phaseolus vulgaris, Opuntia streptacantha,
Spinacea oleracea, Cucumis sativus and Cuminum cyminum decrease significantly
the area under the glucose tolerance curve and the hyperglycemic peak. Brassica
oleracea var. botrytis, Allium cepa and Allium sativum only decrease the
hyperglycemic peak. The glycemic decreases caused by Psidium guajava, Brassica
oleracea and Lactuca sativa var. romana were not significant (P > .05). The
integration of a menu that includes the edible plants with hypoglycemic activity
for the control and prevention of diabetes mellitus may be possible and
recommendable. BACKGROUND: Foods with a low glycemic index are increasingly being acknowledged
as beneficial in relation to the insulin resistance syndrome. Certain organic
acids can lower the glycemic index of bread products. However, the possible
effect of acids in fermented milk products on the glycemic index and on
insulinemic characteristics has not been addressed. The metabolic effects of
fermented milk or pickled products used as additives to mixed meals have also
not been addressed.
OBJECTIVES: One objective was to characterize the glycemic and insulinemic
responses after intake of regular or fermented milk products (study 1). In
addition, the acute metabolic effect of fermented milk (yogurt) and pickled
cucumber as supplements to a traditional breakfast based on a high-glycemic
index bread was evaluated (study 2).
DESIGN: Ten healthy volunteers were served different breakfast meals after an
overnight fast. Capillary blood samples were collected before and during 2
(study 1) or 3 (study 2) h after the meal. White-wheat bread was used as a
reference meal in both studies.
RESULTS: The lactic acid in the fermented milk products did not lower the
glycemic and insulinemic indexes. Despite low glycemic indexes of 15-30, all of
the milk products produced high insulinemic indexes of 90-98, which were not
significantly different from the insulinemic index of the reference bread.
Addition of fermented milk (yogurt) and pickled cucumber to a breakfast with a
high-glycemic index bread significantly lowered postprandial glycemia and
insulinemia compared with the reference meal. In contrast, addition of regular
milk and fresh cucumber had no favorable effect on the metabolic responses.
CONCLUSIONS: Milk products appear insulinotropic as judged from 3-fold to 6-fold
higher insulinemic indexes than expected from the corresponding glycemic
indexes. The presence of organic acids may counteract the insulinotropic effect
of milk in mixed meals. Oxidative stress is implicated as an important mechanism by which diabetes
causes nephropathy. Oxykine is the cantaloupe melon extract rich in vegetal
superoxide dismutase covered by polymeric films of wheat matrix gliadin. In this
study, we examined whether chronic oral administration of oxykine could prevent
the progression of diabetic nephropathy induced by oxidative stress using
preclinical rodent model of type 2 diabetes. We used female db/db mice and their
non-diabetic db/m littermates. The mice were divided into the following three
groups: non-diabetic db/m; diabetic db/db, and diabetic db/db treated with
oxykine. Blood glucose level, body weight, urinary albumin, and urinary
8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments.
Histological and 8-OHdG immunohistochemical studies were preformed on 12 weeks
from the beginning of treatment. After 12 weeks of treatment, the levels of
blood glucose and the body weight were not significantly different between the
oxykine-treated group and the non-treated db/db group, however both groups kept
significantly high levels rather than db/m mice. The relative mesangial area
calculated by mesangial area/total glomerular area ratio was significantly
ameliorated in the oxykine treated group compared with non-treated db/db group.
The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were
significantly inhibited by chronic treatment with oxykine. The 8-OHdG
immunoreactive cells in the glomeruli of non-treated db/db mice were more
numerous than that of oxykine-treated db/db mice. In this study, treatment of
oxykine ameliorated the progression and acceleration of diabetic nephropathy for
rodent model of type 2 diabetes. These results indicated that the oxykine
reduced the diabetes-induced oxidative stress and renal mesangial cell injury.
In conclusion, oxykine might be a novel approach for the prevention of diabetes
nephropathy. The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin
fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from
cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the
liver of rats. In the brain and pancreas, PKC activity was significantly higher
in pectin-treated rats as compared to the control group. Level of blood glucose
was significantly lowered and the level of glycogen in the liver was
significantly increased in pectin-administered rats. Glycogen synthase activity
was enhanced, while glycogen phosphorylase enzyme showed inhibition in
pectin-treated rats. Results indicated that pectin administration might have
caused an increase in the secretion of the insulin, which, in turn, had a
stimulatory effect on the PKC activity in the pancreas. The decreased PKC
activity in the liver and increased PKC activity in the brain and pancreas on
pectin administration indicated enhanced glycogenesis and reduced
glycogenolysis. Hitherto unknown efficacy of the peel extracts of Mangifera indica (MI), Cucumis
melo (CM) and Citrullus vulgaris (CV) fruits in ameliorating the diet-induced
alterations in dyslipidemia, thyroid dysfunction and diabetes mellitus have been
investigated in rats. In one study, out of 4 different doses (50-300 mg/kg), 200
mg/kg of MI and 100 mg/kg for other two peel extracts could inhibit
lipidperoxidation (LPO) maximally in liver. In the second experiment rats were
maintained on pre-standardized atherogenic diet CCT (supplemented with 4%
cholesterol, 1% cholic acid and 0.5% 2-thiouracil) to induce dyslipidemia,
hypothyroidism and diabetes mellitus and the effects of the test peel extracts
(200 mg/kg of MI and 100 mg/kg for CM and CV for 10 consecutive days) were
studied by examining the changes in tissue LPO (in heart, liver and kidney),
concentrations of serum lipids, thyroid hormones, insulin and glucose. Rats,
treated simultaneously with either of the peel extracts reversed the CCT-diet
induced increase in the levels of tissue LPO, serum lipids, glucose, creatinine
kinase-MB and decrease in the levels of thyroid hormones and insulin indicating
their potential to ameliorate the diet induced alterations in serum lipids,
thyroid dysfunctions and hyperglycemia/diabetes mellitus. A phytochemical
analysis indicated the presence of a high amount of polyphenols and ascorbic
acid in the test peel extracts suggesting that the beneficial effects could be
the result of the rich content of polyphenols and ascorbic acid in the studied
peels. ETHNOPHARMACOLOGICAL RELEVANCE: Cucumis trigonus Roxb. (Cucurbitaceae) fruit is
used in the Indian traditional medicine for the treatment of diabetes. Based on
a number of reports on the blood glucose level reduction and the other
complications of diabetes associated with some Cucurbitaceae plants, the
antidiabetic effect of Cucumis trigonus fruit was investigated.
AIM OF THE STUDY: To investigate the antidiabetic action of the aqueous extract
of Cucumis trigonus fruits in the different models of rats.
MATERIALS AND METHODS: The antidiabetic activity of aqueous extract of Cucumis
trigonus fruit was evaluated by using normal and streptozotocin-induced-diabetic
rats. The acute effect of aqueous extract was evaluated by administering 500
mg/kg p.o. to normoglycemic rats. In the chronic model, the aqueous extract was
administered to normal and STZ-induced-diabetic rats at dose of 500 mg/kg p.o.
per day for 21 days. Blood glucose levels and body weights were monitored at
specific intervals and different biochemical parameters were also carried out.
RESULTS: The statistical data indicated the significant increase in the body
weight, liver glycogen and serum insulin level and decrease in the blood
glucose, glycosylated hemoglobin levels, total cholesterol and serum
triglycerides. HDL cholesterol level was significantly increased when treated
with the extract.
CONCLUSION: The aqueous fruit extract of Cucumis trigonus has had beneficial
effects in reducing the elevated blood glucose level and lipid profile of
STZ-induced-diabetic rats. The hitherto unknown glucose regulating role of three vegetable peels from
cucurbitaceae family was evaluated. In a preliminary study, effects of ethanolic
extracts of Cucurbita pepo, Cucumis sativus and Praecitrullus fistulosus peels
were studied at 250 and 500 mg kg(-1) d(-1) for 15 days in the alterations in
serum glucose and in hepatic lipid peroxidation (LPO) in male mice. In the pilot
experiment, the effective and safe concentration of each peel was administered
(p.o.) for 10 consecutive days and then on 11th and 12th days alloxan was
administered along with peel extracts. The treatment was continued up to 15th
day. At the end, alterations in serum glucose, insulin, triiodothyronine,
thyroxine, total cholesterol, triglyceride, high density lipoprotein, low
density lipoprotein, very low density lipoprotein, hepatic lipid peroxidation,
superoxide dismutase and catalase were studied. All the three peel extracts
nearly reversed most of these changes induced by alloxan suggesting their
possible role in ameliorating diabetes mellitus and related changes in serum
lipids. However, Cucurbita pepo peel was found to be the most effective. Total
polyphenols, flavonoids and ascorbic acid contents of the test peels were also
estimated, which appear to be associated with the observed antidiabetic and
antioxidative potentials. The hypoglycemic effects of the fruit extract of C. metuliferus was investigated
in normoglycemic and alloxan-induced hyperglycemic rats. The results showed that
there was an insignificant (P > 0.05) decrease in the blood glucose
concentration of normoglycemic rats treated with oral doses of 1000 and 1500
mg/kg of the extract. On the other hand, 500 mg/kg of the fruit extract produced
an insignificant (P > 0.05) decrease in blood glucose levels of alloxan-treated
rats, while 1000 and 1500 mg/kg oral dose points produced a significant (P <
0.05) decrease in the blood glucose concentration of hyperglycemic rats
comparable to that produced by tolbutamide. From this study, the data suggested
that the fruit extract did not alter the BGC level in normoglycemic rats, but
had a potential hypoglycemic property in alloxan-induced hyperglycemic rats. One therapeutic approach for preventing diabetes mellitus and obesity is to
retard the absorption of glucose via inhibition of α-glucosidase. Two
unsaturated fatty acids with strong α-glucosidase inhibitory activity,
7(Z)-octadecenoic acid (1) and 7(Z),10(Z)-octadecadienoic acid (2), were
purified from the body wall of Stichopus japonicus. IC(50) values of compounds 1
and 2 were 0.51 and 0.67 μg/mL against Saccharomyces cerevisiae α-glucosidase
and 0.49 and 0.60 μg/mL against Bacillus stearothermophilus α-glucosidase,
respectively. These compounds mildly inhibited rat-intestinal sucrase and
maltase. In addition, both compounds showed a mixed type of inhibition against
S. cerevisiae α-glucosidase and were very stable under thermal and acidic
conditions up to 60 min. The K(I) and K(IS) values of compounds 1 and 2 were
0.44 and 0.22 μg/mL, and 0.39 and 0.13 μg/mL, respectively.
PRACTICAL APPLICATION: One therapeutic approach for preventing diabetes mellitus
is to retard the absorption of glucose via inhibition of α-glucosidase. In this
study, 2 fatty acids with strong α-glucosidase-inhibitory activity,
7(Z)-octadecenoic acid and 7(Z),10(Z)-octadecadienoic acid, were purified and
identified from sea cucumber. Therefore, sea cucumber fatty acids can
potentially be developed as a novel natural nutraceutical for the management of
type-2 diabetes. OBJECTIVE: To investigate the effect of sea cucumber cerebroside(SCC) and its
long-chain base(LCB) on lipid and glucose metabolism in obese mice.
METHODS: The mouse obese model was established by feeding high fat diet. The
mice were randomly assigned to 4 groups: control group, model group, SCC group
and LCB group. After 4 weeks, the glucose tolerance test was undertaken. After 5
weeks, the body fat content, organic indexes, serum lipid level, glycemic index
and liver lipid level were determined.
RESULTS: Compared with the model group, the glucose tolerance in the SCC group
and LCB group was ameliorated significantly (P<0.01, P<0.05); glycemic index
(P<0.01, P<0.01), the weight of adipose tissue (P<0.05, P<0.01) and the hepatic
TG were reduced significantly (P<0.05, P<0.05).
CONCLUSION: Sea cucumber cerebroside and its long-chain base can improve the
glucose and lipid metabolism in obese mice. Much attention has been focused on food components that may be beneficial in
preventing lifestyle-related diseases. In this study, we investigated the
effects of saponins of sea cucumber (SSC) on high-fat diet-induced obesity,
insulin resistance, and fatty liver in mice. C57/BL6 mice were fed a high-fat
diet, containing 0.03% SSC, or 0.1% SSC for 8 weeks. Both doses of SSC exhibited
a weight-loss effect and significantly decreased adipose tissue weight, in both
visceral and subcutaneous depots. Furthermore, 0.1% SSC treatment dramatically
decreased the hepatic triglyceride and total cholesterol accumulation. Mice
administrated with 0.1% SSC had significantly decreased serum glucose and
insulin levels, lower homeostatic model assessment for insulin resistance index,
and area under the blood glucose curve, suggesting that insulin sensitivity is
enhanced by dietary SSC. Dietary SSC also prevented adipokine imbalance, by
increasing adiponectin production and decreasing tumor necrosis factor alpha
level caused by high-fat diet. Overall, these data demonstrate that SSC could
improve certain metabolic parameters associated with obesity. |
Which mutations in the cardiac isoform of the ryanodine receptor (RyR2) have been found to be related to CPVT? | Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. A novel RyR2-V2475F mutation is associated with CPVT in humans. 3 CPVT mouse models are: RyR2-R2474S+/-, RyR2-N2386I+/-, and RyR2-L433P+/-. The E189D RyR2 mutation is causative for CPVT. A knock-in mouse model carrier of the R4496C mutation is the mouse equivalent to the R4497C mutations identified in CPVT families. Scanning of 12 Finnish CPVT probands identified three novel RYR2 mutations (V2306I, P4902L, R4959Q), which were absent in unaffected and control individuals. Three CPVT-linked human RyR2 (hRyR2) mutations are: S2246L, N4104K, and R4497C. | Ca2+ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine
receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2
mutations suggested to cause defective Ca2+ channel function have recently been
identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and
arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We
report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S2246L,
N4104K, and R4497C) in HL-1 cardiomyocytes displaying correct targeting to the
endoplasmic reticulum. N4104K also localized to the Golgi apparatus. Phenotypic
characteristics including intracellular Ca2+ handling, proliferation, viability,
RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant
hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca2+ release
was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine
and 4-chloro-m-cresol) or beta-adrenergic stimulation (isoproterenol).
RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation,
but was dramatically disrupted by isoproterenol or forskolin, an activator of
adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar
magnitudes in all cells and were associated with equivalent hyperphosphorylation
of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting
cardiomyocyte phenotype but mediated augmented Ca2+ release on RyR-agonist or
beta-AR stimulation. Furthermore, equivalent interaction between mutant and WT
hRyR2 and FKBP12.6 was demonstrated. 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. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
disease characterized by adrenergically mediated polymorphic ventricular
tachycardia leading to syncope and sudden cardiac death. The autosomal domit
form of CPVT is caused by mutations in the RyR2 gene encoding the cardiac
isoform of the ryanodine receptor. In vitro functional characterization of
mutant RyR2 channels showed altered behavior on adrenergic stimulation and
caffeine administration with enhanced calcium release from the sarcoplasmic
reticulum. As of today no experimental evidence is available to demonstrate that
RyR2 mutations can reproduce the arrhythmias observed in CPVT patients. We
developed a conditional knock-in mouse model carrier of the R4496C mutation, the
mouse equivalent to the R4497C mutations identified in CPVT families, to
evaluate if the animals would develop a CPVT phenotype and if beta blockers
would prevent arrhythmias. Twenty-six mice (12 wild-type (WT) and 14RyR(R4496C))
underwent exercise stress testing followed by epinephrine administration: none
of the WT developed ventricular tachycardia (VT) versus 5/14 RyR(R4496C) mice
(P=0.02). Twenty-one mice (8 WT, 8 RyR(R4496C), and 5 RyR(R4496C) pretreated
with beta-blockers) received epinephrine and caffeine: 4/8 (50%) RyR(R4496C)
mice but none of the WT developed VT (P=0.02); 4/5 RyR(R4496C) mice pretreated
with propranolol developed VT (P=0.56 nonsignificant versus RyR(R4496C) mice).
These data provide the first experimental demonstration that the R4496C RyR2
mutation predisposes the murine heart to VT and VF in response caffeine and/or
adrenergic stimulation. Furthermore, the results show that analogous to what is
observed in patients, beta adrenergic stimulation seems ineffective in
preventing life-threatening arrhythmias. The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for
excitation-contraction coupling in the heart and is also present in the brain.
Mutations in RyR2 have been linked to exercise-induced sudden cardiac death
(catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated
RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of
the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the
channel. We found that mice heterozygous for the R2474S mutation in Ryr2
(Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures
(which occurred in the absence of cardiac arrhythmias), exercise-induced
ventricular arrhythmias, and sudden cardiac death. Treatment with a novel
RyR2-specific compound (S107) that enhances the binding of calstabin2 to the
mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac
arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky
Ryr2-R2474S channels in the brain can cause seizures in mice, independent of
cardiac arrhythmias. Based on these data, we propose that CPVT is a combined
neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy,
and the same leaky channels in the heart cause exercise-induced sudden cardiac
death. RATIONALE: Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been
linked to catecholaminergic polymorphic ventricular tachycardia (CPVT).
CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young
individuals during β-adrenergic stimulation.
OBJECTIVE: This study sought to determine the effects of a novel RyR2-G230C
mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the
channel to luminal calcium (Ca(2+)).
METHODS AND RESULTS: Functional characterizations of recombit human
RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2
mutant channels were generated by site-directed mutagenesis and heterologously
expressed in HEK293 cells together with calstabin2. RyR2 channels were measured
to examine the regulation of the channels by cytosolic versus luminal
sarcoplasmic reticulum Ca(2+). A 50-year-old white man with repeated syncopal
episodes after exercise had a cardiac arrest and harbored the mutation
RyR2-G230C. cAMP-dependent protein kinase-phosphorylated RyR2-G230C channels
exhibited a significantly higher open probability at diastolic Ca(2+)
concentrations, associated with a depletion of calstabin2. The luminal Ca(2+)
sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like.
CONCLUSIONS: The RyR2-G230C mutant exhibits similar biophysical defects compared
with previously characterized CPVT mutations: decreased binding of the
stabilizing subunit calstabin2 and a leftward shift in the Ca(2+) dependence for
activation under conditions that simulate exercise, consistent with a "leaky"
channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca(2+)
activation. Thus, diastolic sarcoplasmic reticulum Ca(2+) leak caused by reduced
calstabin2 binding and a leftward shift in the Ca(2+) dependence for activation
by diastolic levels of cytosolic Ca(2+) is a common mechanism underlying CPVT. RATIONALE: Atrial fibrillation (AF) is the most common cardiac arrhythmia,
however the mechanism(s) causing AF remain poorly understood and therapy is
suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca2+) release
channel on the sarcoplasmic reticulum (SR) required for excitation-contraction
coupling in cardiac muscle.
OBJECTIVE: In the present study, we sought to determine whether intracellular
diastolic SR Ca2+ leak via RyR2 plays a role in triggering AF and whether
inhibiting this leak can prevent AF.
METHODS AND RESULTS: We generated 3 knock-in mice with mutations introduced into
RyR2 that result in leaky channels and cause exercise induced polymorphic
ventricular tachycardia in humans [catecholaminergic polymorphic ventricular
tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse
models harboring RyR2 mutations to explore the role of diastolic SR Ca2+ leak in
AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3
CPVT mouse models (RyR2-R2474S+/-, 70%; RyR2-N2386I+/-, 60%; RyR2-L433P+/-,
35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo
results, there was a significant diastolic SR Ca2+ leak in atrial myocytes
isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit
that stabilizes the closed state of RyR2 and prevents a Ca2+ leak through the
channel. Atrial RyR2 from RyR2-R2474S+/- mice were oxidized, and the RyR2
macromolecular complex was depleted of calstabin2. The Rycal drug S107
stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation
induced dissociation of calstabin2 from the channel. S107 reduced the diastolic
SR Ca2+ leak in atrial myocytes and decreased burst pacing-induced AF in vivo.
S107 did not reduce the increased prevalence of burst pacing-induced AF in
calstabin2-deficient mice, confirming that calstabin2 is required for the
mechanism of action of the drug.
CONCLUSIONS: The present study demonstrates that RyR2-mediated diastolic SR Ca2+
leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal
S107 inhibited diastolic SR Ca2+ leak through RyR2 and pacing-induced AF
associated with CPVT mutations. RATIONALE: Most cardiac ryanodine receptor (RyR2) mutations associated with
catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to
cause a distinctive form of Ca(2+) release dysfunction. Considering the spread
distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity
also was feasible.
OBJECTIVE: To determine the molecular and cellular mechanisms by which a novel
RyR2-V2475F mutation associated with CPVT in humans triggers Ca(2+)-dependent
arrhythmias in whole hearts and intact mice.
METHODS AND RESULTS: Recombit channels harboring CPVT-linked RyR2 mutations
were functionally characterized using tritiated ryanodine binding and
single-channel recordings. Homologous recombination was used to generate a
knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice
heterozygous for the mutation (RyR2-V2475F(+/-)) and their wild-type littermates
were Ca(2+)-imaged by confocal microscopy under conditions that mimic stress.
The propensity of wild-type and RyR2-V2475F(+/-) mice to have development of
arrhythmias was tested at the whole heart level and in intact animals.
Recombit RyR2-V2475F channels displayed increased cytosolic Ca(2+)
activation, abnormal protein kinase A phosphorylation, and increased activation
by luminal Ca(2+). The RyR2-V2475F mutation appears embryonic-lethal in
homozygous mice, but heterozygous mice have no alterations at baseline.
Spontaneous Ca(2+) release events were more frequent and had shorter latency in
isoproterenol-stimulated cardiomyocytes from RyR2-V2475F(+/-) hearts, but their
threshold was unchanged with respect to wild-type. Adrenergically triggered
tachyarrhythmias were more frequent in RyR2-V2475F(+/-) mice.
CONCLUSIONS: The mutation RyR2-V2475F is phenotypically strong among other CPVT
mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living
mice, this mutation appears too severe to be harbored in all RyR2 channels but
remains undetected under basal conditions if expressed at relatively low levels.
β-adrenergic stimulation breaks the delicate Ca(2+) equilibrium of
RyR2-V2475F(+/-) hearts and triggers life-threatening arrhythmias. Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic
polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has
been implicated as a cause of CPVT and atrial fibrillation. The mutation has
been functionally characterized via store-overload-induced Ca(2+) release
(SOICR) and tritium-labelled ryanodine ([(3)H]ryanodine) binding assays. The
S4153R mutation enhanced propensity for spontaneous Ca(2+) release and reduced
SOICR threshold but did not alter Ca(2+) activation of [(3)H]ryanodine binding,
a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that
the S4153R mutation is a gain-of-function RYR2 mutation associated with a
clinical phenotype characterized by both CPVT and atrial fibrillation. CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited
life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed
after-depolarizations) that are induced by spontaneous Ca2+ release during SR
(sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR
(store-overload-induced Ca2+ release). A number of mutations in the cardiac
ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2
mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to
luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2
mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+
sensitivity of single RyR2 channels in lipid bilayers. This observation led to
the suggestion of a SOICR-independent disease mechanism for the G230C mutation.
However, the cellular impact of this mutation on SOICR is yet to be determined.
To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell
lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell
Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity
for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation
increased the sensitivity of single RyR2 channels to both luminal and cytosolic
Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In
addition, the G230C mutation decreased the thermal stability of the N-terminal
region (amino acids 1-547) of RyR2. These data suggest that the G230C mutation
enhances the propensity for SOICR by sensitizing the channel to luminal and
cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on
the N-terminal domains of RyR2. |
Which population has a high frequency of the HLA-B*1502 allele? | HLA-B*1502 has a high frequency in Han Chinese and other Asian populations, except Japanese. (There is a strong association between human leucocyte antigen (HLA)-B*1502 and carbamazepine-induced Stevens-Johnson syndrome (SJS)). | Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but
severe cutaneous adverse drug reactions, which can be caused by a certain number
of specific drugs among which is carbamazepine, an antiepileptic agent. A very
strong association of carbamazepine-induced SJS with HLA-B*1502 has recently
been described in the Han Chinese population. Here in, we report preliminary
results from a European study (RegiSCAR) of 12 carbamazepine-induced SJS/TEN
cases (nine French and three German). Among these only four had a HLA-B*1502
allele. Remarkably, these four patients had an Asian ancestry, whereas the
others did not as far as we have ascertained. This shows that although the HLA
region may contain important genes for SJS, the HLA-B*1502 allele is not a
universal marker for this disease and that ethnicity matters. PURPOSE: Previous studies found a strong association between HLA-B*1502 and
carbamazepine (CBZ)-induced Stevens-Johnson syndrome (SJS) in Han Chinese, but
not in Caucasian populations. Even in Han Chinese, the HLA-B*1502 was not
associated with CBZ-induced maculopapular eruptions (MPE). This study seeks to
identify whether HLA-B*1502 is associated with CBZ- or phenytoin (PHT)-induced
SJS or MPE in a Thai population.
METHODS: Eighty-one Thai epileptic patients between 1994 and 2007 from the
Chulalongkorn Comprehensive Epilepsy Program were recruited. Thirty-one subjects
had antiepileptic drug (AED)-induced SJS or MPE (6 CBZ-SJS, 4 PHT-SJS, 9
CBZ-MPE, 12 PHT-MPE), and 50 were AED-tolerant controls.
RESULTS: For the first time, a strong association between HLA-B*1502 and
PHT-induced SJS was found (p = 0.005). A strong association was also found
between the HLA-B*1502 and CBZ-induced SJS (p = 0.0005), making Thai the first
non-Chinese population demonstrating such an association. Some patients, who
were HLA-B*1502 and suffered from CBZ-induced SJS, could be tolerant to PHT and
vice versa. This suggests that HLA-B*1502 may be a common attribute required for
a Thai patient to develop SJS from these two AEDs; other different elements,
however, are also needed for each AED. In addition, no association between HLA-B
alleles and CBZ- or PHT-induced MPE was found.
CONCLUSIONS: CBZ- and PHT-induced SJS, but not MPE, is associated with
HLA-B*1502 allele in Thai population. Carbamazepine (CBZ) is frequently used for treating epilepsy, but this drug
causes cutaneous adverse drug reactions (cADRs) that may range from mild to
severe. It is reported recently that the human leukocyte antigen HLA-B*1502 is
associated with Stevens-Johnson syndrome (SJS) induced by CBZ in Han Chinese. We
examined HLA class I in 15 Japanese patients who fulfilled the diagnostic
criteria for CBZ-induced cADRs (mild in 10 and severe = SJS in 5). HLA-B*1518,
HLA-B*5901 and HLA-C*0704 alleles showed higher relative risks (above 10.0) for
severe cADRs. The haplotype (HLA-A*2402-B*5901-C*0102) had high relative risk
(16.09) for severe cADRs. In patients with severe cADRs, frequencies of
HLA-A*1101, HLA-A*3303, HLA-B*1501, HLA-B*4403, HLA-B*5101, HLA-B*5201,
HLA-C*0702, and HLA-C*1202 alleles are relatively lower than in the Japanese
population. These data may suggest that HLA-B*5901 is one of the candidate
markers for CBZ-induced SJS in Japanese. Carbamazepine (CBZ) has been reported as the most common culprit drug for
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in several
Asian countries including Thailand. A strong association between HLA-B*1502 and
CBZ-induced SJS/TEN has been reported in Han Chinese but not in Caucasian and
Japanese populations. A case-control study was conducted to determine whether
HLA-B*1502 is a valid pharmacogenetic test for SJS/TEN caused by CBZ in a Thai
population. Among 42 CBZ-induced patients with SJS/TEN, 37 (88.10%) patients
carried the HLA-B*1502 while only 5 (11.90%) of the CBZ-tolerant controls had
this allele. The risk of CBZ-induced SJS/TEN was significantly higher in the
patients with HLA-B*1502, with an odds ratio (OR) of 54.76 [95% confidence
interval (CI) 14.62-205.13, p = 2.89 x 10(-12)]. The sensitivity and specificity
of HLA-B*1502 for prediction of CBZ-induced SJS/TEN were 88.10%. By assuming a
0.27% as a prevalence rate of CBZ-induced SJS/TEN in a Thai population, the
positive predictive value (PPV) and negative predictive value (NPV) of the
HLA-B*1502 were 1.92% and 99.96%. Results from this study suggest that
HLA-B*1502 may be a useful pharmacogenetic test for screening Thai individuals
who may be at risk for CBZ-induced SJS and TEN. BACKGROUND: Recent study demonstrated that HLA-B*1502 was a common risk allele
in aromatic antiepileptic drugs (AEDs) induced Stevens-Johnson syndrome and
toxic epidermal necrolysis in Han Chinese. However, the association of
AEDs-induced mild maculopapular eruption (MPE) with HLA-B*1502 remains unclear
until recently. In the present study, we conducted a pilot study to detect a
possible association of oxcarbazepine (OXC)-induced MPE with HLA-B*1502 allele
in Chinese Han population.
METHODS: We enrolled 90 subjects involving 9 patients with OXC-induced MPE and
two groups of controls, 9 OXC-tolerant and 72 normal controls. High-resolution
HLA genotyping was performed by specific kit. The results of HLA genotyping are
expressed as positive or negative for HLA-B*1502 allele. Differences in genotype
frequencies between groups were assessed by the Fisher's exact test.
RESULTS: Four cases were detected as positive for HLA-B*1502 amongst 9 patients.
However, only 1 subject was positive amongst 9 tolerant controls, and 6 subjects
were positive amongst 72 normal controls. The difference in HLA-B*1502 allele
frequencies between the MPE group and normal controls was statistically
significant (OR: 8.8; 95% CI: 1.853-41.790; P=0.011). In addition, we also
observed an increased frequency of HLA-B*1502 allele in patients (44.44%)
compared with tolerant controls (11.11%), although it failed to reach
statistical significance (P=0.294).
CONCLUSIONS: Our findings indicate that HLA-B*1502 allele may contribute to the
genetic susceptibility to OXC-induced MPE in Chinese Han population. In order to
safer AEDs use, we recommend that HLA-B*1502 allele should be tested for
patients with OXC-induced MPE before changing to other AEDs, and AEDs with
similar chemical structure should be avoided in individuals who test positive
for HLA-B*1502 allele. It should be pointed out that, however, our results may
well be just by chance owing to the small sample size and should be further
confirmed in future studies. Lamotrigine (LTG) is a commonly used antiepileptic drug. However, the use of LTG
is limited because of its cutaneous adverse drug reactions (cADRs) ranging from
mild maculopapular eruption (MPE) to severe Stevens-Johnson syndrome (SJS) and
toxic epidermal necrolysis (TEN). A strong association between HLA-B*1502 and
carbamazepine-induced SJS/TEN has been identified in Chinese and Thai. Although
three of seven cases with HLA-B*1502 have been reported in LTG-induced SJS/TEN
so far, the relationship between HLA-B*1502 and LTG-induced SJS/TEN needs
further investigation. It is also unclear whether there is a specific genetic
marker associated with LTG-induced MPE in Chinese. In this study, we genotyped
43 Han Chinese patients treated with LTG (14 cases with LTG-induced cADRs and 29
LTG-tolerant controls), using PCR-SSP for HLA-B*1502 testing and low-resolution
genotyping, as well as sequencing for four-digit genotyping. The two cases with
SJS were negative for HLA-B*1502, with B1301/1301 and 4601/5610, respectively.
Combining the data with previous studies, there was no significant difference in
the frequency of subjects with HLA-B*1502 between the LTG-induced SJS/TEN group
and the LTG-tolerant group (p = 0.08, OR 4.23, 95% CI 0.94-18.97). In the MPE
group, only one was positive for HLA-B*1502. There was no significant difference
in the frequency of a specific HLA-B allele between the MPE group and the
LTG-tolerant group either. In this study, no significant association between
HLA-B*1502 and LTG-induced SJS or MPE was found. Given the small sample size and
only HLA-B locus genotyping, further large-scale studies are required to explore
genetic associations with LTG-induced cADRs. PURPOSE: The purpose of this study is to examine the association of HLA-B*1502
allele with CBZ-induced SJS/TEN in the mainland Han Chinese population.
METHODS: HLA-B*1502 genotyping with sequence-specific primer polymerase chain
reaction (PCR-SSP) and PCR-sequencing based typing (PCR-SBT) was performed on 17
CBZ-induced SJS/TEN patients, 21 CBZ-tolerant controls, and 185 healthy controls
recruited during 2008-2010.
RESULTS: HLA-B*1502 allele was present in 94.1% (16/17) of CBZ-SJS/TEN patients,
9.5% (2/21) of CBZ-tolerant patients, and 9.2% (17/185) of healthy controls. The
risk of CBZ-induced SJS/TEN was significantly higher (P < 0.01) in the patients
with HLA-B*1502. One CBZ-induced SJS patient tested negative for HLA-B*1502, and
the test result showed HLA-B*3503/B*4601.
CONCLUSIONS: We found a strong association between HLA-B*1502 and CBZ-induced
SJS/TEN in the Han Chinese population from central and northern China. Combined
with previous studies of the southern Han Chinese subpopulation, our results
suggest that HLA-B*1502 is strongly associated with CBZ-induced SJS/TEN in the
whole Han Chinese population. Carbamazepine (CBZ) is the most frequent culprit drug for severe cutaneous
adverse drug reactions (ADR), such as Stevens-Johnson syndrome (SJS), toxic
epidermal necrolysis (TEN) and drug-induced hypersensitivity syndrome (DIHS). A
strong association between human leukocyte antigen (HLA)-B*1502 and CBZ-induced
SJS/TEN has been reported in Han Chinese, Thai, Malaysian and Indian
populations, but not in Caucasian or Japanese populations. Recent studies showed
an association between HLA-A*3101 and CBZ-induced ADR in Caucasian and Japanese
populations. We conducted a case-control study to determine HLA genotyping of
patients with CBZ-induced ADR in a Japanese population. Fifteen patients with
CBZ-induced ADR and 33 subjects who had taken CBZ for more than 3 months without
evidence of any ADR as a control were enrolled. In addition, the results of a
CBZ-induced lymphocyte stimulation test were compared between the groups. A
strong association was found between HLA-A31 and CBZ-induced ADR (P < 0.001),
and a weak association was found between HLA-A11 and HLA-B51 with CBZ-induced
ADR. No HLA-B*1502 was found in either patients or control subjects. The mean
CBZ-induced lymphocyte stimulation index was significantly high in patients with
CBZ-induced ADR compared with CBZ-tolerant patients (P < 0.001); however, no
significant difference was seen between HLA-A31-positive subjects and
HLA-A31-negative subjects in either group. These findings suggest that HLA-A31
is strongly associated with CBZ-induced ADR in the Japanese, but does not
determine CBZ-induced lymphocyte proliferation. |
What are the observations regarding telomere integrity and function in Fanconi anemia? | In Fanconi anemia patients, a higher rate of breakage at TTAGGG sequences in vivo is causing telomere erosion in differentiated cells. Moreover, it has been demonstrated that αIISp is important for telomere maintenance after DNA damage due to interstrand cross-links (ICL), localizing to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FANCA) cells, where αIISp levels are 35-40% of normal, ICL damage results in failure of XPF to localize to telomeres, with markedly increased telomere dysfunction-induced foci, and catastrophic loss of telomeres. | Fanconi anemia (FA) is a fatal inherited disease displaying chromosomal
instability, disturbances in oxygen metabolism and a high burden of
intracellular radical oxygen species. Oxygen radicals can damage DNA including
telomeric regions. Insufficient repair results in single strand breaks that can
induce accelerated telomere shortening. In a longitudinal study we demonstrate
that telomeric DNA is continuously lost at a higher rate in FA fibroblasts
compared to healthy controls. Furthermore, we show that this loss is caused
rather by an increased shortening per cell division in regularly replicating
cells than by apoptosis. Fanconi anemia (FA) is a rare genetic disease characterized by chromosome
instability, progressive pancytopenia and cancer susceptibility. Telomeres are
intimately related to chromosome stability and play an important role in
organismal viability at the hematological level. Since previous works suggested
an accelerated shortening of telomeres in FA, we have studied several markers of
telomere integrity and function in FA patients and age-matched controls to get
insights into the mechanisms and consequences of telomere erosion in FA. A
higher frequency of extra-chromosomic TTAGGG signals and of chromosome ends with
undetectable TTAGGG repeats was observed in FA cells by fluorescence in situ
hybridization (FISH), suggesting intensive breakage at telomeric sequences. This
was proven by measuring the frequency of excess of telomeric signals per cell,
which was 2.8-fold higher in FA. Consistent with previous reports, quantitative
FISH analysis showed an accelerated telomere shortening of 0.68 kb in FA, which
occurred concurrently in both chromosome arms in a similar magnitude. Our data
therefore suggest that the telomere erosion in FA is caused by a higher rate of
breakage at TTAGGG sequences in vivo in differentiated cells, in addition to
mere replicative shortening during lymphocyte proliferation. Consistent with
impaired telomeres in FA patients, we observed a >10-fold increase in chromosome
end fusions in FA compared to normal controls. This observation was independent
of TRF2, a telomere binding factor that protects human telomeres from end
fusions, since immunohistochemistry studies in FA cell lines and corrected
counterparts by retrovirus-mediated transfer of FANCA and FANCD2 cDNA showed
that a functional FA pathway is not required for telomere binding of TRF2. |
Are Notch mutations related to T-cell Acute Lymphoblastic Leukemia (T-ALL)? | Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). | PURPOSE: NOTCH signaling pathway is essential in T-cell development and NOTCH1
mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL).
To gain insight into its clinical significance, NOTCH1 mutation was investigated
in 77 patients with T-ALL.
EXPERIMENTAL DESIGN: Detection of NOTCH1 mutation was done using reverse
transcription-PCR amplification and direct sequencing, and thereby compared
according to the clinical/biological data of the patients.
RESULTS: Thirty-two mutations were identified in 29 patients (with dual
mutations in 3 cases), involving not only the heterodimerization and
proline/glutamic acid/serine/threonine domains as previously reported but also
the transcription activation and ankyrin repeat domains revealed for the first
time. These mutations were significantly associated with elevated WBC count at
diagnosis and independently linked to short survival time. Interestingly, the
statistically significant difference of survival according to NOTCH1 mutations
was only observed in adult patients (>18 years) but not in pediatric patients (<
or = 18 years), possibly due to the relatively good overall response of
childhood T-ALL to the current chemotherapy. NOTCH1 mutations could coexist with
HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation
on prognosis was potentiated by HOX11L2 but was attenuated by HOX11.
CONCLUSION: NOTCH1 mutation is an important prognostic marker in T-ALL and its
predictive value could be even further increased if coevaluated with other
T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with
oncogenic transcriptional factors on T-ALL pathogenesis. Notch signaling is of crucial importance in normal T-cell development and Notch
1 is frequently mutated in T-cell acute lymphoblastic leukemias (T-ALL), leading
to aberrantly high Notch signaling. In this report, we determine whether T-ALL
mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10,
Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is
mutated in leukemic cells from more than 30% of patients with pediatric T-ALL
and derived cell lines. Most hCDC4 mutations found were missense substitutions
at critical arginine residues (Arg(465), Arg(479), and Arg(505)) localized in
the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and T-ALL
cells containing hCDC4 mutations exhibited an increased Notch1 protein
half-life, consistent with the proposed role of hCdc4 in ubiquitin-dependent
proteolysis of Notch1. Furthermore, restoration of wild-type but not mutant
hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation
and decreased Notch1 signaling. These results show that hCdc4 mutations
interfere with normal Notch1 regulation in vivo. Finally, we found that
mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients
carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival.
Collectively, these data show that mutation of hCDC4 is a frequent event in
T-ALL and suggest that hCDC4 mutations and gain-of-function mutations in NOTCH1
might synergize in contributing to the development of pediatric T-ALL
leukemogenesis. The identification of activating mutations in NOTCH1 in over 50% of T-cell acute
lymphoblastic leukemias (T-ALL) has generated major interest in the elucidation
of the mechanisms of transformation downstream of oncogenic NOTCH and in the
targeting of the NOTCH signaling pathway in this disease. Small molecule
gamma-secretase inhibitors (GSIs) block NOTCH1 signaling in T-ALL lymphoblasts,
yet the clinical development of GSIs has been held back by the development of
gastrointestinal toxicity and their weak antileukemic effects against human
T-ALL. However, new therapeutic strategies aiming to optimize the use of
anti-NOTCH1 therapies for T-ALL, including combination therapies with
molecularly targeted drugs and glucocorticoids, have started to emerge as a
result of improved understanding of the molecular mechanisms that mediate the
effects of GSIs in leukemic cells and the intestinal epithelium. This review
focuses on the molecular basis of NOTCH1-induced transformation, the mechanisms
of action of oncogenic NOTCH1 and clinical significance of NOTCH1 mutations in
T-ALL. Activation of the Notch pathway occurs commonly in T acute lymphoblastic
leukemia (T-ALL) because of mutations in Notch1 or Fbw7 and is involved in the
regulation of cell proliferation and survival. Deregulated Notch3 signalling has
also been shown to promote leukemogenesis in transgenic mice, but the targets of
Notch3 in human T-ALL cells remain poorly characterized. Here, we show that
Notch3 controls levels of mitogen-activated protein kinase (MAPK) phosphatase 1
(MKP-1). In a model of T-ALL cell dormancy, both Notch3 activation and MKP-1
expression were upregulated in aggressive compared with dormant tumors, and this
inversely correlated with the levels of phosphorylated p38 and extracellular
signal-regulated kinase1/2 (ERK1/2) MAPKs, two canonical MKP-1 targets. We
demonstrate that MKP-1 protein levels are regulated by Notch3 in T-ALL cell
lines because its silencing by RNA interference or treatment with γ-secretase
inhibitors induced strong MKP-1 reduction whereas activation of Notch3
signalling had the opposite effect. Furthermore, MKP-1 has an important role in
T-ALL cell survival because its attenuation by short hairpin RNA significantly
increased cell death under stress conditions. This protective function has a key
role in vivo, as MKP-1-deficient cells showed impaired tumorigenicity. These
results elucidate a novel mechanism downstream of Notch3 that controls the
survival of T-ALL cells. Notch-1 is a transmembrane receptor protein that directs T-cell differentiation.
Gain-of-function mutations in Notch-1 have been reported in more than 50% of
human T-cell acute lymphoblastic leukemia (T-ALL). The current study was
undertaken to characterize mutations in the heterodimerization (HD) domain and
proline, glutamic acid, serine, threonine-rich (PEST) domain of the Notch-1
receptor. RNA was isolated from peripheral blood/bone marrow of 15 de novo T-ALL
subjects; the Notch-1 HD and PEST regions were amplified and sequenced. Overall
six patients (40%) had at least one Notch-1 mutation, 2/15 (13%) in the HD and
4/15 (27%) in the PEST domain. None of the samples showed simultaneous mutations
in HD and PEST domains. Mutations were seen in 4/10 adult patients (40%); in the
pediatric cohort 2/5 (40%) had mutations both of which were in the PEST domain.
Of the different mutations, two have been previously reported and the other four
are novel. A high incidence of Notch-1 mutations has been seen; unlike other
studies, a higher frequency of mutations was found in PEST domain. The current
study also served to identify four novel mutants that add new insights into the
genetic heterogeneity of T-ALL. More ongoing larger studies are warranted to
elucidate the molecular pathogenesis of T-ALL that arises in this part of the
world. Activating mutations in NOTCH1, an essential regulator of T cell development,
are frequently found in human T cell acute lymphoblastic leukemia (T-ALL).
Despite important advances in our understanding of Notch signal transduction,
the regulation of Notch functions in the nucleus remains unclear. Using
immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL
cells and showed that, beyond the well-characterized core activation complex
(ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the
transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and
the histone demethylases LSD1 and PHF8 acting through their demethylase activity
to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1
functions as a corepressor when associated with CSL-repressor complex and as a
NOTCH1 coactivator upon Notch activation. Our work provides new insights into
the molecular mechanisms that govern Notch transcriptional activity and
represents glimpse into NOTCH1 interaction landscape, which will help in
deciphering mechanisms of NOTCH1 functions and regulation. T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is characterized by
aberrant activation of NOTCH1 in over 60% of T-ALL cases. The high prevalence of
activating NOTCH1 mutations highlights the critical role of NOTCH signaling in
the pathogenesis of this disease and has prompted the development of therapeutic
approaches targeting the NOTCH signaling pathway. Small molecule gamma secretase
inhibitors (GSIs) can effectively inhibit oncogenic NOTCH1 and are in clinical
testing for the treatment of T-ALL. Treatment with GSIs and glucocorticoids are
strongly synergistic and may overcome the gastrointestinal toxicity associated
with systemic inhibition of the NOTCH pathway. In addition, emerging new
anti-NOTCH1 therapies include selective inhibition of NOTCH1 with anti-NOTCH1
antibodies and stapled peptides targeting the NOTCH transcriptional complex in
the nucleus. Growth factor independent 1 (Gfi1) is a transcriptional repressor originally
identified as a gene activated in T-cell leukemias induced by
Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that
is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1
is an important factor in the initiation and maintece of lymphoid leukemias
and its deficiency significantly impedes Notch dependent initiation of T-ALL in
animal models. Here, we show that immature hematopoietic cells require Gfi1 to
competently integrate Notch-activated signaling. Notch1 activation coupled with
Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of
T-cells, whereas activation in later stages leaves development unaffected. In
Gfi1 deficient multipotent precursors, Notch activation induces lethality and is
cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain
Notch1-activated global expression profiles typical for T-lineage precursors. In
agreement with this, we find that both lymphoid-primed multipotent progenitors
(LMPP) and early T lineage progenitors (ETP) do not properly form or function in
Gfi1(-/-) mice. These defects correlate with an inability of Gfi1(-/-)
progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1.
Our data indicate that Gfi1 is required for hematopoietic precursors to
withstand Notch1 activation and to maintain Notch1 dependent transcriptional
programming to determine early T-lymphoid lineage identity. T-cell acute lymphoblastic leukemia (T-ALL) is characterized as a high-risk
stratified disease associated with frequent relapse, chemotherapy resistance,
and a poorer prognostic outlook than B-precursor ALL. Many of the challenges in
treating T-ALL reflect the lack of prognostic cytogenetic or molecular
abnormalities on which to base therapy, including targeted therapy. Notch1
activating mutations were identified in more than 50% of T-ALL cases and can be
therapeutically targeted with γ-secretase inhibitors (GSIs). Mutant Notch1 can
activate cMyc and PI3K-AKT-mTOR1 signaling in T-ALL. In T-ALLs with wild-type
phosphatase and tensin homolog deleted on chromosome ten (PTEN), Notch1
transcriptionally represses PTEN, an effect reversible by GSIs. Notch1 also
promotes growth factor receptor (IGF1R and IL7Rα) signaling to PI3K-AKT. Loss of
PTEN is common in primary T-ALLs due to mutation or posttranslational
inactivation and results in chronic activation of PI3K-AKT-mTOR1 signaling,
GSI-resistance, and repression of p53-mediated apoptosis. Notch1 itself might
regulate posttranslational inactivation of PTEN. PP2A is activated by Notch1 in
PTEN-null T-ALL cells, and GSIs reduce PP2A activity and increase
phosphorylation of AKT, AMPK, and p70S6K. This review focuses on the central
role of the PI3K-AKT-mTOR1 signaling in T-ALL, including its regulation by
Notch1 and potential therapeutic interventions, with emphasis on GSI-resistant
T-ALL. The Notch signaling pathway plays a critical role in maintaining the balance
between cell proliferation, differentiation and apoptosis, and is a highly
conserved signaling pathway that regulates normal development in a context- and
dose-dependent manner. Dysregulation of Notch signaling has been suggested to be
key events in a variety of hematological maligcies. Notch1 signaling appears
to be the central oncogenic trigger in T cell acute lymphoblastic leukemia
(T-ALL), in which the majority of human maligcies have acquired mutations
that lead to constitutive activation of Notch1 signaling. However, emerging
evidence unexpectedly demonstrates that Notch signaling can function as a potent
tumor suppressor in other forms of leukemia. This minireview will summarize
recent advances related to the roles of activated Notch signaling in human
lymphocytic leukemia, myeloid leukemia, stem cells and stromal microenvironment,
and we will discuss the perspectives of Notch signaling as a potential
therapeutic target as well. The Notch signaling pathway has been recognized as a key factor for the
pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high
incidence of activating mutations of Notch1. Notch inhibition could serve as a
new treatment strategy for T-ALL; however, the attempts to perturb Notch
signaling pathways have been unsuccessful so far. In this study, we found that
proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive
activation of Notch1 to a similar extent as myeloma cells. The proteasome
inhibitor bortezomib repressed the transcription of Notch1 and downstream
effectors including Hes1, GATA3, RUNX3 and nuclear factor-κB (NF-κB) (p65 and
p50), coincided with downregulation of the major transactivator Sp1 and its
dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular
domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against
T-ALL cells. Drug combination studies revealed that bortezomib showed
synergistic or additive effects with key drugs for the treatment of T-ALL such
as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily
abolished by NICD overexpression. The synergy of bortezomib and DEX was
confirmed in vivo using a murine xenograft model. Our findings provide a
molecular basis and rationale for the inclusion of proteasome inhibitors in
treatment strategies for T-ALL. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is
frequently associated with activating mutations in NOTCH1 and dysregulation of
MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs
and drug-like small molecules with activity against T-ALL. We developed a
zebrafish system to screen small molecules for toxic activity toward
MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for
small molecules that synergize with Notch inhibitors. We identified the
antipsychotic drug perphenazine in both screens due to its ability to induce
apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity
chromatography coupled with mass spectrometry, we identified protein phosphatase
2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine
exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent
apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated
perphenazine activity, indicating that PP2A mediates the drug's antileukemic
activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed
cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our
findings provide a mechanistic explanation for the recurring identification of
phenothiazines as a class of drugs with anticancer effects. Furthermore, these
data suggest that pharmacologic PP2A activation in T-ALL and other cancers
driven by hyperphosphorylated PP2A substrates has therapeutic potential. The NOTCH1 signaling pathway is essential for hematopoiesis and a critical
regulatory step for T-cell proliferation and maturation. The E3 ubiquitin ligase
FBXW7 controls NOTCH1 protein stability. Mutations in NOTCH1/FBXW7 activate
NOTCH signaling and are of prognostic significance in patients with T-cell acute
lymphoblastic leukemia (T-ALL). In this study we analyzed NOTCH1 and FBXW7
mutations in 50 South Indian T-ALL patients treated by a modified ALL BFM 95
regimen. The hot spot exons (HD-N, HD-C, TAD, and PEST) of NOTCH1 and exons 9 of
the 10 of FBXW7 were polymerase chain reaction amplified and sequenced. In
total, 20 of the 50 (40%) T-ALL patients revealed heterozygous mutations in the
NOTCH1 domains, and a predomice of missense mutations in HD-N (70%) and PEST
(15%) domains. FBXW7 mutations were detected in 5 of the 50 (10%) T-ALL
patients. T-ALL patients with NOTCH1/FBXW7 mutations expressed higher protein
level of NOTCH1 compared with patients without NOTCH1/FBXW7 mutations. Six of
the mutations detected in NOTCH1 were not reported previously. When tested in a
Dual Luciferase Renilla reporter assay some of these conferred increased NOTCH
activity, suggesting that these are activating mutations. Importantly, 13 of the
20 (65%) NOTCH1/FBXW7-mutated T-ALL patients showed a good prednisone response
(P=0.01) and a better clinical outcome compared with NOTCH1/FBXW7 nonmutated
patients (P=0.03). These data suggest that NOTCH1/FBXW7 mutations are present in
T-ALL patients from Southern India and may be useful biomarkers to predict
prognosis in T-ALL. |
What is the role of the Tsix gene during X chromosome inactivation? | One of the two X chromosomes in female mammalian cells is subject to inactivation (XCI) initiated by the Xist gene. Xist works as a functional RNA molecule that recruits repressive chromatin factors towards one of the female Xs for inactivation. The Tsix gene, antisense of Xist, through transcription negatively regulates Xist and protects one X-chromosome in cis from inactivation by Xist. Although, the precise molecular mechanism is still unclear it has been shown that Tsix transcription regulates the chromatin structure by altering histone tail modifications and DNA methylation at the Xist promoter. In addition, Xist and Tsix RNAs form duplexes in vivo and are processed to small RNAs, which have a potential regulatory function. | A counting process senses the X chromosome/autosome ratio and ensures that X
chromosome inactivation (XCI) initiates in the early female (XX) embryo and in
differentiating female ES cells but not in their male (XY) counterparts.
