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Is Fibroblast Growth Factor 23 a phosphaturic hormone? | Yes, fbroblast growth factor 23 (FGF23) is a phosphaturic hormone. | αKlotho is thought to activate the epithelial calcium channel Transient Receptor
Potential Vanilloid-5 (TRPV5) in distal renal tubules through its putative
glucuronidase/sialidase activity, thereby preventing renal calcium loss.
However, αKlotho also functions as the obligatory co-receptor for fibroblast
growth factor-23 (FGF23), a bone-derived phosphaturic hormone. Here, we show
that renal calcium reabsorption and renal membrane abundance of TRPV5 are
reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout
mice. We further demonstrate that αKlotho neither co-localizes with TRPV5 nor is
regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal
tubules and thus renal calcium reabsorption are regulated by FGF23, which binds
the FGF receptor-αKlotho complex and activates a signaling cascade involving
ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a
calcium-conserving hormone in the kidney. Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast
growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)2
vitamin D3 (1,25VitD3). Disruption of FGF23-α-KL signaling is thought to be an
early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL
expression and a reciprocal rise in serum FGF23. It remains unclear, however,
whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated
α-KL expression in renal biopsy samples and measured levels of several
parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and
urinary samples from CKD patients (n = 236). We found that although renal α-KL
levels were significantly reduced and serum FGF23 levels were significantly
elevated in early and intermediate CKD, serum phosphate levels remained within
the normal range. Multiple regression analysis showed that the increases in
FGF23 were significantly associated with reduced renal function and elevated
serum phosphate, but were not associated with loss of renal α-KL. Moreover,
despite falling renal α-KL levels, the increase in FGF23 enhanced urinary
fractional excretion of phosphate and reduced serum 1,25VitD3 levels in early
and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell
significantly over the course of CKD, and renal α-KL was a significant
independent determit of sKL. These results demonstrate that FGF23 levels rise
to compensate for renal failure-related phosphate retention in early and
intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate
balance to be maintained despite the reduction in α-KL. In advanced CKD,
however, renal α-KL declines further. This disrupts FGF23 signaling, and serum
phosphate levels significantly increase, stimulating greater FGF23 secretion.
Our results also suggest the serum sKL concentration may be a useful marker of
renal α-KL expression levels. Rapid and somewhat surprising advances have recently been made toward
understanding the molecular mechanisms causing heritable disorders of
hypophosphatemia. The results of clinical, genetic, and translational studies
have interwoven novel concepts underlying the endocrine control of phosphate
metabolism, with far-reaching implications for treatment of both rare Mendelian
diseases as well as common disorders of blood phosphate excess such as chronic
kidney disease (CKD). In particular, diseases caused by changes in the
expression and proteolytic control of the phosphaturic hormone fibroblast growth
factor-23 (FGF23) have come to the forefront in terms of directing new models
explaining mineral metabolism. These hypophosphatemic disorders as well as
others resulting from independent defects in phosphate transport or metabolism
will be reviewed herein, and implications for emerging therapeutic strategies
based upon these new findings will be discussed. Deranged calcium-phosphate metabolism contributes to the burden of morbidity and
mortality in dialysis patients. This study aimed to assess the association of
the phosphaturic hormone fibroblast growth factor 23 (FGF23) and soluble Klotho
with all-cause mortality. We measured soluble Klotho and FGF23 levels at
enrolment and two weeks later in 239 prevalent hemodialysis patients. The
primary hypothesis was that low Klotho and high FGF23 are associated with
increased mortality. The association between Klotho and atrial fibrillation (AF)
at baseline was explored as secondary outcome. AF was defined as presence of
paroxysmal, persistent or permanent AF. During a median follow-up of 924 days,
59 (25%) patients died from any cause. Lower Klotho levels were not associated
with mortality in a multivariable adjusted analysis when examined either on a
continuous scale (HR 1.25 per SD increase, 95% CI 0.84-1.86) or in tertiles,
with tertile 1 as the reference category (HR for tertile two 0.65, 95% CI
0.26-1.64; HR for tertile three 2.18, 95% CI 0.91-2.23). Higher Klotho levels
were associated with the absence of AF in a muItivariable logistic regression
analysis (OR 0.66 per SD increase, 95% CI 0.41-1.00). Higher FGF23 levels were
associated with mortality risk in a multivariable adjusted analysis when
examined either on a continuous scale (HR 1.45 per SD increase, 95% CI
1.05-1.99) or in tertiles, with the tertile 1 as the reference category (HR for
tertile two 1.63, 95% CI 0.64-4.14; HR for tertile three 3.91, 95% CI
1.28-12.20). FGF23 but not Klotho levels are associated with mortality in
hemodialysis patients. Klotho may be protective against AF. BACKGROUND: Fibroblast growth factor (FGF) 23 is one of the most recently
discovered FGFs. This phosphaturic hormone produced in bones is a risk factor
for cardiovascular diseases and thus mortality. Klotho is an essential
coreceptor for FGF23 and at the same time it is known as a "longevity" hormone.
There are no data considering FGF23 and Klotho roles in heart transplant (HT)
recipients. The aim of this study was to assess Klotho and FGF23 serum
concentration in heart transplant recipients depending on immunosuppressive
therapy regimen and comorbidities.
METHODS: Eighty-four stable heart transplant recipients were enrolled in the
study; 22 healthy volunteers served as control subjects. FGF23 and Klotho
protein concentration, markers of renal function, such as cystatin C and
neutrophil gelatinase-associated lipocalin (NGAL), and heart failure markers,
such as copeptine and N-termiinal pro-B-type natriuretic peptide (NT-proBNP),
were evaluated.
RESULTS: FGF23 concentration was significantly higher in the HT group whereas
Klotho protein was significantly lower. FGF23 correlated with creatinine level
(r = 0.72; P < .001), estimated glomerular filtration rate (eGFR; r = -0.32; P <
.01), cystatin C (r = 0.36; P < .01), NGAL (r = 0.51; P < .001), hemoglobin (r =
-0.39; P < .001), NT-proBNP (r = 0.51; P < .001), high-density lipoprotein (HDL;
r = 0.27; P < .05), intraventricular septum thickness (r = 0.42; P < .01) and
right ventricular systolic pressure (r = 0.34; P < .05). Klotho protein
correlated only with age (r = -0.21; P < .05), creatinine (r = -0.21; P < .05),
and eGFR (r = -0.31; P < .01). FGF23 concentration was significantly higher in
patients with eGFR <60 mL/min whereas Klotho protein was significantly lower.
FGF23 predictors were renal function (creatinine concentration; β = 0.45; P =
.0001), HDL (β = 0.33; P = .003), intraventricular septum thickness (β = 0.38; P
= .0003), and right ventricular systolic pressure (β = 0.34; P = .003),
explaining 70% of FGF23 variability.
CONCLUSIONS: FGF23/Klotho system disorders in HT recipients are related to
cardiovascular system function and kidney failure and could cause increased risk
of cardiovascular disease. PURPOSE OF REVIEW: The purpose of this study is to review current perspectives
regarding the pathogenesis of cardiorenal syndrome (CRS) in chronic kidney
disease (CKD), and current treatment guidelines for this condition.
RECENT FINDINGS: The pathophysiological mechanisms underlying the development of
CRS in CKD include neurohumoral, haemodynamic and CKD-related mechanisms. Recent
evidence suggests that sympathetic nerve activity plays a role in CRS, but the
SYMPLICITY HTN-3 trial failed to show a reduction of blood pressure after
catheter-based renal denervation in patients with resistant hypertension. Kidney
injury in patients with heart failure was previously considered to result from
arterial underfilling due to low cardiac output, but the role of renal venous
hypertension in this process has also recently been investigated. It would be
useful to develop a reliable treatment option for CRS due to haemodynamic
mechanism other than volume control using diuretics. Fibroblast growth factor 23
(FGF23) is a phosphaturic hormone that has recently been identified as a
CKD-related factor affecting CRS. FGF23 treatment has both advantages and
disadvantages in terms of CRS progression.
SUMMARY: Multiple disorders underlie the development of CRS. Current treatment
options include renin-angiotensin system blockade and volume control, but remain
limited. A multidisciplinary approach is required to prevent CRS, including
renal sympathetic denervation, treatment of renal venous hypertension and FGF23
treatment. Phosphate homeostasis is coordinated and regulated by complex cross-organ talk
through delicate hormonal networks. Parathyroid hormone (PTH), secreted in
response to low serum calcium, has an important role in maintaining phosphate
homeostasis by influencing renal synthesis of 1,25-dihydroxyvitamin D, thereby
increasing intestinal phosphate absorption. Moreover, PTH can increase phosphate
efflux from bone and contribute to renal phosphate homeostasis through
phosphaturic effects. In addition, PTH can induce skeletal synthesis of another
potent phosphaturic hormone, fibroblast growth factor 23 (FGF23), which is able
to inhibit renal tubular phosphate reabsorption, thereby increasing urinary
phosphate excretion. FGF23 can also fine-tune vitamin D homeostasis by
suppressing renal expression of 1-alpha hydroxylase (1α(OH)ase). This review
briefly discusses how FGF23, by forming a bone-kidney axis, regulates phosphate
homeostasis, and how its dysregulation can lead to phosphate toxicity that
induces widespread tissue injury. We also provide evidence to explain how
phosphate toxicity related to dietary phosphorus overload may facilitate
incidence of noncommunicable diseases including kidney disease, cardiovascular
disease, cancers and skeletal disorders. |
Which type of cell death is known as anoikis? | Anoikis (Greek for Homelessness) is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonizing of distant organs. Anoikis is important in the normal physiologic development of the human body, as well as in disease states. Adhesion to structural glycoproteins of the extracellular matrix is necessary for survival of the differentiated adherent cells. Cancer cells harbor anoikis resistance allowing spread to occur. | Anoïkis is defined as programmed cell death induced by the loss of cell/matrix
interactions. Adhesion to structural glycoproteins of the extracellular matrix
is necessary for survival of the differentiated adherent cells in the
cardiovascular system, including endothelial cells, smooth muscle cells,
fibroblasts, and cardiac myocytes. Adhesion is also a key factor for the
differentiation of mesenchymal stem cells. In particular, fibronectin is
considered a factor of survival and differentiation for many adherent cells.
Adhesion generates cell tensional integrity (tensegrity) and repression of
apoptotic signals, whereas detachment has the opposite effect. Anoïkis plays a
physiological role by regulating cell homeostasis in tissues. However, anoïkis
can also be involved in pathological processes, as illustrated by the resistance
to anoïkis in cancer and its enhancement in degenerative tissue remodeling.
Extracellular mediators of anoïkis include matrix retraction, leading to loss of
tensegrity in fibroblasts, pharmacological disengagement of integrins by
RGD-like peptides and fragments of fibronectin, and focal adhesion disassembly
by fragments of thrombospondin, plasminogen activator-1, and
high-molecular-weight kininogen. In addition to binding of the RGD peptide by
integrins, the engagement of the heparin binding sites of adhesive glycoproteins
with glycosaminoglycans on the cell surface is also involved in the prevention
of cell detachment-induced apoptosis. Proteases able to degrade adhesive
glycoproteins, such as fibronectin, induce anoïkis of vascular adherent cells.
Active proteases can either be secreted directly by inflammatory cells, as
elastase and cathepsin G by polymorphonuclear leukocytes, chymase and tryptase
by mast cells, and granzymes by lymphocytes, or generated from circulating
zymogens by activation in close contact with the cells. This is the case for the
pericellular conversion of plasminogen to plasmin, which degrades fibronectin
and induces anoïkis of smooth muscle cells. Involvement of proteases has also
been proposed in the apoptotic response of cultured adherent cells to serum
starvation. Anoïkis is probably involved in pathological remodeling of
cardiovascular tissues, including cardiac myocyte detachment in heart failure,
deendothelialization and plaque rupture in atherosclerosis, and smooth muscle
cell disappearance in aneurysms and varicose veins. The absence of cell adhesion
and growth resulting from cleavage of adhesive proteins also represents a major
impediment to cellular healing, including the absence of cell recolonization of
proteolytically injured tissue and the low efficacy of cell transplantation.
However, the exact role of anoïkis in cardiovascular pathologies remains to be
further defined. Cell therapy, in particular liver cell transplantation, holds great therapeutic
potential and is partially hindered by the high rate of apoptosis during cell
isolation, cryopreservation and engraftment. Apoptosis triggered by cell
detachment from the extracellular matrix, which occurs during hepatocyte
isolation, is a phenomenon termed "anoikis". It's importance in the normal
physiologic development of the human body, as well as in disease states, has
been described. Cancer cells harbor anoikis resistance allowing spread to occur.
Activation of the protein Fas associated death domain/MORT1 initiates the
apoptosis cascade, with further downstream activation of caspase 8, Bid,
cytochrome c and the executioner caspases. The anti-apoptotic protein family
(bcl-2) and integrins, in particular beta 1 integrin, balance the pro apoptotic
signals. The family of caspase enzymes, currently including 14 members, is
subdivided by the prodomain length, specific substrate and phylogenetic
analysis, and plays a crucial role in the apoptotic cascade. Therefore,
understanding the molecular biology of apoptosis and specifically the "form"
termed anoikis, has advanced clinical implications in cancer and cell therapy
research. Epithelial cells require attachment to extracellular matrix (ECM) to suppress an
apoptotic cell death program termed anoikis. Here we describe a nonapoptotic
cell death program in matrix-detached cells that is initiated by a previously
unrecognized and unusual process involving the invasion of one cell into
another, leading to a transient state in which a live cell is contained within a
neighboring host cell. Live internalized cells are either degraded by lysosomal
enzymes or released. We term this cell internalization process entosis and
present evidence for entosis as a mechanism underlying the commonly observed
"cell-in-cell" cytological feature in human cancers. Further we propose that
entosis is driven by compaction force associated with adherens junction
formation in the absence of integrin engagement and may represent an intrinsic
tumor suppression mechanism for cells that are detached from ECM. Anoikis is a type of apoptosis due to the detachment from the extracellular
matrix and neighboring cells. In case of cell transplantation therapy for spinal
cord injury, preparation of graft cells includes dissociation of cultured cells,
which may cause anoikis-induced cell death. Thus suppression of anoikis may
increase survival of grafted cells. Here we tested the effect of brain-derived
neurotrophic factor (BDNF) on anoikis-induced cell death of cultured Schwann
cells. Schwann cells were collected and cultured from sciatic nerves of neonatal
Wistar rats. Schwann cells were plated upon a non-adherent polyhydroxyethyl
methacrylate substrate to induce anoikis. BDNF was added into the culture medium
at various concentrations. Twenty-four hours after non-adherent culture,
approximately 40% of Schwann cells died and BDNF significantly decreased the
number of dead cells in that culture condition. Next, Schwann cells were
transplanted with or without BDNF treatment into contused rat spinal cord 1 week
after injury. Five weeks after transplantation, immunohistochemistry revealed
that the number of transplanted cells was significantly larger in the
BDNF-treated group than that of the non-treated group. Suppression of anoikis
may increase survival of grafted cells in case of cell therapy for spinal cord
injury. BACKGROUND: Anoikis is a special type of programmed cell death after loss of
cell-cell and cell-extracellular matrix interactions. Resistance to anoikis is
likely involved in the process of metastasis, specifically during the tumor cell
migration through lymph or vascular channels. We have previously shown that
BCL-2 confers resistance to other forms of programmed cell death (i.e.,
apoptosis); furthermore, the extracellular signaling-regulated kinase (ERK)
signaling pathway regulates BCL-2 expression. We therefore tested the hypothesis
that pancreatic cancer cell lines are resistant to anoikis and this resistance
is due to activation of ERK1/2 and subsequent overexpression of BCL-2.
MATERIALS AND METHODS: Pancreatic cancer cell lines (MIA-PaCa-2 and BxPC-3) were
examined for cell death following loss of adherence to extracellular matrix.
Subclones of the MIA-PaCa-2 cell line (either selected in vivo for increased
metastatic potential [MIA-LM2] or overexpressing BCL-2 [MIA-BCL2]) were also
examined for induction of anoikis following loss of extracellular matrix
adherence. Finally, the effect of the ERK inhibitor (PD98059) on BCL-2
expression and induction of anoikis was examined.
RESULTS: Under conditions of loss of cell-extracellular matrix interaction,
pancreatic cancer cells undergo varying amounts of anoikis. Basal levels of
activated ERK and BCL-2 paralleled the sensitivity to induction of anoikis. The
highly metastatic cell line, MIA-LM2, was more resistant to anoikis than the
parental cell line. Inhibition of ERK down-regulated BCL-2 and was associated
with restoration of sensitivity to anoikis.
CONCLUSIONS: Activation of a signaling pathway from ERK to overexpression of
BCL-2 may confer resistance to anoikis, a critical step in the development of
metastasis. Targeting the ERK/BCL-2 pathway may lead to sensitization of
pancreatic cancer to anoikis, thereby decreasing the ability of these cells to
metastasize. Apoptosis and proliferation are two dynamically and tightly regulated processes
that together maintain the homeostasis of renewable tissues. Anoikis is a
subtype of apoptosis induced by detachment of adherent cells from the
extracellular matrix. By using the defined mTeSR1 medium and collecting freshly
detached cells, we found here that human pluripotent stem (PS) cells including
embryonic stem (ES) cells and induced pluripotent stem cells are subject to
constant anoikis in culture, which is escalated in the absence of basic
fibroblast growth factor (bFGF). Withdrawal of bFGF also promotes apoptosis and
differentiation of the remaining adherent cells without affecting their cell
cycle progression. Insulin-like growth factor 2 (IGF2) has previously been
reported to act downstream of FGF signaling to support self-renewal of human ES
cells. However, we found that IGF2 cannot substitute bFGF in the TeSR1-supported
culture, although endogenous IGF signaling is required to sustain self-renewal
of human ES cells. On the other hand, all of the bFGF withdrawal effects
observed here can be markedly prevented by the caspase inhibitor z-VAD-FMK. We
further demonstrated that the bFGF-repressed anoikis is dependent on activation
of ERK and AKT and associated with inhibition of Bcl-2-interacting mediator of
cell death and the caspase-ROCK1-myosin signaling. Anoikis is independent of
pre-detachment apoptosis and differentiation of the cells. Because previous
studies of human PS cells have been focused on attached cells, our findings
revealed a neglected role of bFGF in sustaining self-renewal of human PS cells:
preventing them from anoikis via inhibition of caspase activation. Anoikis - apoptotic cell death triggered by loss of extracellular matrix (ECM)
contacts - is dysregulated in many chronic debilitating and fatal diseases.
Mechanisms rendering tumor cells resistant to anoikis, although not completely
understood, possess significant therapeutic promise. In death receptor-mediated
anoikis mechanisms, focal adhesion kinase (FAK) and receptor-interacting protein
(RIP) dissociate, leading to association of RIP with Fas, formation of the
death-inducing signaling complex (DISC), activation of caspase-3, and
propagation of anoikis. In contrast, anoikis resistance is accomplished through
constitutive activation of survival pathways that include integrin-dependent
activation of FAK and extracellular-signal-regulated kinase (ERK). In addition,
FAK and RIP association confers anoikis resistance by inhibiting the association
of RIP with Fas and formation of the death signaling complex, which allows cells
to escape anoikis. Up-regulation of CD44 also contributes to survival signals
and promotes anoikis resistance. This review will focus on the roles of death
receptors, prosurvival pathways, and the molecular players involved in anoikis
escalation and resistance in oral squamous cell carcinoma. Anoikis is a programmed cell death occurring upon cell detachment from the
correct extracellular matrix, thus disrupting integrin ligation. It is a
critical mechanism in preventing dysplastic cell growth or attachment to an
inappropriate matrix. Anoikis prevents detached epithelial cells from colonizing
elsewhere and is thus essential for tissue homeostasis and development. As
anchorage-independent growth and epithelial-mesenchymal transition, two features
associated with anoikis resistance, are crucial steps during tumour progression
and metastatic spreading of cancer cells, anoikis deregulation has now evoked
particular attention from the scientific community. The aim of this review is to
analyse the molecular mechanisms governing both anoikis and anoikis resistance,
focusing on their regulation in physiological processes, as well as in several
diseases, including metastatic cancers, cardiovascular diseases and diabetes. As a barrier to metastasis of cancer, cells that lost contact with the
neighbouring cells or extracellular matrix(Extracellular matrix, ECM) will be
subjected to apoptosis. This cell death process has been termed "anoikis". When
normal epithelial cells or solid tumor cells without metastatic potential detach
from the primary site, and then enter into the circulatory system, the anoikis
mechanism will be activated. The significance of anoikis is to prevent the
shedding cells from growing and implanting into other inappropriate sites. Tumor
cells, especially several maligt cells that is prone to transfer to distant
sites, have properties of anti-anoikis, which facilitates metastasis as well as
invasion of tumor cells. The studies found that tumor cells can resist anoikis
through multiple mechanisms: the pro-survival pathways are activated by cells
autocrine growth factors and paracrine factors derived from neighboring cells;
cells change the pattern of integrin expression so that they can receive
survival signals from new environment; reactive oxygen species (ROS) activates
growth factor receptors in a ligand-independent way to avoid apoptosis; and
epithelial-mesenchymal transformation(EMT) is activated etc.. All of these
mechanisms lead to activation of survival signals and inhibition of apoptotic
pathways, and ultimately cause resistance to anoikis as well as metastasis. This
paper summarizes the key mechanisms of the current studies on metastasis, which
also suggest important targets for cancer therapy. |
The small molecule SEA0400 is an inhibitor of which ion antiporter/exchanger? | The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. | The effect of the newly synthesized compound
2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) on the
Na+-Ca2+ exchanger (NCX) was investigated and compared against that of
2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943). In addition, the
effects of SEA0400 on reperfusion injury in vitro and in vivo were examined.
SEA0400 was extremely more potent than KB-R7943 in inhibiting Na+-dependent Ca2+
uptake in cultured neurons, astrocytes, and microglia: IC50s of SEA0400 and
KB-R7943 were 5 to 33 nM and 2 to 4 microM, respectively. SEA0400 at the
concentration range that inhibited NCX exhibited negligible affinities for the
Ca2+ channels, Na+ channels, K+ channels, norepinephrine transporter, and 14
receptors, and did not affect the activities of the Na+/H+ exchanger,
Na+,K+-ATPase, Ca2+-ATPase, and five enzymes. SEA0400, unlike KB-R7943, did not
inhibit the store-operated Ca2+ entry in cultured astrocytes. SEA0400 attenuated
dose- dependently paradoxical Ca2+ challenge-induced production of reactive
oxygen species, DNA ladder formation, and nuclear condensation in cultured
astrocytes, whereas it did not affect thapsigargin-induced cell injury.
Furthermore, administration of SEA0400 reduced infarct volumes after a transient
middle cerebral artery occlusion in rat cerebral cortex and striatum. These
results indicate that SEA0400 is the most potent and selective inhibitor of NCX,
and suggest that the compound may exert protective effects on postischemic brain
damage. The effects of 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline
(SEA0400), a newly synthesized Na(+)-Ca(2+) exchanger (NCX) inhibitor, on the
NCX current and other membrane currents were examined in isolated guinea-pig
ventricular myocytes and compared with those of 2-[2-[4-(4-nitrobenzyloxy)
phenyl]ethyl]isothiourea (KB-R7943). SEA0400 concentration-dependently inhibited
the NCX current with a 10 fold higher potency than that of KB-R7943; 1 microM
SEA0400 and 10 microM KB-R7943 inhibited the NCX current by more than 80%.
KB-R7943, at 10 microM, inhibited the sodium current, L-type calcium current,
delayed rectifier potassium current and inwardly rectifying potassium current by
more than 50%, but SEA0400 (1 microM) had no significant effect on these
currents. These results indicate that SEA0400 is a potent and highly selective
inhibitor of NCX, and would be a powerful tool for further studies on the role
of NCX in the heart and the therapeutic potential of its inhibition. SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has
recently been described as a potent and selective inhibitor of Na(+)-Ca(2+)
exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of
SEA0400 were investigated on the cloned cardiac Na(+)-Ca(2+) exchanger, NCX1.1,
expressed in Xenopus laevis oocytes to gain insight into its inhibitory
mechanism. Na(+)-Ca(2+) exchange currents were measured using the giant excised
patch technique using conditions to evaluate both inward and outward currents.
SEA0400 inhibited outward Na(+)-Ca(2+) exchange currents with high affinity
(IC(50) = 78 +/- 15 and 23 +/- 4 nM for peak and steady-state currents,
respectively). Considerably less inhibitory potency (i.e., micromolar) was
observed for inward currents. The inhibitory profile was reexamined after
proteolytic treatment of excised patches with alpha-chymotrypsin, a procedure
that eliminates ionic regulatory mechanisms. After this treatment, an IC(50)
value of 1.2 +/- 0.6 microM was estimated for outward currents, whereas inward
currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400
on outward exchange currents were evident at both high and low concentrations of
regulatory Ca(2+), although distinct features were noted. SEA0400 accelerated
the inactivation rate of outward currents. Based on paired pulse experiments,
SEA0400 altered the recovery of exchangers from the Na(+)(i)-dependent inactive
state, particularly at higher regulatory Ca(2+)(i) concentrations. Finally, the
inhibitory potency of SEA0400 was strongly dependent on the intracellular Na(+)
concentration. Our data confirm that SEA0400 is the most potent inhibitor of the
cardiac Na(+)-Ca(2+) exchanger described to date and provide a reasonable
explanation for its apparent transport mode selectivity. Activation of the Na+/Ca2+ exchanger may contribute to Ca2+ overload during
reperfusion after transient ischemia. We examined the effects of
2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a
selective inhibitor of Na+/Ca2+ exchange, on a canine model of
ischemia/reperfusion injury (myocardial stunning). Myocardial stunning was
induced by a 15-min occlusion of the left anterior descending coronary artery
followed by a 4-h reperfusion in anesthetized open-chest dogs. Reperfusion
gradually restored myocardial percent segment shortening but remained depressed
during a 4-h reperfusion period. A bolus intravenous injection of SEA0400 (0.3
or 1.0 mg/kg), given 1 min before reperfusion, improved significantly the
recovery of percent segment shortening in the ischemic/reperfused myocardium.
SEA0400 did not affect the hemodynamics and electrocardiogram parameters. In
addition, SEA0400 did not affect reperfusion-induced change in coronary blood
flow. These results suggest that the Na+/Ca2+ exchanger is involved in the
stunned myocardium of dogs after reperfusion, and that SEA0400 has a protective
effect against myocardial stunning in dogs. 1. Using SEA0400, a potent and selective inhibitor of the Na+-Ca2+ exchanger
(NCX), we examined whether NCX is involved in nitric oxide (NO)-induced
disturbance of endoplasmic reticulum (ER) Ca2+ homeostasis followed by apoptosis
in cultured rat microglia. 2. Sodium nitroprusside (SNP), an NO donor, decreased
cell viability in a dose- and time-dependent manner with apoptotic cell death in
cultured microglia. 3. Treatment with SNP decreased the ER Ca2+ levels as
evaluated by measuring the increase in cytosolic Ca2+ level induced by exposing
cells to thapsigargin, an irreversible inhibitor of ER Ca2+-ATPase. 4. The
treatment with SNP also increased mRNA expression of CHOP and GPR78, makers of
ER stress. 5. SEA0400 at 0.3-1.0 microM protected microglia against SNP-induced
apoptosis. 6. SEA0400 blocked not only the SNP-induced decrease in ER Ca2+
levels but also SNP-induced increase in CHOP and GRP78 mRNAs. 7. SEA0400 did not
affect capacitative Ca2+ entry in the presence and absence of SNP. 8. SNP
increased Na+-dependent 45Ca2+ uptake and this increase was blocked by SEA0400.
9. These results suggest that SNP induces apoptosis via the ER stress pathway
and SEA0400 attenuates SNP-induced apoptosis via suppression of the ER stress in
cultured microglia. Our findings imply that NCX plays a role in ER Ca2+
depletion under pathological conditions. The effect of SEA0400, a novel Na+-Ca2+ exchanger inhibitor, on mechanical and
electrophysiological parameters of coronary-perfused guinea-pig right
ventricular tissue preparation was examined during no-flow ischemia and
reperfusion. Contractile force and action potential duration were decreased
during no-flow ischemia, while the resting tension was increased. Upon
reperfusion, transient arrhythmias were observed and contractile force returned
to less than 50% of preischemic values. SEA0400 (1 microM) had no effect on the
decline in contractile force during the no-flow ischemia, but abolished the rise
in resting tension. SEA0400 significantly improved the recovery of contractile
force after reperfusion to about 80% of the preischemic value. SEA0400 had no
effect on the action potential under normal conditions and during ischemia, but
significantly improved the recovery of action potential duration after
reperfusion. Enhancement of the recovery of contractile force during reperfusion
by SEA0400 was also observed when the drug was applied only before and during
the ischemic period and when the drug was applied only during reperfusion. The
present results indicate that inhibition of Na+-Ca2+ exchanger either during
ischemia or during reperfusion exerts cardioprotective effects and enhances the
recovery of myocardial contractile function. The cardioprotective effects of SEA0400, a novel Na(+)-Ca(2+) exchanger
inhibitor, were examined in isolated guinea pig myocardial tissue and
ventricular myocytes. In a coronary-perfused right ventricular tissue
preparation, SEA0400 had no cardiosuppressive effect during normoxia and
experimental ischemia, but enhanced the recovery of contractile force during
reperfusion. SEA0400 had no effect on tissue ATP content during normoxia, but
attenuated its decrease during ischemia. Treatment of ventricular myocytes with
an ischemia mimetic solution (high K(+), glucose free, pH 6.0, gassed with N(2))
resulted in the depolarization of the mitochondrial membrane potential and an
increase in cytoplasmic and mitochondrial Ca(2+) concentration, which had a
similar time course. SEA0400 significantly delayed these changes. These results
suggest that SEA0400 maintains mitochondrial function and tissue ATP content
during ischemia through the inhibition of cytoplasmic and mitochondrial Ca(2+)
overload. The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger
(NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were
examined in bovine adrenal chromaffin cells that were loaded with Na(+) by
treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent
(45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM,
respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and
3.7 microM, respectively. These results indicate that SEA0400 is about 40 times
more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In
intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine
and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of
KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine
and DMPP, respectively, indicating that the potency of SEA0400 is about a half
of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release.
The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400
(IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it
is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on
NCX in bovine adrenal chromaffin cells. Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and
arrhythmogenesis was examined with a specific inhibitor, SEA0400. In right
ventricular papillary muscle isolated from guinea-pig ventricle, 1 microM
SEA0400, which specifically inhibits the Na+/Ca2+ exchanger by 80%, reduced the
ouabain (1 microM)-induced positive inotropy by 40%, but had no effect on the
inotropy induced by 100 microM isobutyl methylxantine. SEA0400 significantly
inhibited the contracture induced by low Na+ solution. In HEK293 cells
expressing the Na+/Ca2+ exchanger, 1 microM ouabain induced an increase in
intracellular Ca2+, which was inhibited by SEA0400. The arrhythmic contractions
induced by 3 microM ouabain were significantly reduced by SEA0400. These results
provide pharmacological evidence that the Na+/Ca2+ exchanger is involved in
ouabain-induced inotropy and arrhythmogenesis. SEA0400 is a selective inhibitor of the Na(+)/Ca(2+) exchanger having equal
potencies to suppress both the forward and reverse mode operation of the
Na(+)/Ca(2+) exchanger. Present experiments were designed to study the effect of
partial blockade of Na(+)/Ca(2+) exchanger on Ca(2+) handling in isolated rat
ventricular myocytes. Intracellular Ca(2+) transient and cell shortening were
measured in ventricular myocytes loaded with Fura-2-AM fluorescent dye. Partial
blockade of Na(+)/Ca(2+) exchanger was induced by superfusion of the cells with
SEA0400 at a concentration of 0.3 microM. Amplitude of the intracellular Ca(2+)
transient and cell shortening was significantly increased by SEA0400 in both
field stimulated and voltage clamped myocytes, without significant elevation of
diastolic Ca(2+) level and the decay time constant of the Ca(2+) transient. In
patch clamped myocytes the SEA0400 induced increase in the Ca(2+) transient and
cell shortening was accompanied by significant reduction of peak L-type Ca(2+)
current. These effects can be explained by the autoregulative nature of cardiac
Ca(2+) handling, as the reduced Ca(2+) efflux from the cell results in an
increased Ca(2+) load to the sarcoplasmic reticulum leading to increased Ca(2+)
release, which in turn may decrease the L-type Ca(2+) current by accelaration of
Ca(2+) dependent inactivation of L-type Ca(2+) current. Our results suggest that
complex changes in the Ca(2+) cycling can occur after selective pharmacological
inhibition of the Na(+)/Ca(2+) exchanger. We examined the involvement of the Na(+)/Ca(2+) exchanger in the automaticity of
the pulmonary vein myocardium with a specific inhibitor, SEA0400. Action
potentials were recorded from the myocardial layer of isolated guinea-pig
pulmonary vein preparations, and Ca(2+) transients were recorded from the
cardiomyocytes. Spontaneous electrical activity was observed in 17.7% of the
preparations, which was inhibited by either SEA0400 or ryanodine. In quiescent
preparations, ouabain induced electrical activity and spontaneous Ca(2+)
transients, which were inhibited by SEA0400, as well as ryanodine. These results
provide pharmacological evidence that the Na(+)/Ca(2+) exchanger underlies the
automaticity of the pulmonary vein myocardium. The sodium-calcium exchanger (NCX) is one of the transporters contributing to
the control of intracellular calcium (Ca(2+)) concentration by normally
mediating net Ca(2+) efflux. However, the reverse mode of the NCX can cause
intracellular Ca(2+) concentration overload, which exacerbates the myocardial
tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has
been shown to therapeutically reduce myocardial injury, no in vivo technique
exists to monitor intracellular Ca(2+) fluctuations produced by this drug.
Cardiac manganese-enhanced MRI (MEMRI) may indirectly assess Ca(2+) efflux by
estimating changes in manganese (Mn(2+)) content in vivo, since Mn(2+) has been
suggested as a surrogate marker for Ca(2+). This study used the MEMRI technique
to examine the temporal features of cardiac Mn(2+) efflux by implementing a
T(1)-mapping method and inhibiting the NCX with SEA0400. The change in (1)H(2)O
longitudinal relaxation rate, Delta R(1), in the left ventricular free wall, was
calculated at different time points following infusion of 190 nmol/g manganese
chloride (MnCl(2)) in healthy adult male mice. The results showed 50% MEMRI
signal attenuation at 3.4 +/- 0.6 h post-MnCl(2) infusion without drug
intervention. Furthermore, treatment with 50 +/- 0.2 mg/kg of SEA0400
significantly reduced the rate of decrease in Delta R(1). At 4.9-5.9 h
post-MnCl(2) infusion, the average Delta R(1) values for the two groups treated
with SEA0400 were 2.46 +/- 0.29 and 1.72 +/- 0.24 s(-1) for 50 and 20 mg/kg
doses, respectively, as compared to the value of 1.27 +/- 0.28 s(-1) for the
control group. When this in vivo data were compared to ex vivo absolute
manganese content data, the MEMRI T(1)-mapping technique was shown to
effectively quantify Mn(2+) efflux rates in the myocardium. Therefore, combining
an NCX inhibitor with MEMRI may be a useful technique for assessing Mn(2+)
transport mechanisms and rates in vivo, which may reflect changes in Ca(2+)
transport. Nitric oxide (NO) is involved in many pathological conditions including
neurodegenerative disorders. We have previously found that sodium nitroprusside
(SNP), an NO donor, stimulates mitogen-activated protein kinases (MAPKs) such as
extracellular signal-regulating kinase (ERK), c-jun N-terminal protein kinase
(JNK) and p38 MAPK, leading to caspase-independent apoptosis in cultured
astrocytes. In view of the previous observation that NO stimulates the activity
of the Na(+)/Ca(2+) exchanger (NCX), this study examines the involvement of NCX
in cytotoxicity. The specific NCX inhibitor SEA0400 blocked SNP-induced
phosphorylation of ERK, JNK and p38 MAPK, and decrease in cell viability.
SNP-induced phosphorylation of ERK, JNK and p38 MAPK was blocked by removal of
external Ca(2+), and SNP treatment caused an increase in (45)Ca(2+) influx. This
increase in (45)Ca(2+) influx was blocked by SEA0400, but not the Ca(2+) channel
blocker nifedipine. In addition, SNP-induced (45)Ca(2+) influx and cytotoxicity
were reduced in NCX1-deficient cells which were transfected with NCX1 siRNA.
Inhibitors of intracellular Ca(2+)-dependent proteins such as calpain and
calmodulin blocked SNP-induced ERK phosphorylation and decrease in cell
viability. Furthermore, the guanylate cyclase inhibitor LY83583 and the
cGMP-dependent protein kinase inhibitor KT5823 blocked SNP-induced cytotoxicity.
These findings suggest that NCX-mediated Ca(2+) influx triggers SNP-induced
apoptosis in astrocytes, which may be mediated by a cGMP-dependent pathway. The Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of intracellular
Ca(2+) levels, and nitric oxide (NO) is involved in many pathological conditions
including neurodegenerative disorders. We have previously found that sodium
nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured
glial cells via NCX-mediated pathways and the mechanism for NO-induced
cytotoxicity is cell type-dependent. The present study examined using the
specific NCX inhibitor
2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX
is involved in NO-induced injury in cultured neuronal cells. The treatment of
neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity
was blocked by the mitogen-activated protein (MAP)/extracellular
signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase
(MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK)
inhibitor SP60012. SNP increased Ca(2+) influx and intracellular Ca(2+) levels.
In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of
reactive oxygen species (ROS) in an extracellular Ca(2+)-dependent manner. These
effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not
affected by inhibitors of the Ca(2+), Na(+) and store-operated/capacitative
channels. Moreover, SNP-induced increase in intracellular Ca(2+) levels, ROS
production and decrease in cell viability were blocked by a cGMP-dependent
protein kinase (PKG) inhibitor. These results suggest that Ca(2+) influx via the
reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and
NO activates the NCX through guanylate cyclase/PKG pathway. We have recently shown that the Na(+)/Ca(2+) exchanger (NCX) is involved in
nitric oxide (NO)-induced cytotoxicity in cultured astrocytes and neurons.
However, there is no in vivo evidence suggesting the role of NCX in
neurodegenerative disorders associated with NO. NO is implicated in the
pathogenesis of neurodegenerative disorders such as Parkinson's disease. This
study examined the effect of SEA0400, the specific NCX inhibitor, on
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic
neurotoxicity, a model of Parkinson's disease, in C57BL/6J mice. MPTP treatment
(10 mg/kg, four times at 2-h intervals) decreased dopamine levels in the
midbrain and impaired motor coordination, and these effects were counteracted by
S-methylthiocitrulline, a selective neuronal NO synthase inhibitor. SEA0400
protected against the dopaminergic neurotoxicity (determined by dopamine levels
in the midbrain and striatum, tyrosine hydroxylase immunoreactivity in the
substantia nigra and striatum, striatal dopamine release, and motor deficits) in
MPTP-treated mice. SEA0400 had no radical-scavenging activity. SEA0400 did not
affect MPTP metabolism and MPTP-induced NO production and microglial activation,
while it attenuated MPTP-induced increases in extracellular signal-regulated
kinase (ERK) phosphorylation and lipid peroxidation product, thiobarbituric acid
reactive substance. These findings suggest that SEA0400 protects against
MPTP-induced neurotoxicity probably by blocking ERK phosphorylation and lipid
peroxidation which are downstream of NCX-mediated Ca(2+) influx. BACKGROUND: Molecular remodeling in heart failure includes slowing of
repolarization, leading to proarrhythmia.
OBJECTIVE: To evaluate the effects of Na(+)/Ca(2+) exchanger (NCX) inhibition on
repolarization as a novel antiarrhythmic concept in chronic heart failure (CHF).
METHODS AND RESULTS: CHF was induced by rapid ventricular pacing in rabbits.
Left ventricular function was assessed by echocardiography. Monophasic action
potentials (MAPs) showed a prolongation of repolarization in CHF after
atrioventricular block and stimulation at different cycle lengths. Sotalol (100
μM, n = 13) or veratridine (0.5 μM; n = 15) resulted in a further significant
increase in the MAP duration. CHF was associated with an increased dispersion of
repolarization, as compared with sotalol-treated (+22 ± 7 ms; P < .05) and
veratridine-treated (+20 ± 6 ms; P < .05) sham hearts. In the presence of a low
potassium concentration, sotalol and veratridine reproducibly induced early
afterdepolarizations (EADs) and polymorphic ventricular tachyarrhythmias (VTs).
SEA0400 (1 μM), a pharmacological inhibitor of NCX, significantly shortened the
MAP duration (P < .01) and reduced dispersion (P < .05). It suppressed EAD in 6
of 13 sotalol-treated failing hearts and in 9 of 10 veratridine-treated failing
hearts, leading to a reduction in VT (60% in sotalol-treated failing hearts and
83% in veratridine-treated failing hearts). Simulations using a mathematical
model showed a reduction in the action potential duration and the number of EADs
by the NCX block in all subgroups.
CONCLUSIONS: In an experimental model of CHF, the acute inhibition of NCX (1)
reduces the MAP duration, (2) decreases dispersion of repolarization, and (3)
suppresses EAD and VT. Our observations indicate for the first time that
pharmacological NCX inhibition increases repolarization reserve and protects
against VTs in heart failure. |
What is Piebaldism? | Piebaldism is a rare autosomal dominant disorder of melanocyte development characterized by a congenital white forelock and multiple symmetrical stable hypopigmented or depigmented macules. | Piebaldism is an autosomal domit genetic disorder of pigmentation
characterized by congenital patches of white skin and hair that lack
melanocytes. Piebaldism results from mutations of the KIT proto-oncogene, which
encodes the cell-surface receptor transmembrane tyrosine kinase for an embryonic
growth factor, Steel factor. Several pathologic mutations of the KIT gene have
now been identified in different patients with piebaldism. Correlation of these
mutations with the associated piebald phenotypes has led to the recognition of a
hierarchy of three classes of mutations that result in a graded series of
piebald phenotypes, and to improved understanding of the mechanisms that
underlie domit genetic disorders. Piebaldism is an autosomal domit genetic disorder of pigmentation
characterized by white patches of skin and hair. Melanocytes are lacking in
these hypopigmented regions, the result of mutations of the KIT gene, which
encodes the cell surface receptor for steel factor (SLF). We describe the
analysis of 26 unrelated patients with piebaldism-like hypopigmentation--17
typical patients, 5 with atypical clinical features or family histories, and 4
with other disorders that involve white spotting. We identified novel pathologic
mutations or deletions of the KIT gene in 10 (59%) of the typical patients, and
in 2 (40%) of the atypical patients. Overall, we have identified pathologic KIT
gene mutations in 21 (75%) of 28 unrelated patients with typical piebaldism we
have studied. Of the patients without apparent KIT mutations, none have apparent
abnormalities of the gene encoding SLF itself (MGF), and genetic linkage
analyses in two of these families are suggestive of linkage of the piebald
phenotype to KIT. Thus, most patients with typical piebaldism appear to have
abnormalities of the KIT gene. Piebaldism is an autosomal domit disorder of melanocyte development
characterized by white skin (leukoderma) and white hair (poliosis). In general,
piebaldism has been distinguished from vitiligo by the presence of lesions from
birth, the hyperpigmented macules of depigmented and normal skin, and the static
course. We hypothesized that an 8-year-old girl and her mother who had unusual
piebaldism of a progressive nature would have a novel mutation of the KIT gene,
the gene that is altered in patients with piebaldism, or of the MITF
(microphthalmia activating transcription factor) gene, which would be expected
to cause type II Waardenburg syndrome, but is associated with a phenotype of
progressive depigmentation in mice. Genomic DNA was extracted from the blood of
affected and unaffected family members, and the KIT and MITF genes were
sequenced. Genetic analysis of genomic DNA from both the mother and daughter
with progressive piebaldism revealed a novel Val620Ala (1859T>C) mutation in the
KIT gene, which was not detected in family members without progressive
piebaldism or in 52 normal control individuals. This KIT mutation affects the
intracellular tyrosine kinase domain and thus predicts a severe phenotype, as
was the case in this family. Although other KIT mutations in the vicinity of
codon 620 lead to the standard phenotype of static piebaldism, the Val620Ala
mutation is novel and may result in a previously undescribed phenotype with
melanocyte instability, leading to progressive loss of pigmentation as well as
the progressive appearance of the hyperpigmented macules. Human piebaldism is a rare autosomal domit disorder that comprises congenital
patchy depigmentation of the scalp, forehead, trunk and limbs. It is caused by
mutations in the cell-surface receptor tyrosine kinase gene (KIT, also c-kit).
We screened three families and three isolated cases of piebaldism from different
countries for mutations in the KIT gene using automated sequencing methods. We
report six novel KIT point mutations: three missense (C788R, W835R, P869S) at
highly conserved amino acid sites; one nonsense (Q347X) that results in
termination of translation of the KIT gene in exon 6; and two splice site
nucleotide substitutions (IVS13+2T>G, IVS17-1G>A) that are predicted to impair
normal splicing. These mutations were not detected in over 100 normal
individuals and are likely to be the cause of piebaldism in our subjects. Piebaldism is a rare disorder present at birth and inherited as an autosomal
domit trait. It results from a mutation in the c-kit proto-oncogene and is
associated with a defect in the migration and differentiation of melanoblasts
from the neural crest. Clinical manifestations and phenotypic severity strongly
correlates with the site of mutation within the KIT gene. Here we report a
3-year-old boy and his 33-year-old father with leukoderma and poliosis
associated with clinical criteria for Neurofibromatosis type 1. Genetic study of
both revealed a p.Gly610Asp mutation in the KIT gene. This familiar mutation has
not yet been reported in the literature. There are rare reports of piebaldism in
association with neurofibromatosis type I. Piebaldism is a rare autosomal domit skin disorder characterized by a white
forelock and depigmented patches of skin, generally located on the forehead,
central chest and abdomen, upper arms, and lower extremities. We report a case
of a 2-year-old girl with a typical presentation and review the literature
concerning this condition. Piebaldism is an autosomal domit disorder characterized by congenital
hypopigmented patches of skin and hair and has been found to be associated with
mutations in the KIT or SLUG genes. Café-au-lait macules (CALM) may occasionally
be seen in piebaldism. There are four reports describing six patients who were
said to have both piebaldism and neurofibromatosis type 1 (NF1) due to the
presence of multiple CALM and intertriginous freckling, but none of these
patients had undergone comprehensive NF1 mutation analysis. We describe a large
family with piebaldism in which two members meet diagnostic criteria for NF1
based on the presence of >5 CALM and intertriginous freckling. Interestingly,
only these two family members are of mixed race, which could be of importance. A
novel complex mutation in the KIT gene was identified in several family members
affected with piebaldism; the proband meeting diagnostic criteria for NF1 also
underwent comprehensive NF1 and SPRED1 testing with no mutations detected. These
findings suggest that piebaldism may occasionally include CALM and
intertriginous freckling, which may create diagnostic confusion especially in
the absence of a family history of piebaldism. However, careful clinical
evaluation and molecular testing if necessary should distinguish these two
disorders. Piebaldism is a rare genodermatosis caused by KIT mutations. We report the case
of a 5-year-old boy who had the white forelock and leukoderma of piebaldism, but
the presence of many café-au-lait macules and axillary and inguinal freckling
complicated the diagnosis. Patients with similar cutaneous findings have been
previously reported, and their disorder has been attributed to an overlap of
piebaldism and neurofibromatosis type 1. Legius syndrome is a recently described
syndrome caused by Sprouty-related, Ena/vasodilator-stimulated phosphoprotein
homology-1 domain containing protein 1 (SPRED1) mutations that also has multiple
café-au-lait macules and intertriginous freckling. Based on our current
understanding of KIT and SPRED1 protein interactions, we propose that
café-au-lait macules and freckling may be seen in some patients with piebaldism
and does not necessarily represent coexistence of neurofibromatosis type 1. Piebaldism is a rare autosomal domit disorder of melanocyte development
characterized by a congenital white forelock and multiple symmetrical stable
hypopigmented or depigmented macules. We report a family with piebaldism
affecting three successive generations and also review the literature. BACKGROUND: Human piebaldism is a rare autosomal domit condition
characterized by congenital white forelock and depigmented patches of skin,
typically on the forehead, anterior trunk and extremities. Mutations in the KIT
gene have been proposed to be responsible for the underlying changes in this
disorder. The aim of this study was to identify gene mutation in a Chinese
family with piebaldism.
METHODS: A Chinese family with piebaldism presenting with white forelock and
large depigmented skin macules on the abdomen, arms and legs was collected. DNA
was isolated from peripheral blood of the family members. The encoding exons
with flanking intron regions of the KIT gene were analyzed by polymerase chain
reactions (PCR) and direct DNA sequencing. Besides, DNA extracted from 100
ethnically matched population individuals was as controls.
RESULTS: A heterozygous missense mutation c.2590T > C was identified in the
patients of the family. This mutation converted a serine residue to proline
(p.Ser864Pro). The mutation was not found in their unaffected family members or
normal controls.
CONCLUSION: A novel missense mutation c.2590 T > C was found and it might play a
significant role in the piebaldism phenotype in the family. |
Which SLC family is FLVCR1 a member of? | Feline leukemia virus subgroup C receptor (FLVCR1) is a member of the SLC49 family. | Heme is critical for a variety of cellular processes, but excess intracellular
heme may result in oxidative stress and membrane injury. Feline leukemia virus
subgroup C receptor (FLVCR1), a member of the SLC49 family of four paralogous
genes, is a cell surface heme exporter, essential for erythropoiesis and
systemic iron homeostasis. Disruption of FLVCR1 function blocks development of
erythroid progenitors, likely due to heme toxicity. Mutations of SLC49A1
encoding FLVCR1 are noted in patients with a rare neurodegenerative disorder:
posterior column ataxia with retinitis pigmentosa. FLVCR2 is highly homologous
to FLVCR1 and may function as a cellular heme importer. Mutations of SLC49A2
encoding FLVCR2 are observed in Fowler syndrome, a rare proliferative vascular
disorder of the brain. The functions of the remaining members of the SLC49
family, MFSD7 and DIRC2 (encoded by the SLC49A3 and SLC49A4 genes), are unknown,
although the latter is implicated in hereditary renal carcinomas. SLC48A1 (heme
responsive gene-1, HRG-1), the sole member of the SLC48 family, is associated
with the endosome and appears to transport heme from the endosome into the
cytosol. |
Is amoxicillin used for treatment of malnutrition in children? | Yes, amoxicillin is used for treatment of malnutrition in children. | OBJETIVO: Revisar sistemáticamente los datos que apoyan las directrices de la
Organización Mundial de la Salud que recomiendan la administración de
antibióticos de amplio espectro en niños con desnutrición grave (NDG).
MÉTODOS: Se realizó una búsqueda en los resúmenes CENTRAL, MEDLINE, EMBASE,
LILACS, POPLINE, CAB y en los registros de ensayos en marcha. Nos pusimos en
contacto con expertos en la materia. Se realizaron búsquedas manuales en listas
de referencia y en actas de congresos. Se incluyeron todo tipo de estudios,
excepto los informes de casos clínicos individuales.
RESULTADOS: Se recuperaron dos ensayos controlados aleatorizados (ECA), un
estudio comparativo del antes y el después y dos informes retrospectivos sobre
eficacia clínica y seguridad, junto con 18 estudios de farmacocinética. La
calidad del ensayo fue generalmente baja y los resultados no se pudieron agrupar
debido a su heterogeneidad. El tratamiento con amoxicilina por vía oral durante
5 días resultó ser tan efectivo como la ceftriaxona intramuscular durante 2 días
(1 ECA). La amoxicilina no superó los beneficios de un placebo para el
tratamiento de niños con desnutrición grave que no presentaban otras
complicaciones (1 estudio retrospectivo). La introducción de un tratamiento
estandarizado con ampicilina y gentamicina redujo de manera significativa la
mortalidad entre los niños hospitalizados (oportunidad relativa, OR: 4,0;
intervalo de confianza del 95%, IC: 1,7–9,8; 1 estudio comparativo del antes y
el después). El tratamiento con cloranfenicol por vía oral resultó ser tan
efectivo como la combinación de trimetoprima y sulfametoxazol en el tratamiento
de niños con neumonía (1 ECA). Los datos de farmacocinética sugieren que las
dosis normales de penicilinas, asociación de trimetroprim y sulfametoxazol y
gentamicina resultan seguras en niños malnutridos, mientras que, en el caso del
cloranfenicol, deberían ajustarse las dosis o la frecuencia de administración.
Las evidencias existentes no son lo suficientemente sólidas para aclarar más las
recomendaciones sobre tratamientos de antibióticos en niños con desnutrición
grave.
CONCLUSIÓN: Es necesario realizar amplios ECA para definir el tratamiento óptimo
con antibióticos de los niños con desnutrición grave con y sin complicaciones.
También es necesario investigar mejor la toxicidad de la gentamicina y el
cloranfenicol y la farmacocinética de la deftriaxona y de la ciprofloxacina. BACKGROUND: Severe acute malnutrition contributes to 1 million deaths among
children annually. Adding routine antibiotic agents to nutritional therapy may
increase recovery rates and decrease mortality among children with severe acute
malnutrition treated in the community.
METHODS: In this randomized, double-blind, placebo-controlled trial, we randomly
assigned Malawian children, 6 to 59 months of age, with severe acute
malnutrition to receive amoxicillin, cefdinir, or placebo for 7 days in addition
to ready-to-use therapeutic food for the outpatient treatment of uncomplicated
severe acute malnutrition. The primary outcomes were the rate of nutritional
recovery and the mortality rate.
RESULTS: A total of 2767 children with severe acute malnutrition were enrolled.
In the amoxicillin, cefdinir, and placebo groups, 88.7%, 90.9%, and 85.1% of the
children recovered, respectively (relative risk of treatment failure with
placebo vs. amoxicillin, 1.32; 95% confidence interval [CI], 1.04 to 1.68;
relative risk with placebo vs. cefdinir, 1.64; 95% CI, 1.27 to 2.11). The
mortality rates for the three groups were 4.8%, 4.1%, and 7.4%, respectively
(relative risk of death with placebo vs. amoxicillin, 1.55; 95% CI, 1.07 to
2.24; relative risk with placebo vs. cefdinir, 1.80; 95% CI, 1.22 to 2.64).
Among children who recovered, the rate of weight gain was increased among those
who received antibiotics. No interaction between type of severe acute
malnutrition and intervention group was observed for either the rate of
nutritional recovery or the mortality rate.
CONCLUSIONS: The addition of antibiotics to therapeutic regimens for
uncomplicated severe acute malnutrition was associated with a significant
improvement in recovery and mortality rates. (Funded by the Hickey Family
Foundation and others; ClinicalTrials.gov number, NCT01000298.). BACKGROUND: Outpatient Therapeutic feeding Program (OTP) brings the services for
management of Severe Acute Malnutrition (SAM) closer to the community by making
services available at decentralized treatment points within the primary health
care settings, through the use of ready-to-use therapeutic foods, community
outreach and mobilization. Little is known about the program outcomes. This
study revealed the levels of program outcome indictors and determit factors
to recovery rate.
METHODS: A retrospective cohort study was conducted on 628 children who had been
managed for SAM under OTP from April/2008 to January/2012. The children were
selected using systematic random sampling from 12 health posts and 4 health
centers. The study relied on information of demographic characteristics,
anthropometries, Plumpy'Nut, medical problems and routine medications intakes.
The results were estimated using Kaplan-Meier survival curves, log-rank test and
Cox-regression.
RESULTS: The recovery, defaulter, mortality and weight gain rates were 61.78%,
13.85%, 3.02% and 5.23 gm/kg/day, respectively. Routine medications were
administered partially and children with medical problems were managed
inappropriately under the program. As a child consumed one more sachet of
Plumpy'Nut, the recovery rate from SAM increased by 4% (HR = 1.04,
95%-CI = 1.03, 1.05, P<0.001). The adjusted hazard ratios to recovery of
children with diarrhea, appetite loss with Plumpy'Nut and failure to gain weight
were 2.20 (HR = 2.20, 95%-CI = 1.31, 3.41, P = 0.001), 4.49 (HR = 1.74,
95%-CI = 1.07, 2.83, P = 0.046) and 3.88 (HR = 1.95, 95%-CI = 1.17, 3.23,
P<0.001), respectively. Children who took amoxicillin and de-worming had 95%
(HR = 1.95, 95%-CI = 1.17, 3.23) and 74% (HR = 1.74, 95%-CI = 1.07, 2.83) more
probability to recover from SAM as compared to those who didn't take them.
CONCLUSIONS: The OTP was partially successful. Management of children with
comorbidities under the program and partial administration of routine drugs were
major threats for the program effectiveness. The stakeholders should focus on
creating the capacity of the OTP providers on proper management of SAM to
achieve fully effective program. |
Where are the orexigenic peptides synthesized? | The orexigenic peptides are sythesized in the hypothalamus. | With advancing age most aspects of the peptidergic regulation of energy balance
are altered. The alteration involves both the peripheral peptides derived from
the adipose tissue or the gastrointestinal tract and the peptides of the central
nervous system (brainstem and hypothalamus). In general, the expression of
orexigenic peptides and their receptors decreases with age, while that of the
anorexic ones rather increases, but not simultaneously and not in a linear
fashion. Apart from such quantitative changes, the efficacy of the related
peptides may also change with age. These changes are not necessarily linear,
either: instead of continuous decline or increase of its effects, the effects of
a peptide may become less pronounced in some phases of aging and much enhanced
in other ones. Comparing the individual peptides, the phasic alterations in
their anabolic or catabolic roles in the regulation of energy balance may
exhibit dissimilar time-patterns. In addition, within the overall anabolic or
catabolic effects, the feeding and metabolic actions of certain peptides may not
change simultaneously. Altogether, as compared with young adults, in middle-aged
animals or individuals the anabolic processes (increased food intake with
decreased energy expenditure) seem to prevail, which processes may contribute to
the explanation of age-related obesity, while in the old ones the catabolic
processes (anorexia with enhanced metabolic rate) dominate, which possibly
explain the aging anorexia, frailty and sarcopenia. While a high-fat diet when compared to low-fat diet is known to produce
overeating and health complications, less is known about the effects produced by
fat-rich diets differing in their specific composition of fat. This study
examined the effects of a high-fat diet containing relatively high levels of
saturated compared to unsaturated fatty acids (HiSat) to a high-fat diet with
higher levels of unsaturated fatty acids (USat). A HiSat compared to USat meal
caused rats to consume more calories in a subsequent chow test meal. The HiSat
meal also increased circulating levels of triglycerides (TG) and expression of
the orexigenic peptides, galanin (GAL) in the hypothalamic paraventricular
nucleus (PVN) and orexin (OX) in the perifornical lateral hypothalamus (PFLH). A
similar increase in TG levels and PVN GAL and PFLH OX was also seen in rats
given chronic access to the HiSat compared to USat diet, while neuropeptide Y
(NPY) and agouti-related protein (AgRP) in the arcuate nucleus showed decreased
expression. The importance of TG in producing these changes was supported by the
finding that the TG-lowering medication gemfibrozil as compared to vehicle, when
peripherally administered before consumption of a HiSat meal, significantly
decreased the expression of OX, while increasing the expression of NPY and AgRP.
These findings substantiate the importance of the fat composition in a diet,
indicating that those rich in saturated compared to unsaturated fatty acids may
promote overeating by increasing circulating lipids and specific hypothalamic
peptides, GAL and OX, known to preferentially stimulate the consumption of a
fat-rich diet. Metformin appears to be involved in altering energy expenditure and
thermogenesis, and could affect hypothalamic feeding circuits. However, it is
not clear whether metformin is able to cross the blood-brain barrier (BBB) to
reach the hypothalamus and exert a direct effect on the central nervous system.
Here we show the presence of metformin in cerebrospinal fluid (CSF) of diabetic
rats administered orally with metformin which was confirmed by detecting the
concentration of metformin with liquid chromatography-tandem mass spectrometry.
Food intake of diabetic rats treated with metformin was reduced, and glucose
homeostasis was gained. Expression of orexigenic peptides neuropeptide Y (NPY)
and agouti-related protein (AgRP) decreased in the hypothalamus of
metformin-treated diabetic rats, though anorexigenic peptides
pro-opiomelanocortin (POMC) did not change significantly. The phosphorylation of
signal transducer and activator of transcription 3 (STAT3) was increased but
phosphorylated AMP-activated kinase (AMPK) was similar in the hypothalamus of
metformin-treated diabetic rats. Our findings suggest that metformin may cross
BBB and play a central mechanism on regulation of food intake in the
hypothalamus. The anorexic effect of metformin may be mediated by inhibition of
NPY and AgRP gene expression through the STAT3 signaling pathway. Aging is often associated with overweight and obesity. There exists a
long-standing debate about whether meal pattern also contributes to the
development of obesity. The orexigenic hormone ghrelin regulates appetite and
satiety by activating its receptor, growth hormone secretagogue receptor
(GHS-R). In mice, circulating ghrelin concentrations and brain GHS-R expression
were shown to increase with aging. To assess whether GHS-R regulates feeding
pattern during aging, we studied meal patterns for the following cohorts of male
mice fed a normal unpurified diet: 1) 3-4 mo, young wild-type (WT) mice; 2) 3-4
mo, young Ghsr-null (Ghsr(-/-)) mice; 3) 12-14 mo, middle-aged WT (WT-M) mice;
4) 12-14 mo, middle-aged Ghsr(-/-) (Ghsr(-/-)-M) mice; 5) 24-26 mo, old WT
(WT-O) mice; and 6) 24-26 mo, old Ghsr(-/-) (Ghsr(-/-)-O) mice. Although the
total daily food intake of Ghsr(-/-) mice was similar to that of WT controls,
Ghsr(-/-)-M and Ghsr(-/-)-O mice had 9% (P = 0.07) and 16% (P < 0.05) less body
weight compared with WT-M and WT-O mice, respectively, primarily due to reduced
fat mass (P < 0.05, WT-M vs. Ghsr(-/-)-M and WT-O vs. Ghsr(-/-)-O).
Intriguingly, Ghsr(-/-)-M mice ate larger meals (on average, Ghsr(-/-)-M mice
ate 0.117 g/meal and WT-M mice ate 0.080 g/meal; P < 0.01) and took a longer
time to eat (Ghsr(-/-)-M, 196.0 s and WT-M, 128.9 s; P < 0.01), but ate less
frequently (Ghsr(-/-)-M, 31.0 times/d and WT-M, 42.3 times/d; P < 0.05) than
WT-M controls. In addition, we found that expression of hypothalamic orexigenic
peptides, neuropeptide Y (NPY) and agouti-related peptide (AgRP), was relatively
lower in aged WT mice (P = 0.09 for NPY and P = 0.06 for AgRP), but anorexic
peptide pro-opiomelanocortin (POMC) expression remained unchanged between the WT
age groups. Interestingly, old Ghsr(-/-) mice had greater hypothalamic NPY
expression (102% higher; P < 0.05) and AgRP expression (P = 0.07) but
significantly lower POMC expression (P < 0.05) when compared with age-matched
WT-O controls. Thus, our results indicate that GHS-R plays an important role in
the regulation of meal pattern and that GHS-R ablation may modulate feeding
behavior through the regulation of hypothalamic neuropeptides. Our results
collectively suggest that ghrelin receptor antagonism may have a beneficial
effect on metabolism during aging. Neuroinflammation is a feedback mechanism against infection, with recent studies
suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2),
and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and
migration in response to damage. Although CCL2 co-localizes with neuropeptides
in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2
is unknown. This led us to consider the possibility that CCL2 acting through
CCR2, under natural conditions, may affect the migration and peptide levels of
hypothalamic neurons that control voluntary behavior. This study used primary
embryonic hypothalamic neurons to examine the effect of CCL2 on migratory
behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment
with CCL2 led to a significant, dose-dependent increase in the number of
migrated neurons and an increase in the velocity and distance traveled. CCL2
also significantly increased the number of ENK-expressing and CCR2/ENK
co-expressing neurons and the percentage of neurons that contain higher levels
of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and
galanin. These results provide evidence for a stimulatory effect of CCL2 on
embryonic hypothalamic neurons involving changes in migratory behavior,
expression, and synthesis of neuropeptides that function in controlling
behavior. Our results demonstrate that the chemokine, CCL2, functions through
its receptor, CCR2, to stimulate the migration and expression of the orexigenic
peptides, enkephalin (ENK) and galanin (GAL), in developing embryonic
hypothalamic neurons that are important for controlling ingestive behavior. This
evidence reveals broad effects of CCL2 in the developing hypothalamus, showing
this chemokine system to be tightly linked to the hypothalamic peptide neurons. SCOPE: It is known that a decline in food intake occurs with aging. In this
study, we investigated changes in parameters associated with food intake in
response to aging, and whether orexigenic peptides stimulated food intake after
peripheral administration even in aged mice.
METHODS AND RESULTS: Food intake and body weight of 27-month-old male C57BL/6N
mice were lower than those of 15-month-old mice. Epididymal and mesenteric fat
mass, blood glucose, triglyceride, and leptin levels were also decreased.
Meanwhile, the hypothalamic mRNA expression of endogenous orexigenic peptides
such as neuropeptide Y (NPY) and agouti-related protein, also called
agouti-related peptide, was increased. Next, we tested responsiveness to
exogenously administered orexigenic peptides coupled to NPY in aged as well as
young mice. Orally administered rubiscolin-6, a δ opioid agonist hexapeptide
derived from a major green leaf protein Rubisco, stimulated food intake in
27-month-old mice. In contrast, ghrelin was ineffective after intraperitoneal
administration to aged mice, suggesting that the NPY system downstream of
ghrelin but not δ opioid receptors might be impaired in aged mice.
CONCLUSION: Orally administered rubiscolin-6 stimulates food intake in aged mice
with ghrelin resistance. Different alcohol drinking patterns, involving either small and frequent
drinking bouts or large and long-lasting bouts, are found to differentially
affect the risk for developing alcohol-related diseases, suggesting that they
have different underlying mechanisms. Such mechanisms may involve orexigenic
peptides known to stimulate alcohol intake through their actions in the
hypothalamic paraventricular nucleus (PVN). These include orexin (OX), which is
expressed in the perifornical lateral hypothalamus, and galanin (GAL) and
enkephalin (ENK), which are expressed within as well as outside the PVN. To
investigate the possibility that these peptides affect different aspects of
consumption, a microstructural analysis of ethanol drinking behavior was
performed in male, Sprague-Dawley rats trained to drink 7% ethanol and implanted
with guide shafts aimed at the PVN. While housed in specialized cages containing
computerized intake monitors (BioDAQ Laboratory Intake Monitoring System,
Research Diets Inc., New Brunswick, NJ) that measure bouts of ethanol drinking,
these rats were given PVN injections of OX (0.9 nmol), GAL (1.0 nmol), or the
ENK analog D-Ala2-met-enkephalinamide (DALA) (14.2 nmol), as compared to saline
vehicle. Results revealed clear differences between the effects of these
peptides. While all 3 stimulated ethanol intake, they had distinct effects on
patterns of drinking, with OX increasing the number of drinking bouts, GAL
increasing the size of the drinking bouts, and DALA increasing both the size and
duration of the bouts. In contrast, these peptides had little impact on water or
food intake. These results support the idea that different peptides can increase
ethanol consumption by promoting distinct aspects of the ethanol drinking
response. The stimulatory effect of OX on drinking frequency may be related to
its neuronally stimulatory properties, while the stimulatory effect of GAL and
ENK on bout size and duration may reflect a suppressive effect of these
neuronally inhibitory peptides on the satiety-controlling PVN. Orexin A and B, orexigenic peptides produced primarily by the lateral
hypothalamus that signal through two G protein-coupled receptors, orexin
receptors 1/2, have been implicated in the regulation of several physiological
processes in mammals. In avian (nonmammalian vertebrates) species; however, the
physiological roles of orexin are not well defined. Here, we provide novel
evidence that not only is orexin and its related receptors 1/2 (ORXR1/2)
expressed in chicken muscle tissue and quail muscle (QM7) cell line, orexin
appears to be a secretory protein in QM7 cells. In vitro administration of
recombit orexin A and B (rORX-A and B) differentially regulated prepro-orexin
expression in a dose-dependent manner with up-regulation for rORX-A (P < 0.05)
and downregulation for rORX-B (P < 0.05) in QM7 cells. While both peptides
upregulated ORXR1 expression, only a high dose of rORX-B decreased the
expression of ORXR2 (P < 0.05). The presence of orexin and its related receptors
and the regulation of its own system in avian muscle cells indicate that orexin
may have autocrine, paracrine, and/or endocrine roles. rORXs differentially
regulated mitochondrial dynamics network. While rORX-A significantly induced the
expression of mitochondrial fission-related genes (DNM1, MTFP1, MTFR1), rORX-B
increased the expression of mitofusin 2, OPA1, and OMA1 genes that are involved
in mitochondrial fusion. Concomitant with these changes, rORXs differentially
regulated the expression of several mitochondrial metabolic genes (av-UCP,
av-ANT, Ski, and NRF-1) and their related transcriptional regulators (PPARγ,
PPARα, PGC-1α, PGC-1β, and FoxO-1) without affecting ATP synthesis. Taken
together, our data represent the first evidence of the presence and secretion of
orexin system in the muscle of nonmammalian species and its role in
mitochondrial fusion and fission, probably through mitochondrial-related genes
and their related transcription factors. |
What is the mechanism of action of APOBEC3G cytidine deaminase to inhibit HIV-1 replication? | During reverse transcription, APOBEC3G deaminates dC to dU in nascent minus-strand viral DNA, resulting in G-to-A hypermutation in the plus strand DNA to inhibit replication of HIV-1, due to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. In an additional line of antiviral defense, APOBEC3G induces deamination of the apical loop cytidine of the trans-activation response (TAR) element, a short stem-loop RNA structure required for binding of elongation factors during HIV-1 transcription elongation, resulting in accumulation of short viral transcripts and production of lower amounts of full-length HIV-1 transcripts in Vif-deficient HIV-1-infected cells. | A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human
immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the
absence of the virally encoded Vif protein (Deltavif virions), at least in part
by causing G-to-A hypermutation. To gain insight into the antiretroviral
function of A3G, we determined the quantities of A3G molecules that are
incorporated in Deltavif virions. We combined three experimental
approaches-reversed-phase high-pressure liquid chromatography (HPLC),
scintillation proximity assay (SPA), and quantitative immunoblotting-to
determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions.
Our studies revealed that the amount of the A3G incorporated into Deltavif
virions was proportional to the level of its expression in the viral producing
cells, and the ratio of the A3G to Gag in the Deltavif virions produced from
activated human peripheral blood mononuclear cells (PBMC) was approximately
1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions
(1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are
incorporated into Deltavif virions produced from human PBMCs. These results
indicate that virion incorporation of only a few molecules of A3G is sufficient
to inhibit HIV-1 replication. The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral
replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins.
These proteins exert their suppressive effects through the inhibition of viral
reverse transcription as well as the induction of cytidine deamination within
nascent viral cDNA. Importantly, these two effects have not been characterized
in detail in human CD4(+) T cells, leading to controversies over their possible
contributions to viral inhibition in the natural cell targets of HIV-1
replication. Here we use wild-type and Vif-deficient viruses derived from the
CD4(+) T cells of multiple donors to examine the consequences of APOBEC3 protein
function at natural levels of expression. We demonstrate that APOBEC3 proteins
impart a profound deficiency to reverse transcription from the initial stages of
cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the
DNAs that are synthesized. Experiments using viruses from transfected cells and
a novel method for mapping the 3' termini of cDNAs indicate that the inhibition
of reverse transcription is not limited to a few specific sites, arguing that
APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation
spectra in viral cDNA strongly imply that one particular APOBEC3 protein,
APOBEC3G, provides the bulk of the antiviral phenotype in CD4(+) T cells, with
the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we
conclude that the dual mechanisms of action of APOBEC3 proteins combine to
deliver more effective restriction of HIV-1 than either function would by
itself. The APOBEC3 deoxycytidine deaminase family functions as host restriction factors
that can block replication of Vif (virus infectivity factor) deficient HIV-1
virions to differing degrees by deaminating cytosines to uracils in
single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the
HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby
inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both
APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are
suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in
contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F
and found that similar to APOBEC3G; it is a processive enzyme and can deaminate
at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F
scanning movement is distinct from APOBEC3G, and relies on jumping rather than
both jumping and sliding. APOBEC3F jumping movements were also different from
APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹²
motif, since insertion of this motif into APOBEC3G decreases its sliding
movements. The APOBEC3G NPM mutant induced significantly less mutations in
comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay
and single-cycle infectivity assay, indicating that differences in DNA scanning
were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F
¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F
¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type
APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates
the influence of sliding on mutagenesis. Overall, we demonstrate key differences
in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that
supports a model in which both the processive DNA scanning mechanism and
preferred deamination motif (APOBEC3F, 5'TTC; APOBEC3G 5'CCC) influences the
mutagenic and gene inactivation potential of an APOBEC3 enzyme. Deamination of cytidine residues in viral DNA is a major mechanism by which
APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1
(HIV-1) replication. dC-to-dU transition following RNase-H activity leads to
viral cDNA degradation, production of non-functional proteins, formation of
undesired stop codons and decreased viral protein synthesis. Here, we
demonstrate that A3G provides an additional layer of defense against HIV-1
infection dependent on inhibition of proviral transcription. HIV-1 transcription
elongation is regulated by the trans-activation response (TAR) element, a short
stem-loop RNA structure required for elongation factors binding. Vif-deficient
HIV-1-infected cells accumulate short viral transcripts and produce lower
amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR
apical loop cytidine, highlighting the requirement for TAR loop integrity in
HIV-1 transcription. We further show that free single-stranded DNA (ssDNA)
termini are not essential for A3G activity and a gap of CCC motif blocked with
juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination.
These results identify A3G as an efficient mutator and that deamination of
(-)SSDNA results in an early block of HIV-1 transcription. |
Which inherited disorder is known to be caused by mutations in the NEMO gene? | Incontinentia pigmenti (IP) is a rare neurocutaneous disorder with a frequency of 1 in 50,000 newborn, and is associated with mutations in IKBKG gene (NEMO) in Xq28, inherited as an X-linked dominant trait | Disruption of the X-linked gene encoding NF-kappa B essential modulator (NEMO)
produces male embryonic lethality, completely blocks NF-kappa B activation by
proinflammatory cytokines, and interferes with the generation and/or persistence
of lymphocytes. Heterozygous female mice develop patchy skin lesions with
massive granulocyte infiltration and hyperproliferation and increased apoptosis
of keratinocytes. Diseased animals present severe growth retardation and early
mortality. Surviving mice recover almost completely, presumably through clearing
the skin of NEMO-deficient keratinocytes. Male lethality and strikingly similar
skin lesions in heterozygous females are hallmarks of the human genetic disorder
incontinentia pigmenti (IP). Together with the recent discovery that mutations
in the human NEMO gene cause IP, our results indicate that we have created a
mouse model for that disease. Amorphic mutations in the NF- kappa B essential modulator (NEMO) cause
X-domit incontinentia pigmenti, which is lethal in males in utero, whereas
hypomorphic mutations cause X-recessive anhidrotic ectodermal dysplasia with
immunodeficiency, a complex developmental disorder and life-threatening primary
immunodeficiency. We characterized the NEMO mutation 110_111insC, which creates
the most-upstream premature translation termination codon (at codon position 49)
of any known NEMO mutation. Surprisingly, this mutation is associated with a
pure immunodeficiency. We solve this paradox by showing that a Kozakian
methionine codon located immediately downstream from the insertion allows the
reinitiation of translation. The residual production of an NH(2)-truncated NEMO
protein was sufficient for normal fetal development and for the subsequent
normal development of skin appendages but was insufficient for the development
of protective immune responses. Incontinentia pigmenti (IP) is a rare inherited multisystem disorder
characterized by a distinctive swirling pattern of the skin; defects of teeth,
hair, and nails; and ophthalmic, central nervous system, and musculoskeletal
abnormalities. It progresses through several well-defined stages. IP is
transmitted as a domit X-linked trait with variable expressivity, but
many--if not most--cases are sporadic. IP has been shown to result from
mutations in the NEMO gene that completely abolish expression of NF-kappaB
essential modulator. The diagnosis of IP typically is made based on
characteristic clinical findings. Molecular analysis of the NEMO gene is now
possible, as is analysis of skewed X-chromosome inactivation, which can further
reduce diagnostic confusion. A number of disorders, including hypomelanosis of
Ito, should be considered in the differential diagnosis. The considerations vary
according to the stage of IP. Careful head-to-toe clinical evaluation is
critical in the evaluation of a child with suspected IP given the frequent
multisystem involvement. A multidisciplinary approach including dermatology,
ophthalmology, neurology, and dental consults is typically warranted. The skin
manifestations of IP do not require specific treatment other than reassurance;
spontaneous resolution of the lesions usually occurs. Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells,
kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential
modulator (NEMO), gene are the most common single cause of incontinentia
pigmenti (IP) in females and anhydrotic ectodermal dysplasia with
immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28
chromosomal region, encodes for the regulatory subunit of the inhibitor of
kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB
pathway. Therefore, the remarkably heterogeneous and often severe clinical
presentation reported in IP is due to the pleiotropic role of this signaling
transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG
gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG
rearrangement found in IP females (which is lethal in males), a total of 69
different small mutations (missense, frameshift, nonsense, and splice-site
mutations) have been reported, including 13 novel ones in this work. The updated
distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene
highlights a secondary hotspot mutation in exon 10, which contains only 11% of
the protein. Furthermore, familial inheritance analysis revealed an unexpectedly
high incidence of sporadic cases (>65%). The sum of the observations can aid
both in determining the molecular basis of IP and EDA-ID allelic diseases, and
in genetic counseling in affected families. Incontinentia pigmenti (IP) is a rare, inherited, multisystem genodermatosis. It
is transmitted as an X-linked domit trait. The disorder is a consequence of
mutations in the NEMO gene (Xq28) that completely abolish expression of the
NF-kappaB essential modulator. Here we present a female infant of healthy
nonconsanguinous, young parents with a clinically evident first phase of IP. PCR
analysis of patient's peripheral blood lymphocytes DNA was done for detection of
NEMO delta4-10 deletion. Skin changes present at birth appertain to first
inflammatory stage. However, a pathohistological feature of the skin biopsy
showed second phase of disease. Genetic testing of diseased child revealed
delta4-10 in NEMO gene. However, the assumption that the female child has
familial IP was rejected as PCR performed on the mother's leukocytes did not
record the presence of the same mutation. Moreover, the existence of a healthy
male infant of the same mother as well as the lack of any phenotypic signs of
the disease in other family members additionally support that IP was not
inherited, but it was a consequence of de novo NEMO gene mutation. In
conclusion, here we describe a Croatian female with clinical IP phenotype having
de novo genomic rearrangements in the NEMO gene. Genetic conditions are increasingly recognised as a cause of multisystem
diseases in children. We report a 6-year-old boy with hypohidrotic ectodermal
dysplasia, immunodeficiency, osteopetrosis and lymphoedema, associated with a
novel mutation in the NF-κβ essential modulator (NEMO) gene. He is the longest
surviving of three reported boys with these clinical features. Hypohidrotic
ectodermal dysplasia, a congenital disorder of teeth, hair and eccrine sweat
glands is most commonly inherited as an X-linked recessive trait. Associated
immunodeficiency (HED-ID) may give rise to serious infections in early life.
Mutations in the NEMO gene give rise to a heterogeneous group of disorders,
including the X-linked domit disorder incontinentia pigmenti. This is
characterised by typical skin changes leading to linear pigmentary change and
variable associated features; in males, prenatal death usually occurs. Our
patient, like one if the previous cases and all of their mothers, demonstrates
features of incontinentia pigmenti. Incontinentia pigmenti (IP) is a rare neurocutaneous disorder with a frequency
of 1 in 50,000 newborn, and is associated with mutations in IKBKG gene (NEMO) in
Xq28, inherited as an X-linked domit trait. Clinical manifestations detected
since the newborn period are highly variable, with 3 well established sequential
or overlapped states and each with a characteristic differential diagnosis. With
PCR+RFLPs, we analyzed the IKBKG gene in 4 patients with different clinical
manifestations and characteristic skin biopsy. In all 4 patients the same
deletion of exons 4 to 10 was identified. In female patients in whom the
dermatological lesions lead to the suspicion of an IP diagnosis, it is important
to have the complete, multidisciplinary and molecular analysis of their first
level female relatives. This should give us a clear diagnosis, which is the
first step to complete genetic counselling. Mendelian susceptibility to mycobacterial disease (MSMD) is a rare syndrome
conferring predisposition to clinical disease caused by weakly virulent
mycobacteria, such as Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccines
and nontuberculous, environmental mycobacteria (EM). Since 1996, MSMD-causing
mutations have been found in six autosomal genes involved in IL-12/23-dependent,
IFN-γ-mediated immunity. The aim of this review is to provide the description of
the two described forms of X-linked recessive (XR) MSMD. Germline mutations in
two genes, NEMO and CYBB, have long been known to cause other human
diseases-incontinentia pigmenti (IP) and anhidrotic ectodermal dysplasia with
immunodeficiency (EDA-ID) (NEMO/IKKG), and X-linked chronic granulomatous
disease (CGD) (CYBB)-but specific mutations in either of these two genes have
recently been shown to cause XR-MSMD. NEMO is an essential component of several
NF-κB-dependent signaling pathways. The MSMD-causing mutations in NEMO
selectively affect the CD40-dependent induction of IL-12 in mononuclear cells.
CYBB encodes gp91(phox) , which is an essential component of the NADPH oxidase
in phagocytes. The MSMD-causing mutation in CYBB selectively affects the
respiratory burst in macrophages. Mutations in NEMO and CYBB may therefore cause
MSMD by selectively exerting their deleterious impact on a single signaling
pathway (CD40-IL-12, NEMO) or a single cell type (macrophages, CYBB). These
experiments of Nature illustrate how specific germline mutations in pleiotropic
genes can dissociate signaling pathways or cell lineages, thereby resulting in
surprisingly narrow clinical phenotypes. Inherited distubances in skin structure and its function are the main cause of
diseases classified as genodermatoses. The following clinical entities are
classified as genodermatoses: epidermolysis bullosa, keratotic disorders,
disorders of skin color, ectodermal genodermatoses, genodermatoses associated
with connective tissue, vascular genodermatoses and genodermatoses with skin
manifestation and elevated cancer risk. One of the most clinically heterogenous
group of genodermatoses, is epidermolysis bullosa. Four main subtypes were
described: simplex, dystrophic, junctional and Kindler syndrome. These diseases
are caused by mutations in the genes encoding proteins forming junctions between
the dermis and epidermis (eg. COL7A1, COL17A1, KRT14, KRT5 or genes coding for
332 laminin). They are inherited in an autosomal recessive or domit manner.
The disease that is inherited as a domit, sex dependent trait, is
incontinenia pigmenti (Bloch-Sulzberger syndrome) characterized by the presence
of extensive pigmentation changes already in the neonatal period. In patients
with incontinenia pigmenti, mutations in the NEMO gene are found. The protein
encoded by NEMO is involved in the negative regulation of activity of the NFκB
transcription factor that is responsible for apoptosis and cell proliferation
control. In the regulation of cell proliferation, the neurofibromin (NF1) - the
suppressor of RAS/MAPK signaling pathway activity, is also involved. The
mutations in the NF1 gene are identified in neurofibromatosis type I - a
genodermatosis with higher risk of cancer development and tumor formation.
Herein, a review of selected genodermatoses in the context of their molecular
pathology is presented. Osteopetrosis, lymphedema, hypohidrotic ectodermal dysplasia, and
immunodeficiency (OL-HED-ID) is a rare X-linked disorder with only three
reported prior cases in the English-language literature. We describe a case of
OL-HED-ID in a male infant who initially presented with congenital lymphedema,
leukocytosis, and thrombocytopenia of unknown etiology at 7 days of age. He
subsequently developed gram-negative sepsis and multiple opportunistic
infections including high-level cytomegalovirus viremia and Pneumocystis
jiroveci pneumonia. The infant was noted to have mildly xerotic skin, fine
sparse hair, and periorbital wrinkling, all features suggestive of ectodermal
dysplasia. Skeletal imaging showed findings consistent with osteopetrosis, and
immunologic investigation revealed hypogammaglobulinemia and mixed T- and B-cell
dysfunction. Genetic testing revealed a novel mutation in the nuclear factor
kappa beta (NF-KB) essential modulator (NEMO) gene, confirming the diagnosis of
OL-HED-ID. Mutations in the NEMO gene have been reported in association with
hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID), OL-HED-ID, and
incontinentia pigmenti. In this case, we report a novel mutation in the NEMO
gene associated with OL-HED-ID. This article highlights the dermatologic
manifestations of a rare disorder, OL-HED-ID, and underscores the importance of
early recognition and prompt intervention to prevent life-threatening
infections. Incontinentia pigmenti (IP) is an X-linked-domit Mendelian disorder caused by
mutation in the IKBKG/NEMO gene, encoding for NEMO/IKKgamma, a regulatory
protein of nuclear factor kappaB (NF-kB) signaling. In more than 80% of cases,
IP is due to recurrent or nonrecurrent deletions causing loss-of-function (LoF)
of NEMO/IKKgamma. We review how the local architecture of the IKBKG/NEMO locus
with segmental duplication and a high frequency of repetitive elements favor de
novo aberrant recombination through different mechanisms producing genomic
microdeletion. We report here a new microindel (c.436_471delinsT, p.Val146X)
arising through a DNA-replication-repair fork-stalling-and-template-switching
and microhomology-mediated-end-joining mechanism in a sporadic IP case. The LoF
mutations of IKBKG/NEMO leading to IP include small insertions/deletions (indel)
causing frameshift and premature stop codons, which account for 10% of cases. We
here present 21 point mutations previously unreported, which further extend the
spectrum of pathologic variants: 14/21 predict LoF because of premature stop
codon (6/14) or frameshift (8/14), whereas 7/21 predict a partial loss of
NEMO/IKKgamma activity (two splicing and five missense). We review how the
analysis of IP-associated IKBKG/NEMO hypomorphic mutants has contributed to the
understanding of the pathophysiological mechanism of IP disease and has provided
important information on affected NF-kB signaling. We built a locus-specific
database listing all IKBKG/NEMO variants, accessible at http://IKBKG.lovd.nl. |
What is ISMARA? | ISMARA (Integrated System for Motif Activity Response Analysis) is a web-based tool that models gene expression or chromatin modifications in terms of genome-wide predictions of regulatory sites. Given only gene expression or chromatin state data across a set of samples as input, ISMARA identifies the key TFs and miRNAs driving expression/chromatin changes and makes detailed predictions regarding their regulatory roles. These include predicted activities of the regulators across the samples, their genome-wide targets, enriched gene categories among the targets, and direct interactions between the regulators. | Accurate reconstruction of the regulatory networks that control gene expression
is one of the key current challenges in molecular biology. Although gene
expression and chromatin state dynamics are ultimately encoded by constellations
of binding sites recognized by regulators such as transcriptions factors (TFs)
and microRNAs (miRNAs), our understanding of this regulatory code and its
context-dependent read-out remains very limited. Given that there are thousands
of potential regulators in mammals, it is not practical to use direct
experimentation to identify which of these play a key role for a particular
system of interest. We developed a methodology that models gene expression or
chromatin modifications in terms of genome-wide predictions of regulatory sites
and completely automated it into a web-based tool called ISMARA (Integrated
System for Motif Activity Response Analysis). Given only gene expression or
chromatin state data across a set of samples as input, ISMARA identifies the key
TFs and miRNAs driving expression/chromatin changes and makes detailed
predictions regarding their regulatory roles. These include predicted activities
of the regulators across the samples, their genome-wide targets, enriched gene
categories among the targets, and direct interactions between the regulators.
Applying ISMARA to data sets from well-studied systems, we show that it
consistently identifies known key regulators ab initio. We also present a number
of novel predictions including regulatory interactions in innate immunity, a
master regulator of mucociliary differentiation, TFs consistently disregulated
in cancer, and TFs that mediate specific chromatin modifications. |
Is it possible to purify pseudopodia to be used for proteomic analysis? | Pseudopodia can be purified, using different strategies, in order to be used in proteomic studies. | Directional cell migration requires the formation of a domit pseudopodium in
the direction toward which the cell migrates. When a migratory cell is
stimulated with a chemoattractant or extracellular matrix (ECM) gradient, it
responds with localized amplification of signals on the side facing the
gradient. The signals mediate reorganization of the actin-myosin cytoskeleton,
leading to morphological polarization of the cell and pseudopodium extension. To
identify these signals, we developed an approach to biochemically isolate the
pseudopodium from the cell body using 3.0-micrometer porous filters for
large-scale quantitative proteomic and phosphoproteomic analysis. Here, we
detail the methodology for pseudopodium purification and proteomic analysis.
This model system should be widely applicable for the analysis of the
pseudopodium proteome from various migratory cell lines, including primary and
cancer cell lines stimulated with a diverse array of chemoattractants, ECM
proteins, or both. Cell migration requires actin/myosin-mediated membrane protrusion of a
pseudopodium (or lamellipodium) and its attachment to the substratum. This
process guides the direction of cell movement through cytoskeletal remodeling
and is regulated by complex signaling networks that act spatially downstream of
integrin adhesion receptors. Understanding how these regulatory networks are
organized in migratory cells is important for many physiological and
pathological processes, including wound healing, immune function, and cancer
metastasis. Here, we describe methods for the immunoaffinity purification of
phosphotyrosine proteins (pY) from pseudopodia that have been isolated from
migratory cells. These methods are compatible with current mass
spectrometry-based protein identification technologies and can be utilized for
the large-scale identification of the pseudopodium pY proteome in various
migratory cell lines, including primary and cancer cells. We developed a novel application to conduct pseudopodia proteomics. Pseudopodia
are ventral actin-rich protrusions and play functional roles in cell migrations.
Identification of pseudopodia proteins leads to a further understanding of
maligt phenotypes of tumor cells and novel therapeutic strategies. In our
application, tumor cells were placed on a fibronectin-coated porous membrane to
form pseudopodia. According to the motile potentials of the cells, the cells
formed pseudopodial microprocesses in the pores. An excimer laser, which was
used for ophthalmic refractive surgeries, horizontally ablated cells at the
membrane surface to remove the cell body. The microscopic observations and the
protein expression studies suggested that the laser treatment caused no apparent
damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were
individually solubilized, labeled with a highly sensitive fluorescent dye, and
separated using two-dimensional difference gel electrophoresis. Among 2508
protein spots observed, 211 had different intensity between whole cells and
pseudopodia fractions (more than fourfold differences and P-value of <0.05). The
protein enrichment depended on the pore size. Mass spectrometric protein
identification revealed 46 pseudopodia-localizing proteins. The localization of
novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin
was confirmed using immunohistochemical examinations. The previous studies
demonstrated that these four proteins may function in the cell migration
process. This method will provide insights into the molecular details of
pseudopodia and a further understanding of maligt phenotypes of tumor cells
and novel therapeutic strategies. Metastatic cancer cells produce invasive membrane protrusions called invadopodia
and pseudopodia, which play a central role in driving cancer cell dissemination
in the body. Maligt cells use these structures to attach to and degrade
extracellular matrix proteins, generate force for cell locomotion, and to
penetrate the vasculature. Recent work using unique subcellular fractionation
methodologies combined with spatial genomic, proteomic, and phosphoproteomic
profiling has provided insight into the invadopodiome and pseudopodiome
signaling networks that control the protrusion of invasive membranes. Here I
highlight how these powerful spatial 'omics' approaches reveal important
signatures of metastatic cancer cells and possible new therapeutic targets aimed
at treating metastatic disease. |
Where does TDP43 localize in ALS neurons? | In control motor neurons, TDP43 was almost exclusively nuclear, whereas in ALS spinal motor neurons, TDP43 was predominantly localized to the cytosol and not the nucleus. | The human TAR DNA-binding protein (TDP43) colocalizes with ubiquitinated
inclusions in motor neurons in amyotrophic lateral sclerosis (ALS). TDP43 is
both a DNA-binding protein with a nuclear export sequence that interacts with
(TG)nTm elements in DNA and an RNA-binding protein that interacts with
(UG)(6-12) motifs in single-stranded RNA. In control motor neurons, TDP43 was
almost exclusively nuclear, whereas in ALS spinal motor neurons, TDP43 was
predomitly localized to the cytosol and not the nucleus. TDP43 was observed
as punctuate immunoreactivity and as dense skeins, with and without
ubiquitinization. We observed that TDP43 stabilizes the human low molecular
weight (hNFL) mRNA through a direct interaction with the 3'UTR. TDP43 is a
unique hNFL mRNA-binding protein that is altered in its somatotopic localization
in ALS spinal motor neurons and potentially contributes to the formation of NF
aggregates in ALS through alterations in NF stoichiometry. |
List interaction partners for the protein GATA1. | GATA-1 interact with factor Gfi-1b, the repressive MeCP1 complex, the chromatin remodeling ACF/WCRF complex, FOG-1, TAL-1, Ldb-1 and LMO2-C. | The Ski oncoprotein dramatically affects cell growth, differentiation, and/or
survival. Recently, Ski was shown to act in distinct signaling pathways
including those involving nuclear receptors, transforming growth factor beta,
and tumor suppressors. These divergent roles of Ski are probably dependent on
Ski's capacity to bind multiple partners with disparate functions. In
particular, Ski alters the growth and differentiation program of erythroid
progenitor cells, leading to maligt leukemia. However, the mechanism
underlying this important effect has remained elusive. Here we show that Ski
interacts with GATA1, a transcription factor essential in erythropoiesis. Using
a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction
is critical for Ski's ability to repress GATA1-mediated transcription and block
erythroid differentiation. Furthermore, the repression of GATA1-mediated
transcription involves Ski's ability to block DNA binding of GATA1. This finding
is in marked contrast to those in previous reports on the mechanism of
repression by Ski, which have described a model involving the recruitment of
corepressors into DNA-bound transcription complexes. We propose that Ski
cooperates in the process of transformation in erythroid cells by interfering
with GATA1 function, thereby contributing to erythroleukemia. 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. Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a
number of tissues during development. It interacts with Gata1, Tal1, E2A and
Lmo2 to form a transcription factor complex regulating late erythroid genes. We
identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells,
in particular the repressor protein Eto-2 (and its family member Mtgr1), the
cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated
knockdowns in zebrafish show these factors to be essential for definitive
haematopoiesis. In accordance with the zebrafish results these factors are
coexpressed in prehaematopoietic cells of the early mouse embryo, although we
originally identified the complex in late erythroid cells. Based on the change
in subcellullar localisation of Eto-2 we postulate that it plays a central role
in the transition from the migration and expansion phase of the
prehaematopoietic cells to the establishment of definitive haematopoietic stem
cells. Protein Inhibitor of Activated Signal Transducer and Activators of Transcription
3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative
properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To
test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we
developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression
results in a distinct transcriptional profile that does not occur with STAT3
knockdown. We identify novel transcription factor binding partners for PIAS3
including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and
regulates their transcriptional effects resulting in alterations in canonical
pathways including Wnt/β-catenin signaling and functions such as cell death and
proliferation. A model is proposed by which PIAS3 effects EGR1 regulated
pathways. |
Is farnesoid X receptor (FXR) a nuclear receptor? | Yes, farnesoid X receptor (FXR) is a nuclear receptor. | Farnesoid X receptor (FXR) is a nuclear receptor that functions as a bile acid
sensor controlling bile acid homeostasis. We investigated the role of FXR in
regulating bone metabolism. We identified the expression of FXR in calvaria and
bone marrow cells, which gradually increased during osteoblastic differentiation
in vitro. In male mice, deletion of FXR (FXR(-/-) ) in vivo resulted in a
significant reduction in bone mineral density by 4.3% to 6.6% in mice 8 to 20
weeks of age compared with FXR(+/+) mice. Histological analysis of the lumbar
spine showed that FXR deficiency reduced the bone formation rate as well as the
trabecular bone volume and thickness. Moreover, tartrate-resistant acid
phosphatase (TRACP) staining of the femurs revealed that both the osteoclast
number and osteoclast surface were significantly increased in FXR(-/-) mice
compared with FXR(+/+) mice. At the cellular level, induction of alkaline
phosphatase (ALP) activities was blunted in primary calvarial cells in FXR(-/-)
mice compared with FXR(+/+) mice in concert with a significant reduction in type
I collagen a1(Col1a1), ALP, and runt-related transcription factor 2 (Runx2) gene
expressions. Cultures of bone marrow-derived macrophages from FXR(-/-) mice
exhibited an increased number of osteoclast formations and protein expression of
nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). In female FXR(-/-)
mice, although bone mineral density (BMD) was not significantly different from
that in FXR(+/+) mice, bone loss was accelerated after an ovariectomy compared
with FXR(+/+) mice. In vitro, activation of FXR by bile acids (chenodeoxycholic
acid [CDCA] or 6-ECDCA) or FXR agonists (GW4064 or Fexaramine) significantly
enhanced osteoblastic differentiation through the upregulation of Runx2 and
enhanced extracellular signal-regulated kinase (ERK) and β-catenin signaling.