Counting depends on the X inactivation center (Xic), which contains the Xist
gene encoding a nuclear RNA, which coats the inactive X chromosome and induces
gene silencing. A 37-kb sequence lying 3' to the Xist gene is known to prevent
initiation of XCI in male differentiating ES cells. This region contains the
major and minor promoters of the Tsix gene, which runs antisense to Xist, and
the DXPas34 tandem repeat lying close to the Tsix major promoter. We have
addressed the role of these elements in counting by using male ES cells.
Targeted deletion of DXPas34 impaired recruitment of RNA-polymerase II and TFIIB
at the Tsix major promoter, resulting in low levels of Tsix expression in ES
cells and moderate ectopic initiation of XCI upon differentiation. A deletion
extending 3' to Xist and including the Tsix major promoter resulted in almost
complete impairment of Tsix transcription and in efficient ectopic XCI upon
differentiation of male ES cells. Internal deletions within the Tsix gene did
not affect significantly the level of antisense transcription within Xist and
had only minor effects upon differentiation. Our results identify a function for
DXPas34 in murine XCI and demonstrate the critical role of Tsix transcription in
preventing XCI in differentiating male ES cells and in normal functioning of the
counting pathway. X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome
dosage imbalance between the XX female and the XY male. In eutherian mammals,
random XCI of the soma requires a master regulatory locus known as the
'X-inactivation center' (XIC/Xic), wherein lies the noncoding XIST/Xist silencer
RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is
imprinted to occur on the paternal X chromosome. To determine whether marsupials
and eutherians share the XIC-driven mechanism, we search for the sequence
equivalents in the genome of the South American opossum, Monodelphis domestica.
Positional cloning and bioinformatic analysis reveal several interesting
findings. First, protein-coding genes that flank the eutherian XIC are
well-conserved in M. domestica, as well as in chicken, frog, and pufferfish.
However, in M. domestica we fail to identify any recognizable XIST or TSIX
equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny
with eutherian mammals and other vertebrates. Therefore, during the evolution of
the marsupial X chromosome, one or more rearrangements broke up an otherwise
evolutionarily conserved block of vertebrate genes. The failure to find
XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to
allow for detection by current methods. Alternatively, the XIC may have arisen
relatively late in mammalian evolution, possibly in eutherians with the
emergence of random XCI. The latter argues that marsupial XCI does not require
XIST and opens the search for alternative mechanisms of dosage compensation. Numerous sense-antisense gene pairs have been discovered in various organisms.
Antisense genes play important roles in establishing parentally imprinted gene
expression patterns in mammals. Typically, protein-coding sense genes are
reciprocally regulated by their non-coding antisense partners. One example for
antisense regulation is the Xist (X-inactive specific transcript) and Tsix gene
pair, which is pivotal in X-inactivation. Xist works as a functional RNA
molecule that recruits repressive chromatin factors towards one of the female Xs
for inactivation. Antisense Tsix transcription negatively regulates Xist and
protects one X-chromosome in cis from inactivation by Xist. Albeit, the precise
molecular mechanism is still obscure it has been shown that Tsix transcription
regulates the chromatin structure by altering histone tail modifications and DNA
methylation at the Xist promoter. In addition, Xist and Tsix RNA form an RNA
duplexes in vivo and are processed to small RNAs, which have a potential
regulatory function. Here we review the latest findings and based on ample
experimental data consider models for antisense-mediated gene regulation in
X-inactivation. One of the two X chromosomes in female mammalian cells is subject to
inactivation (XCI) initiated by the Xist gene. In this study, we examined in
rodents (voles and rat) the conservation of the microsatellite region DXPas34,
the Tsix gene (antisense counterpart of Xist), and enhancer Xite that have been
shown to flank Xist and regulate XCI in mouse. We have found that mouse regions
of the Tsix gene major promoter and minisatellite repeat DXPas34 are conserved
among rodents. We have also shown that in voles and rat the region homologous to
the mouse Tsix major promoter, initiates antisense to Xist transcription and
terminates around the Xist gene start site as is observed with mouse Tsix. A
conservation of Tsix expression pattern in voles, rat and mice suggests a
crucial role of the antisense transcription in regulation of Xist and XIC in
rodents. Most surprisingly, we have found that voles lack the regions homologous
to the regulatory element Xite, which is instead replaced with the Slc7a3 gene
that is unassociated with the X-inactivation centre in any other eutherians
studied. Furthermore, we have not identified any transcription that could have
the same functions as murine Xite in voles. Overall, our data show that not all
the functional elements surrounding Xist in mice are well conserved even within
rodents, thereby suggesting that the regulation of XCI may be at least partially
taxon-specific. |
Is the protein FAK (Focal Adhesion Kinase) phosphorylated? | yes, the protein FAK (Focal Adhesion Kinase) is phosphorylated. | OBJECTIVES: PTPN13 is a new candidate tumor-suppressing gene. To investigate the
PTPN13 expression and its potential function in the invasion and metastasis of
lung squamous cell carcinoma (LSCC), we performed this study in 91 primary LSCC
tissues and the adjacent non-cancerous tissues.
METHODS: The mRNA expression of PTPN13 and FAK was quantitated by reverse
transcription polymerase chain reaction. The protein expression of PTPN13, focal
adhesion kinase (FAK) and phosphorylated FAK (P-FAK) was evaluated using
immunohistochemical staining and western blotting. The association among PTPN13
expression, FAK expression and the clinicopathological parameters were analyzed.
RESULTS: PTPN13 expression was down-regulated in LSCC, and was negatively
correlated with the cancer grade and stage. FAK mRNA, as well as FAK protein
level was elevated in LSCC tissues. P-FAK level, also found increased, had no
association with FAK mRNA and FAK protein expression, but had a negative
correlation with the PTPN13 expression. P-FAK level had a significant positive
correlation with the TNM classification.
CONCLUSION: The over-expression of FAK and increased FAK phosphorylation plays
an important role in the invasion and metastasis of LSCC. OBJECTIVE: To investigate the effect of Ganfukang (GFK) on connective tissue
growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or
Akt) signal pathway in a hepatic fibrosis rat model and to explore the
underlying therapeutic molecular mechanisms of GFK.
METHODS: Fifty SD rats were randomly divided into five groups as follows: the
control group, the model group (repeated subcutaneous injection of CCl4), and
the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric
administration). Reverse transcriptase-polymerase chain reaction (RT-PCR),
Western blotting, and immunohistochemistry were used to examine the expression
of CTGF, integrin α5, integrin β1, FAK/Akt signal pathway, cyclinD1, and
collagen in the different-treated rats.
RESULTS: GFK attenuated the up-regulation of CTGF, integrin α5, and integrin β1
in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the
phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression
of cyclinD1, collagen I, and collagen III was decreased by GFK significantly
(P<0.01).
CONCLUSIONS: CTGF and FAK/Akt signal pathway were activated in the CCl4-induced
hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and
collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due
to its effects against CTGF and FAk/Akt signal pathway. Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho
guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK).
FAK is recruited to adhesions and activated by integrin receptors binding to
matrix proteins, such as fibronectin (FN). Canonical models place Rgnef
downstream of integrin-FAK signaling in regulating Rho GTPase activity and cell
movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK
localization to early peripheral adhesions and promoting FAK activation upon FN
binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion
formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to
peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these
defects, but requires Rgnef-FAK binding. A mutation in the Rgnef pleckstrin
homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397
and paxillin-Y118 phosphorylation without disrupting the Rgnef-FAK interaction.
A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion
localization, but not paxillin-Y118 phosphorylation. This suggests that,
downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a
mechanism distinct from adhesion formation and FAK activation. These results
support a scaffolding role for Rgnef in FAK localization and activation at early
adhesions in a PH-domain-dependent but GEF-activity-independent manner. NEDD9, a focal adhesion scaffolding protein, has been recently proposed to
regulate invasion and metastasis in some cancer types, but unknown in cervical
cancer. The aim of this study was to determine if NEDD9 was involved in the
progression and metastasis of cervical cancer. The experimental results showed
NEDD9 protein was overexpressed in cervical cancer compared with normal cervical
epithelium tissues. Overexpression of NEDD9 was correlated with histological
grading, lymph node metastasis, and FIGO stage of cervical cancer. Silencing
NEDD9 resulted in tyrosine dephosphorylation of FAK and SRC oncoproteins, and
decreased cell migration and invasion in the cervical carcinoma SiHa and HeLa
cells. Overexpression of NEDD9 led to tyrosine phosphorylation of FAK and SRC
oncoproteins, and increased cell migration and invasion. Moreover, tyrosine
phosphorylation of NEDD9 was significantly decreased via suppressing tyrosine
phosphorylation of FAK or SRC, suggesting a positive feedback loop of tyrosine
phosphorylation between NEDD9 and FAK or SRC. In addition, our data showed that
silencing NEDD9 decreased Vimentin expression and increased E-cadherin
expression in cervical cancer cells, and vice versa. E-cadherin was subject to
regulation of NEDD9, FAK and SRC, but altered neither tyrosine-phosphorylated
nor total NEDD9. Our findings suggest that NEDD9 is overexpressed in cervical
cancer tissues and cells, and overexpressed NEDD9 promotes migration and
invasion in cervical carcinoma cells, probably via a positive feedback loop of
tyrosine phosphorylation between NEDD9 and FAK or SRC. By the mid 1980's, it was clear that the transforming activity of oncogenic Src
was linked to the activity of its tyrosine kinase domain and attention turned to
identifying substrates, the putative next level of control in the pathway to
transformation. Among the first to recognize the potential of
phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky
shotgun-based approach that led ultimately to the cDNA cloning and functional
characterization of many of today's best-known Src substrates (for example,
p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and
over 6000 citations later, the original goals of the project may be seen as
secondary to the enormous impact of these protein substrates in many areas of
biology. At the request of the editors, this review is not restricted to the
current status of the substrates, but reflects also on the anatomy of the
project itself and some of the challenges and decisions encountered along the
way. G3BP is a RasGAP binding protein that is overexpressed in many human cancers. We
previously reported that downregulation of G3BP suppressed cell growth and
induced apoptosis in HCT116 cells. Here we report that both transient and stable
knockdown of G3BP suppressed the growth, migration and invasion capability of
human lung carcinoma H1299 cells. Moreover, downregulation of G3BP significantly
inhibited the phosphorylation of Src, FAK and ERK, and the levels of NF-κB were
also markedly decreased in H1299 cells. Knockdown of G3BP also decreased the
expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and plasminogen
activator (uPA), and in vivo data demonstrated that downregulation of G3BP
markedly inhibited the growth of H1299 tumor xenografts. Together, these data
revealed that knockdown of G3BP inhibited the migration and invasion of human
lung carcinoma cells through the inhibition of Src, FAK, ERK and NF-κB and
decreased levels of MMP-2, MMP-9 and uPA. OBJECTIVE: Our poor understanding of how inflammatory mediators can affect
osteoblast behavior led us to investigate the tumor necrosis factor
(TNF)α-induced focal adhesion kinase (FAK) and Src phosphorylation.
MATERIAL AND METHODS: MC3T3-E1 pre-osteoblast cells were harvested at specific
time points after either TNFα treatment or RAW267 stimulated conditioned medium,
and thereafter cell extracts were prepared for Immunoblotting assay. ELISA
detected TNFα content at conditioned medium. Tumor necrosis
factor-α-neutralizing antibodies also were used.
RESULTS: It was possible to show that TNFα provokes attenuation at
Y-phosphorylation of both FAK (at Y397 ) and Src (at Y416 ) proteins (P < 0.05),
suggesting a decrease in their activities. The very similar profile was observed
when osteoblasts were incubated with conditioned medium from lipopolysaccharide
(LPS)-stimulated macrophages, it being significantly different than control (FAK
and Src, P < 0.05). Nevertheless, in order to validate these findings, we
decided to pre-incubate osteoblasts with anti-TNFα neutralizing antibody
(2 μg ml(-1) ) prior exposing to conditioned medium. Importantly, our results
revealed that there was a diminution on those conditioned medium effects when
the same biological parameters were evaluated (P < 0.05). Moreover, we also
showed that TNFα impairs osteoblast adhesion, suggesting an interesting role on
osteoblast performance.
CONCLUSIONS: Altogether, these results suggest that LPS-stimulated macrophage
mediators attenuate both FAK and Src activations in osteoblast, suggesting a
novel role for TNFα on osteoblast performance. Integrin-based adhesion to the extracellular matrix (ECM) plays critical roles
in controlling differentiation, survival, and motility of epithelial cells.
Cells attach to the ECM via dynamic structures called focal adhesions (FA). FA
undergo constant remodeling mediated by vesicle trafficking and fusion. A
soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is
an essential mediator of membrane fusion; however, its roles in regulating ECM
adhesion and cell motility remain unexplored. In this study, we found that
siRNA-mediated knockdown of αSNAP induced detachment of intestinal epithelial
cells, whereas overexpression of αSNAP increased ECM adhesion and inhibited cell
invasion. Loss of αSNAP impaired Golgi-dependent glycosylation and trafficking
of β1 integrin and decreased phosphorylation of focal adhesion kinase (FAK) and
paxillin resulting in FA disassembly. These effects of αSNAP depletion on ECM
adhesion were independent of apoptosis and NSF. In agreement with our previous
reports that Golgi fragmentation mediates cellular effects of αSNAP knockdown,
we found that either pharmacologic or genetic disruption of the Golgi
recapitulated all the effects of αSNAP depletion on ECM adhesion. Furthermore,
our data implicates β1 integrin, FAK, and paxillin in mediating the observed
pro-adhesive effects of αSNAP. These results reveal novel roles for αSNAP in
regulating ECM adhesion and motility of epithelial cells. |
Which is the substrate of the haspin kinase during mitosis? | Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis | Post-translational modifications of conserved N-terminal tail residues in
histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is
highly conserved, but the significance of its phosphorylation is unclear, and
the identity of the corresponding kinase unknown. Immunostaining with
phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation
of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Furthermore
we find that haspin, a member of a distinctive group of protein kinases present
in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly,
depletion of haspin by RNA interference reveals that this kinase is required for
H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal
association, haspin is found at the centrosomes and spindle during mitosis.
Haspin RNA interference causes misalignment of metaphase chromosomes, and
overexpression delays progression through early mitosis. This work reveals a new
kinase involved in composing the histone code and adds haspin to the select
group of kinases that integrate regulation of chromosome and spindle function
during mitosis and meiosis. In vertebrate mitosis, cohesion between sister chromatids is lost in two stages.
In prophase and prometaphase, cohesin release from chromosome arms occurs under
the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to
prevent removal of centromeric cohesin until anaphase. The regulatory enzymes
that act to sustain centromeric cohesion are incompletely described, however.
Haspin/Gsg2 is a histone H3 threonine-3 kinase required for normal mitosis. We
report here that both H3 threonine-3 phosphorylation and cohesin are located at
inner centromeres. Haspin depletion disrupts cohesin binding and sister
chromatid association in mitosis, preventing normal chromosome alignment and
activating the spindle assembly checkpoint, leading to arrest in a
prometaphase-like state. Overexpression of Haspin hinders cohesin release and
stabilizes arm cohesion. We conclude that Haspin is required to maintain
centromeric cohesion during mitosis. We also suggest that Aurora B regulates
cohesin removal through its effect on the localization of Shugoshin. The haspins are divergent members of the eukaryotic protein kinase family that
are conserved in many eukaryotic lineages including animals, fungi, and plants.
Recently-solved crystal structures confirm that the kinase domain of human
haspin has unusual structural features that stabilize a catalytically active
conformation and create a distinctive substrate binding site. Haspin localizes
predomitly to chromosomes and phosphorylates histone H3 at threonine-3 during
mitosis, particularly at inner centromeres. This suggests that haspin directly
regulates chromosome behavior by modifying histones, although it is likely that
additional substrates will be identified in the future. Depletion of haspin by
RNA interference in human cell lines causes premature loss of centromeric
cohesin from chromosomes in mitosis and failure of metaphase chromosome
alignment, leading to activation of the spindle assembly checkpoint and mitotic
arrest. Haspin overexpression stabilizes chromosome arm cohesion. Haspin,
therefore, appears to be required for protection of cohesion at mitotic
centromeres. Saccharomyces cerevisiae homologues of haspin, Alk1 and Alk2, are
also implicated in regulation of mitosis. In mammals, haspin is expressed at
high levels in the testis, particularly in round spermatids, so it seems likely
that haspin has an additional role in post-meiotic spermatogenesis. Haspin is
currently the subject of a number of drug discovery efforts, and the future use
of haspin inhibitors should provide new insight into the cellular functions of
these kinases and help determine the utility of, for example, targeting haspin
for cancer therapy. For proper partitioning of chromosomes in mitosis, the chromosomal passenger
complex (CPC) including Aurora B and survivin must be localized at the center of
paired kinetochores, at the site called the inner centromere. It is largely
unknown what defines the inner centromere and how the CPC is targeted to this
site. Here, we show that the phosphorylation of histone H3-threonine 3 (H3-pT3)
mediated by Haspin cooperates with Bub1-mediated histone 2A-serine 121
(H2A-S121) phosphorylation in targeting the CPC to the inner centromere in
fission yeast and human cells. H3-pT3 promotes nucleosome binding of survivin,
whereas phosphorylated H2A-S121 facilitates the binding of shugoshin, the
centromeric CPC adaptor. Haspin colocalizes with cohesin by associating with
Pds5, whereas Bub1 localizes at kinetochores. Thus, the inner centromere is
defined by intersection of two histone kinases. New evidence from three separate laboratories, published recently in Science,
has shown that centromere positioning of the CPC (chromosomal passenger complex)
during early mitosis is achieved through direct interaction between the CPP
(chromosomal passenger protein) survivin and histone H3. In essence, an acidic
pocket in the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin
binds to the NH2 tail of histone H3 specifically when it is phosphorylated at
threonine 3, a mark that is placed by the mitotic kinase, haspin. These data are
significant, as they describe a fundamental mechanism, conserved throughout
eukaryotes, which is essential for chromosome biorientation and the maintece
of genome stability during mitosis. Successful partitioning of chromosomes in mitosis relies on the bipolar
attachment of sister chromatids at metaphase. For this biorientation, the
chromosomal passenger complex (CPC), composed of catalytic kinase Aurora B and
regulatory components (INCENP, Survivin, and Borealin), must be localized at the
center of paired kinetochores, the site called the inner centromere. It is
largely unknown what defines the inner centromere and how the CPC is targeted to
this site. Recent studies point out that the shugoshin protein (SGO), originally
identified as a cohesin protector, also acts as a conserved centromeric adapter
of the CPC. Phosphorylation of the CPC by Cdk1 promotes direct binding with
shugoshin, thus explaining how the CPC is targeted to the centromere in a timely
manner at prometaphase during the cell cycle. Moreover, the phosphorylation of
histone H3 threonine 3 (H3-pT3) mediated by Haspin cooperates with Bub1-mediated
H2A-S121 phosphorylation in targeting the CPC to the inner centromere. H3-pT3
promotes nucleosome binding of Survivin, whereas H2A-pS121 facilitates the
binding of shugoshin. Haspin colocalizes with cohesin by associating with Pds5,
a cohesin-binding protein, and Bub1 localizes at kinetochores. Thus, the inner
centromere is defined by the spatial intersection of two histone marks mediated
by cohesin- and kinetochore-associated kinases. The transient mitotic histone H3 phosphorylation by various protein kinases
regulates chromosome condensation and segregation, but the counteracting
phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the
major histone H3 phosphatase acting on the mitotically phosphorylated (ph)
residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify
Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of
H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph
dephosphorylation by an indirect mechanism but directly and specifically targets
H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex
opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome
arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the
end of mitosis. Consistent with these findings, Repo-Man modulates in a
PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase
B and its substrate MCAK. Our study defines a novel mechanism by which PP1
counteracts Aurora B. BACKGROUND: Haspin kinases are mitotic kinases that are well-conserved from
yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important
roles in chromosome cohesion during mitosis. Moreover, phosphorylation of
histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required
for accumulation of Aurora B on the centromere, and the subsequent activation of
Aurora B kinase activity for accurate chromosome alignment and segregation.
Although extensive analyses of Haspin have been carried out in yeast and
animals, the function of Haspin in organogenesis remains unclear.
RESULTS: Here, we identified a Haspin kinase, designated AtHaspin, in
Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both
Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in
BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during
prometaphase and metaphase, and around the cell plate during cytokinesis. This
localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11
of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was
expressed in root meristems, shoot meristems, floral meristems, and throughout
the whole embryo at stages of high cell division. Overexpression of a kinase
domain mutant of AtHaspin decreased the size of the root meristem, which delayed
root growth.
CONCLUSIONS: Our results indicated that the Haspin kinase is a histone H3
threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3
and Thr11 in vitro. The expression and domit-negative analysis showed that
AtHaspin may have a role in mitotic cell division during plant growth. Further
analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed
new light on the regulation of chromosome segregation in cell division during
plant growth and development. Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2],
providing a chromatin binding site for the chromosomal passenger complex (CPC)
at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes
increasingly focused at inner centromeres during prometaphase [1, 2], but little
is known about how its level or location and the consequent chromosomal
localization of the CPC are regulated. In addition, CPC binding to shugoshin
proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of
the shugoshins to centromeres requires the phosphorylation of histone H2A at
Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis
for the collaboration of this pathway with H3T3ph has been unclear. Here, we
show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and
that Aurora B kinase activity is required for normal chromosomal localization of
the CPC, indicating an intimate linkage between Aurora B and Haspin functions in
mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback
loop that promotes generation of H3T3ph on chromatin. We also provide evidence
that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the
CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the
well-known accumulation of the CPC in these regions. The approval of histone deacetylase inhibitors for treatment of lymphoma
subtypes has positioned histone modifications as potential targets for the
development of new classes of anticancer drugs. Histones also undergo
phosphorylation events, and Haspin is a protein kinase the only known target of
which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is
necessary for mitosis progression. Mitotic kinases can be blocked by small drugs
and several clinical trials are underway with these agents. As occurs with
Aurora kinase inhibitors, Haspin might be an optimal candidate for the
pharmacological development of these compounds. A high-throughput screening for
Haspin inhibitors identified the CHR-6494 compound as being one promising such
agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent
manner and causes a mitotic catastrophe characterized by metaphase misalignment,
spindle abnormalities and centrosome amplification. From the cellular
standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M
and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its
anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted
nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class
Haspin inhibitor with a wide spectrum of anticancer effects that merits further
preclinical research as a new member of the family of mitotic kinase inhibitors. Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in
mitosis that has emerged as a possible cancer therapeutic target. High
throughput screening of approximately 140,000 compounds identified the
beta-carbolines harmine and harmol as moderately potent haspin kinase
inhibitors. Based on information obtained from a structure-activity relationship
study previously conducted for an acridine series of haspin inhibitors in
conjunction with in silico docking using a recently disclosed crystal structure
of the kinase, harmine analogs were designed that resulted in significantly
increased haspin kinase inhibitory potency. The harmine derivatives also
demonstrated less activity towards DYRK2 compared to the acridine series. In
vitro mouse liver microsome stability and kinase profiling of a representative
member of the harmine series (42, LDN-211898) are also presented. Aurora B localization to mitotic centromeres, which is required for proper
chromosome alignment during mitosis, relies on Haspin-dependent histone H3
phosphorylation and on Bub1-dependent histone H2A phosphorylation--which
interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate
that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances
H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby
promoting Aurora B centromere recruitment in early mitosis. Importantly,
chromosome biorientation defects caused by Mps1 inhibition are improved by
restoring Aurora B centromere recruitment. As Mps1 kinetochore localization
reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment
circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid
centromere accumulation of Aurora B at the onset of mitosis. Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking
site for the Aurora B complex at centromeres. Aurora B functions to correct
improper kinetochore-microtubule attachments and alert the spindle checkpoint to
the presence of misaligned chromosomes. We show that Haspin inhibitors decreased
H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of
centromere and kinetochore Aurora B substrates. Consequently, metaphase
chromosome alignment and spindle checkpoint signaling were compromised. These
effects were phenocopied by microinjection of anti-H3T3ph antibodies.
Retargeting Aurora B to centromeres partially restored checkpoint signaling and
Aurora B-dependent phosphorylation at centromeres and kinetochores, bypassing
the need for Haspin activity. Haspin inhibitors did not obviously affect
phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome
arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed.
Haspin inhibitors did not block Aurora B localization to the spindle midzone in
anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal
centromeric roles of Aurora B in chromosome movement and spindle checkpoint
signaling. By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment
of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC
subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint
(SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a
potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not
Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the
CPC without affecting the bulk of Aurora B activity in HeLa cells.
Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1
and SAC override at high nocodazole concentrations. 5-ITu also impaired
kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed
by concomitant inhibition of phosphatase activity. Forcing localization of
Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but
failed to rescue the SAC override. This result suggests that a target of 5-ITu,
possibly Haspin itself, may further contribute to SAC signaling downstream of
Aurora B. Aurora B is the catalytic subunit of the chromosomal passenger complex (CPC),
which coordinates mitotic processes through phosphorylation of key regulatory
proteins. In prometaphase, the CPC is enriched at the centromeres to regulate
the spindle checkpoint and kinetochore-microtubule interactions. Centromeric CPC
binds to histone H3 that is phosphorylated at T3 (H3T3ph) by Aurora B-stimulated
Haspin. PP1/Repo-Man acts antagonistically to Haspin and dephosphorylates H3T3ph
at the chromosome arms but is somehow prevented from causing a net
dephosphorylation of centromeric H3T3ph during prometaphase. Here, we show that
Aurora B phosphorylates Repo-Man at S893, preventing its recruitment by
histones. We also identify PP2A as a mitotic interactor of Repo-Man that
dephosphorylates S893 and thereby promotes the targeting of Repo-Man to
chromosomes and the dephosphorylation of H3T3ph by PP1. Thus,
Repo-Man-associated PP1 and PP2A collaborate to oppose the chromosomal targeting
of Aurora B. We propose that the reciprocal feedback regulation of Haspin and
Repo-Man by Aurora B generates a robust bistable response that culminates in the
centromeric targeting of the CPC during prometaphase. The mitosis-specific phosphorylation of histone H3 at Thr3 (H3T3ph) plays an
important role in chromosome segregation by recruiting Aurora B. H3T3
phosphorylation is catalyzed by Haspin, an atypical protein kinase whose kinase
domain is intrinsically active without phosphorylation at the activation loop.
Here, we report the molecular basis for Haspin inhibition during interphase and
its reactivation in M phase. We identify a conserved basic segment that
autoinhibits Haspin during interphase. This autoinhibition is neutralized when
Cdk1 phosphorylates the N terminus of Haspin in order to recruit Polo-like
kinase (Plk1/Plx1), which, in turn, further phosphorylates multiple sites at the
Haspin N terminus. Although Plx1, and not Aurora B, is critical for H3T3
phosphorylation in Xenopus egg extracts, Plk1 and Aurora B both promote this
modification in human cells. Thus, M phase-specific H3T3 phosphorylation is
governed by the combinatorial action of mitotic kinases that neutralizes Haspin
autoinhibition through a mechanism dependent on multisite phosphorylation. |
Which are the most widely reported side-effects in the treatment of Crohn's disease? | Leukopenia, paresthesia, psoriasis, alopecia and hemolysis are the most commonly reported side effects depending on the treatment. Severe adverse effects include myelosuppression, liver toxicity and hyperplasia, pancreatitis and pericarditis. The most severe but rare side-effects reported are progressive multifocal leukoencephalopathy (PML), serious infections, and lymphoma. | Hemolytic anemia is a well-recognized complication of sulfasalazine treatment.
17 of 40 (43%) patients with inflammatory bowel disease receiving sulfasalazine
had evidence of hemolysis as detected by starch gel electrophoresis. Only 47%
(8) of patients with hemolysis had Heinz body formation. The hemoglobin was
significantly reduced in patients with hemolysis and 53% had a reticulocyte
count of greater than 5%. A significant correlation was noted between hemolysis
and serum sulfapyridine level, but no correlation was seen with serum
sulfasalazine level. There was no significant difference in disease extent or
activity in patients with hemolysis compared to those without hemolysis.
Hemolysis is not a rare side-effect of sulfasalazine therapy. Heinz body
formation is not invariably found in sulfasalazine-induced hemolysis. It has been suggested that cyclosporin A might be of some benefit to patients
with Crohn's disease. The clinical response and side-effects of cyclosporin A in
Crohn's disease are described in a series of 13 adults. The majority of patients
had ileal disease and all but one were started on an initial oral dose of 15
mg/kg per day. Duration of treatment ranged from 3 to 42 weeks. Of the 13
patients, 6 showed a response to therapy; the remainder showed no response or
deteriorated. The commonest side-effect was hyperaesthesia, but one patient
developed nephrotoxicity and one developed hepatotoxicity. Significant drug
malabsorption occurred in one case. The side-effects were dose dependent and
reversible. Cyclosporin A may have a part to play in the treatment of resistant
Crohn's disease, and in our hands has been associated with a 46 per cent
response rate; however, the precise role of cyclosporin A in the management of
Crohn's disease awaits further study. The present study of metronidazole in perineal Crohn's disease includes 26
patients, and is composed of 17 of 21 patients who were previously reported and
9 additional consecutive patients. The course of these patients was evaluated to
determine if the drug could be reduced or stopped, whether or not it continued
to be effective for prolonged periods, and what long-term side effects were
encountered. Dosage reduction was associated with exacerbation of disease
activity in all patients, but in all, the perineal manifestations of disease
healed promptly when the full dosage of metronidazole was reinstituted. The drug
could be successfully discontinued in only 28% of those in whom cessation was
attempted; in those patients whose perineal disease worsened with cessation of
therapy, rapid healing was achieved if the drug was reintroduced. Sixteen
patients received metronidazole for at least 12 mo including 7 for 18-36 mo.
Eight of these 16 patients, including 4 on and 4 off the drug, remain healed;
the other 8 patients had advanced healing. The only major side effect observed
was paresthesias. These occurred in 50% of the patients and developed in the
patients at a mean of 6.5 mo after the onset of treatment. They appeared to be
dose related and nonprogressive but tended to persist for prolonged periods even
after discontinuance of the drug. BACKGROUND: Treatment with sulphasalazine of patients with mild and moderate
forms of Crohn's disease may result in side effects in some of them.
CASE REPORT: An 11-year-old girl with Crohn's disease was given 40 mg/kd/day of
sulphasalazine after achieving remission with prednisone. She developed
urticaria and eosionophilia 8 days later, then extended skin edema during a
second course of sulphasalazine requiring methyl prednisolone therapy.
Mesalazine administration (25 mg/kg/day), one month later, resulted to watery
stools, vomiting, and fever, a side effect observed again after two further
mesalazine challenges of one-month interval. Eosionophilia was present and the
lymphocyte stimulation test with mesalazine was positive. Successful
desensitization to sulphasalazine could be obtained 9 months later permitting
further safe administration of this drug up to 18 months.
CONCLUSION: Hypersensitivity to sulphasalazine may be successfully overcame by
oral desensitization, especially in those patients with low-grade activity of
Crohn's disease. Mesalamines are slow-release formulations of 5-aminosalicylic acid (5-ASA) and
are effective as primary treatment and maintece therapy in inflammatory bowel
disease. Interstitial nephritis is a recognized side effect. We report two cases
of biopsy-confirmed interstitial nephritis in patients being treated with 5-ASA.
Both had a trial of steroid therapy. One patient had partial recovery of renal
function but the other patient was in chronic renal failure and likely was
approaching the need for dialysis. Interstitial nephritis is an under-recognized
complication of 5-ASA therapy. Early identification and withdrawal of this drug
can lead to a partial or complete reversal of renal dysfunction. Myopericarditis is a rare extraintestinal complication of inflammatory bowel
disease (IBD). It has also been described as a side-effect of the treatment of
IBD. We report a 37-year-old-woman with Crohn's disease who had several mild
episodes of myopericarditis, two of which were associated with a pleural
effusion, and two with conduction abnormalities in the atrioventricular node.
During the last episode, a nodal rhythm was followed by a third-degree
atrioventricular block and a prolonged pause, resulting in loss of consciousness
and convulsions. A permanent pacemaker was implanted. Our patient is also human
lymphocyte antigen (HLA) B27-positive. HLA B27 is known to be associated with
conduction disturbances in the AV node. Recurrent myopericarditis can be a sign
of IBD. BACKGROUND & AIMS: Regulatory assessments of drug risks do not routinely
consider patient preferences, despite evidence that some patients are willing to
accept increased side-effect risk in exchange for therapeutic benefits. The aim
of this study is to estimate the willingness of Crohn's disease (CD) patients to
accept life-threatening adverse event risks in exchange for CD symptom relief.
METHODS: Patients with CD completed choice-format conjoint trade-off tasks
involving hypothetical treatments with varying efficacy and risk levels. The
treatment features included daily symptoms and activity limitations, serious
complications (fistulas, abscesses, bowel obstructions), time between flare-ups,
oral steroid use and risk of 3 serious adverse events (SAEs) known to be
associated with CD treatment (progressive multifocal leukoencephalopathy (PML),
serious infections, and lymphoma). The mean maximum acceptable annual risk (MAR)
for each of the SAEs was calculated for various levels of clinical benefit.
RESULTS: Daily symptom severity was the most important factor in treatment
preferences. Higher MAR was observed for trade-off tasks involving higher levels
of clinical benefit. The MAR was similar across the 3 SAEs. For improvements
from severe daily symptoms to remission and from moderate daily symptoms to
remission, the MARs ranged from 0.69% to 0.81% and from 0.39% to 0.55%,
respectively.
CONCLUSIONS: Patients with CD have well-defined preferences among treatment
attributes and are willing to accept tradeoffs among attributes. The patients
indicated they are willing to accept elevated SAE risks in exchange for clinical
efficacy. The perspective of the patients on the benefit versus risk balance can
assist in making treatment and regulatory decisions. HISTORY: A 48-year-old patient with Crohn's disease was admitted to our hospital
with fatigue, icterus, hepatosplenomegaly and ascites. INVESTIGARTIOS: The whole
blood count revealed a pancytopenia, hyperbilirubinemia and slightly elevated
transaminases. Examination of the liver histology showed areas of enlarged
hyperplastic hepatocytes adjacent to areas of atrophic hepatocytes and dilated
sinusoids.
DIAGNOSIS, TREATMENT AND COURSE: Pancytopenia was most likely
azathioprine-related. Analysis of the liver histology was highly suggestive of
an azathioprine-related, nodular regenerative hyperplasia (NRH). After
discontinuation of azathioprine the patient's condition improved substantially.
CONCLUSIONS: NRH is a rare but potentially serious complication of azathioprine
therapy. Other causes include various rheumatological, vascular and
myeloproliferative diseases. When azathioprine is prescribed it must be borne in
mind that it can cause NRH as a potential adverse effect, and liver enzymes
should be measured at regular follow-up examinations. A 15-year-old, woman, Crohn's disease patient, who carried the TPMT *3C
heterozygous mutant, complained of alopecia 3 days after starting
6-mercaptopurine (6-MP) and then developed severe myelosuppression 6 weeks after
starting 6-MP. The alopecia involved scalp hair only (body hair preserved) and
was domit in the temporal region. Following these side effects, transient
remission of Crohn's disease occurred. Myelosuppression due to 6-MP is a rare
but life-threatening side effect that is difficult to predict despite continuous
monitoring of complete blood cell counts. In the present case, 6-MP-induced
alopecia preceded myelosuppression and progressed rapidly as the
myelosuppression worsened. PURPOSE: To report a case of corneal endothelial deposits associated with
rifabutin therapy for Crohn's disease.
METHOD: Case report, anterior segment photography, and current literature
search.
RESULTS: Persistence of corneal endothelial deposits was found to occur in a
55-year-old gentleman 6 years after cessation of a 3-year course of rifabutin.
The corneal deposits were not associated with lenticular findings or retinal
dysfunction, the latter side effect being published in a recent research
article.
CONCLUSIONS: This is only the second published case of corneal deposits in
conjunction with rifabutin therapy for Crohn's disease. This case uniquely
confirms the persistence of the corneal deposits longer than previously
documented. IMPORTANCE OF THE FIELD: traditional immunosuppressants, including azathioprine,
remain the mainstay of therapy in steroid dependent/refractory patients with
inflammatory bowel diseases (IBD). The main limitations of its use are its side
effects appearing in about a fifth of the patients, including myelosuppression
and liver toxicity. Major complications occur in patients with low
thiopurine-S-methyltransferase (TPMT) enzyme activity; however, the clinical
relevance of these tests remains conflictive.
AREAS COVERED IN THIS REVIEW: in this review, the authors aim to summarize the
new data regarding the relationship between the pharmacology of thiopurines and
pathogenesis of adverse events.
WHAT THE READER WILL GAIN: readers will gain an understanding of the metabolism
of thiopurines, side effect profile, pharmacological background of side effects,
importance of metabolite monitoring, clinical relevance of inherited differences
in drug metabolism and other conditions (e.g., concomitant use of allopurinol)
which can modify enzyme activity. By gaining an understanding of the
pharmacology and metabolism of thiopurines, clinicians will be able to optimize
thiopurine therapy in IBD.
TAKE HOME MESSAGE: TPMT testing and metabolite monitoring are still not
considered the standard of care, and clinicians will continue to choose the
approach that best suits their clinical practice and patient needs. Regardless
of what strategy is chosen, patients need to be carefully monitored and well
informed about the potential risks. The induction of psoriasis as a side effect of treatment with TNF-alpha
inhibitors is one of a few rare complications of treatment, the pathogenic
mechanism of which has not yet been completely clarified. The clinical
presentation of these reactions may show the typical characteristics of
psoriasis, palmoplantar pustulosis and psoriasiform exanthema; the individual
variations of which may combine to give different presentations in individual
patients. We present the case of a patient who, after administration of
infliximab indicated for Crohn's disease, developed not only skin manifestations
but also those of psoriatic arthritis. With the increasing use of anti-tumor necrosis factor α (anti-TNF) biologic
drugs to treat autoimmune diseases, an expanding array of adverse reactions is
emerging. Anti-TNF drug-induced alopecia is a less well-known side effect of
this class of drugs. The aim of this study was to define the clinical and
histopathological features of alopecia arising in the setting of anti-TNF
therapy. Clinical and histopathological features of 3 patients who developed
scalp alopecia during anti-TNF treatment were examined. Two of the 3 patients
also developed psoriasiform lesions outside the scalp, and biopsies from both
scalp and nonscalp sites were reviewed. Clinically, each patient had large scaly
patches associated with the scalp alopecia. All scalp biopsies revealed
psoriasiform epidermal features and alopecia areata-like dermal changes.
Epidermal changes included acanthosis and confluent parakeratosis with
neutrophils and frank pustules. Dermal changes included markedly increased
catagen/telogen and miniaturized hairs and peribulbar lymphocytic inflammation.
Numerous plasma cells and eosinophils were present in all cases. Biopsies from
the nonscalp lesions showed psoriasiform changes and prominent eosinophils and
plasma cells. Two patients showed significant improvement of the alopecia with
topical treatment only. In conclusion, anti-TNF therapy-related alopecia may
closely mimic psoriatic alopecia and alopecia areata but can be histologically
distinguished from alopecia areata by epidermal psoriasiform changes and dermal
plasma cells and from primary psoriasis by the presence of plasma cells and
eosinophils. A correct diagnosis can enable effective treatment and, in some
cases, allow anti-TNF therapy to continue. Treatment with antitumor necrosis factor-alpha (anti-TNF-α) offers a significant
improvement in several immune-based diseases, including Crohn's disease (CD) and
psoriasis. Different cutaneous side effects have been described for anti-TNF-α
therapy such as psoriasis. Previous reports showed that inhibition of TNF-α can
induce over expression of cutaneous IFN-α, which in turn caused a predisposition
to psoriasis. We report a 31-year-old woman with extensive CD and perianal
lesions, without response to conventional treatment. She paradoxically developed
a cutaneous eruption with psoriasiform morphology and distribution during
treatment with both anti-TNF-α approved in Europe for CD, infliximab and
adalimumab. These lesions cleared after topical application of corticosteroids
and cessation of the anti-TNF-α treatment. Due to uneffectiveness of
pharmacological treatment on disease, the patient had to undergo surgery.
TNF-induced psoriasis in patients with CD is rare and has been previously
documented with infliximab or adalimumab. The reason for this apparently
paradoxical effect of the therapy is still unclear. This is the first case of
psoriasis induced first by infliximab and later by adalimumab in the same CD
patient. We would like to review and to draw attention about psoriasis as a
cutaneous side effect with anti-TNF-α treatments. |
Are most driver gene mutations synonymous or non-synonymous? | A common goal of tumor sequencing projects is finding genes whose mutations are selected for during tumor development. This is accomplished by choosing genes that have more non-synonymous mutations than expected from an estimated background mutation frequency. | This is the first report of an insect esterase efficiently expressed in the
methylotrophic yeast Pichia pastoris (so far insect esterases have been produced
only in the baculovirus system). Having isolated a Tribolium castaneum
carboxylesterase cDNA (TCE), we were initially unable to express it in
Escherichia coli or P. pastoris despite significant transcription levels. As
codon usage bias is different in T. castaneum and P. pastoris, we assumed this
was a possible explanation for the translational barrier observed in yeast.
Accordingly, we designed and constructed by recursive PCR a synthetic TCE gene
(synTCE) optimized for heterologous expression in P. pastoris, i.e., a gene in
which certain TCE codons are replaced with synonymous codons 'preferred' in P.
pastoris. When the altered gene was placed under the control of either the P.
pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the
inducible alcohol oxidase (AOX1) promoter and introduced on an expression vector
into P. pastoris, its product was produced intracellularly. We also successfully
explored the possibility of obtaining a secreted product: P. pastoris cells
expressing an in-frame fusion of synTCE with the alpha-factor secretion signal
under the control of the GAP promoter were found to secrete the recombit
esterase into the external medium (to a concentration of 7 mg/L). In addition to
this demonstration of TCE production in yeast, our results suggest that the GAP
promoter could advantageously replace the AOX1 promoter as a driver of synTCE
expression. TCE specific activity was approximately 5 U/mg when p-nitrophenyl
acetate was used as substrate. Previous genome-wide scans of positive natural selection in humans have
identified a number of non-neutrally evolving genes that play important roles in
skin pigmentation, metabolism, or immune function. Recent studies have also
shown that a genome-wide pattern of local adaptation can be detected by
identifying correlations between patterns of allele frequencies and
environmental variables. Despite these observations, the degree to which natural
selection is primarily driven by adaptation to local environments, and the role
of pathogens or other ecological factors as selective agents, is still under
debate. To address this issue, we correlated the spatial allele frequency
distribution of a large sample of SNPs from 55 distinct human populations to a
set of environmental factors that describe local geographical features such as
climate, diet regimes, and pathogen loads. In concordance with previous studies,
we detected a significant enrichment of genic SNPs, and particularly
non-synonymous SNPs associated with local adaptation. Furthermore, we show that
the diversity of the local pathogenic environment is the predomit driver of
local adaptation, and that climate, at least as measured here, only plays a
relatively minor role. While background demography by far makes the strongest
contribution in explaining the genetic variance among populations, we detected
about 100 genes which show an unexpectedly strong correlation between allele
frequencies and pathogenic environment, after correcting for demography.
Conversely, for diet regimes and climatic conditions, no genes show a similar
correlation between the environmental factor and allele frequencies. This result
is validated using low-coverage sequencing data for multiple populations. Among
the loci targeted by pathogen-driven selection, we found an enrichment of genes
associated to autoimmune diseases, such as celiac disease, type 1 diabetes, and
multiples sclerosis, which lends credence to the hypothesis that some
susceptibility alleles for autoimmune diseases may be maintained in human
population due to past selective processes. Cancer cells evolve from normal cells by somatic mutations and natural
selection. Comparing the evolution of cancer cells and that of organisms can
elucidate the genetic basis of cancer. Here we analyse somatic mutations in >400
cancer genomes. We find that the frequency of somatic single-nucleotide
variations increases with replication time during the S phase much more
drastically than germ-line single-nucleotide variations and somatic large-scale
structural alterations, including amplifications and deletions. The ratio of
nonsynonymous to synonymous single-nucleotide variations is higher for cancer
cells than for germ-line cells, suggesting weaker purifying selection against
somatic mutations. Among genes with recurrent mutations only cancer driver genes
show evidence of strong positive selection, and late-replicating regions are
depleted of cancer driver genes, although enriched for recurrently mutated
genes. These observations show that replication timing has a prominent role in
shaping the single-nucleotide variation landscape of cancer cells. BACKGROUND: Gastric adenocarcinoma is a rare diagnosis in childhood. A
14-year-old male patient presented with metastatic gastric adenocarcinoma, and a
strong family history of colon cancer. Clinical sequencing of CDH1 and APC were
negative. Whole exome sequencing was therefore applied to capture the majority
of protein-coding regions for the identification of single-nucleotide variants,
small insertion/deletions, and copy number abnormalities in the patient's
germline as well as primary tumor.
MATERIALS AND METHODS: DNA was extracted from the patient's blood, primary
tumor, and the unaffected mother's blood. DNA libraries were constructed and
sequenced on Illumina HiSeq2000. Data were post-processed using Picard and
Samtools, then analyzed with the Genome Analysis Toolkit. Variants were
annotated using an in-house Ensembl-based program. Copy number was assessed
using ExomeCNV.
RESULTS: Each sample was sequenced to a mean depth of coverage of greater than
120×. A rare non-synonymous coding single-nucleotide variant (SNV) in TP53 was
identified in the germline. There were 10 somatic cancer protein-damaging
variants that were not observed in the unaffected mother genome. ExomeCNV
comparing tumor to the patient's germline, identified abnormal copy number,
spanning 6,946 genes.
CONCLUSION: We present an unusual case of Li-Fraumeni detected by whole exome
sequencing. There were also likely driver somatic mutations in the gastric
adenocarcinoma. These results highlight the need for more thorough and broad
scale germline and cancer analyses to accurately inform patients of inherited
risk to cancer and to identify somatic mutations. Squamous cell lung cancer is a major histotype of non-small cell lung cancer
(NSCLC) that is distinct from lung adenocarcinoma. We used whole-exome
sequencing to identify novel non-synonymous somatic mutations in squamous cell
lung cancer. We identified 101 single-nucleotide variants (SNVs) including 77
non-synonymous SNVs (67 missense and 10 nonsense mutations) and 11 INDELs
causing frameshifts. We also found four SNVs located within splicing sites. We
verified 62 of the SNVs (51 missense, 10 nonsense and 1 splicing-site mutation)
and 10 of the INDELs as somatic mutations in lung cancer tissue. Sixteen of the
mutated genes were also mutated in at least one patient with a different type of
lung cancer in the Catalogue of Somatic Mutation in Cancer (COSMIC) database.
Four genes (LPHN2, TP53, MYH2 and TGM2) were mutated in approximately 10% of the
samples in the COSMIC database. We identified two missense mutations in
C10orf137 and MS4A3 that also occurred in other solid-tumor tissues in the
COSMIC database. We found another somatic mutation in EP300 that was mutated in
4.2% of the 2,020 solid-tumor samples in the COSMIC database. Taken together,
our results implicate TP53, EP300, LPHN2, C10orf137, MYH2, TGM2 and MS4A3 as
potential driver genes of squamous cell lung cancer. We have developed a sequence conservation-based artificial neural network
predictor called NetDiseaseSNP which classifies nsSNPs as disease-causing or
neutral. Our method uses the excellent alignment generation algorithm of SIFT to
identify related sequences and a combination of 31 features assessing sequence
conservation and the predicted surface accessibility to produce a single score
which can be used to rank nsSNPs based on their potential to cause disease.
NetDiseaseSNP classifies successfully disease-causing and neutral mutations. In
addition, we show that NetDiseaseSNP discriminates cancer driver and passenger
mutations satisfactorily. Our method outperforms other state-of-the-art methods
on several disease/neutral datasets as well as on cancer driver/passenger
mutation datasets and can thus be used to pinpoint and prioritize plausible
disease candidates among nsSNPs for further investigation. NetDiseaseSNP is
publicly available as an online tool as well as a web service:
http://www.cbs.dtu.dk/services/NetDiseaseSNP. Neuroblastoma is one of the most genomically heterogeneous childhood maligces
studied to date, and the molecular events that occur during the course of the
disease are not fully understood. Genomic studies in neuroblastoma have showed
only a few recurrent mutations and a low somatic mutation burden. However, none
of these studies has examined the mutations arising during the course of
disease, nor have they systemically examined the expression of mutant genes.
Here we performed genomic analyses on tumors taken during a 3.5 years disease
course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal
primary tumor taken at surgical resection, and a liver metastasis at autopsy).
Whole genome sequencing of the index liver metastasis identified 44
non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large
hemizygous deletion in the ATRX gene which has been recently reported in
neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the
primary tumor and bone marrow biopsy, while the other 30 were unique to the
index tumor, indicating accumulation of de novo mutations during therapy.
Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of
the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of
expression of the mutant alleles, suggesting potential oncogenic driver roles of
these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1
was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant
demonstrated a significantly increased motility through elevated Rho signaling,
but had no effect on growth. Therefore, this study highlights the need for
multiple biopsies and sequencing during progression of a cancer and
combinatorial DNA and RNA sequencing approach for systematic identification of
expressed driver mutations. |
What is the advantage of neutral loss detection in phosphoproteomics? | The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides.
CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group allowing detection and sequencing of phosphopeptides. | Cellular processes such as proliferation, differentiation, and adaptation to
environmental changes are regulated by protein phosphorylation. Development of
sensitive and comprehensive analytical methods for determination of protein
phosphorylation is therefore a necessity in the pursuit of a detailed molecular
view of complex biological processes. We present a quantitative
modification-specific proteomic approach that combines stable isotope labeling
by amino acids in cell culture (SILAC) for quantitation with IMAC for
phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for
identification. This integrated phosphoproteomic technology identified and
quantified phosphorylation in key regulator and effector proteins of a
prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone
response. SILAC encoding of yeast proteomes was achieved by incorporation of
[(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain.
Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control
and lysed, and extracted proteins were digested with trypsin. Phosphopeptides
were enriched by a combination of strong cation exchange chromatography and
IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion
trap-Fourier transform ion cyclotron resoce mass spectrometer. MS/MS and
neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of
phosphopeptides with exceptional accuracy and specificity. Of more than 700
identified phosphopeptides, 139 were differentially regulated at least 2-fold in
response to mating pheromone. Among these regulated proteins were components
belonging to the mitogen-activated protein kinase signaling pathway and to
downstream processes including transcriptional regulation, the establishment of
polarized growth, and the regulation of the cell cycle. Reversible protein phosphorylation is a central cellular regulatory mechanism in
modulating protein activity and propagating signals within cellular pathways and
networks. Development of more effective methods for the simultaneous
identification of phosphorylation sites and quantification of temporal changes
in protein phosphorylation could provide important insights into molecular
signaling mechanisms in various cellular processes. Here we present an
integrated quantitative phosphoproteomics approach and its application for
comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA)
gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O
labeling plus (16)O/ (18)O-methanol esterification for quantitation, a
macro-immobilized metal-ion affinity chromatography trap for phosphopeptide
enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by
neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear
ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were
identified and quantified including receptors, kinases, proteins associated with
small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were
also identified as differentially expressed followed by LPA stimulation, and we
have shown evidence of pseudopodia subcellular localization of one of these
candidate proteins. These results demonstrate the efficiency of this
quantitative phosphoproteomics approach and its application for rapid discovery
of phosphorylation events associated with LPA gradient sensing and cell
chemotaxis. Normal human urine contains large numbers of exosomes, which are 40- to 100-nm
vesicles that originate as the internal vesicles in multivesicular bodies from
every renal epithelial cell type facing the urinary space. Here, we used
LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the
analysis identified 1132 proteins unambiguously, including 177 that are
represented on the Online Mendelian Inheritance in Man database of
disease-related genes, suggesting that exosome analysis is a potential approach
to discover urinary biomarkers. We extended the proteomic analysis to
phosphoproteomic profiling using neutral loss scanning, and this yielded
multiple novel phosphorylation sites, including serine-811 in the
thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use
of exosome analysis to identify a genetic renal disease, we carried out
immunoblotting of exosomes from urine samples of patients with a clinical
diagnosis of Bartter syndrome type I, showing an absence of the
sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are
publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. Here we describe a set of enhanced data processing and filtering methods to
improve significance and coverage of phosphopeptide identifications by mass
spectrometry. We demonstrate that for samples of limited complexity,
spectra-based estimation of false discovery rates will lead to overprediction of
confidently identified phosphorylated peptides due to a bias caused by multiple
fragmentation of highly abundant peptide species. We further provide evidence
that fragmentation of abundant peptides at the tails of their chromatographic
peaks is a major source for false positive peptide matches and that overall
confidence in phosphopeptide identifications can be improved by a
chromatographic peak-based aggregation scheme, intensity rank-based neutral loss
and optimized mass error filters. When replicate runs of a standard sample were
performed using different fragmentation techniques on an Orbitrap mass
spectrometer we observed improvements of 7-31% in phosphopeptide coverage
depending on the fragmentation method and the desired false discovery rate. Accurate determination of protein phosphorylation is challenging, particularly
for researchers who lack access to a high-accuracy mass spectrometer. In this
study, multiple protocols were used to enrich phosphopeptides, and a rigorous
filtering workflow was used to analyze the resulting samples. Phosphopeptides
were enriched from cultured rat renal proximal tubule cells using three commonly
used protocols and a dual method that combines separate immobilized metal
affinity chromatography (IMAC) and titanium dioxide (TiO(2)) chromatography,
termed dual IMAC (DIMAC). Phosphopeptides from all four enrichment strategies
were analyzed by liquid chromatography-multiple levels of mass spectrometry
(LC-MS(n)) neutral-loss scanning using a linear ion trap mass spectrometer.
Initially, the resulting MS(2) and MS(3) spectra were analyzed using
PeptideProphet and database search engine thresholds that produced a false
discovery rate (FDR) of <1.5% when searched against a reverse database. However,
only 40% of the potential phosphopeptides were confirmed by manual validation.
The combined analyses yielded 110 confidently identified phosphopeptides. Using
less-stringent initial filtering thresholds (FDR of 7-9%), followed by rigorous
manual validation, 262 unique phosphopeptides, including 111 novel
phosphorylation sites, were identified confidently. Thus, traditional methods of
data filtering within widely accepted FDRs were inadequate for the analysis of
low-resolution phosphopeptide spectra. However, the combination of a streamlined
front-end enrichment strategy and rigorous manual spectral validation allowed
for confident phosphopeptide identifications from a complex sample using a
low-resolution ion trap mass spectrometer. Phosphoproteomics deals with the identification and quantification of thousands
of phosphopeptides. Localizing the phosphorylation site is however much more
difficult than establishing the identity of a phosphorylated peptide. Further,
recent findings have raised doubts of the validity of the site assignments in
large-scale phosphoproteomics data sets. To improve methods for site
localization, we made use of a synthetic phosphopeptide library and
SILAC-labeled peptides from whole cell lysates and analyzed these with
high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated
gas-phase phosphate rearrangement reactions during collision-induced
dissociation (CID) and used these spectra to devise a quantitative filter that
by comparing signal intensities of putative phosphorylated fragment ions with
their nonphosphorylated counterparts allowed us to accurately pinpoint which
fragment ions contain a phosphorylated residue and which ones do not. We also
evaluated higher-energy collisional dissociation (HCD) and found this to be an
accurate method for correct phosphorylation site localization with no gas-phase
rearrangements observed above noise level. Analyzing a large set of HCD spectra
of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism
that generates a phosphorylation site-specific neutral loss derived x-ion, which
directly pinpoints the phosphorylated residue. Together, these findings
significantly improve phosphorylation site localization confidence. Phosphoproteomics is a powerful analytical platform for identification and
quantification of phosphorylated peptides and assignment of phosphorylation
sites. Bioinformatics tools to identify phosphorylated peptides from their
tandem mass spectra and protein sequence databases are important part of
phosphoproteomics. In this work, we discuss general informatics aspects of
mass-spectrometry-based phosphoproteomics. Some of the specifics of
phosphopeptide identifications stem from the labile nature of phosphor groups
and expanded peptide search space. Allowing for modifications of Ser, Thr, and
Tyr residues exponentially increases effective database size. High mass
resolution and accuracy measurements of precursor mass-to-charge ratios help to
restrict the search space of candidate peptide sequences. The higher-order
fragmentations of neutral loss ions enhance the fragment ion mass spectra of
phosphorylated peptides. We show an example of a phosphopeptide identification
where accounting for fragmentation from neutral loss species improves the
identification scores in a database search algorithm by 50%. The localization of phosphorylation sites in peptide sequences is a challenging
problem in large-scale phosphoproteomics analysis. The intense neutral loss
peaks and the coexistence of multiple serine/threonine and/or tyrosine residues
are limiting factors for objectively scoring site patterns across thousands of
peptides. Various computational approaches for phosphorylation site localization
have been proposed, including Ascore, Mascot Delta score, and ProteinProspector,
yet few address direct estimation of the false localization rate (FLR) in each
experiment. Here we propose LuciPHOr, a modified target-decoy-based approach
that uses mass accuracy and peak intensities for site localization scoring and
FLR estimation. Accurate estimation of the FLR is a difficult task at the
individual-site level because the degree of uncertainty in localization varies
significantly across different peptides. LuciPHOr carries out simultaneous
localization on all candidate sites in each peptide and estimates the FLR based
on the target-decoy framework, where decoy phosphopeptides generated by placing
artificial phosphorylation(s) on non-candidate residues compete with the
non-decoy phosphopeptides. LuciPHOr also reports approximate site-level
confidence scores for all candidate sites as a means to localize additional
sites from multiphosphorylated peptides in which localization can be partially
achieved. Unlike the existing tools, LuciPHOr is compatible with any search
engine output processed through the Trans-Proteomic Pipeline. We evaluated the
performance of LuciPHOr in terms of the sensitivity and accuracy of FLR
estimates using two synthetic phosphopeptide libraries and a phosphoproteomic
dataset generated from complex mouse brain samples. |
Does ghrelin play a role in ischemic stroke? | Yes. It has been shown that serum ghrelin levels are reduced after ischemic stroke and ghrelin is associated with stroke type. Ghrelin can be a useful marker for the prediction of stoke after cardiopulmonary bypass. Ghrelin may be neuroprotective after injury in animal models of cerebral ischemia by inhibiting apoptotic processes, inflammation, nNOS activity and modulating gastrointestinal motility. | This study was performed to evaluate whether cytokines, adhesion molecules,
ghrelin and S-100B are useful markers in predicting the cerebral infarction
after cardiac surgery with cardioplumomary bypass (CPB). The patients (n=20)
were classified into two groups; group A (n=4) showed postoperative organized
cerebral damage, while group B (n=16) consisted of patients without occurrence
of postoperative strokes. Before CPB, serum levels of S-100B in both groups A
and B were low (<0.5 ng/mL), while ghrelin concentrations in group A (all
patients had history of strokes) were much higher than those in group B. After
CPB, when serum levels of S-100B in group A at 24h were higher than those in
group B, ghrelin in group A at same time point showed high levels in comparison
to group B. At 12 and 24h after CPB, levels of tumor necrosis factor
(TNF)-alpha, interleukin-10 and soluble TNF-receptor I in group A were
significantly higher than those in group B. In conclusion, it is considered that
ghrelin as well as S-100B can be a useful marker for the prediction of stoke
after CPB. Increase of TNF-alpha, interleukin-10 and soluble TNF-receptor I
after CPB may be involved in the pathogenesis of stroke after CPB. Ghrelin is known to promote neuronal defense and survival against ischemic
injury by inhibiting apoptotic processes. In the present study, we investigated
the role of prostate apoptosis response-4 (Par-4), a proapoptotic gene the
expression of which is increased after ischemic injury, in ghrelin-mediated
neuroprotection during middle cerebral artery occlusion (MCAO). Both ghrelin and
des-acyl ghrelin protected cortical neurons from ischemic injury. Ghrelin
receptor specific antagonist abolished the protective effects of ghrelin,
whereas those of des-acyl ghrelin were preserved, suggesting the involvement of
a receptor that is distinct from GHS-R1a. The expression of Par-4 was increased
by MCAO, which was attenuated by ghrelin and des-acyl ghrelin treatments. Both
ghrelin and des-acyl ghrelin increased the Bcl-2/Bax ratio, prevented cytochrome
c release, and inhibited caspase-3 activation. Our data indicate that des-acyl
ghrelin, as well as ghrelin, protect cortical neurons against ischemic injury
through the inhibition of Par-4 expression and apoptotic molecules in
mitochondrial pathway. The pathogenesis of stroke involves inflammation, apoptosis, and excitotoxicity,
which is mediated in part by neuronal NO synthase (nNOS) activation. Ghrelin, an
endogenous 28-amino acid peptide, is shown to exert antiapoptotic and
anti-inflammatory properties. However, the effect of ghrelin in permanent focal
cerebral ischemia and the role of the vagus nerve in its action remain unknown.
To study this, male adult Sprague-Dawley rats underwent right-sided permanent
middle cerebral artery occlusion (MCAO) with or without prior bilateral truncal
vagotomy. This was followed by infusion of 4 nmol human ghrelin as treatment or
saline as vehicle. Neurological deficit was assessed at 24 h after MCAO. Rats
were killed thereafter, and brains were rapidly removed and analyzed for infarct
size, markers of inflammation, excitotoxicity, and apoptosis. Compared with
vehicle treatment, human ghrelin treatment in vagus nerve-intact rats after MCAO
showed marked reduction in neurological deficit by 57% and infarct size by 25%.
Middle cerebral artery occlusion resulted in increases in cerebral TNF-α, IL-6,
neutrophil trafficking, matrix metalloproteinase 9 and nNOS gene expression,
nitrotyrosine, and apoptosis. Human ghrelin treatment in vagus nerve-intact rats
significantly decreased the above measurements. Human ghrelin treatment also
improved 7-day survival and significantly decreased neurological deficit over
the entire 7 days after MCAO in vagus nerve-intact rats compared with vehicle.
Prior vagotomy, however, blunted human ghrelin's neuroprotective effects on
neurological deficit, infarct size, TNF-α, neutrophil trafficking,
nitrotyrosine, and apoptosis. Human ghrelin is thus a neuroprotective agent that
inhibits inflammation, nNOS activity, and apoptosis in focal cerebral ischemia
through a vagal pathway. OBJECTIVES: Fat tissue is an important endocrine organ that produces a number of
hormones and cytokines (leptin, adiponectin, resistin, plasminogen activator
inhibitor-1, Tumour necrosis factor TNF α) with essential roles in regulation of
many physiological functions.
METHODS: We targeted implications of adipokines in ischemic stroke patients.
Patients with acute stroke were examined (n=145) and the results were compared
with the control group (n=68). We have examined potential associations between
leptin, adiponectin and ghrelin, and different types of stroke and traditional
risk factors.
RESULTS: Significantly higher levels of leptin and lower levels of adiponectin
and ghrelin were confirmed in the stroke group. The level of leptin in women
with stroke was three-times higher than in men, and the leptin levels positively
correlated with obesity in both sexes. Ghrelin levels correlated mildly with
triglyceride levels, and were domit in men with cardioembolic stroke.
Adiponectin levels were not different between men and women with acute stroke,
and correlated with atherothrombotic and lacunar stroke types in men.
CONCLUSIONS: Adipokines and ghrelin play an important role in ischemic stroke,
but their function in stroke subtypes seems to be different and sex influenced.
More research is required to confirm our results. Ischemic stroke occurs as a result of an obstruction within a blood vessel
supplying blood to the brain. Gastrointestinal mucosal damage not only induces
local and systemic inflammatory reactions but may also result in multiple organ
dysfunction syndrome. We studied whether the changes in serum ghrelin and small
intestinal motility occur in cerebral ischemia. The focal cerebral ischemia rat
models were produced by the middle cerebral artery occlusion (MCAO) method. The
MCAO group was further equally divided into five subgroups at 3, 6, 12, 24, and
48 hr, and the sham operated rats were used as controls. Serum ghrelin level was
analyzed using enzyme-linked immunosorbent assay, and small intestinal motility
was measured by methylene blue staining. The ileum tissue was examined by light
and electron microscopy. The neurologic scores were 0 for all the rats in the
control group and 2-3 for those in the MCAO group, suggesting that rat models
were established successfully. The serum ghrelin level was higher in the MCAO
group when compared with the control group (P < 0.05). However, the impelling
force in MCAO rats was significantly lower than that of the control group (P <
0.05), reaching the lowest level at 24 hr. Damage to the intestinal mucosa,
including villus intestinalis, vacuolar degeneration of organelles, widened
cell-cell junctions, and apoptotic cells could be found under the light and
electron microscopy. Our results showed that higher level of serum ghrelin
decreased gastrointestinal motility and damage to the intestinal mucosa existed
in rats with MCAO. Ghrelin, a gastrointestinal peptide with a major role in regulating feeding and
metabolism, has recently been investigated for its neuroprotective effects. In
this review we discuss pre-clinical evidence suggesting ghrelin may be a useful
therapeutic in protecting the brain against injury after ischemic stroke.
Specifically, we will discuss evidence showing ghrelin administration can
improve neuronal cell survival in animal models of focal cerebral ischemia, as
well as rescue memory deficits. We will also discuss its proposed mechanisms of
action, including anti-apoptotic and anti-inflammatory effects, and suggest
ghrelin treatment may be a useful intervention after stroke in the clinic. |
What is the mechanism of drug-induced gingival overgrowth? | Drug-induced gingival overgrowth (GO) is a frequent and adverse side-effect associated principally with the administration of the immunosuppressive drug cyclosporin A (CsA) and also certain anti-epileptic and anti-hypertensive drugs. It is characterized by a marked increase in the thickness of the epithelial layer and the accumulation of excessive amounts of connective tissue. Keratinocyte growth factor (KGF), which is a potent epithelial cell mitogen that has been implicated in other hyperplastic conditions could be involved in the molecular pathology of GO. Also, since cathepsin-L deficiency was reported to be associated with thickening of the skin, impaired cathepsin-L activity may play a key role in the establishment of skin and gingival abnormalities seen in I-cell disease. In addition, reduced cathepsin-L activity may play an important role in inducing drug-induced gingival overgrowth. Furthermore, the enhanced proliferation of gingival fibroblasts observed in this disease could be caused at least partly by the effects of the drugs on the cell cycle and cyclin expression in these cells. The increase in cell growth that occurs in drug-induced gingival overgrowth may be mediated by over-expression of cyclin B1. | BACKGROUND: Drug-induced gingival overgrowth (GO) is a frequent and adverse
side-effect associated principally with the administration of the
immunosuppressive drug cyclosporin A (CsA) and also certain anti-epileptic and
anti-hypertensive drugs. It is characterized by a marked increase in the
thickness of the epithelial layer and the accumulation of excessive amounts of
connective tissue. Although the mechanism by which the drugs cause GO is not yet
understood, keratinocyte growth factor (KGF), which is a potent epithelial cell
mitogen, has been implicated in other hyperplastic conditions, including mammary
and prostatic hyperplasia, and could also be involved in the molecular pathology
of GO.
METHODS: Immunohistochemistry was used to examine the expression of KGF in
normal gingiva (NG) and GO tissue sections. The relative level of KGF mRNA in GO
tissue and cells was compared with that of NG tissue and fibroblast cells using
the semi-quantitative reverse transcribed-polymerase chain reaction (RT-PCR) and
DNA sequencing was carried out to confirm the identity of the PCR product.
RESULTS: KGF antigen and mRNA were readily detected in the GO tissue
immunohistochemically and by RT-PCR, respectively, but were not expressed in the
NG tissue. Moreover, KGF transcripts were found to be approximately 2 times
higher in the GO than in the NG fibroblasts in vitro, although the difference
was not statistically significant.
CONCLUSIONS: This study has shown, for the first time, that the level of KGF is
elevated in GO and suggests that KGF may have an important role in the enhanced
epithelial proliferation associated with GO. Drug-induced gingival overgrowth, the chronic side effect of calcium
antagonists, is frequently seen due to the increase in patients with
hypertension, although the etiology of the disease is largely unknown. I-cell
disease, which accompanies gingival overgrowth, is characterized by a deficiency
in UDP-N-acetyl-glucosamine and is classified as one of the lysosomal storage
diseases. Here, we hypothesized that a common mechanism may underlie the
etiology of gingival overgrowth seen in patients treated with calcium antagonist
and in patients with I-cell disease. A calcium antagonist, nifedipine,
specifically suppressed cathepsin-L activity and mRNA expression, but not that
of cathepsin-B in cultured gingival fibroblasts. The activity of cathepsin-L was
suppressed up to 50% at 24 hours after treatment of the cells with the reagent.
The selective suppression of cathepsin-L activity appeared not to be dependent
on Ca(2+), since treatment of the cells with thapsigargin suppressed both
cathepsin-B and -L activity. Mice deficient in the cathepsin-L gene manifested
enlarged gingivae. Histological observation of the gingivae demonstrated typical
features of acanthosis, a phenotype very similar to that of experimentally
induced gingival overgrowth. Since cathepsin-L deficiency was reported to be
associated with thickening of the skin, impaired cathepsin-L activity may play a
key role in the establishment of skin and gingival abnormalities seen in I-cell
disease. In addition, reduced cathepsin-L activity may play an important role in
inducing drug-induced gingival overgrowth. Gingival overgrowth is a frequent and adverse side-effect caused by certain
immunosuppressant, anti-convulsant and calcium channel-blocking drugs. Although
the precise mechanism is not yet known, the enhanced proliferation of gingival
fibroblasts observed in this disease could be caused at least partly by the
effects of the drugs on the cell cycle and cyclin expression in these cells. In
the present study, flow cytometry analysis of the effects of the
immunosuppressant drug cyclosporin A showed that it enhanced cell-cycle
progression of gingival fibroblasts in vitro and also up-regulated the
expression of cyclin B1. In addition, reverse transcriptase/polymerase chain
reaction analysis of gingival overgrowth tissues showed markedly elevated
transcription of the cyclin B1 gene. Thus, the increase in cell growth that
occurs in drug-induced gingival overgrowth may be mediated by over-expression of
cyclin B1. It is well-known that the anticonvulsant drug, phenytoin (PHT), induces gingival
overgrowth as a side effect. The mechanism of PHT-induced gingival overgrowth,
however, is not well understood. One reason for this is the lack of an adequate
animal model for the PHT-induced gingival overgrowth. The purpose of this study
was to establish a rat model of the drug-induced gingival overgrowth.
Fourteen-day-old rats were randomly divided into 3 groups (5 rats/group). The
control rats received only the vehicle. The rats in the experimental group were
injected with 50 mg/kg per day (group L) and 100 mg/kg per day (group H) of PHT.
They received a subcutaneous injection of vehicle or PHT twice a day for 42
days. A charge-coupled device (CCD) laser displacement sensor was used for
measurement of the severity of gingival overgrowth of the mandibles. There was
no significant difference in the growth of rats between the PHT-injected and the
control groups. The CCD laser displacement sensor can measure minute changes in
the gingival overgrowth in rats, and a significant extension of the buccal
gingiva was observed in groups L and H. Using the CCD sensor, it is possible to
quantify the change in the gingiva under precise control of the PHT dose. BACKGROUND: Drug-induced gingival overgrowth is a frequent adverse effect
associated principally with administration of the immunosuppressive drug
cyclosporin A and also certain antiepileptic and antihypertensive drugs. It is
characterized by a marked increase in the thickness of the epithelial layer and
accumulation of excessive amounts of connective tissue. The mechanism by which
the drugs cause gingival overgrowth is not yet understood. The purpose of this
study was to compare proliferative activity of normal human gingiva and in
cyclosporine A-induced gingival overgrowth.
METHODS: Gingival samples were collected from 12 generally healthy individuals
and 22 Cyclosporin A-medicated renal transplant recipients. Expression of
proliferating cell nuclear antigen was evaluated in formalin-fixed,
paraffin-embedded gingival samples using an immunoperoxidase technique and a
monoclonal antibody for this antigen.
RESULTS: There were differences between the Cyclosporin A group and control
group in regard to proliferating cell nuclear antigen and epithelial thickness.
In addition, the degree of stromal inflammation was higher in the Cyclosporin A
group when compared with the control group.
CONCLUSION: The results suggest that the increased epithelial thickness observed
in Cyclosporin A-induced gingival overgrowth is associated with increased
proliferative activity in keratinocytes. BACKGROUND: Gingival overgrowth is a serious side-effect that accompanies the
use of Cyclosporin A (CsA). Up to 97% of the transplant recipient children, who
were submitted to CsA therapy, have been reported to suffer from this
side-effect. Several conflicting theories have been proposed to explain the
fibroblast's function in CsA-induced gingival overgrowth. The aim of this study
is to assess the proliferation of gingival fibroblasts and levels of released
cytokines after being exposed to CsA, in both adults and pediatric groups, and
to make a comparison between the results of the two groups.
MATERIALS AND METHODS: The adult fibroblast samples were derived from four
healthy adults, aged 35 to 42 years and pediatric samples were obtained from
four healthy children, age between four and eleven years. Tissue samples were
plated in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine
serum (FBS), Streptomycin and Penicillin. The samples were cultured in 25 cm(2)
plates containing 5% CO2, and incubated at 37°C. The cells used for all the
experiments were at the fourth passage. The concentration of PGE2, IL-1β, IL-6,
IL-8, TNF-α, and TGF-β1 was determined by the enzyme-linked immunosorbent assay
(ELISA) and the proliferation rate was assessed by the MTT assay. Alpha error
levels were set as 0.05.
RESULTS: CsA stimulated significantly higher levels of IL-6, IL-8 and TGF-β1 in
adult gingival fibroblasts than it did in the control group; whereas, the
expression of IL-1β and PGE2 in the fibroblasts exposed to CsA was significantly
weaker (P < 0.05). The fibroblasts in the two groups did not reveal any
noticeable difference in the production of TNF-α. Furthermore, cell
proliferation in the CsA group was not significantly higher than that in the
control group. No significant differences in cytokines TNF-α and IL-1β were
noted between the two groups. The results indicated that CsA stimulated cell
proliferation in the pediatric fibroblast cell line. Comparison between the
results in the adult and pediatric groups demonstrated that the levels of IL-1β,
IL-6, IL-8, and PGE2 were significantly higher in the pediatric group than in
the adult group; however, statistics showed no significant difference in the
levels of TNF-α and TGF-β1 and CsA-induced proliferation between these two
groups.
CONCLUSIONS: The mechanism of a CsA-induced fibroblast overgrowth may converge
on the steps involving fibroblast proliferation and cytokine network including
IL-6, IL-8, IL-1β, TGF-β1, and PGE2, in both adults and pediatrics. As the
prevalence and intensity of drug-induced gingival overgrowth is more serious in
the pediatrics. As group than in adults, we suggest that more studies be
conducted on the pediatric group. |
Is LPS a microbial product? | Yes, the lipopolysaccharide (LPS) is a component of the bacterial cell wall. | In this review, we summarize our investigations concerning the differential
importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid
differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form
lipopolysaccharide (LPS) chemotypes and include the results obtained in studies
with murine and human TLR4-transgenic mice. Furthermore, we present more recent
data on the mechanisms involved in the induction of LPS hypersensitivity by
bacterial and viral infections and on the reactivity of the hypersensitive host
to non-LPS microbial ligands and endogenous mediators. Finally, the effects of
pre-existing hypersensitivity on the course and outcome of a super-infection
with Salmonella typhimurium or Listeria monocytogenes are summarized. Acute lung injury (ALI) and the more severe acute respiratory distress syndrome
are common responses to a variety of infectious and noninfectious insults. We
used a mouse model of ALI induced by intratracheal administration of sterile
bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate
lung microbiota and study microbial community reaction to lung inflammation and
barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice
received LPS via intratracheal injection (n = 6), and another received sterile
water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after
treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag
(V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was
increased fivefold (P = 0.03). The community complexity remained unchanged
(Simpson index, P = 0.7); the Shannon diversity index indicated the increase of
community evenness in response to ALI (P = 0.07). Principal coordinate analysis
and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant
difference between microbiota of control and ALI groups. Bacteria from families
Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as
determined by Metastats test (P < 0.02). In concordance with the 16s-tag data,
Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi
(Brucellaceae) were isolated from lungs of mice from both groups. Metabolic
profiling of BAL detected the presence of bacterial substrates suitable for both
isolates. Additionally, microbiota from LPS-treated mice intensified
IL-6-induced lung inflammation in naive mice. We conclude that the morbid
transformation of ALI microbiota was attributed to the set of inborn
opportunistic pathogens thriving in the environment of inflamed lung, rather
than the external infectious agents. Although the disciplines of bacteriology and virology frequently come together
in the setting of a diagnostic medical microbiology laboratory, the two
scientific fields are usually miles apart. The microbiologists basically form
two non-overlapping groups of scientists, the bacteriologists and virologists,
which go to separate meetings and do not easily intermingle. Some recent
research findings about elegant virus-bacterium interactions may change this
situation. Obviously, interactions between these two microbes can occur only
when they colocalize, which most likely occurs in the gut/intestines where
10(14) commensal bacteria reside (the microbiota). We review findings on the
following enteric microbial tandems: norovirus - Enterobacter cloacae, mouse
mammary tumor virus (MMTV) - bacterial lipopolysaccharide (LPS), poliovirus and
reovirus - intestinal bacteria. The close bacterium-virus interplay may also
present options to develop unique therapeutic strategies for those infected, and
to prevent further virus spread, and thus minimize the risk for the community. |
Which cyclin- dependent kinase inhibitor is regulated by Bmi-1? | p16INK4 (also known as CDKN2A) | Genes of the polycomb group function by silencing homeotic selector genes that
regulate embryogenesis. In mice, downregulation of one of the polycomb genes,
bmi-1, leads to neurological alterations and severe proliferative defects in
lymphoid cells, whilst bmi-1 overexpression, together with upregulation of
myc-1, induces lymphoma. An oncogenic function has been further supported in
primary fibroblast studies where bmi-1 overexpression induces immortalization
due to repression of p16/p19ARF, and where together with H-ras, it readily
transforms MEFs. It was the aim of this study to assess the expression of bmi-1
in resectable non-small cell lung cancer (NSCLC) in association with p16 and
p14ARF (=human p19ARF). Tumours (48 resectable NSCLC (32 squamous, 9 adeno-, 2
large cell, 4 undifferentiated carcinomas and 1 carcinoid); stage I, 29, II, 7,
III, 12; T1, 18, T2, 30; differentiation: G1 12, G2 19, G3 17) were studied by
immunohistochemistry for protein expression and by comparative multiplex PCR for
gene amplification analysis. In tumour-free, normal lung tissue from patients,
weak - moderate bmi-1 staining was seen in some epithelial cells, lymphocytes,
glandular cells and in fibroblasts, whereas blood, endothelial, chondrocytes,
muscle cells and adipocytes did not exhibit any bmi-1 expression. In tumours,
maligt cells were negative/weakly, moderately and strongly positive in 20, 22
and 6 cases, respectively. As assessed by multiplex PCR, bmi-1 gene
amplification was not the reason for high-level bmi-1 expression. Tumours with
moderate or strong bmi-1 expression were more likely to have low levels of p16
and p14ARF (P = 0.02). Similarly, tumours negative for both, p16 and p14ARF,
exhibit moderate-strong bmi-1 staining. 58% of resectable NSCLC exhibit
moderate-high levels of bmi-1 protein. The inverse correlation of bmi-1 and the
INK4 locus proteins expression (p16/p14ARF) supports a possible role for bmi-1
misregulation in lung carcinogenesis. The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor
suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when
WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and
extends replicative life span when overexpressed. Life span extension by Bmi-1
required the pRb, but not p53, tumor suppressor protein. Deletion analysis
showed that the RING finger and helix-turn-helix domains of Bmi-1 were required
for life span extension and suppression of p16. Furthermore, a RING finger
deletion mutant exhibited domit negative activity, inducing p16 and premature
senescence. Interestingly, presenescent cultures of some, but not all, human
fibroblasts contained growth-arrested cells expressing high levels of p16 and
apparently arrested by a p53- and telomere-independent mechanism. Bmi-1
selectively extended the life span of these cultures. Low O(2) concentrations
had no effect on p16 levels or life span extension by Bmi-1 but reduced
expression of the p53 target, p21. We propose that some human fibroblast strains
are more sensitive to stress-induced senescence and have both p16-dependent and
p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1
extends the replicative life span of human fibroblasts by suppressing the
p16-dependent senescence pathway. A central issue in stem cell biology is to understand the mechanisms that
regulate the self-renewal of haematopoietic stem cells (HSCs), which are
required for haematopoiesis to persist for the lifetime of the animal. We found
that adult and fetal mouse and adult human HSCs express the proto-oncogene
Bmi-1. The number of HSCs in the fetal liver of Bmi-1-/- mice was normal. In
postnatal Bmi-1-/- mice, the number of HSCs was markedly reduced. Transplanted
fetal liver and bone marrow cells obtained from Bmi-1-/- mice were able to
contribute only transiently to haematopoiesis. There was no detectable
self-renewal of adult HSCs, indicating a cell autonomous defect in Bmi-1-/-
mice. A gene expression analysis revealed that the expression of stem cell
associated genes, cell survival genes, transcription factors, and genes
modulating proliferation including p16Ink4a and p19Arf was altered in bone
marrow cells of the Bmi-1-/- mice. Expression of p16Ink4a and p19Arf in normal
HSCs resulted in proliferative arrest and p53-dependent cell death,
respectively. Our results indicate that Bmi-1 is essential for the generation of
self-renewing adult HSCs. Stem cells persist throughout life by self-renewing in numerous tissues
including the central and peripheral nervous systems. This raises the issue of
whether there is a conserved mechanism to effect self-renewing divisions.
Deficiency in the polycomb family transcriptional repressor Bmi-1 leads to
progressive postnatal growth retardation and neurological defects. Here we show
that Bmi-1 is required for the self-renewal of stem cells in the peripheral and
central nervous systems but not for their survival or differentiation. The
reduced self-renewal of Bmi-1-deficient neural stem cells leads to their
postnatal depletion. In the absence of Bmi-1, the cyclin-dependent kinase
inhibitor gene p16Ink4a is upregulated in neural stem cells, reducing the rate
of proliferation. p16Ink4a deficiency partially reverses the self-renewal defect
in Bmi-1-/- neural stem cells. This conserved requirement for Bmi-1 to promote
self-renewal and to repress p16Ink4a expression suggests that a common mechanism
regulates the self-renewal and postnatal persistence of diverse types of stem
cell. Restricted neural progenitors from the gut and forebrain proliferate
normally in the absence of Bmi-1. Thus, Bmi-1 dependence distinguishes stem cell
self-renewal from restricted progenitor proliferation in these tissues. To clarify the roles of Bmi-1 in colorectal carcinoma, we examined the
expression of Bmi-1 in 41 samples out of 46 colorectal carcinomas by reverse
transcription-PCR, whereas all 46 were analyzed by immunostaining. In addition,
we analyzed the expression patterns of Bmi-1 in association with p16INK4a and
p14ARF (in mouse p19ARF) in a series of colorectal carcinomas. The level of
Bmi-1 mRNA in the carcinoma tissues was significantly higher than those of the
adjacent non-neoplastic colonic mucosal tissues. Immunohistochemistry for Bmi-1
showed moderate or strong expression levels in 65% (30/46) of colorectal
carcinomas. Colorectal carcinomas with moderate or strong Bmi-1 expression were
more likely to have low levels of the INK4 locus proteins (p16INK4a/p14ARF)
(P<0.07). These results suggested that modulation of Bmi-1 protein might be
involved in human colorectal carcinogenesis by repressing the INK4a/ARF
proteins. Human Polycomb-group (PcG) genes play a crucial role in the regulation of
embryonic development and regulation of the cell cycle and hematopoiesis. PcG
genes encode proteins that form two distinct PcG complexes, involved in
maintece of cell identity and gene silencing patterns. We recently showed
that expression of the BMI-1 and EZH2 PcG genes is separated during normal
B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS)
cells co-express BMI-1 and EZH2. In the current study, we used
immunohistochemistry and immunofluorescence to determine whether the binding
partners of these PcG proteins are also present in H/RS cells and H/RS-derived
cell lines. PcG expression profiles were analyzed in combination with expression
of the cell cycle inhibitor p16INK4a, because experimental model systems
indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and
HL-derived cell lines co-express all core proteins of the two known PcG
complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1,
and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in
normal mature B cells and other maligt lymphomas of B-cell origin, suggesting
that the PcG expression profile of H/RS is unique. In contrast to Bmi-1
transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL
revealed strong nuclear expression of p16INK4a. We propose that abnormal
expression of BMI-1 and its binding partners in H/RS cells contributes to
development of HL. However, abnormal expression of BMI-1 in HL is not
necessarily associated with down-regulation of p16INK4a. It is generally assumed that squamous cell carcinoma develops in a stepwise
manner from normal bronchial epithelium towards cancer by the accumulation of
(epi)genetic alterations. Several mechanisms including mutations and homozygous
deletions or hypermethylation of the p16(INK4a) promoter region can cause loss
of p16 expression. Recent studies suggest overexpression of the polycomb-group
gene BMI-1 might also down-regulate p16 expression. In this study, we analyzed
the p16 expression in relation to the methylation status of the p16 promoter
region of the p16(INK4a) gene and the expression of BMI-1 in bronchial squamous
cell carcinomas (SCC) and its premaligt lesions. Nine (69%) SCC showed loss
of p16 expression and 10 (77%) showed expression of BMI-1. Of four p16 positive
samples two (50%) were BMI-1 positive, whereas among nine p16 negative samples,
eight (89%) revealed BMI-1 staining. Four (44%) p16 negative samples were
hypermethylated at the p16(INK4a) promoter region; the other p16 negative tumors
that showed no hypermethylation revealed BMI-1 staining. Only two premaligt
lesions showed absence of p16 expression, of which one (carcinoma in situ) was
hypermethylated at the p16(INK4a) promoter region and the other (severe
dysplasia) showed BMI-1 expression. In total, 11 precursor lesions (48%)
revealed BMI-1 expression. In conclusion, the results of this study suggest that
loss of p16 expression by promoter hypermethylation is inconsistently and occurs
late in the carcinogenic process at the level of severe dysplasia. To what
extent overexpression of the polycomb-group protein BMI-1 attributes to down
regulating of p16 expression remains unclear. Mantle-cell lymphoma (MCL) is a well-defined subtype of B-cell non-Hodgkin's
lymphomas (B-NHL), accounts for approximately 6% of all lymphoid neoplasms, and
has a median survival of 3 to 4 years. The genetic hallmark of MCL is the
chromosomal translocation t(11;14)(q13;q32) that leads to deregulation and
upregulation of Cyclin D1, an important regulator of the G1 phase of the cell
cycle. This genetic event is present in virtually all cases of MCL, whereas
additional genetic alterations that occur in subsets of MCL have been described.
Most of these alterations appear to disturb the cell cycle machinery/interfere
with the cellular response to DNA damage, thus making MCL a paradigm for cell
cycle and DNA damage response dysregulation in cancer in general. In particular,
Cyclin D1 upregulation, genomic amplification of the cyclin-dependent kinase
(CDK) -4, deletions of the CDK inhibitor p16(INK4a) and overexpression of BMI-1,
a transcriptional repressor of the p16(INK4a) locus, are associated with
dysregulation of the cell cycle machinery in MCL. The DNA damage response
pathway is affected by frequent alterations of the ataxia-telangiectasia mutated
(ATM) kinase as well as occasional inactivation of checkpoint kinase (CHK)-1 and
CHK2 that are kinases that act downstream of ATM in response to detection of DNA
damage. Moreover, p53 is frequently targeted by alterations in MCL. A recent
gene expression profiling study defined the proliferation signature, a
quantitative measure of gene expression of proliferation-associated genes as the
strongest survival predictor available to date allowing the definition of
prognostic MCL subgroups that differ in median survival by more than 5 years. BACKGROUND: Bmi-1 gene determines the proliferative capacity of normal and
leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid
leukemia cells in both humans and mice. This study aimed at assessing the effect
of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16)
expression.
METHODS: A transcriptional repressor, Bmi-1 cDNA was cloned by reverse
transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A
plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design
of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium
after the plasmid was successfully introduced in K562 cells by
lipofectin-mediated DNA transfection. The effects of the antisense expression on
the proliferation of K562 cells were analyzed by using microculture tetrazolium
and colony forming. Cell cycle was analyzed by using flow cytometry. The p16
expression of K562 cells was observed by immunofluorescence histochemical stain.
RESULTS: K562 cells transfected with antisense Bmi-1 plasmid grew significantly
slower than that of controls (the parental K562 and cells transfected with empty
plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected
cells decreased significantly (P < 0.01) compared with controls. The p16
expression of cells transfected with antisense Bmi-1 was upgraded more
apparently than that of controls.
CONCLUSION: The antisense Bmi-1 gene can inhibit the growth of K562 cell and
upgrade expression of p16 in K562 cells. Increased mitogenic signaling by positive effectors such as Ras or Myc can
trigger senescence in normal cells, a response believed to function as a
tumor-suppressive mechanism. We report here the existence of a checkpoint that
monitors hypoproliferative signaling imbalances. Normal human fibroblasts with
one copy of the c-myc gene inactivated by targeted homologous recombination
switched with an increased frequency to a telomere-independent senescent state
mediated by the cyclin-dependent kinase inhibitor p16(INK4a). p16(INK4a)
expression was regulated by the Polycomb group repressor Bmi-1, which we show is
a direct transcriptional target of c-Myc. The Myc-Bmi circuit provides a
mechanism for the conversion of environmental inputs that converge on c-Myc into
discrete cell-fate decisions coupled to cell-cycle recruitment. A mechanism for
limiting the proliferation of damaged or otherwise physiologically compromised
cells would be expected to have important consequences on the generation of
replicatively senescent cells during organismal aging. The Bmi-1 oncoprotein regulates proliferation and oncogenesis in human cells.
Its overexpression leads to senescence bypass in human fibroblasts and
immortalization of human mammary epithelial cells. In this study, we report that
compared with normal nasopharyngeal epithelial cells (NPEC), Bmi-1 is
overexpressed in nasopharyngeal carcinoma cell lines. Importantly, Bmi-1 was
also found to be overexpressed in 29 of 75 nasopharyngeal carcinoma tumors
(38.7%) by immunohistochemical analysis. In contrast to nasopharyngeal
carcinoma, there was no detectable expression of Bmi-1 in noncancerous
nasopharyngeal epithelium. Moreover, high Bmi-1 expression positively correlated
with poor prognosis of nasopharyngeal carcinoma patients. We also report that
the overexpression of Bmi-1 leads to bypass of senescence and immortalization of
NPECs, which normally express p16(INK4a) and exhibit finite replicative life
span. Overexpression of Bmi-1 in NPECs led to the induction of human telomerase
reverse transcriptase activity and reduction of p16(INK4a) expression.
Mutational analysis of Bmi-1 showed that both RING finger and helix-turn-helix
domains of it are required for immortalization of NPECs. Our findings suggest
that Bmi-1 plays an important role in the development and progression of
nasopharyngeal carcinoma, and that Bmi-1 is a valuable marker for assessing the
prognosis of nasopharyngeal carcinoma patients. Furthermore, this study provides
the first cellular proto-oncogene immortalized nasopharyngeal epithelial cell
line, which may serve as a cell model system for studying the mechanisms
involved in the tumorigenesis of nasopharyngeal carcinoma. In response to hyperproliferative signaling elicited by transforming oncogenes
some normal human cells can enter replicative senescence as a tumor defense
mechanism. We recently found that human fibroblasts or endothelial cells with
genetically-engineered reduction of proto-oncogene c-Myc expression switched
with an increased frequency to a senescent state by a telomere-independent
mechanism involving the polycomb group repressor Bmi-1 and the cyclin-dependent
kinase inhibitor p16(INK4a). The same regulatory circuit was triggered upon
exposure to mild oxidative stress. These findings point to the existence of a
mechanism for monitoring hypoproliferative signaling, whose function may be to
limit the proliferation and accretion of physiologically compromised cells. This
mechanism may be another example of antagonistic pleiotropy leading to
organismal aging. Activation of telomerase is sufficient for immortalization of some types of
human cells but additional factors may also be essential. It has been proposed
that stress imposed by inadequate culture conditions induces senescence due to
accumulation of p16(INK4a). Here, we present evidence that many human cell types
undergo senescence by activation of the p16(INK4a)/Rb pathway, and that
introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span
of human epithelial cells derived from skin, mammary gland and lung.
Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1,
suppressed p16(INK4a) expression in human mammary epithelial cells without
promoter methylation, and extended their life span. Subsequent introduction of
hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels
resulted in efficient immortalization of three cell types without crisis or
growth arrest. The majority of the human mammary epithelial cells thus
immortalized showed almost normal ploidy as judged by G-banding and spectral
karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and
introduction of hTERT can immortalize many human cell types with little
chromosomal instability. Stem cell-like cells have recently been identified in melanoma cell lines, but
their relevance for melanoma pathogenesis is controversial. To characterize the
stem cell signature of melanoma, expression of stem cell markers BMI-1 and
nestin was studied in 64 cutaneous melanomas, 165 melanoma metastases as well as
53 melanoma cell lines. Stem cell renewal factor BMI-1 is a transcriptional
repressor of the Ink4a/Arf locus encoding p16(ink4a) and p14(Arf). Increased
nuclear BMI-1 expression was detectable in 41 of 64 (64%) primary melanomas, 117
of 165 melanoma metastases (71%) and 15 of 53 (28%) melanoma cell lines. High
nestin expression was observed in 14 of 56 primary melanomas (25%), 84 of 165
melanoma metastases (50%) and 21 of 53 melanoma cell lines (40%). There was a
significant correlation between BMI-1 and nestin expression in cell lines (p =
0.001) and metastases (p = 0.02). These data indicate that cells in primary
melanomas and their metastases may have stem cell properties. Cell lines
obtained from melanoma metastases showed a significant higher BMI-1 expression
compared to cell lines from primary melanoma (p = 0.001). Further, primary
melanoma lacking lymphatic metastases at presentation (pN0, n = 40) was less
frequently BMI-1 positive than melanomas presenting with lymphatic metastases
(pN1; n = 24; 52% versus 83%; p = 0.01). Therefore, BMI-1 expression appears to
induce a metastatic tendency. Because BMI-1 functions as a transcriptional
repressor of the Ink4a/Arf locus, p16(ink4a) and p14(Arf) expression was also
analyzed. A high BMI-1/low p16(ink4a) expression pattern was a significant
predictor of metastasis by means of logistic regression analysis (p = 0.005).