FXR agonists also suppressed osteoclast differentiation from bone marrow
macrophages. Finally, administration of a farnesol (FOH 1%) diet marginally
prevented ovariectomy (OVX)-induced bone loss and enhanced bone mass gain in
growing C57BL/6J mice. Taken together, these results suggest that FXR positively
regulates bone metabolism through both arms of the bone remodeling pathways; ie,
bone formation and resorption. Liver X receptor-α (LXRα), a member of the nuclear receptor superfamily of
ligand-activated transcription factors, regulates de novo fatty acid synthesis
that leads to stimulate hepatic steatosis. Although, resveratrol has beneficial
effects on metabolic disease, it is not known whether resveratrol affects
LXRα-dependent lipogenic gene expression. This study investigated the effect of
resveratrol in LXRα-mediated lipogenesis and the underlying molecular mechanism.
Resveratrol inhibited the ability of LXRα to activate sterol regulatory element
binding protein-1c (SREBP-1c) and thereby inhibited target gene expression in
hepatocytes. Moreover, resveratrol decreased LXRα-RXRα DNA binding activity and
LXRE-luciferase transactivation. Resveratrol is known to activate Sirtuin 1
(Sirt1) and AMP-activated protein kinase (AMPK), although its precise mechanism
of action remains controversial. We found that the ability of resveratrol to
repress T0901317-induced SREBP-1c expression was not dependent on AMPK and
Sirt1. It is well established that hepatic steatosis is associated with
antioxidant and redox signaling. Our data showing that expression of Sestrin2
(Sesn2), which is a novel antioxidant gene, was significantly down-regulated in
the livers of high-fat diet-fed mice. Moreover, resveratrol up-regulated Sesn2
expression, but not Sesn1 and Sesn3. Sesn2 overexpression repressed
LXRα-activated SREBP-1c expression and LXRE-luciferase activity. Finally, Sesn2
knockdown using siRNA abolished the effect of resveratrol in LXRα-induced FAS
luciferase gene transactivation. We conclude that resveratrol affects Sesn2 gene
induction and contributes to the inhibition of LXRα-mediated hepatic
lipogenesis. The success of pharmacological treatments in primary liver cancers is limited by
the marked efficacy of mechanisms of chemoresistance already present in
hepatocytes. The role of the nuclear receptor FXR is unclear. Although, in
non-treated liver tumors, its expression is reduced, the refractoriness to
anticancer drugs is high. Moreover, the treatment with cisplatin up-regulates
FXR. The aim of this study was to investigate whether FXR is involved in
stimulating chemoprotection/chemoresistance in healthy and tumor liver cells. In
human hepatocytes, the activation of FXR with the agonist GW4064 resulted in a
significant protection against cisplatin-induced toxicity. In human hepatoma
Alexander cells, with negligible endogenous expression of FXR, GW4064 also
protected against cisplatin-induced toxicity, but only if they were previously
transfected with FXR/RXR. Investigation of 109 genes potentially involved in
chemoresistance revealed that only ABCB4, TCEA2, CCL14, CCL15 and KRT13 were
up-regulated by FXR activation both in human hepatocytes and FXR/RXR-expressing
hepatoma cells. In both models, cisplatin, even in the absence of FXR agonists,
such as bile acids and GW4064, was able to up-regulate FXR targets genes, which
was due to FXR-mediated trans-activation of response elements in the promoter
region. FXR-dependent chemoprotection was also efficient against other
DNA-damaging compounds, such as doxorubicin, mitomycin C and potassium
dichromate, but not against non-genotoxic drugs, such as colchicine, paclitaxel,
acetaminophen, artesunate and sorafenib. In conclusion, ligand-dependent and
independent activation of FXR stimulates mechanisms able to enhance the
chemoprotection of hepatocytes against genotoxic compounds and to reduce the
response of liver tumor cells to certain pharmacological treatments. Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol
and vitamin A (retinol) metabolism in the liver. Because previous studies have
shown that human ADH1 enzymes may participate in bile acid metabolism, we
investigated whether the bile acid-activated nuclear receptor farnesoid X
receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous
FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064
increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a
constitutively active form of FXR induced ADH1A and ADH1B expression, whereas
silencing of FXR abolished the effects of FXR agonists on ADH1 expression and
activity. Transient transfection studies and electrophoretic mobility shift
assays revealed functional FXR response elements in the ADH1A and ADH1B proximal
promoters, thus indicating that both genes are direct targets of FXR. These
findings provide the first evidence for direct connection of bile acid signaling
and alcohol metabolism. Bile acids are cholesterol metabolites that have been extensively studied in
recent decades. In addition to having ancestral roles in digestion and fat
solubilization, bile acids have recently been described as signaling molecules
involved in many physiological functions, such as glucose and energy
metabolisms. These signaling pathways involve the activation of the nuclear
receptor farnesoid X receptor (FXRα) or of the G protein-coupled receptor TGR5.
In this review, we will focus on the emerging role of FXRα, suggesting important
functions for the receptor in steroid metabolism. It has been described that
FXRα is expressed in the adrenal glands and testes, where it seems to control
steroid production. FXRα also participates in steroid catabolism in the liver
and interferes with the steroid signaling pathways in target tissues via
crosstalk with steroid receptors. In this review, we discuss the potential
impacts of bile acid (BA), through its interactions with steroid metabolism, on
glucose metabolism, sexual function, and prostate and breast cancers. Although
several of the published reports rely on in vitro studies, they highlight the
need to understand the interactions that may affect health. This effect is
important because BA levels are increased in several pathophysiological
conditions related to liver injuries. Additionally, BA receptors are targeted
clinically using therapeutics to treat liver diseases, diabetes, and cancers. Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are
nuclear receptors that are critical to liver homeostasis. Induction of SHP
serves as a major mechanism of FXR in suppressing gene expression. Both FXR(-/-)
and SHP(-/-) mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one
of the most strongly induced genes by FXR in the liver and is a tumor
suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC
development in the livers of FXR(-/-) mice and therefore, increased SHP
expression in FXR(-/-) mice reduces liver tumorigenesis. To test this
hypothesis, we generated FXR(-/-) mice with overexpression of SHP in hepatocytes
(FXR(-/-)/SHP(Tg)) and determined the contribution of SHP in HCC development in
FXR(-/-) mice. Hepatocyte-specific SHP overexpression did not affect liver tumor
incidence or size in FXR(-/-) mice. However, SHP overexpression led to a lower
grade of dysplasia, reduced indicator cell proliferation and increased
apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6
levels, which was associated with activation of hepatic STAT3. In conclusion,
SHP partially protects FXR(-/-) mice from HCC formation by reducing tumor
maligcy. However, disrupted bile acid homeostasis by FXR deficiency leads to
inflammation and injury, which ultimately results in uncontrolled cell
proliferation and tumorigenesis in the liver. The α-1-acid glycoprotein/orosomucoids (ORMs) are members of the lipocalin
protein family. Encoded by 3 polymorphic genes in mouse (2 in man, 1 in rat),
ORMs are expressed in hepatocytes and function as acute-phase proteins secreted
in plasma under stressful conditions. In addition to their role of ocarrier,
ORMs are involved in several pathophysiological processes such as
immunosuppression, cardioprotection, and inflammatory bowel disease. The nuclear
bile acid receptor farnesoid X receptor (FXR) regulates bile acid homeostasis
and lipid and glucose metabolism and is an important modulator of enterohepatic
functions. Here we report that hepatic FXR deletion in mice affects the
expression of several members of the lipocalin family, among which ORMs are
identified as direct FXR target genes. Indeed, a FXR response element upstream
of the mouse Orm1 promoter was identified to which hepatic, but not ileal, FXR
can bind and activate ORM expression in vitro and in vivo. However, ORMs are
regulated in a species-specific manner because the ORM cluster is regulated by
FXR neither in human nor rat cell lines. Consistent with these data, chromatin
immunoprecipitation sequencing analysis of the FXR genomic binding sites did not
detect any FXR response element in the vicinity of the human or rat ORM gene
cluster. Thus, bile acids and their cognate nuclear receptor, FXR, are
regulators of ORM expression, with potential implications for the
species-specific metabolic and inflammation control by FXR because the
expression of the proinflammatory genes in epididymal white adipose tissue was
dependent on liver FXR activation. Liver X receptors, LXRs, are ligand-activated transcription factors that belong
to the group H nuclear receptor (NR) superfamily. In this study, an LXR (HrLXR)
cDNA was cloned from the ascidian Halocynthia roretzi hepatopancreas and
characterized to examine the functional conservation of ancestral LXRs in
chordates. A phylogenetic analysis of HrLXR showed that it belongs to the
tunicate (urochordate) LXR subgroup, which is distinct from vertebrate LXRs.
Quantitative real-time PCR analysis revealed that HrLXR mRNA was expressed
predomitly in the gills, and highly expressed in unfertilized eggs followed
by decrease at later embryonic and larval stages. Unexpectedly, HrLXR was not
activated by GW3965, whereas a synthetic ligand for a farnesoid X receptor,
GW4064, activated HrLXR. This activation was abolished by the deletion of 51
amino acids from the N-terminus. In a mammalian two-hybrid system, HrLXR
interacted with HrRXR in the presence of GW4064 or 9-cis retinoic acid. The
injection of GW3965 and GW4064 in vivo increased the ATPbinding cassette
sub-family G member 4 and HrLXR mRNA levels in the hepatopancreas and gills.
These results suggest that the mRNA expression and transcriptional properties of
HrLXR are different from those of vertebrate LXRs, although HrLXR is likely
responsive to the related NR ligand, GW4064. Bile acid metabolism is intimately linked to the control of energy homeostasis
and glucose and lipid metabolism. The nuclear receptor farnesoid X receptor
(FXR) plays a major role in the enterohepatic cycling of bile acids, but the
impact of nutrients on bile acid homeostasis is poorly characterized.
Metabolically active hepatocytes cope with increases in intracellular glucose
concentrations by directing glucose into storage (glycogen) or oxidation
(glycolysis) pathways, as well as to the pentose phosphate shunt and the
hexosamine biosynthetic pathway. Here we studied whether the glucose
nonoxidative hexosamine biosynthetic pathway modulates FXR activity. Our results
show that FXR interacts with and is O-GlcNAcylated by O-GlcNAc transferase in
its N-terminal AF1 domain. Increased FXR O-GlcNAcylation enhances FXR gene
expression and protein stability in a cell type-specific manner. High glucose
concentrations increased FXR O-GlcNAcylation, hence its protein stability and
transcriptional activity by inactivating corepressor complexes, which associate
in a ligand-dependent manner with FXR, and increased FXR binding to chromatin.
Finally, in vivo fasting-refeeding experiments show that FXR undergoes
O-GlcNAcylation in fed conditions associated with increased direct FXR target
gene expression and decreased liver bile acid content.
CONCLUSION: FXR activity is regulated by glucose fluxes in hepatocytes through a
direct posttranslational modification catalyzed by the glucose-sensing
hexosamine biosynthetic pathway. Thrombomodulin (TM) serves as a vasoprotective molecule on the surface of
vascular endothelial cells (VECs) to maintain the endothelial microenvironment
by suppressing cellular proliferation, adhesion and inflammatory responses.
Farnesoid X receptor (FXR), a nuclear receptor (NR) and originally considered as
a bile acid-activated transcriptional factor, not only regulates metabolism
homeostasis, but also influences cholesterol transport, vascular tension, and
inflammation. Recent studies have shown that TM expression is upregulated by
several NRs. However, it is unknown whether there is a link between FXR and TM.
Our studies demonstrated that TM expression and activity were up-regulated by
FXR activation in VECs. Reporter assays showed that FXR activation significantly
enhanced the transcriptional activity of human TM gene promoter. Elecrophoretic
mobility-shift and chromatin immunoprecipitation assays indicated that FXR
induced TM expression by binding to a novel FXR-responsive element (FXRE), an
inverted repeat DNA motif, IR8 (-503 AGGTCCtcccaaagTGCCCT-484) in the promoter
region of TM gene. These results suggest that FXR may serve as a novel molecular
target for manipulating TM expression and activity in VECs, which may be helpful
for designing the therapeutic strategies to the treatment of associated diseases
by targeting FXR/TM pathway. The antioxidant tempol reduces obesity in mice. Here we show that tempol alters
the gut microbiome by preferentially reducing the genus Lactobacillus and its
bile salt hydrolase (BSH) activity leading to the accumulation of intestinal
tauro-β-muricholic acid (T-β-MCA). T-β-MCA is an farnesoid X receptor (FXR)
nuclear receptor antagonist, which is involved in the regulation of bile acid,
lipid and glucose metabolism. Its increased levels during tempol treatment
inhibit FXR signalling in the intestine. High-fat diet-fed intestine-specific
Fxr-null (Fxr(ΔIE)) mice show lower diet-induced obesity, similar to
tempol-treated wild-type mice. Further, tempol treatment does not decrease
weight gain in Fxr(ΔIE) mice, suggesting that the intestinal FXR mediates the
anti-obesity effects of tempol. These studies demonstrate a biochemical link
between the microbiome, nuclear receptor signalling and metabolic disorders, and
suggest that inhibition of FXR in the intestine could be a target for
anti-obesity drugs. Cholangiocytes, bile duct lining cells, actively adjust the amount of
cholesterol and bile acids in bile through expression of enzymes and channels
involved in transportation and metabolism of the cholesterol and bile acids.
Herein, we report molecular mechanisms regulating bile acid biosynthesis in
cholangiocytes. Among the cytochrome p450 (Cyp) enzymes involved in bile acid
biosynthesis, sterol 27-hydroxylase (Cyp27) that is the rate-limiting enzyme for
the acidic pathway of bile acid biosynthesis expressed in cholangiocytes.
Expression of other Cyp enzymes for the basic bile acid biosynthesis was hardly
detected. The Cyp27 expression was negatively regulated by a hydrophobic bile
acid through farnesoid X receptor (FXR), a nuclear receptor activated by bile
acid ligands. Activated FXR exerted the negative effects by inducing an
expression of fibroblast growth factor 15/19 (FGF15/19). Similar to its
repressive function against cholesterol 7α-hydroxylase (Cyp7a1) expression in
hepatocytes, secreted FGF15/19 triggered Cyp27 repression in cholangiocytes
through interaction with its cognate receptor fibroblast growth factor receptor
4 (FGFR4). The involvements of FXR and FGFR4 for the bile acid-induced Cyp27
repression were confirmed in vivo using knockout mouse models. Different from
the signaling in hepatocytes, wherein the FGF15/19-induced repression signaling
is mediated by c-Jun N-terminal kinase (JNK), FGF15/19-induced Cyp27 repression
in cholangiocytes was mediated by p38 kinase. Thus, the results collectively
suggest that cholangiocytes may be able to actively regulate bile acid
biosynthesis in cholangiocytes and even hepatocyte by secreting FGF15/19. We
suggest the presence of cholangiocyte-mediated intrahepatic feedback loop in
addition to the enterohepatic feedback loop against bile acid biosynthesis in
the liver. Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through
mouse knockout studies as a tumor suppressor for the development of colon
adenocarcinomas. This study investigates the regulation of FXR in the
development of human colon cancer. We used immunohistochemistry of FXR in normal
tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39,
68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and
Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation
and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA
methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite
sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)
knockdown assessment to investigate FXR regulation in colon cancer development.
Immunohistochemistry and quantitative RT-PCR revealed that expression and
function of FXR was reduced in precancerous lesions and silenced in a majority
of stage I-IV tumors. FXR expression negatively correlated with
phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the
epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12%
colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate
with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both
increased FXR expression. FXR expression is decreased early in human colon
cancer progression, and both DNA methylation and KRAS signaling may be
contributing factors to FXR silencing. FXR potentially suppresses
epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and
restoration of FXR activity, by blocking silencing mechanisms or increasing
residual FXR activity, represents promising therapeutic options for the
treatment of colon cancer. INTRODUCTION: Farnesoid X receptor (FXR) is an ascending target for metabolic
and inflammatory diseases. As a nuclear receptor, FXR exhibits many
physiological effects in transcription control of several genes. Therefore, the
development of synthetic FXR ligands requires elaborate in vitro test systems to
characterize novel ligands and to estimate their in vivo activities.
AREAS COVERED: This work gathers and describes published in vitro test systems
for FXR ligands including cell-based functional assays as well as binding
assays. It also evaluates the information which can be provided by these assays.
EXPERT OPINION: In vitro screening of FXR ligands widely relies on reporter gene
assays. Additionally, some co-activator re-cruitment assays are described and
for the characterization of potent compounds the pattern of affected target
genes is evaluated by qPCR. Compared to other nuclear receptors such as PPARs
the variety of test systems is quite low for FXR and might eventually not be
enough to sufficiently characterize FXR targeting drug candidates. Farnesoid X receptor (FXR) belongs to the ligand-activated nuclear receptor
superfamily, and functions as a transcription factor regulating the
transcription of numerous genes involved in bile acid homeostasis, lipoprotein
and glucose metabolism. In the present study, we identified RECK, a
membrane-anchored inhibitor of matrix metalloproteinases, as a novel target gene
of FXR in mouse liver. We found that FXR agonist substantially augmented hepatic
RECK mRNA and protein expression in vivo and in vitro. FXR regulated the
transcription of RECK through directly binding to FXR response element located
within intron 1 of the mouse RECK gene. Moreover, FXR agonist reversed the
down-regulation of RECK in the livers from mice fed a methionine and choline
deficient diet. In summary, our data suggest that RECK is a novel
transcriptional target of FXR in mouse liver, and provide clues to better
understanding the function of FXR in liver. |
What histone variants play a role in the DNA damage reponse? | Mostly H2A.X, but H2A.Z and H1R have also been associated to DNA damage | The response of eukaryotic cells to the formation of a double-strand break (DSB)
in chromosomal DNA is highly conserved. One of the earliest responses to DSB
formation is phosphorylation of the C-terminal tail of H2A histones located in
nucleosomes near the break. Histone variant H2AX and core histone H2A are
phosphorylated in mammals and budding yeast, respectively. We demonstrate the
DSB-induced phosphorylation of histone variant H2Av in Drosophila melanogaster.
H2Av is a member of the H2AZ family of histone variants. Ser137 within an SQ
motif located near the C- terminus of H2Av was phosphorylated in response to
gamma-irradiation in both tissue culture cells and larvae. Phosphorylation was
detected within 1 min of irradiation and detectable after only 0.3 Gy of
radiation exposure. Photochemically induced DSBs, but not general oxidative
damage or UV-induced nicking of DNA, caused H2Av phosphorylation, suggesting
that phosphorylation is DSB specific. Imaginal disc cells from Drosophila
expressing a mutant allele of H2Av with its C-terminal tail deleted, and
therefore unable to be phosphorylated, were more sensitive to radiation-induced
apoptosis than were wildtype controls, suggesting that phosphorylation of H2Av
is important for repair of radiation-induced DSBs. These observations suggest
that in addition to providing the function of an H2AZ histone, H2Av is also the
functional homolog in Drosophila of H2AX. Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes that have been
implicated in DNA repair. Here we show that Ino80 is required for cell cycle
checkpoint adaptation in response to a persistent DNA double-strand break (DSB).
The failure of cells lacking Ino80 to escape checkpoint arrest correlates with
an inability to maintain high levels of histone H2AX phosphorylation and an
increased incorporation of the Htz1p histone variant into chromatin surrounding
the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p
incorporation and restores H2AX phosphorylation and checkpoint adaptation. We
propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a
DSB, and that they regulate the incorporation of different histone H2A variants
that can either promote or block cell cycle checkpoint adaptation. In Saccharomyces cerevisiae, the linker histone HHO1 is involved in DNA repair.
In higher eukaryotes, multiple variants of linker histone H1 exist but their
involvement in the DNA damage response is unknown. To address this issue, we
examined sensitivity to genotoxic agents in chicken DT40 cells lacking specific
H1 variants. Among the six H1 variant mutants, only H1R(-/-) DT40 cells
exhibited increased sensitivity to the alkylating agent methyl-methanesulfonate
(MMS). The MMS sensitivity of H1R(-/-) cells was not enhanced by inactivation of
Rad54. H1R(-/-) DT40 cells also exhibited: (i) a reduction in gene targeting
efficiencies, (ii) impaired sister chromatid exchange, and (iii) an accumulation
of IR-induced chromosomal aberrations at the G2 phase, all of which indicate the
involvement of H1R in the Rad54-mediated homologous recombination (HR) pathway.
The mobility of H1R but not H1L in the nucleus decreased after MMS treatment and
the repair of double-stranded breaks generated by I-SceI was unaffected in
H1R(-/-) cells, suggesting that H1R integrates into HR-mediated repair pathways
at the chromosome structure level. Together, these findings provide the first
genetic evidence that a specific H1 variant plays a unique and important role in
the DNA damage response in vertebrates. The histone protein family member X (H2AFX) is important in maintaining
chromatin structure and genetic stability. Genetic variants in H2AFX may alter
protein functions and thus cancer risk. In this case-control study, we genotyped
four common single nucleotide polymorphisms (i.e., -1654A > G [rs643788], -1420G
> A [rs8551], and -1187T > C [rs7759] in the H2AFX promoter region and 1057C > T
[rs7350] in the 3' untranslated region (UTR)) in 467 patients with sporadic
breast cancer and 488 cancer-free controls. All female subjects were
non-Hispanic whites aged <or=55 years. We found that significantly increased
risk of breast cancer was associated with variant genotypes in the H2AFX
promoter: adjusted odds ratio [OR] = 1.80, 95% confidence interval [CI] =
1.38-2.34 for -1654AG/GG; OR = 1.40, 95% CI = 1.07-1.83 for -1420GA/AA; and OR =
1.65, 95% CI = 1.26-2.16 for -1187TC/CC. Furthermore, the number of variant
alleles in the promoter haplotypes was associated with increased risks of breast
cancer in a dose-response manner (OR = 6.08, 95% CI = 3.25-11.38; OR = 6.83, 95%
CI = 3.83-12.18; and OR = 23.61, 95% CI = 3.95-140.99 for one, two, and three
variant alleles, respectively) (P (trend) \ < 0.0001). Age at onset of breast
cancer significantly decreased as the number of variant alleles increased (P
(trend) = 0.024). However, these effects were not observed in the 3'UTR 1057C >
T polymorphism. Therefore, we believe that H2AFX promoter polymorphisms may
contribute to the etiology of sporadic breast cancer in young non-Hispanic white
women. Larger association studies and related functional studies are warranted
to confirm these findings. Chromatin structure plays a key role in most processes involving DNA metabolism.
Chromatin modifications implicated in transcriptional regulation are relatively
well characterized and are thought to be the result of a code on the histone
proteins (histone code). This code, involving phosphorylation, ubiquitylation,
sumoylation, acetylation and methylation, is believed to regulate chromatin
accessibility either by disrupting chromatin contacts or by recruiting
non-histone proteins to chromatin. Recent evidences suggest that such mechanisms
are also involved in DNA damage detection and DNA repair. One of the most
well-characterized modifications is caused by the formation of DNA double strand
breaks (DSBs), resulting in phosphorylation of histone H2AX (the so-called
gamma-H2AX) on the chromatin surrounding the DNA lesion. It is generally
believed that histone H2AX phosphorylation is required for the concentration and
stabilization of DNA repair proteins to the damaged chromatin. The
phosphorylation of this histone seems to play a role in both non-homologous
end-joining (NHEJ) and homologous recombination (HR) repair pathways. However,
the choice of the repair pathway might depend on or induce additional
post-translational modifications affecting other histone proteins necessary to
the completion of the entire DNA repair process. Interestingly, even in the
absence of DSBs, histone modifications occur. Indeed, following UV-exposure,
histone acetylation takes place and is believed to facilitate the nucleotide
excision repair (NER) process by promoting chromatin accessibility to the repair
factors. This review focuses on recent data characterizing the function of
histone modification in various repair processes and discusses if the
combination of such modifications can be the trademark of a specific DNA repair
pathway. Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is
incurable because of its ability to differentiate from the rapidly replicating
tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation
pertinent to Toxoplasma differentiation involves histone modification, but very
little is known about the histone proteins in this early branching eukaryote.
Here, we report the characterization of three H2A histones, variants H2AX and
H2AZ, and a canonical H2A1. H2AZ is the minor parasite H2A member. H2A1 and H2AX
both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (where varphi
denotes a hydrophobic residue) known to be phosphorylated in response to DNA
damage. We show that a novel H2B variant interacts with H2AZ and H2A1 but not
with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are
enriched at active genes while H2AX is enriched at repressed genes as well as
the silent TgIRE repeat element. During DNA damage, we detected an increase in
H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We
found that expression of h2ax, but not h2a1 or h2az, increases in bradyzoites
generated in vitro. Similar analysis performed on mature bradyzoites generated
in vivo, which are arrested in G0, showed that h2az and h2ax are expressed but
h2a1 is not, consistent with the idea that h2a1 is the canonical histone
orthologue in the parasite. The increase of H2AX, which localizes to silenced
areas during bradyzoite differentiation, is consistent with the quiescent nature
of this stage of the life cycle. Our results indicate that the early-branching
eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which
is likely to impact multiple facets of parasite biology, including the
clinically important process of bradyzoite differentiation. The fact that eukaryotic DNA is packed into chromatin constitutes a physical
barrier to enzymes and regulatory factors to reach the DNA molecule for
replication, transcription, recombination and repair. Although most studies in
this field have concentrated on how chromatin regulates transcription, there is
a recent emphasis on studying the role of chromatin in the response to DNA
damage. Two main chromatin-remodeling mechanisms have been identified, namely,
ATP-dependent chromatin-remodeling complexes and histone post-translational
modifications (PTMs). PTMs constitute reversible covalent modifications in
aminoacidic residues, such as serine and threonine phosphorylation, lysine
acetylation, lysine and arginine methylation and lysine ubiquitylation, among
others. Moreover, nucleosome composition can be modified by the incorporation of
histone variants, which are assembled into nucleosomes independently of DNA
replication. The phosphorylation of the histone variant H2AX (gammaH2AX) is one
of the best examples of histone PTMs in response to DNA damage induction, but
many others have recently been revealed. In this review, we focus on and
summarize the best-known histone PTMs observed in excision repair (base excision
and nucleotide excision) and double-strand break (non-homologous end-joining and
homologous recombination) repair pathways. In brief, the interplay between
chromatin remodelers and DNA repair factors is discussed in relation to DNA
damage response mechanisms. DNA damage activates signaling pathways that lead to modification of local
chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins
having ubiquitin ligase activity are recruited to sites of DNA damage, where
they ubiquitinate histones and other substrates. This DNA damage-induced histone
ubiquitination is thought to play a critical role in mediating the DNA damage
response. We now report that the polycomb protein BMI1 is rapidly recruited to
sites of DNA damage, where it persists for more than 8 h. The sustained
localization of BMI1 to damage sites is dependent on intact ATM and ATR and
requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA
damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads
to impaired repair of DNA double-strand breaks by homologous recombination and
the accumulation of cells in G(2)/M. These data support a crucial role for BMI1
in the cellular response to DNA damage. DNA repair protects neurons against spontaneous or disease-associated DNA
damage. Dysfunctions of this mechanism underlie a growing list of
neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes
the loss of nna1 expression and is associated with the postnatal degeneration of
PCs. This PC degeneration dramatically affects nuclear architecture and provides
an excellent model to elucidate the nuclear mechanisms involved in a whole array
of neurodegenerative disorders. We used immunocytochemistry for histone variants
and components of the DNA damage response, an in situ transcription assay, and
in situ hybridization for telomeres to analyze changes in chromatin architecture
and function. We demonstrate that the phosphorylation of H2AX, a DNA damage
signal, and the trimethylation of the histone H4K20, a repressive mark, in
extensive domains of genome are epigenetic hallmarks of chromatin in
degenerating PCs. These histone modifications are associated with a large scale
reorganization of chromatin, telomere clustering, and heterochromatin-induced
gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia
telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail
to be concentrated in the damaged chromatin compartments, even though the
expression levels of their coding genes were slightly up-regulated. Although the
mechanism by which Nna1 loss of function leads to PC neurodegeneration is
undefined, the progressive accumulation of DNA damage in chromosome territories
irreversibly compromises global gene transcription and seems to trigger PC
degeneration and death. Epigenetic regulation consists of a multitude of different modifications that
determine active and inactive states of chromatin. Conditions such as cell
differentiation or exposure to environmental stress require concerted changes in
gene expression. To interpret epigenomics data, a spectrum of different
interconnected datasets is needed, ranging from the genome sequence and
positions of histones, together with their modifications and variants, to the
transcriptional output of genomic regions. Here we present a tool, Podbat
(Positioning database and analysis tool), that incorporates data from various
sources and allows detailed dissection of the entire range of chromatin
modifications simultaneously. Podbat can be used to analyze, visualize, store
and share epigenomics data. Among other functions, Podbat allows data-driven
determination of genome regions of differential protein occupancy or RNA
expression using Hidden Markov Models. Comparisons between datasets are
facilitated to enable the study of the comprehensive chromatin modification
system simultaneously, irrespective of data-generating technique. Any organism
with a sequenced genome can be accommodated. We exemplify the power of Podbat by
reanalyzing all to-date published genome-wide data for the histone variant H2A.Z
in fission yeast together with other histone marks and also phenotypic response
data from several sources. This meta-analysis led to the unexpected finding of
H2A.Z incorporation in the coding regions of genes encoding proteins involved in
the regulation of meiosis and genotoxic stress responses. This incorporation was
partly independent of the H2A.Z-incorporating remodeller Swr1. We verified an
Swr1-independent role for H2A.Z following genotoxic stress in vivo. Podbat is
open source software freely downloadable from www.podbat.org, distributed under
the GNU LGPL license. User manuals, test data and instructions are available at
the website, as well as a repository for third party-developed plug-in modules.
Podbat requires Java version 1.6 or higher. Increased oxidative stress has been linked to thyroid carcinogenesis. In this
paper, we investigate whether oxidative DNA damage and DNA repair differ in
follicular adenoma (FA) and follicular thyroid carcinoma (FTC).
7,8-Dihydro-8-oxoguanine (8-OxoG) formation was analysed by immunohistochemistry
in 46 FAs, 52 FTCs and 18 normal thyroid tissues (NTs). mRNA expression of DNA
repair genes OGG1, Mut Y homologue (MUTYH) and endonuclease III (NTHL1) was
analysed by real-time PCR in 19 FAs, 25 FTCs and 19 NTs. Induction and repair of
oxidative DNA damage were studied in rat FRTL-5 cells after u.v. irradiation.
Moreover, activation of DNA damage checkpoints (ataxia telangiectasia mutated
(ATM) and H2A histone family, member X (H2AFX (H2AFX))) and proliferation index
(MIB-1) were quantified in 28 non-oxyphilic and 24 oxyphilic FTCs. Increased
nuclear and cytosolic 8-OxoG formation was detected in FTC compared with
follicular adenoma, whereby cytosolic 8-OxoG formation was found to reflect RNA
oxidation. Significant downregulation of DNA repair enzymes was detected in FTC
compared with FA. In vitro experiments mirrored the findings in FTC with
oxidative stress-induced DNA checkpoint activation and downregulation of OGG1,
MUTYH and NTHL1 in FRTL-5 cells, an effect that, however, was reversible after
24 h. Further analysis of FTC variants showed decreased oxidative DNA damage,
sustained checkpoint activation and decreased proliferation in oxyphilic vs
non-oxyphilic FTC. Our data suggest a pathophysiological scenario of
accumulating unrepaired DNA/RNA damage in FTC vs counterbalanced DNA/RNA damage
and repair in FA. Furthermore, this study provides the first evidence for
differences in oxidative stress defence in FTC variants with possible
implications for therapeutic response and prognostic outcome. Histone H2A variants generate diversity in chromatin structure and functions, as
nucleosomes containing variant H2A histones have altered physical, chemical, and
biological properties. H2A.Z is an evolutionarily ancient and highly conserved
H2A variant that regulates processes ranging from gene expression to the DNA
damage response. Here we find that the unstructured portion of the C-terminal
tail of H2A.Z is required for the normal functions of this histone variant in
budding yeast. We have also identified a novel splice isoform of the human
H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar
to the truncation mutants we identified in yeast. The short forms of H2A.Z in
both yeast and human cells are more loosely associated with chromatin than the
full-length proteins, indicating a conserved function for the H2A.Z C-terminal
tail in regulating the association of H2A.Z with nucleosomes. To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA
synthesis, cells either undergo DNA translesion synthesis (TLS) by specialised
DNA polymerases or tolerate the lesions using homologous recombination
(HR)-based mechanisms. To gain insight into how cells manage UVC-induced stalled
replication forks, we analysed the molecular crosstalk between the TLS DNA
polymerases Polη and Rev1, the double-strand break repair (DSB)-associated
protein MDC1 and the FANC pathway. We describe three novel functional
interactions that occur in response to UVC-induced DNA lesions. First, Polη and
Rev1, whose optimal expression and/or relocalisation depend on the FANC core
complex, act upstream of FANCD2 and are required for the proper relocalisation
of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second, during
S-phase, Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci
simultaneously but independently of each other. Third, Ub-FANCD2 and MDC1 are
independently required for optimal BRCA1 relocalisation. While RPA32
phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with
increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC
pathway-depleted cells, MDC1 depletion was associated with increased UVC-induced
Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the
basis of the previous observations, we propose that the FANC pathway
participates in the rescue of UVC-stalled replication forks in association with
TLS by maintaining the integrity of ssDNA regions and by preserving genome
stability and preventing the formation of DSBs, the resolution of which would
require the intervention of MDC1. Many tumor suppressors play an important role in the DNA damage pathway. Zinc
finger protein 668 (ZNF668) has recently been identified as one of the potential
tumor suppressors in breast cancer, but its function in DNA damage response is
unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668
knockdown impairs cell survival after DNA damage without affecting the ATM/ATR
DNA-damage signaling cascade. However, recruitment of repair proteins to DNA
lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX
interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing
chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment
of repair proteins to DNA lesions, defective homologous recombination (HR)
repair and impaired cell survival after IR. In addition, ZNF668 knockdown
decreased RPA phosphorylation and its recruitment to DNA damage foci in response
to UV. In both IR and UV damage responses, chromatin relaxation counteracted the
impaired loading of repair proteins and DNA repair defects in ZNF668-deficient
U2OS cells, indicating that impeded chromatin accessibility at sites of DNA
breaks caused the DNA repair defects observed in the absence of ZNF668. Our
findings suggest that ZNF668 is a key molecule that links chromatin relaxation
with DNA damage response in DNA repair control. |
List tele monitoring applications of miniaturised sensors | Home-polysomnography (HPSG)
Body weight
Blood pressure control
Heart failure control
Vital signs - disaster relief, dangerous outdoor sports and adventure monitoring, and antiterrorism activities.
Telemetric fetal home monitoring system for recording the trans-abdominal fetal heart signal and the uterine contractions
Vital signs - electrocardiograms (ECGs), temperature (T), and oxygen saturation (SaO2) , breath rate
Step-counting for tele-rehabilitation
Detection of falls in elderly | Fall detection of the elderly is a major public health problem. Thus it has
generated a wide range of applied research and prompted the development of
telemonitoring systems to enable the early diagnosis of fall conditions. This
article is a survey of systems, algorithms and sensors, for the automatic early
detection of the fall of elderly persons. It points out the difficulty to
compare the performances of the different systems due to the lack of a common
framework. It then proposes a procedure for this evaluation. Technological advances in signal processing and in circuits integration offer
numerous perspectives in telemedicine and telemonitoring. Considering the
increase of life expectancy, accurate and reliable assessement of modification
and/or deterioration in the health status of a person is needed. One possible
indicator is the "activity index" of a person. To compute such an index,
previous studies have used accelerometers. Although these sensors are
appropriate for the detection of postural transitions (e.g. Sit To Stand and
Stand To Sit), they do not allow to detect changes of direction of a walking
individual insofar as such activity occurs in a constant gravitation field.
Within this context, the purpose of the present work is to investigate whether
magnetometers can be used to monitor the changes of direction of a person. Remote therapy, involved in a tele-rehabilitation program, should monitor daily
motion activity. Furthermore, patients should have all possible means to improve
rehabilitation care at their disposal, such as suitable prostheses and
biofeedback tools. During stroke rehabilitation at home, the most used equipment
is the AFO prosthesis, which is also called codivilla-spring, as it is used in
cases of both bilateral and mono-lateral damage. A new codivilla-spring
prosthesis with sensors for telemonitoring / telerehabilitation has been
designed and constructed. It provides step counting. It is based on a couple of
force sensing resistors affixed in the plantar area of the prosthesis, and a
wearable unit with a micro-processor microP PIC 16F877 (Microchip, USA). The
data exchange is assured by the Aurel-XTR-434H telemetric system. While
ambulating, FSRs detect the pressure of the foot-tip and heel. The PIC on the
basis of an algorithm furnishes the step-counting. The proposed
sensorized-codivilla-spring (SECOSP) was tested as a step-counter on three
subjects after stroke-rehabilitation with mono-lateral damage at Level 2 of the
Tinetti test of unbalance. These subjects performed 100 steps with two different
instructions (fast, slow). The mean error was lower than 0.6 %. The next phase
will be the optimization of SECOSP for long term medical application in a
patient's home. BACKGROUND: Hypertension is a major risk factor for the long-term complications
of diabetes. Mobile, self-measurement of blood pressure is emerging as a method
to manage blood pressure in general, but its impact in patients with diabetes is
unclear.
METHODS: We randomized 137 patients with diabetes and hypertension to either
mobile telemonitoring (n = 72) or usual care (n = 65). Clinic blood pressure was
recorded at baseline and after 6 months. Patients in the intervention arm
transmitted weekly blood pressure readings wirelessly, using adapted sensors via
mobile phones to a central server. Clinicians received the data in real-time and
using a web-based application provided management advice to the patient and
their physicians.
RESULTS: Systolic blood pressure fell significantly in the patients in the
intervention group (mean [95% confidence interval], -6.5 [-0.8 to -12.2] mm Hg;
P = 0.027) and remained unchanged in the control group (2.1 [9.3 to -5.0] mm Hg;
P = 0.57). Patients within the intervention arm of African origin seemed to
benefit more from the intervention. In addition, those who achieved a systolic
blood pressure of <120 mm Hg had lower average blood sugars than those with
higher readings (7.8 [SD 1.6] vs. 8.9 [SD 2.2] mmol/L; P = 0.02).
CONCLUSIONS: In patients with diabetes, mobile telemonitoring has potential for
delivering intensified care to improve blood pressure control, and its use may
be associated with reduced exposure to hyperglycemia. Recent developments in communications technologies and associated computing and
digital electronics now permit patient data, including routine vital signs, to
be surveyed at a distance. Remote monitoring, or telemonitoring, can be regarded
as a subdivision of telemedicine - the use of electronic and telecommunications
technologies to provide and support health care when distance separates the
participants. Depending on environment and purpose, the patient and the
carer/system surveying, analysing or interpreting the data could be separated by
as little as a few feet or be on different continents. Most telemonitoring
systems will incorporate five components: data acquisition using an appropriate
sensor; transmission of data from patient to clinician; integration of data with
other data describing the state of the patient; synthesis of an appropriate
action, or response or escalation in the care of the patient, and associated
decision support; and storage of data. Telemonitoring is currently being used in
community-based healthcare, at the scene of medical emergencies, by ambulance
services and in hospitals. Current challenges in telemonitoring include: the
lack of a full range of appropriate sensors, the bulk weight and size of the
whole system or its components, battery life, available bandwidth, network
coverage, and the costs of data transmission via public networks. Telemonitoring
also has the ability to produce a mass of data - but this requires
interpretation to be of clinical use and much necessary research work remains to
be done. The purpose of this article is to describe a new, phonocardiography-based fetal
telemonitoring system, which, due to its passive nature, allows long-term
measurements even at the home of the pregt woman. The input element of the
system was the home monitor with two sensors for recording the trans-abdominal
fetal heart signal and the uterine contractions. The recorded signal was
transmitted by mobile network and Internet to an Evaluation Center, where it was
analyzed in detail to obtain information about possible dysfunction of the fetal
heart. The investigations on this system made clear that by advanced processing
of the recorded signal the system captured many additional cardiac features
compared with the traditional ultrasound-based cardiotocographic procedure. This article presents the design and applications of a rescue terminal with
positioning, vital signs sensing, and communicating function for special
environment. The terminal provides three-dimensional positioning functionality
via China's Beidou 1 Navigation Satellite (BD1) System and can collect users'
vital signs with a set of wireless sensors. A controller of the terminal is in
charge of processing data collected from the wireless sensors and communicating
with the monitoring platform. With features such as small sizing, low power
consumption, and accurate positioning, this terminal is very helpful in special
circumstances such as disaster relief, dangerous outdoor sports and adventure
monitoring, and antiterrorism activities. AIM: The rapidly increasing prevalence and poor outcome of congestive heart
failure have stimulated the development of different telemonitoring
technologies. In this study, we monitored remotely self-measured body weight and
blood pressure, in parallel with the data automatically transmitted by
implantable cardioverter-defibrillators. The primary aim of this study was to
evaluate the correlation between different parameters screened by these two
telemonitoring systems.
METHODS AND RESULTS: Thirty-two patients in NYHA class III heart failure were
followed for 164 ± 48 days after cardioverter-defibrillator implantation. In 29
patients, mean heart rate (MHR), resting heart rate (RHR), and patient activity
(determined by implanted devices), weight and blood pressure measurements were
received on 85% of all days when remote data transmissions were expected. Based
on approximately 4,000 daily pairs of measurements pooled for all patients,
weight inversely correlated with activity and with the difference between MHR
and RHR. By contrast, blood pressure did not correlate with weight, activity, or
the difference between MHR and RHR, but it correlated with MHR and RHR
individually.
CONCLUSION: Body weight, patient activity, and the difference between MHR and
RHR are mutually correlated and may reasonably contribute to an algorithm for
predicting heart failure deterioration. Blood pressure appears to offer no
additional value. As both genesis and symptoms of heart failure exacerbation are
non-uniform and complex, the telemonitoring concepts for heart failure patients
should employ continuous monitoring of multiple diagnostic parameters, rather
than rely on a single parameter. Provided that patient compliance is strictly
supervised, reliable data flow from sensors requiring patient involvement is
possible. PURPOSE: Home-polysomnography (HPSG) has been proposed as a cost-effective
alternative for obstructive sleep apnea (OSA) diagnosis. We assessed, in a
feasibility study, whether telematic transmission using the Dream® and Sleepbox®
technologies was associated with low HPSG failure rate.
METHODS: Patients referred by chest physicians for clinical suspicion of OSA
underwent one HPSG, using Dream® and Sleepbox® (Medatec, Belgium), which is a
wireless system able to communicate with Dream®, and with Internet through a
wi-fi/3G interface. It is equipped with a digital infrared camera, and with a
speaker/microphone system for bidirectional audio/video communication via
Skype®. The Sleep Lab nurse performed a remote discontinuous monitoring of the
PSG. In case of sensor loss, she called the patient who had been previously
educated to replace the sensors.
RESULTS: Twenty-one patients have been studied. 90% of the recordings were of
excellent quality. We observed a 10% PSG failure rate: one failure of the
Dream®, and one recording of poor quality. There were 2 successful Skype®
interventions resulting in readjustment of the defective probes (nasal cannula
and EEG). PSG signal visualization was possible in 90% of cases but Skype®
connection was problematic in 19% of cases. However, patients could be reached
by phone to solve the problem.
CONCLUSIONS: Real-time attended HPSG through telematic data transmission is
feasible and could be an interesting perspective to decrease the failure rate of
home sleep studies, even if some technical aspects need to be improved. |
How are ultraconserved elements called when they form clusters? | Ultraconserved non-coding elements (UCNEs) are organized as large clusters, so-called gene regulatory blocks (GRBs) around key developmental genes. Their molecular functions and the reasons for their high degree of conservation remain enigmatic. | MOTIVATION: Genomic context analysis, also known as phylogenetic profiling, is
widely used to infer functional interactions between proteins but rarely applied
to non-coding cis-regulatory DNA elements. We were wondering whether this
approach could provide insights about utlraconserved non-coding elements
(UCNEs). These elements are organized as large clusters, so-called gene
regulatory blocks (GRBs) around key developmental genes. Their molecular
functions and the reasons for their high degree of conservation remain
enigmatic.
RESULTS: In a special setting of genomic context analysis, we analyzed the fate
of GRBs after a whole-genome duplication event in five fish genomes. We found
that in most cases all UCNEs were retained together as a single block, whereas
the corresponding target genes were often retained in two copies, one completely
devoid of UCNEs. This 'winner-takes-all' pattern suggests that UCNEs of a GRB
function in a highly cooperative manner. We propose that the multitude of
interactions between UCNEs is the reason for their extreme sequence
conservation.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online and at http://ccg.vital-it.ch/ucne/ |
What is the basis of the methodology of "functional class scoring" (FCS) for the analysis of gene expression data? | The second method, "functional class scoring" (FCS), examines the statistical distribution of individual gene scores among all genes in the gene ontology class and does not involve an initial gene selection step. | BACKGROUND: It is common for the results of a microarray study to be analyzed in
the context of biologically-motivated groups of genes such as pathways or Gene
Ontology categories. The most common method for such analysis uses the
hypergeometric distribution (or a related technique) to look for
"over-representation" of groups among genes selected as being differentially
expressed or otherwise of interest based on a gene-by-gene analysis. However,
this method suffers from some limitations, and biologist-friendly tools that
implement alternatives have not been reported.
RESULTS: We introduce ErmineJ, a multiplatform user-friendly stand-alone
software tool for the analysis of functionally-relevant sets of genes in the
context of microarray gene expression data. ErmineJ implements multiple
algorithms for gene set analysis, including over-representation and
resampling-based methods that focus on gene scores or correlation of gene
expression profiles. In addition to a graphical user interface, ErmineJ has a
command line interface and an application programming interface that can be used
to automate analyses. The graphical user interface includes tools for creating
and modifying gene sets, visualizing the Gene Ontology as a table or tree, and
visualizing gene expression data. ErmineJ comes with a complete user manual, and
is open-source software licensed under the Gnu Public License.
CONCLUSION: The availability of multiple analysis algorithms, together with a
rich feature set and simple graphical interface, should make ErmineJ a useful
addition to the biologist's informatics toolbox. ErmineJ is available from
http://microarray.cu.genome.org. 3H-1,2-dithiole-3-thione (D3T), an inducer of antioxidant and phase 2 genes, is
known to enhance the detoxification of environmental carcinogens, prevent
neoplasia, and elicit other protective effects. However, a comprehensive view of
the regulatory pathways induced by this compound has not yet been elaborated.
Fischer F344 rats were gavaged daily for 5 days with vehicle or D3T (0.3
mmol/kg). The global changes of gene expression in liver were measured with
Affymetrix RG-U34A chips. With the use of functional class scoring, a
semi-supervised method exploring both the expression pattern and the functional
annotation of the genes, the Gene Ontology classes were ranked according to the
significance of the impact of D3T treatment. Two unexpected functional classes
were identified for the D3T treatment, cytosolic ribosome constituents with 90%
of those genes increased, and cholesterol biosynthesis with 91% of the genes
repressed. In another novel approach, the differentially expressed genes were
evaluated by the Ingenuity computational pathway analysis tool to identify
specific regulatory networks and canonical pathways responsive to D3T treatment.
In addition to the known glutathione metabolism pathway (P = 0.0011), several
other significant pathways were also revealed, including antigen presentation (P
= 0.000476), androgen/estrogen biosynthesis (P = 0.000551), fatty acid (P =
0.000216), and tryptophan metabolism (P = 0.000331) pathways. These findings
showed a profound impact of D3T on lipid metabolism and
anti-inflammatory/immune-suppressive response, indicating a broader
cytoprotective effect of this compound than previously expected. As gene annotation databases continue to evolve and improve, it has become
feasible to incorporate the functional and pathway information about genes,
available in these databases into the analysis of gene expression data, for a
better understanding of the underlying mechanisms. A few methods have been
proposed in the literature to formally convert individual gene results into gene
function results. In this paper, we will compare the various methods, propose
and examine some new ones, and offer a structured approach to incorporating gene
function or pathway information into the analysis of expression data. We study
the performance of the various methods and also compare them on real data, using
a case study from the toxicogenomics area. Our results show that the approaches
based on gene function scores yield a different, and functionally more
interpretable, array of genes than methods that rely solely on individual gene
scores. They also suggest that functional class scoring methods appear to
perform better and more consistently than overrepresentation analysis and
distributional score methods. Identifying the pathways that are significantly impacted in a given condition is
a crucial step in understanding the underlying biological phenomena. All
approaches currently available for this purpose calculate a P-value that aims to
quantify the significance of the involvement of each pathway in the given
phenotype. These P-values were previously thought to be independent. Here we
show that this is not the case, and that many pathways can considerably affect
each other's P-values through a "crosstalk" phenomenon. Although it is intuitive
that various pathways could influence each other, the presence and extent of
this phenomenon have not been rigorously studied and, most importantly, there is
no currently available technique able to quantify the amount of such crosstalk.
Here, we show that all three major categories of pathway analysis methods
(enrichment analysis, functional class scoring, and topology-based methods) are
severely influenced by crosstalk phenomena. Using real pathways and data, we
show that in some cases pathways with significant P-values are not biologically
meaningful, and that some biologically meaningful pathways with nonsignificant
P-values become statistically significant when the crosstalk effects of other
pathways are removed. We describe a technique able to detect, quantify, and
correct crosstalk effects, as well as identify independent functional modules.
We assessed this novel approach on data from four experiments involving three
phenotypes and two species. This method is expected to allow a better
understanding of individual experiment results, as well as a more refined
definition of the existing signaling pathways for specific phenotypes. BACKGROUND: Sets of genes that are known to be associated with each other can be
used to interpret microarray data. This gene set approach to microarray data
analysis can illustrate patterns of gene expression which may be more
informative than analyzing the expression of individual genes. Various
statistical approaches exist for the analysis of gene sets. There are three main
classes of these methods: over-representation analysis, functional class
scoring, and pathway topology based methods.
METHODS: We propose weighted hypergeometric and weighted chi-squared methods in
order to assign a rank to the degree to which each gene participates in the
enrichment. Each gene is assigned a weight determined by the absolute value of
its log fold change, which is then raised to a certain power. The power value
can be adjusted as needed. Datasets from the Gene Expression Omnibus are used to
test the method. The significantly enriched pathways are validated through
searching the literature in order to determine their relevance to the dataset.
RESULTS: Although these methods detect fewer significantly enriched pathways,
they can potentially produce more relevant results. Furthermore, we compare the
results of different enrichment methods on a set of microarray studies all
containing data from various rodent neuropathic pain models.
DISCUSSION: Our method is able to produce more consistent results than other
methods when evaluated on similar datasets. It can also potentially detect
relevant pathways that are not identified by the standard methods. However, the
lack of biological ground truth makes validating the method difficult. |
What is the Timothy syndrome? | Timothy syndrome is a multisystem disorder characterized by cardiac, hand/foot, facial, and neurodevelopmental features. The two forms are type 1 (classic) and type 2, a rare form caused by mutations in a transcript variant of the same gene. Cardiac findings include a rate-corrected QT interval of between 480 ms and 700 ms and congenital heart defects (patent ductus arteriosus, patent foramen ovale, ventricular septal defect, tetralogy of Fallot, hypertrophic cardiomyopathy). Hand/foot findings are unilateral or bilateral cutaneous syndactyly variably involving fingers two (index), three (middle), four (ring), and five (little) and bilateral cutaneous syndactyly of toes two and three. Facial findings include flat nasal bridge, low-set ears, thin upper lip, and round face. Neuropsychiatric involvement includes global developmental delays and autism spectrum disorders. Ventricular tachyarrhythmia is the leading cause of death, followed by infection and complications of intractable hypoglycemia. Average age of death is 2.5 years. | BACKGROUND: Timothy syndrome is a multisystem disorder associated with QT
interval prolongation and ventricular cardiac arrhythmias. The syndrome has been
linked to mutations in Ca(V)1.2 resulting in gain of function of the L-type
calcium current (I(Ca,L)). Ranolazine is an antianginal agent shown to exert an
antiarrhythmic effect in experimental models of long QT syndrome.
OBJECTIVE: The purpose of this study was to develop and characterize an
experimental model of Timothy syndrome by using BayK8644 to mimic the gain of
function of I(Ca,L) and to examine the effects of ranolazine.
METHODS: Action potentials from epicardial and M regions and a
pseudo-electrocardiogram (ECG) were simultaneously recorded from
coronary-perfused left ventricular wedge preparations, before and after addition
of BayK8644 (1 microM).
RESULTS: BayK8644 preferentially prolonged action potential duration of the M
cell, leading to prolongation of the QT interval and an increase in transmural
dispersion of repolarization (from 44.3 +/- 7 ms to 86.5 +/- 25 ms). Stimulation
at cycle lengths of 250-500 ms led to ST-T wave alters due to alternation of
the plateau voltage of the M cell action potential as well as development of
delayed afterdepolarizations in epicardial and M cell action potentials.
Ventricular extrasystoles and tachycardia (monomorphic, bidirectional, or
torsades de pointes) developed spontaneously or after rapid pacing. Peak and
late I(Na) were unaffected by BayK8644. Clinically relevant concentrations of
ranolazine (10 microM) suppressed all actions of BayK8644.
CONCLUSION: A left ventricular wedge model of long QT syndrome created by
augmentation of I(Ca,L) recapitulates the ECG and arrhythmic manifestations of
Timothy syndrome, which can be suppressed by ranolazine. Calcium entry into excitable cells is an important physiological signal,
supported by and highly sensitive to the activity of voltage-gated Ca2+
channels. After membrane depolarization, Ca2+ channels first open but then
undergo various forms of negative feedback regulation including voltage- and
calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+
channel activity is perturbed in a rare yet devastating disorder known as
Timothy syndrome (TS), whose features include autism or autism spectrum disorder
along with severe cardiac arrhythmia and developmental abnormalities. Most cases
of TS arise from a sporadic single nucleotide change that generates a mutation
(G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We
found that the TS mutation powerfully and selectively slows VDI while sparing or
possibly speeding the kinetics of CDI. The deceleration of VDI was observed when
the L-type channels were expressed with beta1 subunits prominent in brain, as
well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI
was further substantiated by measurements of Ca2+ channel gating currents and by
analysis of another channel mutation (I1624A) that hastens VDI, acting upstream
of the step involving Gly406. As highlighted by the TS mutation, CDI does not
proceed to completeness but levels off at approximately 50%, consistent with a
change in gating modes and not an absorbing inactivation process. Thus, the TS
mutation offers a unique perspective on mechanisms of inactivation as well as a
promising starting point for exploring the underlying pathophysiology of autism. The Timothy syndrome is a multisystem disorder associated with the mutation of a
Gly residue (G402 or G406) in the Ca(v)1.2 Ca(2+) channel. G406 is localized at
the end of the IS6 segment and just before the intracellular I-II loop, which is
important for the regulation of channel inactivation and the binding of the
Ca(v)beta subunit. This Gly residue is conserved in all Ca(v)1 and Ca(v)2
channels, and the G to R exchange produces a strong decrease of inactivation not
only in Ca(v)1.2 but also in Ca(v)2.3. Here, we show that the mutation into Arg
or Glu of the homologous Gly residue in Ca(v)2.1 (G363) produces also a slowing
of inactivation. However, the G-to-A exchange that decreases the inactivation
rate in Ca(v)1.2 and Ca(v)2.3 increases inactivation in Ca(v)2.1. Each mutation
affects specifically the gating properties of Ca(v)2.1 that remain nevertheless
modulated by the co-expressed beta subunit as with wild-type channel. The strong
decrease of inactivation produced by the G363R or G363E mutations was
reminiscent to that previously described for a specific splice variant of
Ca(v)2.1 that contains a single Val residue inserted in the I-II loop (V421). We
unexpectedly found that the V421 insertion does not affect the inactivation rate
of Ca(v)2.1 and that the effects previously attributed to this insertion,
including those on G-protein regulation, can be reproduced by the G363E
mutation. Altogether, our results highlight the role of G363 in gating
properties, inactivation kinetics, and G-protein regulation of Ca(v)2.1 and the
lack of effect of V421 insertion on inactivation. Individuals with congenital or acquired prolongation of the QT interval, or long
QT syndrome (LQTS), are at risk of life-threatening ventricular arrhythmia. LQTS
is commonly genetic in origin but can also be caused or exacerbated by
environmental factors. A missense mutation in the L-type calcium channel
Ca(V)1.2 leads to LQTS in patients with Timothy syndrome. To explore the effect
of the Timothy syndrome mutation on the electrical activity and contraction of
human cardiomyocytes, we reprogrammed human skin cells from Timothy syndrome
patients to generate induced pluripotent stem cells, and differentiated these
cells into cardiomyocytes. Electrophysiological recording and calcium (Ca(2+))
imaging studies of these cells revealed irregular contraction, excess Ca(2+)
influx, prolonged action potentials, irregular electrical activity and abnormal
calcium transients in ventricular-like cells. We found that roscovitine, a
compound that increases the voltage-dependent inactivation of Ca(V)1.2 (refs
6-8), restored the electrical and Ca(2+) signalling properties of cardiomyocytes
from Timothy syndrome patients. This study provides new opportunities for
studying the molecular and cellular mechanisms of cardiac arrhythmias in humans,
and provides a robust assay for developing new drugs to treat these diseases. The Timothy syndrome mutations G402S and G406R abolish inactivation of Ca(V)1.2
and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into
the consequences of the G402S mutation on structure and function of the channel,
we systematically mutated the corresponding Gly-432 of the rabbit channel and
applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished
channel inactivation. Homology modeling revealed that Gly-432 forms part of a
highly conserved structure motif (G/A/G/A) of small residues in homologous
positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6),
Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in
contrast, parallel shifts of activation and inactivation curves indicating a
preserved coupling between both processes. Disruption between coupling of
activation and inactivation was specific for mutations of Gly-432 in domain I.
Mutations of Gly-432 removed inactivation irrespective of the changes in
activation. In all four domains residues G/A/G/A are in close contact with
larger bulky amino acids from neighboring S6 helices. These interactions
apparently provide adhesion points, thereby tightly sealing the activation gate
of Ca(V)1.2 in the closed state. Such a structural hypothesis is supported by
changes in activation gating induced by mutations of the G/A/G/A residues. The
structural implications for Ca(V)1.2 activation and inactivation gating are
discussed. RATIONALE: L-type Ca(2+) (Ca(V)1.2) channels shape the cardiac action potential
waveform and are essential for excitation-contraction coupling in heart. A
gain-of-function G406R mutation in a cytoplasmic loop of Ca(V)1.2 channels
causes long QT syndrome 8 (LQT8), a disease also known as Timothy syndrome.
However, the mechanisms by which this mutation enhances Ca(V)1.2-LQT8 currents
and generates lethal arrhythmias are unclear.
OBJECTIVE: To test the hypothesis that the anchoring protein AKAP150 modulates
Ca(V)1.2-LQT8 channel gating in ventricular myocytes.
METHODS AND RESULTS: Using a combination of molecular, imaging, and
electrophysiological approaches, we discovered that Ca(V)1.2-LQT8 channels are
abnormally coupled to AKAP150. A pathophysiological consequence of forming this
aberrant ion channel-anchoring protein complex is enhanced Ca(V)1.2-LQT8
currents. This occurs through a mechanism whereby the anchoring protein
functions like a subunit of Ca(V)1.2-LQT8 channels that stabilizes the open
conformation and augments the probability of coordinated openings of these
channels. Ablation of AKAP150 restores normal gating in Ca(V)1.2-LQT8 channels
and protects the heart from arrhythmias.
CONCLUSION: We propose that AKAP150-dependent changes in Ca(V)1.2-LQT8 channel
gating may constitute a novel general mechanism for Ca(V)1.2-driven arrhythmias. Timothy syndrome type 1 (TS-1) is a rare disorder that affects multiple organ
systems and has a high incidence of sudden death due to profound QT prolongation
and resultant ventricular arrhythmias. All previously described cases of TS-1
are the result of a missense mutation in exon 8A (p.G406R), an alternatively
spliced variant of the L-type calcium channel gene (Ca(v)1.2, CACNA1C). Most
patients reported in the literature represent highly affected individuals who
present early in life with severe cardiac and neurological manifestations. Here,
we describe somatic mosaicism in TS-1 patients with less severe manifestations
than the typical TS-1 patient. These findings suggest that the TS prognosis may
not be as dismal as previously reported. Moreover, our findings have
implications for genetic counseling in that previously described de novo TS
mutations may represent cases of parental mosaicism and warrant careful
genotyping of parental tissue other than peripheral blood lymphocytes. 1. Clin Res Cardiol. 2011 Dec;100(12):1123-7. doi: 10.1007/s00392-011-0358-4.
Epub 2011 Sep 14. Timothy syndrome (TS) is an autosomal domit condition with the constellation
of features including prolonged QT interval, hand and foot abnormalities, and
mental retardation or autism. Splawski et al. [2004] previously described two
phenotypes associated with TS distinguished by two unique and different
mutations within the CACNA1C gene. We report on a newborn who presented with
prolonged QT interval and associated polymorphic ventricular tachycardia,
dysmorphic facial features, syndactyly of the hands and feet, and joint
contractures, suggestive of TS. He developed a stroke, subsequent intractable
seizures, and was found to have cortical blindness and later profound
developmental delay. Initial targeted mutation analysis did not identify either
of the previously described TS associated mutations; however, full gene
sequencing detected a novel CACNA1C gene mutation (p.Ala1473Gly). The clinical
and genetic findings in our case expand both the clinical and molecular
knowledge of TS. Ca(V) channels are multi-subunit protein complexes that enable inward cellular
Ca(2+) currents in response to membrane depolarization. We recently described
structure-function studies of the intracellular α1 subunit domain I-II linker,
directly downstream of domain IS6. The results show the extent of the linker's
helical structure to be subfamily dependent, as dictated by highly conserved
primary sequence differences. Moreover, the difference in structure confers
different biophysical properties, particularly the extent and kinetics of
voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic
disorder due to mutations in the Ca(V)1.2 gene. Here, we explored whether
perturbation of the I-II linker helical structure might provide a mechanistic
explanation for a Timothy syndrome mutant's (human Ca(V)1.2 G406R equivalent)
biophysical effects on inactivation and activation. The results are equivocal,
suggesting that a full mechanistic explanation for this Timothy syndrome
mutation requires further investigation. Voltage-gated, dihydropyridine-sensitive L-type Ca(2+) channels are multimeric
proteins composed of a pore-forming transmembrane α(1) subunit (Ca(v)1.2) and
accessory β, α(2)δ, and γ subunits. Ca(2+) entry via Ca(v)1.2 channels shapes
the action potential (AP) of cardiac myocytes and is required for
excitation-contraction coupling. Two de novo point mutations of Ca(v)1.2 glycine
residues, G406R and G402S, cause a rare multisystem disorder called Timothy
syndrome (TS). Here, we discuss recent work on the mechanisms by which Ca(v)1.2
channels bearing TS mutations display slowed inactivation that leads to
increased Ca(2+) influx, prolonging the cardiac AP and promoting lethal
arrhythmias. Based on these studies, we propose a model in which the scaffolding
protein AKAP79/150 stabilizes the open conformation of Ca(v)1.2-TS channels and
facilitates physical interactions among adjacent channels via their C-tails,
increasing the activity of adjoining channels and amplifying Ca(2+) influx. Timothy syndrome, long QT syndrome type 8, is highly maligt with ventricular
tachyarrhythmia. A 30-month-old boy had sudden cardiac arrest during anesthesia
induction before plastic surgery for bilateral cutaneous syndactyly. After
successful resuscitation, prolonged QT interval (QTc, 0.58-0.60 sec) and T-wave
alters were found in his electrocardiogram. Starting β-blocker to prevent
further tachycardia and collapse event, then there were no more arrhythmic
events. The genes KCNQ1, KCNH2, KCNE1 and 2, and SCN5A were negative for long QT
syndrome. The mutation p.Gly406Arg was confirmed in CACNA1C, which maintains
L-type calcium channel depolarization in the heart and other systems. |
Which disease has been associated to a disruptive ALX1 protein? | Disruption of ALX1 causes extreme microphthalmia and severe facial clefting: expanding the spectrum of autosomal-recessive ALX-related frontonasal dysplasia. | The paired-class homeobox-containing gene, Cart1, is expressed in forebrain
mesenchyme, branchial arches, limb buds and cartilages during embryogenesis.
Here, we show that Cart1-homozygous mutant mice are born alive with acrania and
meroanencephaly but die soon after birth-a phenotype that strikingly resembles a
corresponding human syndrome caused by a neural tube closure defect.
Developmental studies suggest that Cart1 is required for forebrain mesenchyme
survival and that its absence disrupts cranial neural tube morphogenesis by
blocking the initiation of closure in the midbrain region that ultimately leads
to the generation of lethal craniofacial defects. Prenatal treatment of Cart1
homozygous mutants with folic acid suppresses the development of the
acrania/meroanencephaly phenotype. BACKGROUND: Cart1 encodes the paired-like homeodomain in the central portion of
the gene, and plays a crucial role in the developmental lineage of bone and
cartilage, especially in head formation. However, its transactivation mechanism
is still poorly understood, including the target gene. Here, we report
biochemical dissections of Cart1 functional domains and a relationship between
dimerization and transcription activity.
RESULTS: Deletion studies of GAL4-fused Cart1 indicated that the transactivation
domain is located in the middle portion of the C-terminal domain, but the
N-terminal is also required for a full activation of the consensus palindromic
binding site (TAATNNNATTA). Analysis of the basic amino acid residues at both
ends of the homeodomain revealed that both sides act as nuclear localization
signals, and are necessary for the cooperative binding to the palindromic
sequence. In this study, two additional Cart1 isoforms that behave as domit
negatives were identified from rat chondrosarcoma cells. These isoforms
suppressed the transcription activity of the wild-type, despite loss of DNA
binding ability, and could interact with the wild-type in yeast. Finally, we
demonstrated that wild-type Cart1 forms a DNA-independent homodimer in in vivo
conditions, and that the transactivation of wild-type Cart1 was suppressed by
the N- or C-terminal domain which was expressed in the nucleus.
CONCLUSION: These results revealed that homodimerization through direct
interaction is necessary for the potent transcription activity of Cart1. The diverse cellular contributions to the skeletal elements of the vertebrate
shoulder and pelvic girdles during embryonic development complicate the study of
their patterning. Research in avian embryos has recently clarified part of the
embryological basis of shoulder formation. Although dermomyotomal cells provide
the progenitors of the scapular blade, local signals appear to have an essential
guiding role in this process. These signals differ from those that are known to
pattern the more distal appendicular skeleton. We have studied the impact of
Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the
mouse shoulder and pelvic girdles. We observed severe reduction of the scapula
in double and triple mutants of these genes. Analyses of a range of complex
genotypes revealed aspects of their genetic relationship, as well as functions
that had been previously masked due to functional redundancy. Tbx15 and Gli3
appear to have synergistic functions in formation of the scapular blade.
Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates
essential functions for Alx4 and Cart1 in the anterior part of the scapula, as
opposed to Gli3 function being linked to the posterior part. Especially in
Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis
is altered prior to stages when anatomical aberrations are visible in the
shoulder region. This suggests a disorganization of the proximal limb bud and
adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism
providing positional cues to guide progenitor cells to their destination in the
pectoral girdle. Mutations in each of the transcriptional co-activator genes - CBP, p300, Cited2,
Cart1 and Carm1 - result in neural tube defects in mice. The present study thus
furnishes a complete and comparative temporal and spatial expression map of
CBP/p300 and associated transcriptional co-activators, Cited2, Cart1 and Carm1
during the period of murine neural tube development (embryonic days 8.5 to
10.5). Each co-activator except Cart1 was expressed in the dorsal neural folds
on E8.5. Although CBP and p300 are functionally interchangeable in vitro, their
respective expression patterns diverge during embryogenesis before neural fold
fusion is complete. CBP gene expression was lost from the neural folds by E8.75
and was thereafter weakly expressed in the maxillary region and limb buds, while
p300 exhibited strong expression in the first branchial arch, limb bud and
telencephalic regions on E9.5. Cart1 exhibited strong expression in the
forebrain mesenchyme from E9.0 through E10.5. Although CBP, p300, Carm1 and
Cited2 share temporal expression on E8.5, these co-activators have different
spatial expression in mesenchyme and/or the neuroepithelium. Nevertheless,
co-localization to the dorsal neural folds on E8.5 suggests a functional role in
elevation and/or fusion of the neural folds. Target genes, and pathways that
promote cranial neural tube fusion that are activated by
CBP/p300/Carm1/Cited2/Cart1-containing transcriptional complexes await
elucidation. We present an autosomal-recessive frontonasal dysplasia (FND) characterized by
bilateral extreme microphthalmia, bilateral oblique facial cleft, complete cleft
palate, hypertelorism, wide nasal bridge with hypoplasia of the ala nasi, and
low-set, posteriorly rotated ears in two distinct families. Using Affymetrix
250K SNP array genotyping and homozygosity mapping, we mapped this clinical
entity to chromosome 12q21. In one of the families, three siblings were
affected, and CNV analysis of the critical region showed a homozygous 3.7 Mb
deletion containing the ALX1 (CART1) gene, which encodes the aristaless-like
homeobox 1 transcription factor. In the second family we identified a homozygous
donor-splice-site mutation (c.531+1G > A) in the ALX1 gene, providing evidence
that complete loss of function of ALX1 protein causes severe disruption of early
craniofacial development. Unlike loss of its murine ortholog, loss of human ALX1
does not result in neural-tube defects; however, it does severely affect the
orchestrated fusion between frontonasal, nasomedial, nasolateral, and maxillary
processes during early-stage embryogenesis. This study further expands the
spectrum of the recently recognized autosomal-recessive ALX-related FND
phenotype in humans. The genetic pathways underlying shoulder blade development are largely unknown,
as gene networks controlling limb morphogenesis have limited influence on
scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has
suggested their potential roles in girdle development. In this study, by
generating compound mutant mice, we examined the genetic control of scapula
development by Pbx genes and their functional relationship with Emx2. Analyses
of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping
functions in shoulder development and that Pbx1 genetically interacts with Emx2
in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic
interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as
heterodimers. Moreover, the expression of genes crucial for scapula development
is altered in these mutants, indicating that Pbx genes act upstream of essential
pathways for scapula formation. In particular, expression of Alx1, an effector
of scapula blade patterning, is absent in all compound mutants. We demonstrate
that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and
cooperatively activate its transcription via this potential regulatory element.
Our results establish an essential role for Pbx1 in genetic interactions with
its family members and with Emx2 and delineate novel regulatory networks in
shoulder girdle development. |
How many selenoproteins are encoded in the human genome? | 25. 15kDa, DI1, DI2, DI3, GPx1, GPx2, GPx3, GPx4, GPx6, SelH, SelI, SelK, SelM, SelN, SelO, SelP, SelR, SelS, SPS2, SelT, TR1, TR2, TR3, SelV and SelW. | In the genetic code, UGA serves as a stop signal and a selenocysteine codon, but
no computational methods for identifying its coding function are available.
Consequently, most selenoprotein genes are misannotated. We identified
selenoprotein genes in sequenced mammalian genomes by methods that rely on
identification of selenocysteine insertion RNA structures, the coding potential
of UGA codons, and the presence of cysteine-containing homologs. The human
selenoproteome consists of 25 selenoproteins. Selenoproteins are a diverse group of proteins usually misidentified and
misannotated in sequence databases. The presence of an in-frame UGA (stop) codon
in the coding sequence of selenoprotein genes precludes their identification and
correct annotation. The in-frame UGA codons are recoded to cotranslationally
incorporate selenocysteine, a rare selenium-containing amino acid. The
development of ad hoc experimental and, more recently, computational approaches
have allowed the efficient identification and characterization of the
selenoproteomes of a growing number of species. Today, dozens of selenoprotein
families have been described and more are being discovered in recently sequenced
species, but the correct genomic annotation is not available for the majority of
these genes. SelenoDB is a long-term project that aims to provide, through the
collaborative effort of experimental and computational researchers, automatic
and manually curated annotations of selenoprotein genes, proteins and SECIS
elements. Version 1.0 of the database includes an initial set of eukaryotic
genomic annotations, with special emphasis on the human selenoproteome, for
immediate inspection by selenium researchers or incorporation into more general
databases. SelenoDB is freely available at http://www.selenodb.org. BACKGROUND: The selenocysteine (Sec) containing proteins, selenoproteins, are an
important group of proteins present throughout all 3 kingdoms of life. With the
rapid progression of selenoprotein research in the post-genomic era, application
of bioinformatics methods to the identification of selenoproteins in newly
sequenced species has become increasingly important. Although selenoproteins in
human and other vertebrates have been investigated, studies of primitive
invertebrate selenoproteomes are rarely reported outside of insects and
nematodes.
RESULT: A more integrated view of selenoprotein evolution was constructed using
several representative species from different evolutionary eras. Using a
SelGenAmic-based selenoprotein identification method, 178 selenoprotein genes
were identified in 6 invertebrates: Amphimedon queenslandica, Trichoplax
adhaerens, Nematostella vectensis, Lottia gigantean, Capitella teleta, and
Branchiostoma floridae. Amphioxus was found to have the most abundant and
variant selenoproteins of any animal currently characterized, including a
special selenoprotein P (SelP) possessing 3 repeated Trx-like domains and Sec
residues in the N-terminal and 2 Sec residues in the C-terminal. This gene
structure suggests the existence of two different strategies for extension of
Sec numbers in SelP for the preservation and transportation of selenium. In
addition, novel eukaryotic AphC-like selenoproteins were identified in sponges.
CONCLUSION: Comparison of various animal species suggests that even the most
primitive animals possess a selenoproteome range and variety similar to humans.
During evolutionary history, only a few new selenoproteins have emerged and few
were lost. Furthermore, the massive loss of selenoproteins in nematodes and
insects likely occurred independently in isolated partial evolutionary branches. |
In which process Src, Cortactin and MT1-MMP are playing an essential role? | Src was shown to be required for invadopodia formation and function, whereas Cortactin was found to regulate cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation. Finally, membrane type 1 matrix metalloproteinase (MT1-MMP) was demostrated as the key invadopodial enzyme responsible for gelatin matrix degradation in cancer cells. | Metastatic tumor cells that actively migrate and invade surrounding tissues rely
on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are
membrane protrusions that localize enzymes required for ECM degradation. Little
is known about the formation, function, and regulation of invadopodia. Here, we
show that invadopodia have two distinct aspects: (a) structural for organizing
the cellular actin cytoskeleton to form membrane protrusions and (b) functional
for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA
(siRNA) inhibition established that organization of invadopodia structure
requires cortactin, whereas protease inhibitor studies identified membrane type
1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible
for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The
inhibition of invadopodial structure assembly by cortactin depletion resulted in
a block of matrix degradation due to failure of invadopodia formation. Either
protease inhibition or MT1-MMP siRNA depletion moderately decreased the
formation of invadopodial structures that were identified as actin-cortactin
accumulations at the ventral cell membrane adherent to matrix. The invadopodia
that were able to form upon MT1-MMP inhibition or depletion retained
actin-cortactin accumulations but were unable to degrade matrix. Examination of
cells at different time points as well as live-cell imaging revealed four
distinct invadopodial stages: membrane cortactin aggregation at membranes
adherent to matrix, MT1-MMP accumulation at the region of cortactin
accumulation, matrix degradation at the invadopodia region, and subsequent
cortactin dissociation from the area of continued MT1-MMP accumulation
associated with foci of degraded matrix. Based on these results, we propose a
stepwise model of invadopodia formation and function. Invasion of the subendothelial space by vascular smooth muscle cells (VSMCs)
contributes to the development and progression of diverse cardiovascular
diseases. In this report we show that the expression of activated versions of
Src, Cdc42 and Rac1, or a kinase-dead but open form of the p21-activated kinase
(PAK1), induces primary rat aorta VSMCs to form extracellular matrix-degrading
actin-rich protrusions that are morphologically similar to the invadopodia
formed by highly invasive tumor cells. The matrix-degrading structures are
enriched in known markers for invadopodia, including cortactin and
tyrosine-phosphorylated cortactin and contain the matrix metalloproteinases
MMP-9 and MT1-MMP and the urokinase plasminogen activator receptor (uPAR). In
contrast to other cell types, invadopodia formation in VSMCs is only weakly
supported by the phorbol ester PBDu. Invadopodia formation by Src was dependent
on Cdc42, Rac, and ERK, but not on p38 MAPK. Invadopodia formation induced by
kinase-dead PAK1 required Src and ERK activity and a direct interaction with the
exchange factor PIX. VSMCs embedded in a three-dimensional collagen matrix
formed actin- and cortactin-rich extensions that penetrated through holes in the
matrix, suggesting that invadopodia-like structures are formed in a
three-dimensional environment. Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma
cells. The mechanisms regulating invadopodium assembly and maturation are not
understood. We have dissected the stages of invadopodium assembly and maturation
and show that invadopodia use cortactin phosphorylation as a master switch
during these processes. In particular, cortactin phosphorylation was found to
regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin
directly binds cofilin and inhibits its severing activity. Cortactin
phosphorylation is required to release this inhibition so cofilin can sever
actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent
actin polymerization. After barbed end formation, cortactin is dephosphorylated,
which blocks cofilin severing activity thereby stabilizing invadopodia. These
findings identify novel mechanisms for actin polymerization in the invadopodia
of metastatic carcinoma cells and define four distinct stages of invadopodium
assembly and maturation consisting of invadopodium precursor formation, actin
polymerization, stabilization, and matrix degradation. Tumor cell invasion is vital for cancer progression and metastasis. Adhesion,
migration, and degradation of the extracellular matrix are important events
involved in the establishment of cancer cells at a new site, and therefore
molecular targets are sought to inhibit such processes. The effect of a plant
proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on
the adhesion, migration, and invasion of gastric cancer cells was the focus of
this study. EcTI showed no effect on the proliferation of gastric cancer cells
or fibroblasts but inhibited the adhesion, migration, and cell invasion of
gastric cancer cells; however, EcTI had no effect upon the adhesion of
fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular
organization of molecules involved in the formation and maturation of
invadopodia, such as integrin β1, cortactin, neuronal Wiskott-Aldrich syndrome
protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover,
gastric cancer cells treated with EcTI presented a significant decrease in
intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent
cell signaling components. Together, these results indicate that EcTI inhibits
the invasion of gastric cancer cells through alterations in integrin-dependent
cell signaling pathways. SCOPE: Invadopodia are actin-rich membrane protrusions of tumor cells that are
thought to initiate the local migration and invasion during cancer metastasis.
The blockade of invadopodia-associated proteins has been reported as a promising
approach for prevention of tumor metastasis. The aim of this study was to
investigate the modulatory effects of 6-shogaol and pterostilbene on invadopodia
in aggressive breast cancer cells.
METHODS AND RESULTS: By wound-healing, transwell, and gelatin zymography assays,
we found that 6-shogaol and pterostilbene effectively attenuated the motility
and invasion of MDA-MB-231 cells, and suppressed the activities of matrix
metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Further
investigation into the underlying molecular mechanisms revealed that the levels
of key modulators of invadopodium maturation, including c-Src kinase, cortactin,
and membrane type 1-matrix metalloproteinase (MT1-MMP) decreased when cells were
treated with 6-shogaol or pterostilbene.
CONCLUSION: These data suggest that the repression of these factors might affect
the maturation of invadopodia, inhibiting the metastasis of MDA-MB-231 cells. In
conclusion, the present study demonstrates for the first time that 6-shogaol and
pterostilbene can inhibit invadopodium formation and MMP activity in highly
invasive breast cancer cells. We suggest that these compounds may be clinically
useful in chemopreventive treatments for metastatic breast cancer. |
In which yeast chromosome does the rDNA cluster reside? | Chromosome XII context is important for rDNA function in yeast | We have developed a fluorescent in situ hybridization (FISH) method to examine
the structure of both natural chromosomes and small artificial chromosomes
during the mitotic cycle of budding yeast. Our results suggest that the pairing
of sister chromatids: (a) occurs near the centromere and at multiple places
along the chromosome arm as has been observed in other eukaryotic cells; (b) is
maintained in the absence of catenation between sister DNA molecules; and (c) is
independent of large blocks of repetitive DNA commonly associated with
heterochromatin. Condensation of a unique region of chromosome XVI and the
highly repetitive ribosomal DNA (rDNA) cluster from chromosome XII were also
examined in budding yeast. Interphase chromosomes were condensed 80-fold
relative to B form DNA, similar to what has been observed in other eukaryotes,
suggesting that the structure of interphase chromosomes may be conserved among
eukaryotes. While additional condensation of budding yeast chromosomes were
observed during mitosis, the level of condensation was less than that observed
for human mitotic chromosomes. At most stages of the cell cycle, both unique and
repetitive sequences were either condensed or decondensed. However, in cells
arrested in late mitosis (M) by a cdc15 mutation, the unique DNA appeared
decondensed while the repetitive rDNA region appeared condensed, suggesting that
the condensation state of separate regions of the genome may be regulated
differently. The ability to monitor the pairing and condensation of sister
chromatids in budding yeast should facilitate the molecular analysis of these
processes as well as provide two new landmarks for evaluating the function of
important cell cycle regulators like p34 kinases and cyclins. Finally our FISH
method provides a new tool to analyze centromeres, telomeres, and gene
expression in budding yeast. The tools and techniques used in single-cell analysis of DNA damage in yeast
Saccharomyces cerevisiae are limited. In this study, we modified the single cell
gel electrophoresis assay, namely, the single chromosome comet assay based on
DNA break analysis, at the chromosomal level. We studied the largest yeast
chromosome XII, which contains the rDNA locus, and we investigated its
instability using cell cycle checkpoint-, DNA damage- and antioxidative
defence-deficient, and lifespan-deregulated yeast mutant strains. Moreover, we
compared chromosome XII instability with the variability of nucleolar rDNA
fluorescence signals. Three single-gene-deletion strains, cells lacking
single-stranded DNA endonuclease, Rad1p; NAD(+)-dependent histone deacetylase,
Sir2p; and gamma glutamylcysteine synthetase, Gsh1p, were more prone to
chromosome XII instability compared to corresponding wildtype strains,
indicating that DNA damage repair machinery, chromatin silencing and redox
homeostasis may contribute to genome stability. Elevation in the number of DNA
breaks was correlated with a high variability in the levels of nucleolar rDNA in
the Δrad1 background, while unaffected chromosome XII and low variability in
nucleolar rDNA fluorescence signals were observed in the Δtor1 longevity mutant.
Taken together, the single chromosome comet assay may be successfully used to
study DNA damage at the chromosomal level, which might be overlooked using whole
population analysis on DNA breaks with PFGE separation. |
Which is the underlying mechanism for exon skipping used to treat Duchenne muscular dystrophy? | Antisense-mediated exon skipping therapy is a promising therapeutic approach that uses short DNA-like molecules called antisense oligonucleotides (AOs) to skip over/splice out the mutated part of the gene to produce a shortened but functional dystrophin protein. Many Duchenne Muscular Dystrophy patients need exon skipping of multiple exons in order to restore the reading frame, depending on how many base pairs the mutated exon(s) and adjacent exons have. | BACKGROUND: Antisense-mediated exon skipping is currently one of the most
promising therapeutic approaches for Duchenne muscular dystrophy (DMD). Using
antisense oligonucleotides (AONs) targeting specific exons the DMD reading frame
is restored and partially functional dystrophins are produced. Following proof
of concept in cultured muscle cells from patients with various deletions and
point mutations, we now focus on single and multiple exon duplications. These
mutations are in principle ideal targets for this approach since the specific
skipping of duplicated exons would generate original, full-length transcripts.
METHODS: Cultured muscle cells from DMD patients carrying duplications were
transfected with AONs targeting the duplicated exons, and the dystrophin RNA and
protein were analyzed.
RESULTS: For two brothers with an exon 44 duplication, skipping was, even at
suboptimal transfection conditions, so efficient that both exons 44 were
skipped, thus generating, once more, an out-of-frame transcript. In such cases,
one may resort to multi-exon skipping to restore the reading frame, as is shown
here by inducing skipping of exon 43 and both exons 44. By contrast, in cells
from a patient with an exon 45 duplication we were able to induce single exon 45
skipping, which allowed restoration of wild type dystrophin. The correction of a
larger duplication (involving exons 52 to 62), by combinations of AONs targeting
the outer exons, appeared problematic due to inefficient skipping and
mistargeting of original instead of duplicated exons.
CONCLUSION: The correction of DMD duplications by exon skipping depends on the
specific exons targeted. Its options vary from the ideal one, restoring for the
first time the true, wild type dystrophin, to requiring more 'classical'
skipping strategies, while the correction of multi-exon deletions may need the
design of tailored approaches. BACKGROUND: Antisense-mediated exon skipping is a putative treatment for
Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the
disrupted DMD reading frame is restored, allowing generation of partially
functional dystrophin and conversion of a severe Duchenne into a milder Becker
muscular dystrophy phenotype. In vivo studies are mainly performed using
2'-O-methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds
were never directly compared.
METHODS: mdx and humanized (h)DMD mice were injected intramuscularly and
intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human
exons 44, 45, 46 and 51.
RESULTS: Intramuscular injection showed that increasing the length of 2OMePS
AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency
for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and
2OMePS were more comparable for human exons. After intravenous administration,
exon skipping and novel protein was shown in the heart with both chemistries.
Furthermore, PMO showed lower intramuscular concentrations with higher exon 23
skipping levels compared to 2OMePS, which may be due to sequestration in the
extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs
nearly ineffective.
CONCLUSIONS: The results obtained in the present study indicate that increasing
AON length improves skipping efficiency in some but not all cases. It is
feasible to induce exon skipping and dystrophin restoration in the heart after
injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency
between PMO and 2OMePS appear to be sequence and not chemistry dependent.
Finally, the results indicate that PMOs may be less sequence specific than
2OMePS. We previously conducted a proof of principle; dose escalation study in Duchenne
muscular dystrophy (DMD) patients using the morpholino splice-switching
oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon
51 in patients with relevant deletions, restores the open reading frame and
induces dystrophin protein expression after intramuscular (i.m.) injection. We
now show that this dystrophin expression was accompanied by an elevated
expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant
cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is
a component of a different subcomplex of the dystrophin-associated glycoprotein
complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma
in Duchenne patients in whom the dystrophin deletion left the nNOS-binding
domain (exons 42-45) intact, whereas this did not occur in patients with
deletions that involved this domain. Our results indicate that the novel
internally deleted and shorter dystrophin induced by skipping exon 51 in
patients with amenable deletions, can also restore the dystrophin-associated
complex, further suggesting preserved functionality of the newly translated
dystrophin. Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by
mutations in the DMD gene, affecting 1 in 3500 newborn males. Complete loss of
muscle dystrophin protein causes progressive muscle weakness and heart and
respiratory failure, leading to premature death. Antisense oligonucleotides
(AONs) that bind to complementary sequences of the dystrophin pre-mRNA to induce
skipping of the targeted exon by modulating pre-mRNA splicing are promising
therapeutic agents for DMD. Such AONs can restore the open reading frame of the
DMD gene and produce internally deleted, yet partially functional dystrophin
protein isoforms in skeletal muscle. Within the last few years, clinical trials
using AONs have made considerable progress demonstrating the restoration of
functional dystrophin protein and acceptable safety profiles following both
local and systemic delivery in DMD patients. However, improvement of AON
delivery and efficacy, along with the development of multiple AONs to treat as
many DMD patients as possible needs to be addressed for this approach to fulfill
its potential. Here, we review the recent progress made in clinical trials using
AONs to treat DMD and discuss the current challenges to the development of
AON-based therapy for DMD. Duchenne muscular dystrophy (DMD) is one of the most common and lethal genetic
disorders, with 20,000 children per year born with DMD globally. DMD is caused
by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping
therapy is a promising therapeutic approach that uses short DNA-like molecules
called antisense oligonucleotides (AOs) to skip over/splice out the mutated part
of the gene to produce a shortened but functional dystrophin protein. One major
challenge has been its limited applicability. Multiple exon skipping has
recently emerged as a potential solution. Indeed, many DMD patients need exon
skipping of multiple exons in order to restore the reading frame, depending on
how many base pairs the mutated exon(s) and adjacent exons have. Theoretically,
multiple exon skipping could be used to treat approximately 90%, 80%, and 98% of
DMD patients with deletion, duplication, and nonsense mutations, respectively.
In addition, multiple exon skipping could be used to select deletions that
optimize the functionality of the truncated dystrophin protein. The proof of
concept of systemic multiple exon skipping using a cocktail of AOs has been
demonstrated in dystrophic dog and mouse models. Remaining challenges include
the insufficient efficacy of systemic treatment, especially for therapies that
target the heart, and limited long-term safety data. Here we review recent
preclinical developments in AO-mediated multiple exon skipping and discuss the
remaining challenges. |
What is the advantage of using long nano columns in proteomics? | The longer the columns, the longer gradients are applied and finally more proteins (increased peak capacity) are identified in a complex proteomic experiment | In this study, high-efficiency LC-MS/MS separations of complex proteolytic
digests are demonstrated using 50 mm, 250 mm, and 1m long
poly(styrene-co-divinylbenzene) monolithic capillary columns. The
chromatographic performance of the 50 and 250 mm monoliths was compared at the
same gradient steepness for gradient durations between 5 and 150 min. The
maximum peak capacity of 400 obtained with a 50mm column, increased to 485 when
using the 250 mm long column and scaling the gradient duration according column
length. With a 5-fold increase in column length only a 20% increase in peak
capacity was observed, which could be explained by the larger macropore size of
the 250 mm long monolith. When taking into account the total analysis time,
including the dwell time, gradient time and column equilibration time, the 50mm
long monolith yielded better peptide separations than the 250 mm long monolithic
column for gradient times below 80 min (n(c)=370). For more demanding separation
the 250 mm long monolith provided the highest peak production rate and
consequently higher sequence coverage. For the analysis of a proteolytic digest
of Escherichia coli proteins a monolithic capillary column of 1m in length was
used, yielding a peak capacity of 1038 when applying a 600 min gradient. Yeast remains an important model for systems biology and for evaluating
proteomics strategies. In-depth shotgun proteomics studies have reached nearly
comprehensive coverage, and rapid, targeted approaches have been developed for
this organism. Recently, we demonstrated that single LC-MS/MS analysis using
long columns and gradients coupled to a linear ion trap Orbitrap instrument had
an unexpectedly large dynamic range of protein identification (Thakur, S. S.,
Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M.
(2011) Deep and highly sensitive proteome coverage by LC-MS/MS without
prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we
couple an ultra high pressure liquid chromatography system to a novel bench top
Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid,
and robust analysis of the yeast proteome. Single runs of filter-aided sample
preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an
average of 3923 proteins. Combined analysis of six single runs improved these
values to more than 4000 identified proteins/run, close to the total number of
proteins expressed under standard conditions, with median sequence coverage of
23%. Because of the absence of fractionation steps, only minuscule amounts of
sample are required. Thus the yeast model proteome can now largely be covered
within a few hours of measurement time and at high sensitivity. Median coverage
of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10
members was 88%, and pathways not covered were not expected to be active under
the conditions used. To study perturbations of the yeast proteome, we developed
an external, heavy lysine-labeled SILAC yeast standard representing different
proteome states. This spike-in standard was employed to measure the heat shock
response of the yeast proteome. Bioinformatic analysis of the heat shock
response revealed that translation-related functions were down-regulated
prominently, including nucleolar processes. Conversely, stress-related pathways
were up-regulated. The proteomic technology described here is straightforward,
rapid, and robust, potentially enabling widespread use in the yeast and other
biological research communities. Here, we describe an in-house built ultra-high pressure liquid chromatography
(UHPLC) system, with little complexity in design and high separation power
combined with convenience in operation. This system enables the use of long
columns of 40 cm packed with 1.8 μm particles generating pressures below 1000
bar. Furthermore, the system could be operated at flow rates between 50 and 200
nL min(-1) while maintaining its separation power. Several gradients were
optimized ranging from 23 to 458 minutes. With the longest gradient we
identified over 4500 protein groups and more than 26,000 unique peptides from 1
μg of a human cancer cell lysate in a single run using an Orbitrap Velos - a
level of performance often seen solely using multidimensional separation
strategies. Further experiments using a mass spectrometer with faster sequencing
speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups
in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to
the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that
the combination of UHPLC with high resolution mass spectrometry at increased
sequencing speeds enables extensive proteome analysis in single runs. Proteome analyses of human induced pluripotent stem cells (iPSC) were carried
out on a liquid chromatography-tandem mass spectrometry system using meter-scale
monolithic silica-C18 capillary columns without prefractionation. Tryptic
peptides from five different iPSC lysates and three different fibroblast lysates
(4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic
silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20
MPa. We identified 98,977 nonredundant tryptic peptides from 9510 proteins
(corresponding to 8712 genes), including low-abundance protein groups (such as
329 protein kinases) from triplicate measurements within 10 days. The obtained
proteome profiles of the eight cell lysates were categorized into two groups,
iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative
analysis based on an exponentially modified protein abundance index approach
combined with UniProt keyword enrichment analysis revealed that the iPSC group
contains more "transcription regulation"-related proteins, while the fibroblast
group contained more "transport"-related proteins. Our results indicate that
this simplified one-shot proteomics approach with long monolithic columns is
advantageous for rapid, deep, sensitive, and reproducible proteome analysis. |
During which stage of the cell cycle is cohesin deposited on the yeast genome? | In the budding yeast, cohesin is loaded onto the chromosome during the late G1 phase, establishes sister chromatid cohesion concomitant with DNA replication, and dissociates by the telophase. | Sister chromatid cohesion is crucial for chromosome segregation during mitosis.
Loss of cohesion very possibly triggers sister separation at the metaphase -->
anaphase transition. This process depends on the destruction of anaphase
inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a
sister-separating protein Esp1p (Cut1p). By looking for mutants that separate
sister centromeres in the presence of Pds1p, this and a previous study have
identified six proteins essential for establishing or maintaining sister
chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are
subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the
onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin
subunit but it is required for Cohesin's association with chromosomes. The sixth
protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the
establishment of cohesion during DNA replication but not for its maintece
during G2 and M phases. Cohesin complex acts in the formation and maintece of sister chromatid
cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential
subunit, is cleaved and dissociates from chromosomes in anaphase, leading to
sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is
dissociated from chromosomes well before anaphase. The universal role of cohesin
during anaphase thus remains to be determined. We report here initial
characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3,
in fission yeast. They are essential for sister chromatid cohesion.
Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and
Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin
subunits are enriched in broad centromere regions and that the level of
centromere-associated Rad21 did not change from metaphase to anaphase, very
different from budding yeast. In contrast, Rad21 containing similar cleavage
sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage
is essential, although the amount of cleaved product is very small (<5%). Mis4,
another sister chromatid cohesion protein, plays an essential role for loading
Rad21 on chromatin. A simple model is presented to explain the specific behavior
of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to
be cleaved for normal anaphase. In eukaryotic cells, replicated DNA strands remain physically connected until
their segregation to opposite poles of the cell during anaphase. This "sister
chromatid cohesion" is essential for the alignment of chromosomes on the mitotic
spindle during metaphase. Cohesion depends on the multisubunit cohesin complex,
which possibly forms the physical bridges connecting sisters. Proteolytic
cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is
essential for sister chromatid separation and depends on a conserved protein
called separin. We show here that separin is a cysteine protease related to
caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase
arrested cells is sufficient to trigger the separation of sister chromatids and
their segregation to opposite cell poles. At anaphase onset, the protease separase triggers chromosome segregation by
cleaving the chromosomal cohesin complex. Here, we show that cohesin destruction
in metaphase is sufficient for segregation of much of the budding yeast genome,
but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA
in metaphase, unlike most other sequences, remains in an undercondensed and
topologically entangled state. Separase, concomitantly with cleaving cohesin,
activates the phosphatase Cdc14. We find that Cdc14 exerts two effects on rDNA,
both mediated by the condensin complex. Lengthwise condensation of rDNA shortens
the chromosome XII arm sufficiently for segregation. This condensation depends
on the aurora B kinase complex. Independently of condensation, Cdc14 induces
condensin-dependent resolution of cohesin-independent rDNA linkage.
Cdc14-dependent sister chromatid resolution at the rDNA could introduce a
temporal order to chromosome segregation. The 2 microm circle plasmid in Saccharomyces cerevisiae is a model for a stable,
high-copy-number, extrachromosomal "selfish" DNA element. By combining a
partitioning system and an amplification system, the plasmid ensures its stable
propagation and copy number maintece, even though it does not provide any
selective advantage to its host. Recent evidence suggests that the partitioning
system couples plasmid segregation to chromosome segregation. We now demonstrate
an unexpected and unconventional role for the mitotic spindle in the
plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2
microm circle and promotes recruitment of the cohesin complex to the
plasmid-partitioning locus STB. Only the nuclear microtubules, and not the
cytoplasmic ones, are required for loading cohesin at STB. In cells recovering
from nocodazole-induced spindle depolymerization and G(2)/M arrest, cohesin-STB
association can be established coincident with spindle restoration. This
postreplication recruitment of cohesin is not functional in equipartitioning.
However, normally acquired cohesin can be inactivated after replication without
causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid
cosegregates with the spindle and the spindle-associated chromosomes; by
contrast, a substantial number of the chromosomes are not associated with the
spindle. These results are consistent with a model in which the spindle promotes
plasmid segregation in a chromosome-linked fashion. In the budding yeast, cohesin is loaded onto the chromosome during the late G1
phase, establishes sister chromatid cohesion concomitant with DNA replication,
and dissociates by the telophase. Here, using oligonucleotide tiling arrays, we
show that, at the anaphase, nearly all of the cohesin binding sites contain
nucleosome-free regions. The majority of these sites remain nucleosome-free
throughout the cell cycle, consistent with the suggestion of a DNA-binding
anchoring protein present at these sites, although such a region could also
serve as part of a marker for the binding of cohesin in the next cell cycle.
However, a third of these sites are remodeled in the G1 phase, being reoccupied
by nucleosomes by the G1/S boundary, though their subsequent removal in the S
phase appears to be independent of DNA replication. Whether this difference is a
result of other functions of cohesin or of the chromatin remains to be
elucidated. Ability to reproduce is one of the hallmark features of all life forms by which
new organisms are produced from their progenitors. During this process each cell
duplicates its genome and passes a copy of its genome to the daughter cells
along with the cellular matrix. Unlike bacteria, in eukaryotes there is a
definite time gap between when the genome is duplicated and when it is
physically separated. Therefore, for precise halving of the duplicated genome
into two, it is required that each pair of duplicated chromosomes, termed sister
chromatids, should be paired together in a binary fashion from the moment they
are generated. This pairing function between the duplicated genome is primarily
provided by a multimeric protein complex, called cohesin. Thus, genome integrity
largely depends on cohesin as it ensures faithful chromosome segregation by
holding the sister chromatids glued together from S phase to anaphase. In this
review, we have discussed the life cycle of cohesin during both mitotic and
meiotic cell divisions including the structure and architecture of cohesin
complex, relevance of cohesin associated proteins, mechanism of cohesin loading
onto the chromatin, cohesion establishment and the mechanism of cohesin
disassembly during anaphase to separate the sister chromatids. We have also
focused on the role of posttranslational modifications in cohesin biology. For
better understanding of the complexity of the cohesin regulatory network to the
readers, we have presented an interactome profiling of cohesin core subunits in
budding yeast during mitosis and meiosis. Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of
some metazoan genes require the association of cohesins with chromosomes.
Cohesins are deposited by a conserved heterodimeric loading complex composed of
the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4
deposition complex regulates the spatiotemporal association of cohesin with
chromosomes is not understood. We examined Scc2 chromatin association during the
cell division cycle and found that the affinity of Scc2 for chromatin increases
biphasically during the cell cycle, increasing first transiently in late G1
phase and then again later in G2/M. Inactivation of Scc2 following DNA
replication reduces cellular viability, suggesting that this post S-phase
increase in Scc2 chromatin binding affinity is biologically relevant.
Interestingly, high and low Scc2 chromatin binding levels correlate strongly
with the presence of full-length or amino-terminally cleaved forms of Scc2,
respectively, and the appearance of the cleaved Scc2 species is promoted in
vitro either by treatment with specific cell cycle-staged cellular extracts or
by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2-Scc4 physical
interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is
defective for cohesin deposition. These observations suggest a previously
unidentified mechanism for the spatiotemporal regulation of cohesin association
with chromosomes through cell cycle regulation of Scc2 cohesin deposition
activity by Scc2 dephosphorylation and cleavage. |
Which is the neurodevelopmental disorder associated to mutations in the X- linked gene mecp2? | The neurodevelopmental disorder named Rett syndrome, originally termed as cerebroatrophic hyperammonemia. Although most exclusively affects females, has also been found in male patients. | Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett
syndrome (RTT), an X-linked domit neurodevelopmental disorder characterized
by a period of stagnation followed by regression in the development of young
girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT,
seven families with possible familial RTT and five sporadic females with
features suggestive, but not diagnostic of RTT. Long distance PCR coupled with
long-read direct sequencing was employed to sequence the entire MECP2 gene
coding region in all cases. Mutations were identified in 44/55 (80%) unrelated
classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases
with suggestive but non-diagnostic features of RTT. Twenty-one different
mutations were identified (12 missense, four nonsense and five frame-shift
mutations); 14 of these were novel. All missense mutations were located either
in the methyl-CpG-binding domain or in the transcription repression domain. Nine
recurrent mutations were characterized in a total of 33 unrelated cases (73% of
all cases with MECP2 mutations). Significantly milder disease was noted in
patients carrying missense mutations as compared with those with truncating
mutations ( P = 0. 0023), and milder disease was associated with late as
compared with early truncating mutations ( P = 0.0190). Rett syndrome (RTT) is an X-linked domit neurodevelopmental disorder
affecting 1/10,000-15,000 girls. The disease-causing gene was identified as
MECP2 on chromosome Xq28, and mutations have been found in approximately 80% of
patients diagnosed with RTT. Numerous mutations have been identified in de novo
and rare familial cases, and they occur primarily in the methyl-CpG-binding and
transcriptional-repression domains of MeCP2. Our first diagnostic strategy used
bidirectional sequencing of the entire MECP2 coding region. Subsequently, we
implemented a two-tiered strategy that used denaturing high-performance liquid
chromatography (DHPLC) for initial screening of nucleotide variants, followed by
confirmatory sequencing analysis. If a definite mutation was not identified,
then the entire MECP2 coding region was sequenced, to reduce the risk of false
negatives. Collectively, we tested 228 unrelated female patients with a
diagnosis of possible (209) or classic (19) RTT and found MECP2 mutations in 83
(40%) of 209 and 16 (84%) of 19 of the patients, respectively. Thirty-two
different mutations were identified (8 missense, 9 nonsense, 1 splice site, and
14 frameshifts), of which 12 are novel and 9 recurrent in unrelated patients.
Seven unclassified variants and eight polymorphisms were detected in 228
probands. Interestingly, we found that T203M, previously reported as a missense
mutation in an autistic patient, is actually a benign polymorphism, according to
parental analysis performed in a second case identified in this study. These
findings highlight the complexities of missense variant interpretation and
emphasize the importance of parental DNA analysis for establishing an etiologic
relation between a possible mutation and disease. Overall, we found a 98.8%
concordance rate between DHPLC and sequence analyses. One mutation initially
missed by the DHPLC screening was identified by sequencing. Modified conditions
subsequently enabled its detection, underscoring the need for multiple optimized
conditions for DHPLC analysis. We conclude that this two-tiered approach
provides a sensitive, robust, and efficient strategy for RTT molecular
diagnosis. Rett syndrome (RTT) is a neurodevelopmental disorder, which almost exclusively
affects girls, who, after an initial period of apparently normal development,
display gradual loss of speech and purposeful hand use, gait abnormalities and
stereotypical hand movements. In the year 2000, mutations in the gene for the
methyl CpG binding protein 2, MECP2, have been identified in 35-80% of the
patients in three different studies. We have identified 15 different MECP2
mutations in 26 of 30 Danish RTT patients. The mutations included five novel
mutations (one point mutation, three smaller deletions involving identical
regions in the gene, and one duplication). In contrast to the point mutations
and the duplication, which all affect the methyl binding domain or the
transcriptional repressing domain, the three overlapping deletions are clustered
in the 3' end of the gene. We found no consistent correlation between the type
of mutation and the clinical presentation of the patient or the X-inactivation
pattern in peripheral blood. Our high mutation detection rate, compared to two
of the previous studies, underscores the importance of the inclusion criteria of
the patients and supports that MECP2 is the most important, if not the only,
gene responsible for RTT. Rett Syndrome (RTT) is an X-linked domit neurodevelopmental disorder, which
almost exclusively affects girls, with an estimated prevalence of one in
10,000-15,000 female births. Mutations in the methyl CpG binding protein 2 gene
(MECP2) have been identified in roughly 75% of classical Rett girls. The vast
majority of Rett cases (99%) are sporadic in origin, and are due to de novo
mutations. We collected DNA samples from 50 Italian classical Rett girls, and
screened the MECP2 coding region for mutations by denaturing high-performance
liquid chromatography (DHPLC) and subsequent direct sequencing. DHPLC is a
recently developed method for mutation screening which identifies heteroduplexes
formed in DNA samples containing mismatches between wild type and mutant DNA
strands, combining high sensitivity, reduced cost per run, and high throughput.
In our series, 19 different de novo MECP2 mutations, eight of which were
previously unreported, were found in 35 out of 50 Rett girls (70%). Seven
recurrent mutations were characterized in a total of 22 unrelated cases. Initial
DHPLC screening allowed the identification of 17 out of 19 different mutations
(90%); after optimal conditions were established, this figure increased to 100%,
with all recurrent MECP2 mutations generating a characteristic chromatographic
profile. Detailed clinical data were available for 27 out of 35 mutation
carrying Rett girls. Milder disease was detectable in patients carrying nonsense
mutation as compared to patients carrying missense mutations, although this
difference was not statistically significant (P = 0.077). Mutations in the methyl-CpG-binding protein 2 gene (MECP2) are identified in the
majority of females with Rett syndrome (RTT), an X-linked domit
neurodevelopmental disorder. We searched for mutations by sequencing the MECP2
coding region in 45 sporadic cases (35 with classic RTT, eight with variant
forms and two males) and in seven families with two or more affected females.
Following our previous report of mutations in two families and eight sporadic
cases, we here present 18 additional mutations. We found 13 single nucleotide
substitutions, all of which are C-->T transitions at CpG hot spots. Frameshift
mutations, leading to premature termination of translation, include two single
guanine (G) nucleotide deletions from a stretch of contiguous Gs, a novel four
nucleotide deletion, a novel 32 nucleotide deletion in the C-terminal domain and
a novel complex duplication/deletion rearrangement in the same region. When
X-chromosome inactivation patterns were compared in 16 MECP2 mutation-positive
and 23 mutation-negative samples, no significant differences were observed. The
mutational spectrum in our subject population is similar to studies from around
the world. Of over 300 MECP2 mutations reported, two-thirds are truncating
mutations and one-third are missense mutations, mostly in the methyl-binding
domain. Nearly 70% of all identified mutations are C-->T transitions at one of
eight CpG hot spots, and about 10% are intragenic deletions or complex
rearrangements that lead to frameshifts in the C-terminal region. The rate of
mutation detection in the MECP2 coding region ranges from 70 to 85% in
clinically diagnosed RTT and is much lower in diagnostic variants. Rett syndrome is an X-linked domit neurodevelopmental disorder caused by
mutations in the MECP2 gene. Mutations have been demonstrated in more than 80%
of females with typical features of Rett syndrome. We identified mutations in
the MECP2 gene and documented the clinical manifestations in 65 Rett syndrome
patients to characterize the genotype-phenotype spectrum. Bidirectional
sequencing of the entire MECP2 coding region was performed. We diagnosed 65
patients with MECP2 mutations. Of these, 15 mutations had been reported
previously and 13 are novel. Two patients have multiple deletions within the
MECP2 gene. Eight common mutations were found in 43 of 65 patients (66.15%). The
majority of patients with identified mutations have the classic Rett phenotype,
and several had atypical phenotypes. MECP2 analysis identified mutations in
almost all cases of typical Rett syndrome, as well as in some with atypical
phenotypes. Eleven (20.4%) of the 54 patients with defined mutations and in whom
phenotypic data were obtained did not develop acquired microcephaly. Hence,
microcephaly at birth or absence of acquired microcephaly does not obviate the
need for MECP2 analysis. We have initiated cascade testing starting with PCR
analysis for common mutations followed by sequencing, when necessary. Analysis
of common mutations before sequencing the entire gene is anticipated to be the
most efficacious strategy to identify Rett syndrome gene mutations. Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects
almost exclusively girls. Mutations in the X-linked methyl-CpG-binding protein 2
gene (MECP2) have been found to be a cause. In order to study the spectrum of
MECP2 mutations in Chinese patients, we employed PCR and sequencing of the
coding region of MECP2 gene in 31 Chinese cases of classical sporadic RTT.
Mutations in MECP2 were found in about 55%. Twelve different mutations in exon 3
were identified in 17 of these 31 patients; two of these are novel. A novel
missense variant was detected in the C-terminal region in a patient and her
father who was normal. In addition, there was a single nucleotide variant in the
3'UTR. Rett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused
by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this
study, the MECP2 sequences in 121 unrelated Chinese patients with classical or
atypical RTT were screened for deletions and mutations. In all, we identified 45
different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation
(15.7%) was the most common, followed in order of frequency by p. R168X (11.8%),
p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p.
R306C (3.9%). In addition, we identified five novel MECP2 mutations: three
missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and
one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all
identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare
(0.9%). The remaining cases without MECP2 mutations were screened for the
cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance
liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in
exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation
detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases
and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with
homozygous mutations and normal female karyotypes. However, we did not pinpoint
a significant relationship between genotype and phenotype in these cases. Rett syndrome (RTT), an X-linked domit neurodevelopmental disorder in
females, is caused mainly by de novo mutations in the methyl-CpG-binding protein
2 gene (MECP2). Here we report mutation analysis of the MECP2 gene in 87
patients with RTT from the Czech and Slovak Republics, and Ukraine. The
patients, all girls, with classical RTT were investigated for mutations using
bi-directional DNA sequencing and conformation sensitive gel electrophoresis
analysis of the coding sequence and exon/intron boundaries of the MECP2 gene.
Restriction fragment length polymorphism analysis was performed to confirm the
mutations that cause the creation or abolition of the restriction site.
Mutation-negative cases were subsequently examined by multiple
ligation-dependent probe amplification (MLPA) to identify large deletions.
Mutation screening revealed 31 different mutations in 68 patients and 12
non-pathogenic polymorphisms. Six mutations have not been previously published:
two point mutations (323T>A, 904C>T), three deletions (189_190delGA,
816_832del17, 1069delAGC) and one deletion/inversion
(1063_1236del174;1189_1231inv43). MLPA analysis revealed large deletions in two
patients. The detection rate was 78.16%. Our results confirm the high frequency
of MECP2 mutations in females with RTT and provide data concerning the mutation
heterogeneity in the Slavic population. OBJECTIVE: Rett syndrome (RTT) is a neurodevelopmental disorder which affects
1/10,000 girls. The aim of this study is to delineate the molecular
characteristics of Rett syndrome in China based on the largest group of Chinese
patients ever studied.
METHODS: In all, 365 Chinese patients with Rett syndrome were recruited.
Clinical information including the family reproductive history was collected
through interviewing patients and their parents as well as questionnaires.
MECP2, CDKL5, FOXG1 mutational analysis was performed using polymerase chain
reaction (PCR), direct sequencing and multiplex ligation-dependent probe
amplification (MLPA). The parental origin of mutated MECP2 gene, the MECP2 gene
mutation rate in the patients' mothers, and the X-chromosome inactivation
pattern of the mothers who carry the mutation were also analyzed.
RESULTS: Almost all of the patients were sporadic cases except one pair of
twins. The pregcy loss in probands' mothers and sex ratio of offspring in
probands(') generation were available in 352 families and were comparable to the
general population. Out of the 365 cases, 315 had MECP2 gene mutations and 3 had
de novo CDKL5 gene mutations. No patients had FOXG1 mutation. Among the 315
cases with MECP2 mutations, 274 were typical cases and 41 were atypical cases.
All the 3 cases with CDKL5 gene mutations were atypical RTT with early-onset
seizures. The analysis of parental origin of mutated MECP2 gene were performed
on 139 cases, 90 (64.7%) cases were informative for the study. The result showed
94.4% cases with mutations from paternal origin and 5.6% from maternal origin.
Among the cases with paternal mutation, 90.6% had point mutations. C > T was the
most common one, accounting for 85.7% of the point mutations. Only one normal
phenotype mother (0.41%) carried the same p.R133C mutation of MECP2 gene as her
daughter with mild phenotype. The different patterns of X-chromosome
inactivation in the mother and the daughter may explain their different
phenotypes.
CONCLUSION: The high rate of paternal origin of the mutated MECP2 gene may
explain the high occurrence of RTT in female gender. The family cases of RTT are
rare and the recurrence risk of RTT is very low in China. Only 0.41% (1/244)
mothers carry the pathogenic gene. FOXG1 mutations were not found in this group
of Chinese patients. |
What is the treatment of Riedel disease (thyroiditis)? | Riedel thyroiditis is a rare disorder related to a systemic extracervical fibrotic process of unknown origin. The tratment of choice is the surgical treatment: Corticosteroids may be also useful | Chronic invasive fibrous thyroiditis (Riedel's struma) is a very rare disease of
unknown aetiology mainly affecting middle-aged or old female patients. An
aggressive fibrosis partly or totally replaces normal thyroid gland tissue. The
gland becomes stony hard, is not displaceable and, characteristically, the
fibrous tissue penetrates the capsule and infiltrates into surrounding
structures such as muscles, vessels, nerves and even the trachea. Riedel's
struma is often associated with fibrotic processes in other parts of the body.
The preoperative differential diagnosis includes maligt tumours and fibrosing
stages of Hashimoto's disease, as well as subacute thyroiditis de Quervain. This
paper reports the case of a 60-year-old woman suffering from Riedel's struma and
discusses differential diagnostic aspects with regard to preoperative
investigation and pathohistology. Riedel's invasive fibrous thyroiditis is a rare disorder of unknown origin with
progressive extension and invasion of adjacent structures. Clinically it is
impossible to distinguish between Riedel's/thyroiditis and other diseases as
undifferentiated carcinoma, Hashimoto's disease etc... The patients often have
dyspnea, dysphagia, paralysis of the vocal cord; fine needle puncture-aspiration
and biopsy themselves may be insufficient, Surgical treatment depends on the
stage of the disease, when both lobes are involved generous wedge resection of
the isthmus may be the treatment of choice to relieve tracheal compression; in
earlier stages radical operation are considered. Corticosteroid treatment in
Riedel's thyroiditis, as multifocal disease has been successfully used. Other
drugs with antifibrosing actions have also utilised in small groups of patients
with encouraging results. BACKGROUND: Invasive fibrous thyroiditis (IFT) is the rarest form of
thyroiditis, and reports are often limited to case reports and small case
series. In this study, we aimed to summarize our institutional experience with
IFT since 1976.
METHODS: We retrospectively reviewed the cases of all patients with IFT
evaluated at Mayo Clinic, Rochester, Minnesota, from 1976 through 2008, with
special emphasis on clinical presentation, associated risk factors, associated
comorbid conditions, complications, and treatment.
RESULTS: Twenty-one patients met our inclusion criteria of (i) IFT confirmed by
pathologic review at our institution and (ii) evidence of extension of fibrosis
outside the thyroid capsule. Most patients (17, 81%) were women (mean age, 42
years). Presenting symptoms included pain (24%), dysphagia (33%), vocal cord
paralysis (29%), and tracheal narrowing (48%). Three patients had associated
hypoparathyroidism. Sixteen (76%) had a history of tobacco use, and 10 (48%)
were current smokers. Fibrosing mediastinitis was present in four, orbital
fibrosis in one, retroperitoneal fibrosis in three, and pancreatic fibrosis in
one (38% had extracervical fibrotic processes). Eighteen patients had partial
thyroidectomy, 7 (39%) of whom had surgical complications involving vocal cords
and parathyroid. Two required tracheostomy. Thirteen had corticosteroid therapy;
six received tamoxifen. There was no cause-specific mortality, and the fibrotic
process stabilized or partially resolved in all patients.
CONCLUSIONS: IFT often is associated with a systemic extracervical fibrotic
process and tobacco use. Attempted thyroid resection often results in
postoperative complications. Long-term follow-up showed no deaths from IFT and
showed stability of the thyroiditis. Patients with IgG4-related disease (IgG4-RD) typically have elevated serum
concentrations of IgG4 and share histopathologic features that are similar
across affected organ(s). IgG4-RD patients frequently require prolonged
treatment with glucocorticoids and are often unable to taper these medications.
Traditional disease-modifying antirheumatic drugs (DMARDs) are generally
ineffective. We assessed the clinical and serologic responses to B lymphocyte
depletion therapy in 10 consecutive patients with steroid- and DMARD-refractory
IgG4-RD.Ten patients with IgG4-RD were treated with rituximab (RTX) (2 infusions
of 1000 mg, 15 days apart). Clinical improvement was assessed by monitoring the
patient's ability to taper prednisone to discontinuation and to stop DMARDs; by
serial measurements of total IgG and IgG subclasses; and by follow-up radiologic
assessments guided by the patient's particular pattern of organ involvement. We
also developed and retrospectively applied the IgG4-RD Disease Activity Index
and Flare Tool.Organ involvement included the pancreas, biliary tree, aorta,
salivary glands (submandibular and parotid), lacrimal glands, lymph nodes,
thyroid gland, and retroperitoneum. Nine of 10 patients demonstrated striking
clinical improvement within 1 month of starting RTX. One patient with advanced
thyroid fibrosis associated with Riedel thyroiditis and a history of disease in
multiple other organ systems did not have improvement in the thyroid gland, but
the disease did not progress to involve new organs. All 10 patients were able to
discontinue prednisone and DMARDs following RTX therapy. Significant decreases
in IgG concentrations were observed for the IgG4 subclass only. Four patients
were re-treated with RTX after 6 months because of either symptom recurrence and
increasing IgG4 concentration at the time of peripheral B cell reconstitution (n
= 2) or because of physician discretion (n = 2). Repeated courses of RTX
maintained their effectiveness and resulted in further decreases in IgG4
concentrations. In patients who had an increased IgG4 concentration at the time
of presentation, the level of serum IgG4 appeared to be a reliable measure of
disease activity.IgG4-RD is an idiopathic, multiorgan inflammatory disease in
which diverse organ manifestations are linked by characteristic histopathologic
and immunohistochemical features. Treatment with RTX led to prompt clinical and
serologic improvement in refractory IgG4-RD in all patients with active
inflammation. Serial treatments with RTX may lead to progressive declines in
serum IgG4 concentrations and better disease control. Serum IgG4 concentrations
may remain low, and clinical disease activity may remain quiescent even after B
cell reconstitution in a significant proportion of patients. |
What is the link between Dax1 and Esrrb? | Dax1 associates with Esrrb and regulates its function in embryonic stem cells | Self-renewal capacity and pluripotency, which are controlled by the
Oct3/4-centered transcriptional regulatory network, are major characteristics of
embryonic stem (ES) cells. Nuclear hormone receptor Dax1 is one of the crucial
factors in the network. Here, we identified an orphan nuclear receptor, Esrrb
(estrogen-related receptor beta), as a Dax1-interacting protein. Interaction of
Dax1 and Esrrb was mediated through LXXLL motifs of Dax1 and the activation- and
ligand-binding domains of Esrrb. Furthermore, Esrrb enhanced the promoter
activity of the Dax1 gene via direct binding to Esrrb-binding site 1 (ERRE1,
where "ERRE" represents "Esrrb-responsive element") of the promoter. Expression
of Dax1 was suppressed followed by Oct3/4 repression; however, overexpression of
Esrrb maintained expression of Dax1 even in the absence of Oct3/4, indicating
that Dax1 is a direct downstream target of Esrrb and that Esrrb can regulate
Dax1 expression in an Oct3/4-independent manner. We also found that the
transcriptional activity of Esrrb was repressed by Dax1. Furthermore, we
revealed that Oct3/4, Dax1, and Esrrb have a competitive inhibition capacity for
each complex. These data, together with previous findings, suggest that Dax1
functions as a negative regulator of Esrrb and Oct3/4, and these molecules form
a regulatory loop for controlling the pluripotency and self-renewal capacity of
ES cells. |
What is dovitinib? | Dovitinib (TKI258) is a tyrosine kinase receptor inhibitor with potent activity against fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). | PURPOSE: There is no standard of therapy for the treatment of Waldenström
macroglobulinemia (WM), therefore there is a need for the development of new
agents. Fibroblast growth factor receptor 3 (FGFR3) was shown to play a major
role in several types in cancer. Dovitinib, an inhibitor of FGFR3, was effective
in hematologic maligcies. In this study, we tested FGFR3 as a therapeutic
target in WM and tested the effect of dovitinib on cell proliferation and
apoptosis of WM cells in the context of BM microenvironment.
METHODS: The expression of FGFR3 in WM cells was tested using immunofluorescence
and flow cytometry. Cell signaling in response to stimulation with FGF3 and
stromal cells, and its inhibition by dovitinib was performed using
immunoblotting. Cell survival and cell proliferation were assessed by MTT and
BrdU assays. Apoptosis was measured by detection of APO-2.7 and cleavage of
caspase-3 using flow cytometry. Cell cycle was performed by PI staining of cells
and flow cytometry. The combinatory effect of dovitinib with other drugs was
analyzed using Calcusyn software. The effect of dovitinib was tested in vivo.
RESULTS: FGFR3 was overexpressed in WM cells and its activation induced cell
proliferation. Inhibition of FGFR3 with dovitinib decreased cell survival,
increased apoptosis, and induced cell cycle arrest. Inhibition of FGFR3 by
dovitinib reduced the interaction of WM to bone marrow components, and reversed
its proliferative effect. Dovitinib had an additive effect with other drugs.
Moreover, dovitinib reduced WM tumor progression in vivo.
CONCLUSION: We report that FGFR3 is a novel therapeutic target in WM, and
suggest dovitinib for future clinical trial the treatment of patients with WM. PURPOSE: Dovitinib (TKI258) is an orally available inhibitor of fibroblast
growth factor (FGF), VEGF, and platelet-derived growth factor receptors. This
phase I/II dose-escalation study was conducted to evaluate the safety,
pharmacodynamics, and preliminary efficacy of dovitinib in the treatment of
advanced melanoma.
EXPERIMENTAL DESIGN: Patients with advanced melanoma resistant or refractory to
standard therapies or for whom no standard therapy was available were enrolled.
Dovitinib was administered at doses ranging from 200 to 500 mg/d.
RESULTS: Forty-seven patients were enrolled. The most frequently reported
adverse events were fatigue (77%; grade ≥3, 28%), diarrhea (77%; grade ≥3, 11%),
and nausea (77%; grade ≥3, 9%). Six dose-limiting toxicities were observed in
the 400-mg and 500-mg dose cohorts, which consisted of grade 3 nausea, fatigue,
and diarrhea and grade 4 fatigue events. The maximum tolerated dose was 400
mg/d. The best tumor response was stable disease, which was observed in 12
patients. Increases in plasma FGF23, VEGF, and placental growth factor and
decreases in soluble VEGF receptor 2 were noted during the first cycle of
treatment, consistent with FGF receptor (FGFR) and VEGF receptor (VEGFR)
inhibition. Dynamic contrast-enhanced MRI analysis showed a dose-dependent
decrease in tumor blood flow and vascular permeability with dovitinib therapy. A
decrease in FGFR phosphorylation was observed in paired tumor biopsy samples
from a patient treated with dovitinib at a dose of 400 mg/d.
CONCLUSIONS: At a dose of 400 mg/d, dovitinib showed an acceptable safety
profile and limited clinical benefit and inhibited FGFR and VEGFR. The multiple kinase inhibitor dovitinib is currently under clinical
investigation for hepatocellular carcinoma (HCC). Here, we investigated the
mechanistic basis for the effects of dovitinib in HCCs. Dovitinib showed
significant antitumor activity in HCC cell lines PLC5, Hep3B, Sk-Hep1, and
Huh-7. Dovitinib downregulated phospho-STAT3 (p-STAT3) at tyrosine 705 and
subsequently reduced the levels of expression of STAT3-related proteins Mcl-1,
survivin, and cyclin D1 in a time-dependent manner. Ectopic expression of STAT3
abolished the apoptotic effect of dovitinib, indicating that STAT3 is
indispensable in mediating the effect of dovitinib in HCC. SHP-1 inhibitor
reversed downregulation of p-STAT3 and apoptosis induced by dovitinib, and
silencing of SHP-1 by RNA interference abolished the effects of dovitinib on
p-STAT3, indicating that SHP-1, a protein tyrosine phosphatase, mediates the
effects of dovitinib. Notably, dovitinib increased SHP-1 activity in HCC cells.
Incubation of dovitinib with pure SHP-1 protein enhanced its phosphatase
activity, indicating that dovitinib upregulates the activity of SHP-1 via direct
interactions. In addition, dovitinib induced apoptosis in two
sorafenib-resistant cell lines through inhibition of STAT3, and
sorafenib-resistant cells showed significant activation of STAT3, suggesting
that targeting STAT3 may be a useful approach to overcome drug resistance in
HCC. Finally, in vivo, dovitinib significantly suppressed growth of both Huh-7
and PLC5 xenograft tumors and downregulated p-STAT3 by increasing SHP-1
activity. In conclusion, dovitinib induces significant apoptosis in HCC cells
and sorafenib-resistant cells via SHP-1-mediated inhibition of STAT3. Hepatocellular carcinoma (HCC) often displays resistance to recombit tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
Dovitinib, a multiple tyrosine kinase inhibitor, and tigatuzumab, a novel
humanized anti-human death receptor 5 (DR5) agonistic antibody, are both under
clinical investigations in HCC. Here, we report that dovitinib sensitizes
resistant HCC cells to TRAIL- and tigatuzumab-induced apoptosis through
inhibition of signal transducers and activators of transcription 3 (STAT3). Our
data indicate that HCC cells showed significant resistance to TRAIL- and
tigatuzumab-induced apoptosis. The combination of dovitinib and tigatuzumab
restored the sensitivity of HCC cells to TRAIL- and tigatuzumab-induced
apoptosis. Dovitinib down-regulated phospho-STAT3 (Tyr705) (p-STAT3) and
subsequently reduced the protein levels of STAT3-regulated proteins, Mcl-1,
survivin and cylcin D1, in TRAIL-treated HCC cells. Knockdown of STAT3 by
RNA-interference overcame apoptotic resistance to TRAIL in HCC cells, and
ectopic expression of STAT3 in HCC cells abolished the sensitizing effect of
dovitinib on TRAIL-induced apoptosis. Importantly, silencing SHP-1 by
RNA-interference reduced the effects of dovitinib and TRAIL on p-STAT3 and
apoptosis, whereas co-treatment of TRAIL and dovitinib increased the activity of
SHP-1. Moreover, in vivo the combination of tigatuzumab and dovitinib inhibited
Huh-7 xenograft tumor growth. In conclusion, dovitinib sensitizes resistant HCC
cells to TRAIL- and tigatuzumab-induced apoptosis through a novel machinery:
SHP-1 dependent STAT3 inhibition. PURPOSE: This study investigated the metabolism and excretion of dovitinib
(TKI258), a tyrosine kinase inhibitor that inhibits fibroblast, vascular
endothelial, and platelet-derived growth factor receptors, in patients with
advanced solid tumors.
METHODS: Four patients (cohort 1) received a single 500 mg oral dose of
(14)C-dovitinib, followed by the collection of blood, urine, and feces for
≤10 days. Radioactivity concentrations were measured by liquid scintillation
counting and plasma concentrations of dovitinib by liquid chromatography-tandem
mass spectrometry. Both techniques were applied for metabolite profiling and
identification. A continuous-dosing extension phase (nonlabeled dovitinib 400 mg
daily) was conducted with the 3 patients from cohort 1 and 9 additional patients
from cohort 2.
RESULTS: The majority of radioactivity was recovered in feces (mean 61 %; range
52-69 %), as compared with urine (mean 16 %; range 13-21 %). Only 6-19 % of the
radioactivity was recovered in feces as unchanged dovitinib, suggesting high
oral absorption. (14)C-dovitinib was eliminated predomitly via oxidative
metabolism, with prominent primary biotransformations including hydroxylation on
the fluorobenzyl ring and N-oxidation and carbon oxidation on the
methylpiperazine moiety. Dovitinib was the most prominent radioactive component
in plasma. The high apparent volume of distribution (2,160 L) may indicate that
dovitinib distributes extensively to tissues. Adverse events were predomitly
mild to moderate, and most common events included nausea, vomiting,
constipation, diarrhea, and fatigue.
CONCLUSIONS: Dovitinib was well absorbed, extensively distributed, and
eliminated mainly by oxidative metabolism, followed by excretion, predomitly
in feces. The adverse events were as expected for this class of drug. BACKGROUND: Dovitinib is a receptor tyrosine kinase (RTK) inhibitor targeting
vascular endothelial growth factor receptors, fibroblast growth factor receptors
and platelet-derived growth factor receptor β. Dovitinib is currently in
clinical trials for the treatment of hepatocellular carcinoma (HCC).
METHOD: In this study, we used five HCC cell lines and five endothelial cell
lines to validate molecular and cellular targets of dovitinib.
RESULTS: Tumor growth and pulmonary metastasis were significantly suppressed in
an orthotopic HCC model. Immunoblotting revealed that among known dovitinib
targets, only PDGFR-β was expressed in two HCC cell lines, while four of five
endothelial lines expressed PDGFR-β, FGFR-1, and VEGFR-2. Dovitinib inhibited
endothelial cell proliferation and motility at 0.04 μmol/L, a pharmacologically
relevant concentration; it was unable to inhibit the proliferation or motility
of HCC cells at the same concentration. Immunohistochemical analyses showed that
dovitinib significantly decreased the microvessel density of xenograft tumors,
inhibiting proliferation and inducing apoptosis in HCC cells.
CONCLUSION: Our findings indicate that dovitinib inhibits HCC growth and
metastasis preferentially through an antiangiogenic mechanism, not through
direct targeting of HCC cells. PURPOSE: Signaling through the fibroblast growth factor (FGF) pathway may
account for tumor resistance to antiangiogenic therapies targeting the VEGF
pathway. Here, dovitinib (TKI258), a potent oral inhibitor of FGF receptor, VEGF
receptor (VEGFR), and platelet-derived growth factor receptor tyrosine kinases,
is studied in a dose escalation trial.
EXPERIMENTAL DESIGN: Patients with advanced or metastatic renal cell carcinoma
(RCC) with predomit clear cell histology were treated with oral dovitinib 500
or 600 mg/day (5-days-on/2-days-off schedule).
RESULTS: Twenty heavily pretreated patients (median 3 prior regimens) were
enrolled, with 16, 11, and 12 patients having previously received at least 1:
VEGFR inhibitor, mTOR inhibitor, and immunotherapy, respectively. Fifteen and 5
patients were treated in 500- and 600-mg cohorts, respectively. Three patients
experienced dose-limiting toxicities: grade 2 bradycardia (500 mg), grade 4
hypertensive crisis (600 mg), and grade 3 asthenia with grade 2 nausea and
vomiting (600 mg). The most common adverse events related to dovitinib were
nausea (75%), diarrhea (70%), vomiting (70%), and asthenia (50%), the majority
of which were mild (grade 1 or 2), with grade 3 events 5% or less (except
asthenia, 15%) and only one grade 4 event (hypertensive crisis). Two patients
achieved a partial response (500 mg), and 12 patients had stable disease,
including 2 patients with long lasting disease stabilizations (>1 year) in the
500-mg cohort.
CONCLUSIONS: Dovitinib was tolerable and showed antitumor activity at a maximum
tolerated dose of 500 mg on a 5-days-on/2-days-off schedule in heavily
pretreated RCC patients. Dovitinib is an oral multitargeted kinase inhibitor with potent activity against
receptors for vascular endothelial growth factor, platelet-derived growth
factor, and basic fibroblast growth factor. Initial phase 1 to 2 studies of
dovitinib using a continuous daily dosing schedule has shown that dovitinib
exhibits a prolonged and overproportional increase in dose and exposure
relationship above 400 mg/d. To address this, intermittent dosing schedules were
explored using a model-based approach. A semi-mechanistic population
pharmcokinetic/pharmacodynamic (PD) model was developed from 4 dovitinib phase 1
studies with daily dosing schedules. Autoinduction of cytochrome P450 1A (CYP1A)
responsible for dovitinib metabolism was described using an indirect response
model. Simulation of dovitinib plasma concentration profiles following 4
intermittent dosing schedules suggested that intermittent dosing could prevent
prolonged drug accumulation. Based on the simulated plasma profiles, PD
response, and patient compliance, a 5-days-on/2-days-off intermittent dosing
schedule was selected for a phase 1 to 2 clinical study. The observed dovitinib
plasma concentrations in this study confirmed the model predictions.
Furthermore, dovitinib was well tolerated, and antitumor activity was observed
as well in this new study. The 5-days-on/2-days-off dosing schedule is currently
used in a dovitinib registration trial and other clinical trials. Mutations in fibroblast growth factor receptor 2 (FGFR2) have been recently
described as a molecular-specific feature in endometrial carcinomas and the
presence of activated FGFR2 mutations is associated with poor prognosis. For
that reason, inhibition of FGFR2 could be a therapeutic target in the treatment
of endometriod carcinomas. In this work, we investigated the antitumoral
activity of dovitinib (a multiple kinase inhibitor) in human endometrial cancer
cell (ECC) lines. We found that dovitinib caused cell growth arrest, loss of
clonogenic growth, and cell-cycle arrest in FGFR2-mutated ECCs in in vitro and
in vivo experiments. Next, we investigated the mechanistic basis of dovitinib
effects. We could determine that dovitinib modified expression levels of
well-known key cell-cycle regulatory proteins that induce cellular senescence.
To further investigate the role of dovitinib, we analyzed its effect on estrogen
receptor α (ER-α) expression. Surprisingly, we discovered that dovitinib
enhances ER-α expression in FGFR2-mutant ECCs. Because blocking one signaling
pathway is often not sufficient to cause total tumor regression and the
effectiveness of individual inhibitors is often short-lived, we examined the
impact of targeting FGFR2 with dovitinib in combination with a selective ER
antagonist, fulvestrant (ICI182.780). Combination of dovitinib plus ICI182.780
resulted in a significantly higher inhibition of cell growth than dovitinib
treatment alone. These findings suggest that combinatory therapies using
dovitinib plus ICI182.780 treatment can be truly effective in patients with
endometrial carcinomas carrying FGFR2 mutations. BACKGROUND: An unmet medical need exists for patients with metastatic renal cell
carcinoma who have progressed on VEGF-targeted and mTOR-inhibitor therapies.
Fibroblast growth factor (FGF) pathway activation has been proposed as a
mechanism of escape from VEGF-targeted therapies. Dovitinib is an oral
tyrosine-kinase inhibitor that inhibits VEGF and FGF receptors. We therefore
compared dovitinib with sorafenib as third-line targeted therapies in patients
with metastatic renal cell carcinoma.
METHODS: In this multicentre phase 3 study, patients with clear cell metastatic
renal cell carcinoma who received one previous VEGF-targeted therapy and one
previous mTOR inhibitor were randomly assigned through an interactive voice and
web response system to receive open-label dovitinib (500 mg orally according to
a 5-days-on and 2-days-off schedule) or sorafenib (400 mg orally twice daily) in
a 1:1 ratio. Randomisation was stratified by risk group and region. The primary
endpoint was progression-free survival (PFS) assessed by masked central review.
Efficacy was assessed in all patients who were randomly assigned and safety was
assessed in patients who received at least one dose of study drug. This study is
registered with ClinicalTrials.gov, number NCT01223027.
FINDINGS: 284 patients were randomly assigned to the dovitinib group and 286 to
the sorafenib group. Median follow-up was 11·3 months (IQR 7·9-14·6). Median PFS
was 3·7 months (95% CI 3·5-3·9) in the dovitinib group and 3·6 months (3·5-3·7)
in the sorafenib group (hazard ratio 0·86, 95% CI 0·72-1·04; one-sided p=0·063).
280 patients in the dovitinib group and 284 in the sorafenib group received at
least one dose of study drug. Common grade 3 or 4 adverse events included
hypertriglyceridaemia (38 [14%]), fatigue (28 [10%]), hypertension (22 [8%]),
and diarrhoea (20 [7%]) in the dovitinib group, and hypertension (47 [17%]),
fatigue (24 [8%]), dyspnoea (21 [7%]), and palmar-plantar erythrodysaesthesia
(18 [6%]) in the sorafenib group. The most common serious adverse event was
dyspnoea (16 [6%] and 15 [5%] in the dovitinib and sorafenib groups,
respectively).
INTERPRETATION: Dovitinib showed activity, but this was no better than that of
sorafenib in patients with renal cell carcinoma who had progressed on previous
VEGF-targeted therapies and mTOR inhibitors. This trial provides reference
outcome data for future studies of targeted inhibitors in the third-line
setting.
FUNDING: Novartis Pharmaceuticals Corporation. BACKGROUND: Everolimus (mammalian target of rapmaycin (mTOR) inhibitor) and
dovitinib (vascular endothelial growth factor (VEGF) and fibroblast growth
factor 2 (FGF-2) inhibitor) demonstrate activity in metastatic clear cell renal
cancer. The combination of these agents has a broad spectrum of relevant
activity. The combination is explored in this phase Ib study.
METHODS: Patients with metastatic clear cell renal cancer who have failed VEGF
targeted therapy were eligible. Up to four cohorts of three to six patients (3+3
design) were treated with escalating doses of everolimus and dovitinib.
Dose-limiting toxicities (DLTs) were assessed to determine the maximum tolerated
dose (MTD). An expansion cohort (n=15) was investigated to obtain additional
efficacy information. Sequential fluorodeoxyglucose positron emission tomography
(FDG-PET) was used as a surrogate marker of response.
RESULTS: Overall 18 patients were recruited into the study. Fifteen patients
received the MTD, which was everolimus 5mg orally (PO) once daily (OD) and
dovitinib 200mg PO day 1-5/7. The MTD was associated with toxicity, which
included fatigue, mucositis and diarrhoea in 73%, 53% and 53% (Common Toxicity
Criteria (CTC) grade 1-4) of patients, respectively. Frequent biochemical
abnormalities occurred (such as hypertriglyceridaemia in 67%). Higher doses of
the combination were not tolerable due to grade 3 fatigue in 2/3 patients and
grade 3 nausea in 1/3 patients within 1 month of therapy. The response rate at
the MDT was 1/15 (7%) while the progression free survival for the MTD was 7
months (95% confidence interval (CI) 2.2-11 months). Pharmacokinetic data at the
MTD showed stable kinetics with time.
CONCLUSION: Dovitinib and everolimus had modest activity, but did not meet all
of the planned efficacy end-points. Fatigue was the dose limiting toxicity. |
Are DNA helicases involved in progeroid syndromes? | Yes, mutations in genes coding for DNA helicases were found to induce progeroid syndromes, such as Werner syndrome (WS) or Bloom syndrome (BS). | Single-gene mutations can produce human progeroid syndromes--phenotypes that
mimic usual or "normative" aging. These can be divided into two classes--those
that have their impacts upon multiple organs and tissues (segmental progeroid
syndromes) and those that have their major impacts upon a single organ or tissue
(unimodal progeroid syndromes). The prototypic example of the former is the
Werner syndrome, a condition caused by mutations of the RecQ family of DNA
helicases. Research on the Werner syndrome and a surprising number of other
progeroid syndromes support the importance of the maintece of genomic
stability as a partial antidote to aging. The prototypic examples of the latter
are Alzheimer type dementias. The three gene products that cause rare
autosomal-domit early-onset varieties of these disorders all participate in
the modulation of the beta amyloid precursor protein. They thus support the
importance of the maintece of proper protein processing and folding as a
partial antidote to aging. The molecular mechanisms leading to human senescence are still not known mostly
because of the complexity of the process. Different research approaches are used
to study ageing including studies of monogenic segmental progeroid syndromes.
None of the known progerias represents true precocious ageing. Some of them,
including Werner (WS), Bloom (BS), and Rothmund-Thomson syndromes (RTS) as well
as combined xeroderma pigmentosa-Cockayne syndrome (XP-CS) are characterised by
features resembling precocious ageing and the increased risk of maligt
disease. Such phenotypes result from the mutations of the genes encoding
proteins involved in the maintece of genomic integrity, in most cases DNA
helicases. Defective functioning of these proteins affects DNA repair,
recombination, replication and transcription. Other segmental progeroid
syndromes, such as Hutchinson-Gilford progeria (HGPS) and Cockayne syndrome are
not associated with an increased risk of cancer. In this paper we present the
clinical and molecular features of selected progeroid syndromes and describe the
potential implications of these data for studies of ageing and cancer
development. Progeroid syndromes (PSs) constitute a group of disorders characterized by
clinical features mimicking physiological aging at an early age. In some of
these syndromes, biological hallmarks of aging are also present, whereas in
others, a link with physiological aging, if any, remains to be elucidated. These
syndromes are clinically and genetically heterogeneous and most of them,
including Werner syndrome and Hutchinson-Gilford progeria, are known as
'segmental aging syndromes', as they do not feature all aspects usually
associated to physiological aging. However, all the characterized PSs enter in
the field of rare monogenic disorders and several causative genes have been
identified. These can be separated in subcategories corresponding to (i) genes
encoding DNA repair factors, in particular, DNA helicases, and (ii) genes
affecting the structure or post-translational maturation of lamin A, a major
nuclear component. In addition, several animal models featuring premature aging
have abnormal mitochondrial function or signal transduction between membrane
receptors, nuclear regulatory proteins and mitochondria: no human pathological
counterpart of these alterations has been found to date. In recent years,
identification of mutations and their functional characterization have helped to
unravel the cellular processes associated to segmental PSs. Recently, several
studies allowed to establish a functional link between DNA repair and A-type
lamins-associated syndromes, evidencing a relation between these syndromes,
physiological aging and cancer. Here, we review recent data on molecular and
cellular bases of PSs and discuss the mechanisms involved, with a special
emphasis on lamin A-associated progeria and related disorders, for which
therapeutic approaches have started to be developed. |
List the diseases for which there are point-of-care breath tests | Point of care breath tests are available for lung cancer, pulmonary embolism, respiratory distress syndrome, methanol intoxication, kidney diseases, liver diseases, Helicobacter pylori infection, asthma, sepsis, heart failure, diabetes and tuberculosis. | HYPOTHESIS: We hypothesized that late pulmonary dead space fraction (Fd(late))
would be a useful tool to screen for pulmonary embolism (PE) in a group of
surgical patients, including patients who required mechanical ventilation and
patients with adult respiratory distress syndrome.
DESIGN: We prospectively calculated Fd(late) in patients with suspected PE who
underwent pulmonary angiography.
SETTING: University-based, level I trauma center.
MAIN OUTCOME MEASURE: Ability of Fd(late) to identify patients with PE.
RESULTS: Twelve patients had 14 angiograms for suspected PE. The Fd(late) was
0.12 or above in all 5 patients who had PE; 4 required mechanical ventilation.
The Fd(late) values were below 0.12 in 8 of 9 patients without PE. Four patients
had adult respiratory distress syndrome. The Fd(late) had 100% sensitivity and
89% specificity for the detection of PE.
CONCLUSIONS: The Fd(late) is a valuable tool for bedside screening of PE in
surgical patients. We were able to accurately detect all PEs. A portable Fourier transform infrared (FT-IR) multicomponent point-of-care
analyzer was tested for the diagnosis of methanol intoxications. Breath analysis
with FT-IR was fast and easy, and no sample preparation was needed. The analyzer
was adequately sensitive and accurate in detecting and quantitating clinically
relevant amounts of ethanol and methanol in the breath of seriously ill
patients. FT-IR spectrometry was also suitable for nearly on-line monitoring of
the exhaled ethanol and methanol during hemodialysis. The breath analysis
results correlated well with blood samples. The FT-IR method used also has a
traceable calibration to physical properties of the analyte, and the measured
spectra can be saved for later analysis. The 13C-urea breath test provides accurate, noninvasive diagnosis of active
Helicobacter pylori infection and can document posttherapy cure. This study
evaluated point-of-care testing with onsite sample analysis with the use of a
desktop infrared spectrophotometer. Ambulatory patients (N=320) underwent
13C-urea breath testing, and breath samples were analyzed immediately by clinic
staff with no prior breath testing experience. Duplicate samples were sent to a
reference laboratory, and the results of both methods were compared.
Point-of-care testing was simple, with an overall agreement of 99.1%. Accurate
near-patient 13C-urea breath testing is now practical in the primary care
setting even when done by inexperienced personnel. In the daily clinical routine at the bedside, information on effective pulmonary
blood flow (PBF) is limited and requires invasive monitoring, including a
pulmonary artery catheter, to determine both cardiac output and intrapulmonary
shunt. Therefore we evaluated a non-invasive method for the measurement of PBF
in a clinical setting, including 12 patients with acute respiratory failure
(acute respiratory distress syndrome) undergoing prone positioning. PBF was
determined before (baseline), during and after prone positioning, by using a
foreign gas rebreathing method with a new photoacoustic gas analyser. Values
were compared with the cardiac output corrected for intrapulmonary shunt
(COeff). Responders to prone positioning were defined according to the
improvement of arterial oxygenation. A total of 84 measurements were performed.
PBF values correlated well with COeff (R2=0.96; P<0.0001). Bias and limits of
agreement (+/- 2 S.D.) for all measurements were -0.11 +/- 0.76 litre/min. At
baseline, responders showed significantly lower PBF levels than non-responders
(4.8 +/- 1.0 compared with. 6.4 +/- 1.2 litre/min; P=0.03). During prone
positioning, PBF increased continuously in responders and remained high after
patients had been returned to the supine position. PBF was unaffected in
non-responders. Mean total increase in PBF was 1.2 +/- 0.2 litre/min in
responders compared with -0.4 +/- 0.2 litre/min in non-responders (P<0.0001). In
conclusion, the investigated rebreathing system allows for a non-invasive
determination of PBF at the bedside. The accuracy of the measurements is
comparable with the thermodilution method. It is able to reliably reflect
changes in PBF induced by prone positioning. Moreover, measuring PBF might be a
promising tool to identify responders to prone therapy. STUDY OBJECTIVE: To compare the diagnostic performance of volumetric capnography
(VCap), which is the plot of the expired CO(2) partial pressure against the
expired volume during a single breath, with the PaCO(2) to end-tidal CO(2)
(EtCO(2)) gradient, in the case of suspected pulmonary embolism (PE).
DESIGN: Single-center, prospective study.
SETTING: Emergency department of a teaching hospital.
PATIENTS: A total of 45 outpatients with positive enzyme-linked immunosorbent
assay d-dimer levels of > 500 ng/mL. The diagnosis of PE was confirmed in 18
outpatients according to a validated procedure based on the
ventilation-perfusion lung scan and/or spiral CT scanning.
INTERVENTIONS: Curves of VCap were obtained from a compact monitor connected to
a computer. A sequence of four to six stable breaths allowed the calculation of
the following several variables: alveolar dead space fraction; the ratio of
alveolar dead space (VDalv) to airway dead space (VDaw); the VDalv to
physiologic dead space (VDphys) fraction; the slope of phase 3; and the late
dead space fraction (Fdlate) corresponding to the extrapolation of the
capnographic curve to a volume of 15% of the predicted total lung capacity.
RESULTS: The mean (+/- SD) PaCO(2)-EtCO(2) gradient was 5.3 +/- 0.7 mm Hg in the
PE-positive group and 2.8 +/- 0.7 mm Hg in the PE-negative group (p = 0.019).
Four variables of the VCap exhibited a statistical difference between both
groups, as follows: the VDalv/VDaw fraction(;) the slope of phase 3; the
VDalv/VDphys fraction; and the Fdlate, which was 8.2 +/- 3.3% vs -7.7 +/- 2.8%,
respectively (p = 0.000011). The diagnostic performance expressed as the mean
area under a receiver operating characteristic curve comparison was 75.9 +/-
7.4% for the PaCO(2)-EtCO(2) gradient and 87.6 +/- 4.9% for the Fdlate (p =
0.02).
CONCLUSION: Fdlate, a variable of VCap, had a statistically better diagnostic
performance in suspected PE than the PaCO(2)-EtCO(2) gradient. VCap is a
promising computer-assisted bedside application of pulmonary pathophysiology.
Future research should define the place of this technique in the diagnostic
workup of PE, especially in the presence of positive d-dimers. The mass spectrometer (MS) traditionally has been the instrument of choice for
measuring cardiac output (Q (T)) non-invasively using the foreign gas uptake
method. However, the size and cost of the MS has hampered widespread adoption of
this technique outside of the laboratory. Here, we present results, from six
normal human subjects at rest and during exercise, of simultaneous Q (T)
measurements by an MS and a new, portable infrared (IR) device developed in our
laboratories. These measurements are made using on the open-circuit acetylene
uptake method. The IR device measures inspired and end-tidal concentrations of
acetylene, sulfur hexafluoride, and carbon dioxide by IR absorption spectroscopy
with a 10-90% response time of 43 ms; accurate measurements were made down to
sample flow rates of 50 mL min(-1). Excellent correlation [Q (T)(IR)=0.98 Q
(T)(MS), R(2)=0.94] was observed between instruments across the range from rest
to heavy exercise. These results suggest that the IR device, which is small,
light-weight, and rugged may enable the foreign gas uptake method to be used in
clinical, field, and point-of-care settings for Q (T) measurement. BACKGROUND: Hepatologists have long sought to develop a test for assessing liver
function, but this aim has been stalled by the complexity of the liver and its
diverse functions. Results of metabolic tests, including breath tests, correlate
with clinical and histological parameters of patients with liver disorders;
however, these tests tend to be cumbersome and impractical for everyday use. The
recent development of a real-time, point-of-care liver function breath test has
made it straightforward to assess the metabolic function of the liver.
AIM: To review the available data on the use of breath tests for assessing liver
reserve in various conditions and their application in various clinical
hepatology settings.
RESULTS: The (13)C-methacetin breath test enables accurate follow-up of patients
with acute or chronic liver damage, where overall hepatic function is
significantly suppressed by known causes of liver disorders, including acute,
sub-acute or chronic conditions. The metabolic breath test can detect both
gradual and spontaneous improvements in liver function and the effects of
treatment.
CONCLUSIONS: Breath testing that provides continuous quantification of
methacetin metabolism may be a sensitive tool for the diagnosis and follow-up of
patients with liver disorders. OBJECTIVES: A noninvasive method to assess ventilation may aid in management of
children with acute asthma. The purpose of this study was to evaluate the
association between end-tidal carbon dioxide (EtCO2) values and disease severity
among children with acute asthma.
METHODS: This was a prospective, blinded, observational study of children 3-17
years old treated for acute asthma in a pediatric emergency department (ED).
EtCO2 measurements were taken before the initiation of therapy and after each
nebulization treatment (maximum of three). Peak expiratory flow rate (PEFR),
Pediatric Asthma Severity Score (PASS), oxygen saturation, and disposition were
recorded. Treating physicians, unaware of the EtCO2 results, made all treatment
decisions, including disposition.
RESULTS: One hundred children were enrolled. The mean initial EtCO2 value was 35
mm Hg (95% confidence interval = 34.3 to 36.1 mm Hg). The mean disposition EtCO2
value was 33.3 mm Hg (95% confidence interval = 32.6 to 34.4 mm Hg). PEFR
measures were completed on 43 patients and PASS recorded on 100 patients. There
was an overall trend toward lower EtCO2 values during treatment (p < 0.01).
Sixteen patients were admitted. Initial EtCO2 values were lower among children
admitted to the hospital (35.6 mm Hg vs. 32.9 mm Hg; Mann-Whitney U test; p <
0.02). EtCO2 values at disposition did not differ between groups based on PEFR,
PASS, or hospital admission.
CONCLUSIONS: Noninvasive bedside measurement of EtCO2 values among children with
acute asthma is feasible. EtCO2 values did not distinguish children with mild
disease from those with more severe disease. Further data are needed to clarify
the association between EtCO2 values and other indicators of disease severity,
particularly in children with more severe disease. Rapid immunochromatographic tests for Helicobacter pylori infection have been
developed to allow "near-patient" testing. We therefore performed a pilot study
to test a rapid immunochromatographic stool antigen test for the diagnosis of H.
pylori infection in asymptomatic children. We tested stool specimens collected
from children participating in a cohort study in the United States and Mexico.
H. pylori-positive status was defined by positivity on at least 2 tests: a
commercial H. pylori stool antigen enzyme immunoassay, an immunoglobulin G
antibody enzyme immunoassay, and the C-urea breath test. Negative H. pylori
status was defined by negative findings of all of these tests. Of 52 children
(22 girls, 30 boys) 25 were H. pylori-positive, 19 H. pylori-negative, and 8
uncertain (eg, presumably negative; positive findings on 1 of the 3 noninvasive
tests). The sensitivity and specificity of the new stool antigen test for those
with definite H. pylori status were 100% (exact 95% CI 86.3%-100% and
82.4%-100%, respectively). This rapid stool antigen test may prove useful for
point-of-care testing and epidemiological field studies. Larger prospective
studies are needed in symptomatic and asymptomatic children for more precise
estimates. BACKGROUND: Functional residual capacity (FRC) measurements may help to guide
respiratory therapy. Using the oxygen washout technique, FRC can be assessed at
bedside during spontaneous breathing. High repeatability, crucial for
monitoring, has not been shown in ventilated patients. A large step change of
inspiratory fraction of oxygen (FiO(2)) (DeltaFiO(2)) may impede the clinical
use in patients ventilated with high FiO(2). We investigated the repeatability
of FRC measurements and the impact of different DeltaFiO(2) on this
repeatability.
METHODS: The LUFU system (Draeger Medical, Luebeck, Germany) estimates FRC by
oxygen washout, a variant of multiple-breath-nitrogen-washout during a fast
DeltaFiO(2). In 20 postoperative cardiac surgery patients, FRC was measured in
duplicate using DeltaFiO(2) of 0.1, 0.2, and 0.6.
RESULTS: There were no differences between repeated measurements of FRC, neither
using a DeltaFiO(2) of 0.1, 0.2 nor 0.6(Delta0.1: 2.62 L +/- 0.58, 2.62 L +/-
0.59, P = 0.995; Delta0.2: 2.70 L +/- 0.59, 2.66 L +/- 0.56, P = 0.258;
Delta0.6: 2.61 L +/- 0.58, 2.59 L +/- 0.58, P = 0,639). Coefficients of
variation were 6.6%, 5.6%, and 6.6%, respectively.
CONCLUSIONS: FRC can be measured in ventilated patients using the oxygen washout
technique with a clinically acceptable repeatability. Repeatability is not
significantly influenced whether using a DeltaFiO(2) of 0.1, 0.2, or 0.6. BACKGROUND: Exhaled NO (FE(NO)) is a useful biomarker for the monitoring of
asthma control and response to therapy. However, there is a lack of data on
FE(NO) levels and their interpretation in Primary Care asthma population
depending on their treatment and smoking habit. Besides, the majority of current
FE(NO) tests have been done by stationary chemiluminescence analysers whose use
is limited to research laboratories.
METHODS: FE(NO) measurements by the novel hand-held NO monitoring device (NIOX
MINO) were made in 96 asthma patients (32 males, mean age 53+/-12) at five local
General Practices during their scheduled 15-20 min visits for lung function
assessment.
RESULTS: Success rate was 78% and the intra-subject coefficient of variation was
8.7%. Inhaled corticosteroid treatment had an overall reducing effect on the
FE(NO) value (30.5 [19.8-49.8]) vs. patients not on the ICS (26.5 [19-94])
(p<0.044). FE(NO) levels in the ICS treated current or ex-smokers group of
patients were still significantly above the normal values (p<0.0001). FE(NO)
levels were similar in patients receiving ICS whether there were current,
ex-smokers or non-smokers. The highest FE(NO) levels (94 [15.8-151]) were found
in asthmatic current smokers and not receiving treatment with ICS. The most
"normalised" FE(NO) levels (35.3 [13.5-35.3]) were seen in ex-smokers.
CONCLUSIONS: FE(NO) measurements performed with a new hand-held monitoring
device are reproducible and feasible in General Practice in the majority of
patients of different ages and asthma severities. A high percentage of patients
with different severities of asthma and regardless of their treatment with ICS
and current smoking habit (current and/or ex-smokers) had highly elevated FE(NO)
values, suggesting that their current therapy was possibly insufficient to
control the underlying degree of airway inflammation and asthma symptoms. BACKGROUND: Cardiac Output (CO) is an important parameter in the diagnosis and
therapy of heart diseases. Inert gas rebreathing (IGR) and continuous wave
Doppler ultrasound (CWD) are among the most promising newer techniques aiming at
a non-invasive, point of care measurement of CO. A direct comparison of the two
methods has not yet been carried out.
METHODS: 63 consecutive patients were included in the study. CO was measured
twice with both CWD and IGR to assess inter-method agreement and
reproducibility. The statistical comparisons were performed as proposed by Bland
and Altman.
RESULTS: There was a significant correlation between the CO measurements by both
methods (r=0.53, p<0.001). Bland-Altman analysis showed a good agreement of
measurements with a bias of 0.4+/-1.0 l/min (mean+/-standard deviation). Both
methods showed a good reproducibility. CWD measurements were not possible in 14%
of patients while IGR measurements were not possible in 5% of patients (p=0.13).
CONCLUSION: The determination of CO by IGR and CWD revealed a good agreement and
reproducibility with a low rate of impossible measurements, suggesting that IGR
and CWD can be used interchangeably in the clinical setting. End tidal carbon dioxide tension (P(ET,CO(2))) is a surrogate for dead space
ventilation which may be useful in the evaluation of pulmonary embolism (PE). We
aimed to define the optimal P(ET,CO(2)) level to exclude PE in patients
evaluated for possible thromboembolism. 298 patients were enrolled over 6 months
at a single academic centre. P(ET,CO(2)) was measured within 24 h of
contrast-enhanced helical computed tomography, lower extremity duplex or
ventilation/perfusion scan. Performance characteristics were measured by
comparing test results with clinical diagnosis of PE. PE was diagnosed in 39
(13%) patients. Mean P( ET,CO(2)) in healthy volunteers did not differ from P(
ET,CO(2)) in patients without PE (36.3+/-2.8 versus 35.5+/-6.8 mmHg). P(ET,CO(2
)) in patients with PE was 30.5+/-5.5 mmHg (p<0.001 versus patients without PE).
A P(ET,CO(2)) of >or=36 mmHg had optimal sensitivity and specificity (87.2 and
53.0%, respectively) with a negative predictive value of 96.6% (95% CI
92.3-98.5). This increased to 97.6% (95% CI 93.2-99.) when combined with Wells
score <4. A P(ET,CO(2)) of >or=36 mmHg may reliably exclude PE. Accuracy is
augmented by combination with Wells score. P( ET,CO(2)) should be prospectively
compared to D-dimer in accuracy and simplicity to exclude PE. BACKGROUND: Liver failure has remained a major cause of mortality after
hepatectomy, but it is difficult to predict preoperatively. This study describes
the introduction into clinical practice of the new LiMAx test and provides an
algorithm for its use in the clinical management of hepatic tumours.
METHODS: Patients with hepatic tumours and indications for hepatectomy were
investigated perioperatively with the LiMAx test. In one patient, analysis of
liver volume was carried out with preoperative three-dimensional virtual
resection.
RESULTS: A total of 329 patients with hepatic tumours were evaluated for
hepatectomy. Blinded preoperative LiMAx values were significantly higher before
resection (n= 139; mean 351 microg/kg/h, range 285-451 microg/kg/h) than before
refusal (n= 29; mean 299 microg/kg/h, range 223-376 microg/kg/h; P= 0.009).
In-hospital mortality rates were 38.1% (8/21 patients), 10.5% (2/19 patients)
and 1.0% (1/99 patients) for postoperative LiMAx of <80 microg/kg/h, 80-100
microg/kg/h and >100 microg/kg/h, respectively (P < 0.0001). A decision tree was
developed to avoid critical values and its prospective preoperative application
revealed a reduction in mortality from 9.4% to 3.4% (P= 0.019).
DISCUSSION: The LiMAx test can validly determine liver function capacity and is
feasible in every clinical situation. Combination with virtual resection could
enable the calculation of residual liver function. The LiMAx decision tree
algorithm for hepatectomy might significantly improve preoperative evaluation
and postoperative outcome in liver surgery. BACKGROUND/AIMS: Insulin resistance (IR) plays an important role in the
pathogenesis of diabetes and non-alcoholic fatty liver disease (NAFLD). Current
methods for insulin resistance detection are cumbersome, or not sensitive enough
for early detection and follow-up. The BreathID system can continuously analyse
breath samples in real-time at the point-of-care. Here we determined the
efficacy of the BreathID using the 13C-Glucose breath test (GBT) for evaluation
of insulin resistance.
METHODS: Twenty healthy volunteers were orally administered 75 mg of 13C-glucose
1-13C. An oral glucose tolerance test (OGTT) was performed immediately; followed
by serum glucose and insulin level determinations using GBT. GBT and OGTT were
repeated following exercise, which alters insulin resistance levels.
RESULTS: Within-subject correlations of GBT parameters with serum glucose and
serum insulin levels were high. Before and after exercise, between-subjects
correlations were high between the relative insulin levels and the % dose
recoveries at 90 min (PDR 90), and the cumulative PDRs at 60 min (CPDR 60).
Pairwise correlations were identified between pre-exercise Homeostasis Model
Assessment (HOMA) IR at 90 min and PDR 90; HOMA B (for beta cell function) 120
and CPDR 30; HOMA IR 60 and peak time post-exercise; and HOMA B 150 with PDR
150.
CONCLUSIONS: The non-invasive real-time BreathID GBT reliably assesses changes
in liver glucose metabolism, and the degree of insulin resistance. It may serve
as a non-invasive tool for early diagnosis and follow up of patients in
high-risk groups. Today, the assessment of liver function in patients suffering from acute or
chronic liver disease is based on liver biopsy and blood tests including
synthetic function, liver enzymes and viral load, most of which provide only
circumstantial evidence as to the degree of hepatic impairment. Most of these
tests lack the degree of sensitivity to be useful for follow-up of these
patients at the frequency that is needed for decision making in clinical
hepatology. Accurate assessment of liver function is essential to determine both
short- and long-term prognosis, and for making decisions about liver and
non-liver surgery, TIPS, chemoembolization or radiofrequency ablation in
patients with chronic liver disease. Liver function tests can serve as the basis
for accurate decision-making regarding the need for liver transplantation in the
setting of acute failure or in patients with chronic liver disease. The liver
metabolic breath test relies on measuring exhaled (13) C tagged methacetin,
which is metabolized only by the liver. Measuring this liver-specific substrate
by means of molecular correlation spectroscopy is a rapid, non-invasive method
for assessing liver function at the point-of-care. The (13) C methacetin breath
test (MBT) is a powerful tool to aid clinical hepatologists in bedside
decision-making. Our recent findings regarding the ability of point-of-care (13)
C MBT to assess the hepatic functional reserve in patients with acute and
chronic liver disease are reviewed along with suggested treatment algorithms for
common liver disorders. BACKGROUND: Sepsis is one of the main causes of death in adult intensive care
units. The major drawbacks of the different methods used for its diagnosis and
monitoring are their inability to provide fast responses and unsuitability for
bedside use. In this study, performed using a rat sepsis model, we evaluate
breath analysis with Ion Mobility Spectrometry (IMS) as a fast, portable and
non-invasive strategy.
METHODS: This study was carried out on 20 Sprague-Dawley rats. Ten rats were
injected with lipopolysaccharide from Escherichia coli and ten rats were IP
injected with regular saline. After a 24-h period, the rats were anaesthetized
and their exhaled breaths were collected and measured with IMS and SPME-gas
chromatography/mass spectrometry (SPME-GC/MS) and the data were analyzed with
multivariate data processing techniques.
RESULTS: The SPME-GC/MS dataset processing showed 92% accuracy in the
discrimination between the two groups, with a confidence interval of between
90.9% and 92.9%. Percentages for sensitivity and specificity were 98%
(97.5-98.5%) and 85% (84.6-87.6%), respectively. The IMS database processing
generated an accuracy of 99.8% (99.7-99.9%), a specificity of 99.6% (99.5-99.7%)
and a sensitivity of 99.9% (99.8-100%).
CONCLUSIONS: IMS involving fast analysis times, minimum sample handling and
portable instrumentation can be an alternative for continuous bedside
monitoring. IMS spectra require data processing with proper statistical models
for the technique to be used as an alternative to other methods. These animal
model results suggest that exhaled breath can be used as a point-of-care tool
for the diagnosis and monitoring of sepsis. The burden of pediatric asthma remains high with one-third of patients being
under- or overtreated because of the unique challenges in the assessment and
management of childhood asthma. Until recently, there has been no point of care
tool for assessing the underlying airway inflammation (i.e., inflammometry) in
asthma. Recently, fractional exhaled nitric oxide (FeNO) has emerged as an
important biomarker for the assessment and management of asthma. Recent evidence
indicates that FeNO identifies T-helper cell type 2 (Th2)–mediated airway
inflammation with a high positive and negative predictive value for identifying
corticosteroid responsive airway inflammation. This article examines the
evidence for FeNO as a predictor of Th2-mediated inhaled corticosteroid (ICS)
responsive airway inflammation and reviews recent studies evaluating the role of
FeNO, whether helpful or not, in the assessment and management of pediatric
asthma. FeNO is a reliable adjunct to traditional tests in the assessment of
suspected asthma. Importantly, it is useful for identifying and for excluding
ICS-responsive airway inflammation. Although individual study results have
varied, collectively, asthma managed using FeNO is associated with lower
exacerbation rates compared with clinical algorithms alone. Finally, FeNO may be
useful in identifying patients at risk for future impairment or loss of asthma
control during reduction/cessation of ICS treatment. FeNO testing has an
important role in the assessment of pediatric patients with suspected asthma and
in the management of pediatric patients with established asthma. Additional
studies will continue to define the exact role of FeNO testing in pediatric
asthma. Early diagnosis of sepsis is a difficult problem for intensivists and new
biomarkers for early diagnosis have been difficult to come by. Here we discuss
the potential of adapting a technology from the electronics industry, surface
acoustic wave (SAW) sensors, for diagnosis of multiple markers of sepsis in real
time, using non-invasive assays of exhaled breath condensate. The principles and
advantages of the SAW technology are reviewed as well as a proposed plan for
adapting this flexible technology to early sepsis detection. Until recently, no point-of-care tool was available for assessing the underlying
airway inflammation associated with asthma. Fractional exhaled nitric oxide
(FeNO) emerged in the last decade as an important biomarker for asthma
assessment and management. Evidence also indicates that FeNO is most accurately
classified as a marker of T-helper cell type 2 (Th2)-mediated airway
inflammation with a high positive and negative predictive value for identifying
corticosteroid-responsive airway inflammation. This manuscript evaluates the
evidence for FeNO as a predictor of Th2-mediated corticosteroid-responsive
airway inflammation and presents the results of a meta-analysis of three adult
studies comparing asthma exacerbation rates with FeNO-based versus
clinically-based asthma management algorithms, one of which was not included in
a 2012 Cochrane meta-analysis. The primary purpose of the updated meta-analysis
was to evaluate asthma exacerbation rates. The results demonstrate that the rate
of exacerbations was significantly reduced in favor of FeNO-based asthma
management (mean treatment difference = -0.27; 95% CI [-0.42, -0.12] as was the
relative rate of asthma exacerbations (relative rate = 0.57; 95% CI [0.41,
0.80]). In summary, FeNO has value for identifying patients with airway
inflammation who will and will not respond to corticosteroids. Importantly, the
use of FeNO in conjunction with clinical parameters is associated with
significantly lower asthma exacerbation rates compared with asthma managed using
clinical parameters alone. Together these data indicate that FeNO testing has an
important role in the assessment and management of adult asthma. Further studies
will continue to define the exact role of FeNO testing in adult asthma. Continuous monitoring of clinical biomarkers offers the exciting possibility of
new therapies that use biomarker levels to guide treatment in real time. This
review explores recent progress toward this goal. We initially consider
measurements in body fluids by a range of analytical methods. We then discuss
direct tissue measurements performed by implanted sensors; sampling techniques,
including microdialysis and ultrafiltration; and noninvasive methods. A future
directions section considers analytical methods at the cusp of clinical use. A device for measuring human breath ammonia was developed based on a single use,
disposable, inkjet printed ammonia sensor fabricated using polyaniline
oparticles. The device was optimized for sampling ammonia in human breath
samples by addressing issues such as variations in breath sample volume, flow
rate, sources of oral ammonia, temperature and humidity. The resulting system
was capable of measuring ammonia in breath from 40 to 2993 ppbv (r(2 )= 0.99, n
= 3) as correlated with photoacoustic laser spectroscopy and correlation in
normal human breath samples yielded a slope of 0.93 and a Pearson correlation
coefficient of 0.9705 (p < 0.05, n = 11). Measurement of ammonia in the breath
of patients with end-stage kidney disease demonstrated its significant reduction
following dialysis, while also correlating well with blood urea nitrogen (BUN)
(r = 0.61, p < 0.01, n = 96). Excellent intraindividual correlations were
demonstrated between breath ammonia and BUN (0.86 to 0.96), which demonstrates
the possibility of using low cost point of care breath ammonia systems as a
noninvasive means of monitoring kidney dysfunction and treatment. A new non-invasive and potentially inexpensive frontier in the diagnosis of
cancer relies on the detection of volatile organic compounds (VOCs) in exhaled
breath samples. Breath can be sampled and analyzed in real-time, leading to
fascinating and cost-effective clinical diagnostic procedures. Nevertheless,
breath analysis is a very young field of research and faces challenges, mainly
because the biochemical mechanisms behind the cancer-related VOCs are largely
unknown. In this review, we present a list of 115 validated cancer-related VOCs
published in the literature during the past decade, and classify them with
respect to their "fat-to-blood" and "blood-to-air" partition coefficients. These
partition coefficients provide an estimation of the relative concentrations of
VOCs in alveolar breath, in blood and in the fat compartments of the human body.
Additionally, we try to clarify controversial issues concerning possible
experimental malpractice in the field, and propose ways to translate the basic
science results as well as the mechanistic understanding to tools (sensors) that
could serve as point-of-care diagnostics of cancer. We end this review with a
conclusion and a future perspective. |
Is there association of matrix metalloproteinases with behaviour of pituitary adenomas? | Yes, there is evidence to suggest that matrix metalloproteinases are associated with more aggressive of pituitary adenomas. | Beside the digestion of the extracellular matrix during tumor invasion and
metastasis, more recently, new functions for matrix metalloproteinases (MMPs)
have been proposed. We studied the expression and function of these enzymes in
pituitary cells. We observed the activities of MMP-2 and MMP-9 together with
expression of membrane-type MMP and tissue inhibitor of metalloproteinase-1 in
all types of human pituitary adenomas. We found surprisingly high levels of MMP
activity and low levels of tissue inhibitor of metalloproteinases, indicating a
high level of extracellular matrix-degrading activity in pituitary adenomas. To
examine the function of metalloproteinase activity in pituitary cells we used
the synthetic MMP inhibitor batimastat. These studies demonstrate that MMPs
secreted by pituitary cells can release growth factors anchored to the
extracellular matrix that, in turn, control pituitary cell proliferation and
hormone secretion. These results define a new additional mechanism for the
control of pituitary hormone secretion and indicate new potential therapeutic
targets for pituitary adenomas. The matrix metalloproteinases (MMPs) are a family of zinc-containing
endopeptidases that are able to degrade the extracellular matrix and allow
angiogenesis and tumor invasion. The vast majority of pituitary tumors are
benign and do not metastasize to distant sites, although they may invade
locally. The aim of this study was to determine whether expression of the
collagenase MMP-9 may play a role in allowing angiogenesis and invasion by
different pituitary tumor types. Tumor expression of MMP-9 was investigated
using a monoclonal antibody on a series of well-characterized paraffin-embedded
sections of pituitary tumors. Invasive macroprolactinomas (n = 11) were
significantly more likely to express MMP-9 than noninvasive macroprolactinomas
(n = 8) (P = 0.003). Invasive macroprolactinomas showed higher-density MMP-9
staining than noninvasive tumors (P < 0.05). MMP-9 expression did not differ
between noninvasive tumors and normal pituitary gland, or between different
sized prolactinomas. MMP-9 expression was related to aggressive tumor behavior.
It was higher in invasive macroprolactinomas (P = 0.003) when compared with
noninvasive macroprolactinomas or the normal anterior pituitary gland. In
addition, although there was no difference in whether MMP-9 was present or not
when nonfunctioning adenomas that recurred were compared with those that did
not, samples of recurrent tumor at the second presentation were more likely to
express MMP-9 (P = 0.01). Pituitary carcinomas were significantly more likely to
be MMP-9 positive compared with normal anterior pituitary gland (P = 0.05), but
there was no difference from invasive adenomas. Angiogenesis assessed by
vascular density was related to MMP-9 expression (P < 0.05). In summary, we have
shown the presence of MMP-9 expression in some invasive and recurrent pituitary
adenomas, and in the majority of pituitary carcinoma. The mechanisms whereby
MMP-9 expression influences tumor recurrence and invasiveness, and its
association with angiogenesis, remains to be elucidated. However, these
observations suggest that a future potential therapeutic strategy for some
pituitary tumors may be administration of a synthetic MMP-9 inhibitor. BACKGROUND & OBJECTIVE: Usually pituitary adenomas are histological benign and
grow slowly, but a proportion of them will become locally aggressive, and
develop into invasive pituitary adenomas. The reasons for these differences in
tumor behavior are poorly understood. Pituitary adenomas are abounding blood
vessels. Angiogenesis and tumor invasion both require degradation of the
extracellular matrix components to allow cell migration. The matrix
metalloproteinases (MMPs) and their nature inhibitors-the tissue inhibitors of
metalloproteinases (TIMPs) may play a central role in these processes. The
aggressive mechanism of pituitary adenomas was studied through investigating the
expression of MMP-9, MMP-2, TIMP-1, and TIMP-2 in both invasive and non-invasive
adenomas.
METHODS: Sixty-one surgical removed pituitary adenomas (forty-nine cases
invasive and twelve non-invasive adenomas) were investigated.
Immunohistochemistry staining (SP method) was used to detect the expression of
MMP-9, MMP-2, TIMP-1, and TIMP-2 in two groups. The results were treated with
semi-quantitative method and analyzed by using non-parameter rank sum test.
RESULTS: Immunohistochemical staining of tumor cells for MMP-9, TIMP-1, MMP-2,
and TIMP-2 were noted 95.9% (47/49), 57.1% (28/49), 75.5% (37/49) and 89.8%
(44/49) in invasive adenomas, and 100% (12/12), 91.7% (11/12), 66.7% (8/12), and
91.7% (11/12) in non-invasive adenomas, respectively. Invasive tumors were
significantly less expressing TIMP-1 and TIMP-2 (P < 0.05). There was no
significant difference for MMP-9 or MMP-2 between invasive and non-invasive
groups (P > 0.05).
CONCLUSIONS: TIMP-1 and TIMP-2 may play a key role in invasive pituitary
adenomas to biological behavior. Data on the dural invasiveness of pituitary adenomas have been correlated to the
expression of matrix metalloproteinases (e.g. MMP-9). Serine proteases have not
yet been investigated in human pituitary adenomas. In this study,
paraffin-embedded material from 84 human pituitary adenomas (acromegaly n=18,
Cushing's disease n=21, prolactinoma n=18, thyroid-stimulating hormone-secreting
adenoma n=1, nonsecreting adenoma n=26) and 9 nontumourous anterior pituitary
lobes (obtained from patients with prostate cancer) was immunohistochemically
analysed for expression of MMP-2, MMP-9, tissue inhibitor of
metalloproteinases-2 (TIMP-2), urokinase-type plasminogen activator (uPA), uPA
receptor (uPAR), tissue-type plasminogen activator (tPA), plasminogen activator
inhibitor-1 (PAI-1), and interleukin-6 (IL-6). Cavernous sinus invasion was
determined by assessment of preoperative magnetic resoce imaging and
intraoperative inspection (invasive n=50, noninvasive n=34). In pituitary
adenomas, reactions were positive (diffuse expression) to MMP-2 (74% of cases),
MMP-9 (49%), TIMP-2 (88%), uPA (89%), uPAR (90%), tPA (69%), and PAI-1 (87%). A
weak expression of IL-6 was found in 12% of the adenomas. All reactions were
positive (focal expression) in every sample of anterior lobe tissue, except for
uPA (negative in 3 out of 9 cases), and IL-6 (faintly positive in 5 out of 8
cases). Adenomas showed remarkably greater expression of uPA than anterior lobe
tissue (Chi-square P<0.05). Nonsecreting adenomas exhibited a stronger tendency
towards overexpression of uPA in invasive tumours when compared to noninvasive
adenomas (Chi-square P=0.053). We found no correlation of MMP-9 expression and
tumour invasion. TIMP-2 was overexpressed in noninvasive as compared to invasive
adenomas (Chi-square P<0.05). The interrelationship between MMPs and serine
proteinases in pituitary adenomas remains to be elucidated. From our data, a
correlation between IL-6 and an activation of MMP-9 cannot be proven. The
uPA-system may, however, play a role in dural invasion of pituitary adenomas. The preoperative diagnosis of cavernous sinus invasion remains difficult and
controversial, and there are currently no reliable histological or molecular
markers that predict pituitary tumour behaviour and response to treatment. We
evaluated 45 patients with pituitary adenoma. The results have shown that the
sensitivity of MRI for indicating cavernous sinus invasion in this prospective
study was 60%, specificity 85%, positive predictive value 83.33%, negative
predictive value 62.96%. Forty-five specimens of pituitary adenomas were
analyzed for expression of F8, VEGF, Ki-67, c-myc, bcl-2, nm23 and MMP-9
immunoreactivity using immunoperoxidase staining. MVD was assessed using
F8-related antigen. The results have shown that MVD of invasive pituitary
adenomas was significantly higher than that of noninvasive (P < 0.001). There
was an association between the invasion of pituitary adenomas and Ki-67 LI (P =
0.039) or the expression of VEGF (P < 0.001) and MMP-9 (P < 0.001). But c-myc LI
and bcl-2 expression have no association with invasiveness of pituitary adenomas
(P = 0.061 vs. P = 0.201). On the other hand, there is an inverse relationship
between nm23 expression and tumor invasion (P < 0.001). In conclusion,
parasellar extension of pituitary adenomas through the medial wall of the
cavernous sinus diagnosed at surgery, can be determined by radiology with
sensitive gadolinium-enhanced MRI. Although our study has shown that MVD and the
expression of VEGF, Ki-67, nm23 and MMP-9 have associations with invasiveness of
pituitary adenomas, they are lack of specificity. These markers can only provide
some useful informations on the therapeutic strategy of pituitary adenomas. OBJECT: Matrix metalloproteinase (MMP) 2 and 9 are important for tissue
breakdown in the process of tumor invasion. The aim of this study is to evaluate
the relationship between the expression of MMP-2, MMP-9, MIB-1 LI and cavernous
sinus invasion in pituitary adenomas.
METHODS: Tissue samples from 54 patients with pituitary adenomas were studied.
Expression of MMP-2, MMP-9, and MIB-1 labeling index (LI) were evaluated by
immunohistochemical method. In sixteen cases, the expression of MMP-2 and MMP-9
mRNA was also examined by RT-PCR assay.
RESULTS: Thirty-four patients were women and 20 were men, with a mean age of
49.9 years old (range 18-76 years). There were 12 cases with cavernous sinus
invasion, and 42 were noninvasive cases. MMP-2 and MMP-9 score of invasive case
(3.9 +/- 0.5,4.1 +/- 0.4) were significantly higher than those (2.3 +/- 0.2; p <
0.01; 2.6 +/- 0.2; p < 0.01) without invasion. The MIB-1 LI of this study
presented no significantly difference between the invasive and noninvasive
pituitary adenomas. The percentage of MMP-2 mRNA/beta-actin mRNA and MMP-9
mRNA/beta-actin mRNA were also observed significantly higher in invasive
pituitary adenomas (68.2 +/- 15.3%; 59.7 +/- 12.5%) than noninvasive pituitary
adenomas (21.8 +/- 8.2%, p < 0.05; 33.3 +/- 5.4%, p < 0.05).
CONCLUSIONS: Our study suggests that the expression of MMP-2 and MMP-9 may have
a value to assess the invasive pituitary adenomas, and proliferation and
invasion of pituitary adenomas may present a different mechanism. The extracellular matrix is a three-dimensional network of proteins,
glycosaminoglycans and other macromolecules. It has a structural support
function as well as a role in cell adhesion, migration, proliferation,
differentiation, and survival. The extracellular matrix conveys signals through
membrane receptors called integrins and plays an important role in pituitary
physiology and tumorigenesis. There is a differential expression of
extracellular matrix components and integrins during the pituitary development
in the embryo and during tumorigenesis in the adult. Different extracellular
matrix components regulate adrenocorticotropin at the level of the
proopiomelanocortin gene transcription. The extracellular matrix also controls
the proliferation of adrenocorticotropin-secreting tumor cells. On the other
hand, laminin regulates the production of prolactin. Laminin has a dynamic
pattern of expression during prolactinoma development with lower levels in the
early pituitary hyperplasia and a strong reduction in fully grown prolactinomas.
Therefore, the expression of extracellular matrix components plays a role in
pituitary tumorigenesis. On the other hand, the remodeling of the extracellular
matrix affects pituitary cell proliferation. Matrix metalloproteinase activity
is very high in all types of human pituitary adenomas. Matrix metalloproteinase
secreted by pituitary cells can release growth factors from the extracellular
matrix that, in turn, control pituitary cell proliferation and hormone
secretion. In summary, the differential expression of extracellular matrix
components, integrins and matrix metalloproteinase contributes to the control of
pituitary hormone production and cell proliferation during tumorigenesis. The development of estrogen-induced pituitary prolactinoma in Fischer 344 (F344)
rats is associated with enhanced neovascularization. Based on the significance
of matrix metalloproteinases (MMPs) for tumor growth and angiogenesis, we have
studied the effect of batimastat (BB-94), a synthetic MMPs inhibitor (MMPI) on
the progression of prolactin-secreting pituitary adenoma in rats. Pituitary
tumors were induced in male F344 rats by s.c. implantation of Silastic tubes
containing diethylstilbestrol (DES). The effects of chronic treatment with BB-94
(30 mg/kg b.w.) on pituitary weight, cell proliferation, apoptosis and vascular
density were evaluated. We have stated that chronic treatment with batimastat
caused a significant reduction in the pituitary weight. Batimastat has been
found to decrease cell proliferation evaluated by a number of PCNA-positive
stained cell nuclei. A marked increase in the apoptotic index within the
pituitary was observed in the study group. Moreover, the density of microvessels
identified by CD31 was reduced in the group treated with BB-94. The results of
our study provide evidence for an inhibitory effect of batimastat, a synthetic
MMPI, on the growth and angiogenesis in an experimental model of human
prolactinoma. The ability of BB-94 to suppress established pituitary tumor
growth suggests a possible application of MMPIs in the treatment of pituitary
adenomas. Degradation and remodelling of the extracellular matrix has been investigated,
with the main focus on the balance between matrix metalloproteinases (MMP) and
tissue inhibitor of metalloproteinases (TIMP). Recent reports disclose the
presence of a novel MMP-inhibiting cell membrane-anchored glycoprotein
designated 'reversion-inducing cysteine-rich protein with Kazal motifs' (RECK).
Our main aim in this study was to elucidate the role of RECK in cell invasion of
pituitary adenomas and its contribution to signal transduction. The function of
RECK in cell invasion was investigated by comparing data obtained from
full-length RECK clone transfection and gene silencing with RECK mRNA-targeting
siRNA. RECK expression was confirmed using real-time RT-PCR and Western
blotting. Levels of matrix metalloproteinases (MMP-2 and -9) and TIMP-1 were
measured by zymography and reverse zymography, respectively. Cell invasion was
examined with a 3-D invasion assay. The signal cascade was investigated by cDNA
microarray analysis. As expected, expression of RECK was elevated upon cDNA
transfection, and diminished using siRNA. We observed elevation of MMP-2 and -9
expression and consequent 3-D cell invasion in cells under-expressing RECK.
However, TIMP expression was not affected by RECK. Analysis with cDNA microarray
revealed that RECK additionally upregulates growth hormone-releasing hormone
receptor (GHRHR) and latrophilin 2 at the transcriptional level. Our findings
collectively suggest that RECK regulates the cell signalling pathway, playing a
critical neuroendocrinological role in the pituitary adenoma cell line. Pituitary adenomas exhibit a wide range of behaviors. The prediction of
aggressive or maligt behavior in pituitary adenomas remains challenging;
however, the utility of biomarkers is rapidly evolving. In this review, we
discuss potential biomarkers as they relate to aggressive behavior in pituitary
adenomas. While detailed histological subtyping remains the best independent
predictor of aggressive behavior in the majority of cases, evidence suggests
that the additional analyses of FGFR4, MMP, PTTG, Ki-67, p53, and deletions in
chromosome 11 may contribute to decisions concerning management of aggressive
pituitary adenomas. |
What is the evolutionary process described by the "Muller's ratchet" model? | The vast majority of mutations are deleterious and are eliminated by purifying selection. Yet in finite populations, purifying selection cannot completely prevent the accumulation of deleterious mutations. Muller's ratchet is a paradigmatic model for the accumulation of deleterious mutations in a population of finite size, due to genetic drift. Muller's ratchet suggests that population bottlenecks, which may constrain the genetic diversity of a population, could lead to extinction of all individuals with the least number of deleterious mutations, due to a stochastic fluctuation. When the most-fit class of genotypes is lost, it is lost irreversibly. | Why sex exists remains an unsolved problem in biology. If mutations are on the
average deleterious, a high mutation rate can account for the evolution of sex.
One form of this mutational hypothesis is Muller's ratchet. If the mutation rate
is high, mutation-free individuals become rare and they can be lost by genetic
drift in small populations. In asexual populations, as Muller noted, the loss is
irreversible and the load of deleterious mutations increases in a ratchet-like
manner with the successive loss of the least-mutated individuals. Sex can be
advantageous because it increases the fitness of sexual populations by
re-creating mutation-free individuals from mutated individuals and stops (or
slows) Muller's ratchet. Although Muller's ratchet is an appealing hypothesis,
it has been investigated and documented experimentally in only one group of
organisms--ciliated protozoa. I initiated a study to examine the role of
Muller's ratchet on the evolution of sex in RNA viruses and report here a
significant decrease in fitness due to Muller's ratchet in 20 lineages of the
RNA bacteriophage phi 6. These results show that deleterious mutations are
generated at a sufficiently high rate to advance Muller's ratchet in an RNA
virus and that beneficial, backward and compensatory mutations cannot stop the
ratchet in the observed range of fitness decrease. Muller proposed that an asexual organism will inevitably accumulate deleterious
mutations, resulting in an increase of the mutational load and an inexorable,
ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res.