This suggests that BMI-1 mediated repression of p16(ink4a) may contribute to an
increased aggressive behavior of stem cell-like melanoma cells. Self-renewing divisions of normal and cancerous stem cells are responsible for
the initiation and maintece of normal and certain cancerous tissues,
respectively. Recent findings suggest that tumor surveillance mechanisms can
reduce regenerative capacity and frequency of normal stem cells, thereby
contributing to tissue aging. Signaling pathways promoting self-renewal of stem
cells can also drive proliferation in cancer. The BMI-1 proto-oncogene is
required for the maintece of tissue-specific stem cells and is involved in
carcinogenesis within the same tissues. BMI-1 promotes self-renewal of stem
cells largely by interfering with two central cellular tumor suppressor
pathways, p16(Ink4a)/retinoblastoma protein (Rb) and ARF/p53, whose disruption
is a hallmark of cancer. Nucleolin, an Rb-associated protein, is abundant in
proliferating cancerous cells and likely contributes to the maintece of human
CD34-positive stem/progenitor cells of hematopoiesis. Elucidation of the
involvement of proto-oncogenes and tumor suppressors in the maintece of stem
cells might have therapeutic implications. Androgenetic alopecia (AGA), a hereditary disorder that involves the progressive
thinning of hair in a defined pattern, is driven by androgens. The hair follicle
dermal papilla (DP) expresses androgen receptors (AR) and plays an important
role in the control of normal hair growth. In AGA, it has been proposed that the
inhibitory actions of androgens are mediated via the DP although the molecular
nature of these interactions is poorly understood. To investigate mechanisms of
AGA, we cultured DP cells (DPC) from balding and non-balding scalp and confirmed
previous reports that balding DPC grow slower in vitro than non-balding DPC.
Loss of proliferative capacity of balding DPC was associated with changes in
cell morphology, expression of senescence-associated beta-galactosidase, as well
as decreased expression of proliferating cell nuclear antigen and Bmi-1;
upregulation of p16(INK4a)/pRb and nuclear expression of markers of oxidative
stress and DNA damage including heat shock protein-27, super oxide dismutase
catalase, ataxia-telangiectasia-mutated kinase (ATM), and ATM- and Rad3-related
protein. Premature senescence of balding DPC in vitro in association with
expression of p16(INK4a)/pRB suggests that balding DPC are sensitive to
environmental stress and identifies alternative pathways that could lead to
novel therapeutic strategies for treatment of AGA. Thymocytes undergo massive proliferation before T cell receptor (TCR) gene
rearrangement, ensuring the diversification of the TCR repertoire. Because
activated cells are more susceptible to damage, cell-death restraint as well as
promotion of cell-cycle progression is considered important for adequate cell
growth. Although the molecular mechanism of pre-TCR-induced proliferation has
been examined, the mechanisms of protection against cell death during the
proliferation phase remain unknown. Here we show that the survival of activated
pre-T cells induced by pre-TCR signaling required the Polycomb group (PcG) gene
product Bmi-1-mediated repression of Cdkn2A, and that p19Arf expression resulted
in thymocyte cell death and inhibited the transition from CD4(-)CD8(-) (DN) to
CD4(+)CD8(+) (DP) stage upstream of the transcriptional factor p53 pathway. The
expression of Cdkn2A (the gene encoding p19Arf) in immature thymocytes was
directly regulated by PcG complex containing Bmi-1 and M33 through the
maintece of local trimethylated histone H3K27. Our results indicate that this
epigenetic regulation critically contributes to the survival of the activated
pre-T cells, thereby supporting their proliferation during the DN-DP transition. In this issue of Cell Stem Cell, Fasano et al. (2007) investigate the role of
Bmi-1 in neural stem cells in vitro and in vivo. They find a progressively
increasing requirement for Bmi-1 function in stem cells during development that,
surprisingly, seems to involve regulation of the cell-cycle inhibitor p21. Knockout studies have shown that the polycomb gene Bmi-1 is important for
postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have
identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using
lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is
also important for NSC self-renewal in the embryo. We found that neural
progenitors depend increasingly on Bmi-1 for proliferation as development
proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1
reduction causes defects in embryonic and adult NSC proliferation and
self-renewal that, unexpectedly, are mediated by a different cell-cycle
inhibitor, p21. Gene array analyses revealed developmental differences in
Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory
pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs,
potentially with stage-related differences. Understanding stage-related
mechanisms underlying NSC self-renewal has important implications for
development of stem cell-based therapies. The gene that encodes the ATM protein kinase is mutated in ataxia-telangiectasia
(A-T). One of the prominent features of A-T is progressive neurodegeneration. We
have previously reported that primary astrocytes isolated from Atm(-/-) mice
grow slowly and die earlier than control cells in culture. However, the
mechanisms for this remain unclear. We show here that intrinsic elevated
intracellular levels of reactive oxygen species (ROS) are associated with the
senescence-like growth defect of Atm(-/-) astrocytes. This condition is
accompanied by constitutively higher levels of ERK1/2 phosphorylation and
p16(Ink4a) in Atm(-/-) astrocytes. We also observe that ROS-induced
up-regulation of p16(Ink4a) occurs correlatively with ERK1/2-dependent
down-regulation and subsequent dissociation from chromatin of Bmi-1.
Furthermore, both mitogen-activated protein kinase (MAPK)/ERK inhibitor PD98059
and antioxidant N-acetyl-l-cysteine restored normal proliferation of Atm(-/-)
astrocytes. These results suggest that ATM is required for normal astrocyte
growth through its ability to stabilize intracellular redox status and that the
inability to control ROS is the molecular basis of limited cell growth of
Atm(-/-) astrocytes. This defect may be mediated by a mechanism involving ERK1/2
activation and Bmi-1 derepression of p16(Ink4a). These data identify new
potential targets for therapeutic intervention in A-T neurodegeneration. BACKGROUND AND OBJECTIVE: Previous studies have shown that Bmi-1 is
overexpressed in a variety of tumors, suggesting that Bmi-1 plays an important
role in tumorigenesis. In this study, we investigated the effect of Bim-1 siRNA
on cell proliferation, cell cycle, cell apoptosis and migration of human
esophageal carcinoma EC9706 cells, and explored its potential mechanisms.
METHODS: Bmi-1 small interfering RNA (siRNA) was transferred into EC9706 cells.
Then, cell proliferation was measured using cell counting kit-8 (CCK-8), cell
cycle and cell apoptosis were analyzed by flow cytometry, cell migration ability
was detected using Boyden chamber assay, and the mRNA and protein expression
levels of Bmi-1, p16, Bcl-2, Bax, and MMP-2 were determined using real-time
polymerase chain reaction (PCR) and Western blot analysis, respectively.
RESULTS: Bmi-1 siRNA treatment significantly inhibited the expression of Bmi-1
at both mRNA and protein levels in EC9706 cells. Cell proliferation rate
decreased dramatically in the Bmi-1 siRNA treated group than in the untreated
group and in the scrambled siRNA treated group (both P < 0.001). In Bmi-1
treated group, the percentage of cells at G(0)/G(1) stage was 71.93%, which was
higher than that in the untreated group (47.36%) or scramble siRNA treated group
(48.47%) (both P < 0.001). Early cell apoptosis rate also increased
significantly in the Bmi-1 siRNA treated group (both 17.32%) than in the
untreated group (2.61%) and in the scramble siRNA treated group (2.73%) (both P
< 0.001). Further experiment suggested that downregulation of Bmi-1 led to less
cell migration. In EC9706 cells transfected by Bmi-1 siRNA, the expression
levels of p16 and Bax increased, while the expression level of Bcl-2 decreased.
CONCLUSIONS: Bmi-1 downregulation in esophageal carcinoma cells inhibits cell
proliferation, cell cycle, and cell migration, while increases cell apoptosis.
These results suggest that Bmi-1 is a potential molecular target of treating
esophageal cancer. The human oncogene B-cell-specific Moloney murine leukemia virus integration
site 1 (BMI-1) is a member of the mammalian Polycomb group family. The
overexpression of BMI-1 is associated with human maligcies. In this study,
the effects of knockdown of BMI-1 by shRNA-mediated RNA interference on cell
cycle and possible downstream targets in human cervical adenocarcinoma HeLa
cells were investigated. As a result, when the shRNA plasmid was stably
introduced into the cell line, the mRNA and protein of BMI-1 were specifically
down-regulated, and the cells increased in the phase of G1 and cells in S phase
significantly decreased by flow cytometric analysis; the knockdown of BMI-1
expression could lead to significant up-regulation of p16INK4a, HOXA9 and HOXC13
mRNA expression, but hTERT and HOXB4 mRNA expression did not change
significantly. In conclusion, RNAi-mediated knockdown of BMI-1 expression can
induce cell-cycle arrest and up-regulate p16INK4a, HOXA9 and HOXC13 in HeLa
cells. Our results suggest that targeting BMI-1 might be a therapeutic potential
for the treatment of cancer. PURPOSE: Because evasion of tumor suppression is a critical step in cancer
development, cancer cells have developed a variety of mechanisms to circumvent
the influence of tumor suppressive pathways. Thus, genes that negatively
regulate tumor suppressors could be considered novel types of oncogenes such as
Bmi-1 repressing p16Ink4a and inhibiting p53 and were found to be frequently
up-regulated in a variety of cancers. p38 mitogen-activated protein kinase
(MAPK), which reportedly plays a crucial role as a tumor suppressor, is
activated in number of lung adenocarcinomas, which is seemingly at odds with its
role as a tumor suppressor.
METHODS: We examined 10 lung adenocarcinomas and corresponding normal tissues
and determined the expression levels of a variety of tumor suppressor proteins
through real-time polymerase chain reaction and immunohistochemistry and
measured p38 MAPK activity by immunoblotting or immunohistochemistry analysis.
In the in vitro cellular model, p38 activation by H-Ras and consequent
senescence induction was achieved through retro-viral gene transduction.
Similarly, the suppression of p16Ink4a by Bmi-1 after the introduction of H-Ras
was achieved through transient transfection with cationic liposome.
RESULTS: We detected several lung adenocarcinomas that were positive for
activated p38 MAPK but evidenced reduced levels of p16Ink4a expression. The
suppression of p16Ink4a occurred in parallel with an increase in Bmi-1 and/or
p16Ink4a promoter hypermethylation. Consistent with these observations, the
H-Ras-stimulated induction of p16Ink4a was suppressed significantly through the
coexpression of Bmi-1 in vitro.
DISCUSSION: These results demonstrate that the suppression of p16Ink4a by either
the induction of Bmi-1 or the hypermethylation of p16Ink4 may be an important
step in avoiding tumor surveillance by p38 MAPK during the development of lung
cancer. INTRODUCTION: Dental mesenchymal stem cells (dMSCs) might differentiate into
odontoblast-like cells and form mineralized nodules. In the current study, we
investigated the effects of senescence on odontogenic differentiation of dMSCs.
METHODS: dMSCs were serially subcultured until senescence. Telomere lengths and
telomerase activities were determined by quantitative polymerase chain reaction.
Expression of genes involved in cell proliferation and differentiation, eg,
Bmi-1, p16(INK4A), osteocalcin (OC), dentin sialoprotein (DSP), bone
sialoprotein (BSP), and dentin matrix protein-1 (DMP-1) were assayed by Western
blotting and quantitative reverse transcription polymerase chain reaction.
Exogenous Bmi-1 was expressed in dMSCs by using retroviral vectors. Odontogenic
differentiation was assayed by alkaline phosphatase activity.
RESULTS: Subculture-induced replicative senescence of dMSCs led to reduced
expression of Bmi-1, OC, DSP, and BSP compared with rapidly proliferating cells,
whereas p16(INK4A) level increased. The cells exhibited progressive loss of
telomeric DNA during subculture, presumably as a result of lack of telomerase
activity. Bmi-1 transduction did not affect proliferation of cells but enhanced
the expression of OC and DSP in the late passage cultures. Bmi-1-transduced
cells also demonstrated enhanced alkaline phosphatase activity and mineralized
nodule formation.
CONCLUSIONS: These results indicate that dMSCs lose their odontogenic
differentiation potential during senescence, in part by reduced Bmi-1
expression. Recent studies provided strong support for the view that ubiquitin-specific
protease 22 (USP22) plays a central role in cell-cycle progression and also in
pathological processes such as oncogenesis. We have recently shown that USP22
levels are elevated in colorectal carcinoma with associated increase in the
expression of several cell-cycle-related genes. However, the precise mechanism
for these functions of USP22 at molecular level has not been fully elucidated.
Currently, we investigated the role of USP22 in human colorectal cancer (CRC).
We observed that USP22 expression was statistically significantly correlated
positively with that of BMI-1, c-Myc and both, pAkt (Ser473), and pAkt (Thr308),
in primary tumor tissues from 43 CRC patients. Down-regulation of USP22
expression in HCT116 colorectal cancer cells by siRNA resulted in the
accumulation of cells in the G1 phase of the cell cycle. RNAi-knockdown of USP22
in HCT16 cells also led to the repression of BMI-1 and was accompanied by the
up-regulation of p16INK4a and p14ARF, with a consequent decrease in E2F1 and p53
levels. In addition, down-regulation of c-Myc-targeted cyclin D2 was also
noticed in cells treated with USP22-siRNA. Furthermore, our results showed that
USP22 deletion also caused down-regulation of Akt/GSK3β activity, which can also
contribute to the reduction of cyclin D2. Collectively, our current results
suggest that USP22 may act as an oncogene in CRC as it positively regulates cell
cycle via both BMI-1-mediated INK4a/ARF pathway and Akt signaling pathway. |
Which microRNAs are involved in exercise adaptation? | miR-1, miR-133, miR-208a, miR-206, miR-494, miR-146a, miR-222, miR-21, miR-221, miR-20a, miR-133a, miR-133b, miR-23, miR-107 and miR-181 are involved in exercise adaptation | MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA species
involved in post-transcriptional gene regulation. In vitro studies have
identified a small number of skeletal muscle-specific miRNAs which play a
crucial role in myoblast proliferation and differentiation. In skeletal muscle,
an acute bout of endurance exercise results in the up-regulation of
transcriptional networks that regulate mitochondrial biogenesis, glucose and
fatty acid metabolism, and skeletal muscle remodelling. The purpose of this
study was to assess the expressional profile of targeted miRNA species following
an acute bout of endurance exercise and to determine relationships with
previously established endurance exercise responsive transcriptional networks.
C57Bl/6J wild-type male mice (N = 7/group) were randomly assigned to either
sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min)
group. The endurance exercise group was sacrificed three hours following a
single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of
which have been predicted to regulate transcription factors and co-activators
involved in the adaptive response to exercise, was measured in quadriceps
femoris muscle. Endurance exercise significantly increased the expression of
miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced
miR-23 expression by 84% (P<or=0.05 for all), with no change in miR-133.
Importantly, decreased expression of miRNA-23, a putative negative regulator of
PGC-1alpha was consistent with increased expression of PGC-1alpha mRNA and
protein along with several downstream targets of PGC-1alpha including ALAS, CS,
and cytochrome c mRNA. PDK4 protein content remains unaltered despite an
increase in its putative negative regulator, miR-107, and PDK4 mRNA expression.
mRNA expression of miRNA processing machinery (Drosha, Dicer, and DGCR8)
remained unchanged. We conclude that miRNA-mediated post-transcriptional
regulation is potentially involved in the complex regulatory networks that
govern skeletal muscle adaptation to endurance exercise in C57Bl/6J male mice. Muscle specific miRNAs, myomiRs, have been shown to control muscle development
in vitro and are differentially expressed at rest in diabetic skeletal muscle.
Therefore, we investigated the expression of these myomiRs, including miR-1,
miR-133a, miR-133b and miR-206 in muscle biopsies from vastus lateralis of
healthy young males (n = 10) in relation to a hyperinsulinaemic–euglycaemic
clamp as well as acute endurance exercise before and after 12 weeks of endurance
training. The subjects increased their endurance capacity, VO2max (l min−1) by
17.4% (P < 0.001), and improved insulin sensitivity by 19% (P < 0.01). While
myomiR expression remained stable during a hyperinsulinaemic–euglycaemic clamp,
an acute bout of exercise increased mir-1 (P < 0.05) and mir-133a (P < 0.05)
expression before, but not after, training. In resting biopsies, endurance
training for 12 weeks decreased basal expression of all four myomiRs (P < 0.05).
Interestingly, all myomiRs reverted to their pre-training expression levels 14
days after ceasing the training programme. Components of major pathways involved
in endurance adaptation such as MAPK and TGF-β were predicted to be targeted by
the myomiRs examined. Tested predicted target proteins included Cdc42 and ERK
1/2. Although these proteins were downregulated between post-training period and
2 weeks of cessation, an inverse correlation between myomiR and target proteins
was not found. In conclusion, our data suggest myomiRs respond to physiological
stimuli, but their role in regulating human skeletal muscle adaptation remains
unknown. Reprogramming of gene expression contributes to structural and functional
adaptation of muscle tissue in response to altered use. The aim of this study
was to investigate mechanisms for observed improvements in leg extension
strength, gain in relative thigh muscle mass and loss of body and thigh fat
content in response to eccentric and conventional strength training in elderly
men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7
years) by means of structural and molecular analyses. Biopsies were collected
from m. vastus lateralis in the resting state before and after 12 weeks of
training with two weekly resistance exercise sessions (RET) or eccentric
ergometer sessions (EET). Gene expression was analyzed using custom-designed
low-density PCR arrays. Muscle ultrastructure was evaluated using EM
morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle
fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle
growth is likely occurring by the addition of sarcomeres in series or by
hyperplasia. The expression of transcripts encoding factors involved in muscle
growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a
larger extent after EET than RET. MicroRNA 1 expression was decreased
independent of the training modality, and was paralleled by an increased
expression of IGF-1 representing a potential target. IGF-1 is a potent promoter
of muscle growth, and its regulation by microRNA 1 may have contributed to the
gain of muscle mass observed in our subjects. EET depressed genes encoding
mitochondrial and metabolic transcripts. The changes of several metabolic and
mitochondrial transcripts correlated significantly with changes in mitochondrial
volume density. Intramyocellular lipid content was decreased after EET
concomitantly with total body fat. Changes in intramyocellular lipid content
correlated with changes in body fat content with both RET and EET. In the
elderly, RET and EET lead to distinct molecular and structural adaptations which
might contribute to the observed small quantitative differences in functional
tests and body composition parameters. EET seems to be particularly convenient
for the elderly with regard to improvements in body composition and strength but
at the expense of reducing muscular oxidative capacity. MicroRNAs (miRNAs) are intracellular mediators of essential biological
functions. Recently, plasma-based 'circulating' miRNAs (c-miRNAs) have been
shown to control cellular processes, but the c-miRNA response to human exercise
remains unknown. We sought to determine whether c-miRNAs are dynamically
regulated in response to acute exhaustive cycling exercise and sustained rowing
exercise training using a longitudinal, repeated measures study design.
Specifically, c-miRNAs involved in angiogenesis (miR-20a, miR-210, miR-221,
miR-222, miR-328), inflammation (miR-21, miR-146a), skeletal and cardiac muscle
contractility (miR-21, miR-133a), and hypoxia/ischaemia adaptation (miR-21,
miR-146a, and miR-210) were measured at rest and immediately following acute
exhaustive cycling exercise in competitive male rowers (n = 10, age = 19.1 ± 0.6
years) before and after a 90 day period of rowing training. Distinct patterns of
c-miRNA response to exercise were observed and adhered to four major profiles:
(1) c-miRNA up-regulated by acute exercise before and after sustained training
(miR-146a and miR-222), (2) c-miRNA responsive to acute exercise before but not
after sustained training (miR-21 and miR-221), (3) c-miRNA responsive only to
sustained training (miR-20a), and (4) non-responsive c-miRNA (miR-133a, miR-210,
miR-328). Linear correlations were observed between peak exercise levels of
miR-146a and VO2max (r = 0.63, P = 0.003) and between changes in resting miR-20a
and changes in VO2max (pre-training vs. post-training, r = 0.73; P = 0.02).
Although future work is required, these results suggest the potential value of
c-miRNAs as exercise biomarkers and their possible roles as physiological
mediators of exercise-induced cardiovascular adaptation. MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the
most exciting areas of modern medical science. miRNAs modulate a large and
complex regulatory network of gene expression of the majority of the
protein-coding genes. Currently, evidences suggest that miRNAs play a crucial
role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and
miR-208a are highly expressed in the heart and strongly associated with the
development of cardiac hypertrophy. Recent data indicate that these miRNAs as
well as miR-206 change their expression quickly in response to physical
activity. The differential regulation of miRNAs in response to exercise suggests
a potential value of circulating miRNAs (c-miRNAs) as biomarkers of
physiological mediators of the cardiovascular adaptation induced by exercise.
Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as
potential biomarkers in the diagnosis and prognosis of heart failure. On the
other hand, the manipulation of miRNAs levels using techniques such as 'miR
mimics' and 'antagomiRs' is becoming evident the enormous potential of miRNAs as
promising therapeutic strategies in heart failure. |
Has field-programmable gate array (FPGA) technology been used to solve sequence alignment problems? | Yes. Field-Programmable Gate Arrays (FPGAs) are reconfigurable computing platforms that have found several applications in diverse domains, including digital signal processing, medical imaging and bioinformatics. Specific applications of FPGAs for biological sequence alignment have been reported for dynamic programming-based pairwise (local or global) sequence alignment, progressive multiple sequence alignment, profile alignment, Burrows-Wheeler transform (BWT) based alignment, heuristic pairwise alignment. | Aligning hundreds of sequences using progressive alignment tools such as
ClustalW requires several hours on state-of-the-art workstations. We present a
new approach to compute multiple sequence alignments in far shorter time using
reconfigurable hardware. This results in an implementation of ClustalW with
significant runtime savings on a standard off-the-shelf FPGA. Reconfigurable systolic arrays can be adapted to efficiently resolve a wide
spectrum of computational problems; parallelism is naturally explored in
systolic arrays and reconfigurability allows for redefinition of the
interconnections and operations even during run time (dynamically). We present a
reconfigurable systolic architecture that can be applied for the efficient
treatment of several dynamic programming methods for resolving well-known
problems, such as global and local sequence alignment, approximate string
matching and longest common subsequence. The dynamicity of the reconfigurability
was found to be useful for practical applications in the construction of
sequence alignments. A VHDL (VHSIC hardware description language) version of
this new architecture was implemented on an APEX FPGA (Field programmable gate
array). It would be several magnitudes faster than the software algorithm
alternatives. Progressive alignment is a widely used approach to compute multiple sequence
alignments (MSAs). However, aligning several hundred sequences by popular
progressive alignment tools requires hours on sequential computers. Due to the
rapid growth of sequence databases biologists have to compute MSAs in a far
shorter time. In this paper we present a new approach to MSA on reconfigurable
hardware platforms to gain high performance at low cost. We have constructed a
linear systolic array to perform pairwise sequence distance computations using
dynamic programming. This results in an implementation with significant runtime
savings on a standard FPGA. BACKGROUND: This paper describes techniques for accelerating the performance of
the string set matching problem with particular emphasis on applications in
computational proteomics. The process of matching peptide sequences against a
genome translated in six reading frames is part of a proteogenomic mapping
pipeline that is used as a case-study. The Aho-Corasick algorithm is adapted for
execution in field programmable gate array (FPGA) devices in a manner that
optimizes space and performance. In this approach, the traditional Aho-Corasick
finite state machine (FSM) is split into smaller FSMs, operating in parallel,
each of which matches up to 20 peptides in the input translated genome. Each of
the smaller FSMs is further divided into five simpler FSMs such that each simple
FSM operates on a single bit position in the input (five bits are sufficient for
representing all amino acids and special symbols in protein sequences).
RESULTS: This bit-split organization of the Aho-Corasick implementation enables
efficient utilization of the limited random access memory (RAM) resources
available in typical FPGAs. The use of on-chip RAM as opposed to FPGA logic
resources for FSM implementation also enables rapid reconfiguration of the FPGA
without the place and routing delays associated with complex digital designs.
CONCLUSION: Experimental results show storage efficiencies of over 80% for
several data sets. Furthermore, the FPGA implementation executing at 100 MHz is
nearly 20 times faster than an implementation of the traditional Aho-Corasick
algorithm executing on a 2.67 GHz workstation. BACKGROUND: The exponential growth of available biological data has caused
bioinformatics to be rapidly moving towards a data-intensive, computational
science. As a result, the computational power needed by bioinformatics
applications is growing exponentially as well. The recent emergence of
accelerator technologies has made it possible to achieve an excellent
improvement in execution time for many bioinformatics applications, compared to
current general-purpose platforms. In this paper, we demonstrate how the
PlayStation 3, powered by the Cell Broadband Engine, can be used as a
computational platform to accelerate the Smith-Waterman algorithm.
RESULTS: For large datasets, our implementation on the PlayStation 3 provides a
significant improvement in running time compared to other implementations such
as SSEARCH, Striped Smith-Waterman and CUDA. Our implementation achieves a peak
performance of up to 3,646 MCUPS.
CONCLUSION: The results from our experiments demonstrate that the PlayStation 3
console can be used as an efficient low cost computational platform for high
performance sequence alignment applications. BACKGROUND: In the field of RNA secondary structure prediction, the RNAalifold
algorithm is one of the most popular methods using free energy minimization.
However, general-purpose computers including parallel computers or multi-core
computers exhibit parallel efficiency of no more than 50%. Field Programmable
Gate-Array (FPGA) chips provide a new approach to accelerate RNAalifold by
exploiting fine-grained custom design.
RESULTS: RNAalifold shows complicated data dependences, in which the dependence
distance is variable, and the dependence direction is also across two
dimensions. We propose a systolic array structure including one master
Processing Element (PE) and multiple slave PEs for fine grain hardware
implementation on FPGA. We exploit data reuse schemes to reduce the need to load
energy matrices from external memory. We also propose several methods to reduce
energy table parameter size by 80%.
CONCLUSION: To our knowledge, our implementation with 16 PEs is the only FPGA
accelerator implementing the complete RNAalifold algorithm. The experimental
results show a factor of 12.2 speedup over the RNAalifold (ViennaPackage -
1.6.5) software for a group of aligned RNA sequences with 2981-residue running
on a Personal Computer (PC) platform with Pentium 4 2.6 GHz CPU. Molecular biologists use hidden Markov models (HMMs) as a popular tool to
statistically describe biological sequence families. This statistical
description can then be used for sensitive and selective database scanning,
e.g., new protein sequences are compared with a set of HMMs to detect functional
similarities. Efficient dynamic-programming algorithms exist for solving this
problem; however, current solutions still require significant scan times. These
scan time requirements are likely to become even more severe due to the rapid
growth in the size of these databases. This paper shows how reconfigurable
architectures can be used to derive an efficient fine-grained parallelization of
the dynamic programming calculation. We describe how this technique leads to
significant runtime savings for HMM database scanning on a standard
off-the-shelf field-programmable gate array (FPGA). BACKGROUND: Multiple sequence alignment (MSA) is a fundamental analysis method
used in bioinformatics and many comparative genomic applications. Prior MSA
acceleration attempts with reconfigurable computing have only addressed the
first stage of progressive alignment and consequently exhibit performance
limitations according to Amdahl's Law. This work is the first known to
accelerate the third stage of progressive alignment on reconfigurable hardware.
RESULTS: We reduce subgroups of aligned sequences into discrete profiles before
they are pairwise aligned on the accelerator. Using an FPGA accelerator, an
overall speedup of up to 150 has been demonstrated on a large data set when
compared to a 2.4 GHz Core2 processor.
CONCLUSIONS: Our parallel algorithm and architecture accelerates large-scale MSA
with reconfigurable computing and allows researchers to solve the larger
problems that confront biologists today. Program source is available from
http://dna.cs.byu.edu/msa/. |
List inhibtors targeting the mitochondrial permeability transition pore. | Cyclosporine A
Atractyloside
N-metyl-4-isoleucine-cyclosporine
Sanglifehrin A
TRO-19622 | The mitochondrial permeability transition pore is an inducer of cell death.
During the reperfusion phase after cerebral ischemia, calcium accumulates in
mitochondria, and a burst of free radical formation occurs, conditions that
favor the activation of the mitochondrial permeability transition pore. Here the
authors demonstrate that a blocker of the mitochondrial permeability transition
pore, the nonimmunosuppressive cyclosporin A analogue N-methyl-Val-4-cyclosporin
A (10 mg/kg intraperitoneally), administered during reperfusion and at 24 hours
of reperfusion, diminishes infarct size in a rat model of transient focal
ischemia of 2 hours' duration. The mitochondrial permeability transition pore
may be an important target for drugs against stroke. OBJECTIVES: Long-time preservation of the donor heart before transplantation is
associated with mitochondrial damages resulting in functional deterioration
after transplantation, although the exact mechanism behind this is still
uncertain. Here, we have demonstrated the opening of a nonspecific pore in the
inner membrane of the mitochondria, the mitochondrial permeability transition
pore (PTP), as one of the reasons responsible for this functional deterioration.
METHODS: After 30 minutes of perfusion with the Krebs Henseleit buffer at 37
degrees C in working mode, hearts from Wistar rats were arrested with ice-cold
St. Thomas cardioplegic solution and preserved University of Wisconsin solution
at 4 degrees C with or without inhibiting the PTP with cyclosporin A (CsA) (0.2
microM). After 12 hours, the hearts were reperfused for 60 minutes at 37 degrees
C with or without perfusing the hearts during first 15 minutes of reperfusion
with PTP openers lonidamine (30 microM) or atractyloside (20 microM).
RESULTS: Inhibiting PTP with CsA resulted in (a) significant recovery of cardiac
functions, (b) well-preserved myocardial adenosine triphosphate (ATP) levels
(P<0.001), (c) less myocardial water content (P<0.01), (d) less mitochondrial
swelling and cytochrome C release, and (e) up-regulation of Bcl-2 expression
compared with the control hearts without PTP inhibition. These effects are
completely inhibited on opening the PTP before preservation, resulting in poor
recovery of cardiac functions, loss of myocardial ATP, and severe mitochondrial
swelling.
CONCLUSION: The present study demonstrates the potential role of mitochondrial
PTP after long- time hypothermic preservation of the heart, and therefore,
regulation of PTP would prolong the preservation time of donor hearts. Although bradykinin has been demonstrated to protect the heart at reperfusion,
the detailed cellular and molecular mechanisms that mediate the protection
remain elusive. Here we aimed to determine whether bradykinin protects the heart
at reperfusion by modulating the mitochondrial permeability transition pore
(mPTP) opening through glycogen synthase kinase 3beta (GSK-3beta). Bradykinin
given at reperfusion reduced infarct size in isolated rat hearts subjected to 30
min regional ischemia followed by 2 h of reperfusion. The infarct-limiting
effect of bradykinin was reversed by atractyloside, an opener of the mPTP,
suggesting that bradykinin may protect the heart at reperfusion by modulating
the mPTP opening. In support of this observation, bradykinin prevented the
collapse of mitochondrial membrane potential (DeltaPsi(m)), an index of the mPTP
opening. Bradykinin increased GSK-3beta phosphorylation at reperfusion, and the
selective inhibitor of GSK-3beta SB216763 reduced infarct size and prevented the
loss of DeltaPsi(m) by mimicking the effect of bradykinin. The effect of
bradykinin on GSK-3beta phosphorylation was blocked by wortmannin and LY294002,
and bradykinin increased Akt phosphorylation at reperfusion. Further experiments
showed that the MEK inhibitor PD98059 prevented the effect of bradykinin on
GSK-3beta. However, the mTOR/p70s6K pathway inhibitor rapamycin did not alter
bradykinin-induced GSK-3beta phosphorylation and bradykinin failed to alter
phosphorylation of either mTOR or p70s6K at reperfusion. Taken together, these
data suggest that bradykinin protects the heart at reperfusion by modulating the
mPTP opening through inhibition of GSK-3beta. The PI3-kinase/Akt pathway and
ERK, but not the mTOR/p70s6K pathway account for the suppression of GSK-3beta by
bradykinin. The purpose of this study was to determine whether exogenous zinc prevents
cardiac reperfusion injury by targeting the mitochondrial permeability
transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The
treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc
ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation
at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells.
The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol
3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin
(mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that
PI3K but not mTOR or PKC accounts for the action of zinc. In support of this
interpretation, zinc induced a significant increase in Akt but not mTOR
phosphorylation. Further experiments found that zinc also increased
mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the
mitochondria in the action of zinc. The effect of zinc on mitochondrial
GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+
channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell
death in cells subjected to simulated ischemia/reperfusion, indicating that zinc
can prevent reperfusion injury. However, zinc was not able to exert protection
in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA)
mutant, suggesting that zinc prevents reperfusion injury by inactivating
GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta
(GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly
supporting the essential role of GSK-3beta inactivation in cardioprotection.
Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of
GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion
injury by modulating the mPTP opening through the inactivation of GSK-3beta. The
PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by
zinc. BACKGROUND AND AIMS: Hyperglycaemia blocks sevoflurane-induced postconditioning,
and cardioprotection in hyperglycaemic myocardium can be restored by inhibition
of the mitochondrial permeability transition pore (mPTP). We investigated
whether sevoflurane-induced postconditioning is also blocked in the prediabetic
heart and if so, whether cardioprotection could be restored by inhibiting mPTP.
METHODS AND RESULTS: Zucker lean (ZL) and Zucker obese (ZO) rats were assigned
to one of seven groups. Animals underwent 25 min of ischaemia and 120 min of
reperfusion. Control (ZL-/ZO Con) animals were not further treated.
postconditioning groups (ZL-/ZO Sevo-post) received sevoflurane for 5 min
starting 1min prior to the onset of reperfusion. The mPTP inhibitor cyclosporine
A (CsA) was administered intravenously in a concentration of 5 (ZO CsA and ZO
CsA+Sevo-post) or 10 mg/kg (ZO CsA10+Sevo-post) 5 min before the onset of
reperfusion. At the end of reperfusion, infarct sizes were measured by TTC
staining. Blood samples were collected to measure plasma levels of insulin,
cholesterol and triglycerides. Sevoflurane postconditioning reduced infarct size
in ZL rats to 35±12% (p<0.05 vs. ZL Con: 60±6%). In ZO rats sevoflurane
postconditioning was abolished (ZO Sevo-post: 59±12%, n.s. vs. ZO Con: 58±6%). 5
mg and 10 mg CsA could not restore cardioprotection (ZO CsA+Sevo-post: 59±7%, ZO
CsA10+Sevo-post: 57±14%; n.s. vs. ZO Con). In ZO rats insulin, cholesterol and
triglyceride levels were significant higher than in ZL rats (all p<0.05).
CONCLUSION: Inhibition of mPTP with CsA failed to restore cardioprotection in
the prediabetic but normoglycaemic heart of Zucker obese rats in vivo. Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial
dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many
cells. In this study, we have investigated the mechanism by which Bnip3 induces
autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive
autophagy in adult cardiac myocytes. Fluorescent microscopy studies and
ultrastructural analysis revealed selective degradation of mitochondria by
autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased
levels of intracellular Ca(2+) have been reported by others to induce autophagy,
but Bnip3-induced autophagy was not abolished by antioxidant treatment or the
Ca(2+) chelator BAPT A-AM. We also investigated the role of the mitochondrial
permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the
mPTP has previously been implicated in the induction of autophagy and selective
removal of damaged mitochondria by autophagosomes, mitochondria sequestered by
autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability
transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit
mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is
an essential component of the mPTP and Bnip3 induced autophagy to the same
extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice.
These results support a model where Bnip3 induces selective removal of the
mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy
independent of Ca(2+), ROS generation, and mPTP opening. 7-Xylosyl-10-deacetylpaclitaxel is a natural hydrophilic paclitaxel derivative.
It has long been used in Chinese clinics to treat cancer. In order to further
explore the underlying intracellular target of 7-xylosyl-10-deacetylpaclitaxel
towards the PC-3 cell line, the ultra-structural morphology of mitochondria, the
intracellular Ca (2+), the intracellular ATP, the intracellular hydrogen
peroxide and pro-apoptotic Bax and Bcl-2 protein expression were measured.
Additionally, the changes of mitochondrial morphology and membrane potential (
ΔΨm) were analyzed by atomic force microscopy (AFM) and flow cytometry,
respectively. Our results suggest that the intracellular target of
7-xylosyl-10-deacetylpaclitaxel may be the mitochondrial permeability transition
pore (mPTP). To further evaluate this hypothesis, we assessed the effect of a
specific mPTP inhibitor (cyclosporine A) on the toxic action of
7-xylosyl-10-deacetylpaclitaxel. The 7-xylosyl-10-deacetylpaclitaxel-induced
decrease in mitochondrial inner transmembrane potential (ΔΨm) was abolished by
the addition of cyclosporine A (CsA) in PC-3 cells, indicating that
7-xylosyl-10-deacetylpaclitaxel may target mPTP. Furthermore, treatment with
7-xylosyl-10-deacetylpaclitaxel increased ROS levels in PC-3 cells. This effect
was counteracted by 10 µM cyclosporine A. These data indicate that oxidative
damage is involved in mPTP. Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides.
Here we show that antamanide inhibits the mitochondrial permeability transition
pore, a central effector of cell death induction, by targeting the pore
regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by
antamanide is not additive with the cyclophilin D-binding drug cyclosporin A,
(ii) the inhibitory action of antamanide on the pore requires phosphate, as
previously shown for cyclosporin A; (iii) antamanide is ineffective in
mitochondria or cells derived from cyclophilin D null animals, and (iv)
abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability
transition pore inhibition by antamanide needs two critical residues in the
peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on
the pore in a cyclophilin D-independent fashion. Antamanide also abrogates
mitochondrial depolarization and the ensuing cell death caused by two
well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal
peptide. Our findings have implications for the comprehension of cyclophilin D
activity on the permeability transition pore and for the development of novel
pore-targeting drugs exploitable as cell death inhibitors. The protective effects of cyclic dipeptides in alcoholic beverages were
investigated in the perfused guinea-pig hearts subjected to ischemia and
reperfusion. Subsequently, in order to determine the importance of cyclic
dipeptide structure, the effects of cyclo(L-Leu-L-Tyr) (cLY) were compared with
those of the newly synthesized non-cyclic dipeptides, L-Leu-L-Tyr (LY) and
L-Tyr-L-Leu (YL). After reperfusion, pressure recovery (%) in the left ventricle
reached a peak of over 90% in the presence of cLY (10(-6) M and 10(-5) M)
(control: 22.9%). The recovery by LY and YL was significantly lower than that by
cLY, and ATP levels simultaneously monitored using (31)P-NMR were already lower
during the ischemic end period than those observed with cLY treatment. In
perfused mitochondrial preparations, cLY significantly inhibited mitochondrial
Ca(2+) ([Ca(2+)](m)) elevation in a similar way to that of the mitochondrial
permeability transition pore (MPTP) inhibitor cyclosporin A. In vitro electron
paramagnetic resoce (EPR) revealed that the active oxygen radicals quenching
activity of cLY was greater than those of non-cyclic dipeptides. cLY inhibited
caspase-3-induced apoptosis. The cyclic dipeptide structure inhibits opening of
the MPTP by preventing [Ca(2+)](m) overload-induced apoptosis related to
mitochondrial active oxygen radical accumulation in ischemia-reperfusion hearts. AIMS: The present study was designed to examine the impact of chronic Akt
activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical
anomalies, if any, and the underlying mechanism involved.
RESULTS: Wild-type and transgenic mice with cardiac-specific overexpression of
the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer
tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic
(elevated left ventricular end-systolic diameter and reduced fractional
shortening) and cardiomyocyte contractile function, intracellular Ca(2+)
mishandling, and cell survival in wild-type mice associated with mitochondrial
damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER
stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen
species production, carbonyl formation, apoptosis, mitochondrial membrane
potential loss, and mitochondrial permeation pore (mPTP) opening associated with
overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties.
Interestingly, these anomalies were mitigated by chronic Akt activation or the
ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also
dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β
(GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by
Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and
mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A,
GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA.
INNOVATION: This research reported the direct relationship between ER stress and
cardiomyocyte contractile and mitochondrial anomalies for the first time.
CONCLUSION: Taken together, these data suggest that ER stress may compromise
cardiac contractile and intracellular Ca(2+) properties, possibly through the
Akt/GSK3β-dependent impairment of mitochondrial integrity. We have previously shown that mitochondrial membrane potential disruption is
involved in mechanisms underlying differential vulnerabilities to the
excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary
cultured neurons prepared from rat cortex and hippocampus. To further elucidate
the role of mitochondria in the excitotoxicity after activation of NMDA
receptors, neurons were loaded with the fluorescent dye calcein diffusible in
the cytoplasm and organelles for determination of the activity of mitochondrial
permeability transition pore (mPTP) responsible for the leakage of different
mitochondrial molecules. The addition of CoCl(2) similarly quenched the
intracellular fluorescence except mitochondria in both cultured neurons, while
further addition of NMDA led to a leakage of the dye into the cytoplasm in
hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of
viability in hippocampal neurons, while an activator of mPTP induced a similarly
potent loss of viability in cortical and hippocampal neurons. Although NMDA was
more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium
indicator in hippocampal than cortical neurons, a mitochondrial calcium
uniporter inhibitor significantly prevented the NMDA-induced loss of viability
in hippocampal neurons. Expression of mRNA was significantly higher for the
putative uniporter uncoupling protein-2 in hippocampal than cortical neurons.
These results suggest that mitochondrial calcium uniporter would be at least in
part responsible for the NMDA neurotoxicity through a mechanism relevant to
promotion of mPTP orchestration in hippocampal neurons. Ischemic postconditioning (IPost) has been shown to attenuate cerebral
ischemia-reperfusion injury. However, the mechanism remains elusive. Because
opening of the mitochondrial permeability transition pore (MPTP) is a crucial
determit of cell death after ischemia-reperfusion, we hypothesized that the
neuroprotective effect of IPost may be associated with inhibition of MPTP
opening. In part 1 of this study, pentobarbital-anesthetized rats subjected to
middle cerebral artery occlusion for 90 min, followed by reperfusion for 72 h,
were assigned to receive one of the following treatments: three cycles of IPost
(15s each), intracerebroventricular injection of saline (control),
administration of the MPTP inhibitor cyclosporin A (CsA) (2 μmol/L, 15 μL) or
its vehicle alcohol, administration of the MPTP opener atractyloside (Atr) (2
mmol/L, 15 μL), or IPost plus CsA/Atr treatment. Neurological deficit scores
(NDS) and infarct volumes were assessed. Mitochondrial ultrastructure and
swelling were also examined after reperfusion. In part 2, control and IPost
groups underwent ischemia (90 min) and reperfusion (15 min). CsA and Atr groups
were treated as described in part 1. Brain mitochondria were isolated after
reperfusion and MPTP activity was evaluated. IPost or CsA treatment
significantly improved NDS and reduced infarction volume, while Atr reversed the
neuroprotective effects of IPost, and attenuated the decrease in mitochondrial
swelling induced by IPost or CsA. Thus, inhibiting MPTP opening may play a
crucial role in the neuroprotective effects of IPost, which may have potential
clinical value against cerebral ischemia-reperfusion injury. AIMS: Opening of the mitochondrial permeability transition pore (mPTP) is an
important step on the pathway towards cardiomyocyte death, defining the extent
of injury following cardiac ischaemia and reperfusion. In isolated mitochondria,
mPTP opening is triggered by calcium overload facilitated by oxidative stress.
In isolated cells, however, it has been suggested that mPTP opening occurs
before calcium overload and is stimulated by oxidative stress. Our objective was
to establish the events that cause mPTP opening in the intact heart.
METHODS AND RESULTS: We performed multiphoton imaging of Langendorff-perfused
mouse hearts expressing an inducible, Ca(2+)-sensitive reporter (circularly
Permuted GFP and calmodulin (CaM), version 2), to examine the spatiotemporal
relationship between [Ca(2+)](c), redox state, and mPTP opening in the intact
heart during hypoxia and reoxygenation at sub-myocyte resolution. We found that
during reperfusion, calcium waves propagated across multiple cells at 3.3 µm/s.
mPTP opening caused an abrupt loss of mitochondrial membrane potential, measured
using a potentiometric dye, which was invariably preceded by a rise in
[Ca(2+)](c). The probability that localized [Ca(2+)](c) waves led to mPTP
opening was greater early during reoxygenation. During reoxygenation,
coordinated redox changes also occurred across large regions and preceded mPTP
opening on average by 122 ± 38 s. Fewer [Ca(2+)] waves led to mPTP opening in
the presence of mPTP inhibitor cyclosporin A or mitochondrial-targeted scavenger
of reactive oxygen species, MitoQ.
CONCLUSION: These experiments define the spatiotemporal relationship between
changes in [Ca(2+)](c), redox state and mPTP opening during reoxygenation in the
intact heart. Tissue oxidation coincident with localized calcium waves together
conspire to cause mPTP opening and subsequent cell death. OBJECTIVE: Anesthetic preconditioning appears to be a viable strategy to treat
ischemic cerebral injury. Here we investigated 1) whether the protection
conferred by sevoflurane preconditioning sustains in time; 2) whether
sevoflurane preconditioning diminishes mitochondrial dysfunction following
cerebral ischemia; and 3) whether mitochondrial permeability transition pore
plays a crucial role in the sevoflurane preconditioning.
DESIGN: Laboratory investigation.
SETTING: University research laboratory.
SUBJECTS: : Sprague-Dawley rats.
INTERVENTIONS: Rats underwent 2 hrs of focal cerebral ischemia induced by middle
cerebral artery occlusion. Preconditioning was elicited with sevoflurane (2.3%)
for 60 mins at 24 hrs before ischemia. The involvement of mitochondrial
permeability transition pore was determined with a mitochondrial permeability
transition pore opener atractyloside and a specific mitochondrial permeability
transition pore inhibitor cyclosporin A. In vitro study was performed on acutely
isolated mitochondria subjected to calcium overload.
MEASUREMENTS AND MAIN RESULTS: Sevoflurane preconditioning significantly
decreased the infarct size by 35.9% (95% confidence interval 6.5-28.4, p <
.001). This reduction of injury volume was associated with a long-term
improvement of neurological function according to modified neurological severity
score (F = 13.6, p = .001) and sticky-tape test (F = 29.1, p < .001) for 42 days
after ischemia. Furthermore, sevoflurane preconditioning markedly protected
mitochondria, as indicated by preserved respiratory chain complex activities and
membrane potential, lowered mitochondrial hydrogen-peroxide production, and
attenuated mitochondrial permeability transition pore opening. Isolated
mitochondria also demonstrated a reduced sensitivity to Ca-induced mitochondrial
permeability transition pore opening after pre-exposure to sevoflurane in vitro
(95% confidence interval 24.2-196.5,p = .006). Inhibiting mitochondrial
permeability transition pore using cyclosporin A resulted in protective effects
similar to those seen with sevoflurane preconditioning, whereas
pharmacologically opening the mitochondrial permeability transition pore with
atractyloside abrogated all the positive effects of sevoflurane preconditioning
and cyclosporin A, including suppression of mitochondrial permeability
transition pore opening, counteraction of mitochondria-dependent apoptotic
pathway, and subsequent histological and behavioral improvements.
CONCLUSIONS: Sevoflurane preconditioning protects mitochondria from cerebral
ischemia/reperfusion injury and ameliorates long-term neurological deficits.
Inhibition of mitochondrial permeability transition pore opening is a crucial
step in mediating the neuroprotection of sevoflurane preconditioning. Autophagy is a crucial process for cells to maintain homeostasis and survival
through degradation of cellular proteins and organelles, including mitochondria
and endoplasmic reticula (ER). We previously demonstrated that temozolomide
(TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen
species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to
protect glioma cells from apoptosis. In this study, we investigated the role of
mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial
depolarization and mitochondrial permeability transition pore (MPTP) opening
were observed as a prelude to TMZ-induced autophagy, and these were followed by
the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such
as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and
oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A,
decreased mitochondrial damage-mediated autophagy, and therefore increased
TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased
expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein.
ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK)
and Ca(2+) signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA),
an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting
autophagy. Taken together, our data indicate that TMZ induced autophagy through
mitochondrial damage- and ER stress-dependent mechanisms to protect glioma
cells. This study provides evidence that agents targeting mitochondria or ER may
be potential anticancer strategies. Bax, a pro-apoptotic member of the Bcl-2 family of proteins has the ability to
form transmembrane pores large enough to allow cytochrome c (Cyt c) release, as
well as to activate the mitochondrial permeability transition pore (mPTP);
however, no differential study has been conducted to clarify which one of these
mechanisms predominates over the other in the same system. In the present study,
we treated isolated mitochondria from MCF7 cells with recombit protein Bax
and tested the efficacy of the mPTP inhibitor cyclosporin A (CsA) and of the Bax
channel blocker (Bcb) to inhibit cytochrome c release. We also, induced
apoptosis in MCF7 cell cultures with TNF-α plus cycloheximide to determine the
effect of such compounds in apoptosis induction via mPTP or Bax oligomerization.