1, 2-9]. The operation of Muller's ratchet on real populations has been
experimentally demonstrated only in RNA viruses. However, these cases are
exceptional in that the mutation rates of the RNA viruses are extremely high. We
have examined whether Muller's ratchet operates in Salmonella typhimurium, a
DNA-based organism with a more typical genomic mutation rate. Cells were grown
asexually under conditions expected to result in high genetic drift, and the
increase in mutational load was determined. S. typhimurium accumulated mutations
under these conditions such that after 1700 generations, 1% of the 444 lineages
tested had suffered an obvious loss of fitness, as determined by decreased
growth rate. These results suggest that in the absence of sex and with high
genetic drift, genetic mechanisms, such as back or compensatory mutations,
cannot compensate for the accumulation of deleterious mutations. In addition, we
measured the appearance of auxotrophs, which allowed us to calculate an average
spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base
pair per generation. This rate is measured for the largest genetic target
studied so far, a collection of about 200 genes. Theoretical arguments are presented which suggest that each advance of Muller's
ratchet in a haploid asexual population causes the fixation of a deleterious
mutation at a single locus. A similar process operates in a diploid, fully
asexual population under a wide range of parameter values, with respect to
fixation within one of the two haploid genomes. Fixations of deleterious
mutations in asexual species can thus be greatly accelerated in comparison with
a freely recombining genome, if the ratchet is operating. In a diploid with
segregation of a single chromosome, but no crossing over within the chromosome,
the advance of the ratchet can be decoupled from fixation if mutations are
sufficiently close to recessivity. A new analytical approximation for the rate
of advance of the ratchet is proposed. Simulation results are presented that
validate the assertions about fixation. The simulations show that none of the
analytical approximations for the rate of advance of the ratchet are
satisfactory when population size is large. The relevance of these results for
evolutionary processes such as Y chromosome degeneration is discussed. Mitochondria, semi-autonomous organelles possessing their own genetic system,
are commonly accepted to descend from free-living eubacteria, namely
hydrogen-producing alpha-proteobacteria. The progressive loss of genes from the
primitive eubacterium to the nucleus of the eukaryotic cell is strongly
justified by the Muller rachet principle, which postulates that asexual genomes,
like mitochondrial ones, accumulate deleterious and sublethal mutations faster
than sexual genomes, like the nucleus. According to this principle, the
mitochondrial genome would be doomed to death; instead, we observe that the
mitochondrial genome has a variable size and structure in the different
organisms, though it contains more or less the same set of genes. This is an
example of genetic conservation versus structural diversity. From an
evolutionary point of view the genetic system of organelles is clearly under
strong selective pressure and for its survival it needs to utilize strategies to
slow down or halt the ratchet. Anyway, the mitochondrial genome changes with
time, and the rate of evolution is different for both diverse regions of the
mtDNA and between lineages, as demonstrated in the case of mammalian mt genomes.
We report here our data on the evolution of the mitochondrial DNA in mammals
which demonstrate the suitability of mtDNA as a molecular tool for evolutionary
analyses. Muller's ratchet is a principle of evolutionary genetics describing mutant
accumulation in populations that are repeatedly subjected to genetic bottleneck.
The immediate effect of Muller's ratchet, overall loss of fitness, has been
confirmed in several viral systems belonging to different groups. This report
shows that in addition to fitness loss, genetic bottlenecks also have
longer-term effects, namely changes in the capacity of viral populations to
adapt. Thus, vesicular stomatitis virus strains with a history of genetic
bottleneck have lower adaptability than strains maintained at relatively large
population sizes. This lower adaptability is illustrated by their reduced
ability to regain fitness and by their inability to outcompete wild-type
populations in situations where the initial fitness of the bottlenecked mutant
is the same or even higher than the initial fitness of the wild-type. The observation of high mitochondrial mutation rates in human pedigrees has led
to the question of how such an asexual genetic system can survive the
accumulation of slightly deleterious mutations caused by Muller's ratchet. I
define a null model to quantify in unprecedented detail the threat from
extinction caused by Muller's ratchet. This model is general enough to explore
the biological significance of Muller's ratchet in various species where its
operation has been suspected. For increased precision over a wide range of
parameter space I employ individual-based simulations run by evolution@home, the
first global computing system for evolutionary biology. After compiling
realistic values for the key parameters in human mitochondrial DNA (mtDNA) I
find that a surprisingly large range of biologically realistic parameter
combinations would lead to the extinction of the human line over a period of 20
million years - if accepted wisdom about mtDNA and Muller's ratchet is correct.
The resulting genomic decay paradox complements a similar threat from extinction
due to mutation accumulation in nuclear DNA and suggests evaluation of
unconventional explanations for long-term persistence. A substantial list of
potential solutions is given, including compensatory back mutations, mutation
rate heterogeneity and occasional recombination in mtDNA. Future work will have
to explore which of these actually solves the paradox. Nonetheless, the results
presented here provide yet another reason to minimize anthropogenic increase of
mutation rates. Muller's ratchet, the inevitable accumulation of deleterious mutations in
asexual populations, has been proposed as a major factor in genome degradation
of obligate symbiont organisms. Essentially, if left unchecked the ratchet will
with certainty cause extinction due to the ever increasing mutational load. This
paper examines the evolutionary fate of insect symbionts, using mathematical
modelling to simulate the accumulation of deleterious mutations. We investigate
the effects of a hierarchical two level population structure. Since each host
contains its own subpopulation of symbionts, there will be a large number of
small symbiont populations linked indirectly via selection on the host level. We
show that although the separate subpopulations will accumulate deleterious
mutations quickly, the symbiont population as a whole will be protected from
extinction by selection acting on the hosts. As a consequence, the extent of
genome degradation observed in present day symbionts is more likely to represent
loss of functions that were (near-) neutral to the host, rather than a snap shot
of a decline towards complete genetic collapse. Sex and recombination are widespread, but explaining these phenomena has been
one of the most difficult problems in evolutionary biology. Recombination is
advantageous when different individuals in a population carry different
advantageous alleles. By bringing together advantageous alleles onto the same
chromosome, recombination speeds up the process of adaptation and opposes the
fixation of harmful mutations by means of Muller's ratchet. Nevertheless,
adaptive substitutions favour sex and recombination only if the rate of adaptive
mutation is high, and Muller's ratchet operates only in small or asexual
populations. Here, by tracking the fate of modifier alleles that alter the
frequency of sex and recombination, we show that background selection against
deleterious mutant alleles provides a stochastic advantage to sex and
recombination that increases with population size. The advantage arises because,
with low levels of recombination, selection at other loci severely reduces the
effective population size and genetic variance in fitness at a focal locus (the
Hill-Robertson effect), making a population less able to respond to selection
and to rid itself of deleterious mutations. Sex and recombination reveal the
hidden genetic variance in fitness by combining chromosomes of intermediate
fitness to create chromosomes that are relatively free of (or are loaded with)
deleterious mutations. This increase in genetic variance within finite
populations improves the response to selection and generates a substantial
advantage to sex and recombination that is fairly insensitive to the form of
epistatic interactions between deleterious alleles. The mechanism supported by
our results offers a robust and broadly applicable explanation for the
evolutionary advantage of recombination and can explain the spread of costly
sex. Deleterious mutations can accumulate in asexual haploid genomes through the
process known as Muller's ratchet. This process has been described in the
literature mostly for the case where all mutations are assumed to have the same
effect on fitness. In the more realistic situation, deleterious mutations will
affect fitness with a wide range of effects, from almost neutral to lethal. To
elucidate the behavior of the ratchet in this more realistic case, simulations
were carried out in a number of models, one where all mutations have the same
effect on selection [one-dimensional (1D) model], one where the deleterious
mutations can be divided into two groups with different selective effects
[two-dimensional (2D) model], and finally one where the deleterious effects are
distributed. The behavior of these models suggests that deleterious mutations
can be classified into three different categories, such that the behavior of
each can be described in a straightforward way. This makes it possible to
predict the ratchet rate for an arbitrary distribution of fitness effects using
the results for the well-studied 1D model with a single selection coefficient.
The description was tested and shown to work well in simulations where selection
coefficients are derived from an exponential distribution. BACKGROUND: The theory of Muller' Ratchet predicts that small asexual
populations are doomed to accumulate ever-increasing deleterious mutation loads
as a consequence of the magnified power of genetic drift and mutation that
accompanies small population size. Evidence for Muller's Ratchet and knowledge
on its underlying molecular mechanisms, however, are lacking for natural
populations.
RESULTS: We characterized mitochondrial genome evolutionary processes in
Caenorhabditis briggsae natural isolates to show that numerous lineages
experience a high incidence of nonsynonymous substitutions in protein-coding
genes and accumulate unusual deleterious noncoding DNA stretches with associated
heteroplasmic function-disrupting genome deletions. Isolate-specific deletion
proportions correlated negatively with nematode fecundity, suggesting that these
deletions might negatively affect C. briggsae fitness. However, putative
compensatory mutations were also observed that are predicted to reduce
heteroplasmy levels of deleterious deletions. Paradoxically, compensatory
mutations were observed in one major intraspecific C. briggsae clade where
population sizes are estimated to be very small (and selection is predicted to
be relatively weak), but not in a second major clade where population size
estimates are much larger and selection is expected to be more efficient.
CONCLUSION: This study provides evidence that the mitochondrial genomes of
animals evolving in nature are susceptible to Muller's Ratchet, suggests that
context-dependent compensatory mutations can accumulate in small populations,
and predicts that Muller's Ratchet can affect fundamental evolutionary forces
such as the rate of mutation. BACKGROUND: The Amazon molly (Poecilia formosa) is a small unisexual fish that
has been suspected of being threatened by extinction from the stochastic
accumulation of slightly deleterious mutations that is caused by Muller's
ratchet in non-recombining populations. However, no detailed quantification of
the extent of this threat is available.
RESULTS: Here we quantify genomic decay in this fish by using a simple model of
Muller's ratchet with the most realistic parameter combinations available
employing the evolution@home global computing system. We also describe simple
extensions of the standard model of Muller's ratchet that allow us to deal with
selfing diploids, triploids and mitotic recombination. We show that Muller's
ratchet creates a threat of extinction for the Amazon molly for many
biologically realistic parameter combinations. In most cases, extinction is
expected to occur within a time frame that is less than previous estimates of
the age of the species, leading to a genomic decay paradox.
CONCLUSION: How then does the Amazon molly survive? Several biological processes
could individually or in combination solve this genomic decay paradox, including
paternal leakage of undamaged DNA from sexual sister species, compensatory
mutations and many others. More research is needed to quantify the contribution
of these potential solutions towards the survival of the Amazon molly and other
(ancient) asexual species. BACKGROUND: Primary bacterial endosymbionts of insects (p-endosymbionts) are
thought to be undergoing the process of Muller's ratchet where they accrue
slightly deleterious mutations due to genetic drift in small populations with
negligible recombination rates. If this process were to go unchecked over time,
theory predicts mutational meltdown and eventual extinction. Although genome
degradation is common among p-endosymbionts, we do not observe widespread
p-endosymbiont extinction, suggesting that Muller's ratchet may be slowed or
even stopped over time. For example, selection may act to slow the effects of
Muller's ratchet by removing slightly deleterious mutations before they go to
fixation thereby causing a decrease in nucleotide substitutions rates in older
p-endosymbiont lineages.
METHODOLOGY/PRINCIPAL FINDINGS: To determine whether selection is slowing the
effects of Muller's ratchet, we determined the age of the Candidatus
Riesia/sucking louse assemblage and analyzed the nucleotide substitution rates
of several p-endosymbiont lineages that differ in the length of time that they
have been associated with their insect hosts. We find that Riesia is the
youngest p-endosymbiont known to date, and has been associated with its louse
hosts for only 13-25 My. Further, it is the fastest evolving p-endosymbiont with
substitution rates of 19-34% per 50 My. When comparing Riesia to other insect
p-endosymbionts, we find that nucleotide substitution rates decrease
dramatically as the age of endosymbiosis increases.
CONCLUSIONS/SIGNIFICANCE: A decrease in nucleotide substitution rates over time
suggests that selection may be limiting the effects of Muller's ratchet by
removing individuals with the highest mutational loads and decreasing the rate
at which new mutations become fixed. This countering effect of selection could
slow the overall rate of endosymbiont extinction. Recombination restriction between evolving sex chromosomes leads to the
degeneration of the chromosome that is present only in the heterogametic sex
(the Y chromosome in XY species). The evolutionary forces driving Y chromosome
degeneration, however, are still under debate and include positive and negative
selection models. In a recent study, we showed that the rate of accumulation of
loss-of-function mutations on the neo-Y chromosome of Drosophila miranda is
compatible with the process of Muller's ratchet, the stochastic loss of the best
mutational class of individuals from a small asexual population. Purifying
selection at amino acid sites can accelerate the ratchet, and the speed of
degeneration depends on the number of genes still present on the evolving Y
chromosome. Our study shows that Y chromosome degeneration does not require the
action of selective sweeps at linked sites, and can take place under realistic
parameters of purifying selection only. RNA viruses typically occur in genetically diverse populations due to their
error-prone genome replication. Genetic diversity is thought to be important in
allowing RNA viruses to explore sequence space, facilitating adaptation to
changing environments and hosts. Some arboviruses that infect both a mosquito
vector and a mammalian host are known to experience population bottlenecks in
their vectors, which may constrain their genetic diversity and could potentially
lead to extinction events via Muller's ratchet. To examine this potential
challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan
equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector
Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with
a long evolutionary history. Using a mixture of marked VEEV clones to infect C.
taeniopus and real-time RT-PCR to track these clones during mosquito infection
and dissemination, we observed severe bottleneck events that resulted in a
significant drop in the number of clones present. At higher initial doses, the
midgut was readily infected and there was a severe bottleneck at the midgut
escape. Following a lower initial dose, the major bottleneck occurred at initial
midgut infection. A second, less severe bottleneck was identified at the
salivary gland infection stage following intrathoracic inoculation. Our results
suggest that VEEV consistently encounters bottlenecks during infection,
dissemination and transmission by its natural enzootic vector. The potential
impacts of these bottlenecks on viral fitness and transmission, and the viral
mechanisms that prevent genetic drift leading to extinction, deserve further
study. Population bottlenecks can have major effects in the evolution of RNA viruses,
but their possible influence in the evolution of DNA viruses is largely unknown.
Genetic and biological variation of herpes simplex virus type 1 (HSV-1) has been
studied by subjecting 23 biological clones of the virus to 10 plaque-to-plaque
transfers. In contrast to large population passages, plaque transfers led to a
decrease in replicative capacity of HSV-1. Two out of a total of 23 clones did
not survive to the last transfer in 143 TK(-) cells. DNA from three genomic
regions (DNA polymerase, glycoprotein gD and thymidine kinase) from the initial
and passaged clones was sequenced. Nucleotide substitutions were detected in the
TK and gD genes, but not in the DNA polymerase gene. Assuming a uniform
distribution of mutations along the genome, the average rate of fixation of
mutations was about five mutations per viral genome and plaque transfer. This
value is comparable to the range of values calculated for RNA viruses. Four
plaque-transferred populations lost neurovirulence for mice, as compared with
the corresponding initial clones. LD(50) values obtained with the populations
subjected to serial bottlenecks were 4- to 67-fold higher than for their
parental clones. These results equate HSV-1 with RNA viruses regarding fitness
decrease as a result of plaque-to-plaque transfers, and show that population
bottlenecks can modify the pathogenic potential of HSV-1. Implications for the
evolution of complex DNA viruses are discussed. |
Which heat shock protein is found to be upregulated during Hsp90 inhibition? | HSP90 inhibition was found to be associated with induction of HSP70 expression. | OBJECTIVE: Excessive reactive oxygen species contribute to vascular dysfunction.
We have previously shown that heat shock protein (Hsp90) inhibitors potently
suppress Nox 1 to 3 and 5, and the goals of this study were to identify how
molecular chaperones regulate Nox function.
METHODS AND RESULTS: In vitro, protein expression of Nox 1 to 2, 5 was decreased
by Hsp90 inhibitors in multiple cell types (human pulmonary artery endothelial
cells, neutrophils, macrophages, and human saphenous vein). In mice treated with
Hsp90 inhibitors, Nox1 expression was reduced in lung along with reduced
reactive oxygen species from leukocytes. Elevated reactive oxygen species
production in obese (db/db) aorta was suppressed by Hsp90 inhibition. Hsp90
inhibitors did not alter Nox5 micro RNA levels, and proteasome inhibition
prevented Nox2 and 5 protein degradation and increased ubiquitin incorporation.
Inhibition of Hsp90 upregulated the expression of Hsp70 and Hsp70-bound Nox2, 5
and promoted degradation. Silencing Hsp70 prevented Hsp90 inhibitor-mediated
degradation of Nox5. The Hsp70-regulated ubiquitin ligase, carboxyl terminus of
Hsp70-interacting protein (CHIP), also bound Nox5 and promoted increased Nox5
ubiquitination and degradation. The chaperone binding and ubiquitination domains
of CHIP were required, and the silencing of CHIP blunted Hsp90
inhibitor-mediated degradation of Nox2 and 5.
CONCLUSIONS: We conclude that Hsp90 binds to and regulates Nox protein
stability. These actions are opposed by Hsp70 and CHIP, which promote the
ubiquitination and degradation of Nox proteins and reduce reactive oxygen
species production. |
Which gene is responsible for the development of Sotos syndrome? | Sotos syndrome (SoS) is a multiple anomaly, congenital disorder characterized by overgrowth, macrocephaly, distinctive facial features and variable degree of intellectual disability. Haploinsufficiency of the NSD1 gene at 5q35.3, arising from 5q35 microdeletions, point mutations, and partial gene deletions, accounts for a majority of patients with SoS. | Recently, deletions encompassing the nuclear receptor binding SET-Domain 1
(NSD1) gene have been described as the major cause of Japanese patients with the
Sotos syndrome, whereas point mutations have been identified in the majority of
European Sotos syndrome patients. In order to investigate a possible
phenotype-genotype correlation and to further define the predictive value of
NSD1 mutations, we performed mutational analysis of the NSD1 gene in 20 patients
and one familial case with Sotos syndrome, five patients with Weaver syndrome,
six patients with unclassified overgrowth/mental retardation, and six patients
with macrocephaly/mental retardation. We were able to identify mutations within
the NSD1 gene in 18 patients and the familial case with Sotos syndrome (90%).
The mutations (six nonsense, eight frame shifts, three splice site, one
missense, one in-frame deletion) are expected to result in an impairment of NSD1
function. The best correlation between clinical assessment and molecular results
was obtained for the Sotos facial gestalt in conjunction with overgrowth,
macrocephaly, and developmental delay. In contrast to the high mutation
detection rate in Sotos syndrome, none of the patients with Weaver syndrome,
unclassified overgrowth/mental retardation and macrocephaly/mental retardation,
harbored NSD1 mutations. We tested for large deletions by FISH analysis but were
not able to identify any deletion cases. The results indicate that the great
majority of patients with Sotos syndrome are caused by mutations in NSD1.
Deletions covering the NSD1 locus were not found in the patients analyzed here. Sotos syndrome is a genetic disorder characterized by a typical facial
appearance, macrocephaly, accelerated growth, developmental delay, and a
variable range of associated abnormalities. The NSD1 gene was recently found to
be responsible for Sotos syndrome, and more than 150 patients with NSD1
alterations have been identified. A significant ethnic difference is found in
the prevalence of different types of mutation, with a high percentage of
microdeletions identified in Japanese Sotos syndrome patients and with
intragenic mutations in most non-Japanese patients. NSD1 aberrations are rather
specific for Sotos syndrome, but have also been detected in patients lacking one
or more major criteria of the disorder, namely overgrowth, macrocephaly, and
advanced bone age. Thus, new diagnostic criteria should be considered. Studies
have reported different frequencies of mutations versus non-mutations in Sotos
syndrome, thus indicating allelic or locus hetereogeneity. Although some authors
have suggested genotype/phenotype correlations, further studies are needed. Haploinsufficiency of the NSD1 gene due to 5q35 microdeletions or intragenic
mutations is the major cause of Sotos syndrome characterized by generalized
overgrowth, large hands and feet with advanced bone age, craniofacial dysmorphic
features, learning disability, and possible susceptibility to tumors. Here, we
report on a 14-month-old boy with a reverse phenotype of Sotos syndrome due to
the reciprocal duplication of the 5q35.3 region, including the NSD1 gene,
detected by array CGH. The phenotype includes delayed bone age, microcephaly,
seizures, and failure to thrive. Our case suggests that the gene dosage effect
of the NSD1 gene is the likely cause for the reversed phenotype of Sotos
syndrome in this patient. Sotos syndrome is a rare genetic disorder characterized by overgrowth associated
with macrocephaly and delayed psychomotor development. Patients with Sotos
syndrome show 5q35 deletions involving NSD1 or its point mutations. We
identified the common 5q35 deletion in a patient with atypical Sotos syndrome
manifesting extremely severe developmental delay, joint hypermobility, and skin
hyperextensibility, which are recognized as Marfanoid hypermobility syndrome.
Further analyses were performed to identify the genetic cause of these
additional findings. aCGH analysis revealed an additional 862 kb deletion of
Xq22.3 in this patient, which was inherited from his healthy mother. The deleted
region included five genes, including the nik-related kinase gene (NRK), which
would be a candidate gene for the patient's Marfanoid hypermobility, because it
is a member of the glucokinase subfamily that are involved in activating the JNK
pathway, and is expressed in developing skeletal musculature. Severe
developmental delay seen in the patient may be derived from position effect of
the deletion for neighboring interleukin 1 receptor accessory protein-like 2
gene (IL1RAPL2), which is a candidate gene for X-linked mental retardation. Sotos syndrome is a multiple anomaly syndrome characterized by pre- and
postnatal overgrowth with advanced bone age, macrocephaly, developmental delay,
and distinctive facial phenotype. Autosomal domit mutations and deletions of
the nuclear receptor set domain gene (NSD1), which is located at chromosome
5q35, are responsible for most of the cases. We describe a six-year old boy who
had tall stature, macrocephaly, typical facial appearance, learning disability,
megaloencephaly, corpus callosum dysgenesis, and colpocephaly. Although he had
normal bone age, the diagnosis of Sotos syndrome was suspected with these
clinical findings, and fluorescence in situ hybridization analysis of the
patient showed a heterozygous deletion covering the NSD1 region in the 5q35
locus. A brief overview of the syndrome is presented. OBJECTIVE: To present prenatal diagnosis and molecular cytogenetic
characterization of a de novo 5q35 microdeletion associated with Sotos syndrome.
METHODS: This was the first pregcy of a 29-year-old woman. The pregcy was
uneventful until 27 weeks of gestation when left ventriculomegaly was first
noted. At 31 weeks of gestation, polyhydramnios, macrocephaly, and
ventriculomegaly were prominent on ultrasound, and left pyelectasis and
bilateral ventriculomegaly were diagnosed on magnetic resoce imaging. The
woman underwent amniocentesis and cordocentesis at 32 weeks of gestation.
Conventional cytogenetic analysis was performed using cultured amniocytes and
cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was
performed on uncultured amniocytes and parental blood. Metaphase fluorescence in
situ hybridization (FISH) was performed on cultured lymphocytes.
RESULTS: Conventional cytogenetics revealed a karyotype of 46,XX. aCGH on
uncultured amniocytes revealed a de novo 1.07-Mb microdeletion at 5q35.2-q35.3
encompassing NSD1. Metaphase FISH analysis on the cord blood lymphocytes
confirmed the deletion at 5q35.2. The postnatal phenotype was consistent with
Sotos syndrome.
CONCLUSION: Fetuses with Sotos syndrome may present macrocephaly,
polyhydramnios, ventriculomegaly, and pyelectasis in the third trimester. aCGH
and metaphase FISH are useful for rapid diagnosis of 5q35 microdeletion
associated with Sotos syndrome. Sotos syndrome (SoS) is a multiple anomaly, congenital disorder characterized by
overgrowth, macrocephaly, distinctive facial features and variable degree of
intellectual disability. Haploinsufficiency of the NSD1 gene at 5q35.3, arising
from 5q35 microdeletions, point mutations, and partial gene deletions, accounts
for a majority of patients with SoS. Recently, mutations and possible
pathogenetic rare CNVs, both affecting a few candidate genes for overgrowth,
have been reported in patients with Sotos-like overgrowth features. To estimate
the frequency of NSD1 defects in the Brazilian SoS population and possibly
reveal other genes implicated in the etiopathogenesis of this syndrome, we
collected a cohort of 21 Brazilian patients, who fulfilled the diagnostic
criteria for SoS, and analyzed the NSD1 and PTEN genes by means of multiplex
ligation-dependent probe amplification and mutational screening analyses. We
identified a classical NSD1 microdeletion, a novel missense mutation (p.C1593W),
and 2 previously reported truncating mutations: p.R1984X and p.V1760Gfs*2. In
addition, we identified a novel de novo PTEN gene mutation (p.D312Rfs*2) in a
patient with a less severe presentation of SoS phenotype, which did not include
pre- and postnatal overgrowth. For the first time, our study implies PTEN in the
pathogenesis of SoS and further emphasizes the existence of ethno-geographical
differences in NSD1 molecular alterations between patients with SoS from
Europe/North America (70-93%) and those from South America (10-19%). Sotos syndrome is a well-known overgrowth syndrome characterized by excessive
growth during childhood, macrocephaly, distinctive facial appearance and
learning disability. This disorder is caused by mutations or deletions in NSD1
gene. The aim of this study is to examine the relationship between the
neuroimaging and clinical features of children with Sotos syndrome. Six Turkish
children with Sotos syndrome were followed up about 3-7 years. The diagnosis was
confirmed with molecular genetic analysis. We identified the pathogenic NSD1
mutation including three novel in all patients. All the patients had a
characteristic facial gestalt of Sotos syndrome consisting of triangular face
with prominent forehead, frontoparietal sparseness of hair and small nose.
However, the degree of psychomotor and intellectual development was variable.
Severe learning defect and speech delay were remarkable in two patients. The
neuroimaging analysis showed abnormalities in four of six patients including
bilateral large ventricles, thinning of the corpus callosum and persistent cavum
septum pellucidum et vergae. Typical craniofacial appearance is the primary
finding for the diagnosis of the disease even in the infantile period. However,
the degree of psychomotor and intellectual development is very variable and does
not correlate with the neuroimaging findings. |
What is the name for anorexia in gymnasts? | Anorexia athletica | Leptin, the product of the ob-gene, is specifically released by adipocytes. In
addition to its metabolic function it seems to affect the feedback-mechanisms of
the hypothalamic-pituitary-gonadal-axis. We studied 13 female juvenile elite
gymnasts with anorexia athletica (AA) and 9 female patients with anorexia
nervosa (AN) regarding the relation between leptin, fat stores, and the
reproductive hormone levels. Leptin levels in females with anorexia nervosa
(Tanner stage B4 [median]; mean age: 17.8 +/- 1.7 years) were low (2.9 +/- 2.7
microg/L), and were related to body mass index (BMI) (r = 0.71; p = 0.03) and
percentage body fat mass (r = 0.78; p = 0.01). Leptin levels of the elite
gymnasts were even more decreased (1.2 +/- 0.8 microg/L) caused by the low
amount of fat stores. Leptin correlated with BMI (r= 0.77; p = 0.004) and the
percentage body fat mass (r = 0.6; p = 0.04). In elite gymnasts leptin levels
correlated with CA showing an age-dependent increase (r= 0.59; p = 0.04).
Oestradiol was secreted at a low level in both groups (AN: 25.6 +/- 17.4
microg/L; AA: 24.4 +/- 13.5 microg/L). A delay in menarche and a retarded bone
maturation occurred in AA. Our results clearly show that leptin levels are low
in restrained eaters. Leptin levels represent the fat stores in the body and
play a permissive role for female pubertal development. There is evidence that
the mechanisms leading to a dysregulation of the reproductive-axis in patients
with AN are comparable with those leading to delayed puberty in juvenile elite
gymnasts with AA. This implies that AN and AA are overlapping groups and AA can
lead to the development of AN. |
Is Vitamin D deficiency in pregnant women associated with gestational diabetes? | Yes, there are multiple studies reporting an association between low VitD in pregnancy and impaired glucose tolerance, but it is not entirely clear if this translates directly to the increased risk of Gestational diabetes or via maternal obesity and/or genetic polymorphisms. | OBJECTIVE: The goal of this study was to examine whether maternal dietary intake
of vitamin D, omega-3 fatty acids, and omega-6 fatty acids during pregcy is
associated with the appearance of islet autoimmunity (IA) in offspring.
RESEARCH DESIGN AND METHODS: The Diabetes Autoimmunity Study in the Young
(DAISY) is recruiting at birth and following children at increased risk for type
1 diabetes, as determined by HLA-DR genotype or by family history of type 1
diabetes. A total of 233 mothers of newly recruited DAISY subjects were asked to
recall their intake of food and nutritional supplements during the third
trimester of pregcy using the Willett food frequency questionnaire. Children
were followed for an average of 4 years (range 0.8-7.3 years) for the appearance
of insulin, GAD(65), and IA-2 autoantibodies. Sixteen children developed at
least one autoantibody during this period. Unadjusted and adjusted hazard ratios
(HRs) for the development of IA were estimated with survival analysis using a
Weibull distribution.
RESULTS: Maternal intake of vitamin D via food was significantly associated with
a decreased risk of IA appearance in offspring, independent of HLA genotype,
family history of type 1 diabetes, presence of gestational diabetes mellitus,
and ethnicity (adjusted HR = 0.37; 95% CI 0.17-0.78). Vitamin D intake via
supplements, omega-3 fatty acids, and omega-6 fatty acids intake during
pregcy were not associated with appearance of IA in offspring.
CONCLUSIONS: Our findings suggest that maternal intake of vitamin D through food
during pregcy may have a protective effect on the appearance of IA in
offspring. BACKGROUND: The serum level of 25-hydroxyvitamin D deficiency has long been
suspected as a risk factor for glucose intolerance and perhaps
1,25-dihydroxyvitamin D has a role in the regulation of insulin secretion. This
study investigates the relation between 25-hydroxyvitamin D concentrations and
insulin resistance in pregt women.
METHODS: A cross-sectional study was conducted on 741 pregt women referred to
five educating hospital clinics. Universal screening was performed with a GCT-50
g, and those with plasma glucose levels > pr = 7.2 mmol/L were diagnosed as GDM
if they had an impaired GTT-100 g based on Carpenter and Coustan criteria. The
levels of insulin and C-peptide were measured during OGTT-100 g test. The
homeostasis model assessment index (HOMA) equation was used as the insulin
resistance index. The concentrations of 25-hydroxyvitamin D, and PTH were also
measured.
RESULTS: Total prevalence of vitamin D deficiency (<25 nmol/L) was found in
70.6% of pregt women. Prevalence of severe vitamin D deficiency (<12.5) in
GDM patients was higher than in normoglycaemic pregcies. The regression model
revealed a strong correlation between the HOMA index and serum levels of vitamin
D.
CONCLUSIONS: These results show that a positive correlation of 25(OH) vitamin D
concentrations with insulin sensitivity and vitamin D deficiency could be a
confirmative sign of insulin resistance. BACKGROUND/OBJECTIVES: Vitamin D is required for bone growth and normal insulin
secretion. Maternal hypovitaminosis D may impair fetal growth and increase the
risk of gestational diabetes. We have related maternal vitamin D status in
pregcy to maternal and newborn glucose and insulin concentrations, and
newborn size, in a South Indian population.
SUBJECTS/METHODS: Serum 25 hydroxy vitamin D (25(OH)D) concentrations, glucose
tolerance, and plasma insulin, proinsulin and 32-33 split proinsulin
concentrations were measured at 30 weeks gestation in 559 women who delivered at
the Holdsworth Memorial Hospital, Mysore. The babies' anthropometry and cord
plasma glucose, insulin and insulin precursor concentrations were measured.
RESULTS: In total 66% of women had hypovitaminosis D (25(OH)D concentrations <50
nmol l(-1)) and 31% were below 28 nmol l(-1). There was seasonal variation in
25(OH)D concentrations (P<0.0001). There was no association between maternal
25(OH)D and gestational diabetes (incidence 7% in women with and without
hypovitaminosis D). Maternal 25(OH)D concentrations were unrelated to newborn
anthropometry or cord plasma variables. In mothers with hypovitaminosis D,
higher 25(OH)D concentrations were associated with lower 30-min glucose
concentrations (P=0.03) and higher fasting proinsulin concentrations (P=0.04).
CONCLUSIONS: Hypovitaminosis D at 30 weeks gestation is common in Mysore
mothers. It is not associated with an increased risk of gestational diabetes,
impaired fetal growth or altered neonatal cord plasma insulin secretory profile. AIMS: Vitamin D deficiency has been linked to impaired glucose metabolism. We
determined whether serum 25-hydroxyvitamin D (25OHD) is associated with glucose
metabolism in pregt women and the effect of ethnicity on this relationship.
METHODS: We analysed serum 25OHD concentrations in 307 pregt women attending
a metropolitan obstetric clinic between October 2003 and May 2005. Measurements
from 264 of the women were taken at the time of glucose tolerance testing at
mid-gestation, a population therefore at increased risk for gestational
diabetes. Pearson correlation analysis was used to test for univariate linear
relationships between the natural log of serum 25OHD (ln-25OHD) and other
variables. Multiple regression analysis was used to adjust for confounding
factors.
RESULTS: Mean serum 25OHD concentration was 53.8 +/- 23.9 nmol/l (sd). Ln-25OHD
was negatively correlated with serum parathyroid hormone as expected (r -0.24,
confidence intervals -0.35 to -0.12). Ln-25OHD was also negatively correlated
with fasting plasma glucose (r-0.20, -0.31 to -0.08), fasting insulin (r -0.20,
-0.31 to -0.08) and insulin resistance as calculated by homeostasis model
assessment (r -0.21, -0.32 to -0.09). The association between fasting glucose
and log-transformed 25OHD concentration was of borderline significance after
accounting for ethnicity, age and body mass index in multivariate analyses
(-0.13, -0.26 to 0.01). The odds ratio of gestational diabetes in women with
25OHD < 50 nmol/l did not reach statistical significance (1.92, 95% confidence
interval 0.89-4.17).
CONCLUSIONS: Maternal 25OHD concentrations are inversely related to fasting
glucose, although further studies are required to establish whether this is
independent of the effects of ethnic background. BACKGROUND: Evidence is accumulating for a role of vitamin D in maintaining
normal glucose homeostasis. However, studies that prospectively examined
circulating concentrations of 25-hydroxyvitamin D (25-[OH] D) in relation to
diabetes risk are limited. Our objective is to determine the association between
maternal plasma 25-[OH] D concentrations in early pregcy and the risk for
gestational diabetes mellitus (GDM).
METHODS: A nested case-control study was conducted among a prospective cohort of
953 pregt women. Among them, 57 incident GDM cases were ascertained and 114
women who were not diagnosed with GDM were selected as controls. Controls were
frequency matched to cases for the estimated season of conception of the index
pregcy.
RESULTS: Among women who developed GDM, maternal plasma 25-[OH] D concentrations
at an average of 16 weeks of gestation were significantly lower than controls
(24.2 vs. 30.1 ng/ml, P<0.001). This difference remained significant (3.62 ng/ml
lower on average in GDM cases than controls (P value = 0.018)) after the
adjustment for maternal age, race, family history of diabetes, and pre-pregcy
BMI. Approximately 33% of GDM cases, compared with 14% of controls (P<0.001),
had maternal plasma 25-[OH] D concentrations consistent with a pre-specified
diagnosis of vitamin D deficiency (<20 ng/ml). After adjustment for the
aforementioned covariates including BMI, vitamin D deficiency was associated
with a 2.66-fold (OR (95% CI): 2.66 (1.01-7.02)) increased GDM risk. Moreover,
each 5 ng/ml decrease in 25-[OH] D concentrations was related to a 1.29-fold
increase in GDM risk (OR (95% CI): 1.29 (1.05-1.60)). Additional adjustment for
season and physical activity did not change findings substantially.
CONCLUSIONS: Findings from the present study suggest that maternal vitamin D
deficiency in early pregcy is significantly associated with an elevated risk
for GDM. Over the past decade, new evidence has shown that vitamin D deficiency may
contribute to the risk of developing a wide range of common chronic diseases
that are different from the classic action on calcium and bone homeostasis.
Acting through the vitamin D receptor, vitamin D can produce a wide array of
favorable biological effects via genomic, non-genomic or intracrine mechanisms
and, therefore, contributes to the improvement of human health in humans. We
hypothesize that some of these effects may be even more critical during
pregcy. The focus of this paper is to review the data on the classic and
non-classic actions of vitamin D with regards to pregcy. It appears that
vitamin D insufficiency during pregcy is potentially associated with
increased risk of preeclampsia, insulin resistance and gestational diabetes
mellitus. Furthermore, experimental data also anticipate that vitamin D
sufficiency is critical for fetal development, and especially for fetal brain
development and immunological functions. Vitamin D deficiency during pregcy
may, therefore, not only impair maternal skeletal preservation and fetal
skeletal formation but also be vital to the fetal "imprinting" that may affect
chronic disease susceptibility soon after birth as well as later in life. OBJECTIVE: To investigate the association between first-trimester maternal serum
levels of 25-hydroxyvitamin D (25-OH-D) as measured by liquid
chromatography-tandem mass spectrometry and development of gestational diabetes
mellitus (GDM).
RESEARCH DESIGN AND METHODS: We conducted a case-control study involving 248
women in the first-trimester of pregcy, 90 of whom developed GDM and 158
remained normoglycemic.
RESULTS: Although booking 25-OH-D levels correlated negatively with 2-h glucose
post-oral glucose tolerance test and positively with HDL cholesterol, as well as
with ethnicity, obesity, and smoking (all P < 0.05), there were no statistically
significant differences in baseline maternal mean 25-OH-D levels between those
who subsequently developed GDM, 18.9 ng/mL (SD 10.7) and those who remained
normoglycemic, 19.0 ng/mL (10.7) (P = 0.874), even after adjustment for possible
confounders including sampling month (P = 0.784).
CONCLUSIONS: Our large and well-phenotyped prospective study did not find
evidence of an association between first-trimester maternal levels of 25-OH-D
and subsequent development of GDM. OBJECTIVE: To investigate whether maternal serum levels of 25-hydroxyvitamin D
[25(OH)D] in the first trimester are altered in pregt women with pre-existing
type 2 diabetes, women who subsequently develop gestational diabetes mellitus
(GDM) and women who deliver large for gestational age (LGA) neonates compared
with normoglycaemic pregt women who deliver an appropriate for gestational
age (AGA) neonate.
DESIGN: Case-control study.
SETTING: Antenatal clinic.
POPULATION: Singleton pregcies at 11(+0) -13(+6) weeks, including 50 women
with type 2 diabetes, 100 women who subsequently developed GDM, 50 nondiabetic
women who subsequently delivered LGA neonates and 1000 nondiabetic controls who
delivered AGA neonates.
METHODS: Maternal serum total 25(OH)D levels were measured in the four groups of
pregcies. Multiple regression analysis in the controls was used to identify
factors among maternal characteristics with a significant contribution to the
levels of serum 25(OH)D, so that the values in all cases were expressed as a
multiple of the median (MoM) in the controls.
MAIN OUTCOME MEASURES: Comparison of MoM 25(OH)D in the four groups.
RESULTS: In controls, significant independent contributions to the serum level
of 25(OH)D were provided by maternal age, body mass index, smoking status,
racial origin and season of sampling. The median and interquartile range (IQR)
of serum 25(OH)D in the type 2 diabetes group (1.01; IQR, 0.68-1.47 MoM), GDM
group (0.93; IQR, 0.67-1.23 MoM) and LGA group (0.97; IQR, 0.67-1.25 MoM) were
not significantly different from those in the controls (0.99; IQR, 0.71-1.33
MoM) (overall P = 0.643).
CONCLUSIONS: The first-trimester maternal serum level of 25(OH)D is not altered
in women with type 2 diabetes, those who develop GDM or those who deliver LGA
neonates. The aim of the study is evaluating the associations of FokI vitamin D receptor
(VDR) gene polymorphisms with gestational diabetes mellitus (GDM), and its
relations with postpartum metabolic syndrome. In a cohort study, 303 women
referred to outpatient clinic of Shariati Hospital. The VDR FokI genotypes were
determined. All subjects were followed 6?12 weeks after delivery. The
frequencies of Ff, FF, and ff genotypes were 30.4% (49), 63.4% (102), and 6.2%
(10), respectively, in healthy pregcies and 34.5% (49), 54.9% (78), and 10.6%
(15), respectively, in GDM patients. The ff genotype was more common in GDM
patients. Healthy individuals had higher frequency of F allele, suggesting that
F allele may have a role in decreased incidence of GDM. Concerning the GDM risk
factors, f allele had significant association with prepregcy obesity and
family history of diabetes. In postpartum follow-up, women who developed
metabolic syndrome were significantly older with higher prepregcy body mass
index, had more family history of diabetes, and also their ff genotype was two
fold more frequent. Our results indicate a meaningful association between FokI
VDR genotypes and an increase risk of GDM in Iranian population as well as its
effects on postpartum metabolic syndrome. BACKGROUND: Vitamin D deficiency may contribute to impaired glucose metabolism.
There are sparse data regarding vitamin D and the development of gestational
diabetes (GDM). The objective of this study was to assess if first-trimester
vitamin D deficiency is more prevalent in women later diagnosed with GDM
compared with women with uncomplicated pregcies.
METHODS: We conducted a nested case-control study of pregt women who had
previously given blood for routine genetic multiple marker screening and
subsequently delivered at a tertiary hospital between November 2004 and July
2009. From an overall cohort of 4225 women, 60 cases of GDM were matched by
race/ethnicity with 120 women delivering at term (≥37 weeks) with uncomplicated
pregcies. Banked maternal serum was used to measure maternal
25-hydroxyvitamin D [25(OH)D].
RESULTS: The prevalence of first-trimester maternal vitamin D deficiency
(defined as 25(OH)D < 50 nmol/L) was comparable among women with GDM compared
with controls (5/60 vs 8/120, p = 0.90). The median 25(OH)D level for all
subjects was 89 nmol/L (interquartile range, 73-106 nmol/L). Seventy three
percent (117/160) of the cohort had 25(OH)D levels ≥75 nmol/L.
CONCLUSIONS: In a cohort of pregt women with mostly sufficient levels of
serum 25(OH)D, vitamin D deficiency was not associated with GDM. Further studies
are warranted with larger cohorts, especially in populations with lower levels
of vitamin D. AIM: This study was performed to determine the effect of a single, large,
intramuscular injection of vitamin D post-partum on glucose tolerance and
insulin resistance in women with gestational diabetes.
METHODS: Forty-five participants in a randomized controlled trial on gestational
diabetes mellitus were divided into an intervention group and a control group.
Only subjects in the intervention group received one intramuscular injection of
300,000 IU of vitamin D3. HbA(1c), serum 25-hydroxyvitamin D3, fasting insulin
and blood glucose, C-peptide, homeostasis model assessment insulin resistance
index (HOMA-IR), β-cell function, insulin sensitivity and the Quantitative
Insulin Sensitivity Check Index (QUICKI) were measured at baseline and after 3
months of intervention.
RESULTS: Approximately 80% of the mothers had a degree of vitamin D deficiency.
Post-intervention, this was found in 4.2 and 71.4% in the intervention and
control groups, respectively. The medians of HOMA-IR indices before and after
intervention were 0.6 and 0.5 (P = 0.7), respectively, in subjects in the
intervention group, and 0.5 and 0.9 (P = 0.01) in subjects in the control group.
The mean of the QUICKI fell only in the control group (P = 0.008). In the
control group, β-cell function increased by ~8% (P = 0.01) and insulin
sensitivity decreased after 3 months (P = 0.002). Post-intervention, the median
C-peptide decreased in the intervention group and increased in the control
group, but the change was significant only in the control group (P = 0.03).
CONCLUSIONS: A single injection of 300,000 IU of vitamin D3 achieves a 3-month
serum 25-hydroxyvitamin D range of 50-80 nmol/l and is an efficient, effective
and safe procedure for improving the vitamin status and indices of insulin
resistance in mothers with gestational diabetes after delivery. OBJECTIVE: To evaluate the association of maternal serum 25-hydroxyvitamin D
(25[OH]D) status with glucose homeostasis and obstetric and newborn outcomes in
women screened for gestational diabetes mellitus (GDM).
METHODS: Consecutive women were screened for GDM at 24 to 28 weeks' gestation
during the months of maximal sunlight exposure in Spain (June through
September). Serum 25(OH)D levels and parameters of glucose homeostasis were
measured. Outcomes of the delivery and newborn were collected.
RESULTS: Two hundred sixty-six women were screened. Vitamin D deficiency
(25[OH]D <20 ng/mL) was observed in 157 women (59%). We observed an inverse
correlation between 25(OH)D levels and hemoglobin A1c, homeostasis model
assessment of insulin resistance, serum insulin, and fasting and 1-hour oral
glucose tolerance test glucose levels (P<.001). With a 25(OH)D concentration
less than 20 ng/mL, the odds ratios were 3.31 for premature birth (95%
confidence interval, 1.52-7.19; P<.002) and 3.93 for cesarean delivery (95%
confidence interval, 2.00-7.73; P<.001). A 25(OH)D concentration of 20 ng/mL had
79% sensitivity and 51% specificity for cesarean delivery and 80% sensitivity
and 45% specificity for premature birth. The cutoffs with the best combination
of sensitivity and specificity were 16 ng/mL for cesarean delivery (62.9%
sensitivity and 61.2% specificity) and 14 ng/mL for premature birth (66.7%
sensitivity and 71.0% specificity).
CONCLUSIONS: In the population we sampled, vitamin D deficiency is very common
during pregcy. Lower 25(OH)D levels are associated with disorders of glucose
homeostasis and adverse obstetric and newborn outcomes. Vitamin D deficiency during pregcy has been associated with the development
of several adverse health outcomes, e.g., pre-eclampsia, gestational diabetes
mellitus, preterm delivery, low birth weight, birth length, and bone mineral
content. The aims of the present study were to estimate the intake and sources
of vitamin D in Danish pregt women and to examine potential determits of
vitamin D intake of the recommended level (10 µg per day). In 68,447 Danish
pregt women the mean ± SD for vitamin D intake was 9.23 ± 5.60 µg per day
(diet: 3.56 ± 2.05 µg per day, supplements: 5.67 ± 5.20 µg per day). 67.6% of
the women reported use of vitamin D supplements but only 36.9% reported use of
vitamin D supplements of at least 10 µg. Supplements were the primary source of
vitamin D for the two higher quartiles of total vitamin D intake, with diet
being the primary source for the two lower quartiles. Determits of sufficient
total vitamin D intake were: high maternal age, nulliparity, non-smoking, and
filling out of the Food Frequency Questionnaire (FFQ) during summer or fall. We
propose that clinicians encourage vitamin D supplementation among pregt
women, with special focus on vulnerable groups such as the young, smokers and
multiparous women, in order to improve maternal and fetal health both during and
after pregcy. OBJECTIVE: We examined the association of second-trimester maternal plasma
25-hydroxyvitamin D (25[OH]D) during pregcy with gestational diabetes
mellitus (GDM).
STUDY DESIGN: Among 1314 pregt women who participated in Project Viva, a
birth cohort study, we measured 25(OH)D levels at 26-28 weeks gestation during
GDM screening using a 1-hour 50-g glucose challenge test.
RESULTS: We found 25(OH)D levels of <25 nmol/L in 44 of 1087 women (4.0%) with
normal glucose tolerance, 9 of 159 women (5.7%) with impaired glucose tolerance,
and 9 of 68 women (13.2%) with GDM. Analyses that were adjusted for
sociodemographics, season, maternal body mass index, gestational weight gain,
and dietary factors suggested that women with 25(OH)D levels of <25 vs ≥25
nmol/L may have higher odds of experiencing GDM (odds ratio, 2.2; 95% confidence
interval, 0.8-5.5). Glucose levels after the glucose challenge test were
associated inversely with 25(OH)D levels (P < .01).
CONCLUSION: Second-trimester 25(OH)D levels were associated inversely with
glucose levels after 1-hour 50-g glucose challenge test; low 25(OH)D levels may
be associated with increased risk of GDM. OBJECTIVE: To investigate the association between vitamin D deficiency and risk
of gestational diabetes mellitus (GDM) in pregt Chinese women.
METHODS: A nested case-control study was conducted. Clinical and biochemical
data were analyzed for 200 subjects with GDM and 200 subjects with normal
glucose tolerance (NGT).
RESULTS: The median (interquartile range) serum 25-hydroxyvitamin D (25OHD)
levels were 22.39 (17.67, 29.38) and 25.86 (19.09, 34.88) nmol/L in the GDM and
NGT groups, respectively. Rates of 25OHD deficiency or insufficiency were
significantly higher in the GDM group than in the NGT group. Subjects with 25OHD
levels <25 nmol/L had a 1.8-fold higher risk of GDM compared with subjects with
higher vitamin D levels. In the GDM group, serum 25OHD was independently
associated with HbA1c and insulin resistance after adjusting for confounding
factors. In the NGT group, serum 25OHD was independently associated with fasting
plasma glucose and systolic blood pressure after adjusting for maternal age and
other confounding factors.
CONCLUSION: 25OHD insufficiency is very common in Chinese women. Low 25OHD
status may be associated with insulin resistance and act as a risk factor for
GDM. OBJECTIVE: To assess the effect of 25-hydroxyvitamin D (25-OHD) levels on
pregcy outcomes and birth variables.
DESIGN: Systematic review and meta-analysis.
DATA SOURCES: Medline (1966 to August 2012), PubMed (2008 to August 2012),
Embase (1980 to August 2012), CINAHL (1981 to August 2012), the Cochrane
database of systematic reviews, and the Cochrane database of registered clinical
trials.
STUDY SELECTION: Studies reporting on the association between serum 25-OHD
levels during pregcy and the outcomes of interest (pre-eclampsia, gestational
diabetes, bacterial vaginosis, caesarean section, small for gestational age
infants, birth weight, birth length, and head circumference).
DATA EXTRACTION: Two authors independently extracted data from original research
articles, including key indicators of study quality. We pooled the most adjusted
odds ratios and weighted mean differences. Associations were tested in subgroups
representing different patient characteristics and study quality.
RESULTS: 3357 studies were identified and reviewed for eligibility. 31 eligible
studies were included in the final analysis. Insufficient serum levels of 25-OHD
were associated with gestational diabetes (pooled odds ratio 1.49, 95%
confidence interval 1.18 to 1.89), pre-eclampsia (1.79, 1.25 to 2.58), and small
for gestational age infants (1.85, 1.52 to 2.26). Pregt women with low serum
25-OHD levels had an increased risk of bacterial vaginosis and low birthweight
infants but not delivery by caesarean section.
CONCLUSION: Vitamin D insufficiency is associated with an increased risk of
gestational diabetes, pre-eclampsia, and small for gestational age infants.
Pregt women with low 25-OHD levels had an increased risk of bacterial
vaginosis and lower birth weight infants, but not delivery by caesarean section. |
What are the indications for alteplase? | Intravenous alteplase (recombinant tissue plasminogen activator) is the only approved thrombolytic agent at present indicated for acute ischaemic stoke.
Food and Drug Administration approval of alteplase for central venous catheter (CVC) occlusions.
Alteplase is now firmly established as a treatment of choice in the management of acute myocardial infarction. The efficacy of intravenous alteplase in the treatment of pulmonary thromboembolism has also been established and appears to be similar to that of streptokinase and urokinase in this indication and in arterial thrombotic occlusion. However, its use in this latter indication and in other vascular disorders has not been as extensively documented.
Preliminary data suggest efficacy of alteplase of deep vein thrombosis and arterial thrombotic occlusion. | Thirty-two patients with acute, proximal-vein thrombosis were treated with
heparin and alteplase (0.25 versus 0.5 mg/kg/24 h during 3-7 days) in a
randomized, double-blind, multicenter, European (ETTT) trial. The treatment
resulted in a decrease of the venographic Marder's score from 18 (6-25) to 13
(2-24) units (median, range) in Group I (0.25 mg/kg/24 h, n = 15, median
decrease 3.0, p = 0.32) and from 17.5 (3-33) to 15.5 (0-27) in Group II (0.5
mg/kg/24 h, n = 16, median decrease 4.0, p = 0.23). Comparison of the sequential
venograms could be performed in 14 cases of Group I and in 15 cases in Group II.
A minority of patients showed substantial partial recanalization of the
initially obstructed veins on the control venogram (one in each treatment group)
and most of the control venograms showed either thrombus size reductions (5 in
Group I, 7 in Group II) or no change or even deterioration (8 in Group I, 7 in
Group II). Major bleedings were observed in 7 patients (7/32, 22%), 5 of them
occurring in Group II (5/17, 29%). Thus, the results of the ETTT trial show that
the used low dosages of alteplase administered intravenously over 3-7 days in
heparinized patients cannot be recommended as a treatment for patients with deep
venous thrombosis of lower limbs and/or pelvis. Further studies are needed to
define a more suitable dosage regimen of alteplase in this indication. Clinical experience with thrombolytics in non-coronary disorders is limited to
the plasminogen activators streptokinase, urokinase and alteplase; therapeutic
trials with anistreplase (APSAC) are almost, and with saruplase completely,
limited to acute myocardial infarction. In terms of thrombus clearance,
thrombolytic drugs are superior to heparin in patients with recent deep vein
thrombosis in the pelvis or lower limbs. In aggregate, thrombi younger than 8
days are lysed in approximately 60% of patients treated with streptokinase,
urokinase or alteplase. The results of studies assessing the subsequent
development of the postphlebitic syndrome are conflicting, but most suggest that
thrombolytic therapy can reduce symptoms of chronic venous insufficiency.
Currently, the combination of systemic thrombolytic drugs followed by heparin is
recommended for patients with acute major pulmonary embolism who are
haemodynamically unstable. Streptokinase, urokinase and alteplase have all been
shown to accelerate the lysis of pulmonary emboli and to decrease pulmonary
vascular obstruction and pulmonary hypertension. Systemic venous or
intrapulmonary infusions of alteplase offers the same benefit in terms of
angiographic and haemodynamic improvement. A short infusion of 100 mg alteplase
over 2 hours seems to be superior to a 24-hour infusion of urokinase. None of
the thrombolytic trials in pulmonary embolism have been large enough to
demonstrate a reduction in mortality. It is now generally accepted that, unless
contraindicated, thrombolytic therapy is the front-line treatment for patients
with massive pulmonary embolism and major haemodynamic disturbance. The local
treatment of acute arterial occlusion in limb arteries results in rapid clearing
of the artery in 67% of patients treated with streptokinase; the corresponding
success rates for urokinase and alteplase are 81% and 88 to 94%, respectively.
The main question appears to be the identification of patients in whom local
thrombolysis is the treatment of choice, as opposed to established therapeutic
modalities. Thrombolytic treatment following a major ischaemic stroke is
hazardous, although clinical improvement has been noted in a minority of
patients with recanalised cerebral arteries. The safety and efficacy of
thrombolytic treatment remains unproven for this indication, and its use must be
restricted to experimental protocols. Thrombolytic treatment in retinal artery
or vein occlusion has, in practice, been abandoned. Patients with a recent (less than 10 days) proximal deep vein thrombosis of the
leg or pelvis are candidates for thrombolysis as the major benefit over heparin
seems to be the prevention of the postphlebitic limb, an aim which is still not
proven in a satisfactory manner. Nonocclusive thrombi appear to lyse more
readily than occlusive thrombi. For this indication the optimal dose regimens
for the three thrombolytic drugs (streptokinase, urokinase, alteplase) are not
established. Acute massive pulmonary embolism with hypotension or shock should
be treated with thrombolytic drugs and, pending the outcome in the first hour,
be considered for pulmonary embolectomy. Major acute pulmonary embolism with
haemodynamic instability responds well to thrombolysis. Whether thrombolysis is
superior to heparin in subacute intermediate pulmonary embolism has not been
proven unequivocally in terms of mortality or clinically important endpoints.
Systemic administration of thrombolytic drugs for peripheral arterial occlusion
has been abandoned for catheter-directed and intraoperative intra-arterial
repeated bolus or short-term infusions. The efficacy and safety of intravenous
thrombolytic treatment following a major ischaemic stroke is presently being
tested in large scale trials; its use must be restricted to experimental
protocols. Alteplase is the product of recombit DNA technology and is chemically
identical to endogenous tissue-type plasminogen activator: Plasminogen is
converted to plasmin by alteplase, and fibrinolysis of blood thrombi is
subsequently stimulated. Alteplase is now firmly established as a treatment of
choice in the management of acute myocardial infarction. The efficacy of
intravenous alteplase in the treatment of pulmonary thromboembolism has also
been established and appears to be similar to that of streptokinase and
urokinase in this indication and in arterial thrombotic occlusion. However, its
use in this latter indication and in other vascular disorders has not been as
extensively documented. Although trials demonstrating the efficacy of
intravenous alteplase in patients with deep vein thrombosis and intra-arterial
alteplase in patients with arterial thrombotic occlusion exist, reliable data on
the efficacy of the fibrinolytic in ischaemic stroke and intracranial
haemorrhage are scarce. Little clinical benefit is apparent in patients with
unstable angina, although careful use may be warranted in those with definite
pretreatment coronary thrombi. Of concern, there is a suggestion that general
use of alteplase in patients with unstable angina may be associated with
increased incidence of myocardial infarction. The incidence of major haemorrhage
associated with alteplase therapy increases with increasing dose and appears to
be similar to that seen with other fibrinolytic agents. Thus, further
well-designed studies of the use of alteplase in ischaemic stroke and cerebral
haemorrhage are required. However, a small subset of patients with unstable
angina and definite pretreatment coronary thrombi may benefit from alteplase
therapy. Further, preliminary data suggest efficacy in the therapy of deep vein
thrombosis and arterial thrombotic occlusion, and alteplase has a proven place
in the fibrinolytic treatment of pulmonary thromboembolism. OBJECTIVES: Our aim was to design and evaluate a new and easily administered
recombit tissue-type plasminogen activator (rt-PA) regimen for thrombolysis
in acute myocardial infarction (AMI) based on established pharmacokinetic data
that improve the reperfusion success rate.
BACKGROUND: Rapid restoration of Thrombolysis in Myocardial Infarction (TIMI)
grade 3 flow is a primary predictor of mortality after thrombolysis in AMI.
However, TIMI grade 3 patency rates 90 min into thrombolysis of only 50% to 60%
indicate an obvious need for improved thrombolytic regimens.
METHODS: Pharmacokinetic simulations were performed to design a new rt-PA
regimen. We aimed for a plateau tissue-type plasminogen activator (t-PA) plasma
level similar to that of the first plateau of the Neuhaus regimen. These aims
were achieved with a 20-mg rt-PA intravenous (i.v.) bolus followed by an 80-mg
i.v. infusion over 60 min (regimen A). This regimen was tested in a consecutive
comparative trial in 80 patients versus 2.25 10(6) IU of streptokinase/60 min
(B), and 70 mg (C) or 100 mg (D) of rt-PA over 90 min. Subsequently, a
confirmation trial of regimen A in 254 consecutive patients was performed with
angiographic assessment by independent investigators of patency at 90 min.
RESULTS: The comparative phase of the trial yielded, respectively, TIMI grade 3
and total patency (TIMI grades 2 and 3) of 80% and 85% (regimen A), 35% and 50%
(B), 50% and 55% (C) and 60% and 70% (D). In the confirmation phase of the
trial, regimen A yielded 81.1% TIMI grade 3 and 87.0% total patency. At
follow-up angiography 7 (4.1%) of 169 vessels had reoccluded. In-hospital
mortality rate was 1.2%. Nadir levels of fibrinogen, plasminogen and
alpha2-antiplasmin were 3.6 +/- 0.8 mg/ml, 60 +/- 21% and 42 +/- 16%,
respectively (mean +/- SD). Fifty-seven patients (22.4%) suffered from bleeding;
3.5% needed blood transfusions.
CONCLUSIONS: The 60-min alteplase thrombolysis in AMI protocol achieved a TIMI
grade 3 patency rate of 81.1% at 90 min with no indication of an increased
bleeding hazard; it was associated with a 1.2% overall mortality rate. These
results are substantially better than those reported from all currently utilized
regimens. Head to head comparison with established thrombolytic regimens in a
large-scale randomized trial is warranted. Thrombolytic therapy reduces early mortality, preserves left ventricular
function and improves long term prognosis of acute myocardial infarction.
However it is relatively expensive and increasing use will have considerable
ficial consequences. With competing demand for health resources, information
on economic evaluation of this revolutionary therapeutic modality is much
needed. Economic evaluation of thrombolytic therapy of acute myocardial
infarction entails the assessment of all resources consumed (costs) directly and
indirectly in relation to the administration of thrombolytic drugs, versus the
beneficial effects (outcome) on health preservation of the patients. To save 1
year of life, the costs of thrombolytic therapy using intravenous streptokinase,
alteplase (recombit tissue plasminogen activator; rt-PA) or anistreplase
(anisoylated plasminogen streptokinase activator complex) under standard
restricted indication criteria, vary from 1000 pounds British sterling to 1700
pounds British sterling in the UK, SEK3090 to 9660 in Scandinavia and $US35 000
to 800 000 in the US, depending on time delay in starting treatment after pain
onset, size of infarct, thrombolytic agents used, study methodology, lists of
clinical events considered in cost counting and the discount rate. Cost-utility
analyses revealed that the costs of thrombolytic treatment are similar to those
of many other treatments for cardiac or other diseases, but methods for
evaluating quality of life and utility require further refinement and
validation. Economic assessments confirm that thrombolytic treatment of the
elderly ( greater than 70 years) is as cost-effective as treatment of younger
patients and that both early and late thrombolytic therapy (given 6 to 12 hours
after infarction) are beneficial and cost-effective. There are major logistical
problems with prehospital thrombolysis, which despite great initial enthusiasm,
is unlikely to be cost-effective in saving lives unless savings in time are
greater than 1 hour. Cost-effectiveness/utility value of one drug determined
from one study cannot be directly compared with that found in other studies
using different drugs. More direct prospective comparative trials will be needed
in respect of relative benefits and costs with different agents and adjunctive
therapies. (1) Alteplase is the first thrombolytic drug to be approved in France for the
treatment of ischaemic stroke within three hours of symptom onset. (2) The
clinical evaluation dossier contains nine placebo-controlled trials, of which
six were relatively large. In the two NINDS trials (624 patients in total),
treatment was started within the first three hours and it showed no survival
benefit. Near-complete functional recovery was more frequent in the alteplase
group than in the placebo group. In the two ECASS trials (620 and 800 patients)
and the ATLANTIS trials (142 patients and 613 patients), treatment was started
within the first six hours and it showed no significant benefit in terms of
survival or functional recovery. (3) There are two meta-analyses of these
trials. They confirm the lack of a survival benefit with alteplase. Using a
combined endpoint, one meta-analysis showed that treating 1000 patients with
alteplase prevented death or major disability (dependency) in 55 patients. The
other meta-analysis underlined the importance of a short interval between the
onset of symptoms and the beginning of treatment. (4) Intracranial haemorrhage
is the most important adverse effect. One meta-analysis showed that alteplase
caused 62 additional symptomatic intracranial haemorrhages (including 25 deaths)
per 1000 treated patients. (5) Various retrospective subgroup analyses have
tentatively identified subgroups of patients at a particularly high risk of
adverse effects, but subgroup analyses provide only weak evidence. The patients
most likely to benefit from alteplase, started within three hours of symptom
onset, remain to be defined. (6) The current health infrastructure in France
would allow only a small number of stroke patients to be treated with alteplase
under the kind of conditions prevailing in clinical trials (imaging to confirm
ischaemic stroke, and treatment very soon after the onset of symptoms). (7) In
practice, there is a narrow margin between the wanted and unwanted effects of
alteplase. This treatment should be used only by specialised teams and for
strictly selected patients. Research must continue, particularly to identify
those patients most likely to benefit from alteplase, and those most likely to
be harmed in whom thrombolysis is contraindicated. This paper presents a summary of the evidence review group report into the
clinical effectiveness and cost-effectiveness of alteplase for the treatment of
acute ischaemic stroke, in accordance with the licensed indication, based upon
the evidence submission from the manufacturer to the National Institute for
Health and Clinical Excellence (NICE) as part of the single technology appraisal
(STA) process. The submitted clinical evidence included several randomised
controlled trials indicating that, in highly selected patients, alteplase
administered at a licensed dose within 3 hours of the onset of acute ischaemic
stroke is associated with a statistically significant reduction in the risk of
death or dependency at 3 months compared with placebo, despite a significantly
increased risk of symptomatic intracranial haemorrhage within the first 7-10
days. Data from the National Institute of Neurological Disorders and Stroke
(NINDS) trial suggest that the benefit of treatment is sustained at 6 and 12
months. However, data from observational studies suggest that few patients with
acute ischaemic stroke will be eligible for alteplase therapy under the terms of
the current licensing agreement. In particular, many patients will be excluded
by virtue of their age, and many more by the restriction of therapy to patients
in whom treatment can be initiated within 3 hours of symptom onset. The
manufacturer's submission included a state transition model evaluating the
impact of treatment with alteplase within 3 hours of onset of stroke symptoms
compared to standard treatment reporting that, in the base-case analysis,
alteplase was both less costly and more effective than standard treatment. This
increased to a maximum of approximately 4000 pounds upon one-way sensitivity
analysis of the parameters. The probabilistic sensitivity analysis presented
within the submission suggests that the probability that alteplase has a
cost-effectiveness ratio greater than 20,000 pounds per quality-adjusted
life-year (QALY) gained is close to 1 (0.99). The results of the short-term
model demonstrate that alteplase is cost-effective over a 12-month period, with
an incremental cost-effectiveness ratio of 14,026 pounds per QALY gained. This
increased to a maximum of 50,000 pounds upon one-way sensitivity analysis of the
parameters. At 12 months, the probabilistic sensitivity analysis presented
within the submission suggests that the probability that alteplase has a
cost-effectiveness ratio greater than 20,000 pounds per QALY gained is
approximately 0.7. The guidance issued by NICE in April 2007 as a result of the
STA states that alteplase is recommended for the treatment of acute ischaemic
stroke only when used by physicians trained and experienced in the management of
acute stroke and in centres with the required facilities. Stroke is a major cause of mortality and morbidity, and thrombolysis has served
as a catalyst for major changes in the management of acute ischaemic stroke.
Intravenous alteplase (recombit tissue plasminogen activator) is the only
approved thrombolytic agent at present indicated for acute ischaemic stoke.
While the licensed time window extends to 3h from symptom onset, recent data
suggest that the trial window can be extended up to 4.5 h with overall benefit.
Nonetheless, 'time is brain' and every effort must be made to reduce the time
delay to thrombolysis. Intracranial haemorrhage is the major complication
associated with thrombolysis, and key factors increasing risk of haemorrhage
include increasing age, high blood pressure, diabetes and stroke severity.
Currently, there is no direct evidence to support thrombolysis in patients >80
years of age, with a few case series indicating no overt harm. Identification of
viable penumbra based on computed tomography/magnetic resoce imaging may
allow future extension of the time window. Adjuvant transcranial Doppler
ultrasound has the potential to improve reperfusion rates. While intra-arterial
thrombolysis has been in vogue for a few decades, there is no clear advantage
over intravenous thrombolysis. The evidence base for thrombolysis in specific
situations (e.g. dissection, pregcy) is inadequate, and individualized
decisions are needed, with a clear indication to the patient/carer about the
lack of direct evidence, and the risk-benefit balance. Patient-friendly
information leaflets may facilitate the process of consent for thrombolysis.
This article summarizes the recent advances in thrombolysis for acute ischaemic
stroke. Key questions faced by clinicians during the decision-making process are
answered based on the evidence available. RATIONALE: Intravenous thrombolysis with recombit tissue Plasminogen
Activator improves outcomes in patients treated early after stroke but at the
risk of causing intracranial hemorrhage. Restricting recombit tissue
Plasminogen Activator use to patients with evidence of still salvageable tissue,
or with definite arterial occlusion, might help reduce risk, increase benefit
and identify patients for treatment at late time windows.
AIMS: To determine if perfusion or angiographic imaging with computed tomography
or magnetic resoce help identify patients who are more likely to benefit from
recombit tissue Plasminogen Activator in the context of a large multicenter
randomized trial of recombit tissue Plasminogen Activator given within
six-hours of onset of acute ischemic stroke, the Third International Stroke
Trial.
DESIGN: Third International Stroke Trial is a prospective multicenter randomized
controlled trial testing recombit tissue Plasminogen Activator (0·9 mg/kg,
maximum dose 90 mg) started up to six-hours after onset of acute ischemic
stroke, in patients with no clear indication for or contraindication to
recombit tissue Plasminogen Activator. Brain imaging (computed tomography or
magnetic resoce) was mandatory pre-randomization to exclude hemorrhage. Scans
were read centrally, blinded to treatment and clinical information. In centers
where perfusion and/or angiography imaging were used routinely in stroke, these
images were also collected centrally, processed and assessed using validated
visual scores and computational measures.
STUDY OUTCOMES: The primary outcome in Third International Stroke Trial is alive
and independent (Oxford Handicap Score 0-2) at 6 months; secondary outcomes are
symptomatic and fatal intracranial hemorrhage, early and late death. The
perfusion and angiography study additionally will examine interactions between
recombit tissue Plasminogen Activator and clinical outcomes, infarct growth
and recanalization in the presence or absence of perfusion lesions and/or
arterial occlusion at presentation. The study is registered ISRCTN25765518. OBJECTIVE: To review the literature pertaining to the efficacy of alteplase for
restoration of patency of occluded venous and dialysis catheters in pediatric
patients.
DATA SOURCES: A MEDLINE search was conducted and cross-referenced with an EMBASE
search through November 2012. Search terms included alteplase, tissue
plasminogen activator, and catheter.
STUDY SELECTION AND DATA EXTRACTION: Search results were limited to humans,
English language, and ages from neonates to 18 years. Pertinent studies
discussing efficacy of alteplase for restoration of occluded venous or dialysis
catheter function were included. Case reports, review articles, and studies that
specified inclusion of hemophilia patients or more than 75% of children with
maligcy were excluded.
DATA SYNTHESIS: Fibrinolytics are the drug class of choice for restoration of
patency (defined as the ability to withdraw a blood sample) of thrombus-occluded
catheters. The trials used to support Food and Drug Administration approval of
alteplase for central venous catheter (CVC) occlusions generally had low
pediatric enrollment; however, additional small studies are available that
support use of alteplase for this indication in children. Alteplase doses of
0.5-2 mg instilled into the lumen of a CVC with dwell times ranging from 30 to
more than 240 minutes plus the potential for repeat dosing were reported.
Overall efficacy ranged from approximately 50% to 90%, with greater efficacy
generally reported with larger doses and longer dwell times. Alteplase doses of
2-2.5 mg with dwell times of 60-120 minutes were observed in 2 studies of
occluded peritoneal or hemodialysis catheters, in which efficacy was reported in
57-100% of cases. Limitations of current studies of alteplase for catheter
occlusion in children include small study populations and relative lack of
pediatric-specific prospective trials.
CONCLUSIONS: Alteplase appears to show efficacy for treatment of
thrombus-related venous catheter occlusion in pediatric patients; however, data
regarding its use in occluded dialysis catheters are limited. In Japan, intravenous alteplase, a recombit tissue-type plasminogen activator
(rt-PA), was approved for an indication of ischemic stroke in 2005 on the basis
of the results of a clinical trial with a unique dose of the drug (0.6 mg/kg).
The Japan Stroke Society published the guidelines for intravenous application of
rt-PA and organized training sessions for proper use all over Japan in an effort
to promote the safe, widespread use of intravenous alteplase. Seven years
following its approval, clinical experience with intravenous alteplase has
accumulated, additional evidence of intravenous alteplase has been found in
Japan and overseas, and the medical environment has substantially changed,
including approvals for new drugs and medical devices. Notably, the use of
alteplase in the extended therapeutic time window (within 4.5 hours of symptom
onset) became covered by insurance in Japan in August 2012. To address these
changing situations, we have decided to prepare the revised guidelines. In
preparing the second edition, we took care to make its contents more practical
by emphasizing information needed in clinical practice. While the first edition
was developed with emphasis on safety in light of limited clinical experience
with intravenous alteplase in Japan in 2005, this second edition is a
substantial revision of the first edition mainly in terms of eligibility
criteria, on the basis of accumulated evidence and the clinical experience. |
List scaffold proteins of the ERK signaling pathway. | Originally identified in yeast, scaffold proteins are now recognized to contribute to the specificity of MEK/ERK pathways in mammalian cells. These scaffolds include kinase suppressor of Ras (KSR), beta-arrestin, MEK partner-1 (MP-1), Sef and IQ motif-containing GTPase-activating protein 1(IQGAP1). Human disc-large homolog (hDlg) acts as a MEK2-specific scaffold protein for the ERK signaling pathway. Two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2). Several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 an event that boosts the mitogen-activated protein kinase (ERK) pathway. | 14-3-3 proteins may function as adapter or scaffold proteins in signal
transduction pathways. We reported previously that several 14-3-3 isotypes bind
to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van
der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J.
345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK)
pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3
would affect PKC-zeta activity. Using recombit 14-3-3 proteins and purified
PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that
14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to
approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to
their mutual affinity, estimated by co-immunoprecipitation from COS cell
lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding
site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As
14-13-3 proteins are acidic, we tested several other acidic proteins, which
turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral
or basic proteins did not. These effects were not restricted to the atypical
PKC-zeta, but were also found for classical PKC. Together, the results suggest
that the stimulation of PKC activity by 14-3-3 proteins is non-specific and
solely due to the acidic nature of these proteins, quite similar to that known
for acidic lipids. While scaffold proteins are thought to be key components of signaling pathways,
their exact function is unknown. By preassembling multiple components of
signaling cascades, scaffolds are predicted to influence the efficiency and/or
specificity of signaling events. Here we analyze a potential scaffold of the
Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras
(KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal
even though ERK kinase activation was attenuated to a degree sufficient to block
T-cell activation and inhibit tumor development. Consistent with its role as a
scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost
in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that
is not required for but enhances signaling via the Ras/MAPK signaling pathway. Recent studies indicate that kinase suppressor of Ras (KSR)is a scaffold protein
for the Ras/Raf/MEK/ERK signaling cascade in mammals. To help determine the in
vivo function of KSR, we have examined the tissue-specific distribution of this
protein in the embryonic and adult mouse using a rat monoclonal antibody raised
against the mouse protein. Western blot analysis indicates that the protein is
expressed at highest levels in the adult brain. It is also expressed at low
levels in bladder, ovary, testis, and lung, but the protein is not detectable in
any other adult tissue. However, reverse transcription-PCR analysis shows that
Ksr transcripts are detected in all adult tissues except the liver. A variant
containing a differentially spliced exon in the CA4 domain is observed in brain,
cerebellum, ovary, and intestine. The protein is also expressed throughout the
E6.5 embryo and at high levels in the neuroepithelium of the E10.5 embryo. At
this embryonic stage, expression is also detected at lower levels in the limb
and tail buds as well as in the myocardium. Eukaryotic cells use the extracellular signal regulated kinase (ERK) cascade to
connect cell-surface receptors to intracellular targets. Although various
signals are routed through the ERK pathway, cells respond accordingly to a given
stimulus. To regulate proper signal transduction, scaffolds and adaptors are
employed to organize specific signaling units. The scaffold protein MP1 (MEK1
partner) assembles a scaffold complex in the ERK cascade. We show that p14
functions as an adaptor protein, which is required and sufficient to localize
MP1 to endosomes. Reduction of MP1 or p14 protein levels by siRNAi results in
defective signal transduction. Therefore, our results suggest that the endosomal
localization of the p14/MP1-MAPK scaffold complex is crucial for signal
transduction. Signal transduction occurs by the reversible assembly of oligomeric protein
complexes that include both enzymatic proteins and proteins without known
enzymatic activity. These nonenzymatic components can serve as scaffolds or
anchors and regulate the efficiency, specificity, and localization of the
signaling pathway. Here we report the identification of MORG1 (mitogen-activated
protein kinase organizer 1), a member of the WD-40 protein family that was
isolated as a binding partner of the extracellular signal-regulated kinase (ERK)
pathway scaffold protein MP1. MORG1 specifically associates with several
components of the ERK pathway, including MP1, Raf-1, MEK, and ERK, and
stabilizes their assembly into an oligomeric complex. MORG1 facilitates ERK
activation when cells are stimulated with lysophosphatidic acid, phorbol
12-myristate 13-acetate, or serum, but not in response to epidermal growth
factor. Suppression of MORG1 by short interfering RNA leads to a marked
reduction in ERK activity when cells are stimulated with serum. We propose that
MORG1 is a component of a modular scaffold system that participates in the
regulation of agonist-specific ERK signaling. Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the
components of the Raf/MEK/ERK kinase cascade and positively regulates ERK
signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical
regulator of KSR1 subcellular localization and ERK activation. We examined the
role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK
activation and cell proliferation. We hypothesized that KSR1 phosphorylation is
involved in generating signaling specificity through the Raf/MEK/ERK kinase
cascade in response to stimulation by different growth factors. In fibroblasts,
platelet-derived growth factor stimulation induces sustained ERK activation and
promotes S-phase entry. Treatment with epidermal growth factor induces transient
ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and
Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle
progression with either platelet-derived growth factor or epidermal growth
factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA
proliferate two times faster and grow to a higher density than cells expressing
the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than
wild-type KSR1. These data demonstrate that phosphorylation and stability of the
molecular scaffold KSR1 are critical regulators of growth factor-specific
responses that promote cell proliferation. Specificity in signal transduction can be achieved through scaffolds, anchors,
and adapters that assemble generic signal transduction components in specific
combinations and locations. MEK Partner-1 (MP1) was identified as a potential
"scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK)
pathway. To gain insight into the interactions of MP1 with the ERK pathway, we
analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the
regulation of these interactions. Gel filtration of cell lysates revealed two
major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1
mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1
activity, and functioned as a domit negative, consistent with the concept
that MP1 function depends on facilitating these oligomerizations. Activation of
the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1
dimerization or MP1-MEK1 interactions, but caused the dissociation of the
MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did
not restore the complex, suggesting that regulation of complex formation occurs
independently of ERK phosphorylation. These results support the concept that MP1
functions as a regulator of MAP kinase signaling by binding to MEK1 and
regulating its association with a larger signaling complex that may sequentially
service multiple molecules of ERK. Beta-arrestin mediates desensitization and internalization of beta-adrenergic
receptors (betaARs), but also acts as a scaffold protein in extracellular
signal-regulated kinase (ERK) cascade. Thus, we have examined the role of
beta-arrestin2 in the betaAR-mediated ERK signaling pathways. Isoproterenol
stimulation equally activated cytoplasmic and nuclear ERK in COS-7 cells
expressing beta1AR or beta2AR. However, the activity of nuclear ERK was enhanced
by co-expression of beta-arrestin2 in beta2AR-but not beta1AR-expressing cells.
Pertussis toxin treatment and blockade of Gbetagamma action inhibited
beta-arrestin2-enhanced nuclear activation of ERK, suggesting that
beta-arrestin2 promotes nuclear ERK localization in a Gbetagamma dependent
mechanism upon receptor stimulation. beta2AR containing the carboxyl terminal
region of beta1AR lost the beta-arrestin2-promoted nuclear translocation. As the
carboxyl terminal region is important for beta-arrestin binding, these results
demonstrate that recruitment of beta-arrestin2 to carboxyl terminal region of
beta2AR is important for ERK localization to the nucleus. Signal transduction networks allow cells to recognize and respond to changes in
the extracellular environment. All eukaryotic cells have MAPK (mitogen-activated
protein kinase) pathways that participate in diverse cellular functions,
including differentiation, survival, transformation and movement. Five distinct
groups of MAPKs have been characterized in mammals, the most extensively studied
of which is the Ras/Raf/MEK [MAPK/ERK (extracellular-signal-regulated kinase)
kinase]/ERK cascade. Numerous stimuli, including growth factors and phorbol
esters, activate MEK/ERK signalling. How disparate extracellular signals are
translated by MEK/ERK into different cellular functions remains obscure.
Originally identified in yeast, scaffold proteins are now recognized to
contribute to the specificity of MEK/ERK pathways in mammalian cells. These
scaffolds include KSR (kinase suppressor of Ras), beta-arrestin, MEK partner-1,
Sef and IQGAP1. Scaffolds organize multiprotein signalling complexes. This
targets MEK/ERK to specific substrates and facilitates communication with other
pathways, thereby mediating diverse functions. The adaptor proteins regulate the
kinetics, amplitude and localization of MEK/ERK signalling, providing an
efficient mechanism that enables an individual extracellular stimulus to promote
a specific biological response. Cadherin-mediated interactions are integral to synapse formation and
potentiation. Here we show that N-cadherin is required for memory formation and
regulation of a subset of underlying biochemical processes. N-cadherin
antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently
disrupted hippocampal N-cadherin dimerization and impaired the formation of
long-term contextual fear memory while sparing short-term memory, retrieval, and
extinction. HAV-N impaired the learning-induced phosphorylation of a
distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and
altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated
cell adhesion to the cytoskeleton. This effect was accompanied by reduction of
N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures,
HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused
relocation of IQGAP1 from dendritic spines into the shafts. The data suggest
that the newly identified role of hippocampal N-cadherin in memory consolidation
may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling. Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved
transduction pathways involved in many cellular processes. Kinase modules are
associated with scaffold proteins that regulate signaling by providing critical
spatial and temporal specificities. Some of these scaffold proteins have been
shown to be conserved, both in sequence and function. In mouse, the scaffold MP1
(MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we
focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously
during embryonic and larval development. By in vitro and in vivo experiments, we
show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts
with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing
either dMP1 over-expression or dMP1 down-regulation by RNA interference
highlight dMP1 function in the control of cell differentiation during
development of the Drosophila wing. Although the mechanisms underlying striatal neurodegeneration are poorly
understood, we have shown that striatal pathogenesis may be initiated by high
synaptic levels of extracellular dopamine (DA). Here we investigated in rat
striatal primary neurons the mobilization of the mitogen-activated protein
kinase (MAPK) signaling pathways after treatment with DA. Instead of observing
an elevation of the archetypical pro-cytotoxic MAPKs, p-JNK and p-p38 MAPK, we
found that DA, acting through D1 DA receptors, induced a sustained stimulation
of the phosphorylated form of extracellular signal-regulated kinase (p-ERK) via
a cAMP/protein kinase A (PKA)/Rap1/B-Raf / MAPK/ERK kinase (MEK) pathway.
Blockade of D2 DA receptors, beta-adrenergic receptors or N-methyl-D-aspartate
receptors with receptor-specific antagonists had no significant effect on this
process. Activation of D1 DA receptors and PKA by DA caused phosphorylation and
inactivation of the striatal-enriched tyrosine phosphatase, an important
phosphatase for the dephosphorylation and subsequent inactivation of p-ERK in
the striatum. Interestingly, p-ERK was primarily retained in the cytoplasm, with
only low amounts translocated to the nucleus. The scaffold protein
beta-arrestin2 interacted with both p-ERK and D1 DA receptor, triggering the
cytosolic retention of p-ERK and inducing striatal neuronal apoptotic death.
These data provide unique insight into a novel role of p-ERK in striatal
neurodegeneration. Protein scaffolds have emerged as important regulators of MAPK cascades,
facilitating kinase activation and providing crucial spatio/temporal control to
their signaling outputs. Using a proteomics approach to compare the binding
partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2
interact with the kinase components of the ERK cascade and have a common
function in promoting RTK-mediated ERK signaling. Strikingly, we find that the
protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2
uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin
dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus
regulating KSR2 localization and activity. Moreover, we find that depletion of
endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling
responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These
findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new
mechanism whereby Ca2+ impacts Ras to ERK pathway signaling. KSR-1 is a scaffold protein that is essential for Ras-induced activation of the
highly conserved RAF-MEK-ERK kinase module. Previously, we identified a close
homolog of KSR-1, called KSR-2, through structural homology-based data mining.
In order to further understand the role of KSR-2 in MAPK signaling, we undertook
a functional proteomics approach to elucidate the dynamic composition of the
KSR-2 functional complex in HEK-293 cells under conditions with and without
TNF-alpha stimulation. We found nearly 100 proteins that were potentially
associated with KSR-2 complex and 43 proteins that were likely recruited to the
super molecular complex after TNF-alpha treatment. Our results indicate that
KSR-2 may act as a scaffold protein similar as KSR-1 to mediate the MAPK core
(RAF-MEK-ERK) signaling but with a distinct RAF isoform specificity, namely
KSR-2 may only mediate the A-RAF signaling while KSR-1 is responsible for
transducing signals only from c-RAF. In addition, KSR-2 may be involved in the
activation of many MAPK downstream signaling molecules such as p38 MAPK, IKAP,
AIF, and proteins involved in ubiquitin-proteasome, apoptosis, cell cycle
control, and DNA synthesis and repair pathways, as well as mediating crosstalks
between MAPK and several other signaling pathways, including PI3K and insulin
signaling. While interactions with these molecules are not known for KSR-1, it's
reasonable to hypothesize that KSR-1 may also play a similar role in mediating
these downstream signaling pathways. Scaffold proteins regulate intracellular MAP kinase signaling by providing
critical spatial and temporal specificities. We have shown previously that the
scaffold protein MEK1 partner (MP1) is localized to late endosomes by the
adaptor protein p14. Using conditional gene disruption of p14 in livers of mice
(p14(Δhep) ) we analyzed protein and transcript signatures in tissue samples.
Further biological network analysis predicted that the differentially expressed
transcripts and proteins are involved in cell cycle progression and regulation
of cellular proliferation. Although some of the here identified signatures were
previously linked to phospho-ERK activity, most of them were novel targets of
the late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation
defect was confirmed in a chemically induced liver regeneration model in
p14(Δhep) knockout mice. BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK
pathways) are involved in cell fate acquisition during development. These kinase
modules are associated with scaffold proteins that control their activity. In
Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling
during wing development and contributes to intervein and vein cell
differentiation. Functional relationships during wing development between a
chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its
scaffold protein dMP1, are examined here.
RESULTS: Genetic interactions show that corto and dMP1 act together to
antagonize rolled (which encodes ERK) in the future intervein cells, thus
promoting intervein fate. Although Corto, ERK and dMP1 are present in both
cytoplasmic and nucleus compartments, they interact exclusively in nucleus
extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on
polytene chromosomes, suggesting that they regulate gene expression directly on
chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation
pattern differs between cytoplasm and nucleus and changes upon ERK activation.
CONCLUSIONS: Our data therefore suggest that the Enhancer of Trithorax and
Polycomb Corto could participate in regulating vein and intervein genes during
wing tissue development in response to ERK signaling. ERK activation is enhanced by the scaffolding proteins KSR and MP1, localized
near the cell membrane and late endosomes respectively, but little is known
about their dynamic interplay. We develop here a mathematical model with
ordinary differential equations to describe the dynamic activation of EGFR-ERK
signaling under a conventional pathway without scaffolds, a KSR-scaffolded
pathway, and an MP1-scaffolded pathway, and their impacts were examined under
the influence of the endosomal regulators, Cbl-CIN85 and Endophilin A1. This new
integrated model, validated against experimental results and computational
constraints, shows that changes of ERK activation and EGFR endocytosis in
response to EGF concentrations (i.e ligand sensitivity) depend on these scaffold
proteins and regulators. The KSR-scaffolded and the conventional pathways act
synergistically and are sensitive to EGF stimulation. When the KSR level is
high, the sensitivity of ERK activation from this combined pathway remains low
when Cbl-CIN85 level is low. But, such sensitivity can be increased with
increasing levels of Endophilin if Cbl-CIN85 level becomes high. However,
reduced KSR levels already present high sensitivity independent of Endophilin
levels. In contrast, ERK activation by MP1 is additive to that of KSR but it
shows little ligand-sensitivity under high levels of EGF. This can be partly
reversed by increasing level of Endophilin while keeping Cbl-CIN85 level low.
Further analyses showed that high levels of KSR affect ligand-sensitivity of
EGFR endocytosis whereas MP1 ensures the robustness of endosomal ERK activation.
These simulations constitute a multi-dimensional exploration of how
EGF-dependent EGFR endocytosis and ERK activation are dynamically affected by
scaffolds KSR and MP1, co-regulated by Cbl-CIN85 and Endophilin A1. Together,
these results provide a detailed and quantitative demonstration of how
regulators and scaffolds can collaborate to fine-tune the ligand-dependent
sensitivity of EGFR endocytosis and ERK activation which could underlie
differences during normal physiology, disease states and drug responses. Scaffolding proteins add a new layer of complexity to the dynamics of cell
signaling. Above their basic function to bring several components of a signaling
pathway together, recent experimental research has found that scaffolds
influence signaling in a much more complex way: scaffolds can exert some
catalytic function, influence signaling by allosteric mechanisms, are
feedback-regulated, localize signaling activity to distinct regions of the cell
or increase pathway fidelity. Here we review experimental and theoretical
approaches that address the function of two MAPK scaffolds, Ste5, a scaffold of
the yeast mating pathway and KSR1/2, a scaffold of the classical mammalian MAPK
signaling pathway. For the yeast scaffold Ste5, detailed mechanistic models have
been valuable for the understanding of its function. For scaffolds in mammalian
signaling, however, models have been rather generic and sketchy. For example,
these models predicted narrow optimal scaffold concentrations, but when
revisiting these models by assuming typical concentrations, rather a range of
scaffold levels optimally supports signaling. Thus, more realistic models are
needed to understand the role of scaffolds in mammalian signal transduction,
which opens a big opportunity for systems biology. We have previously described a novel modulator of the actin cytoskeleton that
also regulates Ras and mitogen-activated protein kinase activities in
TGFβ-sensitive epithelial cells. Here we examined the functional role of this
signaling regulatory protein (km23-1) in mediating the migration, invasion, and
tumor growth of human colorectal carcinoma (CRC) cells. We show that small
interfering RNA (siRNA) depletion of km23-1 in human CRC cells inhibited
constitutive extracellular signal-regulated kinase (ERK) activation, as well as
pro-invasive ERK effector functions that include phosphorylation of Elk-1,
constitutive regulation of c-Fos-DNA binding, TGFβ1 promoter transactivation,
and TGFβ1 secretion. In addition, knockdown of km23-1 reduced the paracrine
effects of CRC cell-secreted factors in conditioned medium and in fibroblast
co-cultures. Moreover, km23-1 depletion in human CRC cells reduced cell
migration and invasion, as well as expression of the ERK-regulated,
metastasis-associated scaffold protein Ezrin. Finally, km23-1 inhibition
significantly suppressed tumor formation in vivo. Thus, our results implicate
km23-1 as a novel anti-metastasis target for human colon carcinoma cells,
capable of decreasing tumor growth and invasion via a mechanism involving
suppression of various pro-migratory features of CRC. These include a reduction
in ERK signaling, diminished TGFβ1 production, decreased expression of the
plasma membrane-cytoskeletal linker Ezrin, as well as attenuation of the
paracrine effects of colon carcinoma-secreted factors on fibroblast migration
and mitogenesis. As such, km23-1 inhibitors may represent a viable therapeutic
strategy for interfering with colon cancer progression and invasion. The scaffold protein IQGAP1 regulates cell signaling through the RAF/MEK/ERK
pathway. Recent data show that cancer cells in which the RAF/MEK/ERK pathway is
activated are particularly sensitive to the disruption of IQGAP1 function. IQGAP
drugs may be particularly effective in tumors that develop resistance to
existing pathway drugs. Nrf2 plays a critical role in the regulation of cellular oxidative stress.
MEK-ERK activation has been shown to be one of the major pathways resulting in
the activation of Nrf2 and induction of Nrf2 downstream targets, including phase
II detoxifying/antioxidant genes in response to oxidative stress and
xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating
protein 1), a new Nrf2 interaction partner that we have published previously,
was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II
detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids
699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl
isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway.
Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2,
which in turn was associated with a decrease in the nuclear translocation of
Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes.
In the aggregate, these results suggest that IQGAP1 may play an important role
in the MEK-ERK-Nrf2 signaling pathway. BACKGROUND: Scaffold proteins modulate cellular signaling by facilitating
assembly of specific signaling pathways. However, there is at present little
information if and how scaffold proteins functionally interact with each other.
RESULTS: Here, we show that two scaffold proteins, caveolin-1 and IQGAP1, are
required for phosphorylation of the actin associated pool of extracellular
signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C
activation. We show by immunofluorescence and proximity ligation assays, that
IQGAP1 tethers ERK1/2 to actin filaments. Moreover, siRNA experiments
demonstrate that IQGAP1 is required for activation of actin-bound ERK1/2.
Caveolin-1 is also necessary for phosphorylation of actin-bound ERK1/2 in
response to protein kinase C, but is dispensible for ERK1/2 association with
actin. Simultaneous knock down of caveolin-1 and IQGAP1 decreases total phorbol
ester-induced ERK1/2 phosphorylation to the same degree as single knock down of
either caveolin-1 or IQGAP1, indicating that caveolin-1 and IQGAP1 operate in
the same ERK activation pathway. We further show that caveolin-1 knock down, but
not IQGAP1 knock down, reduces C-Raf phosphorylation in response to phorbol
ester stimulation.
CONCLUSIONS: Based on our data, we suggest that caveolin-1 and IQGAP1 assemble
distinct signaling modules, which are then linked in a hierarchical arrangement
to generate a functional ERK1/2 activation pathway. The G protein-coupled receptor kinases (GRKs) are best known for their role in
phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs
also regulate signalling downstream of other families of receptors and have a
number of non-receptor substrates and binding partners. Here we identify RhoAGTP
and Raf1 as novel binding partners of GRK2 and report a previously unsuspected
function for this kinase. GRK2 is a RhoA effector that serves as a
RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of
GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoAGTP binding to the
catalytic domain of the kinase. Exogenous GRK2 has previously been shown to
increase ERK activation downstream of the epidermal growth factor receptor
(EGFR). Here we find that GRK2-mediated ERK activation downstream of the EGFR is
Rho-dependent and that treatment with EGF promotes RhoAGTP binding and ERK
scaffolding by GRK2. Depletion of GRK2 expression by RNAi reveals that GRK2 is
required for EGF-induced, Rho- and ERK-dependent thymidine incorporation in
vascular smooth muscle cells (VSMCs). We therefore hypothesise that
Rho-dependent ERK MAPK scaffolding by GRK2 downstream of the EGFR may have an
important role in the vasculature, where increased levels of both GRK2 and RhoA
have been associated with hypertension. Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is
caused by loss of a large evolutionary conserved protein that serves as a GTPase
Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null
mutant phenotypes, learning/memory deficits and reduced overall growth resemble
human NF1 symptoms. These and other dNf1 defects are relatively insensitive to
manipulations that reduce Ras signaling strength but are suppressed by
increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP)
dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this
pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly
remains controversial. To shed light on this issue we screened 486 1(st) and
2(nd) chromosome deficiencies that uncover >80% of annotated genes for domit
modifiers of the dNf1 pupal size defect, identifying responsible genes in
crosses with mutant alleles or by tissue-specific RNA interference (RNAi)
knockdown. Validating the screen, identified suppressors include the previously
implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two
other genes involved in Ras/ERK signal transduction and several involved in
cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1
growth deficiency include the intersectin-related synaptic scaffold protein
Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin
receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are
among mutants that also suppress a recently identified dNf1 neuromuscular
junction (NMJ) overgrowth phenotype and that manipulations that increase
cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of
the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally,
supporting our previous contention that ALK might be a therapeutic target in
NF1, we report that human ALK is expressed in cells that give rise to NF1 tumors
and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man. Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the
core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and
temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this
mechanism can have a high influence in inhibiting the downstream signaling of
the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of
the studies targeted to prevent the binding of Raf, MEK binding on kinase
suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold
proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding
site with small ligand molecules like flavones and anthocyanidins and analyzed
through insilico docking studies. The binding energy evaluation shows the
importance of hydroxyl groups at various positions on the flavone and
anthocyanidin nucleus. Over all binding interaction shows these ligands occupied
the potential sites of cysteine rich C1 domain of scaffold protein KSR. |
Which are the families of mammalian DNA-(cytosine-5)-methyltransferases? | DNA (cytosine-5)-methyltransferases catalyze the specific transfer of a methyl group to the C5 position of cytosine residues in DNA. Three families of DNA (cytosine-5)-methyltransferases have been identified in mammals: DNMT1, DNMT2 and DNMT3 (including DNMT3a, DNMT3b and DNMT3L isoforms). All of them share homologous catalytic domains. DNMT1 is the ���maintenance” methyltransferase family. DNMT1 is specific for hemi-methylated DNA and ensures the faithful transmission of DNA methylation patterns in every replication cycle. DNMT3 is required for de novo methylation of DNA. DNMT3 targets unmethylated DNA and is responsible for the establishment of new methylation patterns. Dnmt2, in contrast to all other mammalian DNA (cytosine-5)-methyltransferases, does not possess a large N-terminal regulatory domain. The DNA methylation activity of DNMT2 is still controversial. | DNA methylation plays an essential role in genomic imprinting observed in
eutherian mammals and marsupials. In mouse, one of the two de novo DNA
methyltransferases, Dnmt3a, and a related protein, Dnmt3L have been shown to be
essential for imprint establishment in the parental germline. To gain insights
into the evolution of imprinting mechanisms, we have identified and
characterized the DNMT3 family genes in other vertebrate species. We cloned
cDNAs for chicken DNMT3A and DNMT3B, whose putative protein products shared
81.5% and 48.6% amino acid sequence identity with their mouse orthologues. Using
computer-assisted database searches, we also identified DNMT3A and DNMT3B
orthologues in fish (fugu and zebrafish) and marsupials (opossum). We found
that, while opossums had an orthologue for DNMT3L, chickens and fish did not
have this gene. Thus, unlike the other DNMT3 members, DNMT3L was restricted to
the species in which imprinting occurs. The acquisition of DNMT3L by a common
ancestor of eutherians and marsupials might have been closely related to the
evolution of imprinting. DNA methylation is involved in fundamental cellular processes such as silencing
of genes and transposable elements, but the underlying mechanism of regulation
of DNA methylation is largely unknown. DNA methyltransferase 3-like protein
(Dnmt3L), a member of the Dnmt3 family of proteins, is required during the
establishment of DNA methylation patterns in germ cells. Dnmt3L does not possess
enzymatic activity. Rather, in vitro analysis indicates that Dnmt3L stimulates
DNA methylation by both Dnmt3a and Dnmt3b through direct binding to these
proteins. In the current study, we demonstrated that in vivo, Dnmt3L physically
and functionally interacted with the Dnmt3 isoform Dnmt3a2. In wild-type
embryonic stem (ES) cells, but not in cells lacking Dnmt3a, endogenous Dnmt3L
was concentrated in chromatin foci. In ES cells deficient in both Dnmt3a and
Dnmt3b, Dnmt3L was distributed diffusely throughout the nucleus and cytoplasm,
and ectopic expression of Dnmt3a2, but not Dnmt3a or Dnmt3b, restored wild-type
Dnmt3L localization. We showed that endogenous Dnmt3L physically interacted with
Dnmt3a2, but not Dnmt3a or Dnmt3b, in ES cells and embryonic testes. We also
found that specific CpG sites were demethylated upon depletion of either Dnmt3a
or Dnmt3L, but not Dnmt3b, in ES cells. These results provide evidence for a
physical and functional interaction between Dnmt3L and Dnmt3a2 in the nucleus.
We propose that Dnmt3a2 recruits Dnmt3L to chromatin, and induces regional DNA
methylation in germ cells. DNA methylation is a major epigenetic mechanism that has been suggested to
control developmental gene regulation during embryogenesis, but its regulatory
mechanisms remain unclear. In this report, we show that CpG islands associated
with the X-linked homeobox gene cluster Rhox, which is highly expressed in the
extraembryonic trophectoderm, are differentially methylated in a stage- and
lineage-specific manner during the post-implantation development of mice.
Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the
initiation of de novo DNA methylation, abolished the establishment of DNA
methylation and the silencing of Rhox cluster genes in the embryo proper. The
Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large
genomic region ( approximately 1 Mb) containing the Rhox cluster and surrounding
genes. Complementation experiments using embryonic stem (ES) cells deficient in
the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a
and Dnmt3b was restricted within this large genomic region, and did not affect
the neighboring genes outside it, implicating the existence of region-specific
boundaries. Our results suggest that DNA methylation plays important roles in
both long-range gene silencing and lineage-specific silencing in embryogenesis. DNA methyltransferase 1 (DNMT1) has been reported to interact with a wide
variety of factors and to contain intrinsic transcriptional repressor activity.
When a conservative point mutation was introduced at the key catalytic residue,
mutant DNMT1 failed to rescue any of the phenotypes of Dnmt1-null embryonic stem
(ES) cells, which indicated that the biological functions of DNMT1 are exerted
through the methylation of DNA. ES cells that expressed the mutant protein did
not survive differentiation. Intracisternal A-particle family retrotransposons
were no longer methylated and were transcribed at high levels. The proper
localization of DNMT1 depended on normal genomic methylation, and we discuss the
implications of this finding for epigenetic dysregulation in cancer. DNA methylation is an important epigenetic modification regulating various
biological phenomena, including genomic imprinting and transposon silencing. It
is known that methylation of the differentially methylated regions (DMRs)
associated with paternally imprinted genes and of some repetitive elements
occurs during male germ cell development in the mouse. We have performed a
detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2
and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and
Line1] and satellite repeats (major and minor) to determine the timing of this
de novo methylation in male germ cells. Furthermore, we have examined the roles
of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein
(Dnmt3L) in this process. We found that methylation of all DMRs and repeats
occurred progressively in fetal prospermatogonia and was completed by the
newborn stage. Analysis of newborn prospermatogonia from germline-specific
Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19
and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and
Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed
repeats IAP and Line1. Only Dnmt3b was required for the methylation of the
satellite repeats. These results indicate both common and differential target
specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed
moderate to severe hypomethylation in Dnmt3L-deficient prospermatogonia,
indicating the critical function and broad specificity of this factor in de novo
methylation. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many
genes. Recent studies suggest roles of miRNAs in carcinogenesis. We and others
have shown that expression profiles of miRNAs are different in lung cancer vs.
normal lung, although the significance of this aberrant expression is poorly
understood. Among the reported down-regulated miRNAs in lung cancer, the miRNA
(miR)-29 family (29a, 29b, and 29c) has intriguing complementarities to the
3'-UTRs of DNA methyltransferase (DNMT)3A and -3B (de novo methyltransferases),
two key enzymes involved in DNA methylation, that are frequently up-regulated in
lung cancer and associated with poor prognosis. We investigated whether miR-29s
could target DNMT3A and -B and whether restoration of miR-29s could normalize
aberrant patterns of methylation in non-small-cell lung cancer. Here we show
that expression of miR-29s is inversely correlated to DNMT3A and -3B in lung
cancer tissues, and that miR-29s directly target both DNMT3A and -3B. The
enforced expression of miR-29s in lung cancer cell lines restores normal
patterns of DNA methylation, induces reexpression of methylation-silenced tumor
suppressor genes, such as FHIT and WWOX, and inhibits tumorigenicity in vitro
and in vivo. These findings support a role of miR-29s in epigenetic
normalization of NSCLC, providing a rationale for the development of miRNA-based
strategies for the treatment of lung cancer. MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that
regulate gene expression. In animals, miRNAs form imperfect interactions with
sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational
inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with
protein coding regions. Here we show that human miR-148 represses DNA
methyltransferase 3b (Dnmt3b) gene expression through a region in its coding
sequence. This region is evolutionary conserved and present in the Dnmt3b splice
variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed
Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1
expression, short-hairpin RNA-mediated miR-148 repression leads to an increase
in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site
in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA,
which lacks the putative miR-148 target site, is resistant to miR-148-mediated
regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b
mediates regulation by the miR-148 family. More generally, we provide evidence
that coding regions of human genes can be targeted by miRNAs, and that such a
mechanism might play a role in determining the relative abundance of different
splice variants. In a previous genomic analysis, using somatic methyltransferase (DNMT) knockout
cells, we showed that hypomethylation decreased the expression of as many genes
as were observed to increase, suggesting a previously unknown mechanism for
epigenetic regulation. To address this idea, the expression of the BAG family
genes was used as a model. These genes were used because their expression was
decreased in DNMT1(-/-), DNMT3B(-/-), and double knockout cells and increased in
DNMT1-overexpressing and DNMT3B-overexpressing cells. Chromatin
immunoprecipitation analysis of the BAG-1 promoter in DNMT1-overexpressing or
DNMT3B-overexpressing cells showed a permissive dimethyl-H3-K4/dimethyl-H3-K9
chromatin status associated with DNA-binding of CTCFL/BORIS, as well as
increased BAG-1 expression. In contrast, a nonpermissive
dimethyl-H3-K4/dimethyl-H3-K9 chromatin status was associated with CTCF
DNA-binding and decreased BAG-1 expression in the single and double DNMT
knockout cells. BORIS short hairpin RNA knockdown decreased both promoter
DNA-binding, as well as BAG-1 expression, and changed the
dimethyl-H3-K4/dimethyl-H3-K9 ratio to that characteristic of a nonpermissive
chromatin state. These results suggest that DNMT1 and DNMT3B regulate BAG-1
expression via insulator protein DNA-binding and chromatin dynamics by
regulating histone dimethylation. OBJECTIVE: Epigenetic changes in gene expression, including DNA methylation and
histone modifications, might contribute to autoimmunity. DNA methylation is
mediated by a family of DNA methyltransferases. Polymorphisms of the DNA
methyltransferase 3B (DNMT3B) gene may influence DNMT3B activity on DNA
methylation, thereby modulating the susceptibility to some diseases. The purpose
of this study was to investigate the association between the single nucleotide
polymorphism (SNP) in promoter of the DNMT3B gene and the risk for development
of idiopathic thrombocytopenic purpura (ITP).
METHODS: In this hospital-based case-control study, the DNMT3B SNP was genotyped
in 201 patients with ITP and 136 healthy controls by polymerase chain
reaction-restriction fragment length polymorphism.
RESULTS: The C/C genotype was not detected in both the patients with ITP and the
controls. In the controls, the frequencies of T/T and C/T genotypes and T and C
alleles were 97.8%, 2.2%, 98.9%, and 1.1%, respectively. There was no
significant difference in genotype and allele distribution between the patients
with ITP and the controls (P = 0.745 and 0.747, respectively). No significant
difference was observed in genotype and allele distribution between the two
groups when stratified by the age. The similar results were shown among the four
groups of patients with ITP: acute childhood, chronic childhood, acute adult,
and chronic adult.
CONCLUSION: This polymorphism was distributed similarly between the patients
with ITP and the controls. It demonstrated that it may not be used as a
stratification marker to predict the susceptibility to ITP, at least in the
population of North China. CONTEXT AND OBJECTIVE: Epigenetic dysregulation is implicated in pituitary
neoplasia as the cause of silencing of several tumor suppressor genes. However,
the upstream mediators of such events remain unknown.
DESIGN: We examined the three members of the DNA methyltransferase (DNMT) enzyme
family in normal and neoplastic human and mouse pituitary cells.
SETTING: This study was performed at a university-affiliated cancer research
institute.
MAIN OUTCOME MEASURES: Gene expression, promoter DNA methylation, histone
modifications, and cell proliferation were determined.
RESULTS: In contrast to DNMT1 and DNMT3a, DNMT3b was expressed at relatively
higher levels in neoplastic pituitary cells. However, examination of the human
DNMT3b 5' region showed uniformly low DNA methylation levels with little
difference between normal and tumor samples. Through pharmacological methylation
inhibition or histone deacetylation inhibition, we identified that DNMT3b gene
expression is subject to histone modifications. Down-regulation of DNMT3b
resulted in induction of retinoblastoma, p21, and p27, and reduction in cell
proliferation. These targeted effects were associated with enhanced histone 3
acetylation and diminished histone methylation.
CONCLUSION: Our findings identify DNMT3b as a putative mediator of epigenetic
control through histone modifications of gene expression in pituitary cells. DNA methylation is a major epigenetic modification and plays a crucial role in
the regulation of gene expression. Within the family of DNA methyltransferases
(Dnmts), Dnmt3a and 3b establish methylation marks during early development,
while Dnmt1 maintains methylation patterns after DNA replication. The
maintece function of Dnmt1 is regulated by its large regulatory N-terminal
domain that interacts with other chromatin factors and is essential for the
recognition of hemi-methylated DNA. Gelfiltration analysis showed that purified
Dnmt1 elutes at an apparent molecular weight corresponding to the size of a
dimer. With protein interaction assays we could show that Dnmt1 interacts with
itself through its N-terminal regulatory domain. By deletion analysis and
co-immunoprecipitations we mapped the dimerization domain to the targeting
sequence TS that is located in the center of the N-terminal domain (amino acids
310-629) and was previously shown to mediate replication independent association
with heterochromatin at chromocenters. Further mutational analyses suggested
that the dimeric complex has a bipartite interaction interface and is formed in
a head-to-head orientation. Dnmt1 dimer formation could facilitate the
discrimination of hemi-methylated target sites as has been found for other
palindromic DNA sequence recognizing enzymes. These results assign an additional
function to the TS domain and raise the interesting question how these functions
are spatially and temporarily co-ordinated. DNA methyltransferases (DNMTs) are a family of enzymes that methylate DNA at the
C5 position of cytosine residues, and their inhibition is a promising strategy
for the treatment of various developmental and proliferative diseases,
particularly cancers. In the present study, a binding model for hydralazine,
with a validated homology model of human DNMT, was developed by the use of
automated molecular docking and molecular dynamics simulations. The docking
protocol was validated by predicting the binding mode of 2'-deoxycytidine,
5-azacytidine, and 5-aza-2'-deoxycytidine. The inhibitory activity of
hydralazine toward DNMT may be rationalized at the molecular level by similar
interactions within the binding pocket (e.g., by a similar pharmacophore) as
established by substrate-like deoxycytidine analogues. These interactions
involve a complex network of hydrogen bonds with arginine and glutamic acid
residues that also play a major role in the mechanism of DNA methylation.
Despite the different scaffolds of other non-nucleoside DNMT inhibitors such as
procaine and procainamide, the current modeling work reveals that these drugs
exhibit similar interactions within the DNMT1 binding site. These findings are
valuable in guiding the rational design and virtual screening of novel DNMT
inhibitors. OBJECTIVE: Consistent evidence indicated that aberrant DNA methylation may be
involved in the development of schizophrenia. DNA methyltransferase 3B (DNMT3B)
is the key methyltransferase in DNA methylation regulations. In this study, we
investigated the association between DNMT3B polymorphisms and the susceptibility
of early onset schizophrenia in Chinese Han population.
METHODS: Case-control (patients=381 and controls=472) and family based
(trios=103) study was performed through genotyping two tag single nucleotide
polymorphisms (rs2424908 and rs6119954) covering the whole DNMT3B gene. Single
nucleotide polymorphism association and haplotype analysis were performed.
RESULTS: The frequency of G allele of rs6119954 was significantly higher in
patients than that in controls (P=0.017). Genotype distribution of rs6119954 was
significantly different between patients and controls (P=0.046). A
haplotype-wise analysis revealed a higher frequency of the T-G
(rs2424908-rs6119954) haplotype in patients than that in controls (P=0.033). In
the transmission disequilibrium test analysis, G allele of rs6119954 was
preferentially transmitted in the trios (P=0.030).
CONCLUSION: Our findings indicate that DNMT3B may be a candidate gene for
susceptibility to early onset schizophrenia. The genome of extraembryonic tissue, such as the placenta, is hypomethylated
relative to that in somatic tissues. However, the origin and role of this
hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B
are the primary mediators of the establishment and maintece of DNA
methylation in mammals. In this study, we investigated promoter
methylation-mediated epigenetic down-regulation of DNMT genes as a potential
regulator of global methylation levels in placental tissue. Although DNMT3A and
-3B promoters lack methylation in all somatic and extraembryonic tissues tested,
we found specific hypermethylation of the maintece DNA methyltransferase
(DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first
trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic
methylation of DNMT1, with no evidence of imprinting (parent of origin effect).
In vitro reporter experiments confirmed that DNMT1 promoter methylation
attenuates transcriptional activity in trophoblast cells. However, global
hypomethylation in the absence of DNMT1 down-regulation is apparent in
non-primate placentas and in vitro derived human cytotrophoblast stem cells,
suggesting that DNMT1 down-regulation is not an absolute requirement for genomic
hypomethylation in all instances. These data represent the first demonstration
of methylation-mediated regulation of the DNMT1 gene in any system and
demonstrate that the unique epigenome of the human placenta includes
down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene.
This strongly implicates epigenetic regulation of the DNMT gene family in the
establishment of the unique epigenetic profile of extraembryonic tissue in
humans. DNA methylation, an essential regulator of transcription and chromatin
structure, is established and maintained by the coordinated action of three DNA
methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor
DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic
disease. DNMT3B is also highly alternatively spliced in a tissue- and
disease-specific manner. The impact of intra-DNMT3 interactions and alternative
splicing on the function of DNMT3 family members remains unclear. In the present
work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the
consequences of DNMT3B splicing and mutations on its ability to bind DNA,
interact with itself and other DNMT3's, and methylate DNA. Our results show
that, while the C-terminal catalytic domain is critical for most DNMT3B
functions, parts of the N-terminal region, including the PWWP domain, are also
important. Alternative splicing and domain deletions also influence DNMT3B's
cellular localization. Furthermore, our data reveal the existence of extensive
DNMT3B self-interactions that differentially impact on its activity. Finally, we
show that catalytically inactive isoforms of DNMT3B are capable of modulating
the activity of DNMT3A-DNMT3L complexes. Our studies therefore suggest that
seemingly 'inactive' DNMT3B isoforms may influence genomic methylation patterns
in vivo. The DNMT3B de novo DNA methyltransferase (DNMT) plays a major role in
establishing DNA methylation patterns in early mammalian development, but its
catalytic mechanism remains poorly characterized. Here, we provide a
comprehensive biochemical analysis of human DNMT3B function through the
characterization of a series of site-directed DNMT3B variants associated with
immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome.
Our data reveal several novel and important aspects of DNMT3B function. First,
DNMT3B, unlike DNMT3A, requires a DNA cofactor in order to stably bind to
S-adenosyl-l-methionine (SAM), suggesting that it proceeds according to an
ordered catalytic scheme. Second, ICF mutations cause a broad spectrum of
biochemical defects in DNMT3B function, including defects in
homo-oligomerization, SAM binding, SAM utilization, and DNA binding. Third, all
tested ICF mutations, including the A766P and R840Q variants, result in altered
catalytic properties without interfering with DNMT3L-mediated stimulation; this
indicates that DNMT3L is not involved in the pathogenesis of ICF syndrome.
Finally, our study reveals a novel level of coupling between substrate binding,
oligomerization, and catalysis that is likely conserved within the DNMT3 family
of enzymes. DNA methylation is widespread in most species, from bacteria to mammals, and is
crucial for genomic imprinting, gene expression, and embryogenesis. DNA
methylation occurs via two major classes of enzymatic reactions:
maintece-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase
(DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta
(DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ
cells (PGCs) and germ line cells has not been sufficiently established in birds.
Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ
hybridization analyses to examine the structural conservation and conserved
expression patterns of chicken DNMT family genes. We further examined the
regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by
cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a
dual fluorescence reporter assay. All cDNMT family members were differentially
detected during early embryonic development. Of interest, cDNMT3B expression was
highly detected in early embryos and in PGCs. During germ line development and
sexual maturation, cDNMT3B expression was reestablished in a female germ
cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B
expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%),
gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data
highlight the structural conservation and conserved expression patterns of
chicken DNMTs. The miRNAs investigated in this study may induce downregulation
of gene expression in chicken PGCs and germ cells. Mammalian DNA methyltransferase 1 (DNMT1) is essential for maintaining DNA
methylation patterns after cell division. Disruption of DNMT1 catalytic activity
results in whole genome cytosine demethylation of CpG dinucleotides, promoting
severe dysfunctions in somatic cells and during embryonic development. While
these observations indicate that DNMT1-dependent DNA methylation is required for
proper cell function, the possibility that DNMT1 has a role independent of its
catalytic activity is a matter of controversy. Here, we provide evidence that
DNMT1 can support cell functions that do not require the C-terminal catalytic
domain. We report that PCNA and DMAP1 domains in the N-terminal region of DNMT1
are sufficient to modulate E-cadherin expression in the absence of noticeable
changes in DNA methylation patterns in the gene promoters involved. Changes in
E-cadherin expression are directly associated with regulation of
β-catenin-dependent transcription. Present evidence suggests that the DNMT1 acts
on E-cadherin expression through its direct interaction with the E-cadherin
transcriptional repressor SNAIL1. A subset of genes, known as imprinted genes, is present in the mammalian genome.
Genomic imprinting governs the monoallelic expression of these genes, depending
on whether the gene was inherited from the sperm or the egg. This
parent-of-origin specific gene expression is generally dependent on the
epigenetic modification, DNA methylation, and the DNA methylation status of CpG
dinucleotides residing in loci known as differentially methylated regions
(DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3))
groups to the cytosine residue in the CpG dinucleotides are known as DNA
methyltransferases (DNMTs). Correct establishment and maintece of methylation
patterns at imprinted genes has been associated with placental function and
regulation of embryonic/fetal development. Much work has been carried out on
imprinted genes in mouse and human; however, little is known about the
methylation dynamics in the bovine oocyte. The primary objective of the present
study was to characterize the establishment of methylation at maternally
imprinted genes in bovine growing oocytes and to determine if the expression of
the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation
during oocyte development. To this end, a panel of maternally imprinted genes
was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST,
IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene;
the SNRPN DMR has been reported previously. Conventional bisulfite sequencing
revealed that methylation marks were acquired at all five DMRs investigated in
an oocyte size-dependent fashion. This was confirmed for a selection of genes
using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis
revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte
during its growth phase. This study demonstrates for the first time that an
increase in bovine imprinted gene DMR methylation occurs during oocyte growth,
as is observed in mouse. The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote
DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine
(5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine.
Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been
implicated in mESC maintece. Here we demonstrate that, unlike genetic
deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant
reduction in 5hmC and loss of mESC identity. The differentiation phenotype due
to Tet1 depletion positively correlated with the extent of 5hmC loss.
Meta-analyses of genomic data sets suggested interaction between Tet1 and
leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote
self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated
differentiation. Withdrawal of LIF leads to differentiation of mESCs. We
discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene
activation by affecting Stat3's ability to bind to its target sites on
chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known
to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further
supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the
conclusion that analysis of mESCs in the hours/days immediately following
efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining
the pluripotent state that may be subject to homeostatic compensation in genetic
models. Autosomal domit cerebellar ataxia, deafness and narcolepsy (ADCA-DN) is
characterized by late onset (30-40 years old) cerebellar ataxia, sensory
neuronal deafness, narcolepsy-cataplexy and dementia. We performed exome
sequencing in five individuals from three ADCA-DN kindreds and identified DNMT1
as the only gene with mutations found in all five affected individuals. Sanger
sequencing confirmed the de novo mutation p.Ala570Val in one family, and showed
co-segregation of p.Val606Phe and p.Ala570Val, with the ADCA-DN phenotype, in
two other kindreds. An additional ADCA-DN kindred with a p.GLY605Ala mutation
was subsequently identified. Narcolepsy and deafness were the first symptoms to
appear in all pedigrees, followed by ataxia. DNMT1 is a widely expressed DNA
methyltransferase maintaining methylation patterns in development, and mediating
transcriptional repression by direct binding to HDAC2. It is also highly
expressed in immune cells and required for the differentiation of CD4+ into T
regulatory cells. Mutations in exon 20 of this gene were recently reported to
cause hereditary sensory neuropathy with dementia and hearing loss (HSAN1). Our
mutations are all located in exon 21 and in very close spatial proximity,
suggesting distinct phenotypes depending on mutation location within this gene. In eukaryotes, DNA methylation is an important epigenetic modification that is
generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2
family have been shown to have a dual substrate specificity acting on DNA as
well as on three specific tRNAs (tRNA(Asp), tRNA(Val), tRNA(Gly)). Entamoeba
histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth)
that belongs to the DNMT2 family and shows high homology to the human enzyme as
well as to the bacterial DNA MTase M.HhaI. The molecular basis for the
recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is
unknown. Here we present the crystal structure of the
cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with
its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2
MTase, including the active site loop. Mobility shift assays show that in vitro
the full length tRNA is required for stable complex formation with EhMeth. Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive
pathophysiology of this disease, but little is known about the basis for such
changes. In this study, we examined a role for the DNA methyltransferase DNMT3B,
in particular, the truncated isoform DNMT3B7, which is generated frequently in
cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation,
gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B
expression in primary tumors. Higher levels of DNMT3B7 were detected in
differentiated ganglioneuroblastomas compared to undifferentiated
neuroblastomas, suggesting that expression of DNMT3B7 may induce a less
aggressive clinical phenotype. To test this hypothesis, we investigated the
effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a
significant inhibition of cell proliferation in vitro and angiogenesis and tumor
growth in vivo. DNMT3B7-positive cells had higher levels of total genomic
methylation and a dramatic decrease in expression of the FOS and JUN family
members that comprise AP1 transcription factors. Consistent with an established
antagonistic relationship between AP1 expression and retinoic acid receptor
activity, increased differentiation was seen in the DNMT3B7-expressing
neuroblastoma cells following treatment with all-trans retinoic acid (ATRA)
compared to controls. Our results indicate that DNMT3B7 modifies the epigenome
in neuroblastoma cells to induce changes in gene expression, inhibit tumor
growth, and increase sensitivity to ATRA. DNA methylation of cytosine nucleotides is an important epigenetic modification
that occurs in most eukaryotic organisms and is established and maintained by
various DNA methyltransferases together with their co-factors. There are two
major categories of DNA methyltransferases: de novo and maintece. Here, we
report the isolation and functional characterization of a de novo
methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length
coding region of OsDRM2 was cloned and transformed into Escherichia coli and
Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein,
which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and
CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de
novo activity of OsDRM2: (1) a 5'-CCGG-3' containing DNA fragment that had been
pre-treated with OsDRM2 protein expressed in E. coli was protected from
digestion by the CG-methylation-sensitive isoschizomer HpaII; (2)
methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae
genomic DNA from transformants that had been introduced with OsDRM2 revealed CG
and CHG methylation levels of 3.92-9.12%, and 2.88-6.93%, respectively, whereas
the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These
results were further supported by bisulfite sequencing of the 18S rRNA and EAF5
genes of the transformed S. cerevisiae, which exhibited different DNA
methylation patterns, which were observed in the genomic DNA. Our findings
establish that OsDRM2 is an active de novo DNA methyltransferase gene with
conserved activity in both prokaryotic and eukaryotic non-host species. Methylation at the 5-position of DNA cytosine on the vertebrate genomes is
accomplished by the combined catalytic actions of three DNA methyltransferases
(DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintece enzyme DNMT1.
Although several metabolic routes have been suggested for demethylation of the
vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive.
Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates
DNMTs in general, can also act as Ca(2+) ion- and redox state-dependent active
DNA demethylases. This finding suggests new directions for reinvestigation of
the structures and functions of these DNMTs, in particular their roles in Ca(2+)
ion-dependent biological processes, including the genome-wide/local DNA
demethylation during early embryogenesis, cell differentiation, neuronal
activity-regulated gene expression, and carcinogenesis. |
Which classes of endogenous retroelements are known to date? | Endogenous retroelements fall into two distinct classes: retrotransposons containing LTRs (Long Terminal Repeats), and retrostransposons lacking LTRs. | Mammalian LINE-1 (L1) elements belong to the superfamily of autonomously
replicating retrotransposable elements that lack the long terminal repeated
(LTR) sequences typical of retroviruses and retroviral-like retrotransposons.
The non-LTR superfamily is very ancient and L1-like elements are ubiquitous in
nature, having been found in plants, fungi, invertebrates, and various
vertebrate classes from fish to mammals. L1 elements have been replicating and
evolving in mammals for at least the past 100 million years and now constitute
20% or more of some mammalian genomes. Therefore, L1 elements presumably have
had a profound, perhaps defining, effect on the evolution, structure, and
function of mammalian genomes. L1 elements contain regulatory signals and encode
two proteins: one is an RNA-binding protein and the second one presumably
functions as an integrase-replicase, because it has both endonuclease and
reverse transcriptase activities. This work reviews the structure and biological
properties of L1 elements, including their regulation, replication, evolution,
and interaction with their mammalian hosts. Although each of these processes is
incompletely understood, what is known indicates that they represent challenging
and fascinating biological phenomena, the resolution of which will be essential
for fully understanding the biology of mammals. Cassava (Manihot esculenta Crantz), though a major world crop with enormous
potential, is very under studied. Little is known about its genome structure and
organisation. Transposable elements have a key role in the evolution of genome
structure, and can be used as important tools in applied genetics. This paper
sets out to survey the diversity of members of three major classes of
transposable element within the cassava genome and in relation to similar
elements in other plants. Members of two classes of LTR-retrotransposons,
Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator
(En/Spm)-like transposons were isolated and characterised. Analyses revealed 59
families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40
families of En/Spm in the cassava genome. In the comparative analyses, the
predicted amino acid sequences for these transposon classes were compared with
those of related elements from other plant species. These revealed that there
were multiple lineages of Ty1/copia-like retrotransposons in the genome of
cassava and suggested that vertical and horizontal transmission as the source of
cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements
network, two groups of cassava Megyps were evident including the Arabidopsis
Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a
single group within a network of plant En/Spm-like elements. Hybridisation
analysis supported the presence of transposons in the genome of cassava in
medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava
genome was shown to contain diverse members of three major classes of
transposable element; however, the different classes exhibited contrasting
evolutionary histories. |
What is the association between proBNP serum concentrations and stroke outcomes? | ProBNP serum concentrations are elevated in stroke patients relative to healthy controls. Greater proBNP serum concentrations are associated with greater stroke severity and with increased risk for unfvorable functional outcomes. | BACKGROUND: The association between plasma levels of N-terminal pro-brain
natriuretic peptide (NT-proBNP) and prognostic outcomes in patients after
ischemic stroke remains unknown. The present study tested the hypothesis that
NT-proBNP level is noticeably increased after ischemic stroke and that elevated
NT-proBNP is associated with unfavorable clinical outcomes (UFCO).
METHODS AND RESULTS: Blood samples for NT-proBNP levels were collected serially
and examined with sandwich immunoassay after acute ischemic stroke in 86
consecutive patients. The NT-proBNP levels were also measured in 30 healthy
control volunteers and 30 at-risk control subjects. The NT-proBNP levels were
significantly higher at 4 intervals after ischemic stroke than in healthy and
at-risk control subjects (all p<0.001). The NT-proBNP decreased to a
significantly lower level on day 21 and to a substantially lower level on day
90. Additionally, the NT-proBNP level at any of the 4 intervals was
significantly higher in patients with than in patients without UFCO (defined as
combined congestive heart failure > or = class 3, acute myocardial infarction,
recurrent stroke or any cause of death) (all p<0.01). Multivariate analysis
demonstrated that age and NIH Stroke Scale were the 2 strongest independent
predictors of increased NT-proBNP levels (all p<0.01). Furthermore, increased
NT-proBNP (> or = 150 pg/ml) was the strongest independent predictor of
long-term (mean follow-up: 24 months) UFCO (26 patients) (all p<0.05).
CONCLUSIONS: The NT-proBNP level was markedly elevated after acute ischemic
stroke and declined substantially thereafter. An increased NT-proBNP level was
strongly and independently correlated with UFCO in patients after ischemic
stroke. OBJECTIVE: To investigate the significance of N-terminal proBrain natriuretic
peptide (NT-proBNP) in predicting the outcome of acute stroke in relation to
other cardiovascular variables and stroke severity.
DESIGN: Prospective study of previously independent acute ischemic and
hemorrhagic stroke patients admitted to the acute stroke ward.
METHODS: Measurements of NT-proBNP in acute phase in addition to standard
assessment of cardiovascular and neurological details.
MAIN OUTCOME MEASURES: Concentrations and significance of NT-proBNP in patients
who were dead versus alive at 120 days after acute stroke and Cox regression
analysis with stroke severity measured as Scandinavian Stroke Scale (SSS) and
other cardiovascular disease to predict mortality.
RESULTS: One hundred fourteen patients were recruited, median age 74 years. At
120 days, 13 patients had died. Deceased patients had a significantly higher
concentration of NT-proBNP as compared to alive patients (P < .001). All
patients who had died had NT-proBNP concentration above the median for the whole
group. Cox regression analysis revealed that NT-proBNP was the most significant
independent variable predicting mortality (odds ratio [OR] 2.4, 95% confidence
interval [CI] 1.35 to 4.41, P = .003) followed by the SSS (OR 0.95, 95% CI 0.90
to 0.98, P = .005). Higher NT-proBNP and lower SSS predicted mortality. There
was a significant negative correlation of NT-proBNP with SSS score (r = -0.24, P
= .02). There were no significant difference in NT-proBNP concentrations between
ischemic and hemorrhagic strokes (P < .346). At 6 weeks, the dependent patients
had higher concentrations of NT-proBNP than independent patients.
CONCLUSION: NT-proBNP, an index of cardiac impairment, has an independent
prognostic value in acute stroke patients over other cardiovascular variables
and stroke severity. This may provide a channel for interventional therapy in
acute stroke. The prognostic value of the N-amino terminal fragment of the prohormone brain
natriuretic peptide (NT-proBNP) in acute ischemic stroke (AIS) is uncertain. We
sought to determine whether NT-proBNP levels were associated with functional
outcomes after AIS. From August 2012 to October 2013, consecutive first-ever AIS
patients admitted to the Department of Emergency of the First Affiliated
Hospital of Xinxiang Medical University, China, were included in this study.
Plasma NT-proBNP levels were measured from admission. Outcomes were measured as
90-day modified Rankin Scale score ('good outcome'=0-2 vs. 'poor'). Multivariate
logistic regression was used to assess associations between NT-proBNP levels and
outcomes. Predictive performance of NT-proBNP as compared with the clinical
model was assessed by comparing receiver-operating characteristic curves. During
this study period, 217 consecutive patients with AIS were included and completed
90 days of follow-up. There was a strong positive correlation between the plasma
level of NT-proBNP and the National Institutes of Health Stroke Scale score
(r=0.415, P=0.000). Plasma levels of NT-proBNP in patients with an unfavorable
outcome were significantly higher than those in patients with a favorable
outcome [3432 (interquartile range, 1100-54991) vs. 978 (interquartile range,
123-1705) pg/ml; P=0.000]. In multivariate analyses, after adjusting for all
other significant outcome predictors, the NT-proBNP level that remained can be
seen as an independent unfavorable outcome predictor, with an adjusted odds
ratios of 4.14 (95% confidence interval, 2.72-7.99; P=0.000). Our results show
that plasma NT-proBNP levels were significantly elevated in patients with an
unfavorable outcome and might be of clinical importance as a supplementary tool
for the assessment of functional outcomes in patients with AIS. |
What is the effect that EZH2 has on chromatin? | Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms | Gene silencing is required to stably maintain distinct patterns of gene
expression during eukaryotic development and has been correlated with the
induction of chromatin domains that restrict gene activity. We describe the
isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila
Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of
homeotic gene expression implicated in the assembly of repressive protein
complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct
loci in mouse and man, and the two murine Ezh genes display complementary
expression profiles during mouse development. The E(z) gene family reveals a
striking functional conservation in mediating gene repression in eukaryotic
chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic
flies enhance position effect variegation of the heterochromatin-associated
white gene, and expression of either human EZH2 or murine Ezh1 restores gene
repression in Saccharomyces cerevisiae mutants that are impaired in telomeric
silencing. Together, these data provide a functional link between Pc-G-dependent
gene repression and inactive chromatin domains, and indicate that silencing
mechanism(s) may be broadly conserved in eukaryotes. The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine
methyltransferase (HKMT) activity that is associated with transcriptional
repression. We report that Ezh2 expression was developmentally regulated in the
myotome compartment of mouse somites and that its down-regulation coincided with
activation of muscle gene expression and differentiation of
satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle
differentiation, and this property was conferred by its SET domain, required for
the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated
with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with
the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their
presence correlated with methylation of K27 of histone H3. YY1 was required for
Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of
Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and
YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF
were recruited to the chromatin. These findings suggest the existence of a
two-step activation mechanism whereby removal of H3-K27 methylation, conferred
by an active Ezh2-containing protein complex, followed by recruitment of
positive transcriptional regulators at discrete genomic loci are required to
promote muscle gene expression and cell differentiation. Polycomb group proteins are critical to maintaining gene repression established
during Drosophila development. Part of this group forms the PRC2 complex
containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27
(H3K37me2/3), marks repressive to transcription. We report that the mammalian
homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting
repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects
global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In
accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead,
PRC2-Ezh1 directly and robustly represses transcription from chromatinized
templates and compacts chromatin in the absence of the methyltransferase
cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2
genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2
expression is tightly associated with proliferation, as evidenced when analyzing
aging mouse kidney. These results might reflect subfunctionalization of a PcG
protein during evolution. BACKGROUND: The INK4b-ARF-INK4a tumour suppressor locus controls the balance
between progenitor cell renewal and cancer. In this study, we investigated how
higher-order chromatin structure modulates differential expression of the human
INK4b-ARF-INK4a locus during progenitor cell differentiation, cellular ageing
and senescence of cancer cells.
RESULTS: We found that INK4b and INK4a, but not ARF, are upregulated following
the differentiation of haematopoietic progenitor cells, in ageing fibroblasts
and in senescing maligt rhabdoid tumour cells. To investigate the underlying
molecular mechanism we analysed binding of polycomb group (PcG) repressive
complexes (PRCs) and the spatial organization of the INK4b-ARF-INK4a locus. In
agreement with differential derepression, PcG protein binding across the locus
is discontinuous. As we described earlier, PcG repressors bind the INK4a
promoter, but not ARF. Here, we identified a second peak of PcG binding that is
located approximately 3 kb upstream of the INK4b promoter. During progenitor
cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of
PRC binding and transcriptional activation of INK4b and INK4a. The expression
pattern of the locus is reflected by its organization in space. In the repressed
state, the PRC-binding regions are in close proximity, while the intervening
chromatin harbouring ARF loops out. Down regulation of EZH2 causes release of
the approximately 35 kb repressive chromatin loop and induction of both INK4a
and INK4b, whereas ARF expression remains unaltered.
CONCLUSION: PcG silencers bind and coordinately regulate INK4b and INK4a, but
not ARF, during a variety of physiological processes. Developmentally regulated
EZH2 levels are one of the factors that can determine the higher order chromatin
structure and expression pattern of the INK4b-ARF-INK4a locus, coupling human
progenitor cell differentiation to proliferation control. Our results revealed a
chromatin looping mechanism of long-range control and argue against models
involving homogeneous spreading of PcG silencers across the INK4b-ARF-INK4a
locus. In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been
suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2)
is responsible for loss of expression of RUNX3. This is consistent with EZH2
upregulation in multiple cancer types being associated with poor prognosis. We
investigated whether EZH2 influences the expression of RUNX3 in colorectal
cancer (CRC) and whether this is independent of methylation. We determined
protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3
methylation with methylation-specific polymerase chain reaction using 72 human
CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA
interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and
H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation.
Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in
general, no inverse correlation between EZH2 and RUNX3 in paired samples was
found arguing against a major role for histone methylation in silencing RUNX3 in
CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated
with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed
the repressive chromatin marks from RUNX3 but did not result in RUNX3
re-expression. However, it prevented the re-silencing of RUNX3 after the removal
of demethylating agents. In conclusion, DNA methylation is primarily responsible
for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone
methylation are necessary for its methylation-dependent re-silencing after the
removal of demethylating agents. These results would predict that inhibitors of
EZH2 and histone methylation would enhance the effects of demethylating agents
in cancer therapy. BACKGROUND: Polycomb group (PcG) genes code for chromatin multiprotein complexes
that are responsible for maintaining gene silencing of transcriptional programs
during differentiation and in adult tissues. Despite the large amount of
information on PcG function during development and cell identity homeostasis,
little is known regarding the dynamics of PcG complexes and their role during
terminal differentiation.
RESULTS: We show that two distinct polycomb repressive complex (PRC)2 complexes
contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which
is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK)
enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces
PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing
dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is
differentially regulated at the chromatin level by Msk1 dependent methyl/phospho
switch mechanism involving phosphorylation of serine 28 of the H3 histone
(H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2
complex, this pathway does not influence the binding of PRC2-Ezh1 on the
chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the
chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes.
We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG
transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for
myogenic differentiation.
CONCLUSIONS: Our data reveal another important layer of epigenetic control
orchestrating skeletal muscle cell terminal differentiation, and introduce a
novel function of the PRC2-Ezh1 complex in promoter setting. Epigenetic control is crucial for the differentiation of a variety of cells
including oligodendrocytes, the myelinating glial cells of the central nervous
system. However, studies about the implication of epigenetic factors in
peripheral nervous system maturation are just emerging. Here, we demonstrate for
the first time the impact of a histone methyltransferase, encoded by the
enhancer of zeste homolog 2 (EZH2) gene, on Schwann cell differentiation. In
sciatic nerves, EZH2 expression was found in Schwann cells and to peak
perinatally. Suppression of EZH2 expression in cultured primary rat Schwann
cells reduced the length of cell processes. These morphological changes were
accompanied by widespread alterations in the gene expression pattern, including
downregulation of myelin genes and induction of p57kip2, which we have recently
identified as an intrinsic inhibitory regulator of Schwann cell maturation. In
addition, we show that EZH2 suppression in dorsal root ganglion cocultures
interferes with in vitro myelination. Chromatin immunoprecipitation analysis
revealed binding of EZH2 at the p57kip2 promoter and reduction of histone H3K27
trimethylation upon gene suppression. EZH2 suppression-dependent effects on
morphology and myelin genes could be reversed by concomitant suppression of
p57kip2, indicating that p57kip2 is a downstream effector of EZH2. Furthermore,
we describe Hes5 as transcriptional repressor of myelin genes in Schwann cells,
which was induced upon EZH2 suppression and downregulated in p57kip2-suppressed
Schwann cells. Therefore, we have identified a molecular link between histone
methylation and control of Schwann cell differentiation and demonstrate that
this epigenetic mechanism is crucial for glial differentiation to proceed. The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) mediates
trimethylation of lysine 27 in histone 3, which acts as a repressive epigenetic
mark. Ezh2 is essential for maintaining pluripotency of stem cells, but
information on its role in differentiated cells is sparse. Whole-genome mRNA
expression arrays identified 964 genes that were regulated by >2-fold 72 hours
after small interfering RNA-mediated silencing of Ezh2 in human umbilical vein
endothelial cells. Among them, genes associated with the gene ontology terms
cell communication and cell adhesion were significantly overrepresented,
suggesting a functional role for Ezh2 in the regulation of angiogenesis. Indeed,
adhesion, migration, and tube formation assays revealed significantly altered
angiogenic properties of human umbilical vein endothelial cells after silencing
of Ezh2. To identify direct target genes of Ezh2, we performed chromatin
immunoprecipitation experiments followed by whole-genome promoter arrays
(chromatin immunoprecipitation-on-chip) and identified 5585 genes associated
with trimethylation of lysine 27 in histone 3. Comparative analysis with our
mRNA expression data identified 276 genes that met our criteria for putative
Ezh2 target genes, upregulation by >2-fold after Ezh2 silencing and association
with trimethylation of lysine 27 in histone 3. Notably, we observed a striking
overrepresentation of genes involved in wingless-type mouse mammary tumor virus
integration site (WNT) signaling pathways. Epigenetic regulation of several of
these genes by Ezh2 was specifically confirmed by polymerase chain reaction
analysis of DNA enrichment after chromatin immunoprecipitation using an antibody
specific for trimethylation of lysine 27 in histone 3. Combining mRNA expression
arrays and chromatin immunoprecipitation-on-chip analysis, we identified 276
Ezh2 target genes in endothelial cells. Ezh2-dependent repression of genes
involved in cell adhesion and communication contributes to the regulation of
angiogenesis. Loss of Dicer, an enzyme critical for microRNA biogenesis, results in lethality
due to a block in mouse embryonic stem cell (mES) differentiation. Using
ChIP-Seq we found increased H3K9me2 at over 900 CpG islands in the Dicer(-/-)ES
epigenome. Gene ontology analysis revealed that promoters of chromatin
regulators to be among the most impacted by increased CpG island H3K9me2 in ES
(Dicer(-/-)). We therefore, extended the study to include H3K4me3 and H3K27me3
marks for selected genes. We found that the ES (Dicer(-/-)) mutant epigenome was
characterized by a shift in the overall balance between transcriptionally
favorable (H3K4me3) and unfavorable (H3K27me3) marks at key genes regulating ES
cell differentiation. Pluripotency genes Oct4, Sox2 and Nanog were not impacted
in relation to patterns of H3K27me3 and H3K4me3 and showed no changes in the
rates of transcript down-regulation in response to RA. The most striking changes
were observed in regards to genes regulating differentiation and the transition
from self-renewal to differentiation. An increase in H3K4me3 at the promoter of
Lin28b was associated with the down-regulation of this gene at a lower rate in
Dicer(-/-)ES as compared to wild type ES. An increase in H3K27me3 in the
promoters of differentiation genes Hoxa1 and Cdx2 in Dicer(-/-)ES cells was
coincident with an inability to up-regulate these genes at the same rate as ES
upon retinoic acid (RA)-induced differentiation. We found that siRNAs Ezh2 and
post-transcriptional silencing of Ezh2 by let-7 g rescued this effect suggesting
that Ezh2 up-regulation is in part responsible for increased H3K27me3 and
decreased rates of up-regulation of differentiation genes in Dicer(-/-)ES. The androgen receptor (AR) is a pleiotrophic transcription factor that regulates
expression of a large number of genes involved in many diverse cellular
processes. The AR activation pathways have been studied extensively. However,
the molecular mechanism and biological significance of AR inhibitory signals
remain poorly understood. Mukhopadhyay et al. have now identified the nuclear
matrix protein scaffold attachment factor B1 (SAFB1) as a novel AR corepressor.
The authors found that SAFB1 physically associates with AR protein and inhibits
AR transcriptional activity and androgen-sensitive gene expression. SAFB1 had no
effect on chromatin occupancy of AR, but silencing of SAFB1 abolished
recruitment MST1, a known AR repressor, at AR target loci. They also showed that
SAFB1 interacts with EZH2, SUZ12 and EED, three core components of the Polycomb
repressive complex 2 (PRC2), which catalyzes the gene repression histone
modification H3 lysine 27 trimethylation (H3K27me3). The authors further showed
that forced expression of SAFB1 increases H3K27me3 at AR target loci and this
effect requires EZH2. Finally, the authors demonstrated that expression of SAFB1
is downregulated in human prostate cancer (PCa) specimens and SAFB1 knockdown
results in an aggressive phenotype of PCa. These findings identify SAFB1 as an
important node for integration of multiple inhibitory signals of AR, which
represents a viable pathway for therapeutic intervention of PCa. Neural crest cells arise from the border of the neural plate and epidermal
ectoderm, migrate extensively and differentiate into diverse cell types during
vertebrate embryogenesis. Although much has been learnt about growth factor
signals and gene regulatory networks that regulate neural crest development,
limited information is available on how epigenetic mechanisms control this
process. In this study, we show that Polycomb repressive complex 2 (PRC2)
cooperates with the transcription factor Snail2/Slug to modulate neural crest
development in Xenopus. The PRC2 core components Eed, Ezh2 and Suz12 are
expressed in the neural crest cells and are required for neural crest marker
expression. Knockdown of Ezh2, the catalytic subunit of PRC2 for histone H3K27
methylation, results in defects in neural crest specification, migration and
craniofacial cartilage formation. EZH2 interacts directly with Snail2, and
Snail2 fails to expand the neural crest domains in the absence of Ezh2.
Chromatin immunoprecipitation analysis shows that Snail2 regulates EZH2
occupancy and histone H3K27 trimethylation levels at the promoter region of the
Snail2 target E-cadherin. Our results indicate that Snail2 cooperates with EZH2
and PRC2 to control expression of the genes important for neural crest
specification and migration during neural crest development. |
Does Chromatin Immunoprecipitation (ChIP) show a bias for highly expressed loci? | Several issues in the processing and analysis of ChIP-chip data have not been resolved fully, including the effect of background (mock control) subtraction and normalization within and across arrays. We detected a chromatin-state bias: open chromatin regions yielded higher coverage, which led to false positives if not corrected. The localization of unrelated proteins, including the entire silencing complex, to the most highly transcribed genes was highly suggestive of a technical issue with the immunoprecipitations. | We present an algorithm for predicting transcription factor binding sites based
on ChIP-chip and phylogenetic footprinting data. Our algorithm is robust against
low promoter sequence similarity and motif rearrangements, because it does not
depend on multiple sequence alignments. This, in turn, allows us to incorporate
information from more distant species. Representative random data sets are used
to estimate the score significance. Our algorithm is fully automatic, and does
not require human intervention. On a recent S. cerevisiae data set, it achieves
higher accuracy than the previously best algorithms. Adaptive ChIP-chip
threshold and the modular positional bias score are two general features of our
algorithm that increase motif prediction accuracy and could be implemented in
other algorithms as well. In addition, since our algorithm works partly
orthogonally to other algorithms, combining several algorithms can increase
prediction accuracy even further. Specifically, our method finds 6 motifs not
found by the 2nd best algorithm. BACKGROUND: Chromatin immunoprecipitation on tiling arrays (ChIP-chip) has been
widely used to investigate the DNA binding sites for a variety of proteins on a
genome-wide scale. However, several issues in the processing and analysis of
ChIP-chip data have not been resolved fully, including the effect of background
(mock control) subtraction and normalization within and across arrays.
RESULTS: The binding profiles of Drosophila male-specific lethal (MSL) complex
on a tiling array provide a unique opportunity for investigating these topics,
as it is known to bind on the X chromosome but not on the autosomes. These large
bound and control regions on the same array allow clear evaluation of analytical
methods.We introduce a novel normalization scheme specifically designed for
ChIP-chip data from dual-channel arrays and demonstrate that this step is
critical for correcting systematic dye-bias that may exist in the data.
Subtraction of the mock (non-specific antibody or no antibody) control data is
generally needed to eliminate the bias, but appropriate normalization obviates
the need for mock experiments and increases the correlation among replicates.
The idea underlying the normalization can be used subsequently to estimate the
background noise level in each array for normalization across arrays. We
demonstrate the effectiveness of the methods with the MSL complex binding data
and other publicly available data.
CONCLUSION: Proper normalization is essential for ChIP-chip experiments. The
proposed normalization technique can correct systematic errors and compensate
for the lack of mock control data, thus reducing the experimental cost and
producing more accurate results. BACKGROUND: In vivo detection of protein-bound genomic regions can be achieved
by combining chromatin-immunoprecipitation with next-generation sequencing
technology (ChIP-seq). The large amount of sequence data produced by this method
needs to be analyzed in a statistically proper and computationally efficient
manner. The generation of high copy numbers of DNA fragments as an artifact of
the PCR step in ChIP-seq is an important source of bias of this methodology.
RESULTS: We present here an R package for the statistical analysis of ChIP-seq
experiments. Taking the average size of DNA fragments subjected to sequencing
into account, the software calculates single-nucleotide read-enrichment values.
After normalization, sample and control are compared using a test based on the
ratio test or the Poisson distribution. Test statistic thresholds to control the
false discovery rate are obtained through random permutations. Computational
efficiency is achieved by implementing the most time-consuming functions in C++
and integrating these in the R package. An analysis of simulated and
experimental ChIP-seq data is presented to demonstrate the robustness of our
method against PCR-artefacts and its adequate control of the error rate.
CONCLUSIONS: The software ChIP-seq Analysis in R (CSAR) enables fast and
accurate detection of protein-bound genomic regions through the analysis of
ChIP-seq experiments. Compared to existing methods, we found that our package
shows greater robustness against PCR-artefacts and better control of the error
rate. Chromatin immunoprecipitation followed by next-generation sequencing analysis
(ChIP-Seq) is a powerful method with which to investigate the genome-wide
distribution of chromatin-binding proteins and histone modifications in any
genome with a known sequence. The application of this technique to a variety of
developmental and differentiation systems has provided global views of the
cis-regulatory elements, transcription factor function and epigenetic processes
involved in the control of gene transcription. Here we describe several
technical aspects of the ChIP-Seq assay that diminish bias and background noise
and allow the consistent generation of high-quality data. Next-generation sequencing is rapidly transforming our ability to profile the
transcriptional, genetic, and epigenetic states of a cell. In particular,
sequencing DNA from the immunoprecipitation of protein-DNA complexes (ChIP-seq)
and methylated DNA (MeDIP-seq) can reveal the locations of protein binding sites
and epigenetic modifications. These approaches contain numerous biases which may
significantly influence the interpretation of the resulting data. Rigorous
computational methods for detecting and removing such biases are still lacking.
Also, multi-sample normalization still remains an important open problem. This
theoretical paper systematically characterizes the biases and properties of
ChIP-seq data by comparing 62 separate publicly available datasets, using
rigorous statistical models and signal processing techniques. Statistical
methods for separating ChIP-seq signal from background noise, as well as
correcting enrichment test statistics for sequence-dependent and sonication
biases, are presented. Our method effectively separates reads into signal and
background components prior to normalization, improving the signal-to-noise
ratio. Moreover, most peak callers currently use a generic null model which
suffers from low specificity at the sensitivity level requisite for detecting
subtle, but true, ChIP enrichment. The proposed method of determining a cell
type-specific null model, which accounts for cell type-specific biases, is shown
to be capable of achieving a lower false discovery rate at a given significance
threshold than current methods. The advent of high-throughput technologies such as ChIP-seq has made possible
the study of histone modifications. A problem of particular interest is the
identification of regions of the genome where different cell types from the same
organism exhibit different patterns of histone enrichment. This problem turns
out to be surprisingly difficult, even in simple pairwise comparisons, because
of the significant level of noise in ChIP-seq data. In this paper we propose a
two-stage statistical method, called ChIPnorm, to normalize ChIP-seq data, and
to find differential regions in the genome, given two libraries of histone
modifications of different cell types. We show that the ChIPnorm method removes
most of the noise and bias in the data and outperforms other normalization
methods. We correlate the histone marks with gene expression data and confirm
that histone modifications H3K27me3 and H3K4me3 act as respectively a repressor
and an activator of genes. Compared to what was previously reported in the
literature, we find that a substantially higher fraction of bivalent marks in ES
cells for H3K27me3 and H3K4me3 move into a K27-only state. We find that most of
the promoter regions in protein-coding genes have differential
histone-modification sites. The software for this work can be downloaded from
http://lcbb.epfl.ch/software.html. Chromatin immunoprecipitation (ChIP) is the gold-standard technique for
localizing nuclear proteins in the genome. We used ChIP, in combination with
deep sequencing (Seq), to study the genome-wide distribution of the Silent
information regulator (Sir) complex in Saccharomyces cerevisiae. We analyzed
ChIP-Seq peaks of the Sir2, Sir3, and Sir4 silencing proteins and discovered 238
unexpected euchromatic loci that exhibited enrichment of all three.
Surprisingly, published ChIP-Seq datasets for the Ste12 transcription factor and
the centromeric Cse4 protein indicated that these proteins were also enriched in
the same euchromatic regions with the high Sir protein levels. The 238 loci,
termed "hyper-ChIPable", were in highly expressed regions with strong polymerase
II and polymerase III enrichment signals, and the correlation between
transcription level and ChIP enrichment was not limited to these 238 loci but
extended genome-wide. The apparent enrichment of various proteins at
hyper-ChIPable loci was not a consequence of artifacts associated with deep
sequencing methods, as confirmed by ChIP-quantitative PCR. The localization of
unrelated proteins, including the entire silencing complex, to the most highly
transcribed genes was highly suggestive of a technical issue with the
immunoprecipitations. ChIP-Seq on chromatin immunoprecipitated with a
nuclear-localized GFP reproduced the above enrichment in an expression-dependent
manner: induction of the GAL genes resulted in an increased ChIP signal of the
GFP protein at these loci, with presumably no biological relevance. Whereas ChIP
is a broadly valuable technique, some published conclusions based upon ChIP
procedures may merit reevaluation in light of these findings. MOTIVATION: High-throughput sequencing technologies enable the genome-wide
analysis of the impact of genetic variation on molecular phenotypes at
unprecedented resolution. However, although powerful, these technologies can
also introduce unexpected artifacts.
RESULTS: We investigated the impact of library amplification bias on the
identification of allele-specific (AS) molecular events from high-throughput
sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq).
Putative AS DNA binding activity for RNA polymerase II was determined using
ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter
trios. We found that, at high-sequencing depth, many significant AS binding
sites suffered from an amplification bias, as evidenced by a larger number of
clonal reads representing one of the two alleles. To alleviate this bias, we
devised an amplification bias detection strategy, which filters out sites with
low read complexity and sites featuring a significant excess of clonal reads.
This method will be useful for AS analyses involving ChIP-seq and other
functional sequencing assays.
AVAILABILITY: The R package abs filter for library clonality simulations and
detection of amplification-biased sites is available from
http://updepla1srv1.epfl.ch/waszaks/absfilter |
Is clathrin involved in E-cadherin endocytosis? | E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. | The endocytosis of E-cadherin has recently emerged as an important determit
of cadherin function with the potential to participate in remodeling adhesive
contacts. In this study we focused on the initial fate of E-cadherin when it
predomitly exists free on the cell surface prior to adhesive binding or
incorporation into junctions. Surface-labeling techniques were used to define
the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster
ovary cells stably expressing human E-cadherin. We found that in this
experimental system E-cadherin entered a transferrin-negative compartment before
transport to the early endosomal compartment, where it merged with classical
clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by
mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin
internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to
trap endocytosed E-cadherin in large peripheral structures. We conclude that in
isolated cells unbound E-cadherin on the cell surface is predomitly
endocytosed by a clathrin-independent pathway resembling macropinocytotic
internalization, which then fuses with the early endosomal system. Taken with
earlier reports, this suggests the possibility that multiple pathways exist for
E-cadherin entry into cells that are likely to reflect cell context and
regulation. E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the
intercellular adhesion required for tissue cohesion and anchors the
actomyosin-driven tension needed to change cell shape. In the early Drosophila
embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell
junctions and tissue extension. The E-cadherin distribution is also planar
polarized and complementary to the Myosin-II distribution. Here we show that
E-cadherin polarity is controlled by the polarized regulation of clathrin- and
dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell
intercalation defects. We delineate a pathway that controls the initiation of
E-cadherin endocytosis through the regulation of AP2 and clathrin coat
recruitment by E-cadherin. This requires the concerted action of the formin
Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine
exchange factor RhoGEF2, which is planar polarized and absent in
non-intercalating regions. Finally, we provide evidence that Dia and Myo-II
control the initiation of E-cadherin endocytosis by regulating the lateral
clustering of E-cadherin. BACKGROUND: E-cadherin is a cell-cell adhesion molecule and the dysfunction of
which is a common feature of more than 70% of all invasive carcinomas, including
gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric
carcinomas include mutations and silencing at either the DNA or RNA level.
Nevertheless, in a high percentage of gastric carcinoma cases displaying
E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation
is unknown. We have previously demonstrated the existence of a bi-directional
cross-talk between E-cadherin and two major N-glycan processing enzymes,
N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V).
METHODS: In the present study, we have characterized the functional implications
of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin
biological functions and in the molecular assembly and stability of
adherens-junctions in a gastric cancer model. The results were validated in
human gastric carcinoma samples.
RESULTS: We demonstrated that GnT-III induced a stabilizing effect on E-cadherin
at the cell membrane by inducing a delay in the turnover rate of the protein,
contributing for the formation of stable and functional adherens-junctions, and
further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V
promotes the destabilization of E-cadherin, leading to its mislocalization and
unstable adherens-junctions with impairment of cell-cell adhesion.
CONCLUSIONS: This supports the role of GnT-III on E-cadherin-mediated tumor
suppression, and GnT-V on E-cadherin-mediated tumor invasion.
GENERAL SIGNIFICANCE: These results contribute to fill the gap of knowledge of
those human carcinoma cases harboring E-cadherin dysfunction, opening new
insights into the molecular mechanisms underlying E-cadherin regulation in
gastric cancer with potential translational clinical applications. |
What are the generic versions of Viagra | Sildenafil Citrate and Elonza in Thailand are the generic versions of Viagra | OBJECTIVES: to categorise online suppliers of Viagra based on their legal
status, and to quantify the suppliers within each category.
METHODS: Google was used to search for websites offering to sell or supply
either proprietary Viagra tablets or generic versions containing sildenafil
citrate. Relevant websites were classified as falling into one of three
categories, which were further subclassified. Simple descriptive statistics were
calculated.
KEY FINDINGS: the number of relevant sites found within the first 100 Google
hits, following the removal of mirror and affiliate sites, was 44. Only 6.8% of
sites identified were legitimate online pharmacies. Some 34.1% of sites offered
to sell Viagra to patients in the UK without any form of medical consultation.
Whether or not the online consultation offered by 59.1% of sites had to be
completed in order to make a purchase could not be confirmed. The location of
only three pharmacies could be ascertained; the remainder made various claims as
to their location, which could not be verified.
CONCLUSIONS: we have been unable to verify that the questionnaires used for
online consultations are scrutinised by any healthcare practitioners to
determine the appropriateness of the treatment sought. This represents a serious
safety concern for UK residents who procure drugs for erectile dysfunction on
the internet. |
List GATA-1 interacting partners as discovered with the help of the biotinylation tagging approach. | 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 | 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. |
Which diseases are associated with Alu element insertion? | Diseases associated with Alu element insertion are the following: myotonic dystrophy type 2, Friedreich ataxia, spinocerebellar ataxia type 10, autosomal dominant optic atrophy, Menkes disease, hyper-IgM with immunodeficiency syndrome (HIGM), and anterior pituitary aplasia. | Myotonic dystrophy (DM) is associated with abnormal expansions of the CTG
repeats in the 3' untranslated region of its gene. Previous studies in
individuals of European origin demonstrated strong linkage disequilibrium
between different CTG repeat length alleles and an Alu element
insertion/deletion polymorphism in intron 8 of the DM gene: CTG11-13 chromosomes
were almost exclusively associated with the deletion allele, while chromosomes
with five or 19-30 repeats or disease chromosomes were only found on the
insertion allele. One of the models suggested by these results proposed that the
triplet repeats on insertion-associated chromosomes were particularly prone to
mutation. Studies of other triplet repeat disorders have suggested that arrays
of perfect repeats are more prone to instability than those that are
interrupted. We have examined the evolution of this locus by typing a variety of
primates and samples drawn from several different human populations for both CTG
repeat length and the insertion/deletion polymorphism. DM gene sequences from
different primates revealed inter- and intraspecies variability in the number of
CTG repeats. Human chromosomes with five repeats or 11-13 repeats (the two major
modes of the human CTG repeat distributions) showed no evidence of preferential
stabilization of these repeat sizes by imperfect sequences. The insertion and
deletion allele frequencies showed large interpopulation variation and the
degree of association between these alleles and various CTG repeat lengths is
not nearly as complete as was previously supposed. We have found deletion
alleles carrying long normal (> 19) CTG repeats and insertion alleles associated
with CTG11 - 13 in two african populations.(ABSTRACT TRUNCATED AT 250 WORDS) We present the first reported case of Menkes disease caused by an Alu element
insertion mutation that interfered with splicing regulatory elements. A whole
young AluYa5a2 element, which was 382-bp long, was identified within exon 9 of
the ATP7A gene, and all of exon 9 was aberrantly skipped in the cDNA, resulting
in severely truncated proteins. To confirm whether the aberrant skipping
resulted in Alu insertion, an exonic splicing enhancer finder was used. The Alu
element created two new high-score exonic splicing enhancer sequences in the
mutation located near the site of the insertion. Exon 9, which encodes the first
and second transmembrane domains, is necessary for the normal function of the
ATP7A protein. Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused
by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger
protein 9 (ZNF9) gene. The expansions are extremely unstable and variable,
ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic
heterogeneity make molecular diagnosis of DM2 difficult, and yield variable
clinical phenotypes. To better understand the mutational origin and instability
of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking
regions in 26 primate species. The 3'-end of an AluSx element, flanked by target
site duplications (5'-ACTRCCAR-3'or 5'-ACTRCCARTTA-3'), followed the CCTG
repeat, suggesting that the repeat was originally derived from the Alu element
insertion. In addition, our results revealed lineage-specific repetitive motifs:
pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG)
repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and
tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans.
Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A)
tail of the AluSx element, and evolved into unstable CCTG repeats during primate
evolution. Alu elements are known to be the source of microsatellite repeats
responsible for two other repeat expansion disorders: Friedreich ataxia and
spinocerebellar ataxia type 10. Taken together, these findings raise questions
as to the mechanism(s) by which Alu-mediated repeats developed into the large,
extremely unstable expansions common to these three disorders. |
Approximately how many recombination hotspots have been found in the yeast genome? | In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs. | Double-strand DNA breaks (DSBs) occur at recombination hotspots during
Saccharomyces cerevisiae meiosis and are thought to initiate exchange at these
loci. Analysis of DSB sites in three regions of the yeast genome indicated that
breaks occur at or near many potential transcription promoters and that DSBs
initiate most, if not all, meiotic recombination. DSB sites displayed
deoxyribonuclease I hypersensitivity in chromatin from mitotic and meiotic
cells, and changes in chromatin structure produced parallel changes in the
occurrence of DSBs. Thus, features of chromatin structure that are established
before meiosis play a role in determining where meiotic recombination events
initiate. In the yeast, meiotic recombination is initiated by double-strand DNA breaks
(DSBs) which occur at relatively high frequencies in some genomic regions
(hotspots) and relatively low frequencies in others (coldspots). Although
observations concerning individual hot/cold spots have given clues as to the
mechanism of recombination initiation, the prediction of hot/cold spots from DNA
sequence information is a challenging task. In this article, we introduce a
random forest (RF) prediction model to detect recombination hot/cold spots from
yeast genome. The out-of-bag (OOB) estimation of the model indicated that the RF
classifier achieved high prediction performance with 82.05% total accuracy and
0.638 Mattew's correlation coefficient (MCC) value. Compared with an alternative
machine-learning algorithm, support vector machine (SVM), the RF method
outperforms it in both sensitivity and specificity. The prediction model is
implemented as a web server (RF-DYMHC) and it is freely available at
http://www.bioinf.seu.edu.cn/Recombination/rf_dymhc.htm. Given a yeast genome
and prediction parameters (RI-value and non-overlapping window scan size), the
program reports the predicted hot/cold spots and marks them in color. BACKGROUND: Polyadenylated, mRNA-like transcripts with no coding potential are
abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs)
are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks
(DSBs) that initiate homologous recombination. Most meiotic recombination is
positioned at hotspots, but knowledge of the mechanisms is nebulous. In the
fission yeast genome DSBs are located within 194 prominent peaks separated on
average by 65-kbp intervals of DNA that are largely free of DSBs.
METHODOLOGY/PRINCIPAL FINDINGS: We compared the genome-wide distribution of DSB
peaks to that of polyadenylated ncRNA molecules of the prl class. DSB peaks map
to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and
intergenic regions, or most often within intergenic regions. Unconditional
statistical tests revealed that this colocalization is non-random and robust
(P<or=5.5 x 10(-8)). Furthermore, we tested and rejected the hypothesis that the
ncRNA loci and DSB peaks localize preferentially, but independently, to a third
entity on the chromosomes.
CONCLUSIONS/SIGNIFICANCE: Meiotic DSB hotspots are directed to loci that express
polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism
for what directs meiotic recombination to hotspots. It also reveals a likely
biological function for enigmatic ncRNAs. We propose specific mechanisms by
which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA
loci, help to position recombination protein complexes at DSB hotspots within
chromosomes. Deleterious mutations inevitably emerge in any evolutionary process and are
speculated to decisively influence the structure of the genome. Meiosis, which
is thought to play a major role in handling mutations on the population level,
recombines chromosomes via non-randomly distributed hot spots for meiotic
recombination. In many genomes, various types of genetic elements are
distributed in patterns that are currently not well understood. In particular,
important (essential) genes are arranged in clusters, which often cannot be
explained by a functional relationship of the involved genes. Here we show by
computer simulation that essential gene (EG) clustering provides a fitness
benefit in handling deleterious mutations in sexual populations with variable
levels of inbreeding and outbreeding. We find that recessive lethal mutations
enforce a selective pressure towards clustered genome architectures. Our
simulations correctly predict (i) the evolution of non-random distributions of
meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers
and EG clustering, (iii) the evolution of EG enrichment in pericentromeric
regions and (iv) the associated absence of meiotic crossovers (cold
centromeres). Our results furthermore predict optimal crossover rates for yeast
chromosomes, which match the experimentally determined rates. Using a
Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal
phenotypes result predomitly from mutation of single loci and generally do
not impair mating, which leads to an accumulation of mutational load following
meiosis and mating. We hypothesize that purging of deleterious mutations in
essential genes constitutes an important factor driving meiotic crossover.
Therefore, the increased robustness of populations to deleterious mutations,
which arises from clustered genome architectures, may provide a significant
selective force shaping crossover distribution. Our analysis reveals a new
aspect of the evolution of genome architectures that complements insights about
molecular constraints, such as the interference of pericentromeric crossovers
with chromosome segregation. |
How could iPSCs be used for the treatment of diabetes? | One of the promising approaches to cure diabetes is to use induced PCSs (iPSCs) and to differentiate them into insulin-secreting β cells. The induction of iPSC differentiation into insulin-secreting cells can be achieved in several ways, such as with the use of microRNAs, or adenoviral transfection with selected genes. | The pancreas arises from Pdx1-expressing progenitors in developing foregut
endoderm in early embryo. Expression of Ngn3 and NeuroD1 commits the cells to
form endocrine pancreas, and to differentiate into subsets of cells that
constitute islets of Langerhans. β-cells in the islets transcribe gene-encoding
insulin, and subsequently process and secrete insulin, in response to
circulating glucose. Dysfunction of β-cells has profound metabolic consequences
leading to hyperglycemia and diabetes mellitus. β-cells are destroyed via
autoimmune reaction in type 1 diabetes (T1D). Type 2 diabetes (T2D),
characterized by impaired β-cell functions and reduced insulin sensitivity,
accounts for 90% of all diabetic patients. Islet transplantation is a promising
treatment for T1D. Pluripotent stem cells provide an unlimited cell source to
generate new β-cells for patients with T1D. Furthermore, derivation of induced
pluripotent stem cells (iPSCs) from patients captures "disease-in-a-dish" for
autologous cell replacement therapy, disease modeling, and drug screening for
both types of diabetes. This review highlights essential steps in pancreas
development, and potential stem cell applications in cell regeneration therapy
for diabetes mellitus. INTRODUCTION: Anesthetic preconditioning protects cardiomyocytes from oxidative
stress-induced injury, but it is ineffective in patients with diabetes mellitus.
To address the role of hyperglycemia in the inability of diabetic individuals to
be preconditioned, we used human cardiomyocytes differentiated from induced
pluripotent stem cells generated from patients with or without type 2 diabetes
mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of
preconditioning in varying glucose conditions (5, 11, and 25 mM).
METHODS: Induced pluripotent stem cells were induced to generate cardiomyocytes
by directed differentiation. For subsequent studies, cardiomyocytes were
identified by genetic labeling with enhanced green fluorescent protein driven by
a cardiac-specific promoter. Cell viability was analyzed by lactate
dehydrogenase assay. Confocal microscopy was utilized to measure opening of the
mitochondrial permeability transition pore and the mitochondrial adenosine
5'-triphosphate-sensitive potassium channels.
RESULTS: Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs
from oxidative stress-induced lactate dehydrogenase release and mitochondrial
permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane
triggered mitochondrial adenosine-5'-triphosphate-sensitive potassium channel
opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM
glucose; 25 mM glucose disrupted anesthetic preconditioning-mediated protection
in DM-iPSC- and N-iPSC-CMs.
CONCLUSIONS: The opening of mitochondrial adenosine 5'-triphosphate-sensitive
potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and
in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and
patients with a specific disease, such as diabetes in this study, open
possibilities in studying genotype- and phenotype-related pathologies in a
human-relevant model. The landmark discovery of induced pluripotent stem cells (iPSCs) by Shinya
Yamanaka has transformed regenerative biology. Previously, insights into the
pathogenesis of chronic human diseases have been hindered by the inaccessibility
of patient samples. However, scientists are now able to convert patient
fibroblasts into iPSCs and differentiate them into disease-relevant cell types.
This ability opens new avenues for investigating disease pathogenesis and
designing novel treatments. In this review, we highlight the uses of human iPSCs
to uncover the underlying causes and pathological consequences of diabetes and
metabolic syndromes, multifactorial diseases whose etiologies have been
difficult to unravel using traditional methodologies. Islet transplantation is considered as an ultimate option for the treatment of
type I diabetes. Human induced pluripotent stem cells (hiPSCs) have raised the
possibility that patient-specific insulin-secreting cells might be derived from
somatic cells through cell fate reprogramming. However, current protocols mostly
rely on the use of several cytokines and inhibitors for directing
differentiation towards pancreatic fate. Given the high manufacturing cost of
these recombit proteins, this approach is prohibitive for clinical
applications. Knowing that microRNAs (miRNAs) are key players in various stages
of pancreatic development, we present a novel and cost-effective strategy in
which over-expression of miR-375 promotes pancreatic differentiation in hiPSCs
in the absence of any other stimulator. We used a polycistronic viral vector
expressing Sox2, Klf4, c-Myc, and Oct4 to drive hiPSCs from human foreskin
fibroblasts. The established hiPSCs are similar to human embryonic stem cells in
many aspects including morphology, passaging, surface and pluripotency markers,
and gene expression. For differentiation induction, miR-375 was lentivirally
overexpressed in these hiPSCs. Morphological assessment, immunocytochemistry,
and expression analysis of islet marker genes confirmed that islet like cells
were obtained in miR-375 transduced cells compared to controls. Our
differentiated clusters secreted insulin in a glucose-dependant manner, showing
in vitro functionality. We demonstrated for the first time that miRNAs might be
ideal substitutes to induce pancreatic differentiation in hiPSCs. This work
provides a new approach to study the role of miRNAs in pancreatic specification
and increase the feasibility of using patient-specific iPSCs for beta cell
replacement therapy for type I diabetes. Pluripotent stem cells have potential applications in regenerative medicine for
diabetes. Differentiation of stem cells into insulin-producing cells has been
achieved using various protocols. However, both the efficiency of the method and
potency of differentiated cells are insufficient. Oxygen tension, the partial
pressure of oxygen, has been shown to regulate the embryonic development of
several organs, including pancreatic β-cells. In this study, we tried to
establish an effective method for the differentiation of induced pluripotent
stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen
(O2) conditions. Treatment with a high O2 condition in the early stage of
differentiation increased insulin-positive cells at the terminus of
differentiation. We found that a high O2 condition repressed Notch-dependent
gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic
progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α
protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal
stage-specific treatment with a high O2 condition resulted in a significant
increase in insulin production in both mouse embryonic stem cells and human
iPSCs and yielded populations containing up to 10% C-peptide-positive cells in
human iPSCs. These results suggest that culturing in a high O2 condition at a
specific stage is useful for the efficient generation of insulin-producing
cells. Diabetes mellitus is the most prevailing disease with progressive incidence
worldwide. To date, the pathogenesis of diabetes is far to be understood, and
there is no permanent treatment available for diabetes. One of the promising
approaches to understand and cure diabetes is to use pluripotent stem cells
(PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs). ESCs and
iPSCs have a great potential to differentiate into all cell types, and they have
a high ability to differentiate into insulin-secreting β cells. Obtaining PSCs
genetically identical to the patient presenting with diabetes has been a
longstanding dream for the in vitro modeling of disease and ultimately cell
therapy. For several years, somatic cell nuclear transfer (SCNT) was the method
of choice to generate patient-specific ESC lines. However, this technology faces
ethical and practical concerns. Interestingly, the recently established iPSC
technology overcomes the major problems of other stem cell types including the
lack of ethical concern and no risk of immune rejection. Several iPSC lines have
been recently generated from patients with different types of diabetes, and most
of these cell lines are able to differentiate into insulin-secreting β cells. In
this review, we summarize recent advances in the differentiation of pancreatic β
cells from PSCs, and describe the challenges for their clinical use in diabetes
cell therapy. Furthermore, we discuss the potential use of patient-specific PSCs
as an in vitro model, providing new insights into the pathophysiology of
diabetes. Human histocompatibility antigens are quite heterogeneous and promote the
rejection of transplanted tissue. Recent advances in stem cell research that
enable the use of a patient's own stem cells for transplantation are very
important because rejection could be avoided. In particular, Yamanaka's group in
Japan gave new hope to patients with incurable diseases when they developed
induced murine pluripotent stem cells (iPSCs) in 2006 and human iPSCs in 2007.
Whereas embryonic stem cells (ESCs) are derived from the inner cell mass and are
supported in culture by LIF, iPSCs are derived from fetal or adult somatic
cells. Through the application of iPSC technology, adult somatic cells can
develop a pluripotent state. One advantage of using iPSCs instead of ESCs in
regenerative medicine is that (theoretically) immune rejection could be avoided,
although there is some debate about immune rejection of a patient's own iPSCs.
Many diseases occur in elderly patients. In order to use regenerative medicine
with the elderly, it is important to demonstrate that iPSCs can indeed be
generated from older patients. Recent findings have shown that iPSCs can be
established from aged mice and aged humans. These iPSCs can differentiate to
cells from all three germ layers. However, it is not known whether iPSCs from
aged mice or humans show early senescence. Before clinical use of iPSCs, issues
related to copy number variation, tumorigenicity and immunogenicity must be
resolved. It is particularly important that researchers have succeeded in
generating iPSCs that have differentiated to somatic cells related to specific
diseases of the elderly, including atherosclerosis, diabetes, Alzheimer's
disease and Parkinson's disease. These efforts will facilitate the use of
personalized stem cell transplantation therapy for currently incurable diseases. AIMS: The aim of this study was to evaluate the effect of PDX-1 (pancreatic and
duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1) and MafA (V-maf
musculoaponeurotic fibrosarcoma oncogene homolog A) in the differentiation of
induced pluripotent stem cells (iPSCs) into insulin-producing cells and to
explore this new approach of cell transplantation therapy for type 1 diabetes in
mice.
METHODS: iPSCs were infected with adenovirus (Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse
NeuroD1-IRES-GFP and Ad-Mouse Mafa-IRES-GFP) and then differentiated into
insulin-producing cells in vitro. RT-PCR was applied to detect insulin gene
expression, immunofluorescence to identify insulin protein, and mouse insulin
enzyme-linked immunosorbent assay (ELISA) was used to evaluate the amount of
insulin at different concentration of glucose. Insulin-producing cells were
transplanted into the liver parenchyma of diabetic mice. Immunohistochemistry,
intraperitoneal glucose tolerance test (IPGTT) and fasting blood glucose (FBG)
were performed to assess the function of insulin-producing cells.
RESULTS: Insulin biosynthesis and secretion were induced in iPSCs and
insulin-producing cells were responsive to glucose in a dose-dependent manner.
Gene expression of the three-gene-modified embryoid bodies (EBs) was similar to
the mouse pancreatic β cell line MIN6. Transplantation of insulin-producing
cells into type I diabetic mice resulted in hyperglycemia reversal.
CONCLUSIONS: The insulin-producing cells we obtained from three-gene-modified
EBs may be used as seed cells for tissue engineering and may represent a cell
replacement strategy for the production of β cells for the treatment of type 1
diabetes. Successful reprogramming of somatic cells into induced pluripotent stem cells
(iPSCs) has ushered in a new era of regenerative medicine. Several studies on
iPSCs have corroborated their immense promise and potential for use in cell
therapy and disease modeling. However, several shortcomings need to be overcome
before they can be used in clinical therapy. Investigation of iPSC fate and
physiology in vivo and ultimately, the feasibility of their application in cell
transplantation therapy, requires more in-depth studies in living subjects. One
recently established alternative approach to reprogramming involves the direct
conversion of a terminally differentiated somatic cell of one type into another,
without dedifferentiating into a pluripotent state. This direct lineage
reprogramming strategy is significantly faster, has the potential to generate an
enriched population of a specific subtype of cells, and hence, has wide
implications in regenerative cell therapy. Here, we review recent advances in
iPSC technology and summarize the research on the generation of patient-specific
induced cell types using direct lineage conversion. Specifically, we focus on
the scope of application of this approach in autologous cell replacement therapy
for diabetic wound treatment. Insulin resistance is central to diabetes and metabolic syndrome. To define the
consequences of genetic insulin resistance distinct from those secondary to
cellular differentiation or in vivo regulation, we generated induced pluripotent
stem cells (iPSCs) from individuals with insulin receptor mutations and
age-appropriate control subjects and studied insulin signaling and gene
expression compared with the fibroblasts from which they were derived. iPSCs
from patients with genetic insulin resistance exhibited altered insulin
signaling, paralleling that seen in the original fibroblasts. Insulin-stimulated
expression of immediate early genes and proliferation were also potently reduced
in insulin resistant iPSCs. Global gene expression analysis revealed marked
differences in both insulin-resistant iPSCs and corresponding fibroblasts
compared with control iPSCs and fibroblasts. Patterns of gene expression in
patients with genetic insulin resistance were particularly distinct in the two
cell types, indicating dependence on not only receptor activity but also the
cellular context of the mutant insulin receptor. Thus, iPSCs provide a novel
approach to define effects of genetically determined insulin resistance. This
study demonstrates that effects of insulin resistance on gene expression are
modified by cellular context and differentiation state. Moreover, altered
insulin receptor signaling and insulin resistance can modify proliferation and
function of pluripotent stem cell populations. Human histocompatibility antigens are quite heterogeneous and promote the
rejection of transplanted tissue. Recent advances in stem cell research that
enable the use of a patient's own stem cells for transplantation are very
important because rejection could be avoided. In particular, Yamanaka’s group in
Japan gave new hope to patients with incurable diseases when they developed
induced murine pluripotent stem cells (iPSCs) in 2006 and human iPSCs in 2007.
Whereas embryonic stem cells (ESCs) are derived from the inner cell mass and are
supported in culture by LIF, iPSCs are derived from fetal or adult somatic
cells. Through the application of iPSC technology, adult somatic cells can
develop a pluripotent state. One advantage of using iPSCs instead of ESCs in
regenerative medicine is that (theoretically) immune rejection could be avoided,
although there is some debate about immune rejection of a patient's own iPSCs.
Many diseases occur in elderly patients. In order to use regenerative medicine
with the elderly, it is important to demonstrate that iPSCs can indeed be
generated from older patients. Recent findings have shown that iPSCs can be
established from aged mice and aged humans. These iPSCs can differentiate to
cells from all three germ layers. However, it is not known whether iPSCs from
aged mice or humans show early senescence. Before clinical use of iPSCs, issues
related to copy number variation, tumorigenicity and immunogenicity must be
resolved. It is particularly important that researchers have succeeded in
generating iPSCs that have differentiated to somatic cells related to specific
diseases of the elderly, including atherosclerosis, diabetes, Alzheimer's
disease and Parkinson's disease. These efforts will facilitate the use of
personalized stem cell transplantation therapy for currently incurable diseases. |
What is the name of the stem loop present in the 3' end of genes encoding for selenoproteins? | SECIS (selenocysteine insertion sequence) | We investigated the requirements for selenocysteine insertion at single or
multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements
were identified in the 3' untranslated region of the rat selenoprotein P mRNA,
with predicted stem-loops and critical nucleotides similar to those in the SECIS
elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione
peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS
elements confirmed that conserved nucleotides in the loop and in unpaired
regions of the stem are critical for activity. This indicates that multiple
contact sites are required for SECIS function. Stop codon function at any of
five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements
from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of
SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in
UGA codon position. SECIS elements are stem-loop structures located in the 3' untranslated regions
(UTRs) of eukaryotic selenoprotein mRNAs that are required for directing
cotranslational selenocysteine incorporation at UGA codons. In prokaryotes,
stem-loops mediating selenocysteine incorporation are located immediately
downstream of the UGA selenocysteine codon, in the coding region. Previous
characterization studies of the mammalian SECIS elements of type 1 deiodinase,
glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in
the loops and unpaired bulges, and base pairing in the stems are required for
SECIS function. These initial studies utilized approximately 175-230-nt segments
of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional
rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which
corresponds precisely to the 5'-most critical conserved nucleotide identified
previously. We also define base pairing requirements in the stem of this
element. In view of the presence of SECIS elements in the open reading frames
(ORFs) of bacterial selenoproteins, we examine the effects in the type 1
deiodinase of extending the ORF into the SECIS element, and find that this
dramatically inhibits SECIS function. Finally, we define a minimal spacing
requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS
element. Several gene products are involved in co-translational insertion of
selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs
coding for selenoproteins is essential to mediate the selection of the proper
selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this
stem-loop structure, the selenocysteine insertion sequence (SECIS) element,
resides in the 3'-untranslated region, far downstream of the UGA codon. In view
of unravelling the underlying complex mechanism, we have attempted to detect
RNA-binding proteins with specificity for the SECIS element. Using mobility
shift assays, we could show that a protein, present in different types of
mammalian cell extracts, possesses the capacity of binding the SECIS element of
the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein
SBP, for Secis Binding Protein. Competition experiments attested that the
binding is highly specific and UV cross-linking indicated that the protein has
an apparent molecular weight in the range of 60-65 kDa. Finally, some data
suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein,
type I iodothyronine 5' deiodinase, recognize the same SBP protein. This
constitutes the first report of the existence of a 3' UTR binding protein
possibly involved in the eukaryotic selenocysteine insertion mechanism. Selenocysteine, a selenium-containing analog of cysteine, is found in the
prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in
oxidation-reduction reactions. Its biosynthesis and cotranslational insertion
into selenoproteins is performed by an outstanding mechanism, implying the
participation of several gene products. The tRNA(Sec) is one of these. In
eukaryotes, its transcription mode by RNA polymerase III differs from that of
classical tRNA genes, both at the level of the promoter elements and
transcription factors involved. In addition, enhanced transcription is afforded
by a newly characterized zinc finger activator. Not only transcription of the
gene, but also the tRNA(Sec) itself is atypical since its 2D and 3D structures
exhibit features which set it apart from classical tRNAs. Decoding of eukaryotic
selenocysteine UGA codons requires a stem-loop structure in the 3'UTR of mRNAs,
the selenocysteine insertion sequence (SECIS) element. Structure probing and
sequence comparisons led us to propose a 2D structure model for the SECIS
element, containing a novel RNA motif composed of four consecutive
non-Watson-Crick base-pairs. A 3D model, rationalizing the accessibility data,
was elaborated by computer modeling. It yields indicative or suggestive evidence
for the role that could play some conserved residues and/or structural features
in SECIS function. These might act as signals for interaction with SBP, the
SECIS binding protein that we have characterized. SECIS elements form stem-loop structures in the 3' untranslated regions (UTR) of
eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation
of selenocysteine at UGA codons, provided the SECIS element lies a sufficient
distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from
human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS
elements that retained important features common to all known SECIS elements.
Comparative analysis of these SECIS elements showed that in some regions both
predicted secondary structure and nucleotide sequences were conserved, in other
areas secondary structure was maintained using different primary sequence, and
in still other portions, base pairing was not conserved. The rodent and sheep
selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon.
Thus, UGA specified both selenocysteine incorporation and termination in a
single mRNA. The selenoprotein W SECIS elements contained an additional highly
conserved base-paired stem that may prevent inappropriate selenocysteine
incorporation at the UGA stop codons. Mammalian selenium-containing proteins identified thus far contain selenium in
the form of a selenocysteine residue encoded by UGA. These proteins lack common
amino acid sequence motifs, but 3'-untranslated regions of selenoprotein genes
contain a common stem-loop structure, selenocysteine insertion sequence (SECIS)
element, that is necessary for decoding UGA as selenocysteine rather than a stop
signal. We describe here a computer program, SECISearch, that identifies
mammalian selenoprotein genes by recognizing SECIS elements on the basis of
their primary and secondary structures and free energy requirements. When
SECISearch was applied to search human dbEST, two new mammalian selenoproteins,
designated SelT and SelR, were identified. We determined their cDNA sequences
and expressed them in a monkey cell line as fusion proteins with a green
fluorescent protein. Incorporation of selenium into new proteins was confirmed
by metabolic labeling with (75)Se, and expression of SelT was additionally
documented in immunoblot assays. SelT and SelR did not have homology to
previously characterized proteins, but their putative homologs were detected in
various organisms. SelR homologs were present in every organism characterized by
complete genome sequencing. The data suggest applicability of SECISearch for
identification of new selenoprotein genes in nucleotide data bases. In mammals, most of the selenium contained in the body is present as an unusual
amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is
typically recognized as a translation stop signal, it is intriguing how a cell
recognizes and distinguishes a UGA Sec codon from a UGA stop codon. For
eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop
structure designated the Sec insertion sequence (SECIS) in the 3'-untranslated
(3'-UTR) region is required for recognition of UGA as a Sec codon. Some proteins
which bind to SECIS (SBP) have been reported. However, it is not clear how the
SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA
Sec codons. The idea that there must be a signal near the UGA Sec codon is still
considered. Therefore, we searched for a protein which binds to an RNA sequence
surrounding the UGA Sec codon on human glutathione peroxidase (GPx) mRNA. We
found a protein which strongly bound to the RNA fragment upstream of the UGA Sec
codon. However, this protein did not bind to the RNA sequence downstream of the
UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human
GPx, and this binding to SECIS was competed with the RNA fragment upstream of
the UGA Sec codon. Comparison of the RNA fragment with the SECIS fragment
identified the conserved regions, which appeared in the region upstream of the
in-frame UGA Sec codon of Se-protein mRNAs. Thus, this study proposes a novel
model to understand the mechanisms of Sec incorporation at the UGA Sec codon,
especially the regions upstream of the UGA codon of mRNAs of mammalian
selenoproteins. This model explains that the stem-loop structure covering the
UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by
eRF of the peptide releasing factor. Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec
insertion sequence (SECIS) element, which is the stem-loop structure present in
3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is
essential for recognition of UGA as a codon for Sec rather than as a stop
signal. We now report the identification of a new eukaryotic selenoprotein,
designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five
exons and is located on chromosome 22 but has not been correctly identified by
either Celera or the public Human Genome Project. We characterized human and
mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian
cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a
canonical SECIS element. Instead, Sec is incorporated in response to a conserved
mRNA structure, in which cytidines are present in place of the adenosines
previously considered invariant. Substitution of adenosines for cytidines did
not alter Sec incorporation; however, other mutant structures did not support
selenoprotein synthesis, demonstrating that this new form of SECIS element is
functional. SelM is expressed in a variety of tissues, with increased levels in
the brain. It is localized to the perinuclear structures, and its N-terminal
signal peptide is necessary for protein translocation. Incorporation of the 21st amino acid, selenocysteine, into proteins is specified
in all three domains of life by dynamic translational redefinition of UGA
codons. In eukarya and archaea, selenocysteine insertion requires a cis-acting
selenocysteine insertion sequence (SECIS) usually located in the 3'UTR of
selenoprotein mRNAs. Here we present comparative sequence analysis and
experimental data supporting the presence of a second stop codon redefinition
element located adjacent to a selenocysteine-encoding UGA codon in the eukaryal
gene, SEPN1. This element is sufficient to stimulate high-level (6%)
translational redefinition of the SEPN1 UGA codon in human cells. Readthrough
levels further increased to 12% when tested in the presence of the SEPN1 3'UTR
SECIS. Directed mutagenesis and phylogeny of the sequence context strongly
supports the importance of a stem loop starting six nucleotides 3' of the UGA
codon. Sequences capable of forming strong RNA structures were also identified
3' adjacent to, or near, selenocysteine-encoding UGA codons in the Sps2, SelH,
SelO, and SelT selenoprotein genes. Selenoproteins are an elite group of proteins containing a rare amino acid,
selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of
Sec requires a Sec insertion sequence (SECIS) element, a stem-loop structure
located in the 3'-untranslated regions of selenoprotein mRNAs. Here we report
identification of a noncanonical form of SECIS element in Toxoplasma gondii and
Neospora canine, single-celled apicomplexan parasites of humans and domestic
animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of
AUGA previously considered invariant. Using a combination of computational and
molecular techniques, we show that Toxoplasma and Neospora possess both
canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported
Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently
than the natural mammalian SECIS elements tested. In addition, mammalian type I
and type II SECIS elements mutated into the GGGA forms were functional but
manifested decreased Sec insertion efficiency. We carried out computational
searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and
detected five selenoprotein genes, including one coding for a previously
undescribed selenoprotein, designated SelQ, and two containing the GGGA form of
the SECIS element. In contrast, the GGGA-type SECIS elements were not detected
in mammals and nematodes. As a practical outcome of the study, we developed
pSelExpress1, a vector for convenient expression of selenoproteins in mammalian
cells. It contains an SBP2 gene and the most efficient tested SECIS element: an
AUGA mutant of the GGGA-type Toxoplasma SelT structure. Selenocysteine (Sec, U) insertion into proteins is directed by translational
recoding of specific UGA codons located upstream of a stem-loop structure known
as Sec insertion sequence (SECIS) element. Selenoproteins with known functions
are oxidoreductases containing a single redox-active Sec in their active sites.