Cytochrome c release was totally prevented by CsA and partially by Bcb when
apoptosis was induced with recombit Bax in isolated mitochondria from MCF7
cells. CsA increased the number of living cells in cell culture, as compared
with the effect of Bax channel blocker. These results indicate that mPTP
activation is the predomit pathway for Bax-induced cytochrome c release from
MCF7 mitochondria and for apoptosis induction in the whole cell. OBJECTIVE: The NLRP3 (NALP3, cryopyrin) inflammasome, a key component of the
innate immune system, facilitates caspase-1 and interleukin (IL)-1β processing,
which amplifies the inflammatory response. Here, we investigated whether NLRP3
knockdown decreases neutrophil infiltration, reduces brain edema, and improves
neurological function in an intracerebral hemorrhage (ICH) mouse model. We also
determined whether mitochondrial reactive oxygen species (ROS) governed by
mitochondrial permeability transition pores (mPTPs) would trigger NLRP3
inflammasome activation following ICH.
METHODS: ICH was induced by injecting autologous arterial blood (30μl) into a
mouse brain. NLRP3 small interfering RNAs were administered 24 hours before ICH.
A mPTP inhibitor (TRO-19622) or a specific mitochondria ROS scavenger
(Mito-TEMPO) was coinjected with the blood. In naive animals, rotenone, which is
a respiration chain complex I inhibitor, was applied to induce mitochondrial ROS
production, and followed by TRO-19622 or Mito-TEMPO treatment. Neurological
deficits, brain edema, enzyme-linked immunosorbent assay, Western blot, in vivo
chemical cross-linking, ROS assay, and immunofluorescence were evaluated.
RESULTS: ICH activated the NLRP3 inflammasome. NLRP3 knockdown reduced brain
edema and decreased myeloperoxidase (MPO) levels at 24 hours, and improved
neurological functions from 24 to 72 hours following ICH. TRO-19622 or
Mito-TEMPO reduced ROS, NLRP3 inflammasome components, and MPO levels following
ICH. In naive animals, rotenone administration induced mPTP formation, ROS
generation, and NLRP3 inflammasome activation, which were then reduced by
TRO-19622 or Mito-TEMPO.
INTERPRETATION: The NLRP3 inflammasome amplified the inflammatory response by
releasing IL-1β and promoting neutrophil infiltration following ICH.
Mitochondria ROS may be a major trigger of NLRP3 inflammasome activation. The
results of our study suggest that the inhibition of the NLRP3 inflammasome may
effectively reduce the inflammatory response following ICH.ANN NEUROL
2014;75:209-219. Perturbation of calcium signaling that occurs during cell injury and disease,
promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial
permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH)
release, and necrotic cell death that were blocked by cyclosporin A (CsA) and
EGTA. LDH release temporally correlated with arachidonic acid release but did
not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2.
Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient
fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another
serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake.
Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial
Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic
acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated
mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated
mitochondria. Unlike pyrrophenone, the diacylglycerol analog
1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid
release not by preventing mitochondrial calcium uptake but by inhibiting MPTP
formation. In fibroblasts stimulated with thapsigargin, which induces MPTP
formation by a direct effect on mitochondria, LDH and arachidonic acid release
were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or
EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by
blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids
that occurs by a novel pathway during MPTP formation. This work reveals the
potential for development of small molecule cell-permeable serine hydrolase
inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP
formation, and necrotic cell death. The isothiourea derivative, KB-R7943, inhibits the reverse-mode of the plasma
membrane sodium/calcium exchanger and protects against ischemia/reperfusion
injury. The mechanism through which KB-R7943 confers protection, however,
remains controversial. Recently, KB-R7943 has been shown to inhibit
mitochondrial calcium uptake and matrix overload, which may contribute to its
protective effects. While using KB-R7943 for this purpose, we find here no
evidence that KB-R7943 directly blocks mitochondrial calcium uptake. Rather, we
find that KB-R7943 inhibits opening of the mitochondrial permeability transition
pore in permeabilized cells and isolated liver mitochondria. Furthermore, we
find that this observation correlates with protection against calcium
ionophore-induced mitochondrial membrane potential depolarization and cell
death, without detrimental effects to basal mitochondrial membrane potential or
complex I-dependent mitochondrial respiration. Our data reveal another mechanism
through which KB-R7943 may protect against calcium-induced injury, as well as a
novel means to inhibit the mitochondrial permeability transition pore. Tributyltin (TBT), a member of the organotin family, is a known endocrine
disruptor. It persists long in the environment and is widely used in various
industrial applications. This study was planned to understand its toxic
influence on Leydig cells isolated from 28 day old wistar rats. In-vitro
exposure to TBT-Chloride (TBTC) (300-3000 nM) reduced cell viability (DNA
fragmentation, nuclear condensation and MTT assay) and affected testosterone
production. TBTC induced both apoptotic and necrotic cell death (AnnexinV/PI
binding assay). Involvement of calcium (Ca(2+)), redox imbalance (ROS, GSH and
TBARS) and mitochondria in TBTC toxicity was evaluated by using Ca(2+)
inhibitors (BAPTA-AM, EGTA, Ruthenium Red), free radical scavengers (NAC,
C-Phycocyanin) and mitochondrial permeability transition pore inhibitor
(Cyclosporine A). Protein expression analysis of phosphorylated MAPKinases
(ERK1/2, JNK1/2, & p38), steroidogenic proteins (3β-HSD, StAR & TSPO) and
apoptotic proteins (Bax, Bcl2) illustrates the cytotoxic and anti-steroidogenic
activity of TBTC. Traumatic brain injury (TBI) is often caused by accidents that damage the brain.
TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell
death. In this study, we investigated the mechanism of cell death during the
secondary damage caused by TBI in vivo and in vitro, as well as the protective
effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β
(GSK-3β) activation and microtubule-associated protein light chain 3 processing
were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic
and autophagic cell death were induced through glutamate-mediated GSK-3β
activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or
transfection of GSK-3β small-interfering RNA increases cell survival. By
contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity.
Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing
reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which
RV also exerted protective effects in vivo. Mitochondrial damages, including the
opening of mitochondrial permeability transition pore (MPTP) and mitochondrial
depolarization, were induced by glutamate through the ROS/GSK-3β pathway.
Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and
the percentages of cells undergoing autophagy and apoptosis and thereby
increased cell survival. Taken together, our results demonstrated that cell
death occurring after TBI is induced through the ROS/GSK-3β/mitochondria
signaling pathway and that administration of RV can increase cell survival by
suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results
indicated that resveratrol may serve as a potential therapeutic agent in the
treatment of TBI. The currently available experimental data supports the hypothesis that the
neuroprotective effect of dimebon is related to the protection of the
brain-mitochondria from neurodegeneration. In this study, the influence of
dimebon on mitochondria was investigated to gain a better understanding of the
neuroprotective effects of this drug. Here, we demonstrate that dimebon enhances
the resistance of the isolated rat brain and liver mitochondria to the induction
of mitochondrial permeability transition (MPT) by calcium ions even in the
presence of atractyloside, a MPT pore (MPTP) opener, but is ineffective against
atractyloside-induced mitochondria swelling. Unlike cyclosporine A (CsA), a MPTP
inhibitor, Dimebon does not influence the adenine nucleotide translocase (ANT)
conformational changes and is not able to prevent the MPT of de-energized
mitochondria. Using three different assays, and using amyloid-β peptide for
inducing mitochondrial toxicity, we show that the influence of dimebon on the
calcium retention capacity (CRC) of mitochondria depends on the mode of calcium
addition. No obvious influence of dimebon on CRC was observed under the
conditions of calcium infusion in the pump mode but the increase of CRC of rat
brain mitochondria was observed when calcium was added in the bolus mode; the
addition of calcium in the single pulse mode led to the increase of the lag
period of calcium efflux from mitochondria. From these studies it is shown that
dimebon is effective against amyloid-β (Aβ) potentiated mitochondrial swelling
and decrease of calcium retention capacity (CRC) of the brain mitochondria. Selective stimulation of β3 adrenergic-receptor (β3AR) has been shown to reduce
infarct size in a mouse model of myocardial ischemia/reperfusion. However, its
functional long-term effect and the cardioprotective mechanisms at the level of
cardiomyocytes have not been elucidated, and the impact of β3AR stimulation has
not been evaluated in a more translational large animal model. This study aimed
at evaluating pre-perfusion administration of BRL37344 both in small and large
animal models of myocardial ischemia/reperfusion. Pre-reperfusion administration
of the β3AR agonist BRL37344 (5 μg/kg) reduced infarct size at 2-and 24-h
reperfusion in wild-type mice. Long-term (12-weeks) left ventricular (LV)
function assessed by echocardiography and cardiac magnetic resoce (CMR) was
significantly improved in β3AR agonist-treated mice. Incubation with β3AR
agonist (BRL37344, 7 μmol/L) significantly reduced cell death in isolated adult
mouse cardiomyocytes during hypoxia/reoxygenation and decreased susceptibility
to deleterious opening of the mitochondrial permeability transition pore (mPTP),
via a mechanism dependent on the Akt-NO signaling pathway. Pre-reperfusion
BRL37344 administration had no effect on infarct size in cyclophilin-D KO mice,
further implicating mPTP in the mechanism of protection. Large-white pigs
underwent percutaneous coronary ischemia/reperfusion and 3-T CMR at 7 and
45 days post-infarction. Pre-perfusion administration of BRL37344 (5 μg/kg)
decreased infarct size and improved long-term LV contractile function. A
single-dose administration of β3AR agonist before reperfusion decreased infarct
size and resulted in a consistent and long-term improvement in cardiac function,
both in small and large animal models of myocardial ischemia/reperfusion. This
protection appears to be executed through inhibition of mPTP opening in
cardiomyocytes. INTRODUCTION: Operation on the infrarenal aorta and large arteries of the lower
extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may
induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a
mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury,
by inhibiting mitochondrial permeability transition pores (mPTP).
OBJECTIVES: Our aim was to reduce damages in the skeletal muscle and the kidney
after IR of the lower limb with NIM-811.
MATERIALS AND METHODS: Wistar rats underwent 180 minutes of bilateral lower limb
ischemia and 240 minutes of reperfusion. Four animal groups were formed called
Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham
surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and
surgery). Serum, urine and histological samples were taken at the end of
reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations,
laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were
performed. Renal peroxynitrite concentration, serum TNF-α and IL-6 levels were
measured.
RESULTS: Less significant histopathological changes were observable in the
NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were
significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK:
p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be
significantly higher (p = 0.001) and renal function parameters were
significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group
in comparison to the IR group. Serum TNF-α and IL-6 levels were significantly
lower (TNF-α: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite
concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group
than in the IR group.
CONCLUSION: NIM-811 could have the potential of reducing rhabdomyolysis and
impairment of the kidney after lower limb IR injury. AIMS: Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tolerance
against acute myocardial ischaemia-reperfusion injury (IRI). However, the
mechanism through which HIF-1 stabilization actually confers this
cardioprotection is not clear. We investigated whether HIF-1α stabilization
protects the heart against acute IRI by preventing the opening of the
mitochondrial permeability transition pore (MPTP) and the potential mechanisms
involved.
METHODS AND RESULTS: Stabilization of myocardial HIF-1 was achieved by
pharmacological inhibition of prolyl hydroxylase (PHD) domain-containing enzyme
using GSK360A or using cardiac-specific ablation of von Hippel-Lindau protein
(VHL(fl/fl)) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized
HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase
kinase-1 (PDK1) and hexokinase II (HKII), and reprogrammed cell metabolism to
aerobic glycolysis, thereby resulting in the production of less mitochondrial
oxidative stress during IRI, and less MPTP opening, effects which were shown to
be dependent on HKII. These findings were further confirmed when HIF-1
stabilization in the rat and murine heart resulted in smaller myocardial infarct
sizes (both in vivo and ex vivo), decreased mitochondrial oxidative stress, and
inhibited MPTP opening following IRI, effects which were also found to be
dependent on HKII.
CONCLUSIONS: We have demonstrated that acute HIF-1α stabilization using either a
pharmacological or genetic approach protected the heart against acute IRI by
promoting aerobic glycolysis, decreasing mitochondrial oxidative stress,
activating HKII, and inhibiting MPTP opening. This study has shown that purified recombit human α-synuclein (20 μM) causes
membrane depolarization and loss of phosphorylation capacity of isolated
purified rat brain mitochondria by activating permeability transition pore
complex. In intact SHSY5Y (human neuroblastoma cell line) cells, lactacystin (5
μM), a proteasomal inhibitor, causes an accumulation of α-synuclein with
concomitant mitochondrial dysfunction and cell death. The effects of lactacystin
on intact SHSY5Y cells are, however, prevented by knocking down α-synuclein
expression by specific siRNA. Furthermore, in wild-type (non-transfected) SHSY5Y
cells, the effects of lactacystin on mitochondrial function and cell viability
are also prevented by cyclosporin A (1 μM) which blocks the activity of the
mitochondrial permeability transition pore. Likewise, in wild-type SHSY5Y cells,
typical mitochondrial poison like antimycin A (50 nM) produces loss of cell
viability comparable to that of lactacystin (5 μM). These data, in combination
with those from isolated brain mitochondria, strongly suggest that
intracellularly accumulated α-synuclein can interact with mitochondria in intact
SHSY5Y cells causing dysfunction of the organelle which drives the cell death
under our experimental conditions. The results have clear implications in the
pathogenesis of sporadic Parkinson's disease. α-Synuclein is shown to cause
mitochondrial impairment through interaction with permeability transition pore
complex in isolated preparations. Intracellular accumulation of α-synuclein in
SHSY5Y cells following proteasomal inhibition leads to mitochondrial impairment
and cell death which could be prevented by knocking down α-synuclein gene. The
results link mitochondrial dysfunction and α-synuclein accumulation, two key
pathogenic mechanisms of Parkinson's disease, in a common damage pathway. Myocardial dysfunction in sepsis is associated with an increased risk of
mortality. The mitochondrial aldehyde dehydrogenase (ALDH2) enzyme is crucial
for protecting the heart from ischemic injury. The aim of the present study was
to determine the role of ALDH2 in cardiac dysfunction induced by
lipopolysaccharide (LPS). Male rats were treated intraperitoneally with LPS, and
their stroke volume and cardiac output were evaluated using an M‑mode
echocardiograph. The expression levels and activity of ALDH2, nitric oxide
content and inducible nitric oxide synthase (iNOS) activity, and the opening of
the mitochondrial permeability transition pore (MPTP) were also evaluated.
Treatment with LPS (5, 10, or 20 mg/kg) resulted in a steady decrease in cardiac
output and stroke volume. The ALDH2 activity was decreased in a dose‑dependent
manner; however, the ALDH2 protein expression levels remained unchanged. Alda‑1,
a specific activator of ALDH2, increased the activity of ALDH2 and lessened
LPS‑induced cardiac dysfunction. A marked opening of the MPTP was observed 12 h
following treatment with LPS, which was prevented by Alda‑1. The improvement in
cardiac function in response to treatment with Alda‑1, was partially prevented
by treatment with the MPTP inhibitor atractyloside. LPS treatment induced an
increase in iNOS activation and inhibition of ALDH2 activity. The iNOS selective
inhibitor, aminoguanidine, partially reversed the LPS‑induced ALDH2 activity
decrease and MPTP opening. These results indicate that ALDH2 activity may have a
role in protecting against LPS‑induced cardiac dysfunction. The potential
mechanism may involve inhibition of MPTP opening and iNOS expression. BACKGROUND: Protoporphyrin IX (PpIX) and its derivatives are widely used in
photodynamic therapy (PDT) to kill cancer cells. Studies showed that the
application of these drugs could cause systemic toxic effects in human. However,
the molecular pathways involved in PpIX-induced cytotoxicity are not
well-defined. Macrophages represent the primary system for protecting tissues
from toxicants and initiating the resolution of inflammation. Thus, this study
aims to investigate the toxicity of PpIX on macrophages and provide strategies
to prevent the toxic effects.
METHODS: THP-1 macrophages were incubated with PpIX and cell death was measured
by MTT assay and Annexin V-PI staining. Intracellular reactive oxygen species
(ROS) were evaluated by 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and
MitoSOX® Red staining and mitochondrial membrane potential (ΔΨm) was detected by
tetramethylrhodamine methyl ester (TMRM) staining. Mitogen-activated protein
(MAP) kinase activation was assayed by western blotting. Mitochondrial
permeability transition pore (mPTP) opening was measured by calcein
loading/Co(2+) quenching technique and evaluating the release of mitochondrial
content.
RESULTS: PpIX reduced cell viability in a dose- and time-dependent manner. The
cell death was characterized by increasing PI-positive cells, ATP depletion, LDH
releasing and rapid ΔΨm loss favoring necrotic features. In addition, PpIX
successively induced ROS production, c-Jun N-terminal protein kinase (JNK)
activation and mPTP opening. ROS scavengers, N-acetylcysteine (NAC) and
deferoxamine (DFX), JNK inhibitor, SP600125, and mPTP inhibitor, cyclosporin A
(CsA), all significantly rescued this cell death. Furthermore, mPTP opening was
directly regulated by ROS/JNK pathway.
CONCLUSION: PpIX induces a necrotic cell death in THP-1 macrophages through ROS
production, JNK activation, and mPTP opening. It is tempting to speculate that
blocking the pathways involved in the cytotoxic effects of PpIX will alleviate
its side effects. To investigate the influence of the mitochondrial calcium uniporter on the
mitochondrial permeability transition pore, the present study observed
mitochondrial morphology in cortical neurons isolated from adult rats using
transmission electron microscopy, and confirmed the morphology and activity of
isolated mitochondria by detecting succinic dehydrogenase and monoamine oxidase,
two mitochondrial enzymes. Isolated mitochondria were treated with either
ruthenium red, an inhibitor of the uniporter, spermine, an activator of the
uniporter, or in combination with cyclosporin A, an inhibitor of the
mitochondrial permeability transition pore. Results showed that ruthenium red
inhibited CaCl2-induced mitochondrial permeability transition pore opening,
spermine enhanced opening, and cyclosporin A attenuated the effects of spermine.
Results demonstrated that the mitochondrial calcium uniporter plays a role in
regulating the mitochondrial permeability transition pore in mitochondria
isolated from the rat brain cortex. |
What is ceritinib? | Ceritinib is a second generation tyrosine kinase inhibitor, that serves as an effective and approved oral therapy for patients with ALK-rearranged non-small cell lung cancer. | BACKGROUND: Non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma
kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib,
but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor
that has shown greater antitumor potency than crizotinib in preclinical studies.
METHODS: In this phase 1 study, we administered oral ceritinib in doses of 50 to
750 mg once daily to patients with advanced cancers harboring genetic
alterations in ALK. In an expansion phase of the study, patients received the
maximum tolerated dose. Patients were assessed to determine the safety,
pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies
were performed before ceritinib treatment to identify resistance mutations in
ALK in a group of patients with NSCLC who had had disease progression during
treatment with crizotinib.
RESULTS: A total of 59 patients were enrolled in the dose-escalation phase. The
maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic
events included diarrhea, vomiting, dehydration, elevated aminotransferase
levels, and hypophosphatemia. This phase was followed by an expansion phase, in
which an additional 71 patients were treated, for a total of 130 patients
overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib
per day, the overall response rate was 58% (95% confidence interval [CI], 48 to
67). Among 80 patients who had received crizotinib previously, the response rate
was 56% (95% CI, 45 to 67). Responses were observed in patients with various
resistance mutations in ALK and in patients without detectable mutations. Among
patients with NSCLC who received at least 400 mg of ceritinib per day, the
median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5).
CONCLUSIONS: Ceritinib was highly active in patients with advanced,
ALK-rearranged NSCLC, including those who had had disease progression during
crizotinib treatment, regardless of the presence of resistance mutations in ALK.
(Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number,
NCT01283516.). Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK)
gene rearrangements invariably develop resistance to the ALK tyrosine kinase
inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation
of the next-generation ALK TKI, ceritinib (LDK378), in the setting of crizotinib
resistance. An interrogation of in vitro and in vivo models of acquired
resistance to crizotinib, including cell lines established from biopsies of
patients with crizotinib-resistant NSCLC, revealed that ceritinib potently
overcomes crizotinib-resistant mutations. In particular, ceritinib effectively
inhibits ALK harboring L1196M, G1269A, I1171T, and S1206Y mutations, and a
cocrystal structure of ceritinib bound to ALK provides structural bases for this
increased potency. However, we observed that ceritinib did not overcome two
crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these
mutations was identified in 5 of 11 biopsies from patients with acquired
resistance to ceritinib. Altogether, our results demonstrate that ceritinib can
overcome crizotinib resistance, consistent with clinical data showing marked
efficacy of ceritinib in patients with crizotinib-resistant disease.
SIGNIFICANCE: The second-generation ALK inhibitor ceritinib can overcome several
crizotinib-resistant mutations and is potent against several in vitro and in
vivo laboratory models of acquired resistance to crizotinib. These findings
provide the molecular basis for the marked clinical activity of ceritinib in
patients with ALK-positive NSCLC with crizotinib-resistant disease. Cancer
Discov; 4(6); 662-73. ©2014 AACR. See related commentary by Ramalingam and
Khuri, p. 634 This article is highlighted in the In This Issue feature, p. 621. Ceritinib is an oral anaplastic lymphoma kinase (ALK) inhibitor developed by
Novartis for the treatment of tumours characterised by genetic abnormalities in
ALK. ALK is a member of the insulin receptor family of tyrosine kinases that can
become oncogenic when fused to other proteins. Ceritinib has been approved in
the US under 'Breakthrough Therapy' designation for the second-line treatment of
ALK-positive non-small cell lung cancer (NSCLC). Regulatory submissions have
also been made in the EU and other countries. Phase III development is ongoing
worldwide to evaluate ceritinib both as a first- and second-line therapy for
ALK-positive NSCLC. This article summarizes the milestones in the development of
ceritinib leading to this first approval for the treatment of patients with
ALK-positive metastatic NSCLC who have progressed on or are intolerant to
crizotinib. Non-small cell lung cancer (NSCLC) represents the paradigm of personalized
treatment in human cancer. Several oncogenic alterations with druggable
potential have been identified, with ALK rearrangements representing one of the
newest and most appealing. Crizotinib is now recognized as the standard of care
in chemotherapy-pretreated ALK-positive NSCLC due to the positive results of a
recently published trial. Unfortunately, no patient exposed to crizotinib can be
cured, and after a median time of 1 year, resistance inevitably occurs.
Overcoming resistance is the major challenge in clinical oncology and many
molecules are currently under evaluation, including ceritinib (LDK-378).
Ceritinib is an oral, potent, second-generation ALK inhibitor recently approved
by the U.S. Food and Drug Administration. Preclinical data showed impressive
antitumor activity against crizotinib-resistant clones, and based on available
data, ceritinib could represent a suitable option in crizotinib-resistant NSCLC. OBJECTIVE: The echinoderm microtubule associated protein-like 4
(EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients
with non-small cell lung cancer. To the best of our knowledge, there are only
three cell lines harboring the EML4-ALK fusion gene, which have contributed to
the development of therapeutic strategies. Therefore, we tried to establish a
new lung cancer cell line harboring EML4-ALK.
METHODS: A 61-year-old Japanese female presented with chest discomfort. She was
diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was
treated with chemotherapy, her disease progressed with massive pleural effusion.
Because the EML4-ALK rearrangement was found in a biopsied specimen using
fluorescence in situ hybridization, she was treated with crizotinib. She did
well for 3 months.
RESULTS: Tumor cells were obtained from the maligt pleural effusion before
treatment with crizotinib. Cells continued to proliferate substantially for
several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein
and genes were identified in ABC-11 cells using fluorescence in situ
hybridization and immunohistochemistry, respectively. ABC-11 cells were
sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and
AP26113), as determined by a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Phosphorylated ALK protein and its downstream signaling were suppressed by
treatment with crizotinib in western blotting. Furthermore, we could transplant
ABC-11 cells subcutaneously into BALB/c nu/nu mice.
CONCLUSIONS: We successfully established a new lung adenocarcinoma cell line
harboring the EML4-ALK fusion gene. This cell line could contribute to future
research of EML4-ALK-positive lung cancer both in vivo and in vitro. PURPOSE: The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is
a standard therapy for patients with ALK-rearranged non-small cell lung cancer
(NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown
promising activity in crizotinib-resistant patients. As patients still relapse
even on these next-generation ALK-TKIs, we examined mechanisms of resistance to
the next-generation ALK-TKI alectinib and potential strategies to overcome this
resistance.
EXPERIMENTAL DESIGN: We established a cell line model of alectinib resistance,
and analyzed a resistant tumor specimen from a patient who had relapsed on
alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations
and evaluated the potency of other next-generation ALK-TKIs in these models. We
tested the antitumor activity of the next-generation ALK-TKI ceritinib in the
patient with acquired resistance to alectinib. To elucidate structure-activity
relationships of ALK mutations, we performed computational thermodynamic
simulation with MP-CAFEE.
RESULTS: We identified a novel V1180L gatekeeper mutation from the cell line
model and a second novel I1171T mutation from the patient who developed
resistance to alectinib. Both ALK mutations conferred resistance to alectinib as
well as to crizotinib, but were sensitive to ceritinib and other next-generation
ALK-TKIs. Treatment of the patient with ceritinib led to a marked response.
Thermodynamics simulation suggests that both mutations lead to distinct
structural alterations that decrease the binding affinity with alectinib.
CONCLUSIONS: We have identified two novel ALK mutations arising after alectinib
exposure that are sensitive to other next-generation ALK-TKIs. The ability of
ceritinib to overcome alectinib-resistance mutations suggests a potential role
for sequential therapy with multiple next-generation ALK-TKIs. INTRODUCTION: The epidermal growth factor receptor (EGFR) is mutated in 15% of
adenocarcinomas of the lung. In addition, the anaplastic lymphoma kinase (ALK)
is altered in 8% of adenocarcinomas of the lung. Treatment of EGFR mutant and
ALK translocation-positive tumors in NSCLC with tyrosine kinase inhibitors (TKI)
results in a dramatic therapeutic response and has revolutionized therapy.
Unfortunately, resistance to TKIs invariably develops. Many promising new
therapies are under investigation to overcome the resistance.
AREAS COVERED: We analyzed the current primary literature and recent national
meetings to evaluate the clinical characteristics and therapeutic implications
of relevant treatments for EGFR mutant and ALK-positive NSCLC in the first-line,
acquired resistance, and adjuvant settings.
EXPERT OPINION: Treatment with EGFR TKIs in the first-line setting of EGFR
mutant NSCLC results in a significant clinical benefit. Several promising third
generation EGFR TKIs are being evaluated in Phase II and III trials in the
acquired resistance setting. Crizotinib is superior to chemotherapy in the
first-line setting for ALK-positive NSCLC. Ceritinib is effective and approved
for ALK-positive NSCLC in the acquired resistance setting. Continued
investigation is needed to develop novel therapies to overcome acquired
resistance to TKIs. The success in identifying the chromosomal rearrangements involving the
anaplastic lymphoma kinase (ALK) as an oncogenic driver has thoroughly changed
the treatment of non-small-cell lung cancer. In the past decade, targeted drugs
have emerged as an efficient personalized strategy for ALK-rearranged
non-small-cell lung cancer. The accelerated approval of potent ALK inhibitors,
such as crizotinib and more recently ceritinib (LDK378), based on the well
designed phase I/II trials has been a landmark success in clinical cancer
research and contributes a new era of oncogenic targeted therapy characterized
by elegant clinical trial design. In this review, we aim to present the current
knowledge on acquired resistance of crizotinib known as a first-in-class ALK
inhibitor and potential solutions to improve the cost-effectiveness, and to
review the difference between ceritinib and crizotinib; preclinical data and
results of the elegant early clinical trial of ceritinib which promoted its
accelerated approval, pharmacokinetics, safety profile, and tolerability, the
updated results (eg, efficacy on brain metastases), and robust design of ongoing
phase II/III trials, and future directions of ceritinib to be a potent
alternative to crizotinib for ALK-rearranged non-small-cell lung cancer are also
presented. Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a
variety of human cancers and are attractive targets for cancer therapy. The
MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated
promising clinical activity in ROS1 fusion-positive non-small cell lung cancer.
However, emerging clinical evidence has shown that patients can develop
resistance by acquiring secondary point mutations in ROS1 kinase. In this study
we characterized the ROS1 activity of PF-06463922, a novel, orally available,
CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro,
PF-06463922 exhibited subomolar cellular potency against oncogenic ROS1
fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the
ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the
second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922
showed significantly improved inhibitory activity against ROS1 kinase. A crystal
structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions
contributing to the high-affinity binding. In vivo, PF-06463922 showed marked
antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the
CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor
activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken
together, our results indicate that PF-06463922 has potential for treating ROS1
fusion-positive cancers, including those requiring agents with CNS-penetrating
properties, as well as for overcoming crizotinib resistance driven by ROS1
mutation. The anaplastic lymphoma kinase (ALK) is chromosomally rearranged in a subset of
certain cancers, including 2% to 7% of non-small cell lung cancers (NSCLC) and
∼70% of anaplastic large cell lymphomas (ALCL). The ALK kinase inhibitors
crizotinib and ceritinib are approved for relapsed ALK(+) NSCLC, but acquired
resistance to these drugs limits median progression-free survival on average to
∼10 months. Kinase domain mutations are detectable in 25% to 37% of resistant
NSCLC samples, with activation of bypass signaling pathways detected frequently
with or without concurrent ALK mutations. Here we report that, in contrast to
NSCLC cells, drug-resistant ALCL cells show no evidence of bypassing ALK by
activating alternate signaling pathways. Instead, drug resistance selected in
this setting reflects upregulation of ALK itself. Notably, in the absence of
crizotinib or ceritinib, we found that increased ALK signaling rapidly arrested
or killed cells, allowing a prolonged control of drug-resistant tumors in vivo
with the administration of discontinuous rather than continuous regimens of drug
dosing. Furthermore, even when drug resistance mutations were detected in the
kinase domain, overexpression of the mutant ALK was toxic to tumor cells. We
confirmed these findings derived from human ALCL cells in murine pro-B cells
that were transformed to cytokine independence by ectopic expression of an
activated NPM-ALK fusion oncoprotein. In summary, our results show how ALK
activation functions as a double-edged sword for tumor cell viability, with
potential therapeutic implications. |
Which protein is found to be mutated in Friedreich's ataxia? | It is generally accepted that Friedreich's ataxia (FRDA) is caused by a deficiency in frataxin expression, a mitochondrial protein involved in iron homeostasis, which mainly affects the brain, dorsal root ganglia of the spinal cord, heart and in certain cases the pancreas | Frataxin is a mitochondrial protein deficient in Friedreich ataxia (FRDA) and
which is associated with abnormal intramitochondrial iron handling. We
identified the mitochondrial processing peptidase beta (MPPbeta) as a frataxin
protein partner using the yeast two-hybrid assay. In in vitro assays, MPPbeta
binds frataxin which is cleaved by the reconstituted MPP heterodimer. MPP
cleavage of frataxin results in an intermediate form (amino acids 41-210) that
is processed further to the mature form. In vitro and in vivo experiments
suggest that two C-terminal missense mutations found in FRDA patients modulate
interaction with MPPbeta, resulting in a slower maturation process at the normal
cleavage site. The slower processing rate of frataxin carrying such missense
mutations may therefore contribute to frataxin deficiency, in addition to an
impairment of its function. Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused
by a deficiency of frataxin, a conserved mitochondrial protein of unknown
function. Mitochondrial iron accumulation, loss of iron-sulfur
cluster-containing enzymes and increased oxidative damage occur in yeast and
mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA
patients, suggesting that frataxin may be involved in export of iron from the
mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative
damage. We have previously shown that yeast frataxin has structural and
functional features of an iron storage protein. In this study we have
investigated the function of human frataxin in Escherichia coli and
Saccharomyces cerevisiae. When expressed in E.coli, the mature form of human
frataxin assembles into a stable homopolymer that can bind approximately 10
atoms of iron per molecule of frataxin. The iron-loaded homopolymer can be
detected on non-denaturing gels by either protein or iron staining demonstrating
a stable association between frataxin and iron. As analyzed by gel filtration
and electron microscopy, the homopolymer consists of globular particles of
approximately 1 MDa and ordered rod-shaped polymers of these particles that
accumulate small electron-dense cores. When the human frataxin precursor is
expressed in S.cerevisiae, the mitochondrially generated mature form is
separated by gel filtration into monomer and a high molecular weight pool of
>600 kDa. A high molecular weight pool of frataxin is also present in mouse
heart indicating that frataxin can assemble under native conditions. In
radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation
with approximately five atoms of (55)Fe bound per molecule. These findings
suggest that FRDA results from decreased mitochondrial iron storage due to
frataxin deficiency which may impair iron metabolism, promote oxidative damage
and lead to progressive iron accumulation. Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative
disease, is caused by low expression of frataxin, a small mitochondrial protein,
encoded in the nucleus. At the biochemical level, the lack of frataxin leads to
dysregulation of mitochondrial iron homeostasis and oxidative damage, which
eventually causes neuronal death. It is, however, still unclear whether frataxin
is directly involved in iron binding, since the yeast orthologue, but not the
human protein, has been shown to form large aggregates in the presence of large
iron excess. We have compared the properties of three proteins from the frataxin
family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human
frataxin--as representative of organisms of increasing complexity. We show that
the three proteins have the same fold but different thermal stabilities and
iron-binding properties. While human frataxin has no tendency to bind iron, CyaY
forms iron-promoted aggregates with a behaviour similar to that of yeast
frataxin. However, aggregation can be competed by chelator agents or by ionic
strength. At physiological salt conditions, almost no aggregation is observed.
The design of mutants produced to identify the protein surface involved in
iron-promoted aggregation allows us to demonstrate that the process is mediated
by a negatively charged surface ridge. Mutation of three of these residues is
sufficient to convert CyaY in a protein with properties similar to those of
human frataxin. On the other hand, mutation of the exposed surface of the beta
sheet, which contains most of the conserved residues, does not affect
aggregation, suggesting that iron binding is a non-conserved part of a more
complex cellular function of frataxins. Friedreich's ataxia is the most frequent inherited ataxia in Caucasians. It is
caused by deficiency of frataxin, a highly conserved nuclear-encoded protein
localized in mitochondria. The DNA abnormality found in 98% of Friedreich's
ataxia chromosomes is the unstable hyperexpansion of a GAA triplet repeat in the
first intron of the frataxin gene. Most patients are homozygous for this repeat
expansion. The expanded GAA repeat causes frataxin deficiency because it
interferes with the transcription of the gene by adopting a non-B (probably
triple helical) structure. Longer repeats cause a more profound frataxin
deficiency and are associated with earlier onset and increased severity of the
disease. Molecular testing has shown that the phenotypic spectrum of
Friedreich's ataxia is wider than previously thought. Up to 10% of patients with
recessive or sporadic degenerative ataxia who do not fulfill the Friedreich's
ataxia diagnostic criteria are homozygous for expanded alleles at the
Friedreich's ataxia locus. Late age of onset, retained tendon reflexes, and lack
of pyramidal signs are among the atypical features observed in some patients
with a positive molecular test. Yeast cells deficient in the frataxin homologue
accumulate iron in mitochondria and show increased sensitivity to oxidative
stress. This suggests that Friedreich's ataxia is caused by mitochondrial
dysfunction and free radical toxicity, with consequent mitochondrial damage,
axonal degeneration, and cell death. The severe reduction in mRNA and protein levels of the mitochondrial protein
frataxin, encoded by the X25 gene, causes Friedreich ataxia (FRDA), the most
common form of recessive hereditary ataxia. Increasing evidence underlines the
pathogenetic role of oxidative stress in this disease. We generated an in vitro
cellular model of regulated human frataxin overexpression. We identified, by
differential display technique, the mitogen activated protein kinase kinase 4
mRNA down regulation in frataxin overexpressing cells. We studied the stress
kinases pathway in this cellular model and in fibroblasts from FRDA patients.
Frataxin overexpression reduced c-Jun N-terminal kinase phosphorylation.
Furthermore, exposure of FRDA fibroblasts to several forms of environmental
stress caused an up regulation of phospho-JNK and phospho-c-Jun. To understand
if this susceptibility results in cell death, we have investigated the
involvement of caspases. A significantly higher activation of caspase-9 was
observed in FRDA versus control fibroblasts after serum-withdrawal. Our findings
suggest the presence, in FRDA patient cells, of a 'hyperactive' stress signaling
pathway. The role of frataxin in FRDA pathogenesis could be explained, at least
in part, by this hyperactivity. The neurodegenerative disorder FRDA (Friedreich's ataxia) results from a
deficiency in frataxin, a putative iron chaperone, and is due to the presence of
a high number of GAA repeats in the coding regions of both alleles of the
frataxin gene, which impair protein expression. However, some FRDA patients are
heterozygous for this triplet expansion and contain a deleterious point mutation
on the other allele. In the present study, we investigated whether two
particular FRDA-associated frataxin mutants, I154F and W155R, result in unfolded
protein as a consequence of a severe structural modification. A detailed
comparison of the conformational properties of the wild-type and mutant proteins
combining biophysical and biochemical methodologies was undertaken. We show that
the FRDA mutants retain the native fold under physiological conditions, but are
differentially destabilized as reflected both by their reduced thermodynamic
stability and a higher tendency towards proteolytic digestion. The I154F mutant
has the strongest effect on fold stability as expected from the fact that the
mutated residue contributes to the hydrophobic core formation. Functionally, the
iron-binding properties of the mutant frataxins are found to be partly impaired.
The apparently paradoxical situation of having clinically aggressive frataxin
variants which are folded and are only significantly less stable than the
wild-type form in a given adverse physiological stress condition is discussed
and contextualized in terms of a mechanism determining the pathology of FRDA
heterozygous. Friedreich's ataxia results from a deficiency in the mitochondrial protein
frataxin, which carries single point mutations in some patients. In the present
study, we analysed the consequences of different disease-related mutations in
vitro on the stability and dynamics of human frataxin. Two of the mutations,
G130V and D122Y, were investigated for the first time. Analysis by CD
spectroscopy demonstrated a substantial decrease in the thermodynamic stability
of the variants during chemical and thermal unfolding (wild-type > W155R > I154F
> D122Y > G130V), which was reversible in all cases. Protein dynamics was
studied in detail and revealed that the mutants have distinct propensities
towards aggregation. It was observed that the mutants have increased correlation
times and different relative ratios between soluble and insoluble/aggregated
protein. NMR showed that the clinical mutants retained a compact and relatively
rigid globular core despite their decreased stabilities. Limited proteolysis
assays coupled with LC-MS allowed the identification of particularly flexible
regions in the mutants; interestingly, these regions included those involved in
iron-binding. In agreement, the iron metallochaperone activity of the
Friedreich's ataxia mutants was affected: some mutants precipitate upon iron
binding (I154F and W155R) and others have a lower binding stoichiometry (G130V
and D122Y). Our results suggest that, in heterozygous patients, the development
of Friedreich's ataxia may result from a combination of reduced efficiency of
protein folding and accelerated degradation in vivo, leading to lower than
normal concentrations of frataxin. This hypothesis also suggests that, although
quite different from other neurodegenerative diseases involving toxic
aggregation, Friedreich's ataxia could also be linked to a process of protein
misfolding due to specific destabilization of frataxin. Severe mitochondria deficiency leads to a number of devastating degenerative
disorders, yet, mild mitochondrial dysfunction in different species, including
the nematode Caenorhabditis elegans, can have pro-longevity effects. This
apparent paradox indicates that cellular adaptation to partial mitochondrial
stress can induce beneficial responses, but how this is achieved is largely
unknown. Complete absence of frataxin, the mitochondrial protein defective in
patients with Friedreich's ataxia, is lethal in C. elegans, while its partial
deficiency extends animal lifespan in a p53 dependent manner. In this paper we
provide further insight into frataxin control of C. elegans longevity by showing
that a substantial reduction of frataxin protein expression is required to
extend lifespan, affect sensory neurons functionality, remodel lipid metabolism
and trigger autophagy. We find that Beclin and p53 genes are required to induce
autophagy and concurrently reduce lipid storages and extend animal lifespan in
response to frataxin suppression. Reciprocally, frataxin expression modulates
autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts
also display increased autophagy, indicating an evolutionarily conserved
response to reduced frataxin expression. In sum, we demonstrate a causal
connection between induction of autophagy and lifespan extension following
reduced frataxin expression, thus providing the rationale for investigating
autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly
other human mitochondria-associated disorders. In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss
of iron-sulfur (Fe-S) cluster protein activity, accumulation of mitochondrial
iron and leads to the neurodegenerative disease Friedreich's ataxia. In
contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not
to cause any significant phenotype, questioning both its importance and its
actual contribution to Fe-S cluster biogenesis. Because FXN is conserved between
eukaryotes and prokaryotes, this surprising discrepancy prompted us to
reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i)
potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the
maturation of Fe-S cluster-containing proteins and (iii) requires iron-rich
conditions for its contribution to be significant. A genetic interaction was
discovered between cyaY and iscX, the last gene of the isc operon. Deletion of
both genes showed an additive effect on Fe-S cluster protein maturation, which
led, among others, to increased resistance to aminoglycosides and increased
sensitivity to lambda phage infection. Together, these in vivo results establish
the importance of CyaY as a member of the ISC-mediated Fe-S cluster biogenesis
pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona
fide Fe-S cluster biogenesis factor. Friedreich's ataxia is a severe neurodegenerative disease caused by the
decreased expression of frataxin, a mitochondrial protein that stimulates
iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S
cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation
of a persulfide intermediate on NFS1. Here we show that frataxin modulates the
reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds
along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our
data reveal that in the presence of ISCU, frataxin enhances the rate of two
similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the
accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer
to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of
these thiols and ultimately sulfide release. These data raise important
questions on the physiological mechanism of Fe-S cluster assembly and point to a
unique function of frataxin as an enhancer of sulfur transfer within the
NFS1-ISD11-ISCU complex. |
Which is the process that Conserved noncoding elements mostly regulate? | Conserved noncoding elements play a fundamental role in regulating animal development | Fish-mammal genomic comparisons have proved powerful in identifying conserved
noncoding elements likely to be cis-regulatory in nature, and the majority of
those tested in vivo have been shown to act as tissue-specific enhancers
associated with genes involved in transcriptional regulation of development.
Although most of these elements share little sequence identity to each other, a
small number are remarkably similar and appear to be the product of duplication
events. Here, we searched for duplicated conserved noncoding elements in the
human genome, using comparisons with Fugu to select putative cis-regulatory
sequences. We identified 124 families of duplicated elements, each containing
between two and five members, that are highly conserved within and between
vertebrate genomes. In 74% of cases, we were able to assign a specific set of
paralogous genes with annotation relating to transcriptional regulation and/or
development to each family, thus removing much of the ambiguity in identifying
associated genes. We find that duplicate elements have the potential to
up-regulate reporter gene expression in a tissue-specific manner and that
expression domains often overlap, but are not necessarily identical, between
family members. Over two thirds of the families are conserved in duplicate in
fish and appear to predate the large-scale duplication events thought to have
occurred at the origin of vertebrates. We propose a model whereby gene
duplication and the evolution of cis-regulatory elements can be considered in
the context of increased morphological diversity and the emergence of the modern
vertebrate body plan. BACKGROUND: The neuronal synapse is a fundamental functional unit in the central
nervous system of animals. Because synaptic function is evolutionarily
conserved, we reasoned that functional sequences of genes and related genomic
elements known to play important roles in neurotransmitter release would also be
conserved.
RESULTS: Evolutionary rate analysis revealed that presynaptic proteins evolve
slowly, although some members of large gene families exhibit accelerated
evolutionary rates relative to other family members. Comparative sequence
analysis of 46 megabases spanning 150 presynaptic genes identified more than
26,000 elements that are highly conserved in eight vertebrate species, as well
as a small subset of sequences (6%) that are shared among unrelated presynaptic
genes. Analysis of large gene families revealed that upstream and intronic
regions of closely related family members are extremely divergent. We also
identified 504 exceptionally long conserved elements (> or =360 base pairs, > or
=80% pair-wise identity between human and other mammals) in intergenic and
intronic regions of presynaptic genes. Many of these elements form a highly
stable stem-loop RNA structure and consequently are candidates for novel
regulatory elements, whereas some conserved noncoding elements are shown to
correlate with specific gene expression profiles. The SynapseDB online database
integrates these findings and other functional genomic resources for synaptic
genes.
CONCLUSION: Highly conserved elements in nonprotein coding regions of 150
presynaptic genes represent sequences that may be involved in the
transcriptional or post-transcriptional regulation of these genes. Furthermore,
comparative sequence analysis will facilitate selection of genes and noncoding
sequences for future functional studies and analysis of variation studies in
neurodevelopmental and psychiatric disorders. Conserved noncoding elements (CNEs) constitute the majority of sequences under
purifying selection in the human genome, yet their function remains largely
unknown. Experimental evidence suggests that many of these elements play
regulatory roles, but little is known about regulatory motifs contained within
them. Here we describe a systematic approach to discover and characterize
regulatory motifs within mammalian CNEs by searching for long motifs (12-22 nt)
with significant enrichment in CNEs and studying their biochemical and genomic
properties. Our analysis identifies 233 long motifs (LMs), matching a total of
approximately 60,000 conserved instances across the human genome. These motifs
include 16 previously known regulatory elements, such as the histone 3'-UTR
motif and the neuron-restrictive silencer element, as well as striking examples
of novel functional elements. The most highly enriched motif (LM1) corresponds
to the X-box motif known from yeast and nematode. We show that it is bound by
the RFX1 protein and identify thousands of conserved motif instances, suggesting
a broad role for the RFX family in gene regulation. A second group of motifs
(LM2*) does not match any previously known motif. We demonstrate by biochemical
and computational methods that it defines a binding site for the CTCF protein,
which is involved in insulator function to limit the spread of gene activation.
We identify nearly 15,000 conserved sites that likely serve as insulators, and
we show that nearby genes separated by predicted CTCF sites show markedly
reduced correlation in gene expression. These sites may thus partition the human
genome into domains of expression. 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. Sequence conservation has traditionally been used as a means to target
functional regions of complex genomes. In addition to its use in identifying
coding regions of genes, the recent availability of whole genome data for a
number of vertebrates has permitted high-resolution analyses of the noncoding
"dark matter" of the genome. This has resulted in the identification of a large
number of highly conserved sequence elements that appear to be preserved in all
bony vertebrates. Further positional analysis of these conserved noncoding
elements (CNEs) in the genome demonstrates that they cluster around genes
involved in developmental regulation. This chapter describes the identification
and characterization of these elements, with particular reference to their
composition and organization. Pan-vertebrate developmental cis-regulatory elements are discernible as highly
conserved noncoding elements (HCNEs) and are often dispersed over large areas
around the pleiotropic genes whose expression they control. On the loci of two
developmental transcription factor genes, SOX3 and PAX6, we demonstrate that
HCNEs conserved between human and zebrafish can be systematically and reliably
tested for their regulatory function in multiple stable transgenes in zebrafish,
and their genomic reach estimated with confidence using synteny conservation and
HCNE density along these loci. HCNEs of both human and zebrafish function as
specific developmental enhancers in zebrafish. We show that human HCNEs result
in expression patterns in zebrafish equivalent to those in mouse, establishing
zebrafish as a suitable model for large-scale testing of human developmental
enhancers. Orthologous human and zebrafish enhancers underwent functional
evolution within their sequence and often directed related but non-identical
expression patterns. Despite an evolutionary distance of 450 million years, one
pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human
HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting
that multiple regulatory inputs are required to achieve robust and precise
complex expression patterns exhibited by developmental genes. Genomic regulatory blocks are chromosomal regions spanned by long clusters of
highly conserved noncoding elements devoted to long-range regulation of
developmental genes, often immobilizing other, unrelated genes into long-lasting
syntenic arrangements. Synorth http://synorth.genereg.net/ is a web resource for
exploring and categorizing the syntenic relationships in genomic regulatory
blocks across multiple genomes, tracing their evolutionary fate after teleost
whole genome duplication at the level of genomic regulatory block loci,
individual genes, and their phylogenetic context. Animal genomes possess highly conserved cis-regulatory sequences that are often
found near genes that regulate transcription and development. Researchers have
proposed that the strong conservation of these sequences may affect the
evolution of the surrounding genome, both by repressing rearrangement, and
possibly by promoting duplicate gene retention. Conflicting data, however, have
made the validity of these propositions unclear. Here, we use a new
computational method to identify phylogenetically conserved noncoding elements
(PCNEs) in a manner that is not biased by rearrangement and duplication. This
method is powerful enough to identify more than a thousand PCNEs that have been
conserved between vertebrates and the basal chordate amphioxus. We test 42 of
our PCNEs in transgenic zebrafish assays--including examples from vertebrates
and amphioxus--and find that the majority are functional enhancers. We find that
PCNEs are enriched around genes with ancient synteny conservation, and that this
association is strongest for extragenic PCNEs, suggesting that cis-regulatory
interdigitation plays a key role in repressing genome rearrangement. Next, we
classify mouse and zebrafish genes according to association with PCNEs, synteny
conservation, duplication history, and presence in bidirectional promoter pairs,
and use these data to cluster gene functions into a series of distinct
evolutionary patterns. These results demonstrate that subfunctionalization of
conserved cis-regulation has not been the primary determinate of gene duplicate
retention in vertebrates. Instead, the data support the gene balance hypothesis,
which proposes that duplicate retention has been driven by selection against
dosage imbalances in genes with many protein connections. One of the key discoveries of vertebrate genome sequencing projects has been the
identification of highly conserved noncoding elements (CNEs). Some
characteristics of CNEs include their high frequency in mammalian genomes, their
potential regulatory role in gene expression, and their enrichment in gene
deserts nearby master developmental genes. The abnormal development of neural
crest cells (NCCs) leads to a broad spectrum of congenital malformation(s),
termed neurocristopathies, and/or tumor predisposition. Here we review recent
findings that disruptions of CNEs, within or at long distance from the coding
sequences of key genes involved in NCC development, result in neurocristopathies
via the alteration of tissue- or stage-specific long-distance regulation of gene
expression. While most studies on human genetic disorders have focused on
protein-coding sequences, these examples suggest that investigation of genomic
alterations of CNEs will provide a broader understanding of the molecular
etiology of both rare and common human congenital malformations. The vertebrate Lhx2 is a member of the LIM homeobox family of transcription
factors. It is essential for the normal development of the forebrain, eye,
olfactory system and liver as well for the differentiation of lymphoid cells.