In this work, we identified a family of selenoproteins, designated SelL,
containing two Sec separated by two other residues to form a UxxU motif. SelL
proteins show an unusual occurrence, being present in diverse aquatic organisms,
including fish, invertebrates, and marine bacteria. Both eukaryotic and
bacterial SelL genes use single SECIS elements for insertion of two Sec. In
eukaryotes, the SECIS is located in the 3' UTR, whereas the bacterial SelL SECIS
is within a coding region and positioned at a distance that supports the
insertion of either of the two Sec or both of these residues. SelL proteins
possess a thioredoxin-like fold wherein the UxxU motif corresponds to the
catalytic CxxC motif in thioredoxins, suggesting a redox function of SelL
proteins. Distantly related SelL-like proteins were also identified in a variety
of organisms that had either one or both Sec replaced with Cys. Danio rerio
SelL, transiently expressed in mammalian cells, incorporated two Sec and
localized to the cytosol. In these cells, it occurred in an oxidized form and
was not reducible by DTT. In a bacterial expression system, we directly
demonstrated the formation of a diselenide bond between the two Sec,
establishing it as the first diselenide bond found in a natural protein. The expression of selenoproteins requires the translational recoding of the UGA
stop codon to selenocysteine. In eukaryotes, this requires an RNA stem loop
structure in the 3'-untranslated region, termed a selenocysteine insertion
sequence (SECIS), and SECIS-binding protein 2 (SBP2). This study implicates SBP2
in dictating the hierarchy of selenoprotein expression, because it is the first
to show that SBP2 distinguishes between SECIS elements in vitro. Using RNA
electrophoretic mobility shift assays, we demonstrate that a naturally occurring
mutation in SBP2, which correlates with abnormal thyroid hormone function in
humans, lies within a novel, bipartite RNA-binding domain. This mutation alters
the RNA binding affinity of SBP2 such that it no longer stably interacts with a
subset of SECIS elements. Assays performed under competitive conditions to mimic
intracellular conditions suggest that the differential affinity of SBP2 for
various SECIS elements will determine the expression pattern of the
selenoproteome. We hypothesize that the selective loss of a subset of
selenoproteins, including some involved in thyroid hormone homeostasis, is
responsible for the abnormal thyroid hormone metabolism previously observed in
the affected individuals. SUMMARY: Selenoproteins contain the 21st amino acid selenocysteine which is
encoded by an inframe UGA codon, usually read as a stop. In eukaryotes, its
co-translational recoding requires the presence of an RNA stem-loop structure,
the SECIS element in the 3 untranslated region of (UTR) selenoprotein mRNAs.
Despite little sequence conservation, SECIS elements share the same overall
secondary structure. Until recently, the lack of a significantly high number of
selenoprotein mRNA sequences hampered the identification of other potential
sequence conservation. In this work, the web-based tool SECISaln provides for
the first time an extensive structure-based sequence alignment of SECIS elements
resulting from the well-defined secondary structure of the SECIS RNA and the
increased size of the eukaryotic selenoproteome. We have used SECISaln to
improve our knowledge of SECIS secondary structure and to discover novel,
conserved nucleotide positions and we believe it will be a useful tool for the
selenoprotein and RNA scientific communities.
AVAILABILITY: SECISaln is freely available as a web-based tool at
http://genome.crg.es/software/secisaln/. Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a
reprogrammed UGA codon. In mammals, this requires a dedicated machinery
comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the
specific SECIS Binding Protein 2. In this report, disorder-prediction methods
and several biophysical techniques showed that ca. 70% of the SBP2 sequence is
disordered, whereas the RNA binding domain appears to be folded and functional.
These results are consistent with a recent report on the role of the Hsp90
chaperone for the folding of SBP2 and other functionally unrelated proteins
bearing an RNA binding domain homologous to SBP2. Selenium, an essential trace element, is incorporated into selenoproteins as
selenocysteine (Sec), the 21st amino acid. In order to synthesize
selenoproteins, a translational reprogramming event must occur since Sec is
encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on
the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in
the 3' untranslated region of the transcript. The SECIS acts as a platform for
RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV
crosslinking, we identified a 110 kDa protein, which binds with high affinity to
SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity
was purified by RNA affinity chromatography and identified as nucleolin by mass
spectrometry analysis. In vitro binding assays showed that purified nucleolin
discriminates among SECIS elements in the absence of other factors. Based on
siRNA experiments, nucleolin is required for the optimal expression of certain
selenoproteins. There was a good correlation between the affinity of nucleolin
for a SECIS and its effect on selenoprotein expression. As selenoprotein
transcript levels and localization did not change in siRNA-treated cells, our
results suggest that nucleolin selectively enhances the expression of a subset
of selenoproteins at the translational level. The mechanisms regulating the differential selenium (Se)-dependent stability of
selenoprotein mRNAs are partially characterized. To further study the
Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent
reporter to monitor the steady-state mRNA level of an artificial selenoprotein.
Our reporter is a fusion of the Renilla luciferase gene and of the β-globin
gene, but contains features required for incorporation of selenocysteine (SEC),
namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC
incorporation sequence (SECIS). At various levels of Se, the activity of
reporters containing GPX1 or GPX4 SECIS elements is proportional to the
steady-state mRNA level of the reporter construct and reflects the level of the
corresponding endogenous mRNA. In a reporter containing a UGA codon and a
functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in
the cytoplasm, as opposed to the more typical nuclear location. To validate the
reporter system, we used genetic and pharmacologic approaches to inhibit or
promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of
SMG1 altered NMD in this system. Our reporter is derived from a Renilla
luciferase reporter gene fused to an intron containing B-globin gene and is
subject to degradation by NMD when a stop codon is inserted before the second
intron. Selenium (Se) is an essential trace element primarily found in selenoproteins as
the 21st amino acid (selenocysteine, Sec, or U). Selenoproteins play an
important role in growth and proliferation and are typically involved in
cellular redox balance. Selenocysteine is encoded by an in-frame UGA codon
specified by a stem-loop structure, the Sec insertion sequence element (SECIS),
which, in eukaryotes, is located in the 3'-untranslated region (UTR). The
availability of the Naegleria gruberi (ATCC 30224) genome sequence and the use
of this organism as a model system for the pathogenic amoeba N. fowleri allowed
us to investigate the Sec incorporation pathway in this primitive eukaryote.
Using bioinformatics tools, we identified gene sequences encoding PSTK
(O-phosphoseryl-tRNA(Sec) kinase), SepSecS
(O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase), SelD/SPS2 (selenophosphate
synthetase), EFSec (selenocysteine-specific elongation factor) and SBP (SECIS
binding protein). These findings were confirmed by RT-PCR and by sequencing. A
potential tRNA(Ser)Sec (SelC) gene and a putative selenoprotein with sequence
similarity to a mitochondrial thioredoxin reductase (TR3) were also identified.
Our results show that the selenocysteine incorporation machinery is indeed
present in N. gruberi. Interestingly, the SelD/SPS2 gene is 2214 bp in length
and contains two distinct domains. The N-terminal region shows sequence
similarity to predicted methyltransferase proteins, and the C-terminal region is
homologous to prokaryotic SelD/SPS2. Our results suggest the possibility of
novel selenoproteins. Selenoprotein S (SelS) is a 189 amino acid trans-membrane protein that plays an
important yet undefined role in the unfolded protein response. It has been
proposed that SelS may function as a reductase, with the penultimate
selenocysteine (Sec(188)) residue participating in a selenosulfide bond with
cysteine (Cys(174)). Cotranslational incorporation of Sec into SelS depends on
the recoding of the UGA codon, which requires a Selenocysteine Insertion
Sequence (SECIS) element in the 3'UTR of the transcript. Here we identify
multiple mechanisms that regulate the expression of SelS. The human SelS gene
encodes two transcripts (variants 1 and 2), which differ in their 3'UTR
sequences due to an alternative splicing event that removes the SECIS element
from the variant 1 transcript. Both transcripts are widely expressed in human
cell lines, with the SECIS-containing variant 2 mRNA being more abundant. In
vitro experiments demonstrate that the variant 1 3'UTR does not allow
readthrough of the UGA/Sec codon. Thus, this transcript would produce a
truncated protein that does not contain Sec and cannot make the selenosulfide
bond. While the variant 2 3'UTR does support Sec insertion, its activity is
weak. Bioinformatic analysis revealed two highly conserved stem-loop structures,
one in the proximal part of the variant 2 3'UTR and the other immediately
downstream of the SECIS element. The proximal stem-loop promotes Sec insertion
in the native context but not when positioned far from the UGA/Sec codon in a
heterologous mRNA. In contrast, the 140 nucleotides downstream of the SECIS
element inhibit Sec insertion. We also show that endogenous SelS is enriched at
perinuclear speckles, in addition to its known localization in the endoplasmic
reticulum. Our results suggest the expression of endogenous SelS is more complex
than previously appreciated, which has implications for past and future studies
on the function of this protein. Selenoproteins are proteins containing an uncommon amino acid selenocysteine
(Sec). Sec is inserted by a specific translational machinery that recognizes a
stem-loop structure, the SECIS element, at the 3' UTR of selenoprotein genes and
recodes a UGA codon within the coding sequence. As UGA is normally a
translational stop signal, selenoproteins are generally misannotated and
designated tools have to be developed for this class of proteins. Here, we
present two new computational methods for selenoprotein identification and
analysis, which we provide publicly through the web servers at
http://gladyshevlab.org/SelenoproteinPredictionServer or
http://seblastian.crg.es. SECISearch3 replaces its predecessor SECISearch as a
tool for prediction of eukaryotic SECIS elements. Seblastian is a new method for
selenoprotein gene detection that uses SECISearch3 and then predicts
selenoprotein sequences encoded upstream of SECIS elements. Seblastian is able
to both identify known selenoproteins and predict new selenoproteins. By
applying these tools to diverse eukaryotic genomes, we provide a ranked list of
newly predicted selenoproteins together with their annotated cysteine-containing
homologues. An analysis of a representative candidate belonging to the AhpC
family shows how the use of Sec in this protein evolved in bacterial and
eukaryotic lineages. The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus
requiring a specialized machinery to enable its incorporation into
selenoproteins. The machinery comprises the tRNA(Sec), a 3'-UTR mRNA stem-loop
termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for
recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other
proteins. Little is known about the molecular mechanism and, in particular,
when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by
others used the isolated SECIS RNA to address this question. Here, we developed
a novel approach using instead engineered minimal selenoprotein mRNAs containing
SECIS elements derivatized with photoreactive groups. By cross-linking
experiments in rabbit reticulocyte lysate, new information could be gained about
the SBP2 and SECIS contacts with components of the translation machinery at
various translation steps. In particular, we found that SBP2 was bound only to
the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the
complex where the Sec-tRNA(Sec) was accommodated to the A site but
transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS
element as well, and the SECIS had flexible contacts with the 60S ribosomal
subunit involving several ribosomal proteins. Altogether, our findings led to
broadening our understanding about the unique mechanism of selenocysteine
incorporation in mammals. Selenium (Se) is an essential trace element for several organisms and is present
in proteins as selenocysteine (Sec or U), an amino acid that is chemically
distinct from serine and cysteine by a single atom (Se instead of O or S,
respectively). Sec is incorporated into selenoproteins at an in-frame UGA codon
specified by an mRNA stem-loop structure called the selenocysteine incorporating
sequence (SECIS) presented in selenoprotein mRNA and specific selenocysteine
synthesis and incorporation machinery. Selenoproteins are presented in all
domains but are not found in all organisms. Although several functions have been
attributed to this class, the majority of the proteins are involved in oxidative
stress defense. Here, we discuss the kinetoplastid selenocysteine pathway and
how selenium supplementation is able to alter the infection course of
trypanosomatids in detail. These organisms possess the canonical elements
required for selenoprotein production such as phosphoseryl tRNA kinase (PSTK),
selenocysteine synthase (SepSecS), selenophosphase synthase (SelD or SPS), and
elongation factor EFSec (SelB), whereas other important factors presented in
mammal cells, such as SECIS binding protein 2 (SBP) and SecP 43, are absent. The
selenoproteome of trypanosomatids is small, as is the selenoproteome of others
parasites, which is in contrast to the large number of selenoproteins found in
bacteria, aquatic organisms and higher eukaryotes. Trypanosoma and Leishmania
are sensitive to auranofin, a potent selenoprotein inhibitor; however, the
probable drug mechanism is not related to selenoproteins in kinetoplastids.
Selenium supplementation decreases the parasitemia of various Trypanosome
infections and reduces important parameters associated with diseases such as
anemia and parasite-induced organ damage. New experiments are necessary to
determine how selenium acts, but evidence suggests that immune response
modulation and increased host defense against oxidative stress contribute to
control of the parasite infection. |
Is Propofol used for short-term sedation? | Yes. Propofol is the most frequently used sedating agent for patients with expected duration of ICU admission less than 24 hours. There are numerous studies of its efficacy and comparisons with other sedatives. | Sedative-analgesic treatment of patients on long-term artificial ventilation
aims at protection from stress related to their disease or therapy. By
stabilising both the patient's vital functions and psychological state this
treatment may contribute to therapeutic success. The choice of drugs depends
primarily on the nature and course of the underlying disease. Midazolam and
propofol are available as hypnotics for short-term sedation during the
post-operative period. The purpose of this study was to evaluate the effects of
both agents on cardiovascular function, cortisol production, lipometabolism, and
the recovery period following 24-h sedation. METHODS. Twenty female patients
(mean body weight: 72 kg, mean age: 60 years) were randomly assigned to receive
either midazolam or propofol over 24 h following major abdominal surgery.
Balanced anaesthesia (halothane/O2/N2O/fentanyl) was administered for the
surgical procedure. Assisted ventilation was used in all patients during the
post-operative sedation period. Sedation depth was maintained at III-IV on the
Ramsey scale. On arrival in the intensive care unit (ICU), an initial i.v. bolus
of midazolam 0.1 mg/kg or propofol 1 mg/kg was followed by a continuous infusion
(midazolam: 0.1 mg/kg.h; propofol: 2 mg/kg.h). Supplementary boluses of one-half
the initial dose were given if required. Post-operative analgesia was achieved
with 3 mg intravenous piritramide at 2-h intervals. A 7F Swan-Ganz catheter was
inserted in the pulmonary artery and haemodynamic and biochemical parameters
were monitored at 4-h intervals over 24 h starting 2 h after arrival in the ICU.
Catecholamines were measured by high-pressure lipid chromatography (HPLC),
cortisol by radioimmunoassay, midazolam by HPLC and ultraviolet detection, and
propofol by HPLC and fluorescence detection. Data were calculated as means. The
statistical analysis was performed according to the Mann-Whitney test, and
significance was accepted for P less than 0.05. RESULTS. On administration of
the propofol bolus at the onset of sedation, a decrease in blood pressure was
particularly observed in patients with masked hypovolaemia, however, this
decrease was easily controlled by volume administration. Independent of the type
of sedation, the haemodynamic parameters remained unchanged throughout the
observation period. At all times of measurement the mean heart rate was lower in
the propofol group (90/min) when compared with the midazolam group (100/min),
however, this difference did not reach significance. There were also no
significant differences in cardiac index at all times of measurement, although
it increased in both groups within the first 12 h by 0.6 l/min.min2. In both
groups this increase was associated with a reduction in peripheral resistance
and an increase in rectal temperature. To achieve the desired sedation depth,
midazolam was administered at a mean dosage of 0.11 mg/kg.h and propofol at 1.9
mg/kg.h. Catecholamine levels decreased in both groups within the first 8 h:
after 8 h of sedation the plasma levels of noradrenaline and adrenaline were 525
and 65 pg/ml, respectively, in the midazolam group and 327 and 51 pg/ml in the
propofol group. (ABSTRACT TRUNCATED AT 400 WORDS) Sedation in the intensive care unit (ICU) aims to improve patient comfort and
facilitate treatment procedures. Most units still rely on a combination of
opioid and benzodiazepines with the addition of other drugs for specific
requirements. However, the effect of sedative agents in critically ill patients
is often unpredictable, so frequent assessment of the depth of sedation is
essential to match the depth to patient requirements. In the 1990s, heavy
sedation and paralysis is not considered appropriate for many ICU patients; a
minimum sedation approach limits cardiovascular or respiratory depression and
enables earlier weaning and extubation of patients. Administering sedative
agents by continuous infusion is convenient but, unless the level of sedation is
reassessed regularly, many patients may become over-sedated. The use of propofol
for short-term sedation in ICUs has allowed the maintece of sedation to
continue until just a few hours before extubation but the benefits of propofol
for longer-term indications are more debatable. Closer titration of dose and
desired effects could also be achieved by a patient-controlled system. The
technique may not be suitable for a large number of patients, particularly early
in their ICU stay but, for long-term sedation and in the weaning phase--of
sedation as well as ventilation--the utility of a drug delivery system truly
controlled by the patient should be further explored. The ICU has been
succinctly described as an environment in which 'anxiety is prevalent, pain
frequent, rest difficult and sleep impossible'. Sedation in the ICU has the
double objective of relieving patient distress as well as facilitating treatment
procedures.(ABSTRACT TRUNCATED AT 250 WORDS) We conducted a randomized double-blind study to assess the safety and
effectiveness of short-term sedation with propofol in adult patients immediately
after cardiac surgery. Sixty hemodynamically stable adult patients who underwent
cardiac surgery were randomly assigned to receive propofol postoperatively or no
postoperative sedation. The propofol group (n = 30) received propofol infusion
(1 mg.kg-1.hr-1) immediately after they awoke postoperatively. The infusion
rates were adjusted to maintain a sedation level (Ramsay score) of 3. Nurses
assessed the effectiveness of the sedation according to Ramsay scores; the
patients also subjectively evaluated sedation. Postoperative pulmonary
oxygenation dysfunction (PaO2/FiO2 < 300 mm Hg) was noted in 10 patients
receiving propofol and in eight patients in the control group. The dysfunction
was greatly improved after overnight ventilator support, and this improvement
was not affected by propofol sedation. The propofol group received an average of
13.6 +/- 5.3 hours of propofol infusion, at an average infusion rate of 0.96 +/-
0.16 mg.kg-1.hr-1. Patients receiving propofol remained well sedated (Ramsay
score > or = 2) during 99.3% of the study period, while the control patients
remained anxious or agitated (Ramsay score, 1) during 12% of the study period.
Patients receiving propofol were successfully extubated 9 +/- 4 minutes after
cessation of propofol infusion, without complications. The turnover of intensive
care unit beds was not delayed by propofol sedation. Subjective evaluations
(scored on an analog scale: 0 = nil, 10 = extreme) revealed that patients
receiving propofol felt less pain than those in the control group (2.3 +/- 2.7
vs 4.7 +/- 3.1, p < 0.05), had better sleep quality (7.8 +/- 2.9 vs 5.1 +/- 2.9,
p < 0.05), and were more satisfied with the care they received (8.3 +/- 2.2 vs
5.8 +/- 3.9, p < 0.05). These findings suggest that propofol infusion is
effective for short-term sedation of cardiac surgery patients postoperatively
and that an infusion rate adequate to maintain a Ramsay score of 3 may be ideal.
Postoperative sedation should be considered for all cardiac surgery patients,
not only those with outward signs of anxiety or agitation. BACKGROUND: Propofol has been advocated for sedation in intensive care because
of superior recovery characteristics. We hypothesised that the use of two
totally different sedation methods after coronary artery bypass grafting should
result in differences not only in extubation time, but also in breathing pattern
and gas exchange during weaning and after extubation.
METHODS: Thirty patients participated in this randomised and controlled study.
We used propofol infusion and oxycodone-thiopental bolus dosage, titrated to
sedation level 4 or 5 according to Ramsey. Weaning was performed using
protocol-based pressure support trials.
RESULTS: Total (SD) fentanyl dose during operation was 33 (6) microg x kg(-1)
for propofol and 34 (6) microg x kg(-1) for oxycodone-thiopental (ns). The
target sedation was achieved equally with both methods. The time from admission
to intensive care unit to extubation was 494 (100) min for propofol and 521 (98)
min for oxycodone-thiopental (ns). Weaning times were 63 (24) min and 112 (63)
min in the propofol and oxycodone-thiopental groups, respectively (P<0.05).
Breathing frequency increased and tidal volume decreased from weaning to 2 h
postextubation.
CONCLUSION: Propofol infusion and oxycodone-thiopental bolus dosages, titrated
to the same sedation end point, resulted in similar time from admission to
extubation, although the weaning period was shorter in the propofol group. In
terms of breathing pattern, gas exchange, blood gases and haemodynamics, the
methods were similar. Propofol, despite its attractive pharmacological profile,
may offer no clinical benefit in short-term sedation after a moderate dose
fentanyl anaesthesia in cardiac surgery. We have compared the effects of dexmedetomidine and propofol on endocrine,
metabolic, inflammatory and cardiovascular responses in patients in the
intensive care unit (ICU) after major surgery. Twenty patients who were expected
to require 8 h of post-operative sedation and ventilation were allocated
randomly to receive either an infusion of dexmedetomidine 0.2-2.5 microg kg(-1)
h(-1) or propofol 1-3 mg kg(-1) h(-1). Arterial pressure, heart rate and
sequential concentrations of circulating cortisol, adrenocorticotrophic hormone
(ACTH), growth hormone, prolactin, insulin, glucose and interleukin 6 were
measured. An ACTH stimulation test was performed in all patients who received
dexmedetomidine. Heart rate was significantly lower in the dexmedetomidine
patients. There were no differences in arterial pressure, cortisol, ACTH,
prolactin and glucose concentrations between the two groups. A positive response
to the ACTH stimulation test varied depending on the diagnostic criteria used.
The insulin concentration was significantly lower in the dexmedetomidine group
at 2 h (P=0.021), although this did not affect blood glucose concentrations.
Growth hormone concentrations were significantly higher in
dexmedetomidine-treated patients overall (P=0.036), but circulating
concentrations remained in the physiological range. Interleukin 6 decreased in
the dexmedetomidine group. We conclude that dexmedetomidine infusion does not
inhibit adrenal steroidogenesis when used for short-term sedation after surgery. AIMS: This paper describes the pharmacokinetics and effects of propofol in
short-term sedated paediatric patients.
METHODS: Six mechanically ventilated children aged 1-5 years received a 6 h
continuous infusion of propofol 6% at the rate of 2 or 3 mg kg-1 h-1 for
sedation following cardiac surgery. A total of seven arterial blood samples was
collected at various time points during and after the infusion in each patient.
Pharmacokinetic modelling was performed using NONMEM. Effects were assessed on
the basis of the Ramsay sedation score as well as a subjective sedation scale.
RESULTS: The data were best described by a two-compartment pharmacokinetic
model. In the model, body weight was a significant covariate for clearance.
Pharmacokinetic parameters in the weight-proportional model were clearance (CL)
= 35 ml kg-1 min-1, volume of central compartment (V1) = 12 l,
intercompartmental clearance (Q) = 0.35 l min-1 and volume of peripheral
compartment (V2) = 24 l. The interindividual variabilities for these parameters
were 8%, < 1%, 11% and 35%, respectively. Compared with the population
pharmacokinetics in adults following cardiac surgery and when normalized for
body weight, statistically significant differences were observed the parameters
CL and V1 (35 vs 29 ml kg-1 min-1 and 0.78 vs 0.26 l kg-1P < 0.05), whereas the
values for Q and V2 were similar (23 vs 18 ml kg-1 min-1 and 1.6 vs 1.8 l kg-1,
P > 0.05). In children, the percentage of adequately sedated patients was
similar compared with adults (50% vs 67%) despite considerably higher propofol
concentrations (1.3 +/- 0.10 vs 0.51 +/- 0.035 mg l-1, mean +/- s.e. mean),
suggesting a lower pharmacodynamic sensitivity to propofol in children.
CONCLUSIONS: In children aged 1-5 years, a pharmacokinetic model for propofol
was described using sparse data. In contrast to adults, body weight was a
significant covariate for clearance in children. The model may serve as a useful
basis to study the role of covariates in the pharmacokinetics and
pharmacodynamics of propofol in paediatric patients of different ages. Despite the risk of propofol infusion syndrome, a rare but often fatal
complication of propofol infusion in ventilated children and possibly adults,
propofol infusion remains in use in paediatric intensive care units (PICU). This
questionnaire study surveys the current pattern of use of this sedative infusion
in Australian and New Zealand PICUs. Thirty-three of the 45 paediatric intensive
care physicians surveyed (73%), from 12 of the 13 intensive care units, returned
completed questionnaires. The majority of practitioners (82%) use propofol
infusion in children in PICU, the main indication being for short-term sedation
in children requiring procedures. 39% of respondents consider propofol infusion
useful in ventilated children requiring longer-term sedation. 67% of paediatric
intensivists use maximum infusion doses that may be considered dangerously high
(> or = 10 mg/kg/h). Nineteen per cent use propofol infusion for prolonged
periods (> 72 hours). A smaller proportion (15%) of respondents indicate that
they may use both higher doses and prolonged periods of infusion, a practice
likely to lead to a greater chance of serious adverse events. Knowledge of local
protocols for the use of propofol infusion is associated with a significantly
greater level of monitoring for possible adverse events. We suggest that
national guidelines for the use of propofol infusion in children should be
developed. These should include clear indications and contraindications to its
use, a maximum dose rate and maximum period of infusion, with a ceiling placed
on the cumulative dose given and clearly stated minimum monitoring requirements. INTRODUCTION: Sedation and analgesia are provided by using different agents and
techniques in different countries. The goal is to achieve early spontaneous
breathing and to obtain an awake and cooperative pain-free patient. It was the
aim of this study to conduct a survey of the agents and techniques used for
analgesia and sedation in intensive care units in Germany.
METHODS: A survey was sent by mail to 261 hospitals in Germany. The
anesthesiologists running the intensive care unit were asked to fill in the
structured questionnaire about their use of sedation and analgesia.
RESULTS: A total of 220 (84%) questionnaires were completed and returned. The
RAMSAY sedation scale was used in 8% of the hospitals. A written policy was
available in 21% of hospitals. For short-term sedation in most hospitals,
propofol was used in combination with sufentanil or fentanyl. For long-term
sedation, midazolam/fentanyl was preferred. Clonidine was a common part of up to
two-thirds of the regimens. Epidural analgesia was used in up to 68%.
Neuromuscular blocking agents were no longer used.
CONCLUSION: In contrast to the US 'Clinical practice guidelines for the
sustained use of sedatives and analgesics in the critically ill adult', our
survey showed that in Germany different agents, and frequently neuroaxial
techniques, were used. OBJECTIVE: Dexmedetomidine (DEX) may provide a sedation level that enables sleep
and communication, with less amnesia and pain medication requirements, during
mechanical ventilation. Our study directly assessed patient-perceived
satisfaction with coronary artery bypass graft surgery after administration of
DEX or propofol for intensive care unit (ICU) sedation.
DESIGN: Prospective, randomized clinical study with subsequent questionnaire
administration.
SETTING: Tertiary care surgical ICU.
PATIENTS: A total of 89 adult, nonemergent, coronary artery bypass graft
patients with an expected length of intubation of <24 hrs.
INTERVENTIONS: Patients were randomized to either DEX or propofol; drug
administration was performed via standardized anesthesia and nursing protocols.
MEASUREMENTS: Patients reported perceptions of their ICU experience after
mechanical ventilation with a modified numerical-scale Hewitt questionnaire,
validated specifically for ICU patients. Patients were questioned regarding
awareness, recall, generalized comfort, level of pain, ability to interact with
healthcare providers and family, feelings of agitation and anxiety, perceived
ease of extubation, ability to sleep or rest, and satisfaction with ICU
experience.
MAIN RESULTS: Groups were well matched at baseline, with a mean +/- sd age of
63.0 +/- 10.4 yrs and weight of 88.7 +/- 16.7 kg. No difference was observed for
length of surgery, length of intubation, or ICU stay (p > .05). DEX patients
perceived a shorter length of intubation (p = .044). A deeper sedation level was
found in the propofol group (p = .021), with similar morphine and midazolam
requirements (p = .317). Patient-rated level of overall awareness as a marker of
amnesia did not differ between groups (p = .653). The ability to rest or sleep
trended toward significance favoring propofol (p = .051). On evaluation of
questionnaire ratings, DEX patients expressed more discomfort (p = .046), pain
(p = .096), and sleeping difficulty (p = .036). Similar comfort levels were
reported during extubation (p = .179).
CONCLUSIONS: Despite theoretical advantages of DEX to improve overall patient
satisfaction, the two agents provide similar responses to amnesia and pain
control. According to our findings, DEX does not seem to have any advantage
compared with propofol for short-term sedation after coronary artery bypass
graft surgery. INTRODUCTION: The purpose of this study was to evaluate sedation practice in UK
intensive care units (ICUs), particularly the implementation of daily sedation
holding, written sedation guidelines, sedation scoring tools and choice of
agents.
METHODS: A national postal survey was conducted in all UK ICUs.
RESULTS: A total of 192 responses out of 302 addressed units were received
(63.5%). Of the responding ICUs, 88% used a sedation scoring tool, most
frequently the Ramsey Sedation Scale score (66.4%). The majority of units have a
written sedation guideline (80%), and 78% state that daily sedation holding is
practiced. A wide variety of sedating agents is used, with the choice of agent
largely determined by the duration of action rather than cost. The most
frequently used agents were propofol and alfentanil for short-term sedation;
propofol, midazolam and morphine for longer sedation; and propofol for weaning
purposes.
CONCLUSIONS: Most UK ICUs use a sedation guideline and sedation scoring tool.
The concept of sedation holding has been implemented in the majority of units,
and most ICUs have a written sedation guideline. PURPOSE: Assessing feasibility and physiological effects of sedation with
sevoflurane, administered with the anesthetic conserving device (AnaConDa), in
comparison with propofol and remifentanil.
METHODS: Seventeen patients undergoing mechanical ventilation underwent sedation
with sevoflurane delivered with AnaConDa (phase SevAn), preceded and followed by
sedation with propofol and remifentanil (phases ProRe(1), ProRe(2)), with the
same sedation targets.
RESULTS: With both strategies it was possible to achieve the sedation targets.
Time required to sedate and awake patients was greater during SevAn than
ProRe(1): respectively, 3.3 +/- 3.0 versus 8.9 +/- 6.1 and 7.47 +/- 5.05 versus
16.3 +/- 11.4 min. During SevAn the PaCO(2) and minute ventilation increased.
Hemodynamics was stable between ProRe(1) and SevAn, except for an increase in
heart rate in the SevAn phase. Environmental pollution from sevoflurane was
within the safety limits.
CONCLUSIONS: Sevoflurane can be effectively and safely used for short-term
sedation of ICU patients with stable hemodynamic conditions. BACKGROUND: Increased inorganic fluoride levels after methoxyflurane exposure in
the 1970s and prolonged intraoperative sevoflurane use have been suggested to be
potentially nephrotoxic. In the intensive care unit we evaluated the effect on
renal integrity of short-term inhaled postoperative sedation with sevoflurane
using the Anesthetic Conserving Device (ACD) compared with propofol.
METHODS: In this prospective, randomized, single-blinded study, after major
abdominal, vascular or thoracic surgery 125 patients were allocated to receive
either sevoflurane (n = 64) via the ACD (end-tidal 0.5-1 vol%) or i.v. propofol
(n = 61) for postoperative sedation up to 24 h. Urinary
alpha-glutathione-s-transferase as primary outcome variable, urinary
N-acetyl-glucosaminidase, serum creatinine, and inorganic fluoride
concentrations, urine output and fluid management were measured preoperatively,
at the end of surgery, and at 24 and 48 h postoperatively.
RESULTS: The sedation time in the intensive care unit was comparable between the
sevoflurane (9.2 +/- 4.3 h) and the propofol (9.3 +/- 4.7 h) group.
Alpha-glutathione-s-transferase levels were significantly increased at 24 and 48
h postoperatively compared with preoperative values in both groups, without
significant differences between the groups. N-acetyl-glucosaminidase and serum
creatinine remained unchanged in both study groups, and urine output and
creatinine clearance were comparable between the groups throughout the study
period. Inorganic fluoride levels increased significantly (P < 0.001) at 24 h
after sevoflurane exposure (39 +/- 25 micromol/L) compared with propofol (3 +/-
6 micromol/L) and remained elevated 48 h later (33 +/- 26 vs 3 +/- 5
micromol/L). One patient in each group suffered from renal insufficiency,
requiring intensive diuretic therapy, but not dialysis, during hospital stay.
CONCLUSIONS: Short-term sedation with either sevoflurane using ACD or propofol
did not negatively affect renal function postoperatively. Although inorganic
fluoride levels were elevated after sevoflurane exposure, glomerular and tubular
renal integrity were preserved throughout the hospital stay. This study evaluated the correlation and agreement between the Bispectral Index
(BIS) or A-line Autoregressive Index (AAI) and a clinical scoring system, the
Ramsay Sedation Scale (RSS), in 40 patients after elective cardiac surgery and
admission to the intensive care unit. All patients received sedation with
propofol according to the study protocol. BIS, AAI and RSS were documented at
two different levels of sedation: deep sedation RSS 4 - 6; and slight
sedation/extubation RSS 2 - 3. Both the BIS and AAI agreed well with the RSS
(eta-coefficients of 0.902 and 0.836, respectively, for mean overall RSS
stages). The systems agreed well among each other (overall intra-class
correlations of 0.670 for consistency and 0.676 for absolute agreement). There
was significant discrimination between RSS 2 - 3 and RSS 4 - 6 with BIS and AAI
(BIS mean difference of 24.73, 95% confidence intervals [CI] 21.08 - 28.37; AAI
mean difference of 20.90, 95% CI 14.64 - 27.16). In conclusion, BIS and AAI
correlated well with RSS overall and also at different levels of sedation. OBJECTIVE: A variety of agents and techniques are employed in different
countries, settings, and medical specialities in order to provide analgesia and
sedation in intensive care. Several national guidelines have been published in
recent years regarding sedation and analgesia in a general intensive care
patient population; however, to date no data exist for patients with burn
injuries. The aim of the study was to evaluate analgesia and sedation practice
in the intensive care of burn patients in Europe.
DESIGN: A postal survey was sent to 188 burn centers in Europe. The addresses
were provided by the European Burn Association. The heads of the intensive care
units were asked to fill in a structured questionnaire concerning the use of
analgesia and sedation in their units.
RESULTS: The overall response rate was 27.04%; 63% of European burn centers
reported standard operating procedures for sedation and analgesia. A regular
score-based assessment of sedation, analgesia, and delirium is carried out by
58%, 60%, and 5%, respectively, of the units. Propofol is the sedative most
frequently used for short-term sedation and the weaning phase, whereas
benzodiazepines are the preferred substances for medium- and long-term sedation.
α2-agonists are widely used during weaning. Opioids are the analgesics of choice
for approximately two thirds of the patients. Ketamine is preferred for
analgesia in 12% and for sedation in 13% of all substances used. For painful
procedures (eg, dressing changes), a large variety of different combinations of
analgesics and sedatives are used. Half of the responding intensive care units
use neuromuscular blocking agents and supportive nonpharmacological techniques.
Two thirds of the European burn centers perceive the need for change in their
concepts of analgesia and sedation.
CONCLUSION: A wide variety of drugs are used for analgesia and sedation in
European burn centers. This would appear to be due to lack of guidelines or
scientific evidence. The implementation of regular assessment of sedation,
analgesia, and delirium must be improved. The widespread use of neuromuscular
blocking agents should be restricted or even abandoned. Two thirds of the units
identify a need for change in their concepts. Valid scientific data are needed
to develop guidelines for sedation and analgesia of burn patients. Prior reports suggest that dreaming during anaesthesia is dependent on recovery
time. Dreaming during sedation may impact patient satisfaction. The current
study explores the incidence and content of dreaming during short-term sedation
with sevoflurane or propofol and investigates whether dreaming is affected by
recovery time. A total of 200 women undergoing first trimester abortion
(American Society of Anesthesiologists physical status I) participated in the
study. Patients were randomly assigned to receive either sevoflurane or propofol
for short-term sedation. Patients were interviewed upon emergence with the
modified Brice questionnaire. The results showed the incidence of dreaming was
significantly different between anaesthesia groups with 60% (60/100) of the
sevoflurane group and 33% (33/100) of the propofol group (P=0.000). However,
recovery time did not significantly differ between groups. In the sevoflurane
group, a greater number of dreamers could not recall what they had dreamed about
(P=0.02) and more patients reported dreams that had no sound (P=0.03) or
movement (P=0.001) compared with dreamers in the propofol group. Most
participants reported dreams with positive emotional content and this did not
significantly differ between groups. Anaesthesia administered had no effect on
patient satisfaction. The results suggest that the incidence of dreaming was not
affected by recovery time. Patient satisfaction was not influenced by choice of
sedative and/or by the occurrence of dreaming during sevoflurane or propofol
short-term sedation. BACKGROUND: With drug shortages, newer sedative medications, and updates in
research, management of sedation and delirium in patients receiving mechanical
ventilation continues to evolve.
OBJECTIVE: To compare perceived and actual sedation practices for adults
receiving mechanical ventilation in intensive care units (ICUs).
METHODS: This was a multicenter, 2-part study conducted in adult ICUs in US
hospitals. It included a sedation practice survey completed by ICU pharmacists
and an observational study evaluating actual sedation practices over a 24-hour
period.
RESULTS: Surveys were completed for 85 ICUs; observational data for 496 patients
were collected. Preferred sedatives from the survey data were propofol
(short-term); propofol, midazolam, or lorazepam (intermediate); and lorazepam
(long-term). Propofol was the most commonly used agent overall during the
observational period (primarily for short-term and intermediate-length
sedation); midazolam was the most commonly used for long-term sedation. Fentanyl
was the preferred analgesic, and haloperidol and quetiapine were the preferred
antipsychotics. Sedation treatment algorithms were used in only 50% of observed
ICUs. Use of daily interruption of sedation was perceived to be 66% but was only
observed in 36% of patients. Monitoring for delirium was reported among 25% of
those surveyed but was observed in only 10% of patients. Targeted sedation goals
were most frequently achieved when a treatment algorithm was used or when an
opiate infusion was the single agent used for sedative management.
CONCLUSIONS: These data suggest differences in perceived and actual sedation
practice in the US, as well as underutilization of evidence-based interventions.
Most notable was the limited use of sedation treatment algorithms, daily
interruption of sedation, and monitoring for delirium. Individual sedation and
delirium protocols should be evaluated and updated based on evidence-based
recommendations. Metastasis is a major cause of death of patients with maligt tumors. Matrix
metalloproteinases (MMPs) are important for the migration and invasion of
various types of cancer cell. Propofol is a known anesthetic agent, widely used
for short-term anesthesia and for longer-term sedation. Propofol inhibits the
proliferation of a variety of tumor cells, but there is no available information
regarding propofol-inhibited migration and invasion of tumor cells in vitro. In
this study, we investigated the effects of propofol on the migration and
invasion of human lung carcinoma A549 cells. Wound healing assay and Boyden
chamber assays indicated that propofol inhibited the migration and invasion of
A549 cells in vitro. Gelatin zymographic analysis showed the inhibitory effect
of propofol on the activation of expression MMP-2. Western blot analysis also
indicated that propofol suppressed the protein expiration of growth factor
receptor-bound protein 2 (GRB2), Jun N-terminal kinases 1/2 (p-JNK1/2), p-p38,
MMP-2 and MMP-9 in A549 cells. Results from real-time PCR assay also showed that
propofol inhibited the mRNA gene expression of MMP-2, -7 and -9, and enhanced
that of tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 in A549 cells.
Taken together, these data show that propofol inhibits MMP-2 and -9 mRNA and
protein expressions, resulting in suppression of lung cancer cell invasion and
migration in vitro. |
Which proteins are the different members of the NF-kappaB family of transcription factors? | Nuclear factor kappa B (NFκB) is a dimeric transcription factor comprised of five family members RelA (p65), RelB, c-Rel, NF-kB1/p50 and NF-kB2/p52. | Previous studies showed that the binding of p50/NFkappaB1 to particular kappaB
DNA sites altered its conformation in a way that correlated with transcriptional
activation. Here, we investigated the effects of protein-protein interactions on
the transcriptional activity of p50. We show that the association of p50 with a
mutant Rel-homology domain (RHD) defective for DNA binding led to synergistic
activation of kappaB site-dependent transcription, whereas neither protein alone
had any effect. Partial proteolytic analysis showed that the conformation of p50
in these complexes differed from that in wild-type c-Rel-RHD/p50 complexes, and
correlated with activated transcription. These results suggest that the
Rel-homology domain can act as an allosteric effector to promote transcription
by p50/NFkappaB1 and that the configuration of p50 is important for its
activity. This also suggests that Rel proteins can promote transcription by
other Rel-family members without binding to their DNA recognition site. These
studies emphasize the important role of protein-protein interactions in Rel and
NFkappaB-mediated transcription. NF-kappaB is implicated in lymphocyte development, maturation, proliferation and
survival. This inducible transcription factor is widely expressed by virtually
all cell types. In mammals, the genes rela, relb, crel, nfkappaΒ1, and nfkappaB
encode the five NF-kB protein family members RelA (p65), RelB, c-Rel, p50, and
p52, respectively, which form homo- and heterodimeric DNA-binding complexes
capable of regulating target gene transcription of specific biological responses
differentially. NF-kappaB regulates the expression of a wide variety of genes
that play critical roles in innate and adaptive immune responses, is strongly
linked to the inhibition of apoptosis, and contributes to tumor growth,
metastasis, and chemoresistance. Parasites have targeted several parts of the
NF-kappaB pathway, allowing them to interfere with the transcription of immune
response genes. The biology of different parasites is critical in influencing
the patterns and kinetics of NF-kappaB activity and thereby the development of
subsequent immune responses. |
What are the symptoms of Rotor syndrome? | Rotor syndrome is characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics. | BACKGROUND: Rotor's syndrome is a rare congenital disorder characterized by
functional hyperbilirubinemia. Treatment decision may be challenging in a cancer
patient with Rotor's syndrome, since the majority of the antineoplastic agents
are metabolized in the liver and excreted via the biliary system. We report the
first case of paclitaxel administration in a patient with ovarian cancer and
elevated bilirubin levels due to Rotor's syndrome.
CASE: A 50-year-old woman with Rotor's syndrome had an exploratory laparotomy
and was diagnosed to have stage IIIC epithelial ovarian cancer. The baseline
serum bilirubin value was 15.3 mg/dL. She was started on a 50% dose of 87.5
mg/m(2) paclitaxel by 3-h infusion plus carboplatin AUC-6. The paclitaxel dose
was increased by 25% at consecutive cycles until the standard dose of 175
mg/m(2)/3 h was achieved. Six cycles were administered without any metabolic
derangement. The patient was rendered disease free with this treatment.
CONCLUSION: Paclitaxel appears to be safe to administer to cancer patients with
functional hyperbilirubinemia. Rotor syndrome is a rare, benign familial disorder characterized by chronic
fluctuating, nonhemolytic and predomitly conjugated hyperbilirubinemia with
normal hepatic histology. In contrast to Dubin-Johnson syndrome, there is no
liver pigmentation in Rotor syndrome. A 36-year-old man was admitted due to
asymptomatic persistent jaundice. His siblings had jaundice with direct
hyperbilirubinemia. Physical examination revealed icteric sclerae without
hepatosplenomegaly. Laboratory findings showed increased serum bilirubin with
direct bilirubinemia. Hepatic uptake and storage capacity of indocyanine green
was markedly reduced, while excretion into bile was slightly suppressed.
Markedly decreased hepatic uptake and poor visualization of the gallbladder and
biliary tract were shown in 99mTc-DISIDA scan. Histology of the liver showed
mild steatosis without pigmentation. We report a case with the review of
literature. Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic reuptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic re uptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia.Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. |
Does splicing occur co-transcriptionally? | The consensus view, based on four organisms, is that the majority of splicing events take place co-transcriptionally in most cells and tissues. RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. | Nascent transcripts are the true substrates for many splicing events in
mammalian cells. In this review we discuss transcription, splicing, and
alternative splicing in the context of co-transcriptional processing of
pre-mRNA. The realization that splicing occurs co-transcriptionally requires two
important considerations: First, the cis-acting elements in the splicing
substrate are synthesized at different times in a 5' to 3' direction. This
dynamic view of the substrate implies that in a 100 kb intron the 5' splice site
will be synthesized as much as an hour before the 3' splice site. Second, the
transcription machinery and the splicing machinery, which are both complex and
very large, are working in close proximity to each other. It is therefore likely
that these two macromolecular machines interact, and recent data supporting this
notion is discussed. We propose a model for co-transcriptional pre-mRNA
processing that incorporates the concepts of splice site-tethering and dynamic
exon definition. Also, we present a dynamic view of the alternative splicing of
FGF-R2 and suggest that this view could be generally applicable to many
regulated splicing events. The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol
II) is essential for several co-transcriptional pre-messenger RNA processing
events, including capping, 3'-end processing and splicing. We investigated the
role of the CTD of RNA pol II in the coordination of A to I editing and splicing
of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The
auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly
coupled to splicing, as the modification of the dinucleotide AA to AI creates a
new 3' splice site. Unlike other introns, the CTD is not required for efficient
splicing of intron 4 at either the normal 3' splice site or the alternative site
created by editing. However, the CTD is required for efficient
co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD
in site-selective RNA editing by ADAR2 and in coordination of editing with
alternative splicing. Self-splicing of group I intron from the 26S rRNA of Candida albicans is
essential for maturation of the host RNA. Here, we demonstrated that the
co-transcriptional splicing of the intron in vitro was blocked by antisense
oligonucleotides (AONs) targeting the P3-P7 core of the intron. The
core-targeted AON effectively and specifically inhibited the intron splicing
from its host RNA in living C. albicans. Furthermore, flow cytometry experiments
showed that the growth inhibition was caused by a fungicidal effect. For the
first time, we showed that an AON targeting the ribozyme core folding
specifically inhibits the endogenous ribozyme splicing in living cells and
specifically kills the intron-containing fungal strains, which sheds light on
the development of antifungal drugs in the future. Pseudoknots play critical roles in packing the active structure of various
functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I
intron ribozymes has been recently appreciated, while little is known about the
pseudoknot function in co-transcriptional folding. Here we used the Candida
group I intron as a model to address the question. We show that
co-transcriptional folding of the active self-splicing intron is twice as fast
as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional
folding at a rate constant similar to the folding of the active ribozyme, and
folding of both P3-P7 and P1-P10 pseudoknots are inhibited by antisense
oligonucleotides. We conclude that when RNA folding is coupled with
transcription, formation of pseudoknot structures dominates the productive
folding pathway and serves as a rate-limiting step in producing the
self-splicing competent Candida intron. A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous
small exons surrounded by large introns. Abundant evidence indicates that
splicing to excise introns occurs co-transcriptionally, prior to release of the
nascent transcript from RNAP II. Here, we established an efficient model system
for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs
immobilized on beads generate RNAP II transcripts containing two exons and an
intron. Consistent with previous work, our data indicate that elongating nascent
transcripts are tethered to RNAP II on the immobilized DNA template. We show
that nascent transcripts that reach full length, but are still attached to RNAP
II, are efficiently spliced. When the nascent transcript is cleaved within the
intron using RNase H, both the 5' and 3' cleavage fragments are detected in the
bound fraction, where they undergo splicing. Together, our work establishes a
system for co-transcriptional splicing in vitro, in which the spliceosome
containing the 5' and 3' exons are tethered to RNAP II for splicing. RNA processing events that take place on the transcribed pre-mRNA include
capping, splicing, editing, 3' processing, and polyadenylation. Most of these
processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme
is engaged in transcriptional elongation. How Pol II elongation rates are
influenced by splicing is not well understood. We generated a family of
inducible gene constructs containing increasing numbers of introns and exons,
which were stably integrated in human cells to serve as actively transcribing
gene loci. By monitoring the association of the transcription and splicing
machineries on these genes in vivo, we showed that only U1 snRNP localized to
the intronless gene, consistent with a splicing-independent role for U1 snRNP in
transcription. In contrast, all snRNPs accumulated on intron-containing genes,
and increasing the number of introns increased the amount of spliceosome
components recruited. This indicates that nascent RNA can assemble multiple
spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo,
Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors
showed that polymerase elongation rates were uncoupled from ongoing splicing.
This study shows that transcription elongation kinetics proceed independently of
splicing at the model genes studied here. Surprisingly, retention of
polyadenylated mRNA was detected at the transcription site after transcription
termination. This suggests that the polymerase is released from chromatin prior
to the completion of splicing, and the pre-mRNA is post-transcriptionally
processed while still tethered to chromatin near the gene end. Messenger RNA splicing is an essential and complex process for the removal of
intron sequences. Whereas the composition of the splicing machinery is mostly
known, the kinetics of splicing, the catalytic activity of splicing factors and
the interdependency of transcription, splicing and mRNA 3' end formation are
less well understood. We propose a stochastic model of splicing kinetics that
explains data obtained from high-resolution kinetic analyses of transcription,
splicing and 3' end formation during induction of an intron-containing reporter
gene in budding yeast. Modelling reveals co-transcriptional splicing to be the
most probable and most efficient splicing pathway for the reporter transcripts,
due in part to a positive feedback mechanism for co-transcriptional second step
splicing. Model comparison is used to assess the alternative representations of
reactions. Modelling also indicates the functional coupling of transcription and
splicing, because both the rate of initiation of transcription and the
probability that step one of splicing occurs co-transcriptionally are reduced,
when the second step of splicing is abolished in a mutant reporter. Transcriptome sequencing allows for analysis of mature RNAs at base pair
resolution. Here we show that RNA-seq can also be used for studying nascent RNAs
undergoing transcription. We sequenced total RNA from human brain and liver and
found a large fraction of reads (up to 40%) within introns. Intronic RNAs were
abundant in brain tissue, particularly for genes involved in axonal growth and
synaptic transmission. Moreover, we detected significant differences in intronic
RNA levels between fetal and adult brains. We show that the pattern of intronic
sequence read coverage is explained by nascent transcription in combination with
co-transcriptional splicing. Further analysis of co-transcriptional splicing
indicates a correlation between slowly removed introns and alternative splicing.
Our data show that sequencing of total RNA provides unique insight into the
transcriptional processes in the cell, with particular importance for normal
brain development. While studies of alternative pre-mRNA splicing regulation have typically focused
on RNA-binding proteins and their target sequences within nascent message, it is
becoming increasingly evident that mRNA splicing, RNA polymerase II (pol II)
elongation and chromatin structure are intricately intertwined. The majority of
introns in higher eukaryotes are excised prior to transcript release in a manner
that is dependent on transcription through pol II. As a result of
co-transcriptional splicing, variations in pol II elongation influence
alternative splicing patterns, wherein a slower elongation rate is associated
with increased inclusion of alternative exons within mature mRNA. Physiological
barriers to pol II elongation, such as repressive chromatin structure, can
thereby similarly impact splicing decisions. Surprisingly, pre-mRNA splicing can
reciprocally influence pol II elongation and chromatin structure. Here, we
highlight recent advances in co-transcriptional splicing that reveal an
extensive network of coupling between splicing, transcription and chromatin
remodeling complexes. This article is part of a Special Issue entitled:
Chromatin in time and space. Splicing remains an incompletely understood process. Recent findings suggest
that chromatin structure participates in its regulation. Here, we analyze the
RNA from subcellular fractions obtained through RNA-seq in the cell line K562.
We show that in the human genome, splicing occurs predomitly during
transcription. We introduce the coSI measure, based on RNA-seq reads mapping to
exon junctions and borders, to assess the degree of splicing completion around
internal exons. We show that, as expected, splicing is almost fully completed in
cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that
is being transcribed), for 5.6% of exons, the removal of the surrounding introns
is fully completed, compared with 0.3% of exons for which no intron-removal has
occurred. The remaining exons exist as a mixture of spliced and fewer unspliced
molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while
being transcribed: "co-transcriptional splicing." Consistent with
co-transcriptional spliceosome assembly and splicing, we have found significant
enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with
other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores
decrease along the gene, pointing to a "first transcribed, first spliced" rule,
yet more downstream exons carry other characteristics, favoring rapid,
co-transcriptional intron removal. Exons with low coSI values, that is, in the
process of being spliced, are enriched with chromatin marks, consistent with a
role for chromatin in splicing during transcription. For alternative exons and
long noncoding RNAs, splicing tends to occur later, and the latter might remain
unspliced in some cases. Splicing is the removal of intron sequences from pre-mRNA by the spliceosome.
Researchers working in multiple model organisms - notably yeast, insects and
mammalian cells - have shown that pre-mRNA can be spliced during the process of
transcription (i.e. co-transcriptionally), as well as after transcription
termination (i.e. post-transcriptionally). Co-transcriptional splicing does not
assume that transcription and splicing machineries are mechanistically coupled,
yet it raises this possibility. Early studies were based on a limited number of
genes, which were often chosen because of their experimental accessibility.
Since 2010, eight studies have used global datasets as counting tools, in order
to quantify co-transcriptional intron removal. The consensus view, based on four
organisms, is that the majority of splicing events take place
co-transcriptionally in most cells and tissues. Here, we discuss the nature of
the various global datasets and how bioinformatic analyses were conducted.
Considering the broad differences in experimental approach and analysis, the
level of agreement on the prevalence of co-transcriptional splicing is
remarkable. |
List the existing methods for genetic manipulation of cells. | Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations. Based on results there are developed many methods for genetic manipulation of cells such as Microinjection, electroporation, liposomes and via viral vectors. | Introduction and expression of multiple transgenes is frequently required for
basic and applied studies. However, at present, multigene transformation is very
difficult due to technical limitations of existing methods. Here, we describe a
vector system for efficient multigene assembly and transformation. The system
consists of a transformation-competent artificial chromosome (TAC)-based
acceptor vector together with two donor vectors. By exploiting the CreloxP
recombination system and homing endonucleases, multiple rounds of gene assembly
cycling were carried out with alternate use of the donor vectors, and multiple
genes were sequentially delivered into the TAC vector. With this system, we
created constructs containing as many as 10 foreign DNA fragments. Multiple
genes, including six resistant genes stacked in a construct, were transferred
into rice genome by Agrobacterium-mediated transformation. This system extends
the repertoire of molecular genetic studies and biotechnological endeavors by
enabling simultaneous manipulation of multiple genes. Bailey-Wilson JE(1), Almasy L, de Andrade M, Bailey J, Bickeböller H, Cordell
HJ, Daw EW, Goldin L, Goode EL, Gray-McGuire C, Hening W, Jarvik G, Maher BS,
Mendell N, Paterson AD, Rice J, Satten G, Suarez B, Vieland V, Wilcox M, Zhang
H, Ziegler A, MacCluer JW. The plasma membrane of mammalian cells can be transiently permeablized by
optical means and exogenous materials or genes can be introduced into the
cytoplasm of living cells. Until now, few mechanisms were exploited for the
manipulation: laser is directly and tightly focused on the cells for
optoinjection, laser-induced stress waves, photochemical internalization, and
irradiation of selective cell targeting with light-absorbing particles. During
the past few years, extensive progress and numerous breakthroughs have been made
in this area of research. This review covers four different laser-assisted
transfection techniques and their advantages and disadvantages. Universality
towards various cell lines is possibly the main advantage of laser-assisted
optoporation in comparison with presently existing methods of cell transfection. Existing methods for site-directed plasmid mutagenesis are restrained by the
small spectrum of modifications that can be introduced by mutagenic primers and
the amplicon size limitations of in vitro DNA synthesis. As demonstrated here,
the combined use of Red/ET recombination and unique restriction site elimination
enables extensive manipulation regardless of plasmid size and DNA sequence
elements. First, a selectable marker is PCR-amplified with synthetic primers
attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary
restriction site absent in the substrate plasmid. The resulting cassette is
co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli
cells. Following isolation of recombit plasmids, linear nonselectable DNA
replaces the cassette and introduces the desired mutation(s) in a second Red/ET
recombination step. Upon selective digestion of parental plasmids and
retransformation, a 38% mutation efficiency was achieved using a synthetic
97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19
(2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse
Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp). Genome-wide association analyses on large patient cohorts are generating large
sets of candidate disease genes. This is coupled with the availability of
ever-increasing genomic databases and a rapidly expanding repository of
biomedical literature. Computational approaches to disease-gene association
attempt to harness these data sources to identify the most likely disease gene
candidates for further empirical analysis by translational researchers,
resulting in efficient identification of genes of diagnostic, prognostic and
therapeutic value. Existing computational methods analyze gene structure and
sequence, functional annotation of candidate genes, characteristics of known
disease genes, gene regulatory networks, protein-protein interactions, data from
animal models and disease phenotype. To date, a few studies have successfully
applied computational analysis of clinical phenotype data for specific diseases
and shown genetic associations. In the near future, computational strategies
will be facilitated by improved integration of clinical and computational
research, and by increased availability of clinical phenotype data in a format
accessible to computational approaches. Plant Biotechnology involves manipulation of genetic material to develop better
crops. Keeping in view the challenges being faced by humanity in terms of
shortage of food and other resources, we need to continuously upgrade the
genomic technologies and fine tune the existing methods. For efficient genetic
transformation, Agrobacterium-mediated as well as direct delivery methods have
been used successfully. However, these methods suffer from many disadvantages
especially in terms of transfer of large genes, gene complexes and gene
silencing. To overcome these problems, recently, some efforts have been made to
develop genetic transformation systems based on engineered plant chromosomes
called minichromosomes or plant artificial chromosomes. Two approaches namely,
"top-down" or "bottom-up" have been used for minichromosomes. The former
involves engineering of the existing chromosomes within a cell and the latter de
novo assembling of chromosomes from the basic constituents. While some success
has been achieved using these chromosomes as vectors for genetic transformation
in maize, however, more studies are needed to extend this technology to crop
plants. The present review attempts to trace the genesis of minichromosomes and
discusses their potential of development into plant artificial chromosome
vectors. The use of these vectors in genetic transformation will greatly
ameliorate the food problem and help to achieve the UN Millennium development
goals. Microinjection has a long and distinguished history in Xenopus and has been used
to introduce a surprisingly diverse array of agents into embryos by both intra-
and intercellular means. In addition to nuclei, investigators have variously
injected peptides, antibodies, biologically active chemicals, lineage markers,
mRNA, DNA, morpholinos, and enzymes. While enumerating many of the different
microinjection approaches that can be taken, we will focus upon the mechanical
operations and options available to introduce mRNA, DNA, and morpholinos
intracellularly into early stage embryos for the study of neurogenesis. INTRODUCTION: The overexpression of transferrin (Tf) receptors on cancer cells
renders them a useful target for the delivery of small-molecule drugs and
nucleic acid therapeutics to these cells. This approach could alleviate the
non-target effects of the drugs.
AREAS COVERED: The function of the Tf receptor, the development of Tf-lipid-DNA
complexes (Tf lipoplexes), therapeutic use of lipoplexes and polymer-DNA
complexes (poylplexes), and the therapeutic use of Tf-lipoplexes and
anti-Tf-receptor antibody-lipoplexes are outlined. The literature search for
this review was based primarily on the terms 'lipoplexes,' 'lipopolyplexes'
'transferrin,' 'transferrin receptor,' and 'gene therapy.' However, the review
was not intended to be comprehensive.
EXPERT OPINION: Complexes of Tf with cationic liposomes and nucleic acids, or
liposomes with covalently attached Tf or anti-transferrin receptor antibodies
have been used for the delivery of therapeutic genes, antisense
oligodeoxynucleotides, and short interfering RNA. Although such targeted
nonviral delivery vehicles may benefit from further enhancement of their
efficacy, current achievements at the cell culture and animal model level should
be translated into clinical applications, restricted initially to localized
delivery into accessible tissues to avoid potential systemic side-effects and
non-target delivery. A novel series of carbamate-linked cationic lipids containing hydroxyl headgroup
were synthesized and included in formulations for transfection assays. The
DNA-lipid complexes were characterized for their ability to bind DNA, their
size, ζ-potential and cytotoxicity. Compared with our previously reported
cationic transfection lipid DDCDMA lacking the hydroxyl group and the
commercially available, these cationic liposomes exhibited relatively higher
transfection efficiency. Recombit adeno-associated viral vectors (rAAV) have now been used in several
clinical trials to treat a variety of diseases, and are currently the preferred
choice of many investigators in the field, due to both their low pathogenicity
and immunogenicity compared with other viral vectors, as well as localized
long-term gene expression, despite their limitations of DNA size packaging and
speed of expression. Recently, a number of advances have led to new generations
of rAAV vectors, with improved features. This review addresses the various
strategies employed to such effect, namely exploring distinct serotype tropisms,
the production of mosaic and chimeric capsids, the selection of vectors through
directed evolution, the development of self-complementary vectors, the use of
pharmacological adjuvants and the induction of specific capsid mutations. Such
approaches are expected to help the establishment of rAAV-based clinical gene
therapy in the near future. Numerous viral vectors have been developed for the delivery of transgenes to
specific target cells. For persistent transgene expression, vectors based on
retroviruses are attractive delivery vehicles because of their ability to stably
integrate their DNA into the host cell genome. Initially, vectors based on
simple retroviruses were the vector of choice for such applications. However,
these vectors can only transduce actively dividing cells. Therefore, much
interest has turned to retroviral vectors based on the lentivirus genus because
of their ability to transduce both dividing and non-dividing cells. The best
characterized lentiviral vectors are derived from the human immunodeficiency
virus type 1 (HIV-1). This chapter describes the basic features of the HIV-1
replication cycle and the many improvements reported for the lentiviral vector
systems to increase the safety and efficiency. We also provide practical
information on the production of HIV-1 derived lentiviral vectors, the cell
transduction protocol and a method to determine the transduction titers of a
lentiviral vector. We established a high-efficiency nuclear transformation method for the diatom
Phaeodactylum tricornutum using an electroporation system. Based on a universal
electroporation protocol, the conditions for the introduction of exogenous DNA
including electric field strength and plasmid form were optimized. Following
optimization, the diatom cells could be transformed with exogenous gene easily,
the maximum transformation frequency obtained was 2.8×10(-5) cells. The
cotransformation of P. tricornutum with a non-selective GUS gene together with
the selectable resistance gene has also been achieved using our new method and
found to be very efficient (up to 60%). The electroporation procedure described
in this article offers a number of advantages, including simplicity, general
utility, low-cost and high efficiency. The described method also provides some
clue for developing electroporation transformation system in other eukaryotic
microalgae. This study seeks correlations between the molecular structures of cationic and
neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form
with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic
cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were
co-formulated (3:2) with the neutral lipids
1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All
lipoplex formulations exhibited plasmid DNA binding and a level of protection
from DNase I degradation. Composition-dependent transfection (beta-galactosidase
and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The
most active formulations containing the pyridinium lipids were less cytotoxic
but of comparable activity to a Lipofectamine 2000™ control. Molecular structure
parameters and partition coefficients were calculated for all lipids using
fragment additive methods. The derived shape parameter values correctly
correlated with observed hexagonal lipid phase behavior of lipoplexes as derived
from small-angle X-ray scattering experiments. A transfection index applicable
to hexagonal phase lipoplexes derived from calculated parameters of the lipid
mixture (partition coefficient, shape parameter, lipoplex packing) produced a
direct correlation with transfection efficiency. Here we present a quantitative mechanism-based investigation aimed at comparing
the cell uptake, intracellular trafficking, endosomal escape and final fate of
lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) oparticles
(NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid
formulations were used for comparison. The first formulation is made of the
cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the
zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is
made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol
(DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine
(DOPE). Our findings indicate that lipoplexes are efficiently taken up through
fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs
through a combination of both macropinocytosis and clathrin-dependent pathways.
Inside the cell, both lipoplexes and LPD NPs are actively transported towards
the cell nucleus, as quantitatively addressed by spatio-temporal image
correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape
from endosomes more efficiently than lipoplexes. When cells were treated with
DOTAP-DOPC-containing systems the majority of the DNA was trapped in the
lysosome compartment, suggesting that extensive lysosomal degradation was the
rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side,
escape from endosomes is large for DC-Chol-DOPE-containing systems most likely
due to DOPE and cholesterol-like molecules, which are able to destabilize the
endosomal membrane. The lipid-dependent and structure-dependent enhancement of
transfection activity suggests that DNA is delivered to the nucleus
synergistically: the process requires both the membrane-fusogenic activity of
the ocarrier envelope and the employment of lipid species with intrinsic
endosomal rupture ability. |
Are there clinical trials on stem cells in multiple sclerosis | Yes. Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for multiple sclerosis. Several small pilot clinical trials in subjects with advanced MS have demonstrated that MSC administration is safe and provided an early signal of clinical effectiveness. The current aim of clinicians and scientists interested in the development of MSC-based strategies for the treatment of MS is to have the ultimate demonstration in large clinical trials that MSC can inhibit CNS inflammation and foster tissue repair. | Currently available therapies for multiple sclerosis (MS) delay disease
progression via immunomodulation or immunosuppression. A persisting neurological
deficit is mostly irreversible. Thus, a reparative treatment is urgently
warranted. After positive results in animal models, clinical trials to promote
endogenous remyelination with intravenous immunoglobulins (IVIg) or the growth
factor IGF-1 were performed, unfortunately without clinical improvement. Another
possibility to achieve remyelination is the transplantation of myelinating cells
into the central nervous system. Proof of principle and demonstration of the
functionality were shown in numerous experiments, and a first clinical trial in
patients with MS has started. Although there are still several open questions,
many are specific to MS and can not be answered in an animal model. This first
trial will show if cell transplantation is a feasible concept in MS and whether
the transplanted cells will survive and form new myelin. Schwann cells are
currently the most promising cells to be transplanted, due to the advantages of
an autologous transplantation from the patient's sural nerve biopsy, possibility
to expand the cells in culture, and the possibility that they may escape the
ongoing inflammatory reaction in MS. Other cell types are available, including
stem cells, which are in the centre of a lively discussion. The results of the
ongoing trial must be awaited before other transplant studies are performed to
tackle other yet unresolved problems. At the time, it seems unlikely that cell
transplantation will become clinical practice in the near future. Stem cell transplantation approaches offer for the first time the opportunity to
design therapeutic approaches for multiple sclerosis (MS) with curative intent.
Here we discuss key observations and questions emerging from clinical trials of
hematopoietic stem cell transplantation for MS and from studies of myelin/neural
repair in experimental models of demyelinating disorders. Stem cells offer the potential for regeneration of lost tissue in neurological
disease, including multiple sclerosis (MS). Their development in vitro and their
use in vivo in animal models of degenerative neurological disease and recent
first efforts in human clinical trials were the topics of a recent international
meeting sponsored by the Multiple Sclerosis International Federation and the
National Multiple Sclerosis Society on "Stem Cells & MS: Prospects and
Strategies" Participants reviewed the current state of knowledge about the
potential use of stem and progenitor cells in MS and other degenerative
neurological disorders and outlined a series of urgent fundamental and applied
clinical research priorities that should allow the potential of regeneration of
damaged tissue in MS to be assessed and pursued. BACKGROUND: No treatments are currently available that slow, stop, or reverse
disease progression in established multiple sclerosis (MS). The Mesenchymal Stem
Cells in Multiple Sclerosis (MSCIMS) trial tests the safety and feasibility of
treatment with a candidate cell-based therapy, and will inform the wider
challenge of designing early phase clinical trials to evaluate putative
neuroprotective therapies in progressive MS. Illustrated by the MSCIMS trial
protocol, we describe a novel methodology based on detailed assessment of the
anterior visual pathway as a model of wider disease processes--the "sentinel
lesion approach".
METHODS/DESIGN: MSCIMS is a phase IIA study of autologous mesenchymal stem cells
(MSCs) in secondary progressive MS. A pre-test : post-test design is used with
healthy controls providing normative data for inter-session variability.
Complementary eligibility criteria and outcomes are used to select participants
with disease affecting the anterior visual pathway.
RESULTS: Ten participants with MS and eight healthy controls were recruited
between October 2008 and March 2009. Mesenchymal stem cells were successfully
isolated, expanded and characterised in vitro for all participants in the
treatment arm.
CONCLUSIONS: In addition to determining the safety and feasibility of the
intervention and informing design of future studies to address efficacy, MSCIMS
adopts a novel strategy for testing neuroprotective agents in MS--the sentinel
lesion approach--serving as proof of principle for its future wider
applicability.
TRIAL REGISTRATION: ClinicalTrials.gov (NCT00395200). Autologous bone marrow stromal cells (BMSCs) offer significant practical
advantages for potential clinical applications in multiple sclerosis (MS). Based
on recent experimental data, a number of clinical trials have been designed for
the intravenous (IV) and/or intrathecal (ITH) administration of BMSCs in MS
patients. Delivery of BMSCs in the cerebrospinal fluid via
intracerebroventricular (ICV) transplantation is a useful tool to identify
mechanisms underlying the migration and function of these cells. In the current
study, BMSCs were ICV administered in severe and mild EAE, as well as naive
animals; neural precursor cells (NPCs) served as cellular controls. Our data
indicated that ICV-transplanted BMSCs significantly ameliorated mild though not
severe EAE. Moreover, BMSCs exerted significant anti-inflammatory effect on
spinal cord with concomitant reduced axonopathy only in the mild EAE model.
BMSCs migrated into the brain parenchyma and, depending on their cellular
density, within brain parenchyma formed cellular masses characterized by focal
inflammation, demyelination, axonal loss and increased collagen-fibronectin
deposition. These masses were present in 64% of ICV BMASC-transplanted severe
EAE animals whereas neither BMSCs transplanted in mild EAE cases nor the NPCs
exhibited similar behavior. BMSCs possibly exerted their fibrogenic effect via
both paracrine and autocrine manner, at least partly due to up-regulation of
connective tissue growth factor (CTGF) under the trigger of TGFb1. Our findings
are of substantial relevance for clinical trials in MS, particularly regarding
the possibility that ICV transplanted BMSCs entering the inflamed central
nervous system may exhibit - under conditions - a local pathology of yet unknown
consequences. Mesenchymal stem cells have been demonstrated to ameliorate experimental
autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, prompting
clinical trials in multiple sclerosis which are currently ongoing. An important
question is whether this therapeutic effect generalises to other autoimmune
neurological diseases. We performed two trials of efficacy of MSCs in
experimental autoimmune neuritis (EAN) in Lewis (LEW/Han (M)Hsd) rats, a model
of human autoimmune inflammatory neuropathies. No differences between the groups
were found in clinical, histological or electrophysiological outcome measures.
This was despite the ability of mesenchymal stem cells to inhibit proliferation
of CD4+ T-cells in vitro. Therefore the efficacy of MSCs observed in autoimmune
CNS demyelination models do not necessarily generalise to the treatment of other
forms of neurological autoimmunity. Mesenchymal stem cells (MSC) promote functional recovery in experimental models
of central nervous system (CNS) pathology and are currently being tested in
clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial
effects are attributed to the activation of endogenous CNS protection and repair
processes as well as immune regulation but their mechanisms of action are poorly
understood. Here we investigated the neuroprotective effects of mouse MSC in
rodent MSC-neuron co-cultures and mice using models of glutamate excitotoxicity.
A 24h pre-culture of mouse primary cortical neurons with MSC protected them
against glutamate (NMDA) receptor-induced death and conditioned medium from MSC
(MSC CM) was sufficient for this effect. Protection by MSC CM was associated
with reduced mRNA levels of genes encoding NMDA receptor subunits, and increased
levels for genes associated with non-neuronal and stem cell types, as shown by
RT-PCR and cDNA microarray analyses. Changes in gene expression were not
associated with alterations in cell lineage representation within the cultures.
Further, MSC CM-mediated neuroprotection in rat retinal ganglion cells was
associated with reduced glutamate-induced calcium influx. The adoptive transfer
of EGFP(+)MSC in a mouse kainic acid epilepsy model also provided
neuroprotection against glutamate excitotoxicity in vivo, as shown by reduced
neuron damage and glial cell activation in the hippocampus. These results show
that MSC mediate direct neuroprotection by reducing neuronal sensitivity to
glutamate receptor ligands and altering gene expression, and suggest a link
between the therapeutic effects of MSC and the activation of cell plasticity in
the damaged CNS. The unmet need for therapies capable of repairing the central nervous system
(CNS) damage occurring in many diseases including multiple sclerosis (MS) has
sparked the interest of the neurological community for stem cell-based
therapies. An exhaustive amount of preclinical data has shown that the
intravenous administration of mesenchymal stem cells (MSC), a subset of
progenitor cells isolated from many mesodermal tissues, effectively ameliorates
experimental autoimmune encephalomyelitis (EAE), a model of MS, through the
release of anti-inflammatory and neuroprotective molecules. Based on these
results, several small pilot clinical trials in subjects with advanced MS have
demonstrated that MSC administration is safe and provided an early signal of
clinical effectiveness. The current aim of clinicians and scientists interested
in the development of MSC-based strategies for the treatment of MS is to have
the ultimate demonstration in large clinical trials that MSC can inhibit CNS
inflammation and foster tissue repair as realized clinically, with functional
recovery, or visualized by magnetic resoce imaging (MRI). BACKGROUND: Adult stem cells come from many sources and have the capacity to
differentiate into many cell types, including those of the skin. The most
commonly studied stem cells are those termed mesenchymal stem cells (MSCs),
which are easily isolated from bone marrow and adipose tissue. Mesenchymal stem
cells are known to produce a wide array of cytokines that modulate the
regeneration process. The ease of collection, propagation and use of these MSCs
in therapy of traumatic, ischaemic and immune-mediated skin conditions is
emerging.
APPROACH AND EVIDENCE: In traumatic and ischaemic skin damage, MSCs are used in
tissue-engineered skin and by direct injection into damaged tissue. For
immune-mediated diseases, systemic administration of stem cells can modulate the
immune system. The earliest clinical work has been with autologous stem cell
sources, such as adipose tissue and bone marrow. In immune-mediated diseases,
the MSCs are used to downregulate production of inflammatory cytokines and to
block T-cell activation. Cells are generally given intravenously. Multiple
sclerosis, rheumatoid arthritis and lupus have been successfully treated in
human clinical trials. Mesenchymal stem cells can also stimulate resident local
cells, such as keratinocytes and progenitor cells, to proliferate, migrate and
repair skin injury and disease.
LOOKING AHEAD: The discovery of the MSC in adipose tissue has spawned a global
effort to utilize these cells in therapy of a wide range of diseases of the
skin. Reconstructive surgery, scar blocking and resolution and skin regeneration
have all been shown to be possible in human and animal studies. |
Which SWI/SNF protein complex subunit has been demonstrated to interact with the FANCA gene product? | The Fanconi anemia protein FANCA has been shown to interact with the brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. | Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic
failure, birth defects and cancer. We identified an interaction between the FA
protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the
SWI/SNF complex, which remodels chromatin structure through a DNA-dependent
ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF
complex. We also found a significant increase in the molecular chaperone,
glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from
a FANCA-mutant cell line, which was not seen in either a normal control cell
line or the mutant line complemented by wild-type FANCA. Despite this specific
difference, FANCA did not appear to be absolutely required for in vitro
chromatin remodeling. Finally, we demonstrated co-localization in the nucleus
between transfected FANCA and BRG1. The physiological action of FANCA on the
SWI/SNF complex remains to be clarified, but our work suggests that FANCA may
recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear
functions such as transcription and DNA repair. |
Are piRNAs involved in gene silencing? | Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state. piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire. Transcriptional silencing implies a piRNA-mediated formation of repressive chromatin which diminishes the transcriptional capacity of the target locus. | Genetic studies have shown that Aubergine (Aub), one of the Piwi subfamily of
Argonautes in Drosophila, is essential for germ cell formation and maintaining
fertility. aub mutations lead to the accumulation of retrotransposons in ovaries
and testes, and Stellate transcripts in testes. Aub in ovaries associates with a
variety of Piwi-interacting RNAs (piRNAs) derived from repetitive intergenic
elements including retrotransposons. Here we found that Aub in testes also
associates with various kinds of piRNAs. Although in ovaries Aub-associated
piRNA populations are quite diverse, piRNAs with Aub in testes show a strong
bias. The most abundant piRNAs were those corresponding to antisense transcripts
of Suppressor of Stellate [Su(Ste)] genes known to be involved in Stellate gene
silencing. The second most abundant class was made up of those from chromosome X
and showed strong complementarity to vasa transcripts. Immunopurified Aub-piRNA
complexes from testes displayed activity in cleaving target RNA containing
sequences complementary to Stellate and vasa transcripts. These results provide
the first biochemical insights into gene silencing mechanisms mediated by Aub
and piRNAs in fly testes. Germline silencing of transposable elements is essential for the maintece of
genome integrity. Recent results indicate that this repression is largely
achieved through a RNA silencing pathway that involves Piwi-interacting RNAs
(piRNAs). However the repressive mechanisms are not well understood. To address
this question, we used the possibility to disrupt the repression of the
Drosophila I element retrotransposon by hybrid dysgenesis. We show here that the
repression of the functional I elements that are located in euchromatin requires
proteins of the piRNA pathway, and that the amount of ovarian I element piRNAs
correlates with the strength of the repression in the female germline. Antisense
RNAs, which are likely used to produce antisense piRNAs, are transcribed by
heterochromatic defective I elements, but efficient production of these
antisense small RNAs requires the presence in the genome of euchromatic
functional I elements. Finally, we demonstrate that the piRNA-induced silencing
of the functional I elements is at least partially posttranscriptional. In a
repressive background, these elements are still transcribed, but some of their
sense transcripts are kept in nurse cell nuclear foci together with those of the
Doc retrotransposon. In the absence of I element piRNAs, either in dysgenic
females or in mutants of the piRNA silencing pathway, sense I element
transcripts are transported toward the oocyte where retrotransposition occurs.
Our results indicate that piRNAs are involved in a posttranscriptional
gene-silencing mechanism resulting in RNA nuclear accumulation. PIWI interacting RNAs (piRNAs) are a subset of small RNAs predomitly
expressed in the germlines of various species. In Drosophila, the main sources
of piRNAs are transcripts of mobile DNA elements, including active transposons
and their wreckage, found on the genome. After maturing from the primary
transcripts, piRNAs are specifically loaded onto germline-specific Argonaute
proteins - Argonaute3, Aubergine and Piwi - collectively referred to as PIWI
proteins. Loss of function of PIWI proteins and/or the piRNA loci on the genome
lead to derepression of transposons and causes severe defects in gametogenesis
and fertility. The necessity for both PIWI proteins and piRNAs in protecting the
genome of the gametes from mischievous mobile genomic elements is thus obvious.
There have been extensive biochemical and genetic studies on PIWI proteins and
piRNAs. These have shed light not only on the molecular mechanisms of gene
silencing mediated by piRNAs and PIWI proteins, but also on their intriguing
relationship with cellular genes that have been shown to be important for
gametogenesis and fertility. In the Drosophila germline, retrotransposons are silenced by the
PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and
TAHRE, which are involved in telomere maintece in Drosophila, are also the
targets of piRNA-mediated silencing. We have demonstrated that expression of
reporter genes driven by the HeT-A promoter is under the control of the piRNA
silencing pathway independent of the transgene location. In order to test
directly whether piRNAs affect the transcriptional state of retrotransposons we
performed a nuclear run-on (NRO) assay and revealed increased density of the
active RNA polymerase complexes at the sequences of endogenous HeT-A and TART
telomeric retroelements as well as HeT-A-containing constructs in the ovaries of
spn-E mutants and in flies with piwi knockdown. This strongly correlates with
enrichment of two histone H3 modifications (dimethylation of lysine 79 and
dimethylation of lysine 4), which mark transcriptionally active chromatin, on
the same sequences in the piRNA pathway mutants. spn-E mutation and piwi
knockdown results in transcriptional activation of some other non-telomeric
retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore
piRNA-mediated transcriptional mode of silencing is involved in the control of
retrotransposon expression in the Drosophila germline. Proteins of the PIWI subfamily Aub and AGO3 associated with the
germline-specific perinuclear granules (nuage) are involved in the silencing of
retrotransposons and other selfish repetitive elements in the Drosophila genome.
PIWI proteins and their 25- to 30-nt PIWI-interacting RNA (piRNAs) are
considered as key participants of the piRNA pathway. Using immunostaining, we
found a large, nuage-associated organelle in the testes, the piNG-body (piRNA
nuage giant body), which was significantly more massive than an ordinary nuage
granule. This body contains known ovarian nuage proteins, including Vasa, Aub,
AGO3, Tud, Spn-E, Bel, Squ, and Cuff, as well as AGO1, the key component of the
microRNA pathway. piNG-bodies emerge at the primary spermatocyte stage of
spermatogenesis during the period of active transcription. Aub, Vasa, and Tud
are located at the periphery of the piNG-body, whereas AGO3 is found in its
core. Mutational analysis revealed that Vasa, Aub, and AGO3 were crucial for
both the maintece of the piNG-body structure and the silencing of selfish
Stellate repeats. The piNG-body destruction caused by csul mutations that
abolish specific posttranslational symmetrical arginine methylation of PIWI
proteins is accompanied by strong derepression of Stellate genes known to be
silenced via the piRNA pathway. PIWI-interacting RNAs (piRNAs) are endogenous small noncoding RNAs that act as
guardians of the genome, protecting it from invasive transposable elements in
the germline. Animals lacking piRNA functions show defects in gametogenesis and
exhibit sterility. Their descendants are also predisposed to inheriting
mutations. Thus, the piRNA pathway has evolved to repress transposons
post-transcriptionally and/or transcriptionally. A growing number of studies on
piRNAs have investigated piRNA-mediated gene silencing, including piRNA
biogenesis. However, piRNAs remain the most enigmatic among all of the
silencing-inducing small RNAs because of their complexity and uniqueness.
Although piRNAs have been previously suggested to be germline-specific, recent
studies have shown that piRNAs also play crucial roles in nongonadal cells.
Furthermore, piRNAs have also recently been shown to have roles in
multigenerational epigenetic phenomena in worms. The purpose of this review is
to highlight new piRNA factors and novel insights in the piRNA world. In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs
(piRNAs) have an important role in silencing retrotransposons, which are vicious
genomic parasites, through transcriptional and post-transcriptional mechanisms.
To examine whether the piRNA pathway can be used to silence genes of interest in
germ cells, we have generated knock-in mice in which a foreign DNA fragment was
inserted into a region generating pachytene piRNAs. The knock-in sequence was
transcribed, and the resulting RNA was processed to yield piRNAs in postnatal
testes. When reporter genes possessing a sequence complementary to portions of
the knock-in sequence were introduced, they were greatly repressed after the
time of pachytene piRNA generation. This repression mainly occurred at the
post-transcriptional level, as degradation of the reporter RNAs was accelerated.
Our results show that the piRNA pathway can be used as a tool for
sequence-specific gene silencing in germ cells and support the idea that the
piRNA generating regions serve as traps for retrotransposons, enabling the host
cell to generate piRNAs against active retrotransposons. In the metazoan germline, piwi proteins and associated piwi-interacting RNAs
(piRNAs) provide a defense system against the expression of transposable
elements. In the cytoplasm, piRNA sequences guide piwi complexes to destroy
complementary transposon transcripts by endonucleolytic cleavage. However, some
piwi family members are nuclear, raising the possibility of alternative pathways
for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi
is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on
polytene chromosomes. Knockdown of Piwi in the germline increases expression of
transposable elements that are targeted by piRNAs, whereas protein-coding genes
remain largely unaffected. Derepression of transposons upon Piwi depletion
correlates with increased occupancy of Pol II on their promoters. Expression of
piRNAs that target a reporter construct results in a decrease in Pol II
occupancy and an increase in repressive H3K9me3 marks and heterochromatin
protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies
targets complementary to the associated piRNA and induces transcriptional
repression by establishing a repressive chromatin state when correct targets are
found. Piwi-interacting RNAs (piRNAs) were reported in 2006 as a novel class of small
non-coding RNAs associated with Piwi proteins of the Argonaute/Piwi family.
Recent studies have revealed not only the biogenesis of piRNAs and their roles
in transposon silencing, but also the function of the Piwi-piRNA pathway in
epigenetic and post-transcriptional regulation of gene expression. In addition,
the function of this pathway in somatic cells has also been more systematically
characterized. The new findings reveal the Piwi-piRNA pathway as a more general
mechanism of gene regulation. The Piwi-interacting RNA (piRNA) pathway defends animal genomes against the
harmful consequences of transposable element (TE) infection by imposing
small-RNA-mediated silencing. Because silencing is targeted by TE-derived
piRNAs, piRNA production is posited to be central to the evolution of genome
defense. We harnessed genomic data sets from Drosophila melanogaster, including
genome-wide measures of piRNA, mRNA, and genomic abundance, along with estimates
of age structure and risk of ectopic recombination, to address fundamental
questions about the functional and evolutionary relationships between TE
families and their regulatory piRNAs. We demonstrate that mRNA transcript
abundance, robustness of "ping-pong" amplification, and representation in piRNA
clusters together explain the majority of variation in piRNA abundance between
TE families, providing the first robust statistical support for the prevailing
model of piRNA biogenesis. Intriguingly, we also discover that the most
transpositionally active TE families, with the greatest capacity to induce
harmful mutations or disrupt gametogenesis, are not necessarily the most
abundant among piRNAs. Rather, the level of piRNA targeting is largely
independent of recent transposition rate for active TE families, but is rapidly
lost for inactive TEs. These observations are consistent with population genetic
theory that suggests a limited selective advantage for host repression of
transposition. Additionally, we find no evidence that piRNA targeting responds
to selection against a second major cost of TE infection: ectopic recombination
between TE insertions. Our observations confirm the pivotal role of
piRNA-mediated silencing in defending the genome against selfish transposition,
yet also suggest limits to the optimization of host genome defense. In the Drosophila germline, retrotransposons are silenced by the
PIWI-interacting RNA (piRNA) pathway. piRNA pathway mutations lead to
overexpression and mobilization of retrotransposons in the germline. In
different organisms, small RNAs were shown to be implicated in the
posttranscriptional degradation of mRNA and/or transcriptional repression of the
homologous locus. In Drosophila, the mechanism of piRNA-mediated silencing is
still far from being understood. Transcriptional silencing implies a
piRNA-mediated formation of repressive chromatin which diminishes the
transcriptional capacity of the target locus. Nuclear Run-On (NRO) assay allows
a direct estimation of the density of transcribing polymerases at specific
genomic regions. Here we describe the NRO protocol on Drosophila ovarian tissues
which can be useful for investigation of the transcriptional silencing in the
female germline. During Drosophila oogenesis, transposable element (TE) repression involves the
Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next
generation. We developed a transgenic model to study repression of the Idefix
retrotransposon in the germline. Using a candidate gene KD-approach, we
identified differences in the spatio-temporal requirements of the piRNA pathway
components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are
necessary in very early stages of oogenesis within the germarium and appear to
be less important for efficient TE silencing thereafter. Others (Piwi, Ago3,
Mael) are required at all stages of oogenesis. Moreover, during early oogenesis,
in the dividing cysts within the germarium, Idefix anti-sense transgenes escape
host control, and this is associated with very low piwi expression. Silencing of
P-element-based transgenes is also strongly weakened in these cysts. This
region, termed the 'Piwiless pocket' or Pilp, may ensure that new TE insertions
occur and are transmitted to the next generation, thereby contributing to genome
dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells
in which TE mobilization is tightly repressed ensuring the continued production
of viable germline cysts. Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose
a constant threat to genomic integrity. This threat is particularly critical in
germ cells, as changes in the genome that are induced by TEs will be transmitted
to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize
and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon
repression correlates with establishment of CpG DNA methylation on their
sequences, yet the mechanism and the spectrum of genomic targets of piRNA
silencing are unknown. Here we show that in addition to DNA methylation, the
piRNA pathway is required to maintain a high level of the repressive H3K9me3
histone modification on long interspersed nuclear elements (LINEs) in germ
cells. piRNA-dependent chromatin repression targets exclusively full-length
elements of actively transposing LINE families, demonstrating the remarkable
ability of the piRNA pathway to recognize active elements among the large number
of genomic transposon fragments. PIWI-interacting small non-coding RNAs (piRNAs) are genetic and epigenetic
regulatory factors in germline cells, where they maintain genome stability, are
involved in RNA silencing and regulate gene expression. We found that the piRNA
biogenesis and effector pathway are present in human breast cancer (BC) cells
and, analyzing smallRNA-Seq data generated from BC cell lines and tumor
biopsies, we identified >100 BC piRNAs, including some very abundant and/or
differentially expressed in mammary epithelial compared to BC cells, where this
was influenced by estrogen or estrogen receptor β, and in cancer respect to
normal breast tissues. A search for mRNAs targeted by the BC piRNome revealed
that eight piRNAs showing a specific expression pattern in breast tumors target
key cancer cell pathways. Evidence of an active piRNA pathway in BC suggests
that these small non-coding RNAs do exert transcriptional and
post-transcriptional gene regulatory actions also in cancer cells. Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress
transposable elements (TEs), it is unclear how the highly diverse piRNA
populations direct Piwi proteins to silence TE targets without silencing the
entire transcriptome. To determine the capacity of piRNA-mediated silencing, we
introduced reporter genes into Drosophila OSS cells, which express microRNAs
(miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in
gene regulation. Reporter constructs containing several target sites that were
robustly silenced by miRNAs were not silenced to the same degrees by piRNAs.
However, another set of reporters we designed to enable a large number of both
TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA
complex in OSS cells. These reporters show that a bulk of piRNAs are required to
pair to the reporter's transcripts and not the reporter's DNA sequence to engage
PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated
targets in OSS cells, we assessed candidate gene elements with our reporter
platform. These results suggest TE sequences are the most direct of PIWI
regulatory targets while coding genes are less directly affected by PIWI
targeting. Finally, our study suggests that the PIWI transcriptional silencing
mechanism triggers robust chromatin changes on targets with sufficient piRNA
binding, and preferentially regulates TE transcripts because protein-coding
transcripts lack a threshold of targeting by piRNA populations. This reporter
platform will facilitate future dissections of the PIWI-targeting mechanism. Here we investigated dynamic properties of the piNG-body, large perinuclear
granule that was discovered previously in spermatocytes of Drosophila. The
piNG-body contains ribonucleoprotein complexes involved in piRNA-silencing of
genome repeats including transposons in premeiotic spermatocytes with aid of
short piRNAs. Confocal microscopy of fixed and native preparations demonstrates
that the piNG-body is mobile structure which does not occupy a stationary
position near nuclear surface relative to chromosomal territories. FRAP-analysis
reveals a high exchange rate of RNA helicase Vasa in the piNG-body and small
perinuclear granules with the cytozol Vasa pool. Disruption of microtubule
assembly of cytoskeleton does not affect to stability of the piNG-body and small
granules. We suppose that the combination of piNG-body mobility and permanent
molecular exchange of Vasa protein provides an efficient "scanning" of total
volume of the cytoplasm of primary spermatocytes and timely recognition and
destruction of unwanted transcripts of the repetitive elements of genome. piRNAs silence foreign genes, such as transposons, to preserve genome integrity,
but they also target endogenous mRNAs by mechanisms that are poorly understood.
Caenorhabditis elegans piRNAs interact with both transposon and nontransposon
mRNAs to initiate sustained silencing via the RNAi pathway. To assess the
dysregulation of gene silencing caused by lack of piRNAs, we restored RNA
silencing in RNAi-defective animals in the presence or absence of piRNAs. In the
absence of piRNAs and a cellular memory of piRNA activity, essential and
conserved genes are misrouted into the RNAi pathway to produce siRNAs that bind
the nuclear Argonaute HRDE-1, resulting in dramatic defects in germ cell
proliferation and function such that the animals are sterile. Inactivation of
RNAi suppresses sterility, indicating that aberrant siRNAs produced in the
absence of piRNAs target essential genes for silencing. Thus, by reanimating
RNAi, we uncovered a role for piRNAs in protecting essential genes from RNA
silencing. PIWI clade Argonaute proteins silence transposon expression in animal gonads.
Their target specificity is defined by bound ∼23- to 30-nucleotide (nt)
PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor
transcripts via two distinct pathways. Primary piRNAs are defined by the
endonuclease Zucchini, while biogenesis of secondary piRNAs depends on
piRNA-guided transcript cleavage and results in piRNA amplification. Here, we
analyze the interdependencies between these piRNA biogenesis pathways in
developing Drosophila ovaries. We show that secondary piRNA-guided target
slicing is the predomit mechanism that specifies transcripts—including those
from piRNA clusters—as primary piRNA precursors and defines the spectrum of
Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the
cytoplasm therefore enforces nuclear transcriptional target silencing, which
ensures the tight suppression of transposons during oogenesis. As target slicing
also defines the nuclear piRNA pool during mouse spermatogenesis, our findings
uncover an unexpected conceptual similarity between the mouse and fly piRNA
pathways. |
List representatives of the major fungal hypoxanthine-adenine-guanine transporter families. | AzgA and Fcy21p are prototypes of the two major fungal hypoxanthine-adenine-guanine transporter families. | The azgA gene of Aspergillus nidulans encodes a hypoxanthine-adenine-guanine
transporter. It has been cloned by a novel transposon methodology. The null
phenotype of azgA was defined by a number of mutations, including a large
deletion. In mycelia, the azgA gene is, like other genes of purine catabolism,
induced by uric acid and repressed by ammonium. Its transcription depends on the
pathway-specific UaY zinc binuclear cluster protein and the broad domain AreA
GATA factor. AzgA is not closely related to any other characterized membrane
protein, but many close homologues of unknown function are present in fungi,
plants, and prokaryotes but not metazoa. Two of three data bases and the
phylogeny presented in this article places proteins of this family in a cluster
clearly separated (but perhaps phylogenetically related) from the NAT family
that includes other eukaryotic and prokaryotic nucleobase transporters. Thus
AzgA is the first characterized member of this family or subfamily of membrane
proteins. In fungi, uptake of salvageable purines is carried out by members of two
evolutionarily distinct protein families, the Purine-Related Transporters
(PRT/NCS1) and the AzgA-like Transporters. We carried out a comparative kinetic
analysis of two prototypes of these transporter families. The first was Fcy21p,
a herein characterized protein of Candida albicans, and the second was AzgA, a
transporter of Aspergillus nidulans. Our results showed that: (i) AzgA and
Fcy21p are equally efficient high-affinity, high-capacity, purine transporters,
(ii) Fcy21p, but not AzgA, is an efficient cytosine and 5-fluorocytosine
transporter, interacting with =O2 and C4-NH2 of the pyrimidine ring, (iii) the
major interactions of AzgA and Fcy21p with the purine ring are similar, but not
identical, involving in all cases positions 6 and 7, and for some substrates,
positions 1 and 9 as well, and (iv) in AzgA, bulky groups at position N3 have a
detrimental steric effect on substrate binding, while similar substitutions at
C2 or N9 are fully or partially tolerated. In contrast, in Fcy21p, C2 and N9
bulky substitutions abolish substrate binding, while similar substitutions in N3
are fully tolerated. These results suggest that all fungal purine transporters
might have evolved from a single ancestral protein, and show that fungal
transporters use different substrate interactions compared to the analogous
protozoan or mammalian proteins. Finally, results are also discussed in respect
of the possibility of using fungal purine transporters as specific gateways for
the development of targeted antifungal pharmacological therapies. The AzgA purine/H(+) symporter of Aspergillus nidulans is the founding member of
a functionally and phylogenetically distinct transporter family present in
fungi, bacteria and plants. Here a valid AzgA topological model is built based
on the crystal structure of the Escherichia coli uracil transporter UraA, a
member of the nucleobase-ascorbate transporter (NAT/NCS2) family. The model
consists of 14 transmembrane, mostly α-helical, segments (TMSs) and cytoplasmic
N- and C-tails. A distinct compact core of 8 TMSs, made of two intertwined
inverted repeats (TMSs 1-4 and 8-11), is topologically distinct from a flexible
domain (TMSs 5-7 and 12-14). A putative substrate binding cavity is visible
between the core and the gate domains. Substrate docking, molecular dynamics and
mutational analysis identified several residues critical for purine binding
and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339
(TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates,
while Asp342 (TMS8) might be involved in subsequent substrate translocation,
through H(+) binding and symport. Thus, AzgA and other NAT transporters use
topologically similar TMSs and amino acid residues for substrate binding and
transport, which in turn implies that AzgA-like proteins constitute a distant
subgroup of the ubiquitous NAT family. |
Which proteins have been identified as RET ligands? | RET is activated by members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands, which include GDNF, neurturin, artemin, and persephin. | Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two
structurally related, potent survival factors for sympathetic, sensory and
central nervous system neurons. GDNF mediates its actions through a
multicomponent receptor system composed of a ligand-binding
glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) and
the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by
which the NTN signal is transmitted is not well understood. Here we describe the
identification and tissue distribution of a GPI-linked protein (designated
NTNR-alpha) that is structurally related to GDNFR-alpha. We further demonstrate
that NTNR-alpha binds NTN (K[d] approximately 10 pM) but not GDNF with high
affinity; that GDNFR-alpha binds to GDNF but not NTN with high affinity; and
that cellular responses to NTN require the presence of NTNR-alpha. Finally, we
show that NTN, in the presence of NTNR-alpha, induces tyrosine-phosphorylation
of Ret, and that NTN, NTNR-alpha and Ret form a physical complex on the cell
surface. These findings identify Ret and NTNR-alpha as signalling and
ligand-binding components, respectively, of a receptor for NTN and define a
novel family of receptors for neurotrophic and differentiation factors composed
of a shared transmembrane protein tyrosine kinase and a ligand-specific
GPI-linked protein. BACKGROUND/PURPOSE: In 1996, the glial cell line-derived neurotrophic factor
(GDNF) was identified as one of the ligands of the RET transmembrane receptor.
In the same year, GDNF mutations were found in association with RET
protooncogene mutations in Hirschsprung patients. Mutations in GDNF per se are
thought neither necessary nor sufficient to cause Hirschsprung's disease (HD).
To date, our study group has identified GDNF mutations only in 2 of 98 cases of
intestinal dysganglionosis. The aim of our study was to investigate a possible
expression deficit of GDNF in the enteric nervous system of Hirschsprung
patients not mutated for the GDNF gene.
METHODS: We used rabbit polyclonal antibodies raised against a peptide
corresponding to amino acids 186-205 mapping within the carboxy-terminal domain
of human GDNF. GDNF expression was studied immunohistochemically in surgical
specimens from 30 HD cases (27 classic forms and 3 ultralong forms) and from 10
age-matched controls. Serial sections from the same full-thickness specimens
were investigated with the following histochemical and immunohistochemical
techniques: acetylcholinesterase, lactate dehydrogenase, succinic dehydrogenase,
alpha-naphthyl-esterase, glial fibrillary acid protein, S-100 protein, and
neuron-specific enolase.
RESULTS: A high level of GDNF expression was found in normal intestine and in
Hirschsprung ganglionic segment. Satellite elements of myenteric ganglia
presented a strong immunoreactivity to GDNF. Conversely, the aganglionic segment
showed cholinergic hyperinnervation and hypertrophic trunks of nerve fibers in
the muscular interstitium with complete absence of GDNF expression. The small
ganglia of the hypoganglionic segment showed a reduced GDNF immunoreactivity.
CONCLUSIONS: GDNF, a distantly related member of the transforming growth
factor-beta superfamily, is a potent neurotrophic and survival factor for
neurons and enteric ganglion cells. Mutations of the GDNF gene or GDNF
expression deficit interrupt the faithful GDNF signaling via Ret, contributing
to HD pathogenesis. Hirschsprung disease (HSCR) is a frequent neurocristopathy characterized by the
absence of submucosal and myenteric plexuses in a variable length of the
gastrointestinal tract. Pedigrees and segregation analyses suggested the
involvement of one or several domit genes with low penetrance in HSCR.
Considering that RET and glial cell line-derived neurotrophic factor (GDNF)
mutations have been reported in the disease, we regarded the other RET ligand,
neurturin (NTN), as an attractive candidate gene, especially as it shares large
homologies with GDNF. Here, we report on the finding of a heterozygous missense
NTN mutation in a large non-consanguineous family including four children
affected with a severe aganglionosis phenotype extending up to the small
intestine. Interestingly, it appears that the NTN mutation reported here is not
sufficient to cause HSCR, and this multiplex family also segregates a RET
mutation. This cascade of independent and additive genetic events fits well with
the multigenic pattern of inheritance expected in HSCR, and further support the
role of RET ligands in development of the enteric nervous system. The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which
plays a role in neural crest and kidney development. Ret ligands have been
recently identified as the neuron survival factor GDNF (Glial-Derived
Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated
RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In
addition, distinct germ-line mutations of RET gene have been associated with the
inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC
(Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon
or Hirschsprung's disease, thus enlightening a significant role of this receptor
gene in diverse human pathologic conditions. In this study, by performing
classical inhibition experiments using synthetic phosphopeptides and by
site-directed mutagenesis of the putative docking site, we have determined that
for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform
(corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the
interaction of Grb2 with the two short and long Ret isoforms expressed
separately is of similar strength, thus suggesting that Ret short isoform
interaction with Grb2 could be mediated not only by Shc but also by a molecule
that binds preferentially to this isoform. This possibility is supported by the
evidence that the mutant Ret/ptc2Y620F long isoform displays a weak
coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site
for Grb2 but owing all the others phosphotyrosines, surprisingly displays a
reduced transforming activity compared to that of the two WTs oncogenes. We thus
conclude that in intact cells both Ret isoforms bind to Grb2, although with
different modalities. In addition, the present results are in agreement with the
possibility that different signal transduction pathways are associated with the
two isoforms of Ret. Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Three susceptibility
genes have been recently identified in HSCR, namely the RET proto-oncogene, the
endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene
mutations were found in significant proportions of familial (50%) and sporadic
(15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the
rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous
EDNRB and EDN3 missense mutations have been reported in isolated HSCR patients.
Some of these results were obtained after the identification of mouse genes
whose natural or site-directed mutations resulted in megacolon and coat color
spotting. There is also conclusive evidence for the involvement of other
independent loci in HSCR. In particular, the recent identification of
neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide
additional candidate genes for HSCR. The dissection of the genetic etiology of
HSCR disease may then provide a unique opportunity to distinguish between a
polygenic and a genetically heterogeneous disease, thereby helping to understand
other complex disorders and congenital malformations hitherto considered as
multifactorial in origin. Finally, the study of the molecular bases of HSCR is
also a step towards the understanding of developmental genetics of the enteric
nervous system giving support to the role of the tyrosine kinase and
endothelin-signaling pathways in the development of neural crest-derived enteric
neurons in human. The ret proto-oncogene encodes a receptor tyrosine kinase whose ligands belong
to the glial cell line-derived neurotrophic factor (GDNF) protein family. Its
germline mutations are responsible for the development of multiple endocrine
neoplasia (MEN) types 2A and 2B and Hirschsprung's disease (HSCR). MEN2A and
MEN2B mutations result in the constitutive activation of Ret by different
molecular mechanisms. MEN2A mutations involve cysteine residues present in the
Ret extracellular domain and induce disulfide-linked Ret dimerization on the
cell surface. MEN2B mutations were identified in methionine 918 in the tyrosine
kinase domain and activate Ret without dimerization, probably due to a
conformational change of its catalytic core region. In contrast to MEN2
mutations, HSCR mutations represent loss of function mutations. We found that
most of HSCR mutations detected in the extracellular domain impair the Ret cell
surface expression. More interestingly, ret mutations in cysteines 618 and 620
were reported in several families who developed both MEN2A and HSCR. It was
suggested that these mutations might have two biological effects on Ret
function, leading to the development of different clinical phenotypes in the
same patients. The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs)
(GDNF, neurturin, artemin, and persephin) are critical regulators of
neurodevelopment and support the survival of midbrain dopaminergic and spinal
motor neurons in vitro and in animal disease models making them attractive
therapeutic candidates for treatment of neurodegenerative diseases. The GFLs
signal through a multicomponent receptor complex comprised of a high affinity
binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4))
and the receptor tyrosine kinase RET. To begin characterization of GFL receptor
specificity at the molecular level, we performed comprehensive
homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs.
Replacing short segments of GDNF with the homologous segments from persephin
(PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified
sites along the second finger of GDNF critical for activating the GFRalpha1.RET
and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions
from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient
for activating GFRalpha1. RET, but additional determits are required for
interaction with the other GFRalphas. This difference in the molecular basis of
GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific
agonists and provides a foundation for understanding of GFL-GFRalpha.RET
signaling at the molecular level. Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal
obstruction due to an absence of intramural ganglia along variable lengths of
the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF
gene, and encoding one of the RET ligands, either alone or in combination with
RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3,
EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however,
are more or less restricted to HSCR associated with specific phenotypes. We have
developed a novel comprehensive mutation detection system to analyse all but
three amplicons of the RET and GDNF genes, based on denaturing gradient gel
electrophoresis. We make use of two urea-formamide gradients on top of each
other, allowing mutation detection over a broad range of melting temperatures.
For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and
constant denaturing gel electrophoresis (CDGE). These two dual gel systems
substantially facilitate mutation scanning of RET and GDNF, and may also serve
as a model to develop mutation detection systems for other disease genes. In a
screening of 95 HSCR patients, RET mutations were found in nine out of 17
familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic
cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a
sporadic case. OBJECTIVE: Mutations in the RET proto-oncogene are found in about one third of
sporadic medullary thyroid carcinomas (MTCs), mostly affecting codon 918. Glial
cell line derived neurotropic factor (GDNF) and its membrane-bound GDNF family
receptor alpha (GFRalpha-1), as well as neurturin (NTN) and its membrane-bound
receptor GFRalpha-2 form a complex with the RET product, a receptor tyrosine
kinase, resulting in downstream signaling to the nucleus.
DESIGN: To elucidate the role of these RET ligands in MTC tumorigenesis, their
expression was determined in 15 MTC samples, one papillary thyroid carcinoma
(PTC) and three normal thyroid tissue specimens.
METHODS: The mRNA expression of RET, GDNF, GFRalpha-1, NTN and GFRalpha-2 was
investigated by mRNA in situ hybridization, and confirmed by reverse
transcription-PCR analysis.
RESULTS: None of the five genes was expressed in the normal thyroids or in the
PTC. All MTCs showed expression of RET, 13 expressed GDNF, 12 expressed
GFRalpha-1 and 9 expressed NTN and GFRalpha-2. In 7 of the tumors RET, GDNF and
GFRalpha-1 were expressed at high levels, and in five of these seven tumors NTN
and GFRalpha-2 genes were also expressed at high levels. The high level of
expression was preferentially seen in tumor cells adjacent to stroma and
connective tissue. All MTCs without expression of the RET ligands harbored the
RET codon 918 mutation.
CONCLUSIONS: The results suggest that this signaling pathway is important for
MTC development, and that it may be activated by expression of the RET ligand
complexes by the tumor cells themselves. Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Three susceptibility
genes have been recently identified in HSCR, namely the RET proto-oncogene, the
endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene
mutations were found in significant proportions of familial (50%) and sporadic
(15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the
rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous
EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients.
Some of these results were obtained after the identification of mouse genes
whose natural or site-directed mutations resulted in megacolon and coat color
spotting. There is also conclusive evidence for the involvement of other
independent loci in HSCR. In particular, the recent identification of
neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide
additional candidate genes for HSCR. The dissection of the genetic etiology of
HSCR disease may then provide a unique opportunity to distinguish between a
polygenic and a genetically heterogeneous disease, thereby helping to understand
other complex disorders and congenital malformations hitherto considered as
multifactorial in origin. Finally, the study of the molecular bases of HSCR is
also a step towards the understanding of developmental genetics of the enteric
nervous system giving support to the role of the tyrosine kinase and
endothelin-signaling pathways in the development of neural crest-derived enteric
neurons in human. The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha
(GFRalpha) are cell surface bound glycoproteins that mediate interactions of the
GDNF ligand family with the RET receptor. These interactions are crucial to the
development of the kidney and some peripheral nerve lineages. In humans,
mutations of RET or RET ligands are associated with the congenital abnormality
Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are
absent. As the GFRalpha family are required for normal activation of the RET
receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is
required for the development of the myenteric nerve plexus, is an excellent
candidate gene for HSCR. In this study, we cloned the human GFRA2 locus,
characterized the gene structure, and compared it with other GFRA family
members. We further investigated the GFRA2 gene for mutations in a panel of HSCR
patients. GFRA2 has nine coding exons that are similar in size and organization
to those of other GFRA family genes. We identified six sequence variants of
GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2
protein. Two further changes that resulted in amino acid substitutions were
found in exon 9 and were predicted to lie in the amino acid sequence encoding
the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no
difference in frequency of any of the sequence variants between control and HSCR
populations. Our data indicate that members of the GFRA gene family are closely
related in intron/exon structure and in sequence. We have not detected any
correlation between sequence variants of GFRA2 and the HSCR phenotype. Hirschsprung disease (HSCR) is a complex disorder characterised by aganglia of
distal gastrointestinal tracts. The highest proportion of both familial and
sporadic cases is due to mutations of the RET proto-oncogene. Five germline
mutations in the glial cell-line-derived neurotrophic factor (GDNF) gene, one of
the RET ligands, have been detected in HSCR patients. Pedigrees analysis and the
observed association between these GDNF alterations and RET variants in the same
patients raised the question of whether the GDNF gene plays any
causative/predisposing role in HSCR pathogenesis. In the present work, we have
studied the ability of GDNF proteins, each bearing one of the reported
mutations, to activate RET by performing a functional test in cultured
neuroblastoma cells. Consistently with the lack of genotype/phenotype
correlation in human subjects, our results indicate absence of detectable
alterations of mutant GDNF induced RET activation. To clarify the role of Ret signaling components in enteric nervous system (ENS)
development, we evaluated ENS anatomy and intestinal contractility in mice
heterozygous for Ret, GFRalpha1 and Ret ligands. These analyses demonstrate that
glial cell line-derived neurotrophic factor (GDNF) and neurturin are important
for different aspects of ENS development. Neurturin is essential for maintaining
the size of mature enteric neurons and the extent of neuronal projections, but
does not influence enteric neuron number. GDNF availability determines enteric
neuron number by controlling ENS precursor proliferation. However, we were
unable to find evidence of programmed cell death in the wild type ENS by
immunohistochemistry for activated caspase 3. In addition, enteric neuron number
is normal in Bax(-/-) and Bid(-/-) mice, suggesting that, in contrast to most of
the rest of the nervous system, programmed cell death is not important for
determining enteric neuron numbers. Only mild reductions in neuron size and
neuronal fiber counts occur in Ret(+/-) and Gfra1(+/-) mice. All of these
heterozygous mice, however, have striking problems with intestinal contractility
and neurotransmitter release, demonstrating that Ret signaling is critical for
both ENS structure and function. Glial cell line-derived neurotrophic factor (GDNF), a ligand of RET tyrosine
kinase, and its family ligands promote the survival and differentiation of a
variety of neurons. Gene ablation studies have revealed that the GDNF-RET
receptor system is essential for the development of kidney and peripheral
neurons, including sympathetic, parasympathetic and enteric neurons. RET can
activate various signaling pathways such as RAS/extracellular signal-regulated
kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/AKT, p38 mitogen-activated
protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. These
signaling pathways are activated via binding of adaptor proteins to
intracellular tyrosine residues of RET phosphorylated by its own kinase
activity. The RET is profoundly involved in the development of several human
neuroendocrine diseases. The constitutive activation of the RET by somatic
rearrangement with other partner genes or germ-line mutations causes a
considerable population of human papillary thyroid carcinomas or multiple
endocrine neoplasia (MEN) type 2A and 2B, respectively, whereas the dysfunction
of RET by germ-line missense and/or nonsense mutations causes Hirschsprung's
disease. Biological properties of mutant RET protein determine the disease
phenotype. For example, the MEN 2B mutation alters the substrate specificity of
RET tyrosine kinase and RET carrying the MEN 2B mutation hereby induces the
different set of genes from that carrying the MEN 2A mutation. In this review,
we describe the current knowledge about the molecular mechanism of RET
activation in human neuroendocrine tumors as well as the physiological roles and
signal transduction of RET tyrosine kinase. The c-ret protooncogene, RET, encodes a receptor tyrosine kinase. RET is
activated by members of the glial cell line-derived neurotrophic factor (GDNF)
family of ligands, which include GDNF, neurturin, artemin, and persephin. The
ligands bind RET through GDNF family receptor alpha, termed GFRalpha1-4. Despite
the importance of RET signaling in the development of the enteric nervous system
and the kidney, the differential signaling mechanisms between RET ligands are
poorly established. It has been suggested that signal specificity is achieved
through binding of the ligand to its preferred GFRalpha. To compare the
signaling profiles of GDNF and neurturin, we have identified a cell line,
NG108-15, which endogenously expresses RET and GFRalpha1 but not GFRalpha2-4.
Immunoblot data showed that GDNF caused a transient activation, whereas
neurturin caused a sustained activation, of both p44/p42 MAP kinases and
PLCgamma. Under serum starvation, NG108-15 cells differentiate and form
neurites. Neurturin but not GDNF stimulated neurite outgrowth, which could be
blocked by the selective PLC inhibitor U73122. On the other hand, GDNF but not
neurturin promoted cell survival, and this could be blocked by the p44/p42 MAP
kinase inhibitor PD98059. Our findings not only show the differential signaling
of GDNF and neurturin but also suggest that this can be achieved through binding
to the same GFRalpha subtype, leading to distinct biological responses. The RET kinase has emerged as a promising target for the therapy of medullary
thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487,
a N,N'-diphenyl urea with an IC(50) of 0.88 mumol/L on RET kinase, inhibited RET
autophosphorylation and activation of downstream effectors, and potently
inhibited the growth of human thyroid cancer cell lines with activating
mutations of RET but not of lines without RET mutations. NVP-AST487 induced a
dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing
oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete
calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels
derived from the TT cell xenografts were inhibited shortly after treatment, when
tumor volume was still unchanged, indicating that the effects of RET kinase
inhibition on calcitonin secretion were temporally dissociated from its
tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene
expression in vitro in TT cells, in part, through decreased gene transcription.
These data point to a previously unknown physiologic role of RET signaling on
calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly
stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487.
Antagonists of RET kinase activity in patients with MTC may result in effects on
plasma calcitonin that are either disproportionate or dissociated from the
effects on tumor burden, because RET kinase mediates a physiologic pathway
controlling calcitonin secretion. The role of traditional tumor biomarkers may
need to be reassessed as targeted therapies designed against oncoproteins with
key roles in pathogenesis are implemented. The AP-1 transcription factor c-Jun is a master regulator of the axonal response
in neurons. c-Jun also functions as a negative regulator of myelination in
Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We
demonstrate here that, after injury, the absence of c-Jun specifically in SCs
caused impaired axonal regeneration and severely increased neuronal cell death.
c-Jun deficiency resulted in decreased expression of several neurotrophic
factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor
tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic
inactivation of Ret specifically in neurons resulted in regeneration defects
without affecting motoneuron survival and, conversely, administration of
recombit GDNF and Artemin protein substantially ameliorated impaired
regeneration caused by c-Jun deficiency. These results reveal an unexpected
function for c-Jun in SCs in response to axonal injury, and identify paracrine
Ret signaling as an important mediator of c-Jun function in SCs during
regeneration. |
Which substances are dangerous to g6PD deficient individuals? | Antimalarial drugs (primaquine, pamaquine, chloriquine), fava beans, sulfonamides, some antibiotics( nalidixic acid, nitrofurantoin, isoniazid, dapsone, and furazolidone) and henna | BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is important in the control
of oxidant stress in erythrocytes, the host cells for Plasmodium falciparum.
Mutations in this enzyme produce X-linked deficiency states associated with
protection against malaria, notably in Africa where the A- form of G6PD
deficiency is widespread. Some reports have proposed that heterozygous females
with mosaic populations of normal and deficient erythrocytes (due to random X
chromosome inactivation) have malaria resistance similar to or greater than
hemizygous males with populations of uniformly deficient erythrocytes. These
proposals are paradoxical, and they are not consistent with currently
hypothesized mechanisms of protection.
METHODS AND FINDINGS: We conducted large case-control studies of the A- form of
G6PD deficiency in cases of severe or uncomplicated malaria among two ethnic
populations of rural Mali, West Africa, where malaria is hyperendemic. Our
results indicate that the uniform state of G6PD deficiency in hemizygous male
children conferred significant protection against severe, life-threatening
malaria, and that it may have likewise protected homozygous female children. No
such protection was evident from the mosaic state of G6PD deficiency in
heterozygous females. We also found no significant differences in the parasite
densities of males and females with differences in G6PD status. Pooled odds
ratios from meta-analysis of our data and data from a previous study confirmed
highly significant protection against severe malaria in hemizygous males but not
in heterozygous females. Among the different forms of severe malaria, protection
was principally evident against cerebral malaria, the most frequent form of
life-threatening malaria in these studies.
CONCLUSIONS: The A- form of G6PD deficiency in Africa is under strong natural
selection from the preferential protection it provides to hemizygous males
against life-threatening malaria. Little or no such protection is present among
heterozygous females. Although these conclusions are consistent with data from
at least one previous study, they have not heretofore been realized to our
knowledge, and they therefore give fresh perspectives on malaria protection by
G6PD deficiency as an X-linked trait. BACKGROUND: G6PD deficiency is common in malaria endemic regions and is
estimated to affect more than 400 million people worldwide. Treatment of malaria
patients with the anti-malarial drug primaquine or other 8-aminoquinolines may
be associated with potential haemolytic anaemia. The aim of the present study
was to investigate the prevalence of G6PD variants in Thai population who
resided in malaria endemic areas (western, northern, north-eastern, southern,
eastern and central regions) of Thailand, as well as the Burmese population who
resided in areas along the Thai-Myanmar border.
METHODS: The ten common G6PD variants were investigated in dried blood spot
samples collected from 317 Thai (84 males, 233 females) and 183 Burmese (11
males, 172 females) populations residing in malaria endemic areas of Thailand
using PCR-RFLP method.
RESULTS: Four and seven G6PD variants were observed in samples collected from
Burmese and Thai population, with prevalence of 6.6% (21/317) and 14.2%
(26/183), respectively. Almost all (96.2%) of G6PD mutation samples collected
from Burmese population carried G6PD Mahidol variant; only one sample (3.8%)
carried G6PD Kaiping variant. For the Thai population, G6PD Mahidol (8/21:
38.1%) was the most common variant detected, followed by G6PD Viangchan (4/21:
19.0%), G6PD Chinese 4 (3/21: 14.3%), G6PD Canton (2/21: 9.5%), G6PD Union
(2/21: 9.5%), G6PD Kaiping (1/21: 4.8%), and G6PD Gaohe (1/21: 4.8%). No G6PD
Chinese 3, Chinese 5 and Coimbra variants were found. With this limited sample
size, there appeared to be variation in G6PD mutation variants in samples
obtained from Thai population in different regions particularly in the western
region.
CONCLUSIONS: Results indicate difference in the prevalence and distribution of
G6PD gene variants among the Thai and Burmese populations in different malaria
endemic areas. Dosage regimen of primaquine for treatment of both Plasmodium
falciparum and Plasmodium vivax malaria may need to be optimized, based on
endemic areas with supporting data on G6PD variants. Larger sample size from
different malaria endemic is required to obtain accurate genetic mapping of G6PD
variants in Burmese and Thai population residing in malaria endemic areas of
Thailand. We screened 423 patients referred to our laboratory after hemolysis triggered by
fava beans ingestion, neonatal jaundice or drug hemolysis. Others were
asymptomatic but belonged to a family with a history of G6PD deficiency. The
determination of enzymatic activity using spectrophotometric method, revealed
293 deficient (143 males and 150 females). The molecular analysis was performed
by a combination of PCR-RFLP and DNA sequencing to characterize the mutations
causing G6PD deficiency. 14 different genotypes have been identified : G6PD A(-)
(376A>G;202G>A) (46.07%) and G6PD Med (33.10%) were the most common variants
followed by G6PD Santamaria (5.80%), G6PD Kaiping (3.75%), the association
[c.1311T and IVS11 93c] (3.75%), G6PD Chatham (2.04%), G6PD Aures (1.70%), G6PD
A(-) Betica (0.68%), the association [ 376G;c.1311T;IVS11 93c] (0.68%), G6PD
Malaga, G6PD Canton and G6PD Abeno respectively (0.34%). Two novel missense
mutations were identified (c.920A>C: p.307Gln>Pro and c.968T>C: p.323 Leu>Pro).
We designated these two class III variants as G6PD Tunis and G6PD Nefza. A
mechanism which could account for the defective activity is discussed. We performed a study to evaluate the role of three single nucleotide
polymorphisms (SNPs), factor V Leiden G1691A (FVL), prothrombin gene mutation
G20210A (PRT or FII-G20210A) and methylenotetrahydrofolate reductase variant
C677T (MTHFRC677T), as risk factors for G6PD in Saudi populations. Our results
did not show any association with the three Thrombophilic genes with FVL gene,
no statistical analysis have shown any association with either allele or
genotype frequencies OR=0.566, p=.0.667, (95% CI=0.014-22.48) and OR=0.569,
p=0.251¸ (95% CI=0.014-22.96).In PRT gene G20210A for G Vs A, p=0.774; OR=0.566
(95%CI; 0.011-29.6); AA+GA Vs GG; p=0.502; OR=0.569 (95%CI=0.010-2969). G and A
allele frequencies were similar between cases and controls with no statistical
significance. In the MTHFR gene none of the genotypes or allele frequency cannot
show any association OR=1.281, p=.0.667, (95% CI=0.414-3.958) and OR=1.1.172,
p=0.800¸ (95% CI=0.343-4.008). Similarly, the difference of T allele frequencies
between patients and controls was not found any association. In conclusion, our
finding indicates that the prevalence of G1691A, G20210A and C677T mutations in
G6PD deficient individuals is not statistically different compared to normal
subjects and G6PD is not associated with these thrombophilic mutations in Saudi
population. We report a 67-year-old man presenting with abdominal pain of acute onset,
pallor, jaundice and behavioral changes after ingestion of fava beans. In the
initial evaluation he appeared acutely ill and had resting dyspnea, edema and
jaundice. His initial laboratory assessment disclosed azotemia, elevated lactate
dehydrogenase levels, a low hemoglobin concentration (4.9 /dL) and a high
corrected reticulocyte count (4,7%) with negative direct and indirect Coombs'
test. The patient was transferred to the ICU, where he received support therapy
with hemodialysis, mechanical ventilation, vasoactive drugs and transfusions of
packed red cells. The evolution after 1 month was favorable and he was
discharged without anemia and with normal renal function. Three months after
discharge, the glucose-6-phosphate-dehydrogenase screening study did not
demonstrate detectable enzymatic activity. BACKGROUND: G6PD deficiency is the most common enzymopathy of red blood cells.
The clinical symptoms of favism are jaundice, hematuria and haemolytic anaemia
that seem to affect liver and kidney in long term. Thus we evaluate kidney and
liver function of favism patients in an endemic area of the disease with a high
rate of fava beans cultivation.
METHODS: This study was performed on favism patients and healthy controls
referring to Iranshahr central hospital. Liver and kidney function tests were
performed.
RESULTS: The results showed a statistically significant difference between these
two groups (p <0.05) for liver function tests, (AST, ALT and ALP), but not for
renal tests (BUN and creatinine) (p >0.05).
CONCLUSION: Due to abnormalities were seen in the liver function tests of these
patients, we suggest that these tests be regularly performed for favism patients
who are constantly exposed to oxidant agents. BACKGROUND AND OBJECTIVES: The etiology of favism remains unclear and the fate
of favic patients has not previously been studied. Therefore, individuals who
had experienced an episode of favism were studied regarding subsequent fava bean
ingestion, including the reason for fava bean ingestion after the initial favic
attack and any adverse reactions. In addition, a new hypothesis for the etiology
of favism is proposed.
PATIENTS AND METHODS: From June 2005 to June 2012, a total of 38 patients with a
history of favism were included in this study. Circumstances regarding the
initial favic attack were obtained from medical records and patient interviews,
and subsequent fava bean ingestion and recurrence of symptoms were investigated.
RESULTS: Three of the 38 patients (7.9%) were female, and 35 (92.1%) were male.
The mean age was 27.9 years (14-63 years). The first attack of favism occurred
before 10 years of age for 31 patients (81.6%) and in the springtime for 35
patients (92.1%). Thirty-three patients (86.7%) regularly ate fava beans before
the attack, and 35 (92.1%) resumed eating fava beans within 1-17 years after the
attack without symptoms. Two patients (5.2%) experienced a single recurrence of
symptoms. No evidence of hemolysis was found in the four patients checked after
fava bean re-ingestion.
CONCLUSIONS: Patients resumed eating fava bean for various reasons, and the
recurrence of symptoms was uncommon. An infectious agent such as a virus may
play a role in the development of favism. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common
human enzyme deficiencies in the world. It is particularly common in populations
living in malaria-endemic areas, affecting more than 400 million people
worldwide. This present study was conducted with the aim of determining the
prevalence of G6PD deficiency among children visiting the Emergency Paediatric
Unit of Usmanu Danfodiyo University Teaching Hospital for pediatric-related
care. The study included 118 children, made up of 77 (65.3%) males and 41
(34.7%) females aged ≤5 years with mean age of 3.26 ± 1.90 years. Randox G6PD
quantitative in vitro test screening was used for the diagnosis of G6PD
deficiency. Of the 118 children tested, 17 (14.4%) were G6PD-deficient.
Prevalence of G6PD deficiency was concentrated predomitly among male children
(22.1%). Male sex was significantly correlated with G6PD deficiency among the
children studied (r = 7.85, P = 0.01). The highest prevalence occurred among
children in the 2- to 5-year age-group. Of the 17 G6PD-deficient children,
twelve (70.2%) were moderately deficient, while five (29.4%) were severely
deficient. Blood film from G6PD-deficient children indicated the following
morphological changes; Heinz bodies, schistocytes, target cells, nucleated red
cells, spherocytes, and polychromasia. This present study has shown a high
prevalence of G6PD deficiency among children residing in Sokoto in the
northwestern geopolitical zone of Nigeria. The study indicated a male sex bias
in the prevalence of G6PD deficiency among the children studied. There is a need
for the routine screening of children for G6PD deficiency in our environment, to
allow for evidence-based management of these children and to ensure the
avoidance of food, drugs, and infective agents that can potentially predispose
these children to oxidative stress as well as diseases that deplete
micronutrients that protect against oxidative stress. There is need to build
capacity in our setting among pediatricians to ensure the effective management
of children with G6PD deficiency. |
Which mutations of SCN5A gene are implicated in Brugada syndrome? | The following mutations of SCN5A gene have been linked to Brugada syndrome:I137M, p.W1095X; c.3284G>A, R27H, E901K, G1743R, V728I, N1443S, E1152X, c.664C>T; p.Arg222X, Ala2>Thr, Ala735, Ala735>Thr, Val1340>Ile, IVS18-1G>A, E1784K (14x), F861WfsX90 (11x), D356N (8x), G1408R (7x), G400A, H558R, W822X, Q55X, V95I, A1649V, delF1617, c.4810+3_4810+6dupGGGT, K1527R, A1569P, R367H, A735V, R1192Q, D1795. | Brugada syndrome is a hereditary cardiac disease causing abnormal ST segment
elevation in the ECG, right bundle branch block, ventricular fibrillation and
sudden death. In this study we characterized a new mutation in the SCN5A gene
(T1620M), causing the Brugada syndrome. The mutated channels were expressed in
both Xenopus leavis oocytes and in mammalian tsA201 cells with and without the
beta-subunit and studied using the patch clamp technique. Opposite phenotypes
were observed depending on the expression system. T1620M mutation led to a
faster recovery from inactivation and a shift of steady-state inactivation to
more positive voltages when expressed in Xenopus oocytes. However, using the
mammalian expression system no effect on steady-state inactivation was observed,
but this mutation led to a slower recovery from inactivation. Our finding
supports the idea that the slower recovery from inactivation of the cardiac
sodium channels seen in our mammalian expression system could decrease the
density of sodium channels during the cardiac cycle explaining the in vivo
arrhythmogenesis in patients with Brugada syndrome. BACKGROUND: Primary dysrhythmias other than those associated with the long QT
syndrome, are increasingly recognized. One of these are represented by patients
with a history of resuscitation from cardiac arrest but without any structural
heart disease. These patients exhibit a distinct electrocardiographic (ECG)
pattern consisting of a persistent ST-segment elevation in the right precordial
leads often but not always accompanied by a right bundle branch block (Brugada
syndrome). This syndrome is associated with a high mortality rate and has been
shown to display familial occurrence.
METHODS AND RESULTS: Pharmacological sodium channel blockade elicits or worsens
the electrocardiographic features associated with this syndrome. Hence, a
candidate gene approach directed towards SCN5A, the gene encoding the
alpha-subunit of the cardiac sodium channel, was followed in six affected
individuals. In two patients missense mutations were identified in the coding
region of the gene: R1512W in the DIII-DIV cytoplasmic linker and A1924T in the
C-terminal cytoplasmic domain. In two other patients mutations were detected
near intron/exon junctions. To assess the functional consequences of the R1512W
and A1924T mutations, wild-type and mutant sodium channel proteins were
expressed in Xenopus oocytes. Both missense mutations affected channel function,
most notably a 4-5 mV negative voltage shift of the steady-state activation and
inactivation curves in R1512W and a 9 mV negative voltage shift of the
steady-state activation curve in A1924T, measured at 22 degrees C. Recovery from
inactivation was slightly prolonged for R1512W channels. The time dependent
kinetics of activation and inactivation at -20 mV were not significantly
affected by either mutation.
CONCLUSIONS: Two SCN5A mutations associated with the Brugada syndrome,
significantly affect cardiac sodium channel characteristics. The alterations
seem to be associated with an increase in inward sodium current during the
action potential upstroke. Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada
syndrome has so far been associated with only one gene, SCN5A, which encodes the
cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes,
including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and
the insertion (D1795) induces both LQTS and Brugada syndromes in carrier
patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the
tsA201 human cell line and characterized using the patch clamp technique in
whole-cell configuration. Our data revealed a persistent inward sodium current
of about 6% at -30 mV for both D1790G and insD1795, and a reduction of 62% of
channel expression for the insD1795. Moreover, a shift of steady-state
inactivation curve in both mutants was also observed. Our findings uphold the
idea that LQT3 is related to a persistent sodium current whereas reduction in
the expression level of cardiac sodium channels is one of the biophysical
characteristics of Brugada syndrome. The SCN5A gene encodes the alpha subunit of the human heart sodium channel
(hH1), which plays a critical role in cardiac excitability. Mutations of SCN5A
underlie Brugada syndrome, an inherited disorder that leads to ventricular
fibrillation and sudden death. This study describes changes in cellular
localization and functional expression of hH1 in a naturally occurring SCN5A
mutation (R1432G) reported for Brugada syndrome. Using patch-clamp experiments,
we show that there is an abolition of functional hH1 expression in R1432G
mutants expressed in human tsA201 cells but not in Xenopus oocytes. In tsA201
cells, a conservative positively charged mutant, R1432K, produced sodium
currents with normal gating properties, whereas other mutations at this site
abolished functional sodium channel expression. Immunofluorescent staining and
confocal microscopy showed that the wild-type alpha subunit expressed in tsA201
cells was localized to the cell surface, whereas the R1432G mutant was
colocalized with calnexin within the endoplasmic reticulum. The beta(1) subunit
was also localized to the cell surface in the presence of the alpha subunit;
however, in its absence, the beta(1) subunit was restricted to a perinuclear
localization. These results demonstrate that the disruption of SCN5A
cell-surface localization is one mechanism that can account for the loss of
functional sodium channels in Brugada syndrome. The full text of this article is
available at http://www.circresaha.org. BACKGROUND: The SCN5A gene encoding the human cardiac sodium channel alpha
subunit plays a key role in cardiac electrophysiology. Mutations in SCN5A lead
to a large spectrum of phenotypes, including long-QT syndrome, Brugada syndrome,
and isolated progressive cardiac conduction defect (Lenègre disease).
METHODS AND RESULTS: In the present study, we report the identification of a
novel single SCN5A missense mutation causing either Brugada syndrome or an
isolated cardiac conduction defect in the same family. A G-to-T mutation at
position 4372 was identified by direct sequencing and was predicted to change a
glycine for an arginine (G1406R) between the DIII-S5 and DIII-S6 domain of the
sodium channel protein. Among 45 family members, 13 were carrying the G1406R
SCN5A mutation. Four individuals from 2 family collateral branches showed
typical Brugada phenotypes, including ST-segment elevation in the right
precordial leads and right bundle branch block. One symptomatic patient with the
Brugada phenotype required implantation of a cardioverter-defibrillator. Seven
individuals from 3 other family collateral branches had isolated cardiac
conduction defects but no Brugada phenotype. Three flecainide test were
negative. One patient with an isolated cardiac conduction defect had an episode
of syncope and required pacemaker implantation. An expression study of the
G1406R-mutated SCN5A showed no detectable Na(+) current but normal protein
trafficking.
CONCLUSIONS: We conclude that the same mutation in the SCN5A gene can lead
either to Brugada syndrome or to an isolated cardiac conduction defect. Our
findings suggest that modifier gene(s) may influence the phenotypic consequences
of a SCN5A mutation. Sudden unexplained nocturnal death syndrome (SUNDS), a disorder found in
southeast Asia, is characterized by an abnormal electrocardiogram with
ST-segment elevation in leads V1-V3 and sudden death due to ventricular
fibrillation, identical to that seen in Brugada syndrome. We screened patients
with SUNDS for mutations in SCN5A, the gene known to cause Brugada syndrome, as
well as genes encoding ion channels associated with the long-QT syndrome. Ten
families were enrolled, and screened for mutations using single-strand DNA
conformation polymorphism analysis, denaturing high-performance liquid
chromatography and DNA sequencing. Mutations were identified in SCN5A in three
families. One mutation, R367H, lies in the first P segment of the pore-lining
region between the DIS5 and DIS6 transmembrane segments of SCN5A. A second
mutation, A735V, lies in the first transmembrane segment of domain II (DIIS1)
close to the first extracellular loop between DIIS1 and DIIS2, whereas the third
mutation, R1192Q, lies in domain III. Analysis of these mutations in Xenopus
oocytes showed that the R367H mutant channel did not express any current and the
likely effect of this mutation is to depress peak current due to the loss of one
functional allele. The A735V mutant expressed currents with steady state
activation voltage shifted to more positive potentials. The R1192Q mutation
accelerated the inactivation of the sodium channel current. Both mutations
resulted in reduced sodium channel current (I(Na)) at a time corresponding to
the end of phase 1 of the action potential, as described previously in the
Brugada syndrome. Based upon these observations we suggest that SUNDS and
Brugada syndrome are phenotypically, genetically and functionally the same
disorder. Idiopathic ventricular fibrillation in patients with an electrocardiogram (ECG)
pattern of right bundle branch block and ST-segment elevation in leads V1 to V3
(now frequently called Brugada syndrome) is associated with a high incidence of
syncopal episodes or sudden death. The disease is inherited as an autosomal
domit trait. Mutations in SCN5A, a cardiac sodium channel gene, have been
recently associated with Brugada syndrome. We have analyzed 7 patients from
Israel affected with Brugada syndrome. The families of these patients are
characterized by a small number of symptomatic members. Sequencing analysis of
SCN5A revealed two novel mutations, G35S and R104Q, in two Brugada patients, and
a possible R34C polymorphism in two unrelated controls. No mutations were
detected in 5 other patients, suggesting genetic heterogeneity. Low penetrance
is probably the cause for the small number of symptomatic members in the two
families positive for the SCN5A mutations. OBJECTIVE: To perform PCR-based site-directed mutagenesis of a new SCN5A
mutation (K317N) identified in a Chinese family with Brugada syndrome and
construct the recombit expression plasmid pRc/CMV-Hh1 containing the human
cardiac sodium channel alpha subunit (hH1), mutant cDNA.
METHODS: A pair of primers was designed according to the restricted sites Sse
8387I and Age I of the SCN5A sequence with the mismatches introduced into
primers. Mutagenesis was performed in a single-step PCR, and the fragments
amplified by PCR containing the mutation site were subcloned into the
pRc/CMV-hH1 vector.
RESULTS: Sequence analysis confirmed the presence of the desired mutation site,
and a mutation from K (Lys) to N (Asn) in codon 317 was identified in the SCN5A
gene, indicating the successful induction of the mutation at K317N of the SCN5A
gene.
CONCLUSION: PCR site-directed mutagenesis is accurate and highly efficient, and
the successfully constructed recombit expression plasmid pRc/CMV-hH1 (K317N)
may provide a molecular basis for further functional and genomic investigation
of SCN5A. BACKGROUND: Brugada syndrome is a form of idiopathic ventricular fibrillation
characterized by right bundle-branch block pattern and ST elevation in the right
precordial leads of the ECG. The SCN5A gene encodes the alpha-subunit of the
human heart sodium channel, which plays a critical role in cardiac excitability,
and mutations of SCN5A could underlie Brugada syndrome.
METHODS AND RESULTS: To detect mutations of SCN5A, DNA samples from 12 Japanese
patients with Brugada syndrome were analyzed using direct sequencing. Two
patients had novel mutations, G292S and S835L, but no other mutations of SCN5A
were detected in the remaining patients. The first mutation, G292S, was
identified adjacent to the pore-lining region between the DIS5 and DIS6
transmembrane segments of SCN5A, and the second mutation, S835L, was in the
intracellular loop connecting the DIIS4 to DIIS5. Both mutations were not
detected in 100 unrelated control subjects.
CONCLUSION: Two novel SCN5A mutations have been found in Japanese patients with
Brugada syndrome. The SCN5A gene encodes the alpha subunit of the human cardiac voltage-gated
sodium channel. Mutations in SCN5A are responsible for Brugada syndrome, an
inherited cardiac disease that leads to idiopathic ventricular fibrillation
(IVF) and sudden death. In this study, we screened nine individuals from a
single family and 12 sporadic patients who were clinically diagnosed with
Brugada syndrome. Using PCR-SSCP, DHPLC, and DNA sequencing analysis, we
identified a novel single missense mutation associated with Brugada syndrome in
the family and detected a C5607T polymorphism in Korean subjects. A single
nucleotide substitution of G to A at nucleotide position 3934 changed the coding
sense of exon 21 of the SCN5A from glycine to serine (G1262S) in segment 2 of
domain III (DIII-S2). Four individuals in the family carried the identical
mutation in the SCN5A gene, but none of the 12 sporadic patients did. This
mutation was not found in 150 unrelated normal individuals. This finding is the
first report of a novel mutation in SCN5A associated with Brugada syndrome in
Koreans. BACKGROUND: Mutations in the cardiac sodium channel, SCN5A, have been associated
with one type of long-QT syndrome, with isolated cardiac conduction defects and
Brugada syndrome. The sodium channelopathies exhibit marked variation in
clinical phenotypes. The mechanisms underlying the phenotypical diversity,
however, remain unknown. Exonic SCN5A mutations can be detected in 20% of
Brugada syndrome patients.
RESULTS: An intronic mutation (c.4810+3_4810+6dupGGGT) in the SCN5A gene,
located outside the consensus splice site, was detected in this study in a
family with a highly variable clinical phenotype of Brugada syndrome and/or
conduction disease and in a patient with Brugada syndrome. The mutation was not
found in a control panel of 100 (200 alleles) ethnically matched normal control
subjects. We provide in vivo and in vitro evidence that the mutation can disrupt
the splice donor site, activate a cryptic splice site, and create a novel splice
site. Notably, our data show that normal transcripts can be also derived from
the mutant allele.
CONCLUSIONS: This is the first report of an unconventional intronic splice site
mutation in the SCN5A gene leading to cardiac sodium channelopathy. We speculate
that its phenotypical diversity might be determined by the ratio of
normal/abnormal transcripts derived from the mutant allele. Brugada syndrome is an inherited cardiac disorder caused by mutations in the
SCN5A gene encoding the cardiac sodium channel alpha subunit, which can lead
ventricular fibrillation and sudden death. Inattentive use of antiarrhythmic
drugs potentially triggers fatal cardiac arrhythmias through further reduction
of sodium current (I(Na)). We studied the molecular mechanism underlying a case
of Brugada syndrome that showed no response to a class Ic antiarrhythmic drug.
Molecular genetic studies of a patient with Brugada syndrome identified a novel
mutation in SCN5A, which causes substitution of serine for asparagine (N406S) in
S6 of domain I (IS6). The provocation test with pilsicainide, a class Ic
antiarrhythmic drug, failed to exacerbate ST-segment elevation in this case.
Electrophysiological analyses of the N406S-mutant channel expressed together
with the beta1 subunit in HEK293 cells showed that the voltage dependence of
activation was positively shifted by 16 mV and that intermediate inactivation
was enhanced. Whereas tonic block by pilsicainide was not changed in the N406S
channel, use-dependent block by pilsicainide was almost completely abolished,
consistent with the clinical findings of the negative provocation test. In
contrast, the N406S channel showed stronger use-dependent block by quinidine
than the wild-type channel. We demonstrate a novel Brugada mutation N406S, which
is associated with the discordant effects on blocking actions of antiarrhythmic
drugs as well as the multiple channel gating defects. We emphasis that an
antiarrhythmic drug may exert unpredicted effects in patients with channel
mutations. OBJECTIVES: We carried out a complete screening of the SCN5A gene in 38 Japanese
patients with Brugada syndrome to investigate the genotype-phenotype
relationship.
BACKGROUND: The gene SCN5A encodes the pore-forming alpha-subunit of
voltage-gated cardiac sodium (Na) channel, which plays an important role in
heart excitation/contraction. Mutations of SCN5A have been identified in 15% of
patients with Brugada syndrome.
METHODS: In 38 unrelated patients with clinically diagnosed Brugada syndrome, we
screened for SCN5A gene mutations using denaturing high-performance liquid
chromatography and direct sequencing, and conducted a functional assay for
identified mutations using whole-cell patch-clamp in heterologous expression
system.
RESULTS: Four heterozygous mutations were identified (T187I, D356N, K1578fs/52,
and R1623X) in 4 of the 38 patients. All of them had bradyarrhythmic
complications: three with sick sinus syndrome (SSS) and the other (D356N) with
paroxysmal complete atrioventricular block. SCN5A-linked Brugada patients were
associated with a higher incidence of bradyarrhythmia (4 of 4) than
non-SCN5A-linked Brugada patients (2 of 34). Families with T187I and K1578fs/52
had widespread penetrance of SSS. Notably, the patient with K1578fs/52, who had
been diagnosed as having familial SSS without any clinical signs of Brugada
syndrome, showed a Brugada-type ST-segment elevation after intravenous
administration of pilsicainide and programmed electrical stimulation-induced
ventricular tachycardia. All of the mutations encoded non-functional Na
channels, and thus were suggested to cause impulse propagation defect underlying
bradyarrhythmias.
CONCLUSIONS: Our findings suggest that loss-of-function SCN5A mutations
resulting in Brugada syndrome are distinguished by profound bradyarrhythmias. BACKGROUND: Loss-of-function mutations in SCN5A have been associated with the
Brugada syndrome. We report the first Brugada syndrome family with compound
heterozygous mutations in SCN5A. The proband inherited 1 mutation from each
parent and transmitted 1 to each daughter.
METHODS AND RESULTS: The effects of the mutations on the function of the sodium
channel were evaluated with heterologous expression in TSA201 cells, patch-clamp
study, and confocal microscopy. Genetic analysis revealed that the proband
carried 2 heterozygous missense mutations (P336L and I1660V) on separate
alleles. He displayed a coved-type ST-segment elevation and a prolonged PR
interval (280 ms). One daughter inherited P336L and exhibited a prolonged PR
(210 ms). The other daughter inherited mutation I1660V and displayed a normal PR
interval. Both daughters had a slightly elevated, upsloping ST-segment
elevation. The parents had normal ECGs. Patch-clamp analysis showed that the
P336L mutation reduced I(Na) by 85% relative to wild type. The I1660V mutation
produced little measurable current, which was rescued by room temperature
incubation for 48 hours. Sodium channel blockers also rescued the I1660V
current, with mexiletine proving to be the most effective. Confocal
immunofluorescence showed that I1660V channels conjugated to green fluorescent
protein remained trapped in intracellular organelles.
CONCLUSIONS: Mutation P336L produced a reduction in cardiac I(Na), whereas
I1660V abolished it. Only the proband carrying both mutations displayed the
Brugada syndrome phenotype, whereas neither mutation alone produced the clinical
phenotype. I1660V channels could be rescued pharmacologically and by incubation
at room temperature. The present data highlight the role of compound
heterozygosity in modulating the phenotypic expression and penetrance of Brugada
syndrome. OBJECTIVE: Brugada syndrome is an inherited channelopathy that characterized by
ST-segment elevation in the right precordial lead (V(1)-V(3)) on the
electrocardiogram with or without right bundle branch block and related with
high risk of sudden cardiac death and structurally normal hearts. The first and
only gene linked to this disease is SCN5A, a gene encodes for alpha subunit of
the cardiac sodium channel. The objective of this study is to explore SCN5A gene
mutations in Chinese patients with Brugada syndrome.
METHODS: Four patients diagnosed as Brugada syndrome and nine patients with
suspected Brugada syndrome were chosen for the study. The exons in the
functional regions of SCN5A gene were amplified with polymerase chain reaction
and the amplified products were sequenced with Sanger method. If a mutation was
identified, patient's family members were also screened.
RESULTS: Two heterozygous mutations were found in one family diagnosed as
Brugada syndrome. One missense mutation was a G-->A transition in the first
nucleotide of codon 95 in SCN5A gene exon 3, which was predicted to result in
substitution of Valine with Isoleucine (V95I). The other missense mutation was a
C-->T transition in the second nucleotide of codon 1649 in SCN5A gene exon 28,
which was predicted to result in substitution of Alanine with Valine (A1649V). A
heterozygous mutation was identified in one family suspected to have the
disease. The mutation was a three nucleotides (TCT) deletion that caused
Phenylalanine deletion in codon 1617 in SCN5A gene exon 28. The three mutations
were not detected in 100 control chromosomes.
CONCLUSIONS: Mutation in SCN5A gene is one of the causes of Brugada syndrome in
Chinese. Three novel SCN5A gene mutations were identified in Chinese with
Brugada syndrome, which expands the spectrum of SCN5A mutations associated with
the disease. AIMS: To describe a patient showing monomorphic ventricular tachycardia, ECG
aspect of Brugada syndrome, and structural heart abnormalities due to a
homozygous missense mutation in SCN5A.
METHODS AND RESULTS: Thirteen subjects (six males, seven females, mean age 46
+/- 22 years) belonging to the same family underwent physical examination, basal
biochemical marker detection, 12-lead ECG, Holter ECG, signal-averaged ECG,
echocardiogram and genetic analysis. The proband underwent a stress test
together with left and right ventricular angiography and electrophysiological
study. Three subjects (the proband, his mother, and one brother) showed on ECG
an ST-segment elevation in the right precordial leads with coved type aspect.
Moreover, the proband presented a sustained monomorphic ventricular tachycardia
(left bundle branch block aspect with superior axis), whereas all other family
members were asymptomatic. Imaging techniques documented right ventricular
structural abnormalities only in the proband. Mutation screening in SCN5A gene
was performed in the proband and in available family members. The proband
carries a novel SCN5A mutation, R814Q, in homozygous, whereas the parents and
four siblings were heterozygous carriers of the same mutation.
CONCLUSION: This study provides the first evidence of a homozygous missense
mutation in SCN5A associated with atypical ventricular arrhythmias and right
structural abnormalities. The Brugada syndrome is characterized by ST segment elevation in the right
precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular
fibrillation causing syncope or even sudden death. The molecular and cellular
mechanisms that lead to Brugada syndrome are not yet completely understood.
However, SCN5A is the most well known responsible gene that causes Brugada
syndrome. Until now, more than a hundred mutations in SCN5A responsible for
Brugada syndrome have been described. Functional studies of some of the
mutations have been performed and show that a reduction of human cardiac sodium
current accounts for the pathogenesis of Brugada syndrome. Here we reported
three novel SCN5A mutations identified in patients with Brugada syndrome in
Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological
properties were altered by patch clamp analysis. The p.I848fs mutant generated
no sodium current. The p.R965C and p.1876insM mutants produced channels with
steady state inactivation shifted to a more negative potential (9.4 mV and 8.5
mV respectively), and slower recovery from inactivation. Besides, the steady
state activation of p.1876insM was altered and was shifted to a more positive
potential (7.69 mV). In conclusion, the SCN5A channel defect related to Brugada
syndrome might be diverse but all resulted in a decrease of sodium current. Ventricular tachycardia and fibrillation (VT/VF) complicating Brugada syndrome,
a genetic disorder linked to SCN5A mutations, and VF complicating acute
myocardial infarction (AMI) have both been linked to phase 2 reentry. Because of
these mechanistic similarities in arrhythmogenesis, we examined the contribution
of SCN5A mutations to VT/VF complicating AMI. Nineteen consecutive patients
developing VF during AMI were enrolled. Wild-type (WT) and mutant SCN5A genes
were co-expressed with SCN1B in TSA201 cells and studied using whole-cell
patch-clamp techniques. One missense mutation (G400A) in SCN5A was detected in a
conserved region among the cohort of 19 patients. A H558R polymorphism was
detected on the same allele. Unlike the other 18 patients who each developed 1-2
VF episodes during acute MI, the mutation carrier developed six episodes of
VT/VF within the first 12 hours. All VT/VF episodes were associated with ST
segment changes and were initiated by short-coupled extrasystoles. We describe
the first sodium channel mutation to be associated with the development of an
arrhythmic storm during acute ischemia. These findings suggest that a loss of
function in SCN5A may predispose to ischemia induced arrhythmic storm. These
results could be very useful for forensic implications regarding genetic
screening in relatives. Brugada syndrome (BrS) is an inherited cardiac arrhythmia that may lead to
sudden death in patients with structurally normal heart. Mutations in the alpha
subunit of the cardiac sodium channel SCN5A gene are found in approximately 20%
of cases. We clinically evaluated and genetically screened 7 patients that fully
satisfied the clinical diagnostic criteria for the syndrome and 8 patients with
a partial clinical diagnosis, for mutations in the SCN5A gene in order to
explore the genetic status of BrS patients from Greece for whom there are no
published data available. Genetic testing was positive in 3 out of the 7
patients with a definite diagnosis. The probands carried 1 nonsense (p.Trp301X)
and 2 missense (p.Ala1949Pro and p.Arg808Cys) mutations. All 3 mutations were
novel. Furthermore, genetic testing was negative in all 8 clinically suspected
cases. Additionally, 10 single nucleotide polymorphisms (SNPs) were detected, 2
of which are novel. We report on the genetic status of BrS patients of Greek
origin amongst whom novel SCN5A mutations were a frequent underlying cause of
the syndrome. BACKGROUND: Brugada syndrome (BrS) is a common heritable channelopathy.
Mutations in the SCN5A-encoded sodium channel (BrS1) culminate in the most
common genotype.
OBJECTIVE: This study sought to perform a retrospective analysis of BrS
databases from 9 centers that have each genotyped >100 unrelated cases of
suspected BrS.
METHODS: Mutational analysis of all 27 translated exons in SCN5A was performed.
Mutation frequency, type, and localization were compared among cases and 1,300
ostensibly healthy volunteers including 649 white subjects and 651 nonwhite
subjects (blacks, Asians, Hispanics, and others) that were genotyped previously.
RESULTS: A total of 2,111 unrelated patients (78% male, mean age 39 +/- 15
years) were referred for BrS genetic testing. Rare mutations/variants were more
common among BrS cases than control subjects (438/2,111, 21% vs. 11/649, 1.7%
white subjects and 31/651, 4.8% nonwhite subjects, respectively, P <10(-53)).
The yield of BrS1 genetic testing ranged from 11% to 28% (P = .0017). Overall,
293 distinct mutations were identified in SCN5A: 193 missense, 32 nonsense, 38
frameshift, 21 splice-site, and 9 in-frame deletions/insertions. The 4 most
frequent BrS1-associated mutations were E1784K (14x), F861WfsX90 (11x), D356N
(8x), and G1408R (7x). Most mutations localized to the transmembrane-spanning
regions.
CONCLUSION: This international consortium of BrS genetic testing centers has
added 200 new BrS1-associated mutations to the public domain. Overall, 21% of
BrS probands have mutations in SCN5A compared to the 2% to 5% background rate of
rare variants reported in healthy control subjects. Additional studies drawing
on the data presented here may help further distinguish pathogenic mutations
from similarly rare but otherwise innocuous ones found in cases. Brugada syndrome is characterized by right bundle branch block and ST-segment
elevation in the right precordial ECG leads. Familial transmission is frequent
and approximately 25% of cases exhibit mutations in the SCN5A gene. We analyzed
the sequence of this gene in 25 Spanish patients with Brugada syndrome. In 4
(16%), we found mutations that had not previously been described: three were
amino acid changes (i.e. Ala2>Thr, Ala735>Thr and Val1340>Ile) and one was an
intron mutation that affected messenger RNA processing (i.e. IVS18-1G>A). These
four patients had relatives who were also mutation carriers, several of whom had
normal ECGs, even on flecainide challenge. Our study suggests that genetic
analysis could be helpful in the presymptomatic diagnosis of Brugada syndrome,
but may be less useful for stratifying the risk of adverse events. Mutations in SCN5A are linked to Brugada syndrome in approximately 20% of all
cases (BrS1). Several dozen distinct SCN5A mutations in BrS1 have been
associated with the increased risk of cardiac arrhythmias. However, the
genotype-phenotype relationship remains elusive. The current study analyzed the
SCN5A gene to elucidate the potential variability of clinical features in
Japanese BrS1 subjects. Subjects of the present study included 30 probands (25
male subjects, 45 ± 15 years of age) with Brugada-pattern ECG. Seven patients
had been resuscitated from cardiopulmonary arrest (CPA group). Another 10
patients had a history of syncope (Sy group), and 13 more remain asymptomatic
(Asy group). We identified 8 different SCN5A mutations, including 6 novel
mutations (CPA group: 1/7, Sy group: 3/10, Asy group: 4/13). An A735E mutation
(located at segment (S)1 in domain (D)2) was identified in the CPA group. A
novel splice acceptor site mutation (c.393-1c>t), which may produce a
prematurely truncated protein, was identified in the Sy group. An E1784K
mutation (C-terminus) and a novel mutation V1951M (C-terminus) were also
identified in the Sy group. Four novel missense mutations, A586T (D1-D2 linker),
R689H (D1-D2 linker), S1553R (S1-S2 in D4), and Q1706H (S5-Pore in D4) were
identified in the Asy group. These data may help us understand the genetic
heterogeneity of BrS1, which is more prevalent in Japanese than in whites and
other ethnic groups. BACKGROUND: Cardiac sodium channel alterations have been identified as the
underlying condition in patients with Brugada syndrome.
OBJECTIVE: This study identified a novel mutation of the SCN5A gene in a family
with Brugada syndrome.
METHODS: Blood was drawn from the children and mother for genetic analysis. All
exons of the SCN5A gene were amplified by polymerase chain reaction, and a
sequence analysis was performed.
RESULTS: The mutation was detected in 1 symptomatic and 2 asymptomatic family
members. The deletion of base 4066_4068delTT leads to a shift in the amino acid
sequence and a premature stop of the protein translation. The clinical diagnosis
of Brugada syndrome in this family was supported by the detection of the new
mutation.
CONCLUSION: We describe a family partly with Brugada syndrome and a novel
mutation in the exon 23 of the SCN5A gene leading to a deletion of 2 thymidine
bases. This mutation results in an early termination of the encoded protein and
possibly in a nonfunctional channel protein. Mutations in the α-subunit of cardiac sodium channel gene SCN5A can lead to the
overlapping phenotypes of both the Brugada and type 3 long QT syndromes.
However, the combination of Brugada and a short QT phenotype resulting from
mutation in SCN5A has not previously been described. A man with concomitant
Brugada-like and short QT electrocardiogram (ECG) was identified and the SCN5A
gene was sequenced. Whole-cell patch clamp analysis of human embryo kidney (HEK)
293 cells expressing a SCN5A channel with the patient's sequence was used to
investigate the biophysical properties of the channel. The patient with the
family history of sudden death showed Brugada-like and short QT interval ECG.
Sequence analysis of the coding region of the SCN5A gene, identified a G to A
heterozygous missense mutation at nucleotide site 2066 that resulted in a
amino-acid substitution of arginine to histidine at amino-acid site 689 (R689H).
Patch clamp analysis showed that the R689H failed to generate current when
heterologously expressed in HEK293 cells, indicating it was a loss-of-function
mutation. Our finding firstly shows that a heterozygous missense mutation R689H
in SCN5A gene results in the loss of protein function and the coexistents of the
Brugada-like and short QT interval ECG phenotypes. We aim to study the SCN5A gene in a cohort of Brugada syndrome (BS) patients and
evaluate the genotype-phenotype correlation. BS is caused by mutations in up to
10 different genes, SCN5A being the most frequently involved. Large genomic
rearrangements in SCN5A have been associated with conduction disease, but its
prevalence in BS is unknown. Seventy-six non-related patients with BS were
studied. Clinical characteristics and family risk profile were recorded. Direct
sequencing and multiplex ligation-dependent probe amplification (MLPA) of the
SCN5A gene for identification of mutations and larger rearrangements were
performed, respectively. Eight patients (10.5%) had point mutations (R27H,
E901K, G1743R (detected in three families), V728I, N1443S and E1152X). Patients
with mutations had a trend toward a higher proportion of spontaneous type I
Brugada electrocardiogram (ECG) (87.5% vs 52.9%, p = 0.06) and had evidence of
familial disease (62.5%, vs 23.5%, p = 0.03). The symptoms and risk profile of
the carriers were not different from wild-type probands. There were
non-significant differences in the prevalence of type I ECG, syncope and history
of arrhythmia in carriers of selected polymorphisms. None of the patients had
any deletion/duplication in the SCN5A gene. In conclusion, 10.5% of our patients
had mutations in the SCN5A gene. Patients with mutations seemed to have more
spontaneous type I ECG, but no differences in syncope or arrhythmic events
compared with patients without mutations. Larger studies are needed to evaluate
the role of polymorphisms in the SCN5A in the expression of the phenotype and
prognosis. Large rearrangements were not identified in the SCN5A gene using the
MLPA technique. BACKGROUND: We identified 2 compound heterozygous mutations (p.D1690N and
p.G1748D) in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) in a
proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele
encoding the p.D1690N mutation, the p.H558R polymorphism was also detected.
OBJECTIVE: The purpose of this study was to analyze the functional properties of
the mutated channels as well as the putative modulator effects produced by the
presence of the polymorphism.
METHODS: Wild-type and mutated human Nav1.5 channels were expressed in Chinese
hamster ovary cells and recorded using whole-cell patch-clamp technique.
RESULTS: Separately, both p.D1690N and p.G1748D mutations produced a marked
reduction in peak Na(+) current density (>80%), mainly due to their limited
trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly
affected channel gating. Both p.D1690N and p.G1748D produced a marked domit
negative effect when cotransfected with either wild-type or p.H558R channels.
Conversely, p.H558R was able to rescue defective trafficking of p.D1690N
channels toward the membrane when both polymorphism and mutation were in the
same construct. Surprisingly, cotransfection with p.D1690N, either alone or
together with the polymorphism (p.H558R-D1690N), completely restored the
profound gating defects exhibited by p.G1748D channels but only slightly rescued
their trafficking.
CONCLUSIONS: Our results add further support to the hypothesis that Nav1.5
subunits interact among them before trafficking toward the membrane and suggest
that a missense mutation can "rescue" the defective gating produced by another
missense mutation. Cardiac arrhythmias are associated with abnormal channel function due to
mutations in ion channel genes. Epilepsy is a disorder of neuronal function also
involving abnormal channel function. It is increasingly demonstrated that the
etiologies of long QT syndrome and epilepsy may partly overlap. However, only a
few genetic studies have addressed a possible link between cardiac and neural
channelopathies. We describe a family showing the association between Brugada
syndrome and epilepsy in which a known mutation in the SCN5A gene (p.W1095X,
c.3284G>A) was identified. We suggest that this mutation can be responsible for
cardiac and brain involvement, probably at different developmental age in the
same individual. This observation confirms the possibility that SCN5A mutations
may confer susceptibility for recurrent seizure activity, supporting the
emerging concept of a genetically determined cardiocerebral channelopathy. BACKGROUND: Arrhythmogenic right ventricular dysplasia (ARVD) is a genetically
determined disorder, characterized by two components: cardiomyopathy and
arrhythmia. To date, the ion channel-related pathogenesis underlying this
phenomenon has been poorly understood. The aim of this study was to
systematically evaluate the sodium channel variants in Chinese patients with
ARVD.
PATIENTS AND METHODS: Patients meeting the diagnostic guidelines of ARVD revised
in 2010 were enrolled. All exons and exon-intron boundaries of the SCN5A gene
and desmosomal genes known to be associated with ARVD, including DSC2, DSG2,
DSP, JUP, and PKP2, were sequenced by direct DNA sequencing. A total of 12
unrelated index patients were included in the study.
RESULTS: Eight of the patients developed ventricular tachycardia (VT) and
ventricular fibrillation (VF), one of them showed epsilon wave, one of them
showed type-1 Brugada wave, seven of them exhibited syncope or dizziness, and
none of the patients had a family history of SCD. A new missense heterozygote
mutation, I137M, in SCN5A was found in proband 5 with recurrent palpitations and
a high incidence of VT. I137M is in exon 4 of SCN5A, at the S1 segment in domain
I of Nav1.5, which predicted a substitution of isoleucine for methionine at
codon site 137 (p. Ile137Met, I137M). I137M was not detected in 400 healthy
control chromosomes from individuals of the same ethnic background, which
indicated that this mutation was a conservative site in the SCN5A gene, and the
encoded protein Nav1.5 might have a functional defect resulting in arrhythmia.
CONCLUSION: This was the first study to systematically investigate sodium
channel variants in Chinese patients with ARVD; a new SCN5A mutation, I137M, was
found. This finding may provide new evidence of the genetic pathogenesis of ARVD
in Chinese patients, implying that the SCN5A gene should be screened in patients
with ARVD and VT/VF. |
What is Sotos syndrome? | Sotos syndrome is a well-known overgrowth syndrome characterized by excessive growth during childhood, macrocephaly, distinctive facial appearance and learning disability | Sotos' syndrome, or cerebral gigantism, is a disorder of growth regulation.
Tumours have occasionally been reported in children with Sotos' syndrome, but it
is uncertain whether this is a coincidence, or whether it is aetiologically
related to the underlying disorder of growth. We report a 15 month old child
with a paraspinal neuroblastoma and Sotos' syndrome and suggest that children
with this condition may be at higher risk for developing tumours than the
general population. Sotos syndrome is a well-described multiple anomaly syndrome characterized by
overgrowth, distinctive craniofacial appearance, and variable learning
disabilities. The diagnosis of Sotos syndrome relied solely on these clinical
criteria until haploinsufficiency of the NSD1 gene was identified as causative.
We describe a 63-year-old woman with classic features and a pathogenic NSD1
mutation, who we believe to be the oldest reported person with Sotos syndrome.
She is notable for the diagnosis of Sotos syndrome late in life, mild cognitive
limitation, and chronic kidney disease attributed to fibromuscular dysplasia for
which she recently received a transplant. She has basal cell and squamous cell
carcinoma for which her lifetime of sun exposure and fair cutaneous phototype
are viewed as risk factors. We also reviewed previous literature reports
(n = 11) for adults with Sotos syndrome, and studied patients ascertained in the
Spanish Overgrowth Syndrome Registry (n = 15). Analysis was limited to 21/27
(78%) total patients who had molecular confirmation of Sotos syndrome (15 with a
mutation, 6 with a microdeletion). With a mean age of 26 years, the most common
features were learning disabilities (90%), scoliosis (52%), eye problems (43%),
psychiatric issues (30%), and brain imaging anomalies (28%). Learning
disabilities were more severe in patients with a microdeletion than in those
with a point mutation. From this small study with heterogeneous ascertainment we
suggest modest adjustments to the general healthcare monitoring of individuals
with Sotos syndrome. Although this series includes neoplasia in four cases, this
should not be interpreted as incidence. Age-appropriate cancer surveillance
should be maintained. Sotos syndrome is characterised by excessive pre and postnatal growth, a
variable degree of learning difficulties and a recognisable facial appearance.
This report highlights the difficulty in making the diagnosis where failure to
thrive is the presenting feature and documents a previously undescribed
association with recurrent parotitis. Sotos syndrome (OMIM #117550) is a congenital syndrome characterized by
overgrowth with advanced bone age, macrocephaly, and learning difficulties.
Endocrine complications of this syndrome have not yet been fully described in
previous reports. We here investigated the clinical manifestations of Sotos
syndrome in Japanese patients who presented with hyperinsulinemic hypoglycemia
of infancy. We recruited patients diagnosed as having Sotos syndrome who
presented with the complication of hyperinsulinemia during the neonatal period
using a survey of the abstracts of Pediatric Meetings in domestic areas of Japan
from 2007 to 2011. As a result, five patients (four females and one male) were
recruited to evaluate the clinical presentation of Sotos syndrome by reference
to the clinical record of each patient. A 5q35 deletion including the NSD1 gene
was detected in all patients. Major anomalies in the central nervous,
cardiovascular, and genito-urinary systems were frequently found. Hypoglycemia
occurred between 0.5 and 3 hr after birth and high levels of insulin were
initially found within 3 days of birth. The patients were treated with
intravenous glucose infusion at a maximum rate of 4.6-11.0 mg/kg/min for 12-49
days. Three of the five patients required nasal tube feeding. One patient
received medical treatment with diazoxide. This study shows that patients with
Sotos syndrome may present with transient hyperinsulinemic hypoglycemia in the
neonatal period. CONTEXT: Sotos syndrome is a rare genetic disorder with a distinct phenotypic
spectrum including overgrowth and learning difficulties. Here we describe a new
case of Sotos syndrome with a 5q35 microdeletion, affecting the fibroblast
growth factor receptor 4 (FGFR4) gene, presenting with infantile hypercalcemia.
OBJECTIVE: We strove to elucidate the evanescent nature of the observed
hypercalcemia by studying the ontogenesis of FGFR3 and FGFR4, which are both
associated with fibroblast growth factor (FGF) 23-mediated mineral homeostasis,
in the developing human kidney.
DESIGN: Quantitative RT-PCR and immunohistochemical analyses were used on
archival human kidney samples to investigate the expression of the FGFR
signaling pathway during renal development.
RESULTS: We demonstrated that renal gene and protein expression of both FGFRs
increased during fetal development between the gestational ages (GAs) of 14-40
weeks. Yet FGFR4 expression increased more rapidly as compared with FGFR3 (slope
0.047 vs 0.0075, P = .0018). Moreover, gene and protein expression of the
essential FGFR coreceptor, Klotho, also increased with a significant positive
correlation between FGFR and Klotho mRNA expression during renal development.
Interestingly, we found that perinatal FGFR4 expression (GA 38-40 wk) was 7-fold
higher as compared with FGFR3 (P = .0035), whereas in adult kidney tissues,
FGFR4 gene expression level was more than 2-fold lower compared with FGFR3 (P =
.0029), thus identifying a molecular developmental switch of FGFR isoforms.
CONCLUSION: We propose that the heterozygous FGFR4 deletion, as observed in the
Sotos syndrome patient, leads to a compromised FGF23 signaling during infancy
accounting for transient hypercalcemia. These findings represent a novel and
intriguing view on FGF23 mediated calcium homeostasis. Sotos syndrome (SoS) is a multiple anomaly, congenital disorder characterized by
overgrowth, macrocephaly, distinctive facial features and variable degree of
intellectual disability. Haploinsufficiency of the NSD1 gene at 5q35.3, arising
from 5q35 microdeletions, point mutations, and partial gene deletions, accounts
for a majority of patients with SoS. Recently, mutations and possible
pathogenetic rare CNVs, both affecting a few candidate genes for overgrowth,
have been reported in patients with Sotos-like overgrowth features. To estimate
the frequency of NSD1 defects in the Brazilian SoS population and possibly
reveal other genes implicated in the etiopathogenesis of this syndrome, we
collected a cohort of 21 Brazilian patients, who fulfilled the diagnostic
criteria for SoS, and analyzed the NSD1 and PTEN genes by means of multiplex
ligation-dependent probe amplification and mutational screening analyses. We
identified a classical NSD1 microdeletion, a novel missense mutation (p.C1593W),
and 2 previously reported truncating mutations: p.R1984X and p.V1760Gfs*2. In
addition, we identified a novel de novo PTEN gene mutation (p.D312Rfs*2) in a
patient with a less severe presentation of SoS phenotype, which did not include
pre- and postnatal overgrowth. For the first time, our study implies PTEN in the
pathogenesis of SoS and further emphasizes the existence of ethno-geographical
differences in NSD1 molecular alterations between patients with SoS from
Europe/North America (70-93%) and those from South America (10-19%). Sotos syndrome is a well-known overgrowth syndrome characterized by excessive
growth during childhood, macrocephaly, distinctive facial appearance and
learning disability. This disorder is caused by mutations or deletions in NSD1
gene. The aim of this study is to examine the relationship between the
neuroimaging and clinical features of children with Sotos syndrome. Six Turkish
children with Sotos syndrome were followed up about 3-7 years. The diagnosis was
confirmed with molecular genetic analysis. We identified the pathogenic NSD1
mutation including three novel in all patients. All the patients had a
characteristic facial gestalt of Sotos syndrome consisting of triangular face
with prominent forehead, frontoparietal sparseness of hair and small nose.
However, the degree of psychomotor and intellectual development was variable.
Severe learning defect and speech delay were remarkable in two patients. The
neuroimaging analysis showed abnormalities in four of six patients including
bilateral large ventricles, thinning of the corpus callosum and persistent cavum
septum pellucidum et vergae. Typical craniofacial appearance is the primary
finding for the diagnosis of the disease even in the infantile period. However,
the degree of psychomotor and intellectual development is very variable and does
not correlate with the neuroimaging findings. |
What is the CRAPome database? | The CRAPome is a contaminant repository for affinity purification-mass spectrometry data. | Affinity purification coupled with mass spectrometry (AP-MS) is a widely used
approach for the identification of protein-protein interactions. However, for
any given protein of interest, determining which of the identified polypeptides
represent bona fide interactors versus those that are background contamits
(for example, proteins that interact with the solid-phase support, affinity
reagent or epitope tag) is a challenging task. The standard approach is to
identify nonspecific interactions using one or more negative-control
purifications, but many small-scale AP-MS studies do not capture a complete,
accurate background protein set when available controls are limited.
Fortunately, negative controls are largely bait independent. Hence, aggregating
negative controls from multiple AP-MS studies can increase coverage and improve
the characterization of background associated with a given experimental
protocol. Here we present the contamit repository for affinity purification
(the CRAPome) and describe its use for scoring protein-protein interactions. The
repository (currently available for Homo sapiens and Saccharomyces cerevisiae)
and computational tools are freely accessible at http://www.crapome.org/. |
List all articles on network meta-analysis for smoking cessation | Cardiovascular events associated with smoking cessation pharmacotherapies: a network meta-analysis.
Pharmacological interventions for smoking cessation: an overview and network meta-analysis
Effectiveness and cost-effectiveness of computer and other electronic aids for smoking cessation: a systematic review and network meta-analysis.
Smoking cessation interventions in COPD: a network meta-analysis of randomised trials. | The aim of this study was to rank order the effectiveness of smoking cessation
interventions for chronic obstructive pulmonary disease (COPD) patients. We
searched 10 databases to identify randomised trials of smoking cessation
counselling (SCC) with or without pharmacotherapy or nicotine replacement
therapy (NRT). We conducted a network meta-analysis using logistic regression
analyses to assess the comparative effectiveness of smoking cessation
interventions while preserving randomisation of each trial. The analysis of
7,372 COPD patients from six out of eight identified trials showed that SCC in
combination with NRT had the greatest effect on prolonged abstinence rates
versus usual care (OR 5.08, p<0.0001) versus SCC alone (2.80, p = 0.001) and
versus SCC combined with an antidepressant (1.53, p = 0.28). The second most
effective intervention was SCC combined with an antidepressant (3.32, p = 0.002)
versus SCC alone (1.83, p = 0.007), with no difference between antidepressants.
SCC alone was of borderline superiority compared with usual care (1.81, p =
0.07). A small body of evidence suggests that SCC combined with NRT is more
effective than other combinations and single smoking cessation treatments in
COPD, but substantially more research is needed for this most important COPD
treatment. BACKGROUND: Network meta-analysis is becoming increasingly popular for
establishing comparative effectiveness among multiple interventions for the same
disease. Network meta-analysis inherits all methodological challenges of
standard pairwise meta-analysis, but with increased complexity due to the
multitude of intervention comparisons. One issue that is now widely recognized
in pairwise meta-analysis is the issue of sample size and statistical power.
This issue, however, has so far only received little attention in network
meta-analysis. To date, no approaches have been proposed for evaluating the
adequacy of the sample size, and thus power, in a treatment network.
FINDINGS: In this article, we develop easy-to-use flexible methods for
estimating the 'effective sample size' in indirect comparison meta-analysis and
network meta-analysis. The effective sample size for a particular treatment
comparison can be interpreted as the number of patients in a pairwise
meta-analysis that would provide the same degree and strength of evidence as
that which is provided in the indirect comparison or network meta-analysis. We
further develop methods for retrospectively estimating the statistical power for
each comparison in a network meta-analysis. We illustrate the performance of the
proposed methods for estimating effective sample size and statistical power
using data from a network meta-analysis on interventions for smoking cessation
including over 100 trials.
CONCLUSION: The proposed methods are easy to use and will be of high value to
regulatory agencies and decision makers who must assess the strength of the
evidence supporting comparative effectiveness estimates. BACKGROUND: Smoking is the leading preventable cause of illness and premature
death worldwide. Some medications have been proven to help people to quit, with
three licensed for this purpose in Europe and the USA: nicotine replacement
therapy (NRT), bupropion, and varenicline. Cytisine (a treatment
pharmacologically similar to varenicline) is also licensed for use in Russia and
some of the former socialist economy countries. Other therapies, including
nortriptyline, have also been tested for effectiveness.
OBJECTIVES: How do NRT, bupropion and varenicline compare with placebo and with
each other in achieving long-term abstinence (six months or longer)? How do the
remaining treatments compare with placebo in achieving long-term abstinence? How
do the risks of adverse and serious adverse events (SAEs) compare between the
treatments, and are there instances where the harms may outweigh the benefits?
METHODS: The overview is restricted to Cochrane reviews, all of which include
randomised trials. Participants are usually adult smokers, but we exclude
reviews of smoking cessation for pregt women and in particular disease groups
or specific settings. We cover nicotine replacement therapy (NRT),
antidepressants (bupropion and nortriptyline), nicotine receptor partial
agonists (varenicline and cytisine), anxiolytics, selective type 1 cannabinoid
receptor antagonists (rimonabant), clonidine, lobeline, dianicline,
mecamylamine, Nicobrevin, opioid antagonists, nicotine vaccines, and silver
acetate. Our outcome for benefit is continuous or prolonged abstinence at least
six months from the start of treatment. Our outcome for harms is the incidence
of serious adverse events associated with each of the treatments. We searched
the Cochrane Database of Systematic Reviews (CDSR) in The Cochrane Library, for
any reviews with 'smoking' in the title, abstract or keyword fields. The last
search was conducted in November 2012. We assessed methodological quality using
a revised version of the AMSTAR scale. For NRT, bupropion and varenicline we
conducted network meta-analyses, comparing each with the others and with placebo
for benefit, and varenicline and bupropion for risks of serious adverse events.
MAIN RESULTS: We identified 12 treatment-specific reviews. The analyses covered
267 studies, involving 101,804 participants. Both NRT and bupropion were
superior to placebo (odds ratios (OR) 1.84; 95% credible interval (CredI) 1.71
to 1.99, and 1.82; 95% CredI 1.60 to 2.06 respectively). Varenicline increased
the odds of quitting compared with placebo (OR 2.88; 95% CredI 2.40 to 3.47).
Head-to-head comparisons between bupropion and NRT showed equal efficacy (OR
0.99; 95% CredI 0.86 to 1.13). Varenicline was superior to single forms of NRT
(OR 1.57; 95% CredI 1.29 to 1.91), and to bupropion (OR 1.59; 95% CredI 1.29 to
1.96). Varenicline was more effective than nicotine patch (OR 1.51; 95% CredI
1.22 to 1.87), than nicotine gum (OR 1.72; 95% CredI 1.38 to 2.13), and than
'other' NRT (inhaler, spray, tablets, lozenges; OR 1.42; 95% CredI 1.12 to
1.79), but was not more effective than combination NRT (OR 1.06; 95% CredI 0.75
to 1.48). Combination NRT also outperformed single formulations. The four
categories of NRT performed similarly against each other, apart from 'other'
NRT, which was marginally more effective than NRT gum (OR 1.21; 95% CredI 1.01
to 1.46). Cytisine (a nicotine receptor partial agonist) returned positive
findings (risk ratio (RR) 3.98; 95% CI 2.01 to 7.87), without significant
adverse events or SAEs. Across the 82 included and excluded bupropion trials,
our estimate of six seizures in the bupropion arms versus none in the placebo
arms was lower than the expected rate (1:1000), at about 1:1500. SAE
meta-analysis of the bupropion studies demonstrated no excess of
neuropsychiatric (RR 0.88; 95% CI 0.31 to 2.50) or cardiovascular events (RR
0.77; 95% CI 0.37 to 1.59). SAE meta-analysis of 14 varenicline trials found no
difference between the varenicline and placebo arms (RR 1.06; 95% CI 0.72 to
1.55), and subgroup analyses detected no significant excess of neuropsychiatric
events (RR 0.53; 95% CI 0.17 to 1.67), or of cardiac events (RR 1.26; 95% CI
0.62 to 2.56). Nortriptyline increased the chances of quitting (RR 2.03; 95% CI
1.48 to 2.78). Neither nortriptyline nor bupropion were shown to enhance the
effect of NRT compared with NRT alone. Clonidine increased the chances of
quitting (RR 1.63; 95% CI 1.22 to 2.18), but this was offset by a dose-dependent
rise in adverse events. Mecamylamine in combination with NRT may increase the
chances of quitting, but the current evidence is inconclusive. Other treatments
failed to demonstrate a benefit compared with placebo. Nicotine vaccines are not
yet licensed for use as an aid to smoking cessation or relapse prevention.
Nicobrevin's UK license is now revoked, and the manufacturers of rimonabant,
taranabant and dianicline are no longer supporting the development or testing of
these treatments.
AUTHORS' CONCLUSIONS: NRT, bupropion, varenicline and cytisine have been shown
to improve the chances of quitting. Combination NRT and varenicline are equally
effective as quitting aids. Nortriptyline also improves the chances of quitting.
On current evidence, none of the treatments appear to have an incidence of
adverse events that would mitigate their use. Further research is warranted into
the safety of varenicline and into cytisine's potential as an effective and
affordable treatment, but not into the efficacy and safety of NRT. BACKGROUND: Stopping smoking is associated with many important improvements in
health and quality of life. The use of cessation medications is recommended to
increase the likelihood of quitting. However, there is historical and renewed
concern that smoking cessation therapies may increase the risk of cardiovascular
disease events associated within the quitting period. We aimed to examine
whether the 3 licensed smoking cessation therapies-nicotine replacement therapy,
bupropion, and varenicline-were associated with an increased risk of
cardiovascular disease events using a network meta-analysis.
METHODS AND RESULTS: We searched 10 electronic databases, were in communication
with authors of published randomized, clinical trials (RCTs), and accessed
internal US Food and Drug Administration reports. We included any RCT of the 3
treatments that reported cardiovascular disease outcomes. Among 63 eligible RCTs
involving 21 nicotine replacement therapy RCTs, 28 bupropion RCTs, and 18
varenicline RCTs, we found no increase in the risk of all cardiovascular disease
events with bupropion (relative risk [RR], 0.98; 95% confidence interval [CI],
0.54-1.73) or varenicline (RR, 1.30; 95% CI, 0.79-2.23). There was an elevated
risk associated with nicotine replacement therapy that was driven predomitly
by less serious events (RR, 2.29; 95% CI, 1.39-3.82). When we examined major
adverse cardiovascular events, we found a protective effect with bupropion (RR,
0.45; 95% CI, 0.21-0.85) and no clear evidence of harm with varenicline (RR,
1.34; 95% CI, 0.66-2.66) or nicotine replacement therapy (RR, 1.95; 95% CI,
0.26-4.30).
CONCLUSION: Smoking cessation therapies do not appear to raise the risk of
serious cardiovascular disease events. |
Can RG7112 inhibit MDM2? | Yes, RG7112 is a small molecule MDM2 antagonist. | Increasing knowledge of the relationship between p53 and MDM2 has led to
development of potential small molecule inhibitors useful for clinical studies.
Herein, we discuss the patented (2006-2010) inhibitors of p53-MDM2 interaction.
The anticancer agents discussed in this review belong to several different
chemical classes including benzodiazepinediones, cis-imidazolines, oxindoles,
spiro-oxindoles, and numerous miscellaneous groups. This review also provides
comprehensive information on inhibitors of p53-MDM2 interaction that are
currently being tested in clinical trials. It is important to note that many of
the disclosed inhibitors need further validation to be considered as bona fide
inhibitors of p53-MDM2 interaction and some will not be further considered for
future studies. On the other hand, JNJ-26854165, a novel tryptamine derivative
and RG7112, a cis-imidazoline representative have shown promising results in
early phases of trials in cancer patients. AT-219, a spiroindolinone in late
stage preclinical studies is a likely candidate to proceed into clinical trials.
It remains to be seen how these inhibitors will perform in future clinical
studies as single agents and in combination with the currently approved
chemotherapeutic agents. BACKGROUND: RG7112 is a selective inhibitor of p53-MDM2 binding that frees p53
from negative control, activating the p53 pathway in cancer cells leading to
cell cycle arrest and apoptosis. RG7112 was selected for evaluation by the
Pediatric Preclinical Testing Program (PPTP) due to the relatively low incidence
of p53 mutations in pediatric cancers compared with adult maligcies.
PROCEDURES: RG7112 and its inactive etiomer RG7112i were evaluated against
the 23 cell lines of the PPTP in vitro panel using 96 hours exposure (1 nM to 10
µM). It was tested against the PPTP in vivo panel focusing on p53 wild-type (WT)
xenografts at a dose of 100 mg/kg daily for 14 days followed by 4 weeks of
observation. Response outcomes were related to MDM2 and p53 expression datasets
(http://pptp.nchresearch.org/data.html).
RESULTS: RG7112 demonstrated cytotoxic activity with a lower median IC(50) for
p53 WT versus p53 mutant cell lines (approximately 0.4 µM vs. >10 µM,
respectively). RG7112 induced tumor growth inhibition meeting criteria for
intermediate activity (EFS T/C > 2) in 10 of 26 (38%) solid tumor xenografts.
Objective responses included medulloblastoma, alveolar rhabdomyosarcoma, Wilms,
rhabdoid and Ewing sarcoma xenografts. For the ALL panel, there was one partial
response, five complete responses and one maintained complete response. The ALL
xenografts expressed the highest levels of p53 among the PPTP panels.
CONCLUSIONS: RG7112 induced tumor regressions in solid tumors from different
histotype panels, and exhibited consistent high-level activity against ALL
xenografts. This high level of activity supports prioritization of RG7112 for
further evaluation. BACKGROUND: We report a proof-of-mechanism study of RG7112, a small-molecule
MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed
well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were
eligible for resection.
METHODS: Patients with well-differentiated or dedifferentiated liposarcoma were
enrolled at four centres in France. Patients received up to three 28-day
neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a
patient progressed at any point after the first cycle, the lesion was resected
or, if unresectable, an end-of-study biopsy was done. The primary endpoint was
to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation
(P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and
apoptosis). All analyses were per protocol. This trial is registered with
EudraCT, number 2009-015522-10.
RESULTS: Between June 3, and Dec 14, 2010, 20 patients were enrolled and
completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type
tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had
MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations,
assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR
4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day
8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression
had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline.
The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR
-12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of
MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed
partial response and 14 had stable disease. All patients experienced at least
one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia
(nine patients), diarrhoea (nine patients), and thrombocytopenia (eight
patients). There were 12 serious adverse events in eight patients, the most
common of which were neutropenia (six patients) and thrombocytopenia (three
patients).
DISCUSSION: MDM2 inhibition activates the P53 pathway and decreases cell
proliferation in MDM2-amplified liposarcoma. This study suggests that it is
feasible to undertake neoadjuvant biopsy-driven biomarker studies in
liposarcoma.
FUNDING: F Hoffmann-La Roche. MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2
as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding
that can reactivate p53 in cancer cells may offer an effective approach for
cancer therapy. RG7112 is a potent and selective member of the nutlin family of
MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with
high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A
crystal structure of the RG7112-MDM2 complex revealed that the small molecule
binds in the p53 pocket of MDM2, mimicking the interactions of critical p53
amino acid residues. Treatment of cancer cells expressing wild-type p53 with
RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis.
RG7112 showed potent antitumor activity against a panel of solid tumor cell
lines. However, its apoptotic activity varied widely with the best response
observed in osteosarcoma cells with MDM2 gene amplification. Interestingly,
inhibition of caspase activity did not change the kinetics of p53-induced cell
death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic
concentrations caused dose-dependent changes in proliferation/apoptosis
biomarkers as well as tumor inhibition and regression. Notably, RG7112 was
highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our
findings offer a preclinical proof-of-concept that RG7112 is effective in
treatment of solid tumors expressing wild-type p53. Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been
considered an attractive approach for cancer treatment. However, the hydrophobic
protein-protein interaction surface represents a significant challenge for the
development of small-molecule inhibitors with desirable pharmacological
profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical
development. Here, we report the discovery and characterization of a second
generation clinical MDM2 inhibitor, RG7388, with superior potency and
selectivity. The tumor suppressor p53 is thought to play a role in megakaryocyte (MK)
development. To assess the influence of the p53 regulatory pathway further, we
studied the effect of RG7112, a small molecule MDM2 antagonist that activates
p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and
platelet production. This drug has been previously been evaluated in clinical
trials of cancer patients where thrombocytopenia was one of the major
dose-limiting toxicities. In this study, we demonstrated that administration of
RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we
identified two distinct mechanisms by which RG7112-mediated activation of p53
affected human megakaryocytopoiesis and platelet production in vitro. RG7112
promoted apoptosis of MK progenitor cells, resulting in a reduction of their
numbers and RG7112 affected mature MK by blocking DNA synthesis during
endomitosis and impairing platelet production. Together, the disruption of these
events provides an explanation for RG7112-induced thrombocytopenia and insight
into the role of the p53-MDM2 auto-regulatory loop in normal
megakaryocytopoiesis. PURPOSE: Antitumor clinical activity has been demonstrated for the MDM2
antagonist RG7112, but patient tolerability for the necessary daily dosing was
poor. Here, utilizing RG7388, a second-generation nutlin with superior
selectivity and potency, we determine the feasibility of intermittent dosing to
guide the selection of initial phase I scheduling regimens.
EXPERIMENTAL DESIGN: A pharmacokinetic-pharmacodynamic (PKPD) model was
developed on the basis of preclinical data to determine alternative dosing
schedule requirements for optimal RG7388-induced antitumor activity. This PKPD
model was used to investigate the pharmacokinetics of RG7388 linked to the
time-course of the antitumor effect in an osteosarcoma xenograft model in mice.
These data were used to prospectively predict intermittent and continuous dosing
regimens, resulting in tumor stasis in the same model system.
RESULTS: RG7388-induced apoptosis was delayed relative to drug exposure with
continuous treatment not required. In initial efficacy testing, daily dosing at
30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically
equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was
equivalent to 10 mg/kg given daily. The implementation of modeling and
simulation on these data suggested several possible intermittent clinical dosing
schedules. Further preclinical analyses confirmed these schedules as viable
options.