However, despite the highly restricted spatio-temporal expression pattern of
Lhx2, nothing is known about its transcriptional regulation. In mammals and
chicken, Crb2, Dennd1a and Lhx2 constitute a conserved linkage block, while the
intervening Dennd1a is lost in the fugu Lhx2 locus. To identify functional
enhancers of Lhx2, we predicted conserved noncoding elements (CNEs) in the
human, mouse and fugu Crb2-Lhx2 loci and assayed their function in transgenic
mouse at E11.5. Four of the eight CNE constructs tested functioned as
tissue-specific enhancers in specific regions of the central nervous system and
the dorsal root ganglia (DRG), recapitulating partial and overlapping expression
patterns of Lhx2 and Crb2 genes. There was considerable overlap in the
expression domains of the CNEs, which suggests that the CNEs are either
redundant enhancers or regulating different genes in the locus. Using a large
set of CNEs (810 CNEs) associated with transcription factor-encoding genes that
express predomitly in the central nervous system, we predicted four
over-represented 8-mer motifs that are likely to be associated with expression
in the central nervous system. Mutation of one of them in a CNE that drove
reporter expression in the neural tube and DRG abolished expression in both
domains indicating that this motif is essential for expression in these domains.
The failure of the four functional enhancers to recapitulate the complete
expression pattern of Lhx2 at E11.5 indicates that there must be other Lhx2
enhancers that are either located outside the region investigated or divergent
in mammals and fishes. Other approaches such as sequence comparison between
multiple mammals are required to identify and characterize such enhancers. |
What is the main mechanism by which human papillomavirus proteins E6 and E7 contribute to cell transformation? | Although they may have other targets, human papillomavirus proteins E6 and E7 interact with and block the function of p53 and pRb, respectively, therefore deregulating cell cycle and leading to cellular transformation. | Studies of adenovirus E1A oncoprotein mutants suggest that the association of
E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated
transformation. Mutational analysis of E1A indicates that two regions of pRB are
required for E1A to form stable complexes with the retinoblastoma protein. In
addition to pRB binding, these regions are necessary for E1A association with
several other cellular proteins, including p130, p107, cyclin A, and p33cdk2.
Here we show that short synthetic peptides containing the pRB-binding sequences
of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7
protein of human papillomavirus type 16 contains an element that binds to pRB
and appears to be functionally homologous to the E1A sequences. Peptides
containing this region of the E7 protein were able to interact with p107, cyclin
A, and p130 in addition to pRB. These findings suggest that the common mechanism
of transformation used by these viral oncogenes involves their association with
a set of polypeptides. Transformation by the human papillomavirus (HPV) early gene products, E6 and E7,
involves their interaction with cellular proteins p53 and Rb. Using glutathione
S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and
that the relative efficiency of binding varied such that the GST-HPV type 16 E6
(16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6,
and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53
purified from a baculovirus expression system as well as in vitro translation
sources, while no association was observed with GST-18E7 or a GST-16E6 mutant
bearing a five-amino-acid deletion in E6. When the site-specific DNA binding
activity of p53 was examined in the presence of GST-E6 proteins, an inhibition
of DNA binding was observed. The degree of inhibition correlated with the
relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most
potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least
effective. Prevention of p53 DNA binding is likely to play a role in the
abrogation of the transcriptional activity of p53 by HPV E6 and provides a
further mechanism for E6 disruption of p53 growth suppressor function in
addition to its role in directing specific degradation of p53 through the
ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with
the various E6 proteins may thus contribute to the differences in oncogenic
potential seen among the HPV types. The loss of the tumor-suppressor activity of p53, either by mutation or by
interaction with the human papillomavirus (HPV) E6 protein, is considered to be
an important mechanism in the carcinogenesis of cervical cancer. We have studied
the cytological distribution of these proteins in human cervical carcinoma cell
lines using polyclonal anti-p53 and monoclonal anti-E6 antibodies. The antibody
specificity was confirmed by immunoblot and immunocompetition analyses. The
intracellular localization of p53 and E6 was detected using the techniques of
conventional and three-dimensional confocal microscopy. In the HPV-18 or -16
integrated cell lines, HeLa, CaSki and SiHa, viral oncoprotein E6 and endogenous
tumor-suppressor protein, p53, were observed by immunofluorescence in the
cytoplasm; p53 also had a weak punctate staining in the nuclei of HeLa and CaSki
cells. In the HPV-negative cervical carcinoma cell lines, C-33A and HT-3, which
have mutated p53, p53 was localized predomitly to the nucleus, with C-33A
cells having elevated levels of p53 compared with the other cell lines. High
spatial resolution imaging, using confocal microscopy, was performed on the
cells after double fluorescence staining for p53 (fluorescein) and E6
(rhodamine). The images showed that both p53 and E6 had similar cytoplasmic
distributions, which implied that these two proteins may exist as a cytoplasmic
complex. To substantiate this implication, fluorescence resoce energy
transfer microscopy was performed, which provided direct evidence of a close
association between p53 and E6 within individual HeLa cells. The results from
this study support the theory that p53 protein binds HPV-16/18 E6 protein in the
cell cytoplasm, thus preventing p53 from exerting its tumor-suppressor function
in the nucleus. Hence, inactivation of wild-type p53 by p53-E6 complex formation
in cervical cancer may be a critical step in maligt transformation. High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with
the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7,
are commonly found to be expressed in these cancers. The presence of HPV-16 E7
is sufficient to induce epidermal hyperplasia and epithelial tumors in
transgenic mice. In this study, we have performed experiments in transgenic mice
to determine which domains of E7 contribute to these in vivo properties. The
human keratin 14 promoter was used to direct expression of mutant E7 genes to
stratified squamous epithelia in mice. The E7 mutants chosen had either an
in-frame deletion in the conserved region 2 (CR2) domain, which is required for
binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like
proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain
contributes to cellular transformation at a level other than pRb binding. Four
lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five
lines harboring a CR2 deletion were generated and were observed for overt and
histological phenotypes. A detailed time course analysis was performed to
monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of
high-level expression. In the transgenic mice with the wild-type E7 gene,
age-dependent expression of HPV-16 E7 correlated with the severity of epidermal
hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes
failed to result in any phenotypes. In addition, the transgenic mice with a
mutant E7 gene did not develop tumors. These experiments indicate that binding
and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are
necessary for induction of epidermal hyperplasia and carcinogenesis in mouse
skin and also suggest a role for the CR1 domain in the induction of these
phenotypes through as-yet-uncharacterized mechanisms. Studies on human papillomavirus type 16 have demonstrated that the product of
the early gene, E7, plays a key role in the immortalization and maligt
transformation of the host cell. Several of the biological activities of HPV16
E7 are mediated by inactivation of the members of the pocket protein family,
pRb, p107 and p130. In this study, we have characterized the in vitro properties
of five E7 proteins from benign and maligt HPV types (10, 32, 48, 54, 77). We
show that these E7 proteins associate with pRb and p107 with different
efficiencies. All E7s increased the proliferative rate of immortalized rodent
fibroblasts cultured in 10% calf serum containing medium. This property is
completely independent of their ability to associate with the pocket proteins.
Furthermore, all E7s, except HPV10 E7, stimulate G1/S progression and activated
the cyclin E and cyclin A promoter in the absence of growth factors. This
activity also does not correlate with the E7-efficiency of binding the pocket
proteins. Together these data provide evidence that different E7s alter the
regulation of the cell cycle by diverse mechanism(s). Finally, this comparative
analysis of the different E7 proteins demonstrates that the oncogenicity of a
HPV type is not determined by the ability of E7 to associate with the pocket
proteins. The high risk human papillomaviruses (HPVs) are associated with carcinomas of
cervix and other genital tumors. Previous studies have identified two viral
oncoproteins E6 and E7, which are expressed in the majority of HPV-associated
carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary
epithelial cells has provided a single gene model to study the mechanisms of
E6-induced oncogenic transformation. In recent years, it has become clear that
in addition to E6-induced degradation of p53 tumor suppressor protein, other
targets of E6 are required for mammary epithelial cells immortalization. Using
the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6
with protein kinase PKN, a fatty acid- and Rho small G protein-activated
serine/threonine kinase with a catalytic domain highly homologous to protein
kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in
wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins
of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all
the immortalization-competent and many immortalization-non-competent E6 mutants
bind PKN. These data suggest that binding to PKN may be required but not
sufficient for immortalizing normal mammary epithelial cells. Finally, we show
that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a
phosphoprotein. Our finding suggests a novel link between HPV E6 mediated
oncogenesis and regulation of a well known phosphorylation cascade. Mutational activation of the K-Ras oncogene is well established as a key genetic
step in the development and growth of pancreatic adenocarcinomas. However, the
mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor
cell growth remains to be fully elucidated. The recent use of primary human
cells to study Ras-mediated oncogenesis provides important model cell systems to
dissect this mechanism. We have used a model of telomerase-immortalized human
pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth
transformation. First, we found that human papillomavirus E6 and E7 oncogenes,
which block the function of the p53 and Rb tumor suppressors, respectively, and
SV40 small t antigen were required to allow mutant K-Ras(12D) growth
transformation. Second, K-Ras(12D) caused growth transformation in vitro,
including enhanced growth rate and loss of density dependency for growth,
anchorage independence, and invasion through reconstituted basement membrane
proteins, and tumorigenic transformation in vivo. Third, we determined that the
Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange
factor effector pathways were activated, although extracellular signal-regulated
kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic
inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling
impaired K-Ras-induced anchorage-independent growth and invasion. In summary,
our studies established, characterized, and validated E6/E7/st cells for the
study of Ras-induced oncogenesis. The recognition of a causal relationship between human papillomaviruses and
cancer almost 30 years ago led to a rapid expansion of knowledge in the field,
resulting in the description of the main mediators of HPV-induced
carcinogenesis, the viral proteins E6 and E7. These oncoproteins show a
remarkable pleiotropism in binding host-cell proteins, with the tumour
suppressor genes p53 and pRb as their major targets. These interactions induce
proliferation, immortalization and maligt transformation of infected cells.
The link between HPV and cervical cancer led to the development of molecular
methods, often based on the detection of E6 and E7, for screening and diagnosis.
Therapeutic vaccines and gene therapy are primarily directed at E6 and E7.
Although prophylactic vaccines are available, further understanding of the viral
life cycle and the mechanisms underlying HPV-induced oncogenesis is necessary to
face the many challenges in the field of HPV and cancer. DNA tumor viruses ensure genome amplification by hijacking the cellular
replication machinery and forcing infected cells to enter the S phase. The
retinoblastoma (Rb) protein controls the G1/S checkpoint, and is targeted by
several viral oncoproteins, among these the E7 protein from human
papillomaviruses (HPVs). A quantitative investigation of the interaction
mechanism between the HPV16 E7 protein and the RbAB domain in solution revealed
that 90% of the binding energy is determined by the LxCxE motif, with an
additional binding determit (1.0 kcal.mol(-1)) located in the C-terminal
domain of E7, establishing a dual-contact mode. The stoichiometry and
subomolar affinity of E7 indicated that it can bind RbAB as a monomer. The
low-risk HPV11 E7 protein bound 2.0 kcal.mol(-1) more weakly than the high-risk
HPV16 and HPV18 type counterparts, but the modularity and binding mode were
conserved. Phosphorylation at a conserved casein kinase II site in the natively
unfolded N-terminal domain of E7 affected the local conformation by increasing
the polyproline II content and stabilizing an extended conformation, which
allowed for a tighter interaction with the Rb protein. Thus, the E7-RbAB
interaction involves multiple motifs within the N-terminal domain of E7 and at
least two conserved interaction surfaces in RbAB. We discussed a mechanistic
model of the interaction of the Rb protein with a viral target in solution,
integrated with structural data and the analysis of other cellular and viral
proteins, which provided information about the balance of interactions involving
the Rb protein and how these determine the progression into either the normal
cell cycle or transformation. Cervical cancer is one of the leading causes of cancer death in women worldwide.
Human papillomavirus (HPV) infection is necessary but not sufficient for the
development of cervical cancer. Genomic instability caused by HPV allows cells
to acquire additional mutations required for maligt transformation. Genomic
instability in the form of polyploidy has been implicated in a causal role in
cervical carcinogenesis. Polyploidy not only occurs as an early event during
cervical carcinogenesis but also predisposes cervical cells to aneuploidy, an
important hallmark of human cancers. Cell cycle progression is regulated at
several checkpoints whose defects contribute to genomic instability.The
high-risk HPVs encode two oncogenes, E6 and E7, which are essential for cellular
transformation in HPV-positive cells. The ability of high-risk HPV E6 and E7
protein to promote the degradation of p53 and pRb, respectively, has been
suggested as a mechanism by which HPV oncogenes induce cellular transformation.
E6 and E7 abrogate cell cycle checkpoints and induce genomic instability that
leads to maligt conversion.Although the prophylactic HPV vaccine has recently
become available, it will not be effective for immunosuppressed individuals or
those who are already infected. Therefore, understanding the molecular basis for
HPV-associated cancers is still clinically relevant. Studies on genomic
instability will shed light on mechanisms by which HPV induces cancer and hold
promise for the identification of targets for drug development. Cervical carcinomas result from cellular transformation by the human
papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in
cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53
target genes as shown previously in the cervical carcinoma cell line HeLa. Here
we show that the TAp63β isoform of the p63 transcription factor is also a target
of E6. The p63 gene plays an essential role in skin homeostasis and is expressed
as at least six isoforms. One of these isoforms, ΔNp63α, has been found
overexpressed in squamous cell carcinomas and is shown here to be constitutively
expressed in Caski cells associated with HPV16. We therefore explored the role
of p63 in these cells by performing microarray analyses after repression of
endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of
p53 target genes was found activated together with many p63 target genes related
to cell adhesion. However, through siRNA silencing and ectopic expression of
various p63 isoforms we demonstrated that TAp63β is involved in activation of
this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β.
Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA
silencing, that E6 induces an accelerated degradation of TAp63β although not
through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6
transcription also induces stabilization of endogenous TAp63β in cervical
carcinoma cells that lead to an increased concentration of focal adhesions at
the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6
in cervical carcinoma as demonstrated previously for p53. Down-modulation of
focal adhesions through disruption of TAp63β therefore appears as a novel
E6-dependent pathway in transformation. These findings identify a major
physiological role for TAp63β in anchorage independent growth that might
represent a new critical pathway in human carcinogenesis. |
List bacterial species identified in the iceman tissues. | Spirochete Treponema denticola
Clostridium perfringens
Clostridium ghonii
Clostridium sordellii
Eubacterium tenue
Bacteroides sp
Vibrio
Sphingomonas
Afipia
Curtobacterium
Microbacterium
Agromyces | About 80 sequences (16s ribosomal RNA gene) of bacterial DNA in samples of skin
and muscle taken directly from the Tyrolean iceman (3350-3100 years B.C.) or
recovered during the 1992 archaeological expedition at the Alpine site were
analyzed to obtain clues to the natural mummification process that allowed the
corpse of the Neolithic shepherd/hunter to be preserved for more than 5,000
years. The investigation was made more complex by the fact that the surface of
the mummy had been swabbed with phenol soon after the discovery (September 19,
1991). Our results show that no trace of microbial DNA is left on the actual
surface of the body, while the untreated skin still bears the remains of large
numbers of bacteria belonging to the genera Sphingomonas, Afipia,
Curtobacterium, Microbacterium, Agromyces, and others. Compared to the untreated
skin, the iceman's muscle is also very rich in bacterial DNA. However, this DNA
comes, with few exceptions, from the species Clostridium algidicarnis. The sharp
difference in the bacterial DNA composition of skin and muscle suggests that the
remains of the original cadaveric microflora of the latter have not disappeared
during the iceman's taphonomic history. On the other hand, the massive presence
of C. algidicarnis, a cold-adapted sporigenous, the DNA of which was previously
(Ubaldi et al. [1998] Am. J. Phys. Anthropol. 107:285-295) found in the soft
tissue of a naturally desiccated Andean mummy, indicates that the hypothesis
that the iceman's corpse underwent rapid dehydration by the effect of a warm
wind (föhn) is no longer plausible. The results best fit with the hypothesis
(Bereuter et al. [1997] Chem. Eur. J. 7:1032-1038) that the body was first
covered by snow and ice, and then underwent thawing and, finally, desiccation. Ancient hominoid genome studies can be regarded by definition as metagenomic
analyses since they represent a mixture of both hominoid and microbial sequences
in an environment. Here, we report the molecular detection of the oral
spirochete Treponema denticola in ancient human tissue biopsies of the Iceman, a
5,300-year-old Copper Age natural ice mummy. Initially, the metagenomic data of
the Iceman's genomic survey was screened for bacterial ribosomal RNA (rRNA)
specific reads. Through ranking the reads by abundance a relatively high number
of rRNA reads most similar to T. denticola was detected. Mapping of the
metagenome sequences against the T. denticola genome revealed additional reads
most similar to this opportunistic pathogen. The DNA damage pattern of
specifically mapped reads suggests an ancient origin of these sequences. The
haematogenous spread of bacteria of the oral microbiome often reported in the
recent literature could already explain the presence of metagenomic reads
specific for T. denticola in the Iceman's bone biopsy. We extended, however, our
survey to an Iceman gingival tissue sample and a mouth swab sample and could
thereby detect T. denticola and Porphyrimonas gingivalis, another important
member of the human commensal oral microflora. Taken together, this study
clearly underlines the opportunity to detect disease-associated microorganisms
when applying metagenomics-enabled approaches on datasets of ancient human
remains. |
Which properties of the mRNA does N6-methyladenosine (m6A) affect? | N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. m6A predominantly and directly reduces mRNA stability. | We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA
of Rous sarcoma virus (RSV). The results of that study and of experiments using
inhibitors of methylation suggest that m6A might be involved in mRNA processing
events. We describe an approach for directly analyzing the function of m6A in
RNA and for studying the sequence specificity of the m6A methylase. Two sites of
methylation in RSV (nucleotides 7414 and 7424) were altered by
oligonucleotide-directed mutagenesis. The highly conserved GAC consensus
sequence at those sites was changed to GAU. The new sequences were no longer
methylated in the RSV genomic RNA; the GAC sequence was required for efficient
base modification at those two adenosines. The altered m6A pattern did not
affect viral RNA processing or the viral life cycle within infected cells. Cycloleucine, a competitive inhibitor of methionine transferase was used to
generate in vivo partially methylated mRNA in SV40-infected BSC-1 cells.
Cycloleucine at 0.5 mg/ml causes more than a 30% decrease in internal m6As of
late SV40 mRNA with only minor effect on the dimethyladenosine of the 5' caps
m7GpppmAm. After treatment with 2 and 5 mg/ml of cycloleucine, internal m6As
were reduced by 10- and 100-fold, respectively. The inhibition of BSC-1 mRNA
methylations paralleled that observed for late SV40 mRNAs. In cells exposed to 2
mg/ml cycloleucine production of late SV40 mRNA was inhibited by 80% whereas the
amount of SV40 nuclear RNA was only slightly reduced. Size fractionation of SV40
nuclear RNA from cycloleucine-treated cells revealed a loss of SV40 19 S RNA
with a corresponding increase of fragmented RNA sedimenting between 11 to 5 S,
so that the total amount of SV40 RNA in the nucleus was almost unchanged.
Analysis of viral transcription complexes from cells treated with cycloleucine
indicated that SV40 transcription was not affected by cycloleucine.
SV40-transformed cells, in contrast to BSC-1 cells, were able to process and
transport undermethylated RNA. When transformed cells were treated with 2 mg/ml
cycloleucine no changes in quantities or size of cytoplasmic and nuclear RNA
were detected. The data argues for a role of internal m6A moieties in modulating
the processing-linked transport of mRNA from the nucleus to the cytoplasm of
nontransformed cells. Transformed cells may escape these controls due to
structural alterations in their perinuclear regions. N6-Methyladenosine (m6A) residues, which are found internally in viral and
cellular mRNA populations at the sequences Apm6ApC and Gpm6ApC, have been
proposed to play a role in mRNA processing and transport. We have developed a
sensitive approach to analyze the level and location of m6A in specific purified
cellular mRNAs in an attempt to correlate m6A location with function.
Polyadenylylated mRNA is hybridized to cDNA clones representing the full size
mRNA under study or fragments of it, and the protected RNA is digested and
labeled with polynucleotide kinase in vitro. After enrichment for m6A with
anti-m6A antibody, the [32P]-pm6A is separated on TLC plates, and compared with
the total amount of radiolabeled nucleotides. Using this combination of in vitro
RNA labeling and antibody selection, we were able to detect m6A in purified
stable mRNAs that cannot be readily labeled in cells with greater sensitivity
than was possible by previous techniques. We applied this technique to bovine
prolactin mRNA and showed that this mRNA contains m6A. Moreover, all of the m6A
residues in this message are found within the 3' two-thirds of the molecule and
are highly concentrated (61%) within a sequence of 108 nucleotides at the 3'
noncoding region of the message. The nonrandom distribution of m6A in a specific
cellular mRNA, as demonstrated for bovine prolactin, will have to be taken into
account when designing a model for m6A function. It has been repeatedly shown that chronic stress changes dendrites, spines and
modulates expression of synaptic molecules. These effects all may impair
information transfer between neurons. The present study shows that chronic
stress also regulates expression of M6a, a glycoprotein which is localised in
axonal membranes. We have previously demonstrated that M6a is a component of
glutamatergic axons. The present data reveal that it is the splice variant
M6a-Ib, not M6a-Ia, which is strongly expressed in the brain. Chronic stress in
male rats (3 weeks daily restraint) has regional effects: quantitative in situ
hybridization demonstrated that M6a-Ib mRNA in dentate gyrus granule neurons and
in CA3 pyramidal neurons is downregulated, whereas M6a-Ib mRNA in the medial
prefrontal cortex is upregulated by chronic stress. This is the first study
showing that expression of an axonal membrane molecule is differentially
affected by stress in a region-dependent manner. Therefore, one may speculate
that diminished expression of the glycoprotein in the hippocampus leads to
altered output in the corresponding cortical projection areas. Enhanced M6a-Ib
expression in the medial prefrontal cortex (in areas prelimbic and infralimbic
cortex) might be interpreted as a compensatory mechanism in response to changes
in axonal projections from the hippocampus. Our findings provide evidence that
in addition to alterations in dendrites and spines chronic stress also changes
the integrity of axons and may thus impair information transfer even between
distant brain regions. An extensive repertoire of modifications is known to underlie the versatile
coding, structural and catalytic functions of RNA, but it remains largely
uncharted territory. Although biochemical studies indicate that
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in
messenger RNA, an in-depth study of its distribution and functions has been
impeded by a lack of robust analytical methods. Here we present the human and
mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel
approach, m(6)A-seq, based on antibody-mediated capture and massively parallel
sequencing. We identify over 12,000 m(6)A sites characterized by a typical
consensus in the transcripts of more than 7,000 human genes. Sites
preferentially appear in two distinct landmarks--around stop codons and within
long internal exons--and are highly conserved between human and mouse. Although
most sites are well preserved across normal and cancerous tissues and in
response to various stimuli, a subset of stimulus-dependent, dynamically
modulated sites is identified. Silencing the m(6)A methyltransferase
significantly affects gene expression and alternative splicing patterns,
resulting in modulation of the p53 (also known as TP53) signalling pathway and
apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a
fundamental role in regulation of gene expression. Author information:
(1)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
(2)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead
Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
(3)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Graduate
Program in Immunology, Division of Medical Sciences, Harvard Medical School,
Boston, MA 02115, USA.
(4)Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
(5)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem
Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard
University, Cambridge, MA 02138, USA.
(6)Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA;
Department of Radiation Oncology, Dana-Farber Cancer Institute, 450 Brookline
Avenue, Boston, MA 02215, USA.
(7)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for
Immunology and Inflammatory Diseases, Massachusetts General Hospital,
Charlestown, MA 02129, USA.
(8)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern
Institute for Brain Research, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA; Department of Brain and Cognitive Sciences and Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
(9)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;
Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA.
Electronic address: [email protected].
(10)Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard
Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA.
Electronic address: [email protected]. Recent discoveries of reversible N(6)-methyladenosine (m(6)A) methylation on
messenger RNA (mRNA) and mapping of m(6)A methylomes in mammals and yeast have
revealed potential regulatory functions of this RNA modification. In plants,
defects in m(6)A methyltransferase cause an embryo-lethal phenotype, suggesting
a critical role of m(6)A in plant development. Here, we profile m(6)A
transcriptome-wide in two accessions of Arabidopsis thaliana and reveal that
m(6)A is a highly conserved modification of mRNA in plants. Distinct from
mammals, m(6)A in A. thaliana is enriched not only around the stop codon and
within 3'-untranslated regions, but also around the start codon. Gene ontology
analysis indicates that the unique distribution pattern of m(6)A in A. thaliana
is associated with plant-specific pathways involving the chloroplast. We also
discover a positive correlation between m(6)A deposition and mRNA abundance,
suggesting a regulatory role of m(6)A in plant gene expression. Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and
ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) residues
in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains
a challenge, however. Here, we have identified meclofenamic acid (MA) as a
highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug
that mechanistic studies indicate competes with FTO binding for the
m(6)A-containing nucleic acid. The structure of FTO/MA has revealed much about
the inhibitory function of FTO. Our newfound understanding, revealed herein, of
the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped
elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of
HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of
m(6)A modification in mRNA. Our collective results highlight the development of
functional probes of the FTO enzyme that will (i) enable future biological
studies and (ii) pave the way for the rational design of potent and specific
inhibitors of FTO for use in medicine. Polymorphism of the FTO gene encoding an N(6)-methyladenosine (m(6)A) RNA
demethylase was robustly associated with human obesity; however, the mechanism
by which FTO affects metabolism, considering its emerging role in RNA
modification, is still poorly understood. A new study published in Cell Research
reports novel functions implicating FTO in the regulation of mRNA alternative
splicing in the control of adipogenesis. Author information:
(1)The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel.
(2)Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel, and
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
(3)Department of Chemistry and Institute for Biophysical Dynamics, The
University of Chicago, Chicago, IL 60637, USA.
(4)Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat
Gan, Israel.
(5)The Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.
(6)The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel. The Department of Pediatrics and the Pediatric Immunology Unit, Rambam
Medical Center, and the B. Rappaport Faculty of Medicine, Technion, Haifa,
Israel.
(7)Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel, and
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. Mina and
Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan,
Israel.
(8)The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot,
Israel. [email protected] [email protected]
[email protected].
(9)Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer, Israel, and
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
[email protected] [email protected]
[email protected]. N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA
and has been linked to diverse effects on mRNA fate. Current mapping approaches
localize m6A residues to transcript regions 100-200 nt long but cannot identify
precise m6A positions on a transcriptome-wide level. Here we developed m6A
individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP)
and used it to demonstrate that antibodies to m6A can induce specific mutational
signatures at m6A residues after ultraviolet light-induced antibody-RNA
cross-linking and reverse transcription. We found that these antibodies
similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a
modification found at the first nucleotide of certain mRNAs. Using these
signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and
mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of
m6A-containing non-coding RNAs (ncRNAs). |
What is the implication of histone lysine methylation in medulloblastoma? | Aberrant patterns of H3K4, H3K9, and H3K27 histone lysine methylation were shown to result in histone code alterations, which induce changes in gene expression, and affect the proliferation rate of cells in medulloblastoma. | We used high-resolution SNP genotyping to identify regions of genomic gain and
loss in the genomes of 212 medulloblastomas, maligt pediatric brain tumors.
We found focal amplifications of 15 known oncogenes and focal deletions of 20
known tumor suppressor genes (TSG), most not previously implicated in
medulloblastoma. Notably, we identified previously unknown amplifications and
homozygous deletions, including recurrent, mutually exclusive, highly focal
genetic events in genes targeting histone lysine methylation, particularly that
of histone 3, lysine 9 (H3K9). Post-translational modification of histone
proteins is critical for regulation of gene expression, can participate in
determination of stem cell fates and has been implicated in carcinogenesis.
Consistent with our genetic data, restoration of expression of genes controlling
H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro.
Copy number aberrations of genes with critical roles in writing, reading,
removing and blocking the state of histone lysine methylation, particularly at
H3K9, suggest that defective control of the histone code contributes to the
pathogenesis of medulloblastoma. |
What is the Orco protein in mosquitos? | Odorant co-receptor. | In insects, odor cues are discriminated through a divergent family of odorant
receptors (ORs). A functional OR complex consists of both a conventional
odorant-binding OR and a nonconventional coreceptor (Orco) that is highly
conserved across insect taxa. Recent reports have characterized insect ORs as
ion channels, but the precise mechanism of signaling remains unclear. We report
the identification and characterization of an Orco family agonist, VUAA1, using
the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies
reveal that the Orco family can form functional ion channels in the absence of
an odor-binding OR, and in addition, demonstrate a first-in-class agonist to
further research in insect OR signaling. In light of the extraordinary
conservation and widespread expression of the Orco family, VUAA1 represents a
powerful new family of compounds that can be used to disrupt the destructive
behaviors of nuisance insects, agricultural pests, and disease vectors alike. BACKGROUND: Insect odorant receptors (ORs) function as odorant-gated ion
channels consisting of a conventional, odorant-binding OR and the Orco
coreceptor. While Orco can function as a homomeric ion channel, the role(s) of
the conventional OR in heteromeric OR complexes has largely focused only on
odorant recognition.
RESULTS: To investigate other roles of odorant-binding ORs, we have employed
patch clamp electrophysiology to investigate the properties of the channel pore
of several OR complexes formed by a range of different odorant-specific
Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal
significant differences in cation permeability and ruthenium red susceptibility
among different AgOr complexes.
CONCLUSIONS: With observable differences in channel function, the data support a
model in which the odorant-binding OR also affects the channel pore. The
variable effect contributed by the conventional OR on the conductive properties
of odorant-gated sensory channels adds additional complexity to insect olfactory
signaling, with differences in odor coding beginning with ORs on the periphery
of the olfactory system. Carbon dioxide (CO(2)) elicits an attractive host-seeking response from
mosquitos yet is innately aversive to Drosophila melanogaster despite being a
plentiful byproduct of attractive fermenting food sources. Prior studies used
walking flies exclusively, yet adults track distant food sources on the wing.
Here we show that a fly tethered within a magnetic field allowing free rotation
about the yaw axis actively seeks a narrow CO(2) plume during flight. Genetic
disruption of the canonical CO(2)-sensing olfactory neurons does not alter
in-flight attraction to CO(2); however, antennal ablation and genetic disruption
of the Ir64a acid sensor do. Surprisingly, mutation of the obligate olfactory
coreceptor (Orco) does not abolish CO(2) aversion during walking yet eliminates
CO(2) tracking in flight. The biogenic amine octopamine regulates critical
physiological processes during flight, and blocking synaptic output from
octopamine neurons inverts the valence assigned to CO(2) and elicits an aversive
response in flight. Combined, our results suggest that a novel Orco-mediated
olfactory pathway that gains sensitivity to CO(2) in flight via changes in
octopamine levels, along with Ir64a, quickly switches the valence of a key
environmental stimulus in a behavioral-state-dependent manner. Insect odorant receptors function as heteromeric odorant-gated cation channels
comprising a conventional odorant-sensitive tuning receptor, and a conserved
co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric
and homomeric Orco-containing channels. Very little is known about specific
residues in Orco that contribute to cation permeability and gating. We
investigated the importance of two conserved Asp residues, one in each of
transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila
melanogaster Orco and its substitution mutants were expressed in HEK cells and
VUAA1-stimulated channel activity was determined by Ca(2+) influx and whole-cell
patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with
amino acids other than glutamic acid resulted in a substantial reduction in
channel activity. The D466E Orco substitution mutant was ~2 times more sensitive
to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged
relative to wild-type Orco. When D466E Orco is co-expressed with a conventional
tuning odorant receptor, the heteromeric complex also shows increased
sensitivity to an odorant. Thus, the effect of the D466E mutation is not
specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest
the gain-of-activation characteristic of the D466E mutant identifies an amino
acid that is likely to be important for activation of both heteromeric and
homomeric insect odorant receptor channels. |
List some ways to reverse Tau hyperphosphorylation in Tauopathies? | Different ways have been used to try to reverse Tau hyperphosphorylation through administration of inhibitors such as: 7-nitroindazole, memantine, glycogen synthase kinase-3 inhibitors.
Other approaches are transplantation of Human umbilical cord blood-derived mesenchymal stem cells and administration of M1 muscarinic agonists. | The M1 muscarinic agonists AF102B (Cevimeline, EVOXACTM: prescribed in USA and
Japan for Sjogren's Syndrome), AF150(S) and AF267B--1) are neurotrophic and
synergistic with neurotrophins such as nerve growth factor and epidermal growth
factor; 2) elevate the non-amyloidogenic amyloid precursor protein (alpha-APPs)
in vitro and decrease beta-amyloid (A beta) levels in vitro and in vivo; and 3)
inhibit A beta- and oxidative-stress-induced cell death and apoptosis in PC12
cells transfected with the M1 muscarinic receptor. These effects can be combined
with the beneficial effects of these compounds on some other major hallmarks of
Alzheimer's disease (AD) (e.g. tau hyperphosphorylation and paired helical
filaments [PHF]; and loss of cholinergic function conducive to cognitive
impairments.) These drugs restored cognitive impairments in several animal
models for AD, mimicking different aspects of AD, with a high safety margin
(e.g. AF150[S] >1500 and AF267B >4500). Notably, these compounds show a high
bioavailability and a remarkable preference for the brain vs. plasma following
p.o. administration. In mice with small hippocampi, unlike rivastigmine and
nicotine, AF150(S) and AF267B restored cognitive impairments also on escape
latency in a Morris water maze paradigm in reversal learning. Furthermore, in
aged and cognitively impaired microcebes (a natural animal model that mimics AD
pathology and cognitive impairments), prolonged treatment with AF150(S) restored
cognitive and behavioral impairments and decreased tau hyperphosphorylation, PHF
and astrogliosis. Our M1 agonists, alone or in polypharmacy, may present a
unique therapy in AD due to their beneficial effects on major hallmarks of AD. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine
kinase that is particularly abundant in the CNS. Dysregulation of GSK-3 activity
is believed to play a key role in the pathogenesis of CNS chronic disorders such
as Alzheimer's disease (AD), bipolar disorder, and Huntington's disease, and of
metabolic disorders such as type II diabetes. Accordingly, GSK-3 inhibitors have
been postulated as therapeutic tools for these diseases. Interestingly,
pathophysiological and pharmacological regulation of GSK-3 is affected by an
amplification mechanism that applies both to inhibition and activation. The
possibility therefore exists that sustained inhibition or activation might
persist after cessation of the initial trigger. Regarding AD, GSK-3 has been
shown to accumulate in pretangle neurons. Furthermore, GSK-3 phosphorylates tau
in most serine and threonine residues hyperphosphorylated in PHF (paired helical
filament)-tau and GSK-3 activity contributes both to beta-amyloid production and
to beta-amyloid-mediated neuronal death. In good agreement, mice with
conditional overexpression of GSK-3 in forebrain neurons (Tet/GSK-3beta mice)
recapitulate aspects of AD neuropathology such as tau hyperphosphorylation,
apoptotic neuronal death, and reactive astrocytosis as well as spatial learning
deficit. Here, we exploit the conditional system used to generate Tet/GSK-3beta
mice to explore whether the biochemical, histopathological, and behavioral
consequences of increased GSK-3 activity are susceptible to revert after
restoration of normal GSK-3 levels. Here, we show that transgene shutdown in
symptomatic mice leads to normal GSK-3 activity, normal phospho-tau levels,
diminished neuronal death, and suppression of the cognitive deficit, thus
further supporting the potential of GSK-3 inhibitors for AD therapeutics. The activity of protein phosphatase (PP)-2A, which regulates tau
phosphorylation, is compromised in Alzheimer disease brain. Here we show that
the transient transfection of PC12 cells with inhibitor-2 (I2PP2A) of PP2A
causes abnormal hyperphosphorylation of tau at Ser396/Ser404 and Ser262/Ser356.
This hyperphosphorylation of tau is observed only when a sub-cellular shift of
I2PP2A takes place from the nucleus to the cytoplasm and is accompanied by
cleavage of I2PP2A into a 20 kDa fragment. Memantine, an un-competitive
inhibitor of N-methyl-D-aspartate receptors, inhibits this abnormal
phosphorylation of tau and cell death and prevents the I2PP2A-induced inhibition
of PP2A activity in vitro. These findings demonstrate novel mechanisms by which
I2PP2A regulates the intracellular activity of PP2A and phosphorylation of tau,
and by which Memantine modulates PP2A signaling and inhibits neurofibrillary
degeneration. Effects of chronic stress are not completely understood. They may underlie
depression and dementia. This study assessed the association between chronic
stress, glutamate levels, tau-protein phosphorylation, and nitric-oxide in old
rats exposed to chronic mild stress (CMS). Old (>15 months) male Wistar rats
were exposed to CMS. Comparison groups included old and young control rats,
young CMS-exposed, and old CMS-exposed rats treated with the neuronal
nitric-oxide synthase (nNOS) enzyme inhibitor, 7-nitroindazole (20 mg/kg/day
i.p.). Hippocampal glutamate levels and glutamate decarboxylase (GAD) activity
were determined and tau protein phosphorylation was assessed. Age was a
significant (p=0.025) source of variation in glutamate level [811.71+/-218.1,
665.9+/-124.9 micromol/g tissue protein (M+/-SD) in young and old control rats,
respectively]. Old rats exposed to CMS were characterized by an increased risk
to develop anhedonia. There was significant (p=0.035) decrease in GAD enzyme
activity (-60.06%) and increased tau protein hyperphosphorylation in old rats
exposed to CMS compared to control. Administration of 7-nitroindazole to
CMS-exposed old rats significantly (p=0.002) increased GAD activity, decreased
glutamate levels (7.19+/-3.19 vs. 763.9+/-91 micromol/g tissue protein;
p=0.0005), and decreased phosphorylation of tau proteins compared to CMS exposed
rats. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) have a
potential therapeutic role in the treatment of neurological disorders, but their
current clinical usage and mechanism of action has yet to be ascertained in
Alzheimer's disease (AD). Here we report that hUCB-MSC transplantation into
amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic mice
significantly improved spatial learning and memory decline. Furthermore,
amyloid-β peptide (Aβ) deposition, β-secretase 1 (BACE-1) levels, and tau
hyperphosphorylation were dramatically reduced in hUCB-MSC transplanted APP/PS1
mice. Interestingly, these effects were associated with reversal of
disease-associated microglial neuroinflammation, as evidenced by decreased
microglia-induced proinflammatory cytokines, elevated alternatively activated
microglia, and increased anti-inflammatory cytokines. These findings lead us to
suggest that hUCB-MSC produced their sustained neuroprotective effect by
inducing a feed-forward loop involving alternative activation of microglial
neuroinflammation, thereby ameliorating disease pathophysiology and reversing
the cognitive decline associated with Aβ deposition in AD mice. |
What is the role of 5hmC (5 hydroxy-methyl-Cytocine) in differentiation? | The balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. | DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a
key epigenetic event critical for various cellular processes. The ten-eleven
translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to
5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA
methylation. Here we report that Tet1 binds to unmodified C or 5mC- or
5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1
and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse
embryonic stem cells (mESCs). We also uncover a comprehensive gene network
influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA
methylation both by binding to CpG-rich regions to prevent unwanted DNA
methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase
activity. This Tet1-mediated antagonism of CpG methylation imparts differential
maintece of DNA methylation status at Tet1 targets, ultimately contributing
to mESC differentiation and the onset of embryonic development. 5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in
embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is
generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes
and may be an intermediate to further oxidation and finally demethylation of 5
mC. We have used immunohistochemistry (IHC) and isotope-based liquid
chromatography mass spectrometry (LC-MS) to investigate the presence and
distribution of 5 hmC in human brain and brain tumors. In the normal adult
brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC
in white matter (WM) areas. In tumors, positive staining of cells ranged from
1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas
(pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed
highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the
cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor
differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO
Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC
measurements were unrelated to 5 mC values. We find that the number of 5 hmC
positive cells and the amount of 5 hmC/dG in the genome that has been proposed
to be related to pluripotency and lineage commitment in embryonic stem cells is
also associated with brain tumor differentiation and anaplasia. The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell
differentiation and development through epigenetic gene regulation. 5mC can be
converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet
methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between
5hmC and 5mC in the genome is linked with cell-differentiation processes such as
pluripotency and lineage commitment. We have previously reported that the
maternal factor PGC7 (also known as Dppa3, Stella) is required for the
maintece of DNA methylation in early embryogenesis, and protects 5mC from
conversion to 5hmC in the maternal genome. Here we show that PGC7 protects 5mC
from Tet3-mediated conversion to 5hmC by binding to maternal chromatin
containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition,
imprinted loci that are marked with H3K9me2 in mature sperm are protected by
PGC7 binding in early embryogenesis. This type of regulatory mechanism could be
involved in DNA modifications in somatic cells as well as in early embryos. Enhancers are developmentally controlled transcriptional regulatory regions
whose activities are modulated through histone modifications or histone variant
deposition. In this study, we show by genome-wide mapping that the newly
discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine
(5hmC) is dynamically associated with transcription factor binding to distal
regulatory sites during neural differentiation of mouse P19 cells and during
adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals
that regions gaining 5hmC are associated with genes expressed either in neural
tissues when P19 cells undergo neural differentiation or in adipose tissue when
3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions
gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as
differentiation-dependent transcriptional enhancers. Identified regions are
enriched in motifs for transcription factors regulating specific cell fates such
as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of
hydroxymethylated Meis1 sites were associated with a dynamic engagement of the
5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine
hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation
is an early event of enhancer activation. Hence, acquisition of 5hmC in
cell-specific distal regulatory regions may represent a major event of enhancer
progression toward an active state and participate in selective activation of
tissue-specific genes. 5-hydroxy methyl cytosine (5hmC) is a modification identified in vertebrates
several decades ago. More recently, a possible role of 5hmC as an epigenetic
modifier and/or transcriptional regulator has started to emerge, with altered
levels in early embryonic development, embryonic stem (ES) cell differentiation
and tumours (Tahiliani et al, 2009; Yang et al, 2012). The balance between 5hmC
and 5-methyl cytosine (5mC) at gene promoters and CpG islands in the genome
appears to be linked to pluripotency and lineage commitment of a cell (Ito et
al, 2010). However, proteins with 5hmC binding capability have not yet been
identified, and it has been proposed that 5hmC may only be a reaction
intermediate in the process of demethylation (He et al, 2011; Ito et al, 2011).
Over the last few years, ten-eleven translocation (Tet) family proteins have
been shown to be responsible for the conversion of 5mC to 5hmC (Iyer et al,
2009; Loenarz and Schofield, 2009; Tahiliani et al, 2009). However, how Tet
family proteins and 5hmC are linked to transcriptional regulation is currently
not clear. 5-Hydroxymethylcytosine (5hmC), converted from 5-methylcytocine (5mC) by Tet
family of dioxygenases (Tet1, Tet2, and Tet3), is enriched in the embryonic stem
cells (ESCs) and in the brain. However, the role of 5hmC and Tet family in the
process of ESC differentiation especially neuronal differentiation remains
elusive. Here, we showed the evidence that Tet3 is critical in neural progenitor
cell (NPC) maintece and terminal differentiation of neurons. We found that
Tet3 expression is basically undetectable in ESCs, but its level increases
rapidly during neuronal differentiation. Tet3 knockout ESCs appear normal in
self-renewal and maintece but impaired in neuronal differentiation. NPCs
could be induced efficiently from Tet3 knockout ESCs, as the expression of NPC
marker Pax6 and nestin is comparable with NPCs from wild-type ESCs, but undergo
apoptosis rapidly, and the terminal differentiation of neurons is greatly
reduced. Our results indicate that Tet3 is important for NPC maintece and
terminal differentiation of neurons. Author information:
(1)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;
(2)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Department of Information and Computer
Science, Aalto University School of Science, FI-00076 Aalto, Finland;
(3)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Sanford Consortium for Regenerative Medicine,
La Jolla, CA 92037;
(4)Howard Hughes Medical Institute,Department of Molecular, Cell, and
Developmental Biology, andEli and Edythe Broad Center of Regenerative Medicine
and Stem Cell Research, University of California, Los Angeles, CA 90095; and.
(5)Department of Information and Computer Science, Aalto University School of
Science, FI-00076 Aalto, Finland;
(6)Department of Signaling and Gene Expression, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92034;Sanford Consortium for Regenerative Medicine,
La Jolla, CA 92037;Department of Pharmacology and Moores Cancer Center,
University of California, San Diego, La Jolla, CA 92093 [email protected]. 5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine
(5mC), has been implicated as an important epigenetic regulator of mammalian
development. Current procedures use DNA sequencing methods to discriminate 5hmC
from 5mC, limiting their accessibility to the scientific community. Here we
report a method that combines TET-assisted bisulfite conversion with Illumina
450K DNA methylation arrays for a low-cost high-throughput approach that
distinguishes 5hmC and 5mC signals at base resolution. Implementing this
approach, termed "TAB-array", we assessed DNA methylation dynamics in the
differentiation of human pluripotent stem cells into cardiovascular progenitors
and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we
identified a large number of novel dynamically methylated genomic regions that
are implicated in the development of these lineages. The increased resolution
and accuracy afforded by this approach provides a powerful means to investigate
the distinct contributions of 5mC and 5hmC in human development and disease. Author information:
(1)Department of Orthodontics, Peking University School & Hospital of
Stomatology, #22 Zhongguancun South Avenue, Beijing 100081, China; Department of
Anatomy and Cell Biology, University of Pennsylvania, School of Dental Medicine,
240 South 40(th) Street, Philadelphia, PA 19104, USA; Center for Craniofacial
Molecular Biology, Ostrow School of Dentistry, University of Southern
California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA.
(2)Center for Craniofacial Molecular Biology, Ostrow School of Dentistry,
University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA
90033, USA.
(3)Department of Molecular Microbiology and Immunology, University of Southern
California, 2011 Zonal Avenue, Los Angeles, CA 90033, USA.
(4)National Institute of Dental and Craniofacial Research, NIH, 30 Convent
Drive, MSC 4352 Bethesda, MD 20892, USA.
(5)Laboratory of Tissue Regeneration and Immunology and Department of
Periodontics, Capital Medical University School of Stomatology, #4 Tiantanxili
Avenue, Beijing 100050, China.
(6)Department of Anatomy and Cell Biology, University of Pennsylvania, School of
Dental Medicine, 240 South 40(th) Street, Philadelphia, PA 19104, USA; Center
for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of
Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA.
(7)Department of Orthodontics, Peking University School & Hospital of
Stomatology, #22 Zhongguancun South Avenue, Beijing 100081, China.
(8)Norris Medical Library, Keck School of Medicine, University of Southern
California, 2003 Zonal Avenue, Los Angeles, CA 90033, USA.
(9)Department of Orthodontics, Peking University School & Hospital of
Stomatology, #22 Zhongguancun South Avenue, Beijing 100081, China. Electronic
address: [email protected].
(10)Department of Anatomy and Cell Biology, University of Pennsylvania, School
of Dental Medicine, 240 South 40(th) Street, Philadelphia, PA 19104, USA; Center
for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of
Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA.
Electronic address: [email protected]. |
Which is the histone residue methylated by MLL1? | Histone H3 at lysine 4 (H3K4) | The mixed-lineage leukemia (MLL1/ALL-1/HRX) histone methyltransferase is
involved in the epigenetic maintece of transcriptional memory and the
pathogenesis of human leukemias. To understand its role in cell type
specification, we determined the human genomic binding sites of MLL1. We found
that MLL1 functions as a human equivalent of yeast Set1. Like Set1, MLL1
localizes with RNA polymerase II (Pol II) to the 5' end of actively transcribed
genes, where histone H3 lysine 4 trimethylation occurs. Consistent with this
global role in transcription, MLL1 also localizes to microRNA (miRNA) loci that
are involved in leukemia and hematopoiesis. In contrast to the 5' proximal
binding behavior at most protein-coding genes, MLL1 occupies an extensive domain
within a transcriptionally active region of the HoxA cluster. The ability of
MLL1 to serve as a start site-specific global transcriptional regulator and to
participate in larger chromatin domains at the Hox genes reveals dual roles for
MLL1 in maintece of cellular identity. The product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor
gene, menin, is an integral component of MLL1/MLL2 histone methyltransferase
complexes specific for Lys4 of histone H3 (H3K4). We show that menin is a
transcriptional coactivator of the nuclear receptors for estrogen and vitamin D.
Activation of the endogenous estrogen-responsive TFF1 (pS2) gene results in
promoter recruitment of menin and in elevated trimethylation of H3K4. Knockdown
of menin reduces both activated TFF1 (pS2) transcription and H3K4
trimethylation. In addition, menin can directly interact with the estrogen
receptor-alpha (ERalpha) in a hormone-dependent manner. The majority of
disease-related MEN1 mutations prevent menin-ERalpha interaction. Importantly,
ERalpha-interacting mutants are also defective in coactivator function. Our
results indicate that menin is a critical link between recruitment of histone
methyltransferase complexes and nuclear receptor-mediated transcription. The transcriptional networks that regulate embryonic stem (ES) cell pluripotency
and lineage specification are the subject of considerable attention. To date
such studies have focused almost exclusively on protein-coding transcripts.
However, recent transcriptome analyses show that the mammalian genome contains
thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed
in a developmentally regulated manner. The functions of these remain untested.
To identify ncRNAs involved in ES cell biology, we used a custom-designed
microarray to examine the expression profiles of mouse ES cells differentiating
as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs
expressed during EB differentiation, of which 174 were differentially expressed,
many correlating with pluripotency or specific differentiation events. Candidate
ncRNAs were identified for further characterization by an integrated examination
of expression profiles, genomic context, chromatin state, and promoter analysis.
Many ncRNAs showed coordinated expression with genomically associated
developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel
developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from
homeotic loci and share similar expression patterns and localization in mouse
embryos with their associated protein-coding genes. Using chromatin
immunoprecipitation, we provide evidence that both ncRNAs are associated with
trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a
role in epigenetic regulation of homeotic loci during ES cell differentiation.
Taken together, our data indicate that long ncRNAs are likely to be important in
processes directing pluripotency and alternative differentiation programs, in
some cases through engagement of the epigenetic machinery. The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an
essential role in early development and hematopoiesis. The biological function
of MLL1 is mediated by the histone H3K4 methyltransferase activity of the
carboxyl-terminal SET domain. We have determined the crystal structure of the
MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3
peptide. This structure indicates that, in order to form a well-ordered active
site, a highly variable but essential component of the SET domain must be
repositioned. To test this idea, we compared the effect of the addition of MLL
complex members on methyltransferase activity and show that both RbBP5 and Ash2L
but not Wdr5 stimulate activity. Additionally, we have determined the effect of
posttranslational modifications on histone H3 residues downstream and upstream
from the target lysine and provide a structural explanation for why H3T3
phosphorylation and H3K9 acetylation regulate activity. Constitutive nuclear factor (NF)-kappaB activation in haematological
maligcies is caused in several cases by loss of function mutations within the
coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100.
Hut-78, a truncated form of p100, constitutively generates p52 and contributes
to the development of T-cell lymphomas but the molecular mechanism underlying
this oncogenic potential remains unclear. We show here that MMP9 gene expression
is induced through the alternative NF-kappaB-activating pathway in fibroblasts
and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma
cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78
mutant as well as other truncated NF-kappaB2 proteins that are not processed
into p52 failed to induce the expression of this metalloproteinase. Conversely,
MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly,
MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9
but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity
recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells.
Moreover, MLL1 and MLL2 are associated with Hut-78 in a native
chromatin-enriched extract. Thus, we identified a molecular mechanism by which
the recruitment of a H3K4 histone methyltransferase complex on the promoter of a
NF-kappaB-dependent gene induces its expression and potentially the invasive
potential of lymphoma cells harbouring constitutive activity of the alternative
NF-kappaB-activating pathway. The conserved DPY-30 is an essential component of the dosage compensation
complex that balances the X-linked gene expression by regulation of the complex
formation in Caenorhabditis elegans. The human DPY-30-like protein (DPY-30L)
homolog is a conserved member of certain histone methyltransferase (HMT)
complexes. In the human MLL1 (mixed-lineage leukemia-1) HMT complex, DPY-30L
binds to the BRE2 homolog ASH2L in order to regulate histone 3-lysine 4
trimethylation. We have determined the 1.2-A crystal structure of the human
DPY-30L C-terminal domain (DPY-30L(C)). The DPY-30L(C) structure, harboring the
conserved DPY-30 motif, is composed of two alpha-helices linked by a sharp loop
and forms a typical X-type four-helix bundle required for dimer formation.
DPY-30L(C) dimer formation is largely mediated by an extensive hydrophobic
interface with some additional polar interactions. The oligomerization of
DPY-30L(C) in solution, together with its reported binding to ASH2L, leads us to
propose that the hydrophobic surface of the dimer may provide a platform for
interaction with ASH2L in the MLL1 HMT complex. The Ski-interacting protein SKIP/SNW1 associates with the P-TEFb/CDK9 elongation
factor and coactivates inducible genes, including HIV-1. We show here that SKIP
also associates with c-Myc and Menin, a subunit of the MLL1 histone
methyltransferase (H3K4me3) complex and that HIV-1 Tat transactivation requires
c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP
acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold
for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is
recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter in a
step that is bypassed by Tat and downregulated by c-Myc. Of interest, we find
that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription,
which is strongly upregulated by treating cells with the CDK9 inhibitor
flavopiridol. Thus, SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb
transcription at an elongation step that is bypassed under stress. Reversible methylation of histone tails serves as either a positive signal
recognized by transcriptional assemblies or a negative signal that result in
repression. Invading viral pathogens that depend upon the host cell's
transcriptional apparatus are also subject to the regulatory impact of chromatin
assembly and modifications. Here we show that infection by the
alpha-herpesviruses, herpes simplex virus (HSV) and varicella zoster virus
(VZV), results in the rapid accumulation of chromatin bearing repressive histone
H3 Lys9 methylation. To enable expression of viral immediate early (IE) genes,
both viruses use the cellular transcriptional coactivator host cell factor-1
(HCF-1) to recruit the lysine-specific demethylase-1 (LSD1) to the viral
immediate early promoters. Depletion of LSD1 or inhibition of its activity with
monoamine oxidase inhibitors (MAOIs) results in the accumulation of repressive
chromatin and a block to viral gene expression. As HCF-1 is a component of the
Set1 and MLL1 histone H3 Lys4 methyltransferase complexes, it thus coordinates
modulation of repressive H3 Lys9 methylation levels with addition of activating
H3 Lys4 trimethylation marks. Strikingly, MAOIs also block the reactivation of
HSV from latency in sensory neurons, indicating that the HCF-1 complex is a
crucial component of the reactivation mechanism. The results support
pharmaceutical control of histone modifying enzymes as a strategy for
controlling herpesvirus infections. The multidrug resistance 1 gene (MDR1) encodes P-glycoprotein (Pgp), a member of
the ATP-binding cassette (ABC) transporter family that confers tumor drug
resistance by actively effluxing a number of antitumor agents. We had previously
shown that MDR1 transcription is regulated by epigenetic events such as histone
acetylation, and had identified the histone acetylase P/CAF and the
transcription factor NF-Y as the factors mediating the enzymatic and
DNA-anchoring functions, respectively, at the MDR1 promoter. It has also been
shown that MDR1 activation is accompanied by increased methylation on lysine 4
of histone H3 (H3K4). In this study, we further investigated histone methylation
in MDR1 regulation and function. We show that the mixed lineage leukemia 1
(MLL1) protein, a histone methyltransferase specific for H3K4, is required for
MDR1 promoter methylation, as knockdown of MLL1 resulted in a decrease in MDR1
expression. The regulation of MDR1 by MLL1 has functional consequences in that
downregulation of MLL1 led to increased retention of the Pgp-specific substrate
DIOC(2)(3), as well as increased cellular sensitivity to several Pgp substrates.
Regulation of MDR1 by MLL1 was dependent on the CCAAT box within the proximal
MDR1 promoter, similar to what we had shown for MDR1 promoter acetylation, and
also requires NF-Y. Finally, overexpression of the most prevalent MLL fusion
protein, MLL-AF4, led to increased MDR1 expression. This is the first
identification of a histone methyltransferase and its leukemogenic rearrangement
that regulates expression of an ABC drug transporter, suggesting a new target
for circumvention of tumor multidrug resistance. The classical view of the molecular clock is based on interlocked
transcriptional-translational feedback loops. Because a substantial fraction of
the mammalian genome is expressed in a circadian manner, chromatin remodeling
has been proposed to be crucial in clock function. Here we show that Lys4 (K4)
trimethylation of histone H3 is rhythmic and follows the same profile as
previously described H3 acetylation on circadian promoters. MLL1, a mammalian
homolog of Drosophila trithorax, is an H3K4-specific methyltransferase
implicated in transcriptional control. We demonstrate that MLL1 is essential for
circadian transcription and cyclic H3K4 trimethylation. MLL1 is in a complex
with CLOCK-BMAL1 and contributes to its rhythmic recruitment to circadian
promoters and to H3 acetylation. Yet MLL1 fails to interact with CLOCKΔ19,
providing an explanation for this mutation's domit negative phenotype. Our
results favor a scenario in which H3K4 trimethylation by MLL1 is required to
establish a permissive chromatin state for circadian transcription. Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with
transcription activation and is mainly catalyzed by the MLL/SET1 family histone
methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the
presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic
subunit containing a SET domain. Unlike most other histone lysine
methyltransferases, all four proteins are required for efficient H3 K4
methylation. Despite extensive efforts, mechanisms for how three core components
regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a
heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity.
This activity requires the highly conserved SPRY domain of Ash2L and a short
peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are
capable of binding to S-adenosyl-L- [methyl-(3)H] methionine in the MLL1 core
complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4
methylation also compromise SAM binding by Ash2L. Taken together, our results
show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall
catalysis of MLL1 mediated H3 K4 methylation. The results we describe here
provide mechanistic insights for unique regulation of the MLL1 methyltransferase
activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct
contacts with the substrates and contribute to the formation of a joint
catalytic center. Given the shared core configuration among all MLL/SET1 family
HMTs, it will be interesting to test whether the mechanism we describe here can
be generalized to other MLL/SET1 family members in the future. Change in gene expression associated with pancreatic cancer could be attributed
to the variation in histone posttranslational modifications leading to
subsequent remodeling of the chromatin template during transcription. However,
the interconnected network of molecules involved in regulating such processes
remains elusive. hPaf1/PD2, a subunit of the human PAF-complex, involved in the
regulation of transcriptional elongation has oncogenic potential. Our study
explores the possibility that regulation of histone methylation by hPaf1 can
contribute towards alteration in gene expression by nucleosomal rearrangement.
Here, we show that knockdown of hPaf1/PD2 leads to decreased di- and
tri-methylation at histone H3 lysine 4 residues in pancreatic cancer cells.
Interestingly, hPaf1/PD2 colocalizes with MLL1 (Mixed Lineage Leukemia 1), a
histone methyltransferase that methylates H3K4 residues. Also, a reduction in
hPaf1 level resulted in reduced MLL1 expression and a corresponding decrease in
the level of CHD1 (Chromohelicase DNA-binding protein 1), an ATPase dependent
chromatin remodeling enzyme that specifically binds to H3K4 di and trimethyl
marks. hPaf1/PD2 was also found to interact and colocalize with CHD1 in both
cytoplasmic and nuclear extracts of pancreatic cancer cells. Further, reduced
level of CHD1 localization in the nucleus in hPaf1/PD2 Knockdown cells could be
rescued by ectopic expression of hPaf1/PD2. Micrococcal nuclease digestion
showed an altered chromatin structure in hPaf1/PD2-KD cells. Overall, our
results suggest that hPaf1/PD2 in association with MLL1 regulates methylation of
H3K4 residues, as well as interacts and regulates nuclear shuttling of chromatin
remodeling protein CHD1, facilitating its function in pancreatic cancer cells. Transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast-like
phenotype is the pivotal event in liver fibrosis. The dramatic change in
phenotype associated with transdifferentiation is underpinned by a global change
in gene expression. Orchestrated changes in gene expression take place at the
level of chromatin packaging which is regulated by enzymatic activity of
epigenetic regulators that in turn affect histone modifications. Using
expression profiling of epigenetic regulators in quiescent and activated primary
HSCs we found a number of histone methyltransferases including MLL1, MLL5, Set1
and ASH1 to be highly up-regulated during transdifferentiation of HSCs. All of
these histone methyltransferases regulate methylation of lysine 4 of histone H3,
which is a signature of actively transcribed genes. We therefore postulated that
one or more of these enzymes may be involved in positively influencing
expression of profibrogenic genes.
CONCLUSION: We find that ASH1 directly binds to the regulatory regions of alpha
smooth muscle actin (αSMA), collagen I, tissue inhibitor of metalloproteinase-1
(TIMP1) and transforming growth factor beta1 (TGFβ1) in activated HSCs while
depletion of ASH1 caused broad suppression of fibrogenic gene expression. We
also discovered that MeCP2 positively regulates ASH1 expression and therefore
identify ASH1 as a key transcriptional activator component of the MeCP2
epigenetic relay pathway that orchestrates coordinated induction of multiple
profibrogenic genes. Mice with a mutation in the Clock gene (ClockΔ19) have been identified as a
model of mania; however, the mechanisms that underlie this phenotype, and the
changes in the brain that are necessary for lithium's effectiveness on these
mice remain unclear. Here, we find that cholecystokinin (Cck) is a direct
transcriptional target of CLOCK and levels of Cck are reduced in the ventral
tegmental area (VTA) of ClockΔ19 mice. Selective knockdown of Cck expression via
RNA interference in the VTA of wild-type mice produces a manic-like phenotype.
Moreover, chronic treatment with lithium restores Cck expression to near
wild-type and this increase is necessary for the therapeutic actions of lithium.
The decrease in Cck expression in the ClockΔ19 mice appears to be due to a lack
of interaction with the histone methyltransferase, MLL1, resulting in decreased
histone H3K4me3 and gene transcription, an effect reversed by lithium. Human
postmortem tissue from bipolar subjects reveals a similar increase in Cck
expression in the VTA with mood stabilizer treatment. These studies identify a
key role for Cck in the development and treatment of mania, and describe some of
the molecular mechanisms by which lithium may act as an effective antimanic
agent. |
What is CRISPRi? | Clustered regularly interspaced palindromic repeats interference (CRISPRi). This discovery tool is is used for genetic screening based on loss-of-function phenotypes. | Sequence-specific control of gene expression on a genome-wide scale is an
important approach for understanding gene functions and for engineering genetic
regulatory systems. We have recently described an RNA-based method, CRISPR
interference (CRISPRi), for targeted silencing of transcription in bacteria and
human cells. The CRISPRi system is derived from the Streptococcus pyogenes
CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring
only the coexpression of a catalytically inactive Cas9 protein and a
customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA
elements complementary to the sgRNA and causes a steric block that halts
transcript elongation by RNA polymerase, resulting in the repression of the
target gene. Here we provide a protocol for the design, construction and
expression of customized sgRNAs for transcriptional repression of any gene of
interest. We also provide details for testing the repression activity of CRISPRi
using quantitative fluorescence assays and native elongating transcript
sequencing. CRISPRi provides a simplified approach for rapid gene repression
within 1-2 weeks. The method can also be adapted for high-throughput
interrogation of genome-wide gene functions and genetic interactions, thus
providing a complementary approach to RNA interference, which can be used in a
wider variety of organisms. The use of synthetic non-coding RNAs for post-transcriptional regulation of gene
expression has not only become a standard laboratory tool for gene functional
studies but it has also opened up new perspectives in the design of new and
potentially promising therapeutic strategies. Bioinformatics has provided
researchers with a variety of tools for the design, the analysis, and the
evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin
RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a
new system for genome engineering based on the bacterial CRISPR-Cas9 system
(Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have
the potential to also regulate gene expression at both transcriptional and
post-transcriptional level in a more specific way. In this mini review, we
present RNAi and CRISPRi design principles and discuss the advantages and
limitations of the current design approaches. T cell proliferation is critical for immune responses; however, the molecular
mechanisms that mediate the proliferative response are poorly understood.
MicroRNAs (miRs) regulate various molecular processes, including development and
function of the immune system. Here, utilizing multiple complementary genetic
and molecular approaches, we investigated the contribution of a
hematopoietic-specific miR, miR-142, in regulating T cell responses. T cell
development was not affected in animals with a targeted deletion of Mir142;
however, T cell proliferation was markedly reduced following stimulation both in
vitro and in multiple murine models of graft-versus-host disease (GVHD).
miR-142-deficient T cells demonstrated substantial cell-cycling defects, and
microarray and bioinformatics analyses revealed upregulation of genes involved
in cell cycling. Moreover, 2 predicted miR-142 target genes, the atypical E2F
transcription factors E2f7 and E2f8, were most highly upregulated in
miR-142-deficient cells. Clustered regularly interspaced short palindromic
repeat interference-mediated (CRISPRi-mediated) silencing of E2F7 and E2F8 in
miR-142-deficient T cells ameliorated cell-cycling defects and reduced GVHD, and
overexpression of these factors in WT T cells inhibited the proliferative
response. Together, these results identify a link between hematopoietic-specific
miR-142 and atypical E2F transcription factors in the regulation of mature T
cell cycling and suggest that targeting this interaction may be relevant for
mitigating GVHD. |
Which type of cells is affected in Amyotrophic Lateral Sclerosis? | Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which motor neurons are affected. | Stem cell research raises hopes for incurable neurodegenerative diseases. In
amyotrophic lateral sclerosis (ALS), affecting the motoneurones of the central
nervous system (CNS), stem cell-based therapy aims to replace dying host
motoneurones by transplantation of cells in disease-affected regions. Moreover,
transplanted stem cells can serve as a source of trophic factors providing
neuroprotection, slowing down neuronal degeneration and disease progression.
AIM: To determine the profile of seven trophic factors expressed by mesenchymal
stem cells (MSC) and neural stem cells (NSC) upon stimulation with CNS protein
extracts from SOD1-linked ALS rat model.
METHODS: Culture of rat MSC, NSC and fibroblasts were incubated with brain and
spinal cord extracts from SOD1(G93A) transgenic rats and mRNA expression of
seven growth factors was measured by quantitative PCR.
RESULTS: MSC, NSC and fibroblasts exhibited different expression patterns. Nerve
growth factor and brain-derived neurotropic factor were significantly
upregulated in both NSC and MSC cultures upon stimulation with SOD1(G93A) CNS
extracts. Fibroblast growth factor 2, insulin-like growth factor and
glial-derived neurotropic factor were upregulated in NSC, while the same factors
were downregulated in MSC. Vascular endothelial growth factor A upregulation was
restricted to MSC and fibroblasts. Surprisingly, SOD1(G93A) spinal cord, but not
the brain extract, upregulated brain-derived neurotropic factor in MSC and
glial-derived neurotropic factor in NSC.
CONCLUSIONS: These results suggest that inherent characteristics of different
stem cell populations define their healing potential and raise the concept of
ALS environment in stem cell transplantation. Spasticity is a common and disabling symptom observed in patients with central
nervous system diseases, including amyotrophic lateral sclerosis, a disease
affecting both upper and lower motor neurons. In amyotrophic lateral sclerosis,
spasticity is traditionally thought to be the result of degeneration of the
upper motor neurons in the cerebral cortex, although degeneration of other
neuronal types, in particular serotonergic neurons, might also represent a cause
of spasticity. We performed a pathology study in seven patients with amyotrophic
lateral sclerosis and six control subjects and observed that central
serotonergic neurons suffer from a degenerative process with prominent neuritic
degeneration, and sometimes loss of cell bodies in patients with amyotrophic
lateral sclerosis. Moreover, distal serotonergic projections to spinal cord
motor neurons and hippocampus systematically degenerated in patients with
amyotrophic lateral sclerosis. In SOD1 (G86R) mice, a transgenic model of
amyotrophic lateral sclerosis, serotonin levels were decreased in brainstem and
spinal cord before onset of motor symptoms. Furthermore, there was noticeable
atrophy of serotonin neuronal cell bodies along with neuritic degeneration at
disease onset. We hypothesized that degeneration of serotonergic neurons could
underlie spasticity in amyotrophic lateral sclerosis and investigated this
hypothesis in vivo using tail muscle spastic-like contractions in response to
mechanical stimulation as a measure of spasticity. In SOD1 (G86R) mice, tail
muscle spastic-like contractions were observed at end-stage. Importantly, they
were abolished by 5-hydroxytryptamine-2b/c receptors inverse agonists. In line
with this, 5-hydroxytryptamine-2b receptor expression was strongly increased at
disease onset. In all, we show that serotonergic neurons degenerate during
amyotrophic lateral sclerosis, and that this might underlie spasticity in mice.
Further research is needed to determine whether inverse agonists of
5-hydroxytryptamine-2b/c receptors could be of interest in treating spasticity
in patients with amyotrophic lateral sclerosis. Activation of microglia, CNS resident immune cells, is a pathological hallmark
of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting
motor neurons. Despite evidence that microglia contribute to disease
progression, the exact role of these cells in ALS pathology remains unknown. We
immunomagnetically isolated microglia from different CNS regions of SOD1(G93A)
rats at three different points in disease progression: presymptomatic, symptom
onset and end-stage. We observed no differences in microglial number or
phenotype in presymptomatic rats compared to wild-type controls. Although after
disease onset there was no macrophage infiltration, there were significant
increases in microglial numbers in the spinal cord, but not cortex. At disease
end-stage, microglia were characterized by high expression of galectin-3,
osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6,
BDNF and arginase-1. Flow cytometry revealed the presence of at least two
phenotypically distinct microglial populations in the spinal cord.
Immunohistochemistry showed that galectin-3/osteopontin positive microglia were
restricted to the ventral horns of the spinal cord, regions with severe motor
neuron degeneration. End-stage SOD1(G93A) microglia from the cortex, a less
affected region, displayed similar gene expression profiles to microglia from
wild-type rats, and displayed normal responses to systemic inflammation induced
by LPS. On the other hand, end-stage SOD1(G93A) spinal microglia had blunted
responses to systemic LPS suggesting that in addition to their phenotypic
changes, they may also be functionally impaired. Thus, after disease onset,
microglia acquired unique characteristics that do not conform to typical M1
(inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was
observed only in the most affected CNS regions, suggesting that overexpression
of mutated hSOD1 is not sufficient to trigger these changes in microglia. These
novel observations suggest that microglial regional and phenotypic heterogeneity
may be an important consideration when designing new therapeutic strategies
targeting microglia and neuroinflammation in ALS. Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial
amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and
aggregate to form intracellular inclusions. We report that both small
ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant
proteins at lysine 75 in a motoneuronal cell line, the cell type affected in
ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9
of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather
than by SUMO1, significantly increased the stability of the proteins and
accelerated intracellular aggregate formation. These findings suggest the
contribution of sumoylation, particularly by SUMO3, to the protein aggregation
process underlying the pathogenesis 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. |
Is Achondroplasia associated with hearing loss? | Yes, there is hearing deficit in achondroplasia | Increased intracranial pressure and ventricular and subarachnoidal dilatation
are common manifestations in achondroplasia. They rarely lead to major
neurologic and/or psychomotor deficits and neurosurgical intervention is seldom
needed. The present study was undertaken to detect signs of minor cerebral
dysfunction and discuss possibilities of their prevention. Thirty children with
achondroplasia were compared to 3 control groups: their next-born sibs, 30
children with other forms of dwarfism, and 30 children with normal height. Early
development was assessed by means of questionnaires. Cognitive skills were
evaluated with the German version of the Cognitive Abilities Test and the
Lorge-Thorndike Intelligence Test. Personality data were tested using
standardized neuroticism, extraversion, and anxiety scales. Children with
achondroplasia had more frequent histories of delayed motor development,
retarded speech development, and lower school grades in language-related
specialties. Psychometric testing disclosed total and subtest scores in the
population-based normal range. In comparison with their sibs and matched
controls children with achondroplasia had significantly lower total scores
mainly caused by low scores in the subtest "verbal comprehension." We conclude
that verbal comprehension is significantly impaired in children with
achondroplasia. This partial deficiency is probably related to frequent middle
ear infections and resulting conductive hearing loss. Hypotonia with delayed
oropharyngeal muscle coordination and parental response to an altered, more
infantile instinctive releasing pattern may be contributing factors. Characteristic temporal bone changes have recently been defined by high
resolution CT in nine patients with achondroplasia (Cobb et al., Am J
Neuroradiol 9:1195, 1988). These included narrowing of the skull base and
"towering" petrous ridges resulting in abnormal orientation of the inner and
middle ear structures. In order to determine whether these morphologic changes
are the cause of the hearing deficit in achondroplasia, audiometric studies and
ENT evaluation were performed in eight of the nine patients. All had a history
of frequent otitis media and four had experienced tympanic membrane tube
insertion. Three patients had significant sensorineural hearing loss, two had
conductive hearing loss and one patient had combined hearing loss. None of the
temporal bone morphologic changes were found to be correlated with the degree of
either sensorineural or conductive hearing loss. Fusion of the ossicular chain
was not present in any of our cases. Appropriate treatment of frequent acute
otitis media and early awareness of middle ear effusions and conductive hearing
loss in children with achondroplasia may be of great importance in preventing
permanent hearing loss. Achondroplasia is the most prevalent chondrodysplasia and numerous authors have
documented the varied social and medical complications that may compromise a
full and productive life. Complications include cervicomedullary compression,
spinal stenosis, restrictive and obstructive lung disease, otitis media, and
tibial bowing, among others. These known complications have led to
recommendations for the anticipatory management of such patients. There are
relatively few data on the actual rates and timing of these problems. This paper
reports data on the rates and age of occurrence of several of these
complications based on a review of recorded chart information of 193 patients
ascertained from several well established genetic centres with a known interest
in the chondrodysplasias. The length of follow up varied and the rates of
occurrence at specific age intervals were used to estimate the cumulative
percentage affected for each complication. The report includes information on
otitis media, ventilation tubes, hearing loss, tonsillectomy, speech problems,
tibial bowing and osteotomy, ventricular shunting, apnoea, cervicomedullary
decompression, and neurological signs attributable to spinal stenosis. OBJECTIVE: To determine the prevalence of hearing loss and abnormal tympanometry
in children with skeletal dysplasia.
DESIGN: Clinical screening program.
SETTING: National convention of the Little People of America.
PATIENTS: Convenience sample of volunteers aged 18 years or younger with
skeletal dysplasias.
INTERVENTIONS: Hearing screening with behavioral testing and/or otoacoustic
emissions, otoscopy, and tympanometry.
MAIN OUTCOME MEASURES: A failed hearing screen was defined as hearing 35 dB HL
(hearing level) or greater at 1 or more tested frequencies or by a "fail"
otoacoustic emissions response. Types B and C tympanograms were considered
abnormal.
RESULTS: A total of 58 children (aged ≤18 years) with skeletal dysplasia
enrolled, and 56 completed hearing screening. Forty-one children had normal
hearing (71%); 9 failed in 1 ear (16%); and 6 failed in both ears (10%).
Forty-four children had achondroplasia, and 31 had normal hearing in both ears
(71%); 8 failed hearing screening in 1 ear (18%), and 3 in both ears (7%).
Tympanometry was performed in 45 children, with normal tympanograms found in 21
(47%), bilateral abnormal tympanograms in 15 (33%), and unilateral abnormal
tympanograms in 9 (20%). Fourteen children with achondroplasia had normal
tympanograms (42%); 11 had bilateral abnormal tympanograms (33%); and 8 had
unilateral abnormal tympanograms (24%). For those children without functioning
tympanostomy tubes, there was a 9.5 times greater odds of hearing loss if there
was abnormal tympanometry (P = .03).
CONCLUSIONS: Hearing loss and middle-ear disease are both highly prevalent in
children with skeletal dysplasias. Abnormal tympanometry is highly associated
with the presence of hearing loss, as expected in children with eustachian tube
dysfunction. Hearing screening with medical intervention is recommended for
these children. A hearing screening program was performed to determine the prevalence of hearing
loss and abnormal tympanometry in individuals with short-stature skeletal
dysplasias attending a national meeting. Behavioral audiometry, otoacoustic
emission testing, and tympanometry were used to assess hearing. Failed hearing
screen was defined as hearing ≥ 35 dB at one or more frequencies or by "fail" on
otoacoustic emissions. One hundred ten of 112 subjects completed the screening.
58 (51.8%) were children. Seventy-three (65.2%) had achondroplasia, 34 (30.4%)
had one of 11 other diagnoses, and 5(4.4%) were undiagnosed. 25.8% of children
failed hearing screening in one or both ears, while 46.3% of adults failed in
one or both ears. 55.1% of adults and 25.0% of children with achondroplasia
failed screening. Abnormal hearing was also found in the some patients with
spondyloepiphyseal dysplasia congenital (SEDC; 75%), diastrophic dysplasia
(66%), and Morquio (66%). Hearing was normal in those with hypochondroplasia,
pseudoachondroplasia, and microcephalic osteodysplastic primordial dwarfism.
Tympanometry was abnormal in at least one ear in 53.3% of children and 38.5% of
adults. Abnormal tympanometry in the absence of functioning tympanostomy tubes
was associated with 9.5 greater odds of hearing loss in children and 2.8 greater
odds of hearing loss in the total cohort. Only 3 (2.7%) respondents reported the
use of hearing aids. Hearing loss and middle ear disease are common in both
children and adults with skeletal dysplasia. Adults were more likely to fail
hearing screening than children. Abnormal tympanometry is associated with
hearing loss. Hearing screening with appropriate intervention is recommended for
these patients. |
Is the JNK pathway activated during liver regeneration? | Yes, the Jun-N-terminal kinase (JNK) pathway is strongly activated after partial hepatectomy. | Many growth factors and cytokines are involved in liver regeneration. Of them,
only hepatopoietin (HPO)/ALR (augmenter of liver regeneration) is a specifically
hepatotrophic factor originally identified from the cytosol of regenerating or
hyperplastic hepatic cells. Previous reports indicate that extracellular HPO
triggers the MAPK pathway by binding its specific receptor on the cell surface.
However, its function in the cytosol of hepatocytes is unclear. Here we
identified that JAB1 (Jun activation domain-binding protein 1), a co-activator
of AP-1, which is essential for liver regeneration, specifically interacts with
intracellular HPO. JAB1 colocalizes with HPO in nuclei of hepatic cells or COS-7
cells. As an intracrine factor, the intracellular function of HPO is to increase
c-Jun phosphorylation independent of c-Jun amino-terminal kinase (JNK),
extracellular signal-regulated kinase (ERK) -1 and -2, and leads to potentiation
of JAB1-mediated AP-1 activation. Amino acids 1-63 of HPO molecule are
sufficient to bind to JAB1, but the full-length HPO is necessary for its
intracellular signaling. Taken together, these results elucidate a novel
mechanism of intracrine cytokine signaling by specifically modulating the AP-1
pathway through JAB1, in a MAPK-independent fashion. The c-Jun-N-terminal kinase (JNK) pathway is strongly activated after partial
hepatectomy (PH), but its role in hepatocyte proliferation is not known. In this
study, JNK activity was blocked with the small molecule inhibitor JNK SP600125
in vivo and in vitro as shown by a reduction of c-Jun phosphorylation, AP-1 DNA
binding activity, and c-jun messenger RNA (mRNA) expression. SP600125 inhibited
proliferating cell nuclear antigen (PCNA) expression, cyclin D1 mRNA and protein
expression and reduced mitotic figures after PH. Survival was reduced
significantly 3 days after PH in SP600125-treated versus vehicle-treated rats (3
of 11 vs. 8 of 9, P <.01). In epidermal growth factor (EGF)-treated primary
cultures of rat hepatocytes, SP600125 decreased (3)H-thymidine uptake, cyclin D1
mRNA and protein expression, and inhibited the EGF-induced transcription of a
cyclin D1 promoter-driven reporter gene. The defective regeneration and the
decreased survival in SP600125-treated rats did not result from a major increase
in apoptosis as shown by normal levels of caspase 3 activity and only slight
increases in apoptotic figures. In conclusion, our data show that JNK drives G0
to G1 transition in hepatocytes and that cyclin D1 is a downstream target of the
JNK pathway during liver regeneration. To investigate the effects of arsenite on cell proliferation and the signal
transduction in hapatocytes in vivo, rats received a single injection of sodium
arsenite immediately after partial hepatectomy. Characteristic DNA fragmentation
was observed at 4h after the arsenite-injection in partially hepatectomized
liver, while it was not detected either in the control (partial hepatectomy
only) or arsenite-injected normal (without partial hepatectomy) liver. The
effect of the arsenite-injection on the activation of extracellular
signal-regulated kinase (ERK) was not observed in the normal or the partially
hepatectomized liver. The activity of p38 mitogen-activated protein kinase
(MAPK) markedly increased after 15min to 2h after the arsenite-injection in
partially hepatectomized liver while no or a less increase was observed in the
arsenite-injected normal or the control, respectively. The Jun N-terminal kinase
(JNK) was activated to a maximal level, about six-fold the maximum of the
control, at 15min after the injection with partial hepatectomy. The
arsenite-injection markedly increased the phosphorylated forms of c-Jun and
ATF-2 and the protein levels of c-Jun, p53 and p21(WAF1/CIP1) in the partially
hepatectomized liver. These results suggested that arsenite induced apoptosis in
the hepatocytes in vivo, through the enhancement of the activation of JNK and
p38 MAPK caused by partial hepatectomy and the p53-dependent p21(WAF1/CIP1)
protein expression. During the development and organogenesis of all multicellular organisms, cell
fate decisions determine whether cells undergo proliferation, differentiation,
or aging. Two independent stress kinase signaling pathways, p38-MAPK, and JNKs,
have evolved that relay developmental and environmental cues to determine cell
responses. Although multiple stimuli can activate these two stress kinase
pathways, the functional interactions and molecular cross-talks between these
common second signaling cascades are poorly elucidated. Here we report that JNK
and p38-MAPK pathways antagonistically control cellular senescence, oncogenic
transformation, and proliferation in primary mouse embryonic fibroblasts (MEFs).
Similarly, genetic inactivation of the JNK pathway results in impaired
proliferation of fetal hepatoblasts in vitro and defective adult liver
regeneration in vivo, which is rescued by inhibition of the p38-MAPK pathway.
Thus, the balance between the two stress-signaling pathways, MKK7-JNK and
MKK3/6-p38-MAPK, determines cell fate and links environmental and developmental
stress to cell cycle arrest, senescence, oncogenic transformation, and adult
tissue regeneration. In the liver, the JNK cascade is induced downstream of TNF receptors (TNFRs) in
response to inflammatory, microbial, and toxic challenges. Sustained activation
of JNK triggers programmed cell death (PCD), and hepatocyte survival during
these challenges requires induction of the NF-kappaB pathway, which antagonizes
this activation by upregulating target genes. Thus, modulation of JNK activity
is crucial to the liver response to TNFR-mediated challenge. The basis for this
modulation, however, is unknown. Here, we investigated the role of the NF-kappaB
target Gadd45b in the regulation of hepatocyte fate during liver regeneration
after partial hepatectomy. We generated Gadd45b(-/-) mice and found that they
exhibited decreased hepatocyte proliferation and increased PCD during liver
regeneration. Notably, JNK activity was markedly increased and sustained in
livers of Gadd45b(-/-) mice compared with control animals after partial
hepatectomy. Furthermore, imposition of a Jnk2-null mutation, attenuating JNK
activity, completely rescued the regenerative response in Gadd45b(-/-) mice.
Interestingly, Gadd45beta ablation did not affect hepatotoxic JNK signaling
after a TNFR-mediated immune challenge, suggesting specificity in the inducible
hepatic program for JNK restraint activated during distinct TNFR-mediated
challenges. These data provide a basis for JNK suppression during liver
regeneration and identify Gadd45beta as a potential therapeutic target in liver
diseases. The c-Jun N-terminal kinase (JNK) pathway enhances graft injury after liver
transplantation (LT). We hypothesized that the JNK2 isoform promotes graft
injury via the mitochondrial permeability transition (MPT). Livers of C57BL/6J
(wild-type, WT) and JNK2 knockout (KO) mice were transplanted into WT recipients
after 30 h of cold storage in UW solution. Injury after implantation was
assessed by serum ALT, histological necrosis, TUNEL, Caspase 3 activity, 30-day
survival, and cytochrome c and 4-hydroxynonenal immunostaining. Multiphoton
microscopy after LT monitored mitochondrial membrane potential in vivo. After
LT, ALT increased three times more in WT compared to KO (p < 0.05). Necrosis and
TUNEL were more than two times greater in WT than KO (p < 0.05). Immunostaining
showed a >80% decrease of mitochondrial cytochrome c release in KO compared to
WT (p < 0.01). Lipid peroxidation was similarly decreased. Every KO graft but
one survived longer than all WT grafts (p < 0.05, Kaplan-Meier). After LT,
depolarization of mitochondria occurred in 73% of WT hepatocytes, which
decreased to 28% in KO (p < 0.05). In conclusion, donor JNK2 promotes injury
after mouse LT via the MPT. MPT inhibition using specific JNK2 inhibitors may be
useful in protecting grafts against adverse outcomes from ischemia/reperfusion
injury. BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion
resulting in ischemia/reperfusion injury in the remt liver. Here, we
developed a rat model of selective lobe occlusion to isolate reperfusion stress
from ischemia and to analyze its effect on liver regeneration.
METHODS: Left lateral and median lobes of liver were either mobilized or
subjected twice for 10min to ischemia followed by 5min reperfusion prior to
resection while the regenerative lobes were only subjected to reperfusion.
RESULTS: Although intermittent reperfusion stress induced higher levels of serum
transaminases, analysis of cell cycle regulators revealed accelerated
regenerative response compared to standard partial hepatectomy. The G0/G1
transition occurred before tissue resection, as evidenced by c-fos, junB, and
IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition,
and cell division occurred earlier than normal. Unexpectedly, liver
mobilization, a component of the clamping procedure, also resulted in earlier
G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal
Kinase pathway and was associated with an oxidative stress response as evidenced
by the expression of inducible nitric oxide synthase.
CONCLUSION: Intermittent selective clamping of lobes to be resected induced
reperfusion stress on remt liver that was beneficial for liver regeneration,
suggesting this procedure could be applied in clinical practice. |
Is STAT3 transcription factor regulated by mTORC1? | mTORC1 was found to regulate STAT3 activity in, at least, three ways: 1) after induction by IL6, 2) by direct phosphorylation during hypoxia, to promote HIF-1α mRNA transcription, and 3) after activation by excess amino acids, which then positively regulate Notch1 expression through STAT3 activation. | Aberrant activation of mammalian target of rapamycin complex 1 (mTORC1), caused
by loss or inactivation of TSC1/TSC2 protein complex, leads to negative feedback
inhibition of Akt. The exact mechanisms of this process are still not fully
understood. Here we present evidence for the involvement of STAT3, a known
mTORC1 regulated transcription factor, in this process. We demonstrate that
STAT3 promotes the transcription of PTEN by directly binding on the PTEN
promoter. Elevated PTEN then inhibits the proliferation of Tsc1(-/-) or
Tsc2(-/-) cells through down-regulation of Akt signaling. Activation of PTEN in
this pathway may thus serve as a protective mechanism against hyper-activated
mTORC1 mediated tumorigenesis and contribute to the benign nature of tumors
caused by loss of either TSC1 or TSC2. The mechanistic (or mammalian) target of rapamycin (mTOR) is a kinase that
regulates key cellular functions linked to the promotion of cell growth and
metabolism. This kinase, which is part of two protein complexes termed mTOR
complex 1 (mTORC1) and 2 (mTORC2), has a fundamental role in coordinating
anabolic and catabolic processes in response to growth factors and nutrients. Of
the two mTOR complexes, mTORC1 is by far the best characterized. When active,
mTORC1 triggers cell growth and proliferation by promoting protein synthesis,
lipid biogenesis, and metabolism, and by reducing autophagy. The fact that
mTORC1 deregulation is associated with several human diseases, such as type 2
diabetes, cancer, obesity and neurodegeneration, highlights its importance in
the maintece of cellular homeostasis. Over the last years, several groups
observed that mTORC1 inhibition, in addition to reducing protein synthesis,
deeply affects gene transcription. Here, we review the connections between
mTORC1 and gene transcription by focusing on its impact in regulating the
activation of specific transcription factors including including STAT3, SREBPs,
PPARγ, PPARα, HIF1α, YY1–PGC1α and TFEB. We also discuss the importance of these
transcription factors in mediating the effects of mTORC1 on various cellular
processes in physiological and pathological contexts. Nutrient overload is associated with the development of obesity, insulin
resistance, and type 2 diabetes. However, the underlying mechanisms for
developing insulin resistance in the presence of excess nutrients are
incompletely understood. We investigated whether activation of AMP-activated
protein kinase (AMPK) prevents the hepatic insulin resistance that is induced by
the consumption of a high-protein diet (HPD) and the presence of excess amino
acids. Exposure of HepG2 cells to excess amino acids reduced AMPK
phosphorylation, upregulated Notch1 expression, and impaired the
insulin-stimulated phosphorylation of Akt Ser(473) and insulin receptor
substrate-1 (IRS-1) Tyr(612). Inhibition of Notch1 prevented amino acid-induced
insulin resistance, which was accompanied by reduced expression of Rbp-Jk, hairy
and enhancer of split-1, and forkhead box O1. Mechanistically, mTORC1 signaling
was activated by excess amino acids, which then positively regulated Notch1
expression through the activation of the signal transducer and activator of
transcription 3 (STAT3). Activation of AMPK by metformin inhibited mTORC1-STAT3
signaling, thereby preventing excess amino acid-impaired insulin signaling.
Finally, HPD feeding suppressed AMPK activity, activated mTORC1/STAT3/Notch1
signaling, and induced insulin resistance. Chronic administration of either
metformin or rapamycin inhibited the HPD-activated mTORC1/STAT3/Notch1 signaling
pathway and prevented hepatic insulin resistance. We conclude that the
upregulation of Notch1 expression by hyperactive mTORC1 signaling is an
essential event in the development of hepatic insulin resistance in the presence
of excess amino acids. Activation of AMPK prevents amino acid-induced insulin
resistance through the suppression of the mTORC1/STAT3/Notch1 signaling pathway. Recent clinical trials using rapalogues in tuberous sclerosis complex show
regression in volume of typically vascularised tumours including angiomyolipomas
and subependymal giant cell astrocytomas. By blocking mechanistic/mammalian
target of rapamycin complex 1 (mTORC1) signalling, rapalogue efficacy is likely
to occur, in part, through suppression of hypoxia-inducible factors (HIFs) and
vascular endothelial growth factors (VEGFs). We show that rapamycin reduces
HIF-1α protein levels, and to a lesser extent VEGF-A levels, in renal
cystadenoma cells in a Tsc2+/- mouse model. We established that mTORC1 drives
HIF-1α protein accumulation through enhanced transcription of HIF-1α mRNA, a
process that is blocked by either inhibition or knockdown of signal transducer
and activation of transcription 3 (STAT3). Furthermore, we demonstrated that
STAT3 is directly phosphorylated by mTORC1 on Ser727 during hypoxia, promoting
HIF-1α mRNA transcription. mTORC1 also regulates HIF-1α synthesis on a
translational level via co-operative regulation of both initiation factor
4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase-1 (S6K1), whereas
HIF-1α degradation remains unaffected. We therefore proposed that mTORC1 drives
HIF-1α synthesis in a multifaceted manner through 4E-BP1/eIF4E, S6K1 and STAT3.
Interestingly, we observed a disconnect between HIF-1α protein levels and VEGF-A
expression. Although both S6K1 and 4E-BP1 regulate HIF-1α translation, VEGF-A is
primarily under the control of 4E-BP1/eIF4E. S6K1 inhibition reduces HIF-1α but
not VEGF-A expression, suggesting that mTORC1 mediates VEGF-A expression via
both HIF-1α-dependent and -independent mechanisms. Our work has important
implications for the treatment of vascularised tumours, where mTORC1 acts as a
central mediator of STAT3, HIF-1α, VEGF-A and angiogenesis via multiple
signalling mechanisms. The mammalian target of rapamycin (mTOR) signaling pathway integrates
environmental cues to regulate cell growth and survival through various
mechanisms. However, how mTORC1 responds to acute inflammatory signals to
regulate bowel regeneration is still obscure. In this study, we investigated the
role of mTORC1 in acute inflammatory bowel disease. Inhibition of mTORC1
activity by rapamycin treatment or haploinsufficiency of Rheb through genetic
modification in mice impaired intestinal cell proliferation and induced cell
apoptosis, leading to high mortality in dextran sodium sulfate- and
2,4,6-trinitrobenzene sulfonic acid-induced colitis models. Through bone marrow
transplantation, we found that mTORC1 in nonhematopoietic cells played a major
role in protecting mice from colitis. Reactivation of mTORC1 activity by amino
acids had a positive therapeutic effect in mTORC1-deficient Rheb(+/-) mice.
Mechanistically, mTORC1 mediated IL-6-induced Stat3 activation in intestinal
epithelial cells to stimulate the expression of downstream targets essential for
cell proliferation and tissue regeneration. Therefore, mTORC1 signaling
critically protects against inflammatory bowel disease through modulation of
inflammation-induced Stat3 activity. As mTORC1 is an important therapeutic
target for multiple diseases, our findings will have important implications for
the clinical usage of mTORC1 inhibitors in patients with acute inflammatory
bowel disease. |
How is mTORC1 involved in the regulation of heat stress? | mTORC1 attenuates stress response by inhibiting cap-independent Hsp70 translation. | Protein synthesis is a key regulated cellular process that links nutrient
availability and organismal growth. It has long been known that some cellular
proteins continue to be synthesized under conditions where global translation is
severely compromised. One prominent example is the selective translation of heat
shock proteins (Hsps) under stress conditions. Although the transcriptional
regulation of Hsp genes has been well established, neither the specific
translation-promoting features nor the regulatory mechanism of the translation
machinery have been clearly defined. Here we show that the stress-induced
preferential translation of Hsp70 mRNA is negatively regulated by PI3K-mTORC1
signaling. Despite the transcriptional up-regulation, the translation of Hsp70
mRNA is deficient in cells lacking tuberous sclerosis complex 2. Conversely,
Hsp70 synthesis is enhanced under the reduced PI3K-mTORC1 signaling. We found
that the 5' UTR of Hsp70 mRNA contributes to cap-independent translation without
exhibiting typical features of internal ribosome entry site. Our findings imply
a plausible mechanism for how persistent PI3K-mTORC1 signaling favors the
development of age-related pathologies by attenuating stress resistance. |
List components of the CRSP/Med complex. | Mediator of RNA polymerase II transcription subunit 7
Mediator of RNA polymerase II transcription subunit 14
Mediator of RNA polymerase II transcription subunit 17
Mediator of RNA polymerase II transcription subunit 23
Mediator of RNA polymerase II transcription subunit 24
Mediator of RNA polymerase II transcription subunit 26
Mediator of RNA polymerase II transcription subunit 27 | Activation of gene transcription in metazoans is a multistep process that is
triggered by factors that recognize transcriptional enhancer sites in DNA. These
factors work with co-activators to direct transcriptional initiation by the RNA
polymerase II apparatus. One class of co-activator, the TAF(II) subunits of
transcription factor TFIID, can serve as targets of activators and as proteins
that recognize core promoter sequences necessary for transcription initiation.
Transcriptional activation by enhancer-binding factors such as Sp1 requires
TFIID, but the identity of other necessary cofactors has remained unknown. Here
we describe a new human factor, CRSP, that is required together with the
TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies
a complex of approximate relative molecular mass 700,000 (M(r) approximately
700K) that contains nine subunits with M(r) values ranging from 33K to 200K.
Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of
the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in
yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear
hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new
proteins, but the amino terminus of CRSP70 is homologous to elongation factor
TFIIS. Immunodepletion studies confirm that these subunits have an essential
cofactor function. The presence of common subunits in distinct cofactor
complexes suggests a combinatorial mechanism of co-activator assembly during
transcriptional activation. Transcription of protein coding genes in metazoans involves the concerted action
of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk
between these two classes of transcription factors is mediated by an elaborate
set of cofactor complexes. For the activation of transcription by the promoter
specificity protein 1 (Sp1), TATA binding protein-associated factors in the
TFIID complex originally were identified as necessary coactivators, but the
identity of additional cofactors required for activated transcription was
unknown. Recently, we have reported the isolation and properties of a cofactor
complex, CRSP (cofactor required for Sp1), which functions in conjunction with
the TATA binding protein-associated factors to promote efficient activation of
transcription by Sp1. CRSP contains unique subunits as well as polypeptides that
are shared with other cofactor complexes. Here, we report a detailed
purification protocol for the isolation of CRSP from human HeLa cells. Our
purification strategy takes advantage of the ability of CRSP to bind
Ni2+-nitrilotriacetic acid-agarose resin as well as other conventional
chromatographic resins. We also describe a streamlined purification protocol
that allows a more rapid and efficient means to isolate active CRSP. The human cofactor complexes ARC (activator-recruited cofactor) and CRSP
(cofactor required for Sp1 activation) mediate activator-dependent transcription
in vitro. Although these complexes share several common subunits, their
structural and functional relationships remain unknown. Here, we report that
affinity-purified ARC consists of two distinct multisubunit complexes: a larger
complex, denoted ARC-L, and a smaller coactivator, CRSP. Reconstituted in vitro
transcription with biochemically separated ARC-L and CRSP reveals differential
cofactor functions. The ARC-L complex is transcriptionally inactive, whereas the
CRSP complex is highly active. Structural determination by electron microscopy
(EM) and three-dimensional reconstruction indicate substantial differences in
size and shape between ARC-L and CRSP. Moreover, EM analysis of independently
derived CRSP complexes reveals distinct conformations induced by different
activators. These results suggest that CRSP may potentiate transcription via
specific activator-induced conformational changes. The interferon (IFN)-induced signal transduction and transcription activation
complex, ISGF3, is assembled from three proteins, STAT1, STAT2, and IRF9. Of
these components, STAT2 provides a fundamental and essential transcriptional
activation function for ISGF3. In the present study, we show that ISGF3-mediated
transcription is dependent on STAT2 interactions with DRIP150, a subunit of the
multimeric Mediator coactivator complex. Other Mediator subunits, DRIP77 and
DRIP130, were found either to bind STAT2 without augmenting ISGF3
transcriptional activity or to enhance ISGF3 transcription without binding
STAT2, but only DRIP150 both enhanced IFN-dependent transcription and
coimmunoprecipitated with STAT2. Endogenous DRIP150 and STAT2 were able to
interact in solution, and DNA affinity chromatography and chromatin
immunoprecipitation assays demonstrated that DRIP150 binds to the mature,
activated ISGF3-DNA complex and is recruited to target gene promoters in an
IFN-dependent fashion. IFN-dependent recruitment of DRIP130 to an ISGF3 target
promoter and SRB10-STAT2 coprecipitation suggest indirect association with a
multisubunit Mediator complex. The site of STAT2 interaction was mapped to
DRIP150 residues 188 to 566, which are necessary and sufficient for interaction
with STAT2. Expression of this DRIP150 fragment, but not DRIP150 fragments
outside the STAT2 interaction region, suppressed ISGF3-mediated transcriptional
activity in a domit-negative fashion, suggesting a direct functional role of
this domain in mediating STAT2-DRIP150 interactions. These findings indicate
that the IFN-activated ISGF3 transcription factor regulates transcription
through contact with DRIP150 and implicate the Mediator coactivator complex in
IFN-activated gene regulation. C/EBPbeta is an intrinsically repressed transcription factor that regulates
genes involved in differentiation, proliferation, tumorigenesis, and apoptosis.
C/EBPbeta acts as a repressor that is turned into an activator by the Ras
oncoprotein through phosphorylation of a MAPK site. C/EBPbeta activation is
accompanied by a conformational change. Active and repressive C/EBPbeta
interacts with multisubunit Mediator complexes through the CRSP130/Sur2 subunit.
The CRSP130/Sur2 subunit is common to two distinct types of Mediator complexes,
characterized by CRSP70 and CDK8 proteins as transcriptionally active and
inactive Mediator, respectively. Knockdown of CRSP130/Sur2 prevents Mediator
binding and transactivation through C/EBPbeta. Oncogenic Ras signaling or
activating mutations in C/EBPbeta selects the transcriptionally active Mediator
complex that also associates with RNA polymerase II. These results show that a
Ras-induced structural alteration of C/EBPbeta determines differential gene
activation through selective interaction with distinct Mediator complexes. Sequence-specific DNA-binding activators, key regulators of gene expression,
stimulate transcription in part by targeting the core promoter recognition TFIID
complex and aiding in its recruitment to promoter DNA. Although it has been
established that activators can interact with multiple components of TFIID, it
is unknown whether common or distinct surfaces within TFIID are targeted by
activators and what changes if any in the structure of TFIID may occur upon
binding activators. As a first step toward structurally dissecting
activator/TFIID interactions, we determined the three-dimensional structures of
TFIID bound to three distinct activators (i.e., the tumor suppressor p53
protein, glutamine-rich Sp1 and the oncoprotein c-Jun) and compared their
structures as determined by electron microscopy and single-particle
reconstruction. By a combination of EM and biochemical mapping analysis, our
results uncover distinct contact regions within TFIID bound by each activator.
Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global
structural changes upon activator binding, instead, a rather confined set of
local conserved structural changes were observed when each activator binds
holo-TFIID. These results suggest that activator contact may induce unique
structural features of TFIID, thus providing oscale information on
activator-dependent TFIID assembly and transcription initiation. Human MED26 was originally purified in the cofactor required for the Sp1
activation complex (CRSP) as a 70-kDa component named CRSP70. This polypeptide
was specific to metazoans and the “small” form of the Mediator complex. We
report here that a Drosophila melanogaster homologue of MED26 similarly
interacts with other components of the core Drosophila Mediator complex but not
with the kinase module and is recruited to genes upon activation. Using a null
allele of Med26, we show that Med26 is required for organismal viability but not
for cell proliferation or survival. Clones lacking Med26 in the wing disc lead
to loss of the adult wing margin and reduced expression of genes involved in
wing margin formation. Surprisingly, when polytene chromosomes from the salivary
gland were examined using antibodies to Med26, it was apparent that a fraction
of the protein was associated with the chromocenter, which contains pericentric
heterochromatin. This staining colocalizes with heterochromatin protein 1 (HP1).
Immunoprecipitation experiments show that Med26 interacts with HP1. The
interaction is mediated through the chromoshadow domain of HP1 and through the
conserved motif in the carboxy terminus of the Med26 protein. This work is the
first characterization of the metazoan-specific Mediator subunit in an animal
model. |
Patients of which disease could be treated by utilizing knowledge obtained from experiments suppressing TDP-43 toxicity in yeast? | Amyotrophic lateral sclerosis (ALS). | Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease
primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause
some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in
degenerating neurons of most individuals with ALS. Thus, strategies aimed at
targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here,
we report results from two genome-wide loss-of-function TDP-43 toxicity
suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was
deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that,
in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the
cytoplasm and likely act as decoys to sequester TDP-43, preventing it from
interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of
Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient
to rescue TDP-43 toxicity. Our findings provide insight into TDP-43-mediated
cytotoxicity and suggest that decreasing Dbr1 activity could be a potential
therapeutic approach for ALS. |
Is calcium overload involved in the development of diabetic cardiomyopathy? | Yes. | The use of insulin by diabetics has largely removed the threat of death from
ketotic coma but cardiovascular dysfunction remains a major cause of death in
patients with diabetes. Recent research has indicated a generalized membrane
defect, which may cause abnormalities of calcium metabolism in nerves, cardiac
and smooth muscle as well as endothelial cells and thus may lead respectively to
the development of neuropathy, primary cardiomyopathy, microangiopathy and
atherosclerosis in the diabetic population. Each of these pathogenic processes,
which are associated with insulin deficiency, alone or in combination with
others, may result in cardiac dysfunction in chronic diabetes. Activation of the
sympathetic nervous system and abnormalities in catecholamine metabolism have
been identified in diabetes; their involvement in the genesis of cardiac pump
failure as well as large and small vessel disease is likely. The membrane
defects as indicated by changes in both plasma membrane and glycocalyx in
diabetic cardiomyopathy appear to be complex and may involve alterations in the
metabolism of lipids and pyrimidine nucleotides. It seems that intracellular
calcium overload is intimately involved in the development of diabetic
cardiomyopathy; however, a concentrated research effort is required to
understand the primary biochemical lesion in the pathogenesis of cardiac
dysfunction in diabetes. In the meantime, a heightened awareness on the part of
clinicians concerning the susceptibility of diabetic patients to cardiovascular
problems may help in reducing mortality and morbidity in the diabetic
population. In order to determine if cardiac membrane Ca(2+)-transport activities are
altered in chronic diabetes induced by alloxan, rats were given an intravenous
injection of 65 mg/kg and the hearts were used 8 weeks later. Some 4 weeks,
diabetic animals were injected with insulin (3 U/day) for 4 weeks. Both
sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes were isolated from
the ventricular tissue and their Ca(2+)-transporting activities were determined.
SL Na(+)-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca(2+)-stimulated
ATPase activities were depressed in the diabetic heart. Likewise, SR
ATP-dependent Ca2+ uptake activity in the diabetic heart was markedly decreased
in comparison to the control preparations. These defects in diabetic SL and SR
Ca(2+)-transport activities were prevented by treatment of diabetic animals with
insulin. The results from the alloxan-rat model of diabetes support the view
that membrane abnormalities with respect to Ca2+ handling may lead to the
occurrence of intracellular Ca2+ overload and the development of diabetic
cardiomyopathy. BACKGROUND: It has been suggested that intracellular Ca2+ overload in cardiac
myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an
insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has
been shown to prevent diabetes-induced myocardial changes. To elucidate the
underlying mechanism of troglitazone action on cardiac myocytes, the effects of
troglitazone on voltage-dependent Ca2+ currents were examined and compared with
classic Ca2+ antagonists (verapamil and nifedipine).
METHODS AND RESULTS: Whole-cell voltage-clamp techniques were applied in single
guinea pig atrial myocytes. Under control conditions with CsCl internal
solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T)
and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude
of ICa,L in a concentration-dependent manner. Troglitazone also suppressed
ICa,T, but the effect of troglitazone on ICa,T was less potent than that on
ICa,L. The current-voltage relationships for ICa,L and the reversal potential
for ICa,L were not altered by troglitazone. The half-maximal inhibitory
concentration of troglitazone on ICa,L measured at a holding potential of -40 mV
was 6.3 micromol/L, and 30 micromol/L troglitazone almost completely inhibited
ICa,L. Troglitazone 10 micromol/L did not affect the time courses for
inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion,
without shifting the voltage-dependency of inactivation. This effect was
different from those of verapamil and nifedipine. Troglitazone also reduced
isoproterenol- or cAMP-enhanced ICa,L.
CONCLUSIONS: These results demonstrate that troglitazone inhibits
voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the
effects of isoproterenol in cardiac myocytes, thus possibly playing a role in
preventing diabetes-induced intracellular Ca2+ overload and subsequent
myocardial changes. |
What is the mechanism of viroid replication? | The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation. Replication of these minimal genomes, composed exclusively by a circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. | Contrary to earlier beliefs, viruses are not the smallest causative agents of
infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in
certain higher plants and cause more than a dozen crop diseases. These RNAs have
been termed viroids. Despite their extremely limited information content,
viroids replicate autonomously in susceptible cells--that is, they do not
require helper functions from simultaneously replicating conventional viruses.
Viroids are covalently closed circular molecules with a characteristic rodlike
secondary structure in which short helical regions are interrupted by internal
and bulge loops. Viroids are not translated; they are replicated by a host
enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA
intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble
viroids in size and molecular structure, but are found within the capsids of
specific helper viruses on which they depend for their own replication. These
RNAs are of great interest to molecular biology for at least two reasons: 1)
they are the smallest and simplest replicating molecules known, and 2) they may
represent living fossils of precellular evolution in a hypothetical RNA world. On the basis of sequence homology a model is proposed for five structural and
functional domains in viroids. These domains include (i) a conserved central
region capable of forming two alternative structures that may regulate two
phases of the viroid replication cycle, (ii) a region associated with
pathogenicity, (iii) a domain with high sequence variability, (iv and v) two
terminal domains that are interchangeable between viroids. That the evolution of
viroids has involved RNA rearrangements of domains is supported by the partial
duplication of coconut cadang cadang viroid, which arises de novo during each
infection. Similar RNA rearrangements have been established for animal viral
defective interfering RNAs, which arise by some form of discontinuous
transcription. This mechanism could account for the origin of viroids and also
RNA viruses, whereby modules of genetic information may have undergone repeated
exchange between RNA pathogens and the RNA of their hosts. 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. 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. Nucleotides within the pathogenicity domain of potato spindle tuber viroid
(PSTVd) are known to play an important role in regulating symptom expression,
but the underlying molecular mechanism is unknown. In order to determine more
precisely how structural features within the pathogenicity domain regulate
symptom expression, we have characterized a series of mutations that
progressively stabilize premelting region 1 and the rest of the "virulence
modulating" region. The structural effects of these mutations were monitored by
temperature gradient gel electrophoresis of circularized RNA transcripts, and
their biological effects were assessed by quantitative bioassays in tomato.
Closure of a 4-nucleotide loop within the premelting region 1 virtually
abolished PSTVd infectivity, especially when a nearby 2-nucleotide loop was also
closed. Although RNA transcripts containing less stabilizing mutations were
readily infectious, none of the four single and one double substitutions
examined were stably maintained in vivo. The pattern of spontaneous, apparently
compensatory sequence changes observed in the progeny suggests that PSTVd
variants with less stable secondary structures enjoy a selective advantage.
Mutations which stabilize the pathogenicity domain of PSTVd in vitro also
suppressed symptom expression, but at least one other mutation having no obvious
structural effects was associated with a similar phenotype. Conformational
stability appears to be only one of several factors regulating PSTVd replication
and pathogenicity. Although some viroid-like satellite RNAs possess self-cleavage and self-ligation
activities, we show that the peach latent mosaic viroid (PLMVd) is unique among
all known viroids since it also has such activities. These catalytic activities
should have important roles in the rolling circle replication of PLMVd.
According to this proposed mechanism, self-cleavage of the multimeric strands
occurs via hammerhead structures producing monomers possessing 2',3'-cyclic
phosphate and 5'-hydroxyl termini. In the most stable predicted secondary
structure for PLMVd these two extremities are juxtaposed, in order for
self-ligation to occur. To establish the nature of the phosphodiester bond
produced by self-ligation, we followed the classical procedure of complete
enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation.
Using this procedure, we report that the self-ligation of PLMVd transcripts
produces almost exclusively the 2',5' isomer (>96%). Primer extension assays
also revealed that reverse transcriptase can read througth this 2', 5' linkage,
suggesting that it does not prevent further replication of the viroid. Moreover,
we have observed that this 2',5' linkage is resistant to the debranching
activity contained in nuclear extracts, as well as being capable of preventing
further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo,
the resulting 2', 5'-phosphodiester bond could contribute to the stability of
these RNA species. Finally, an analysis of both the sequence and the structural
requirements for hammerhead self-cleavage and self-ligation suggests that these
two RNA processes may be interrelated. We hypothesize that the intramolecular
self-ligation which produces circular conformers may contribute to the
circularization step of the rolling circle replication, while the intermolecular
non-enzymatic ligation is a potential mechanism for the sequence reassortment of
viroids and viroid-like species. The replication of many viral and subviral pathogens as well as the
amplification of certain cellular genes proceeds via a rolling circle mechanism.
For potato spindle tuber (PSTVd) and related viroids, the possible role of a
circular (-)strand RNA as a template for synthesis of (+)strand progeny is
unclear. Infected plants appear to contain only multimeric linear (-)strand
RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally
have failed. To examine critically the infectivity of monomeric (-)strand viroid
RNAs, we have developed a ribozyme-based expression system for the production of
precisely full length (-)strand RNAs whose termini are capable of undergoing
facile circularization in vitro. Mechanical inoculation of tomato seedlings with
electrophoretically purified (-)PSTVd RNA led to a small fraction of plants
becoming infected whereas parallel assays with an analogous tomato planta macho
viroid (-)RNA resulted in a much larger fraction of infected plants.
Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the
appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd
progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally
infected plants by using either ribonuclease protection or electrophoresis under
partially denaturing conditions. Although not a component of the normal
replicative pathway, precisely full length (-)PSTVd RNA appears to contain all
of the structural and regulatory elements necessary for initiation of viroid
replication. Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires
extensive rearrangement of the highly base-paired native structure. For Potato
spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by
the change from a multibranched tetraloop structure to a loop E conformation.
Here we present evidence that processing of Citrus viroid III (CVd-III), a
member of a related group of viroids that also replicate in the nucleus, may
proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs
with DMS and CMCT revealed that the loop E motifs of these two viroids have
quite different tertiary structures. As shown by temperature gradient gel
electrophoresis, the presence of two likely Watson-Crick GC pairs results in a
significant overall stabilization of the CVd-III loop E-like motif. Unlike
PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA
tetraloop, and comparison of suboptimal structures indicates that the initial
cleavage event could occur on the 5' side of the only GU wobble pair in a helix
involving a nearby pair of inverted repeats. According to our model,
rearrangement of 3' sequences into a hairpin stem containing an identical
arrangement of GC, GU, and CG base pairs and a second cleavage event is followed
by formation of loop E, which serves to align the 5' and 3' termini of the
CVd-III monomer prior to ligation. Because ligation would occur within loop E
itself, stabilization of this motif may be needed to hold the 5' and 3' termini
of CVd-III in position for the host ligase. Viroids, as a consequence of not encoding any protein, are extremely dependent
on their hosts. Replication of these minimal genomes, composed exclusively by a
circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in
the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism
with three steps: (1) synthesis of longer-than-unit strands catalyzed by host
DNA-dependent RNA polymerases recruited and redirected to transcribe RNA
templates, (2) cleavage to unit-length, which in family Avsunviroidae is
mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase
or autocatalytically. This consistent but still fragmentary picture has emerged
from a combination of studies with in vitro systems (analysis of RNA
preparations from infected plants, transcription assays with nuclear and
chloroplastic fractions, characterization of enzymes and ribozymes mediating
cleavage and ligation of viroid strands, dissection of 5' terminal groups of
viroid strands, and in situ hybridization and microscopy of subcellular
fractions and tissues), and in vivo systems (tissue infiltration studies,
protoplasts, studies in planta and use of transgenic plants expressing viroid
RNAs). |
Which mitochondrial genes are regulated by thyroid hormone? | subunit 6 of ATP synthase, ATPase-6, mitochondrial II and III subunits of cytochrome-c oxidase, NADH dehydrogenase subunit 3 | Biogenesis of mitochondria involves the expression of genes located on nuclear
chromosomes as well as on mitochondrial DNA. We studied the coordination of the
two genomes by measuring transcript levels for nuclear (IV, Va, and VIc) and
mitochondrial (II and III) subunits of cytochrome-c oxidase after altering the
mitochondrial content of rat muscle and liver by altering the thyroid state of
the animals. Tissue levels of these mRNAs were generally decreased in
hypothyroid animals and were up-regulated again after thyroid hormone (T3)
treatment. However, significant increases in the levels of all nuclear
transcripts were observed in the liver 24 h after T3 treatment, but were delayed
or remained unaltered (VIc) in muscle. In contrast, levels of mitochondrial
transcripts were elevated early in muscle and late in liver. The abundance of
the corresponding polypeptides, which were analyzed by immunoblotting, changed
in direction and magnitude according to the changes in their mRNAs, indicating
pretranslational control. We conclude that the two genomes are regulated by T3
not through a common coordinating mechanism, but via two separate pathways,
which respond to T3 with tissue-specific kinetics. S1-nuclease protection
analysis showed that probably only one transcript for subunit VIc is present in
both tissues, thus excluding the possibility that the tissue-specific response
is due to the expression of two isogenes. The abundance of mitochondrial DNA was
unaltered despite the observed changes in mitochondrial transcripts, indicating
that mitochondrial gene expression is regulated by transcriptional mechanisms
and not by gene dosage as has been postulated by others. We previously described a modification of the whole genome PCR method which
allowed us to characterize several genes whose expression is regulated by
thyroid hormone in the mouse liver. Following this procedure, we now report the
identification of the mitochondrial NADH dehydrogenase subunit 3 (ND3) gene as
target of thyroid hormone. ND3 gene expression is regulated by thyroid hormone
in rat brain and heart. Sequencing and electrophoretic mobility shift assays
confirmed the presence of a thyroid hormone receptor (TR)/c-erbA specific
binding site in the mitochondrial ND3 gene. Hypothyroidism decreases ND3 mRNA
levels in several brain areas such as cortex and hippocampus during the early
postnatal development. In line with the recent findings showing the presence of
TR/c-erbA alpha and beta proteins inside the mitochondria, our results suggest
the possibility of direct transcriptional regulation of mitochondrial genes by
thyroid hormone. Thyroid hormone plays an important role in bone development and metabolism. We
used a polymerase chain reaction (PCR)-based mRNA differential display (DD)
analysis to obtain a profile of thyroid hormone-responsive genes in
osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M
triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated,
reverse-transcribed, and amplified using a total of 72 combinations (2 hours)
and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point,
1 true-positive novel clone was identified and shown to be the mitochondrial
gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3
differentially expressed (DE) mRNAs were confirmed as true-positives including;
nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone.
T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours,
but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was
increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC
mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and
induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC
and on the mitochondrial gene ATPase 6, represent novel targets and potential
mediators of thyroid hormone action on bone. Cell type, and the extent of cell
differentiation, influences T(3) regulation of genes in osteoblast-derived
cells. |
Mutation of which gene is associated with McLeod syndrome? | Mutation of XK gene is associated with McLeod syndrome. The XK gene is an X-chromosomal gene. The McLeod phenotype is derived from various forms of XK gene defects that result in the absence of XK protein, and is defined hematologically by the absence of Kx antigen, weakening of Kell system antigens, and red cell acanthocytosis. | McLeod syndrome, characterized by acanthocytosis and the absence of a
red-blood-cell Kell antigen (Kx), is a multisystem disorder involving a
late-onset myopathy, splenomegaly, and neurological defects. The locus for this
syndrome has been mapped, by deletion analysis, to a region between the loci for
Duchenne muscular dystrophy (DMD) and chronic granulomatous disease (CGD). In
this study, we describe a new marker, 3BH/R 0.3 (DXS 709), isolated by cloning
the deletion breakpoint of a DMD patient. A long-range restriction map of Xp21,
encompassing the gene loci for McLeod and CGD, was constructed, and multiple CpG
islands were found clustered in a 700-kb region. Using the new marker, we have
limited the McLeod syndrome critical region to 150-380-kb. Within this interval,
two CpG-rich islands which may represent candidate sites for the McLeod gene
were identified. In a patient suffering from X-linked chronic granulomatous disease (X-CGD)--a
disorder of phagocytesuperoxide generation--and McLeod syndrome, characterized
by the absence of the red cell Kell antigen, we identified a deletion of the
entire X-CGD gene by means of DNA hybridization with a cDNA probe. Our findings
suggest that the X-CGD and McLeod loci are physically close in the p21 region of
the X chromosome proximal to the Duchenne muscular dystrophy locus. McLeod syndrome is an X-linked multisystem disorder characterized by
abnormalities in the neuromuscular and hematopoietic systems. We have assembled
a cosmid contig of 360 kb that encompasses the McLeod gene locus. A 50 kb
deletion was detected by screening DNA from patients with radiolabeled whole
cosmids, and two transcription units were identified within this deletion. The
mRNA expression pattern of one of them, designated as XK, correlates closely to
the McLeod phenotype. XK encodes a novel protein with structural characteristics
of prokaryotic and eukaryotic membrane transport proteins. Nucleotide sequence
analysis of XK from two unrelated McLeod patients has identified point mutations
at conserved splice donor and acceptor sites. These findings provide direct
evidence that XK is responsible for McLeod syndrome. We report a novel mutation in the XK gene (XK) in a Japanese patient with McLeod
syndrome. A 50-year-old man showed progressive muscular atrophy, choreic
movement, elevated level of serum creatinine kinase, and acanthocytosis. The
expression level of all the Kell antigens in erythrocyte was decreased and
molecular analysis revealed a single-base (T) deletion at the nucleotide
position 1095 in XK. This deletion caused a frameshift in translation, leading
to a premature stop codon at the amino acid position 408. We conclude this
single-base deletion causes defective Kx protein, which is responsible for the
McLeod phenotype in this patient. The McLeod syndrome is a rare X-linked recessive disorder characterized by blood
group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene
defects and may include large deletions in the Xp21 region. Analysis of three
unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and
OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th
exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient
Bi) and from the XK to CYBB loci (patient Lh). All three patients normally
expressed the Lutheran (Lu) red cell antigens, thus excluding the interval
between the RPGR and DMD genes as site of the XS locus, previously mapped to the
Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood
group gene on chromosome 19. Loss-of-function mutations in the gene ced-8 lead to the late appearance of cell
corpses during embryonic development in C. elegans. ced-8 functions downstream
of or in parallel to-the regulatory cell death gene ced-9 and may function as a
cell death effector downstream of the caspase encoded by the programmed cell
death killer gene ced-3. In ced-8 mutants, embryonic programmed cell death
probably initiates normally but proceeds slowly. ced-8 encodes a transmembrane
protein that appears to be localized to the plasma membrane. The CED-8 protein
is similar to human XK, a putative membrane transport protein implicated in
McLeod Syndrome, a form of hereditary neuroacanthocytosis. McLeod syndrome is a rare X-linked hematologic and neuromuscular disorder
manifested by chorea, myopathy, cardiomyopathy, areflexia, hyperCKemia, and
acanthocytosis. Only four mutations have been reported in the gene responsible
for McLeod syndrome. We report a novel gene mutation in a Japanese family.
Direct sequencing of the PCR-amplified genomic DNA revealed the mutation was a
single C-nucleotide insertion at codon 151 in exon 2 of the XK gene, which
resulted in a 3'-frameshift. Study of family members revealed that the patient's
mother was a manifesting carrier heterozygous for this mutation. BACKGROUND: Kx is lacking in the RBCs of patients with the McLeod syndrome. This
condition is sometimes associated with chronic granulomatous disease (CGD). If
given allogeneic RBCs, CGD patients with the McLeod phenotype may produce
anti-Kx and anti-Km, and only phenotypically matched McLeod blood would be
compatible. McLeod phenotype persons without CGD have made anti-Km but not
anti-Kx (2 examples), and thus both McLeod and K(O) blood would be compatible.
CASE REPORT: RBCs from a transfused patient with the McLeod phenotype but not
with CGD (non-CGD McLeod) were typed for the Kell blood group antigens, and the
plasma was analyzed for the presence of antibody by agglutination. The molecular
basis was determined by analyzing for XK protein on RBC membranes by Western
immunoblotting, by sequencing the XK gene, and by RFLP.
RESULTS: The RBCs did not react with anti-Kx + anti-Km and showed weakening of
Kell system antigens. The patient's plasma reacted moderately (2+) with RBCs of
common Kell type and strongly (4+) with K(O) RBCs and RBCs of common Kell type
treated with dithiothreitol, and did not react with McLeod RBCs. XK protein was
absent from the RBC membranes. The XK gene had a point mutation in the donor
splice site of intron 1 (G>C).
CONCLUSION: This is the first report describing the molecular alteration in a
non-CGD McLeod patient who has made anti-Kx. The immune response of people with
the McLeod phenotype can vary, and K(O) blood may not always be compatible. The McLeod syndrome is an X-linked neuroacanthocytosis manifesting with myopathy
and progressive chorea. It is caused by mutations of the XK gene encoding the XK
protein, a putative membrane transport protein of yet unknown function. In
erythroid tissues, XK forms a functional complex with the Kell glycoprotein.
Here, we present an immunohistochemical study in skeletal muscle of normal
controls and a McLeod patient with a XK gene point mutation (C977T) using
affinity-purified antibodies against XK and Kell proteins. Histological
examination of the affected muscle revealed the typical pattern of McLeod
myopathy including type 2 fiber atrophy. In control muscles, Kell
immunohistochemistry stained sarcoplasmic membranes. XK immunohistochemistry
resulted in a type 2 fiber-specific intracellular staining that was most
probably confined to the sarcoplasmic reticulum. In contrast, there was only a
weak background signal without a specific staining pattern for XK and Kell in
the McLeod muscle. Our results demonstrate that the lack of physiological XK
expression correlates to the type 2 fiber atrophy in McLeod myopathy, and
suggest that the XK protein represents a crucial factor for the maintece of
normal muscle structure and function. A 29-year-old man with a history of elevated creatine kinase and necrotizing
myopathy was reviewed. Prominent red cell acanthocytosis in association with
reduced Kell antigen expression was present, findings consistent with the McLeod
syndrome. Investigation of the patient's XK gene revealed a novel TGG- to-TAG
transition at position 1023 in exon 3. This point mutation creates an in-frame
stop codon (W314X), and predicts a truncated XK protein of 313 amino acids,
compared with 444 amino acids in the normal XK protein. The mutation was not
identified in the patient's mother or sister indicating that this mutation was
spontaneous. Among the movement disorders associated with acanthocytosis, McLeod syndrome
(McKusick 314850) is the one that is best characterized on the molecular level.
Its defining feature is low reactivity of Kell erythrocyte antigens. This is due
to absence of membrane protein KX that forms a complex with the Kell protein. KX
is coded for by the XK gene on the X-chromosome. We present six males (aged 29
to 60 years), with proven XK mutations, to discuss the chorea associated with
McLeod syndrome. The movement disorder commonly develops in the fifth decade and
is progressive. It affects the limbs, the trunk and the face. In addition to
facial grimacing, involuntary vocalization can be present. In early stages there
may only be some restlessness or slight involuntary distal movements of ankles
and fingers. Lip-biting and facial tics seem more common in autosomal recessive
choreoacanthocytosis linked to chromosome 9. This, together with the absence of
dysphagia in McLeod syndrome, may help in differential diagnosis. Recent
findings suggest a role for the endothelin system of the striatum in the
pathogenesis of McLeod syndrome. McLeod syndrome and chorea-acanthocytosis are classified with the so-called
neuroacanthocytosis group of syndromes. Both lead to progressive basal ganglia
degeneration and were not easily distinguished in the past. With the discovery
of their molecular bases, mutations of the X-linked gene XK and autosomal
recessive mutations of the gene coding for chorein, respectively, the two
phenotypes can now be differentiated and extend the diagnostic spectrum in
patients presenting with chorea. The present review compares the two conditions
and proposes a practical approach to diagnosis and treatment. Better-defined
disease concepts should eventually replace the umbrella term of
"neuroacanthocytosis." Animal models are needed to understand the underlying
mechanisms. A final common pathway is likely for the pathogenesis of these
conditions and is most probably shared with Huntington's disease. McLeod Syndrome (MLS) is a rare X-linked disorder characterized by haemopoietic
abnormalities and late-onset neurological and muscular defects. The McLeod blood
group phenotype is typically associated with erythrocyte acanthocytosis, absence
of the Kx antigen and reduced expression of Kell system antigens. MLS is caused
by hemizygosity for mutations in the XK gene. We describe a patient with MLS who
first showed symptoms in 1989 (aged 51 years). As the disease progressed, the
patient developed a slight dementia, aggressive behaviour and choreatic
movements. A cardiomyopathy was also diagnosed. An electroneuromyography showed
neuropathic and myopathic changes. Liver enzymes were elevated and a blood smear
showed acanthocytes. MLS was confirmed by serological analysis of the Kell
antigens. Analysis of red blood cells by flow cytometry revealed the patient and
his grandson to have reduced Kell antigen expression. The patient's daughters
had two populations of red cells, consistent with them being heterozygous for an
XK0 allele. The molecular basis of MLS in this family is a novel mutation
consisting of a 7453-bp deletion that includes exon 2 of the XK gene. This
confirms that the patient's 7-year-old grandson, who is currently asymptomatic,
also has the XK0 allele and is therefore likely to develop MLS. A 50-year-old man presented with worsening, virtually lifelong, chorea and
progressive behavioural disturbance, involving disinhibition and hoarding, over
10 years. Clinical assessment revealed chorea, dysarthria, areflexia, an
inappropriately jovial, impulsive manner and neuropsychological evidence of
frontosubcortical dysfunction. Investigation results included an elevated
creatine kinase, caudate atrophy and hypoperfusion, acanthocytes in the
peripheral blood and the McLeod phenotype. DNA studies demonstrated a
single-base deletion at position 172 in exon 1 of the XK gene, giving rise to a
premature stop codon at position 129 in exon 2. BACKGROUND: McLeod neuroacanthocytosis syndrome is a late-onset X-linked
multisystem disorder affecting the peripheral and central nervous systems, red
blood cells (RBCs), and internal organs. A variety of mutations have been found
in the responsible gene (XK) including single nonsense and missense mutations,
nucleotide mutations at or near the splice junctions of introns of XK, and
different deletion mutations. To date no clear phenotype-genotype correlation is
apparent. The clinical details of one case of McLeod phenotype without apparent
neuromuscular abnormalities have been reported. Here the clinical details of two
additional cases are presented, of which the genetic details have previously
been published.
STUDY DESIGN AND METHODS: Two asymptomatic or minimally symptomatic cases at
ages expected to manifest the McLeod syndrome (MLS) were evaluated. The first
case had been authenticated as a genuine McLeod both by serology and by
genotyping (R222G missense mutation) and the second case had a mutation in XK
(IVS2+5G>A) and by serology exhibited very weak Kx antigen and no detectable
Kell antigens, except extremely low k antigen by adsorption-elution technique.
The patients were examined for hematologic, neurologic, and other clinical
abnormalities.
RESULTS: Despite documented McLeod phenotype on RBCs, and identified mutations
of XK, neurologic and other clinical findings were minimal at ages expected to
manifest MLS.
CONCLUSIONS: The different XK mutations may have different effects upon the XK
gene product and thus may account for the variable phenotype. The X-linked McLeod neuroacanthocytosis syndrome strongly resembles Huntington's
disease and has been reported in various countries world-wide. Herein, we report
two Chilean brothers with predomit psychiatric features at disease onset
including schizophrenia-like psychosis and obsessive compulsive disorder.
Molecular genetic analysis revealed a small deletion in the XK gene
(938-942delCTCTA), which has been already described in a North American patient
of Anglo-Saxon descent and a Japanese family, presenting with seizures, muscle
atrophy or chorea yet absence of psychiatric features. These findings argue
against a founder effect and indicate a profound phenotypic variability
associated with the 938-942delCTCTA deletion. Our report supports the inclusion
of McLeod syndrome in the differential diagnosis of Huntington's disease as well
as acute psychosis in male subjects. A 45-year-old man developed chorea, behavioural changes, moderate amyotrophy and
polyneuropathy. Hypertrophic cardiomyopathy and increased serum lactate
dehydrogenase and creatine kinase (CK) were found. Acanthocytes were not
detected. The absence of XK protein and faintly expressed Kell antigens on
erythrocytes were found. Genetic test revealed a R133X mutation of the XK gene,
confirming the McLeod syndrome. After 7 years he suddenly developed delirium
followed by severe hypoglycaemia, hyperthermia, rhabdomyolysis, hepatic and
renal failure. Maligt arrhythmia caused death. BACKGROUND AND OBJECTIVES: Kell antigens are encoded by the KEL gene on the long
arm of chromosome 7. Kx antigen is encoded by the XK gene on the short arm of
the X chromosome. Kell and Kx proteins in the red cell membrane are covalently
linked by a disulphide bond. The McLeod phenotype is characterized by weakened
expression of antigens in the Kell blood group system, absence of Km and Kx
antigens, and acanthocytosis. It has an X-linked mode of inheritance with
transmission through carrier females. Some males with the McLeod syndrome also
have chronic granulomatous disease (CGD). It is generally believed that patients
with non-CGD McLeod may develop anti-Km but not anti-Kx, but that those with CGD
McLeod can develop both anti-Km and anti-Kx.
MATERIALS AND METHODS: We present serological data, DNA genotyping and gene
sequencing, monocyte monolayer assay and neutrophil oxidative burst test from a
patient with the McLeod phenotype without clinical evidence of CGD.
RESULTS: We report here the second example of a patient with non-CGD McLeod who
developed anti-Kx in addition to anti-Km. Sequencing of our patient's XK gene
confirmed the presence of a mutation resulting in a premature stop codon and
lack of Kx protein in the red cell membrane, which is consistent with the
diagnosis of McLeod syndrome. Neutrophil oxidative burst test was normal,
indicating that our patient did not have CGD. The challenge of providing 10
compatible blood units for multiple surgeries was met.
CONCLUSION: The second case of a rare entity, a patient with non-CGD McLeod who
developed anti-Kx and anti-Km, was managed successfully with a combination of
autologous donations and procurement of compatible units from national and
international sources. BACKGROUND: McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with
hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused
by mutations in the XK gene whose product is expressed at the red blood cell
(RBC) surface but whose function is currently unknown. A variety of XK mutations
has been reported but no clear phenotype-genotype correlation has been found,
especially for the point mutations affecting splicing sites.
STUDY DESIGN AND METHODS: A man suspected of neuroacanthocytosis was evaluated
by neurologic examination, electromyography, muscle biopsy, muscle computed
tomography, and cerebral magnetic resoce imaging. The McLeod RBC phenotype
was disclosed by blood smear and immunohematology analyses and then confirmed at
the biochemical level by Western blot analysis. The responsible XK mutation was
characterized at the mRNA level by reverse transcription-polymerase chain
reaction (PCR), identified by genomic DNA sequencing, and verified by
allele-specific PCR.
RESULTS: A novel XK splice site mutation (IVS1-1G>A) has been identified in a
McLeod patient who has developed hematologic, neuromuscular, and neurologic
symptoms. This is the first reported example of a XK point mutation affecting
the 3' acceptor splice site of Intron 1, and it was demonstrated that this
mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at
the RBC membrane.
CONCLUSION: The detailed characterization at the molecular biology level of this
novel XK splice site mutation associated with the clinical description of the
patient contributes to a better understanding of the phenotype-genotype
correlation in the McLeod syndrome. Neuroacanthocytosis syndromes are mainly comprised of two diseases:
chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS). There is a high
incidence of psychiatric disorders such as mood disorder and schizophrenia among
neuroacanthocytosis patients. We hypothesized that
neuroacanthocytosis-related-genes might be associated with susceptibility to
these psychiatric disorders. We performed a comprehensive mutation screen of
VPS13A and XK, the gene responsible for ChAc and MLS, respectively, in 85 mood
disorder subjects and XK in 86 schizophrenia subjects and compared the variants
to 100 or more control alleles. We also performed copy number variation (CNV)
analysis in 72 mood disorder subjects and 86 schizophrenia subjects. We
identified three non-synonymous, two synonymous and six intron variants in mood
disorder subjects and a novel GAT triplet repeat polymorphism in VPS13A. By CNV
analysis, we identified a heterozygous exon 60-61 deletion in VPS13A in one mood
disorder subject. We identified one non-synonymous and one intron variant in
mood disorder and schizophrenia subjects, respectively, in XK. The presence of a
pathogenic mutation or a potentially functional variant in mood disorder or
schizophrenia subjects suggests that neuroacanthocytosis-related-genes might be
involved in the pathogenesis of these psychiatric disorders. We report the first case of McLeod syndrome (MLS) in a 47-year-old Chinese man
who presented with progressive limb weakness, chorea of feet, red blood cell
acanthocytosis, absence of Kx red blood cell antigen and weak expression of Kell
antigens. The diagnosis of MLS was confirmed by genetic testing showing a
hemizygous mutation of XK gene. We review literature on neuroacanthocytosis in
the Chinese population. This study investigates the effects of XKR4, a recently identified candidate
gene for Attention-Deficit/Hyperactivity Disorder (ADHD), birth weight, and
their interaction on brain volume in ADHD. XKR4 is expressed in cerebellum and
low birth weight has been associated both with changes in cerebellum and with
ADHD, probably due to its relation with prenatal adversity. Anatomical MRI scans
were acquired in 58 children with ADHD and 64 typically developing controls and
processed to obtain volumes of cerebrum, cerebellum and gray and white matter in
each structure. DNA was collected from saliva. Analyses including data on birth
weight were conducted in a subset of 37 children with ADHD and 51 controls where
these data were retrospectively collected using questionnaires. There was an
interaction between genotype and birth weight for cerebellum gray matter volume
(p = .020). The combination of homozygosity for the G-allele (the allele
previously found to be overtransmitted in ADHD) and higher birth weight was
associated with smaller volume. Furthermore, birth weight was positively
associated with cerebellar white matter volume in controls, but not ADHD
(interaction: p = .021). The interaction of genotype with birth weight affecting
cerebellum gray matter is consistent with models that emphasize increased
influence of genetic risk-factors in an otherwise favorable prenatal
environment. The absence of an association between birth weight and cerebellum
white matter volume in ADHD suggests that other genetic or environmental effects
may be at play, unrelated to XKR4. These results underscore the importance of
considering environmental effects in imaging genetics studies. |
Is RIP1 (RIP-1) part of the necrosome? | Yes, RIP1 is part of the necrosome. | Necrosis, a form of death characterized by rupture of the cell membrane, is
closely interlinked with inflammation. Cellular components released during
necrotic death can trigger inflammation. Conversely, inflammation often yields
tissue damage and, as a consequence, cell death. Which occurs first--necrosis or
inflammation--in specific in vivo situations is currently difficult to tell. A
way out of this 'chicken-and-egg' conundrum may be found via the recent finding
that both necrotic cell death and inflammation can be initiated by a distinct
set of signaling proteins, the 'necrosome', that includes receptor-interacting
protein (RIP)1, RIP3 and caspase-8. Further clarifying the function of these
signaling proteins should make it possible to establish when they induce
inflammation directly and when inflammation is caused by necrotic cell death. Glioblastoma constitute the most frequent and deadliest brain tumors of
astrocytic origin. They are resistant to all current therapies and are
associated with a high rate of recurrence. Glioblastoma were previously shown to
respond to treatments by 5-aminolevulinic acid (5-ALA)-based photodynamic
therapy (PDT) mainly by activating a necrotic type of cell death. The
receptor-interacting protein 3 (RIP3) has recently been outlined as a key
mediator of this caspase-independent form of programmed cell death. In the
present study, we analyzed the necrotic mechanism induced by 5-ALA-PDT in human
glioblastoma cells and explored the role of RIP3 in this context. Our results
show that PDT-induced necrosis is dependent on RIP3, which forms aggregates and
colocalizes with RIP1 following photosensitization. We demonstrate that
PDT-mediated singlet oxygen production is the cause of RIP3-dependent necrotic
pathway activation. We also prove that PDT induces the formation of a
pro-necrotic complex containing RIP3 and RIP1 but lacking caspase-8 and FADD,
two proteins usually part of the necrosome when TNF-α is used as a stimulus.
Thus, we hypothesize that PDT might lead to the formation of a different
necrosome whose components, besides RIP1 and RIP3, are still unknown. In most
cases, glioblastoma are characterized by a constitutive activation of NF-κB.
This factor is a key regulator of various processes, such as inflammation,
immune response, cell growth or apoptosis. Its inhibition was shown to further
sensitize glioblastoma cells to PDT-induced necrosis, however, no difference in
RIP3 upshift or aggregation could be observed when NF-κB was inhibited. Tumor necrosis factor (TNF) is an important inflammatory cytokine and induces
many cellular responses, including inflammation, cell proliferation, apoptosis,
and necrosis. It is known that receptor interacting protein (RIP) kinases, RIP1
and RIP3, are key effectors of TNF-induced necrosis, but little is known about
how these two RIP kinases mediate this process, although reactive oxygen species
(ROS) generation and JNK activation have been suggested to be two downstream
events of RIP kinases. Here we report the identification of mixed lineage kinase
domain-like, MLKL, as a key RIP3 downstream component of TNF-induced necrosis.
Through screening a kinase/phosphatase shRNA library in human colon
adenocarcinoma HT-29 cells, we found that knockdown of MLKL blocked TNF-induced
necrosis. Our data suggest that MLKL functions downstream of RIP1 and RIP3 and
is recruited to the necrosome through its interaction with RIP3. Finally, we
found that MLKL is required for the generation of ROS and the late-phase
activation of JNK during TNF-induced necrosis. However, because these two events
are not involved in TNF-induced necrosis in HT-29 cells, the target of MLKL
during TNF-induced necrosis remains elusive. Taken together, our study suggests
that MLKL is a key RIP3 downstream component of TNF-induced necrotic cell death. Cellular inhibitor of apoptosis proteins (cIAPs) have emerged as important
anti-cell death mediators, particularly in cancer. Although they are known to be
expressed in immune tissue, their specific immune function remains unclear. We
observed that degradation of cIAPs with SMAC mimetic (SM) results in death of
primary bone-marrow-derived macrophages. SM-induced death of macrophages
occurred by programmed necrosis (necroptosis), which was dependent on TNF
receptor expression. Consistent with necroptosis, SM-induced death of
macrophages was abrogated by inhibition of receptor interacting protein 1 (Rip1)
kinase signaling or by receptor interacting protein 3 (Rip3) knockdown.
SM-induced necroptosis was also dependent on inhibition of SM-induced apoptosis
due to the expression of the endogenous caspase inhibitor, xIAP. We found that
cIAPs limit Rip3, and to a lesser extent Rip1, expression via
post-transcriptional mechanisms, leading to inhibition of the Rip1-Rip3 death
complex (necrosome). Reduced cIAP activity in vivo, via SM treatment or specific
knockout of either cIAP, resulted in elevated macrophage cell death and
compromised control of an intracellular bacterium, Listeria monocytogenes. These
results show that cIAPs have an important role in limiting programmed necrosis
of macrophages, which facilitates effective control of a pathogen. RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis.
Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and
RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils
exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT)
and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray
diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1
and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3
complex isolated from necrotic cells binds ThT, is ultrastable, and has a
fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR,
and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are
defective in the interaction compromise cluster formation, kinase activation,
and programmed necrosis in vivo. The current study provides insight into the
structural changes that occur when RIP kinases are triggered to execute
different signaling outcomes and expands the realm of amyloids to complex
formation and signaling. Receptor interacting protein 3 (RIP3) is a protein kinase essential for
TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is
required for its interaction with human mixed lineage kinase domain-like (MLKL)
in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3
mediate necrosome aggregation leading to the formation of amyloid-like signaling
complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in
mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact
with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is
not conserved in hRIP3. Although the RIP3-MLKL interaction is required for
necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL
and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL
interaction is primarily determined by the sequence differences in the
phosphorylation sites and the flanking sequence around the phosphorylation sites
in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected
as an evolutionarily conserved mechanism in mediating necroptosis signaling
despite that differing structural and mechanistic bases for this interaction
emerged simultaneously in different organisms. In addition, we further revealed
that the interaction of RIP3 with MLKL prevented massive abnormal RIP3
aggregation, and therefore should be crucial for formation of the amyloid
signaling complex of necrosomes. We also found that the interaction between RIP3
and MLKL is required for the translocation of necrosomes to
mitochondria-associated membranes. Our data demonstrate the importance of the
RIP3-MLKL interaction in the formation of functional necrosomes and suggest that
translocation of necrosomes to mitochondria-associated membranes is essential
for necroptosis signaling. Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral
properties, but less is known about how they induce cell death. Here, we show
that both type I (α/β) and type II (γ) IFNs induce precipitous
receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the
adaptor protein Fas-associated death domain (FADD) is lost or disabled by
phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced
necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex
that requires Jak1/STAT1-dependent transcription, but does not need the kinase
activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive
protein kinase PKR, which then interacts with RIP1 to initiate necrosome
formation and trigger necrosis. Although IFNs are powerful activators of
necrosis when FADD is absent, these cytokines are likely not the domit
inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We
also identify phosphorylation on serine 191 as a mechanism that disables FADD
and collaborates with caspase inactivation to allow IFN-activated necrosis.
Collectively, these findings outline a mechanism of IFN-induced RIP
kinase-dependent necrotic cell death and identify FADD and caspases as negative
regulators of this process. Programmed necrosis or necroptosis is controlled by the action of two
serine/threonine kinases, RIP1 (receptor-interacting serine/threonine protein
kinase 1; also known as RIPK1) and RIP3. The phosphorylation of RIP1 and RIP3 is
critical for assembly of the necrosome, an amyloid-like complex that initiates
transmission of the pro-necrotic signal. In the present study, we used
site-directed mutagenesis to systematically examine the effects of putative
phosphoacceptor sites on RIP1 and RIP3 on TNF (tumour necrosis factor)-induced
programmed necrosis. We found that mutation of individual serine residues in the
kinase domain of RIP1 had little effect on RIP1 kinase activity and TNF-induced
programmed necrosis. Surprisingly, an alanine residue substitution for Ser(89)
enhanced RIP1 kinase activity and TNF-induced programmed necrosis without
affecting RIP1-RIP3 necrosome formation. This indicates that Ser(89) is an
inhibitory phosphoacceptor site that can dampen the pro-necrotic function of
RIP1. In addition, we show that a phosphomimetic mutant of RIP3, S204D, led to
programmed necrosis that was refractory to RIP1 siRNA and insensitive to
necrostatin-1 inhibition. Our results show that programmed necrosis is regulated
by positive and inhibitory phosphorylation events. |
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