CONCLUSION: Besides chronic administration, antitumor activity can be achieved
with intermittent schedules of RG7388, as predicted through modeling and
simulation. These alternative regimens may potentially ameliorate tolerability
issues seen with chronic administration of RG7112, while providing clinical
benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd)
schedules were selected for RG7388 clinical testing. In this issue of Blood, Lu et al describe the cooperation between an orally
bioavailable mouse double minute 2 (MDM2) antagonist (RG7112) and the pegylated
interferon α (Peg-IFNα 2a) to target JAK2V617F hematopoietic progenitors and
stem cells. Their work provides a rationale for the treatment of patients
suffering from myeloproliferative neoplasms (MPNs). |
Which clotting factor is inhibited by betrixaban? | Betrixaban is an orally administered direct clotting factor Xa inhibitor. | Systematic SAR studies of in vitro factor Xa inhibitory activity around compound
1 were performed by modifying each of the three phenyl rings. A class of highly
potent, selective, efficacious and orally bioavailable direct factor Xa
inhibitors was discovered. These compounds were screened in hERG binding assays
to examine the effects of substitution groups on the hERG channel affinity. From
the leading compounds, betrixaban (compound 11, PRT054021) has been selected as
the clinical candidate for development. Anticoagulation is recommended for prophylaxis and treatment of venous
thromboembolism (VTE) (deep vein thrombosis and pulmonary embolism) and/or
arterial thromboembolism. The therapeutic arsenal of anticoagulants available to
clinicians is mainly composed by unfractionated heparin (UFH),
low-molecular-weight heparin (LMWH), fondaparinux and oral vitamin K antagonists
(VKA) (i.e. warfarin and acenocumarol). These anticoagulants are effective, but
they require parenteral administration (UFH, LMWH, fondaparinux) and/or frequent
anticoagulant monitoring (intravenous UFH, oral VKA). Novel anticoagulants in
clinical testing include orally active direct factor II inhibitors [dabigatran
etexilate (BIBR 1048), AZD0837)], parenteral direct factor II inhibitors
(flovagatran sodium), orally active direct factor X inhibitors [rivaroxaban (BAY
59-7939), apixaban, betrixaban, YM150, DU-176b, LY-517717, GW813893, TAK-442, PD
0348292] and new parenteral FXa inhibitors [idraparinux, idrabiotaparinux
(biotinilated idraparinux; SSR 126517), ultra-low-molecular-weight heparins
(ULMWH: AVE5026, RO-14)]. These new compounds have the potential to complement
heparins and fondaparinux for short-term anticoagulation and/or to replace VKA
for long-term anticoagulation in most patients. Dabigatran and rivaroxaban have
been the firsts of the new oral anticoagulants to be licensed for the prevention
of VTE after hip and knee replacement surgery. In the present review, we discuss
the pharmacology of new anticoagulants, the key points necessary for
interpreting the results of studies on VTE prophylaxis and treatment, the
results of clinical trials testing these new compounds and their potential
advantages and drawbacks over existing therapies. Atrial fibrillation is already the most common clinically significant cardiac
arrhythmia and a common cause of stroke. Vitamin K antagonists are very
effective for the prevention of cardioembolic stroke but have numerous
limitations that limit their uptake in eligible patients with AF and reduce
their effectiveness in treated patients. Multiple new anticoagulants are under
development as potential replacements for vitamin K antagonists. Most are small
synthetic molecules that target factor IIa (e.g., dabigatran etexilate,
AZD-0837) or factor Xa (e.g., rivaroxaban, apixaban, betrixaban, DU176b,
idrabiotaparinux). These drugs have predictable pharmacokinetics that allow
fixed dosing without laboratory monitoring, and are being compared with vitamin
K antagonists or aspirin in phase III clinical trials [corrected]. A new vitamin
K antagonist (ATI-5923) with improved pharmacological properties compared with
warfarin is also being evaluated in a phase III trial. None of the new agents
have as yet been approved for clinical use. Venous thromboembolism (VTE), which includes deep vein thrombosis and pulmonary
embolism, is a major cause of morbidity and mortality in patients undergoing
major orthopedic surgery, and routine thromboprophylaxis has been the standard
of care over the last 20 years. Currently available options for the prevention
of VTE in major orthopedic surgery include low-molecular-weight heparins,
vitamin K antagonists and, more recently, the synthetic pentasaccharide
fondaparinux. Although effective, these drugs have several limitations, and new
oral antithrombotics offering predictable, effective and safe anticoagulation
are strongly needed. This overview focuses on the most advanced oral direct
inhibitors of factor Xa, rivaroxaban, apixaban, LY517717, YM150 and betrixaban.
Specifically, the results of phase II and III studies and the designs of ongoing
clinical trials in patients undergoing elective hip and knee replacement are
reviewed. Interpatient variability in the safety and efficacy of oral anticoagulation with
warfarin presents several challenges to clinicians, thus underscoring the
emergent need for new orally available anticoagulants with predictable
pharmacokinetic and pharmacodynamic profiles and ability to target circulating
clotting factors. Seven compounds including rivaroxaban, apixaban, betrixaban,
and eribaxaban are orally available direct inhibitors of activated factor X
currently in development for the prevention and treatment of venous
thromboembolism and for thromboprophylaxis in patients with atrial fibrillation
or following an acute coronary syndrome. At doses used in phase 2 and 3 clinical
trials, rivaroxaban and apixaban demonstrated a predictable onset of effect,
maximal plasma concentration, and half-life that was unaffected by age, renal,
or hepatic disease. In clinical trials for the treatment and prevention of
venous thromboembolism, rivaroxaban and apixaban produced equivalent or superior
reductions in the development or progression of venous thromboembolism compared
with either low molecular weight heparin or warfarin. Trials comparing the
efficacy of rivaroxaban or apixaban to standard therapy for stroke prophylaxis
in patients with atrial fibrillation are in process. Rivaroxaban, the sentinel
compound in this class, is already approved in the European Union and Canada. It
is likely to be approved for use in the United States in 2010. PURPOSE OF REVIEW: Prevention of stroke and systemic emboli is paramount in the
management of atrial fibrillation. Although warfarin is the predomit
anticoagulant used in patients with atrial fibrillation, it has significant
limitations that have impeded appropriate use of stroke prophylaxis in eligible
patients with atrial fibrillation. Consequently, much research has been focused
on finding an alternative to warfarin. We review the potential alternatives in
development and evaluate the current evidence concerning their safety and
efficacy.
RECENT FINDINGS: Oral direct factor Xa inhibitors are potentially well tolerated
and effective replacements for warfarin. These agents do not require cofactors
and offer selective inhibition at a critical step of amplification in the
coagulation cascade. Multiple direct anti-factor Xa agents are currently
undergoing evaluation in phase I, II, and III trials. Early results suggest that
these novel anticoagulants have favorable pharmacokinetic and pharmacodynamic
profiles with minimal-to-no requirements for therapeutic monitoring. Two direct
factor Xa inhibitors are emerging from phase II trials (betrixaban and YM150)
and three are being evaluated in phase III trials (apixaban, edoxaban, and
rivaroxaban) for the prevention of stroke and systemic emboli in patients with
atrial fibrillation. The phase III trials of apixaban and rivaroxaban have
completed enrollment and are in the follow-up phase.
SUMMARY: Given the growing population of patients with atrial fibrillation,
there is a great interest in finding new therapies for oral anticoagulation. The
direct factor Xa inhibitors may offer several promising alternatives to warfarin
therapy. Heparin and low molecular weight heparins have limitations in their efficacy and
safety for the prevention and treatment of venous thromboembolism (VTE). New
synthetic antithrombotic drugs, designed with the intention of improving the
therapeutic window for prophylaxis and treatment, are in various stages of
development. Synthetic pentasaccharides include fondaparinux and its long-acting
analogue idraparinux. Dabigatran is a direct thrombin inhibitor that has
undergone clinical trials for VTE prophylaxis and treatment. Direct factor Xa
inhibitors include rivaroxiban, which has shown promising results for VTE
prophylaxis and is being studied for VTE treatment, as well as apixaban and
betrixaban, which are at earlier stages of clinical validation. These newer
agents may represent viable options for prophylaxis and therapy as further
clinical studies are performed. Atrial fibrillation (AF) is the most common cardiac rhythm disorder and a major
risk factor for ischemic stroke. Antithrombotic therapy using aspirin or vitamin
K antagonists (VKA) is currently prescribed for prevention for ischemic stroke
in patients with AF. A narrow therapeutic range and the need of regular
monitoring of its anticoagulatory effect impair effectiveness and safety of VKA,
causing a need for alternative anticoagulant drugs. Recently developed
anticoagulants include direct thrombin antagonists such as dabigatran or factor
Xa inhibitors such as rivaroxaban, apixaban, betrixaban, and edoxaban.
Currently, data from a phase III clinical trial are available for dabigatran
only, which show the direct thrombin antagonist to be at least noninferior in
efficacy to VKA for the prevention of stroke and systemic embolism in patients
with AF. This review focuses on current advances in the development of directly
acting oral anticoagulant drugs and their potential to replace the VKA class of
drugs in patients with AF. Warfarin has been the effective treatment in the prophylaxis of cardioembolism,
in particular in patients with atrial fibrillation, for more than 50 years.
Nevertheless, many patients with atrial fibrillation are not currently treated
because of the numerous limits of oral anticoagulation and in those treated the
quality of anticoagulation is often poor. Novel oral anticoagulant drugs, the
direct thrombin antagonist dabigatran and factor Xa inhibitors such as
rivaroxaban, apixaban, edoxaban, and betrixaban are more predictable and
convenient anticoagulants in comparison with warfarin, mainly because of the
non-requirement of regular laboratory monitoring and dose adjustments. Current
data from phase III clinical trials are available for dabigatran, rivaroxaban
and apixaban, which show to be at least noninferior in efficacy to warfarin for
the prevention of stroke in patients with atrial fibrillation. This review
focuses on the potential of novel anticoagulants to replace warfarin in patients
with atrial fibrillation. Also the place in therapy and the potential
limitations of the new agents in clinical practice represent important issues to
be considered. The promise of new oral anticoagulants gives us the hope that
warfarin will finally be replaced in a near future, but more importantly that
anticoagulant undertreatment of atrial fibrillation will be partially overcome. Safe and effective stroke prevention in atrial fibrillation (AF) is crucial as
the number of patients with this condition continues to increase. Several novel
oral anticoagulants are being developed as replacements for warfarin for this
indication. Direct factor Xa inhibitors comprise the largest class of oral
anticoagulants in development; the inhibition of factor Xa is recognized to be a
promising target for therapeutic anticoagulation, partly because of its location
in the coagulation cascade. Apixaban, betrixaban, edoxaban, and rivaroxaban are
small-molecule, selective inhibitors that directly and reversibly bind to the
active site of factor Xa. Their pharmacokinetic and pharmacodynamic profiles
vary, which might allow patient-specific therapy. Several of these agents have
been tested in clinical trials for various indications, including AF, with
favorable results. In particular, apixaban and rivaroxaban have shown
superiority and noninferiority, respectively, to warfarin in phase III clinical
trials for stroke prevention in AF. These agents have also been shown to be safe
in terms of bleeding risk. Despite these advantages, factor Xa inhibitors have
several characteristics, such as potential interactions with other drugs
(inhibitors of cytochrome P450 and P-glycoprotein) and the inability to reverse
their anticoagulant effects, as well as concerns about poor patient compliance,
which must be considered when initiating patients on a novel factor Xa
inhibitor. Atrial fibrillation (AF), the most common, clinically significant, cardiac
arrhythmia affects 1% of the general population and has important hemodynamic
and thromboembolic complications that contribute to elevated morbidity and
mortality. AF increases the overall risk of stroke five-fold, accounting for
approximately 15% of all strokes and is associated with particularly severe
stroke. For the last 50 years, long-term anticoagulation with vitamin K
antagonists has been the most effective therapy for preventing stroke and
systemic embolism in patients with AF and other risk factors, but their use has
a lot of limitations and drawbacks (frequent monitoring and dose adjustment,
food and drug interactions, delayed onset of action etc). Nowadays, new oral
anticoagulants have emerged that seem to overcome those limitations. Direct
thrombin inhibitor dabigatran and factor Xa inhibitors rivaroxaban and apixaban
have proven, in large, multicenter, randomized, phase III, clinical studies, to
be at least as efficient as warfarin in stroke prevention in patients with AF.
RELY and ROCKET AF trials have contributed to market approval of dabigatran and
rivaroxaban, respectively and made them available to clinical practice. Another
factor Xa inhibitor, edoxaban, is under evaluation in an ongoing phase III
clinical trial and others such as AZD0837, betrixaban and darexaban are still in
safety and tolerability phase II studies. The oral anticoagulation landscape is
changing rapidly and these new agents seem to be very promising. However future
post-marketing studies and registries will help clarify their efficacy and
safety. Warfarin, heparin and their derivatives have been the traditional anticoagulants
used for prophylaxis and treatment of venous thromboembolism. While the modern
clinician is familiar with the efficacy and pharmacokinetics of these agents,
their adverse effects have provided the impetus for the development of newer
anticoagulants with improved safety, ease of administration, more predictable
pharmacodynamics and comparable efficacy. Research into haemostasis and the
coagulation cascade has made the development of these newer anticoagulants
possible. These drugs include the factor Xa inhibitors and IIa (thrombin)
inhibitors. Direct and indirect factor Xa inhibitors are being developed with a
relative rapid onset of action and stable pharmacokinetic profiles negating the
need for close monitoring; this potentially makes them a more attractive option
than heparin or warfarin. Examples of direct factor Xa inhibitors include
apixaban, rivaroxaban, otamixaban, betrixaban and edoxaban. Examples of indirect
factor Xa inhibitors include fondaparinux, idraparinux and idrabiotaparinux.
Direct thrombin inhibitors (factor IIa inhibitors) were developed with the
limitations of standard heparin and warfarin in mind. Examples include
recombit hirudin (lepirudin), bivalirudin, ximelagatran, argatroban, and
dabigatran etexilate. This review will discuss emerging novel anticoagulants and
their use for the prophylaxis and management of venous thromboembolism, for
stroke prevention in nonvalvular atrial fibrillation and for coronary artery
disease. Following the clinical approval of novel oral anticoagulants as alternatives to
the vitamin K antagonists, many additional novel oral anticoagulant drugs are
currently in early and advanced stages of clinical development. The majority of
the drugs in development belong to the class of direct factor Xa inhibitors (the
-xabans). These include betrixaban, letaxaban, darexaban, eribaxaban, and
LY517717. Another representative of the class of orally available direct
thrombin inhibitors (the -gatrans) is known as AZD0837. Furthermore other
coagulation factors with central roles within the coagulation cascade are
currently investigated as potential targets for the development of novel oral
anticoagulant drugs. Among those, the first direct oral factor IXa inhibitor
TTP889 has entered the clinical phase of development. A short summary of novel
oral anticoagulant currently in earlier stages of clinical development is
provided. Introduction Over the past 60 years, clinicians have used vitamin K antagonists,
primarily warfarin, as the sole oral anticoagulants for managing a variety of
thrombotic disorders. Warfarin, which requires frequent monitoring, has a
variable dose response, a narrow therapeutic index, and numerous drug and
dietary interactions. However, intravenous and subcutaneous agents, such as
unfractionated heparin, low-molecular-weight heparin, direct thrombin
inhibitors, and pentasaccharide, have been introduced over the past 30 years for
managing thromboembolic disorders. Recently, 5 new oral anticoagulants,
dabigatran, rivaroxaban, apixaban, endoxaban, and betrixaban, have been
introduced into clinical trials. Apixaban, rivaroxaban, endoxaban, and
betrixaban are specific direct inhibitors of factor Xa, while dabigatran
inhibits factor IIa. These drugs have a pharmacological profile that does not
require monitoring in order to adjust therapy, which is the mainstay of warfarin
management. In addition, these new medications have not shown any major issues
regarding food interactions; rather, they demonstrate the potential for limited
drug-drug interactions due to their limited metabolism through the cytochrome
P450 system. This unique pharmacokinetic profile may provide clinicians with a
new era of managing thromboembolic disorders. Two of these agents, dabigatran
and rivaroxaban, have been approved by the US Food and Drug Administration (FDA)
for stroke prevention in patients with nonvalvular atrial fibrillation (AF); in
addition, rivaroxaban can be used in the prevention of venous thromboembolism
(VTE) in total hip and knee arthroplasty during the acute and extended periods
of risk. However, the challenge for hospital formularies will be the appropriate
use and management of these new medications as they become integrated into
outpatient care. In order to better understand the issues that pharmacy and
therapeutics committees will encounter, a review of the 2 FDA-approved oral
anticoagulants will be evaluated. Atrial fibrillation is a major risk factor for first and recurrent ischaemic
stroke, and anticoagulation, mainly by use of coumarin medications, is an
effective strategy for reducing ischaemic stroke occurrence in these patients.
However, the coumarin medications have disadvantages. Over the past decade,
important strides have been made towards developing improved anticoagulant
medications. This review discusses these new developments and what they mean for
the future of primary and secondary ischaemic stroke prevention in patients with
atrial fibrillation. Relevant papers were identified with electronic searches of
the Medline and EMBASE databases. Ongoing trials were checked using the Trials
Results Centre website. The direct thrombin inhibitors, and the factor Xa
inhibitors are the two major new anticoagulant drug classes under development at
present. In phase III trials, dabigatran and rivaroxaban demonstrated at least
as good performance as warfarin at reducing the rate of ischaemic stroke,
systemic embolus, and haemorrhagic ischaemic stroke, whilst maintaining a
comparable or lower rate of major bleeding events. Drug level monitoring was not
required due to stable pharmacodynamics. AZD0837, apixaban, YM-150, edoxaban and
betrixaban all showed promising results in phase II trials, as did S35972 in
animal, in vitro and ex vivo models. The future of these new anticoagulants
looks encouraging, although there are still some significant challenges to
overcome. We need to consider the accumulation of long-term safety and efficacy
data, and the development of effective means of reversal of anticoagulation for
the direct thrombin inhibitors and factor Xa inhibitors. AIMS: Patients with atrial fibrillation (AF) are at increased risk of stroke.
Betrixaban is a novel oral factor Xa inhibitor administered once daily, mostly
excreted unchanged in the bile and with low (17%) renal excretion.
METHODS AND RESULTS: Patients with AF and more than one risk factor for stroke
were randomized to one of three blinded doses of betrixaban (40, 60, or 80 mg
once daily) or unblinded warfarin, adjusted to an international normalized ratio
of 2.0-3.0. The primary outcome was major or clinically relevant non-major
bleeding. The mean follow-up was 147 days. Among 508 patients randomized, the
mean CHADS2 score was 2.2; 87% of patients had previously received vitamin K
antagonist therapy. The time in therapeutic range on warfarin was 63.4%. There
were one, five, five, and seven patients with a primary outcome on betrixaban
40, 60, 80 mg daily, or warfarin, respectively. The rate of the primary outcome
was lowest on betrixaban 40 mg (hazard ratio compared with warfarin = 0.14,
exact stratified log-rank P-value 0.04, unadjusted for multiple testing). Rates
of the primary outcome with betrixaban 60 or 80 mg were more similar to those of
wafarin. Two ischaemic strokes occurred, one each on betrixaban 60 and 80 mg
daily. There were two vascular deaths, one each on betrixaban 40 mg and
warfarin. Betrixaban was associated with higher rates of diarrhoea than
warfarin.
CONCLUSION: Betrixaban was well tolerated and had similar or lower rates of
bleeding compared with well-controlled warfarin in patients with AF at risk for
stroke. New oral anticoagulants are directed towards a single target, essentially factor
Xa (FXa) or factor IIa. They do not require routine coagulation monitoring.
However, in special clinical settings (emergency surgery, bleeding, thrombosis,
control of the patient's compliance, suspected overdose, potential drug
interference, and so on), measurement of plasma levels is needed. Several
available anti-FXa assays are used for monitoring anticoagulant activity of
heparins and fondaparinux. They must be modified and standardized for the
measurement of direct FXa inhibitors (rivaroxaban, apixaban, edoxaban,
betrixaban and others). The use of calibrators (lyophilized plasma with a known
concentration of drug) allows an expression of the results in ng per ml of
plasma. Two categories of assays - endogenous and exogenous assays are
available. Endogenous assays are useful in pharmaceutical research, while
exogenous assays are used in clinical laboratories. The preferred anti-FXa assay
is a specific method in contrast to prothrombin time and activated partial
thromboplastin time, but it is not available everywhere at any time. A specific
measurement of direct FXa inhibitors is feasible with the use of a new test
developed by the authors' group. The physicians must be aware of the possibility
to measure the plasma concentration of FXa inhibitors in patients at high risk
of bleeding and in several other special clinical situations. Venous thromboembolism (VTE) is a serious disease that is often neglected, and
effective and safe antithrombotic treatments are a public health priority. New
antithrombotics such as rivaroxaban, apixaban, betrixaban, edoxaban, darexaban,
TAK-442, LY517717, eribaxaban, otamixaban are being developed to overcome
current therapeutic limitations. The new oral anticoagulants and parenteral
otamixaban are under evaluation in clinical trials for VTE treatment, for VTE
prevention in orthopedic surgery, for stroke prevention in patients with atrial
fibrillation and for cardiovascular event prevention in patients with acute
coronary syndrome. These antithrombotic agents directly and selectively inhibit
factor Xa, and do not require coagulation monitoring and dose adjustment.
Several of these drugs have shown promising results and have the potential to
either replace or act as alternatives to traditional anticoagulants (heparins,
vitamin K antagonists). INTRODUCTION: The introduction of novel anticoagulants has had contrasting
effects on the agents in the pipeline, fueling the development of some and
sinking the others. The complexity of the coagulation cascade offers interesting
inhibition choices that might become valid treatment options.
AREAS COVERED: This review will highlight some of the anticoagulants in the
pipeline. Following the success of the direct thrombin and FXa inhibitors
already in the market, new agents are being tested. These include AZD0837,
betrixaban, letaxaban, darexaban, and LY517717. Targeting other components of
the hemostatic pathway might lead to better safety profiles without influencing
efficacy. Inhibitors to FVIIa-tissue factor (FVIIa/TF) complex, FIX, FXI, and
FXII are being assessed. New inspiring inhibitors are antisense oligonucleotides
(ASOs) and aptamers. These are highly specific agents with readily reversible
effect and might be engineered to inhibit any coagulation factor. Currently
tested ASOs and aptamers are inhibitors of FXI, FXII, thrombin, FIXa, and
platelet GPIV.
EXPERT OPINION: Some of the agents in the pipeline offer valid treatment option
for long-term therapy, overcoming some of the drawbacks of the novel
anticoagulants. Research is being driven by an expanding market in the
anticoagulation field that has been unexploited for a long time. Heparins and vitamin K antagonists (VKA) used commonly are the standard
treatment of venous and arterial thromboses. They are very efficient and safe,
but have some limitations: iatrogenicity, laboratory monitoring, parenteral use
for heparins and fondaparinux. Nowadays, four new inhibitors of factor Xa are
used orally (rivaroxaban, apixaban, edoxaban, betrixaban), and they are at least
as efficient as heparins and vitamin K antagonists. The objective is to
substitute these indirect inhibitors of factor Xa (heparins, low molecular
weight heparins and fondaparinux) in the prevention of venous and arterial
thromboembolic episodes. The new direct inhibitors do not require routine
laboratory monitoring of blood coagulation. They inhibit the extrinsic and the
intrinsic pathways of blood coagulation. Rivaroxaban and apixaban are
efficacious and safe in the prevention of cerebral infarcts in patients with
non-valvular fibrillation. Apixaban is another direct inhibitor of factor Xa
used orally which is developed in the same indications as rivaroxaban. Edoxaban
and betrixaban are also in development. The objective of this work is to study
the pharmacodynamic, pharmacokinetic, the efficacy and safety of these four oral
direct factor Xa inhibitors. |
Which are the most frequent syndromes associated with inherited bone marrow failure? | The inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders that share the inability of the bone marrow to produce an adequate number of blood cells. The 4 most frequent syndromes are Fanconi anemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anemia (DBA), and Shwachman-Diamond syndrome (SDS) | Aplastic anemia may be inherited or acquired. The distinction between these lies
not in the age of the patient, but in the clinical and laboratory diagnoses.
Adult hematologists must consider adult presentations of the inherited
disorders, in order to avoid incorrect management of their patients. Physicians
for adult patients must also realize that children with inherited disorders now
survive to transition into their care. The major inherited bone marrow failure
syndromes associated with development of pancytopenia include Fanconi anemia,
dyskeratosis congenita, Shwachman-Diamond syndrome, and amegakaryocytic
thrombocytopenia. The ages at presentation are highly variable, but often
include individuals of adult age who have previously undiagnosed Fanconi anemia
or dyskeratosis congenita. Many of the genes responsible for these disorders
have been identified (12 Fanconi anemia genes, 3 dyskeratosis congenita genes,
and 1 each for Shwachman-Diamond syndrome and amegakaryocytic thrombocytopenia).
A high index of suspicion and specific testing of children or adults with what
appears to be acquired aplastic anemia may identify inherited disorders. Correct
classification of patients with aplastic anemia of any age is mandatory for
their appropriate management. |
How is yellow fever virus transmitted? | Yellow fever virus is transmitted by mosquitoes and is restricted to Africa, Central and South America and the Caribbean.
Yellow fever virus is a flavivirus, and there is only one antigenic type. It was taken to the Americas by the early slave traders, and nowadays reported in Africa, America, Asia and Europe. Yellow fever virus is transmitted by two different cycles:
-from human to human by the mosquito Aedes aegypti; which is well-adapted to breeding around human habitations; the infection can be maintained in this way as ‘urban’ yellow fever.
-from infected monkeys to humans by mosquitoes such as Haemagogus. This is ‘jungle’ yellow fever and is seen in Africa and South America.
Yellow fever is not transmitted directly from human to human by day-to-day contact, but transmission from ill patients to healthcare workers has been reported, notably after needlestick injury. | After a 15-year period of low incidence, dengue has reemerged in Singapore in
the past decade. We identify potential causes of this resurgence. A combination
of lowered herd immunity, virus transmission outside the home, an increase in
the age of infection, and the adoption of a case-reactive approach to vector
control contribute to the increased dengue incidence. Singapore's experience
with dengue indicates that prevention efforts may not be sustainable. For
renewed success, Singapore needs to return to a vector control program that is
based on carefully collected entomologic and epidemiologic data. Singapore's
taking on a leadership role in strengthening disease surveillance and control in
Southeast Asia may also be useful in reducing virus importation. Yellow fever virus (YFV) is the prototype member of the genus Flavivirus, a
group of viruses that are transmitted between vertebrates by arthropod vectors.
The virus is found in tropical regions of Africa and South America and is
transmitted to primates by mosquitoes: Aedes spp. in Africa and Haemagogus and
Sabethes spp. in South America. Despite the availability of an effective
vaccine, yellow fever (YF) is considered a reemerging disease owing to its
increased incidence in the past 25 years. Molecular epidemiologic data suggest
there are seven genotypes of YFV that are geographically separated, and
outbreaks of disease are more associated with particular genotypes. In addition,
the risk of urban YF, owing to transmission of the virus by Aedes aegypti, is
increasing in Africa, as is the potential of urban YF returning to South
America. Both present serious potential public health problems to large
population centers. INTRODUCTION: Yellow fever is a serious illness public health importance and is
transmitted by mosquitoes of the genera Haemagogus and Sabethes in the rural and
forest environments, and by Aedes aegypti in the urban setting. In Colombia,
Haemagogus janthinomys and H. equinus are considered efficient vectors of this
viral disease.
OBJECTIVE: The presence of the mosquito Haemagogus equinus was recorded over an
8 year period, in the periurban areas of the Soledad and Malambo municipalities
(Atlantico Province) of northern Colombia.
MATERIALS AND METHODS: The data was obtained from records of the entomological
collections from two collection sites: (1) 14 larva traps located at the
Erneasto Cortissoz airport in the municipality of Soledad between 1997--2005 and
(2) 10 larva traps located at Vergara and Velasco Batallion in the municipality
of Malambo in 2005.
RESULTS: Haemogogus equinus was reported for the first time in Soledad in 1998.
In the following 8 years, 197 larvae were reported. The individuals were found
sharing the trap with Aedes aegypti, Culex nigripalpus and Uranotaenia lowii. In
Malambo, the first discovery of H.equinus occurred in 2005, with a total of 641
larvae. No other Culicidae were associated with it.
CONCLUSION: The presence of H. equinus in larvitraps located near the urban
zone, shows adaptation to the use of artificial containers as larval habitats,
Urbanization of this species in zones with a high Ae. aegypti infestation index
increases the potential introduction of sylvan yellow fever virus and
constitutes a risk for re-emergence of urban cycles of yellow fever. Yellow fever (YF) is a viral disease, endemic to tropical regions of Africa and
the Americas, which principally affects humans and nonhuman primates and is
transmitted via the bite of infected mosquitoes. Yellow fever virus (YFV) can
cause devastating epidemics of potentially fatal, hemorrhagic disease. Despite
mass vaccination campaigns to prevent and control these outbreaks, the risk of
major YF epidemics, especially in densely populated, poor urban settings, both
in Africa and South America, has greatly increased. Consequently, YF is
considered an emerging, or reemerging disease of considerable importance. This
article comprehensively reviews the history, microbiology, epidemiology,
clinical presentation, diagnosis, and treatment of YFV, as well as the vaccines
produced to combat YF. In 2008, residents of the United States made 12 million visits to developing
countries in Asia, South America, Central America, Oceania, the Middle East, and
Africa. Due to the presence of Anopheles, Aedes, and Culex mosquitoes, travel to
these destinations poses a risk for diseases such as malaria, yellow fever, and
Japanese encephalitis that cause significant morbidity and mortality. To gain a
better understanding of the major emerging and established travel-related
infectious diseases transmitted principally by mosquitoes and the measures for
their prevention in U.S. residents who travel to these developing countries, we
performed a literature search of the PubMed and MEDLINE databases (January
1950-February 2010). Information from the Centers for Disease Control and
Prevention and the World Health Organization and relevant references from the
publications identified were also reviewed. Vaccines for the prevention of
Japanese encephalitis and yellow fever are commercially available to U.S.
travelers and should be administered when indicated. However, the prevention of
malaria, dengue fever, chikungunya, and West Nile virus relies on personal
insect protection measures and chemoprophylaxis for malaria. As the rate of
international travel continues to rise, individuals traveling overseas should be
made aware of the risk of various infectious diseases and the importance of
prevention. Physicians, pharmacists, nurses, and other practitioners can play a
vital role in disease education and prevention, including the administration of
vaccines and provision of chemoprophylactic drugs. Flaviviral infections have a re-emerging impact on the health situation in
developing countries with several billions of people living at risk. In the
present review, we focus on three members of the genus Flavivirus belonging to
the Flaviviridae family. They are transmitted to humans by mosquito bites,
namely those viruses leading to Dengue Fever, Yellow Fever and mosquito-borne
Japanese encephalitis. All three virus groups have a spherical structure with a
diameter of approximately 50 nm. Although sharing a similar genomic structure
and intracellular life cycle, they show different clinical manifestations.
Infections are incurable, as there is no antiviral treatment available for
either of the three viruses. Thus, prevention and vaccination are the best
defenses. The most promising vaccines are live attenuated vaccines (LAVs), such
as the YF17D strain against Yellow Fever or the SA-14-14-2 strain against
Japanese encephalitis. Additionally, recombit vaccines for Japanese
encephalitis are in development. Although Dengue Fever is the most prevalent
arthropode-borne flaviviral infection and a lot of research to develop a vaccine
against all four Dengue Fever serotypes was undertaken, no vaccine is available
on the market yet. Promising tetravalent vaccine candidates are currently
undergoing clinical phase trials, including LAVs, recombit and chimeric
candidates as well as non-replicating vaccine approaches. Additionally,
encouraging anti-flaviviral approaches target non-structural proteins,
virus-specific proteases essential for cellular maturation of viral particles.
Peptide inhibitors against the highly conserved NS2B and NS3 proteases are
attractive as pan-flaviviral drug candidates. At least 27 alphaviruses and 68 flaviviruses have been recognized, approximately
one-third of which are medically important human pathogens. They vary widely in
their basic ecology; each virus occupies a distinct ecologic niche, often with
restricted geographic and biologic distribution. As shown in Tables 54-1 and
54-2, alphaviruses and flaviviruses can cause various syndromes, ranging from
benign febrile illnesses to severe systemic diseases with hemorrhagic
manifestations or major organ involvement. The neurotropic alphaviruses and
flaviviruses can produce severe destructive central nervous system disease with
serious sequelae. Several alphaviruses (chikungunya, Mayaro, and Ross River)
cause painful arthritis that persists for weeks or months after the initial
febrile illness. Yellow fever virus has unique hepatotropic properties that
cause a clinically and pathologically distinct form of hepatitis with a
hemorrhagic diathesis. The dengue viruses, which cause more human illness than
all other members of their family, may produce a serious, sometimes fatal,
immunopathologic disease in which shock and hemorrhage occur. Hepatitis C virus
(Chapter 70) may be a flavivirus. Alphavirus is one of the two genera in the
family Togaviridae; the other genus (Rubivirus) has rubella virus (Chapter 55)
as its only member. Flavivirus, once classified in the Togaviridae, now
constitutes one of three genera in the family Flaviviridae; the other two genera
are Pestivirus and “Hepatitis C-like viruses”. Pestivirus includes animal
pathogens (bovine viral diarrhea and hog cholera viruses) that are of
considerable economic importance, but contains no known human pathogens.
Hepatitis C virus is described in Chapter 70. All alphaviruses and flaviviruses
that cause disease in humans are arthropod-borne viruses (arboviruses). In the
original classification scheme based on antigenic relationships, alphaviruses
and flaviviruses were termed group A and group B arboviruses, respectively. Most
alphaviruses and flaviviruses survive in nature by replicating alternately in a
vertebrate host and a hematophagous arthropod (mosquitoes or, for some
flaviviruses, ticks). Arthropod vectors acquire the viral infection by biting a
viremic host, and after an extrinsic incubation period during which the virus
replicates in the vector's tissues, they transmit virus through salivary
secretions to another vertebrate host. Virus replicates in the vertebrate host,
causing viremia and sometimes illness. The ability to infect and replicate in
both vertebrate and arthropod cells is an essential quality of alphaviruses and
flaviviruses. The principal vertebrate hosts for most are various species of
wild mammals or birds. The natural zoonotic cycles that maintain the virus do
not usually involve humans. However, a few viruses (yellow fever virus, dengue
virus types 1, 2, 3 and 4 and chikungunya virus) can be transmitted in a
human-mosquito-human cycle. As a result of being pathogenic for humans and
capable of transmission in heavily populated areas, these viruses can cause
widespread and serious epidemics. Because of their high transmission potential,
these viruses are major public health problems in many tropical and subtropical
regions of the world where appropriate mosquito vectors are present. Because
some of these agents are dangerous human pathogens and are highly infectious,
special containment and safety precautions in the laboratory are required. Yellow fever is a mosquito-transmitted hemorrhagic viral disease that is endemic
to tropical regions in South America and Africa. It remains a significant health
concern for deploying military personnel, accordingly vaccination is frequently
performed on troops. Although the vaccine is generally administered with only
minor complications, rare severe complications are also reported. Herein, we
report a mild case of yellow fever vaccine-associated viscerotropic disease 4
days after administration of the vaccine. The various complications of the
vaccine and their pathogenesis are also reviewed. BACKGROUND: The causative agent of yellow fever is an arbovirus of the
Flaviviridae family transmitted by infected Aedes mosquitoes, particularly in
Africa. In the Central African Republic since 2006, cases have been notified in
the provinces of Ombella-Mpoko, Ouham-Pende, Basse-Kotto, Haute-Kotto and in
Bangui the capital. As the presence of a vector of yellow fever virus (YFV)
represents a risk for spread of the disease, we undertook entomological
investigations at these sites to identify potential vectors of YFV and their
abundance.
FINDINGS: Between 2006 and 2010, 5066 mosquitoes belonging to six genera and 43
species were identified. The 20 species of the Aedes genus identified included
Ae. aegypti, the main vector of YFV in urban settings, and species found in
tropical forests, such as Ae. africanus, Ae. simpsoni, Ae. luteocephalus, Ae.
vittatus and Ae. opok. These species were not distributed uniformly in the
various sites studied. Thus, the predomit Aedes species was Ae. aegypti in
Bangui (90.7 %) and Basse-Kotto (42.2 %), Ae. africanus in Ombella-Mpoko
(67.4 %) and Haute-Kotto (77.8 %) and Ae. vittatus in Ouham-Pende (62.2 %). Ae.
albopictus was also found in Bangui. The distribution of these domit species
differed significantly according to study site (P < 0.0001). None of the pooled
homogenates of Aedes mosquitoes analysed by polymerase chain reaction contained
the YFV genome.
CONCLUSION: The results indicate a wide diversity of vector species for YFV in
the Central African Republic. The establishment of surveillance and vector
control programs should take into account the ecological specificity of each
species. There are 219 virus species that are known to be able to infect humans. The
first of these to be discovered was yellow fever virus in 1901, and three to
four new species are still being found every year. Extrapolation of the
discovery curve suggests that there is still a substantial pool of undiscovered
human virus species, although an apparent slow-down in the rate of discovery of
species from different families may indicate bounds to the potential range of
diversity. More than two-thirds of human viruses can also infect non-human
hosts, mainly mammals, and sometimes birds. Many specialist human viruses also
have mammalian or avian origins. Indeed, a substantial proportion of mammalian
viruses may be capable of crossing the species barrier into humans, although
only around half of these are capable of being transmitted by humans and around
half again of transmitting well enough to cause major outbreaks. A few possible
predictors of species jumps can be identified, including the use of
phylogenetically conserved cell receptors. It seems almost inevitable that new
human viruses will continue to emerge, mainly from other mammals and birds, for
the foreseeable future. For this reason, an effective global surveillance system
for novel viruses is needed. Flaviviruses comprise important arthropod-transmitted human pathogens, including
yellow fever (YF), dengue (Den), Japanese encephalitis (JE), West Nile (WN) and
tick-borne encephalitis (TBE) viruses that have the potential of expanding their
endemic areas due to global climatic, ecological and socio-economic changes.
While effective vaccines against YF, JE and TBE are in widespread use, the
development of a dengue vaccine has been hampered for a long time because of
concerns of immunopathological consequences of vaccination. Phase III clinical
trials with a recombit chimeric live vaccine are now ongoing and will show
whether the enormous problem of dengue can be resolved or at least reduced by
vaccination in the future. Unprecedented details of the flavivirus particle
structure have become available through the combined use of X-ray
crystallography and cryo-electron microscopy that led to novel and surprising
insights into the antigenic structure of these viruses. Recent studies provided
evidence for an important role of virus maturation as well as particle dynamics
in virus neutralization by antibodies and thus added previously unknown layers
of complexity to our understanding of flavivirus immune protection. This
information is invaluable for interpreting current investigations on the
functional activities of polyclonal antibody responses to flavivirus infections
and vaccinations and may open new avenues for studies on flavivirus cell biology
and vaccine design. Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever
(YFV) viruses is increasing in many parts of the world. The viruses are
primarily transmitted by Aedes aegypti, a highly domesticated mosquito species
that is notoriously difficult to control. When transinfected into Ae. aegypti,
the intracellular bacterium Wolbachia has recently been shown to inhibit
replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing
a potentially powerful biocontrol strategy for human pathogens. Because the
extent of pathogen reduction can be influenced by the strain of bacterium, we
examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection
in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and
dissemination rates were significantly reduced in mosquitoes with the wMel
strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar
rates of infection and dissemination were observed in wMel infected and
non-infected Ae. aegypti when intrathoracic inoculation was used to deliver
virus. YFV infection, dissemination and replication were similar in
wMel-infected and control mosquitoes following intrathoracic inoculations. In
contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a
10(4) times reduction in YFV RNA copies compared to controls. The extent of
reduction in virus infection depended on Wolbachia strain, titer and strain of
the virus, and mode of exposure. Although originally proposed for dengue
biocontrol, our results indicate a Wolbachia-based strategy also holds
considerable promise for YFV and CHIKV suppression. Vector-borne disease is one of the greatest contributors to human mortality and
morbidity throughout the tropics. Mosquito-transmitted diseases such as malaria,
dengue, yellow fever and filariasis are the main contributors to this burden.
Although insecticides have historically been used to try to control vector
populations, over the past 15 years, substantial progress has been made in
developing alternative vector control strategies ranging from biocontrol methods
through to genetic modification of wild insect populations. Here, we review
recent advances concerning these strategies and consider the potential
impediments to their deployment, including the challenges of obtaining
regulatory approval and community acceptance. Two different species of flaviviruses, dengue virus (DENV) and yellow fever
virus (YFV), that originated in sylvatic cycles maintained in non-human primates
and forest-dwelling mosquitoes have emerged repeatedly into sustained
human-to-human transmission by Aedes aegypti mosquitoes. Sylvatic cycles of both
viruses remain active, and where the two viruses overlap in West Africa they
utilize similar suites of monkeys and Aedes mosquitoes. These extensive
similarities render the differences in the biogeography and epidemiology of the
two viruses all the more striking. First, the sylvatic cycle of YFV originated
in Africa and was introduced into the New World, probably as a result of the
slave trade, but is absent in Asia; in contrast, sylvatic DENV likely originated
in Asia and has spread to Africa but not to the New World. Second, while
sylvatic YFV can emerge into extensive urban outbreaks in humans, these
invariably die out, whereas four different types of DENV have established human
transmission cycles that are ecologically and evolutionarily distinct from their
sylvatic ancestors. Finally, transmission of YFV among humans has been
documented only in Africa and the Americas, whereas DENV is transmitted among
humans across most of the range of competent Aedes vectors, which in the last
decade has included every continent save Antarctica. This review summarizes
current understanding of sylvatic transmission cycles of YFV and DENV, considers
possible explanations for their disjunct distributions, and speculates on the
potential consequences of future establishment of a sylvatic cycle of DENV in
the Americas. INTRODUCTION: Yellow fever is a non-contagious infectious disease, highly
lethal, transmitted by the Aedes, Haemagogus and Sabethes.
METHODS: Descriptive retrospective study of the yellow fever cases in Amazonas,
between 1996 and 2009.
RESULTS: Forty two cases of yellow fever were confirmed, with 30 deaths, 10% of
which were foreigners.
CONCLUSIONS: The presence of Aedes aegypti and Aedes albopictus in both rural
Amazonas and its capital demonstrates the dispersion of these vectors and
underscores the need for better and continuous epidemiological and entomological
control. Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod
vectors and vertebrate hosts. While barriers limiting arbovirus population
diversity have been observed in mosquitoes, whether barriers exist in vertebrate
hosts is unclear. To investigate whether arboviruses encounter bottlenecks
during dissemination in the vertebrate host, we infected immunocompetent mice
and immune-deficient mice lacking alpha/beta interferon (IFN-α/β) receptors
(IFNAR⁻/⁻ mice) with a pool of genetically marked viruses to evaluate
dissemination and host barriers. We used the live attenuated vaccine strain
YFV-17D, which contains many mutations compared with virulent YFV. We found that
intramuscularly injected immunocompetent mice did not develop disease and that
viral dissemination was restricted. Conversely, 32% of intramuscularly injected
IFNAR⁻/⁻ mice developed disease. By following the genetically marked viruses
over time, we found broad dissemination in IFNAR⁻/⁻ mice followed by clearance.
The patterns of viral dissemination were similar in mice that developed disease
and mice that did not develop disease. Unlike our previous results with
poliovirus, these results suggest that YFV-17D encounters no major barriers
during dissemination within a vertebrate host in the absence of the type I IFN
response. Yellow fever (YF) is an acute viral communicable disease transmitted by an
arbovirus of the Flavivirus genus. It is primarily a zoonotic disease,
especially the monkeys. Worldwide, an estimated 200,000 cases of yellow fever
occurred each year, and the case-fatality rate is ~15%. Forty-five endemic
countries in Africa and Latin America, with a population of close to 1 billion,
are at risk. Up to 50% of severely affected persons from YF die without
treatment. During 2009, 55 cases and 18 deaths were reported from Brazil,
Colombia, and Peru. Brazil reported the maximum number of cases and death, i.e.,
42 cases with 11 deaths. From January 2010 to March 2011, outbreaks of YF were
reported to the WHO by Cameroon, Democratic Republic of Congo, Cote d'Ivoire,
Guinea, Sierra Leone, Senegal, and Uganda. Cases were also reported in three
northern districts of Abim, Agago, and Kitugun near the border with South Sudan.
YF usually causes fever, muscle pain with prominent backache, headache, shivers,
loss of appetite, and nausea or vomiting. Most patients improve, and their
symptoms disappear after 3 to 4 d. Half of the patients who enter the toxic
phase die within 10-14 d, while the rest recover without significant organ
damage. Vaccination has been the single most important measure for preventing
YF. The 17D-204 YF vaccine is a freeze-dried, live attenuated, highly effective
vaccine. It is available in single-dose or multi-dose vials and should be stored
at 2-8 °C. It is reconstituted with normal saline and should be used within 1 h
of reconstitution. The 0.5 mL dose is delivered subcutaneously. Revaccination is
recommended every 10 y for people at continued risk of exposure to yellow fever
virus (YFV). This vaccine is available worldwide. Travelers, especially to
Africa or Latin America from Asia, must have a certificate documenting YF
vaccination, which is required by certain countries for entry under the
International Health Regulations (IHR) of the WHO. |
Are there any specific antidotes for dabigatran? | No specific antidote currently exists for dabigatran | Dabigatran etexilate is an oral, reversible direct thrombin inhibitor that is
approved in the EU and several other countries for the prevention of venous
thromboembolism after elective hip and knee replacement, and is in advanced
clinical development for other thromboembolic disorders. Dabigatran has a
predictable pharmacokinetic profile, allowing for a fixed-dose regimen without
the need for routine coagulation monitoring. In certain clinical situations such
as serious bleeding into critical organs (e.g. intracerebral bleeding),
potential overdose and emergency surgery, clinicians will need to make an
assessment of the anticoagulant status of a patient receiving dabigatran before
deciding on future management strategies. If available, thrombin clotting time
(TT), ecarin clotting time (ECT) and TT determined by Hemoclot thrombin
inhibitor assay are sensitive tests to evaluate the anticoagulant effects of
dabigatran. Prothrombin time (INR) is less sensitive than other assays and
cannot be recommended. The activated partial thromboplastin time (aPTT) can
provide a useful qualitative assessment of anticoagulant activity but is less
sensitive at supratherapeutic dabigatran levels. There are limited data for
activated clotting time (ACT). Overall, the aPTT and TT are the most accessible
qualitative methods for determining the presence or absence of anticoagulant
effect. Although there is no specific antidote to antagonise the anticoagulant
effect of dabigatran, due to its short duration of effect drug discontinuation
is usually sufficient to reverse any excessive anticoagulant activity. In case
of potential overdose, the feasibility of early administration of activated
charcoal and subsequent charcoal filtration are undergoing preclinical
evaluation. Dabigatran can also be dialysed in patients with renal impairment.
In instances of life-threatening bleeding, where conventional measures have
failed or are unavailable, other non-specific prohaemostatic agents such as
recombit activated factor VII and prothrombin complex concentrates can be
considered. An increasing number of patients receive anticoagulant therapy to prevent and
treat arterial or venous thromboembolism. The major complication of
anticoagulant therapy is the increase of the individual bleeding risk. All
anticoagulant drugs can cause haemorrhages, that can sometimes be
life-threatening. Although heparins and the vitamin K antagonists have been the
most widely used anticoagulants for decades, the correct management of bleeding
complications associated with these agents has been poorly studied. More
recently, new anticoagulant drugs, both parenteral and oral, have been approved
for clinical use. Currently, none of these new agents has a specific antidote,
and little advise can be given on how to manage a major bleeding event. The aim
of this article is to describe the haemorrhagic risk and the management of
bleeding complications associated with the principal anticoagulant drugs. Amongst numerous promising anticoagulant molecules, rivaroxaban (Xarelto(®)),
dabigatran (Pradaxa(®)) and apixaban (Eliquis(®)) have been registered outside
the USA in the prevention of thromboembolic events in patients undergoing total
hip or knee prosthetic replacement. Rivaroxaban however has been granted
authorisation by the FDA for the thromboprophylaxis after surgery for total hip
or knee surgery. Dabigatran has been granted authorisation by the FDA in
non-valvular atrial fibrillation (RE-LY trial) while rivaroxaban is expecting
approval in this same indication (ROCKET trial). Phase III results in the
treatment and in the secondary prevention of established venous thrombosis and
pulmonary embolism are encouraging. These small molecules are obtained by
chemical synthesis, their molecular weight is lower than 500 daltons. Many
coagulation tests may be affected by these molecules. Those modifications should
be known in order to avoid misinterpretation of the tests but could also be used
to measure plasma concentrations of these products. The choice of a non specific
global and readily available test has been documented (Quick time for
rivaroxaban and aPTT for dabigatran). Anti-Xa (for rivaroxaban) and anti-IIa
(for dabigatran) activities should however be preferred, expressed in ng/ml with
calibrated plasmas (containing predetermined concentration of the tested drug).
The half-life is around 8 to 12 hours, with a peak activity 2 to 4 hours after
ingestion. Dabigatran is mainly eliminated via the kidney, hence requiring
dose-adjustment in case of moderate renal insufficiency, and contra-indicated in
case of severe renal insufficiency. Rivaroxaban being excreted via kidney and
liver, some precautions should apply in case of liver insufficiency. No data are
available in pregcy or pediatrics, clinical trials are ongoing. There are few
interactions with concomitant drugs, which should not be ignored. The short
half-life of these new agents compensates for the lack of any specific antidote
in many instances. Their oral administration, without the need for dose
adjustment, and without requirement for a laboratory monitoring will increase
their use in a large number of patients, in those indications for which an
approval has been granted by health authorities. The range of anticoagulants has been very active recently with the development
of new compounds including injectable anti-Xa such as fondaparinux, and new oral
drugs which can be divided into anti-IIa with dabigatran, and anti-Xa, such as
rivaroxaban and apixaban still in the development stage. Others are coming
forward. They are more convenient to use and do not require routine coagulation
monitoring. However, several points need to be clarified and the place for each
drug remains to be determined. In case of massive bleeding, management is
unclear and none of these newer agents has a specific antidote that completely
reverses its anticoagulant effect. For more than 60 years, vitamin K antagonists have been the only available oral
anticoagulants for the prevention of stroke and systemic embolism in atrial
fibrillation (AF). Several new molecules, with a favorable pharmacokinetic
profile and avoiding routine monitoring, have been recently developed, opening a
new era in anticoagulation. The oral direct thrombin inhibitor, dabigatran, and
the oral activated factor X inhibitors, rivaroxaban and apixaban, are the novel
oral anticoagulants with data from large randomized clinical trials showing that
these drugs are noninferior to warfarin in the prevention of stroke and
thromboembolic complications of AF, with the advantage of less hemorrhagic
stroke and intracranial bleeding. While these trial data are extremely
encouraging, several practical issues (e.g., lack of specific antidote, safety
of long-term treatment or cost-effectiveness in "real-life" clinical practice)
still need to be elucidated. A patient on oral anticoagulation with dabigatran presented with chest pain and
dyspnoea. A CT scan of the chest revealed an intramural haematoma of the
ascending aorta with a large pericardial effusion. The patient underwent a
modified Bentall procedure. In the absence of a specific antidote for this novel
oral anticoagulant medication, even in an emergency situation, successful
surgical treatment was possible with an aggressive use of available
prohaemostatic agents. Novel oral anticoagulants (NOACs) that directly inhibit thrombin (dabigatran) or
factor Xa (rivaroxaban, apixaban, edoxaban) are effective therapies for the
prevention and treatment of thromboembolism with reduced bleeding complications
compared with warfarin for some indications. However, specific antidotes to
reverse the anticoagulant activity of NOACs in the event of major bleeding are
not available. Evidence supporting non-specific prohemostatic therapies
(prothrombin complex concentrate [PCC], activated prothrombin complex
concentrate [aPCC], recombit factor VIIa) in this setting is limited to
healthy human volunteers, animal models, and in vitro studies. Clinical outcome
data are lacking. Administration of PCC or aPCC may be considered in addition to
supportive measures for patients with severe or life-threatening bleeding.
Clinical studies are needed to establish the efficacy and safety of these
treatments. Target-specific antidotes are in development and hold promise for
NOAC reversal, but require further investigation. OBJECT: The direct thrombin inhibitor dabigatran has recently been approved in
the US as an alternative to warfarin. The lack of guidelines, protocols, and an
established specific antidote to reverse the anticoagulation effect of
dabigatran potentially increases the rates of morbidity and mortality in
patients with closed head injury (CHI). Confronted with this new problem, the
authors reviewed their initial clinical experience.
METHODS: The authors retrospectively reviewed all cases of adult patients (age ≥
18 years) who sustained CHI secondary to ground-level falls and who presented to
the authors' provisional regional Level I trauma center between February 2011
and May 2011. The authors divided these patients into 3 groups based on
anticoagulant therapy: dabigatran, warfarin, and no anticoagulants.
RESULTS: Between February 2011 and May 2011, CHIs from ground-level falls were
sustained by 5 patients while on dabigatran, by 15 patients on warfarin, and by
25 patients who were not on anticoagulants. The treatment of the patients on
dabigatran at the authors' institution had great diversity. Repeat CT scans
obtained during reversal showed 4 of 5 patients with new or expanded hemorrhages
in the dabigatran group, whereas the warfarin group had 3 of 15 (p = 0.03). The
overall mortality rate for patients sustaining CHI on dabigatran was 2 (40%) of
5, whereas that of the warfarin group was 0 (0%) of 15 (p = 0.05).
CONCLUSIONS: It is critical for physicians involved in the care of patients with
CHI on dabigatran to be aware of an elevated mortality rate if no treatment
protocol or guideline is in place. The authors will soon implement a reversal
management protocol for patients with CHI on dabigatran at their institution in
an attempt to improve efficacy and safety in their treatment approach. The target-specific oral anticoagulants represent the first new oral
anti-thrombotic therapy in over 50 years and have the potential to make therapy
easier and hence more accessible to many patients. Like any new therapy, the
potential benefits must be weighed against the potential challenges and one of
the most concerning aspects of the new target-specific oral anticoagulants is
the lack of a proven method to reverse their effect. Unlike the vitamin K
antagonist, i.e. warfarin, there is no specific antidote for these medications.
This paper will review the limited data on the use of non-specific therapies to
reverse anticoagulation for the new agents. We hope to prepare clinicians who
are faced with a patient who has serious bleeding or needs emergent surgery
while taking dabigatran, rivaroxaban or apixaban. New oral anticoagulants which specifically inhibit factor Xa (FXa) or thrombin
(FIIa) do not require routine laboratory monitoring. However, they induce a
state of hypocoagulation and increase the risk of bleeding. In some clinical
situations, such as emergency surgery, hemorrhagic episodes, or recurrent
stroke, coagulation monitoring may be useful. A significant number of
publications have reported uncontrollable hemorrhagic complications and deaths
in patients treated with these new anticoagulants. The selection of the most
appropriate clotting assay is based on the drug used and the availability of the
test. The new anticoagulants influence all global clot-based tests. Prothrombin
time and partial thromboplastin time measured before and after treatment are
considered as qualitative tests since they are not specific. Specific anti-Xa
and anti-IIa assays are available and results can be expressed in ogram per
milliliter of plasma using calibrated plasmas containing well-established
amounts of drug. The fact that there is no specific antidote to reverse the
anticoagulant action of the new anticoagulants can impair management of
hemorrhagic complications; clinical experience is still limited. Pro-hemostatic
treatment with non-activated or activated prothrombin complexes (FEIBA(®)), or
as a last recourse with FVIIa concentrates (NovoSeven(®)), has been used with
variable results. Some suggestions for the management of patients with bleeding
have been published but there is still little clinical evidence for these
interventions. Direct new oral anticoagulants (NOACs) - inhibitors of thrombin or factor Xa -
are intended to be used largely in the treatment of venous thromboembolic
disease or the prevention of systematic embolism in atrial fibrillation, instead
of vitamin K antagonists. Like any anticoagulant treatment, they are associated
with spontaneous or provoked haemorrhagic risk. Furthermore, a significant
proportion of treated patients are likely to be exposed to emergency surgery or
invasive procedures. Given the absence of a specific antidote, the action to be
taken in these situations must be defined. The lack of data means that it is
only possible to issue proposals rather than recommendations, which will evolve
according to accumulated experience. The proposals presented here apply to
dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)); data for apixaban and
edoxaban are still scarce. For urgent surgery with haemorrhagic risk, the drug
plasma concentration should be less or equal to 30ng/mL for dabigatran and
rivaroxaban should enable surgery associated with a high bleeding risk. Beyond
that, if possible, the intervention should be postponed by monitoring the drug
concentration. The course to follow is then defined according to the NOAC and
its concentration. If the anticoagulant dosage is not immediately available,
worse propositions, based on the usual tests (prothrombin time and activated
partial thromboplastin time), are presented. However, these tests do not really
assess drug concentration or the risk of bleeding that depends on it. In case of
serious bleeding in a critical organ, the effect of anticoagulant therapy should
be reduced using a non-specific procoagulant drug as a first-line approach:
activated prothrombin complex concentrate (aPCC) (FEIBA(®) 30-50U/kg) or
non-activated PCC (50U/kg). In addition, for any other type of severe
haemorrhage, the administration of a procoagulant drug, which is potentially
thrombogenic in these patients, is discussed according to the NOAC concentration
and the possibilities of mechanical haemostasis. |
What is the outcome of TAF10 interacting with the GATA1 transcription factor? | TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis. | The ordered assembly of a functional preinitiation complex (PIC), composed of
general transcription factors (GTFs), is a prerequisite for the transcription of
protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding
protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought
to recognize the promoter sequences allowing site-specific PIC assembly.
Transcriptional cofactors, such as SAGA, are also necessary for tightly
regulated transcription initiation. The contribution of the two TAF10-containing
complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10
specifically in erythroid cells in vivo, we observed a differentiation block
accompanied by deregulated GATA1 target genes, including Gata1 itself,
suggesting functional cross talk between GATA1 and TAF10. Additionally, we
analyzed by mass spectrometry the composition of TFIID and SAGA complexes in
mouse and human cells and found that their global integrity is maintained, with
minor changes, during erythroid cell differentiation and development. In
agreement with our functional data, we show that TAF10 interacts directly with
GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid
cells. Thus, our findings demonstrate a cross talk between canonical TFIID and
SAGA complexes and cell-specific transcription activators during development and
differentiation. |
How does TNF affect thyroid hormone receptors? | TNF-alpha inhibits the T3-induced expression of thyroid hormone receptor-beta | Amphibian metamorphosis induced by T(3) involves programmed cell death and the
differentiation of various types of cells in degenerated and reconstructed
tissues. However, the signaling pathway that directs the T(3)-dependent
cell-fate determinations remains unclear. TNF-alpha is a pleiotropic cytokine
that affects diverse cellular responses. Engagement of TNF-alpha with its
receptor (TNFR1) causes intracellular apoptotic and/or survival signaling. To
investigate TNF signaling functions during anuran metamorphosis, we first
identified Xenopus laevis orthologs of TNF (xTNF)-alpha and its receptor. We
found that xTNF-alpha activated nuclear factor-kappaB in X. laevis A6 cells
through the Fas-associated death domain and receptor-interacting protein 1.
Interestingly, xTNF-alpha mRNA in blood cells showed prominent expression at
prometamorphosis during metamorphosis. Next, to elucidate the apoptotic and/or
survival signaling induced by xTNF-alpha in an in vitro model of metamorphosis,
we established a vascular endothelial cell line, XLgoo, from X. laevis tadpole
tail. XLgoo cells formed actin stress fibers and elongated in response to
xTNF-alpha. T(3) induced apoptosis in these cells, but the addition of
xTNF-alpha blocked the T(3)-induced apoptosis. In addition, treatment of the
cells with T(3) for 2 d induced the expression of thyroid hormone receptor-beta
and caspase-3, and this thyroid hormone receptor-beta induction was drastically
repressed by xTNF-alpha. Furthermore, in organ culture of the tail, xTNF-alpha
significantly attenuated the tail degeneration induced by T(3). These findings
suggested that xTNF-alpha could protect vascular endothelial cells from
apoptotic cell death induced by T(3) during metamorphosis and thereby
participate in the regulation of cell fate. |
Which ones are the ESKAPE organisms? | The 6 ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. | BACKGROUND: The aim of this study was to test in the clinic whether
antimicrobial diversity affects resistance of Enterococcus faecium,
Staphylococcus aureus, Klebsiella species, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter species (ESKAPE) pathogens in ventilator-associated
pneumonia (VAP).
METHODS: Three different strategies of empirical antimicrobial prescription for
VAP were consecutively implemented in an ICU: patient specific (10 months);
scheduling, including sequential quarterly prioritization (12 months) and
restriction (12 months) of antimicrobials; and mixing (10 months). Periods were
compared, measuring the antimicrobial heterogeneity index (AHI). Incidence and
resistance patterns of VAP caused by ESKAPE were compared.
RESULTS: Overall, 127 microbiologic VAP episodes were documented. ESKAPE VAP
increased significantly during scheduling (AHI, 0.65) compared with
patient-specific (AHI, 0.88) and mixing (AHI, 0.87) periods (relative risk, 2.67
and 3.84, respectively). This finding was associated with a significant (P <
.05) increase of carbapenem-resistant A baumannii during the scheduling period
(15.0%) compared with the patient-specific (2.4%) and mixing (0%) periods. ICU
mortality of resistant patients with ESKAPE VAP was doubled that of patients
without ESKAPE VAP (relative risk, 2.25; 95% CI, 1.67-9.48). Thirty-day
mechanical ventilation-free days was significantly increased (5 days) in
patients with resistant ESKAPE VAP.
CONCLUSIONS: Antibiotic strategies promoting diversity may prevent the emergence
of resistance of ESKAPE organisms, improving use of health-care resources. PURPOSE OF REVIEW: Despite important geographical variations, Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa and Enterobacter species (ESKAPE) pathogens constitute
more than 80% of ventilator-associated pneumonia (VAP) episodes. Their clinical
importance relies on their virulence and ability in developing mechanisms to
decrease susceptibility to antimicrobials, increasing inappropriate therapy and
affecting negatively on ICU patients' outcome. This review updates information
on VAP due to ESKAPE pathogens.
RECENT FINDINGS: Although methicillin-resistant Staphylococcus aureus VAP may be
clinically similar to that caused by susceptible strains, it is associated with
poorer outcomes despite adequate treatment. Local colonization determines
treatment options. The contribution of tracheobronchitis is an important issue.
Minimum inhibitory concentration should be considered for nonfermentative
Gram-negative bacteria VAP to prescribe extended infusion β-lactam treatment due
to an increase of resistant strains. Strategies promoting antimicrobial
diversity may protect against emergence and spread of resistance by ESKAPE
pathogens.
SUMMARY: VAP due to ESKAPE pathogens represents a global challenge that can be
prevented using stewardship programmes promoting diversity. BACKGROUND: There is a lack of consensus regarding the definition of risk
factors for healthcare-associated infection (HCAI). The purpose of this study
was to identify additional risk factors for HCAI, which are not included in the
current definition of HCAI, associated with infection by multidrug-resistant
(MDR) pathogens, in all hospitalized infected patients from the community.
METHODS: This 1-year prospective cohort study included all patients with
infection admitted to a large, tertiary care, university hospital. Risk factors
not included in the HCAI definition, and independently associated with MDR
pathogen infection, namely MDR Gram-negative (MDR-GN) and ESKAPE microorganisms
(vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus
aureus, extended-spectrum beta-lactamase-producing Escherichia coli and
Klebsiella species, carbapenem-hydrolyzing Klebsiella pneumonia and MDR
Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species), were
identified by logistic regression among patients admitted from the community
(either with community-acquired or HCAI).
RESULTS: There were 1035 patients with infection, 718 from the community. Of
these, 439 (61%) had microbiologic documentation; 123 were MDR (28%). Among MDR:
104 (85%) had MDR-GN and 41 (33%) had an ESKAPE infection. Independent risk
factors associated with MDR and MDR-GN infection were: age (adjusted odds ratio
(OR) = 1.7 and 1.5, p = 0.001 and p = 0.009, respectively), and hospitalization
in the previous year (between 4 and 12 months previously) (adjusted OR = 2.0 and
1,7, p = 0.008 and p = 0.048, respectively). Infection by pathogens from the
ESKAPE group was independently associated with previous antibiotic therapy
(adjusted OR = 7.2, p < 0.001) and a Karnofsky index <70 (adjusted OR = 3.7, p =
0.003). Patients with infection by MDR, MDR-GN and pathogens from the ESKAPE
group had significantly higher rates of inadequate antibiotic therapy than those
without (46% vs 7%, 44% vs 10%, 61% vs 15%, respectively, p < 0.001).
CONCLUSIONS: This study suggests that the inclusion of additional risk factors
in the current definition of HCAI for MDR pathogen infection, namely age >60
years, Karnofsky index <70, hospitalization in the previous year, and previous
antibiotic therapy, may be clinically beneficial for early diagnosis, which may
decrease the rate of inadequate antibiotic therapy among these patients. BACKGROUND: Bacterial species from the ESKAPE group (i.e. Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa and Enterobacter species) are frequently resistant to
antibiotics. The purpose of this study was to monitor the incidence of ESKAPE
pathogens at the intensive care unit (ICU) of a tertiary care hospital in
Monterrey, Mexico.
METHODS: All clinically relevant organisms isolated from June 2011 to June 2012
were included. Identification and susceptibility testing was performed using
panels from Sensititre. Resistance to oxacillin, for S. aureus, and the
production of extended spectrum β-lactamases (ESBLs), for K. pneumonia, were
determined as defined by the Clinical Laboratory Standards Institute. Also, the
presence of vanA and vanB genes was determined in E. faecium vancomycin
(VAN)-resistant isolates.
RESULTS: The majority of pathogens (64.5%) isolated in the ICU unit were from
the ESKAPE group. The organisms most frequently isolated were A. baumannii
(15.8%) and P. aeruginosa (14.3%). A high resistance to carbapenems was detected
for A. baumannii (75.3%) while 62% of S. aureus isolates were confirmed to be
methicillin resistant. Of the K. pneumoniae isolates, 36.9% were ESBL producers.
We detected three E. faecium VAN-resistant isolates, all of which contained the
vanA gene.
CONCLUSION: The presence of the ESKAPE group of pathogens is a major problem in
the ICU setting. The results of this study support the implementation of special
antimicrobial strategies to specifically target these microorganisms. The widespread use of antibiotics has been associated with the emergence of
antimicrobial resistance among bacteria. 'ESKAPE' (Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acintobacter baumannii,
Pseudomonas aeruginosa and Enterobacter spp.) pathogens play a major role in the
rapidly changing scenario of antimicrobial resistance in the 21st century.
Chloramphenicol is a broad spectrum antibiotic that was abandoned in developed
countries due to its association with fatal aplastic anemia. However, it is
still widely used in the developing world. In light of the emerging problem of
multi-drug resistant pathogens, its role should be reassessed. Our paper reviews
in vitro data on the activity of chloramphenicol against ESKAPE pathogens.
Susceptibility patterns for Gram-positives were good, although less favorable
for Gram-negatives. However, in combination with colistin, chloramphenicol was
found to have synergistic activity. The risk-benefit related to chloramphenicol
toxicity has not been analyzed. Therefore, extra precautions should be taken
when prescribing this agent. The rapid emergence of multidrug-resistant (MDR) bacterial pathogens poses a
major threat for human health. In recent years, genome sequencing has unveiled
many poorly expressed antibiotic clusters in actinomycetes. Here, we report a
well-defined ecological collection of >800 actinomycetes obtained from sites in
the Himalaya and Qinling mountains, and we used these in a concept study to see
how efficiently antibiotics can be elicited against MDR pathogens isolated
recently from the clinic. Using 40 different growth conditions, 96 actinomycetes
were identified - predomitly Streptomyces - that produced antibiotics with
efficacy against the MDR clinical isolates referred to as ESKAPE pathogens:
Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa and/or Enterobacter cloacae.
Antimicrobial activities that fluctuated strongly with growth conditions were
correlated with specific compounds, including borrelidin, resistomycin,
carbomethoxy-phenazine, and 6,7,8- and 5,6,8-trimethoxy-3-methylisocoumarin, of
which the latter was not described previously. Our work provided insights into
the potential of actinomycetes as producers of drugs with efficacy against
clinical isolates that have emerged recently and also underlined the importance
of targeting a specific pathogen. BACKGROUND: Although bacteremias caused by the 6 ESKAPE pathogens (Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter species) have recently been highlighted
as a serious complication in solid organ transplant (SOT), more information is
urgently needed. We sought to investigate the frequency and clinical outcomes of
ESKAPE bacteremia in SOT and determine the risk factors for mortality.
METHODS: A retrospective analysis of bacteremia after SOT was reviewed. Risk
factors for mortality caused by ESKAPE bacteremia were identified.
RESULTS: Eighty-four episodes of bacteremia were caused by ESKAPE strains. Of
these strains, 41 were caused by resistant ESKAPE (rESKAPE) organisms. The only
factor for bacteremia-related mortality independently associated with ESKAPE was
septic shock (odds ratio [OR] = 21.017, 95% confidence interval [CI] =
5.038-87.682, P < 0.001). The factors for bacteremia-related mortality
independently associated with rESKAPE bacteremia were septic shock (OR = 16.558,
95% CI = 6.620-104.668, P = 0.003) and age ≥40 years (OR = 7.521, 95% CI =
1.196-47.292, P = 0.031).
CONCLUSIONS: To improve the outcomes of transplantation, more effective
therapeutic treatments are of paramount importance when older SOT recipients
with bacteremia due to ESKAPE/rESKAPE organisms present with septic shock. Author information:
(1)Department of Clinical Microbiology, Kalmar County Hospital, Kalmar, Sweden
Department of Medicine and Optometry, Linnaeus University, Kalmar, Sweden
[email protected].
(2)Department of Clinical Microbiology and Department of Clinical and
Experimental Medicine, Linköping University, Linköping, Sweden.
(3)The Public Health Agency of Sweden, Solna, Sweden.
(4)Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
Linköping University, Linköping, Sweden.
(5)Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar,
Sweden.
(6)Department of Clinical Microbiology, Kalmar County Hospital, Kalmar, Sweden
Department of Medicine and Optometry, Linnaeus University, Kalmar, Sweden
Department of Clinical Microbiology and Department of Clinical and Experimental
Medicine, Linköping University, Linköping, Sweden. BACKGROUND: Although infections caused by the pathogens Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter spp (ESKAPE) have recently been
identified as serious emerging problems in solid organ transplant, no
information in liver transplant (LT) recipients is available. We sought to
investigate the risk factors for associated mortality in LT recipients with
ESKAPE infections.
METHODS: A retrospective analysis of infection after LT was reviewed. Risk
factors for mortality caused by ESKAPE infection were identified.
RESULTS: Fifty-three episodes of infections caused by ESKAPE were documented in
51 LT recipients. The main sites of infection were the bloodstream (49.0%), the
lungs (33.3%), and the intra-abdominal/biliary tract (17.6%). The risk factors
for mortality independently associated with ESKAPE infection were female sex
(odds ratio [OR] = 6.6, 95% confidence interval [CI] = 1.1-40.8, P = .042),
septic shock (OR = 30.1, 95% CI = 3.7-244.8, P = .001), and lymphocyte
counts <300/mm(3) (OR = 20.2, 95% CI = 2.9-142.2, P = .003).
CONCLUSIONS: To improve the results of LT, more effective therapeutic treatments
are of paramount importance when female LT recipients with ESKAPE infection
present with septic shock and decreased lymphocyte counts. Antimicrobial drug resistance is a growing threat to global public health.
Multidrug resistance among the 'ESKAPE' organisms - encompassing Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa and Enterobacter spp. - is of particular concern because
they are responsible for many serious infections in hospitals. Although some
promising agents are in the pipeline, there is an urgent need for new antibiotic
scaffolds. However, antibacterial researchers have struggled to identify new
small molecules with meaningful cellular activity, especially those effective
against multidrug-resistant Gram-negative pathogens. This difficulty ultimately
stems from an incomplete understanding of efflux systems and compound permeation
through bacterial membranes. This Opinion article describes findings from
target-based and phenotypic screening efforts carried out at AstraZeneca over
the past decade, discusses some of the subsequent chemistry challenges and
concludes with a description of new approaches comprising a combination of
computational modelling and advanced biological tools which may pave the way
towards the discovery of new antibacterial agents. |
What is the effect of dovitinib on the cell cycle? | Dovitinib triggers a G2 /M arrest. It promotes a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induce a G2 arrest similar to the G2 DNA damage checkpoint. | PURPOSE: There is no standard of therapy for the treatment of Waldenström
macroglobulinemia (WM), therefore there is a need for the development of new
agents. Fibroblast growth factor receptor 3 (FGFR3) was shown to play a major
role in several types in cancer. Dovitinib, an inhibitor of FGFR3, was effective
in hematologic maligcies. In this study, we tested FGFR3 as a therapeutic
target in WM and tested the effect of dovitinib on cell proliferation and
apoptosis of WM cells in the context of BM microenvironment.
METHODS: The expression of FGFR3 in WM cells was tested using immunofluorescence
and flow cytometry. Cell signaling in response to stimulation with FGF3 and
stromal cells, and its inhibition by dovitinib was performed using
immunoblotting. Cell survival and cell proliferation were assessed by MTT and
BrdU assays. Apoptosis was measured by detection of APO-2.7 and cleavage of
caspase-3 using flow cytometry. Cell cycle was performed by PI staining of cells
and flow cytometry. The combinatory effect of dovitinib with other drugs was
analyzed using Calcusyn software. The effect of dovitinib was tested in vivo.
RESULTS: FGFR3 was overexpressed in WM cells and its activation induced cell
proliferation. Inhibition of FGFR3 with dovitinib decreased cell survival,
increased apoptosis, and induced cell cycle arrest. Inhibition of FGFR3 by
dovitinib reduced the interaction of WM to bone marrow components, and reversed
its proliferative effect. Dovitinib had an additive effect with other drugs.
Moreover, dovitinib reduced WM tumor progression in vivo.
CONCLUSION: We report that FGFR3 is a novel therapeutic target in WM, and
suggest dovitinib for future clinical trial the treatment of patients with WM. BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of
cancer death. Although sorafenib has been shown to improve survival of patients
with advanced HCC, this improvement is modest and patients eventually have
refractory disease. This study aims at investigating the antitumor,
antiangiogenesis and antimetastatic activities of dovitinib in preclinical
models of HCC.
METHODS: 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were
employed to study the antitumor effect of dovitinib. Changes of biomarkers
relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting.
Microvessel density, apoptosis and cell proliferation were analyzed by
immunohistochemistry.
RESULTS: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle
arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced
cell migration. Dovitinib inhibited basal expression and FGF-induced
phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently
inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated
with inactivation of FGFR/PDGFR-β/VEGFR-2 signaling pathways. Dovitinib also
caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and
p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction
in cellular proliferation and the induction of tumor cell apoptosis. In an
orthotopic model, dovitinib potently inhibited primary tumor growth and lung
metastasis and significantly prolonged mouse survival.
CONCLUSIONS: Dovitinib demonstrated significant antitumor and antimetastatic
activities in HCC xenograft models. This study provides a compelling rationale
for clinical investigation in patients with advanced HCC. |
What is the involvement of PDGFRB in metastatic medulloblastoma? | Platelet-derived growth factor (PDGF) receptor B (PDGFRB) expression was shown to correlate with metastatic medulloblastoma, while PDGFRB tyrosine kinase activity was demonstrated to be critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR. | Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates
with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells
phosphorylates extracellular signal-regulated kinase (ERK) and promotes
migration. We sought to determine whether blocking PDGFR activity effectively
inhibits signaling required for medulloblastoma cell migration and invasion.
DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate
(Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small
interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR
phosphorylation and expression, respectively. PDGFR cell signaling, migration,
invasion, survival, and proliferation following PDGF-BB stimulation, with and
without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased
PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor
receptor (EGFR), which correlated with enhanced migration, survival, and
proliferation. Imatinib (1 μmol/L) treatment of DAOY and D556 cells inhibited
PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively,
and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but
increased PTEN expression and activity. Imatinib treatment also induced DAOY
cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h.
siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival
and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR
transactivation, indicating that the effects of imatinib treatment are specific
to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase
activity is critical for migration and invasion of medulloblastoma cells
possibly by transactivating EGFR; thus, imatinib may represent an important
novel therapeutic agent for the treatment of medulloblastoma. |
What is known as Calcium Induced Calcium Release (CICR) and its role in cardiomyocyte contractility? | the cicr mechanism has been understood mainly based on binding of cytosolic ca(2+) with ryanodine receptors (ryrs) and inducing ca(2+) release from the sarcoplasmic reticulum (sr). l-type ca(2+) channels activate ryrs to produce cicr in smooth muscle cells in the form of ca(2+) sparks and propagated ca(2+) waves. in heart cells, a tight coupling between the gating of single l-type ca(2+) channels and ryanodine receptors (ryrs) underlies calcium release. the importance of ca-induced ca release in excitation-contraction coupling in the heart. waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. in mammals, ca(2+) influx as l-type ca(2+) current (ica) triggers the release of ca(2+) from sarcoplasmic reticulum (sr) and ca(2+)-induced ca(2+) release (cicr) is critical for excitation-contraction coupling. | Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as
elementary calcium release events (calcium sparks) associated with the opening
of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating
of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies
calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs
to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and
propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel
opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type
Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+)
sparks are often observed after channel closure. CICR is a function of the net
flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude
of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium
release is completely eliminated by cytosolic calcium buffering. Thus, L-type
Ca(2+) channels are loosely coupled to RYR through an increase in global
[Ca(2+)] due to an increase in the effective distance between L-type Ca(2+)
channels and RYR, resulting in an uncoupling of the obligate relationship that
exists in striated muscle between the action potential and calcium release. The majority of contractile calcium in cardiac muscle is released from stores in
the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release
(CICR) through ryanodine receptors. Because CICR is intrinsically
self-reinforcing, the stability of and graded regulation of cardiac EC coupling
appear paradoxical. It is now well established that this gradation results from
the stochastic recruitment of varying numbers of elementary local release
events, which may themselves be regenerative, and which can be directly observed
as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices,
and most calcium sparks are now believed to involve activation of multiple RyRs.
This implies that local CICR is regenerative, requiring a mechanism to terminate
it. It was initially assumed that this mechanism was inactivation of the RyR,
but during the decade since the discovery of sparks, no sufficiently strong
inactivation mechanism has been demonstrated in vitro and all empirically
determined gating schemes for the RyR give unstable EC coupling in Monte Carlo
simulations. We consider here possible release termination mechanisms.
Stochastic attrition is the spontaneous decay of active clusters due to random
channel closure; calculations show that it is much too slow unless assisted by
another process. Calcium-dependent RyR inactivation involving third-party
proteins remains a viable but speculative mechanism; current candidates include
calmodulin and sorcin. Local depletion of SR release terminal calcium could
terminate release, however calculations and measurements leave it uncertain
whether a sufficient diffusion resistance exists within the SR to sustain such
depletion. Depletion could be assisted by dependence of RyR activity on SR
lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but
it is not clear if it is a strong enough effect to account for the robust
termination of sparks. The existence of direct interactions among clustered RyRs
might account for the discrepancy between the inactivation properties of
isolated RyRs and intact clusters. Such coupled gating remains controversial.
Determining the mechanism of release termination is the outstanding unsolved
problem of cardiac EC coupling, and will probably require extensive genetic
manipulation of the EC coupling apparatus in its native environment to unravel
the solution. Pressure overload-induced hypertrophy is a key step leading to heart failure.
The Ca(2+)-induced Ca(2+) release (CICR) process that governs cardiac
contractility is defective in hypertrophy/heart failure, but the molecular
mechanisms remain elusive. To examine the intermolecular aspects of CICR during
hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling
between a single L-type Ca(2+) channel (LCC) and ryanodine receptors (RyRs) in
aortic stenosis rat models of compensated (CHT) and decompensated (DHT)
hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49%
prolongation in coupling latency, a 47% decrease in chance of hit, and a 72%
increase in chance of miss in DHT, demonstrating a state of "intermolecular
failure." Unexpectedly, these modifications also occurred robustly in CHT due at
least partially to decreased expression of junctophilin, indicating that
intermolecular failure occurs prior to cellular manifestations. As a result,
cell-wide Ca(2+) release, visualized as "Ca(2+) spikes," became desynchronized,
which contrasted sharply with unaltered spike integrals and whole-cell Ca(2+)
transients in CHT. These data suggested that, within a certain limit, termed the
"stability margin," mild intermolecular failure does not damage the cellular
integrity of excitation-contraction coupling. Only when the modification steps
beyond the stability margin does global failure occur. The discovery of "hidden"
intermolecular failure in CHT has important clinical implications. Calcium (Ca(2+))-induced Ca(2+) release (CICR) is widely accepted as the
principal mechanism linking electrical excitation and mechanical contraction in
cardiac cells. The CICR mechanism has been understood mainly based on binding of
cytosolic Ca(2+) with ryanodine receptors (RyRs) and inducing Ca(2+) release
from the sarcoplasmic reticulum (SR). However, recent experiments suggest that
SR lumenal Ca(2+) may also participate in regulating RyR gating through
calsequestrin (CSQ), the SR lumenal Ca(2+) buffer. We investigate how SR Ca(2+)
release via RyR is regulated by Ca(2+) and calsequestrin (CSQ). First, a
mathematical model of RyR kinetics is derived based on experimental evidence. We
assume that the RyR has three binding sites, two cytosolic sites for Ca(2+)
activation and inactivation, and one SR lumenal site for CSQ binding. The open
probability (P(o)) of the RyR is found by simulation under controlled cytosolic
and SR lumenal Ca(2+). Both peak and steady-state P(o) effectively increase as
SR lumenal Ca(2+) increases. Second, we incorporate the RyR model into a CICR
model that has both a diadic space and the junctional SR (jSR). At low jSR
Ca(2+) loads, CSQs are more likely to bind with the RyR and act to inhibit jSR
Ca(2+) release, while at high SR loads CSQs are more likely to detach from the
RyR, thereby increasing jSR Ca(2+) release. Furthermore, this CICR model
produces a nonlinear relationship between fractional jSR Ca(2+) release and jSR
load. These findings agree with experimental observations in lipid bilayers and
cardiac myocytes. PURPOSE: Release of calcium (Ca(2+)) from the sarcoplasmic reticulum (SR)
induced by Ca(2+) influx through voltage-dependent sarcolemmal L-type Ca(2+)
channels (CICR) in cardiac muscle cells has been implicated as a potential
target contributing to anesthetic-induced myocardial depression. In an earlier
study, we found that (1) a half-logistic (h-L) function, which represents a
half-curve of a sigmoid logistic function with a boundary at the inflection
point, curve-fits the first half of the ascending phases of the isometric
myocardial tension and isovolumic left ventricular (LV) pressure waveforms
better than a mono-exponential (m-E) function and (2) the h-L time constants are
useful as inotropic indices. We report here our investigation of the potential
application of an h-L function to the analysis of the first half of the
ascending phase of the Ca(2+) transient curve (faCaT) that precedes and
initiates myocardial contraction and the increase in LV pressure.
METHODS: Ca(2+) transients (CaT) were measured using the Ca(2+)-sensitive
photoprotein aequorin, which was microinjected into seven isolated rabbit right
ventricular and 15 isolated mouse LV papillary muscles. The faCaT data from the
beginning of twitch stimulation to the maximum of the first-order time
derivative of Ca(2+) concentration (dCa/dt(max)) was curve-fitted by the
least-squares method using h-L and m-E function equations.
RESULTS: The mean correlation coefficient (r) values of the h-L and m-E
curve-fits for the faCaTs were 0.9740 and 0.9654 (P < 0.05) in the rabbit and
0.9895 and 0.9812 (P < 0.0001) in the mouse.
CONCLUSION: The h-L curves tracked the amplitudes and time courses of the faCaTs
in cardiac muscles more accurately than m-E functions. Based on this result, we
suggest that the h-L time constant may be a more reliable index than the m-E
time constant for evaluating the rate of CICR from the SR in myocardial Ca(2+)
handling. The h-L approach may provide a more useful model for the study of CICR
during the contraction process induced by anesthetic agents. Calcium-induced calcium release (CICR) is an inherently regenerative process due
to the Ca(2+)-dependent gating of ryanodine receptors (RyRs) in the
sarco/endoplasmic reticulum (SR) and is critical for cardiac
excitation-contraction coupling. This process is seen as Ca(2+) sparks, which
reflect the concerted gating of groups of RyRs in the dyad, a specialised
junctional signalling domain between the SR and surface membrane. However, the
mechanism(s) responsible for the termination of regenerative CICR during the
evolution of Ca(2+) sparks remain uncertain. Rat cardiac RyR gating was recorded
at physiological Ca(2+), Mg(2+) and ATP levels and incorporated into a 3D model
of the cardiac dyad which reproduced the time-course of Ca(2+) sparks, Ca(2+)
blinks and Ca(2+) spark restitution. Model CICR termination was robust,
relatively insensitive to the number of dyadic RyRs and automatic. This emergent
behaviour arose from the rapid development and dissolution of oscopic Ca(2+)
gradients within the dyad. These simulations show that CICR does not require
intrinsic inactivation or SR calcium sensing mechanisms for stability and
cessation of regeneration that arises from local control at the molecular scale
via a process we call 'induction decay'. Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In
mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of
Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+) release (CICR)
is critical for excitation-contraction coupling. In fish, the relative
contribution of external and internal Ca(2+) is unclear. Here, we characterized
the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular
myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded
with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol
conditions. In the absence of β-adrenergic stimulation, the rate of ICa
inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs.
30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was
significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was
the charge carrier, inactivation was significantly slower in both conditions
(61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed
that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+),
while with isoproterenol, inactivation was Ca(2+)-dependent (∼65%) and highly
reliant on SR Ca(2+) (∼46%). Thus, SR Ca(2+) is not released in basal
conditions, and ICa is the main trigger of contraction, whereas during a stress
response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not
attributed to differences in SR Ca(2+) load because caffeine-induced transients
were not different in both conditions. Therefore, Ca(2+) stored in SR of trout
cardiomyocytes may act as a safety mechanism, allowing greater contraction when
higher contractility is required, such as stress or exercise. |
What is the action of molindone? | Molindone is a short-acting antipsychotic.
Molindone, along with other antipsychotic drugs which elicit little or no Parkinsonism, bind more loosely than dopamine to D2 receptors. Compared to the tightly bound antipsychotic drugs, the more loosely bound antipsychotics generally require higher clinical doses, require fewer days for clinical adjustment, but may dissociate from the D2 receptor more rapidly and could lead to clinical relapse somewhat earlier than that found with the traditional tightly bound antipsychotic drugs.
Molindone is D2-selective in vitro and has a dual D1-D2 receptor profile in vivo.
Molindone can selectively block the presynaptic DA receptors.
Molindone causes a statistically significant up-regulation of both the long and short isoforms of the D2 receptor mRNAs in the prefrontal and temporal cortex, but has no effect on D4 mRNA levels in either cortical or striatal tissue.
Molindone elevates Fos-like immunoreactivity (FLI) in the dorsolateral striatum.
Molindone exhibits selectivity for cortical serotonin-stimulated cyclase versus dopamine-stimulated cyclase.
Molindone in low intravenous doses (0.4-0.8 mg/kg) was found to reverse d-amphetamine and apomorphine induced depression of DA neurons and to block apomorphine induced depression of these cells. Molindone was also found to increase dopamine synthesis and dihydroxyphenylactic acid levels in the striatum and olfacotry tubercles. In all of these respects molindone behaves identically to most classical neuroleptics. However, unlike most antipsychotic drugs previously tested, molindone failed to increase the baseline firing rate of DA cells and blocked haloperidol induced increases in DA neuron activity. In this regard molindone most closely resembles thioridazine and clozapine. | The effect of molindone on the activity of dopaminergic (DA) neurons in the rat
midbrain and on DA metabolism in the striatum and olfactory tubercles was
studied using extracellular single unit recording and biochemical techniques
respectively. Molindone in low intravenous doses (0.4-0.8 mg/kg) was found to
reverse d-amphetamine and apomorphine induced depression of DA neurons and to
block apomorphine induced depression of these cells. Molindone was also found to
increase dopamine synthesis and dihydroxyphenylactic acid levels in the striatum
and olfacotry tubercles. In all of these respects molindone behaves identically
to most classical neuroleptics. However, unlike most antipsychotic drugs
previously tested, molindone failed to increase the baseline firing rate of DA
cells and blocked haloperidol induced increases in DA neuron activity. In this
regard molindone most closely resembles thioridazine and clozapine. Possible
mechanisms of action of molindone are discussed based on these findings. In vivo binding of the benzamide derivative [3H]raclopride was studied in mouse
brain. The binding was saturable, reversible and stereospecific. Non-specific
binding was 5-15% of the total binding. Pharmacological characterization of the
binding indicated labelling of dopamine D2 receptors since the binding was
potently inhibited by compounds with high affinity for this receptor in vitro.
On the other hand, compounds with low affinity in vitro i.e., dopamine
D1-selective compounds were weak or inactive as inhibitors of [3H]raclopride
binding. A comparison of the pharmacological characteristics of [3H]raclopride
and [3H]SCH 23390 binding in vivo indicated that compounds with selectivity in
vitro retained this selectivity in vivo. Thus, spiroperidol, haloperidol,
1-sulpiride, clebopride, LY 171555 and (-)-NPA ((-)-N-propyl-norapomorphine)
were D2 selective while SCH 23390, SKF 38393 and SKF 75670 were D1 selective.
Clozapine, tilozepine, cis-flupentixol, chlorpromazine and butaclamol were
non-selective both in vitro and in vivo. However, a few compounds changed
profile in vivo compared to in vitro. Thus, fluperlapine and fluphenazine had a
dual D1-D2 receptor profile in vitro but were D1- or D2-selective in vivo,
respectively. Pergolide and molindone which were D2-selective in vitro both had
a dual D1-D2 receptor profile in vivo. In conclusion, [3H]raclopride, in vivo,
selectively labels the dopamine D2 receptor. Comparison of the pharmacological
characteristics of [3H]raclopride and [3H]SCH 23390 binding in vivo supported
the that the dopamine D1 receptor is an important target for a variety of
neuroleptics, especially of the clozapine type. This may indicate that blockade
of the dopamine D1 receptor conveys antipsychotic action. The ergot alkaloids studied do exert selective effects on monoamine receptor
systems. Lisuride acts as a very potent stimulator of adenylate cyclase in
cortical brain regions, and may function as a mixed agonist-antagonist at high
concentrations. It is most likely that in cortex, lisuride effects both dopamine
and serotonin receptors, but predomitly serotonin receptors coupled to
adenylate cyclase. The antagonist molindone exhibits selectivity for cortical
serotonin-stimulated cyclase versus dopamine-stimulated cyclase and may prove
useful for further elucidating the sites of lisuride action. LSD interacts with
serotonin-stimulated cortical adenylate cyclase at higher concentrations than
are needed for lisuride stimulation but, nevertheless, at lower concentrations
than for serotonin itself (2-4). Bromocriptine, lergotrile and ergonovine may
also act as agonists in stimulating adenylate cyclase, but with considerably
less potency, and with differences in regional specificity for this stimulation,
from lisuride and LSD. Each of these ergots may act as a mixed
agonist-antagonist at high concentrations. With respect to the regions studied,
antagonist effects on cyclase appear to be more prominent in striatum than in
the cortical regions. The greater specificity of lisuride for serotonergic
cortical receptors should make this compound useful in further studies of this
system. Small doses of apomorphine (AP, 31.25-125 micrograms/ kg IP) induced
dose-dependent catalepsy in rats. However, unlike the stereotyped behavior
induced by high doses of AP which has a rapid onset and is short-lasting, the
cataleptic effect induced by small doses of AP was evident 30 min after AP
injection and was unusually long-lasting. Further, AP (31.25-125 micrograms/kg)
administered 60 min before methamphetamine was found to significantly antagonize
the methamphetamine-induced stereotyped behavior. Pretreatment with molindone
(0.45 and 0.8 mg/kg IP), in doses reported to selectively block the presynaptic
DA receptors, not only decreased the cataleptic effect of AP but also reversed
the AP antagonism of methamphetamine stereotypy. The results suggest that small
doses of AP induce catalepsy and antagonize methamphetamine stereotypy probably
by an action at presynaptic DA receptor sites. Psychotic symptoms related to mental and medical disorders can pose a medical
emergency. Selecting an appropriate antipsychotic medication to treat this
emergency is based on the clinical situation, preferred route of administration,
pharmacokinetic profile of the antipsychotic and the medications currently being
taken by the patient. Intramuscular preparations are usually preferred over oral
medication when the patients are not co-operative and require drugs with a
faster onset of action and good bioavailability. High potency antipsychotics
such as haloperidol and fluphenazine are effective in stabilising patients with
psychotic symptoms quickly. Loxapine is an alternative when sedation is
necessary and molindone is useful if a short-acting antipsychotic is required.
Rapid neuroleptisation with intramuscular preparations of antipsychotic achieves
therapeutic drug concentrations more rapidly, and also provides optimal control
of psychotic symptoms. If the patient is cooperative, liquid oral preparations
can be used; they are as effective as intramuscular formulations. If long term
treatment with an antipsychotic in necessary and patients are stabilised, they
can be switched from intramuscular to oral preparations. The oral dose is
usually 1.5 to 5 times the total intramuscular dose per day, based on the
bioavailability of the antipsychotic medication. If the patient is currently
taking antipsychotic medication when the emergency situation occurs, it is
usually adequate to increase the dose of antipsychotic drug. Appropriate dose
adjustment or antipsychotic selection is necessary when drug interactions are
expected. An in-depth knowledge of the pharmacokinetic profile and drug
interaction profile of antipsychotic in necessary for the selection of the
appropriate antipsychotic for any given emergency situation. The RNase Protection Assay was used to examine the regulation of D2 and D4
dopamine receptor mRNAs in the cerebral cortex and neostriatum of nonhuman
primates after chronic treatment with a wide spectrum of antipsychotic
medications (chlorpromazine, clozapine, haloperidol, molindone, olanzapine,
pimozide, remoxipride and risperidone). Tiapride, a D2 antagonist that lacks
antipsychotic activity, was also included. All drugs were administered orally
for 6 months at doses recommended for humans. All antipsychotic drug treatments
examined in this study caused a statistically significant up-regulation of both
the long and short isoforms of the D2 receptor mRNAs in the prefrontal and
temporal cortex. Tiapride, in contrast, significantly up-regulated only the
level of D2-long mRNA in these areas. The same drug treatments produced less
uniform effects in the neostriatum than in the cortex: clozapine and olanzapine
failed to significantly elevate either D2-long or D2-short receptor messages in
this structure unlike all other drugs, including tiapride. In both the cerebral
cortex and striatum, D4 receptor mRNA was upregulated by certain typical
(chlorpromazine and haloperidol) and certain atypical (clozapine, olanzapine and
risperidone) antipsychotic agents as well as by tiapride. Other drugs of the
typical (molindone and pimozide) and atypical (remoxipride) classes had no
effect on D4 mRNA levels in either cortical or striatal tissue. The finding that
up-regulation of D2 dopamine receptor mRNAs was a consistently observed effect
of a wide range of antipsychotic agents in the cerebral cortex but not in the
neostriatum, coupled with the fact that the D2-short isoforms in the cortex were
not regulated by a notipsychotic D2 antagonist, tiapride, draws attention to
the importance of the D2 dopamine receptor in the cerebral cortex as a
potentially critical, common site of action of antipsychotic medications. This review addresses two questions. First, why does clozapine apparently occupy
low levels of dopamine D2 receptors in patients, in contrast to all other
antipsychotic drugs which occupy 70-80% of brain dopamine D2 receptors? Second,
what is the receptor basis of action of antipsychotic drugs which elicit low
levels of Parkinsonism? Antipsychotic doses of clozapine occupy between 0% and
50% of D2 receptors, as measured in patients by a variety of radioligands. It
has recently been found, however, that the percent occupancy of a receptor by a
drug depends on the radioligand used to measure that receptor. Based on this new
finding, this review concludes that clozapine clinically occupies high levels of
D2 receptors in the absence of any radioligand. This occupancy is estimated to
be of the order of 70-80% in the dopamine-rich region of the human striatum, and
even higher in the limbic D2-containing regions which are low in endogenous
synaptic dopamine. This conclusion arises from two different approaches. One
approach is to relate the reported clozapine occupancies in the human striatum
with the dissociation constants of the various radioligands at the D2 receptor.
This relation extrapolates to approximately 70-80% occupancy by clozapine when
clozapine competes with endogenous dopamine at the D2 receptor. The second
approach is to calculate the D2 occupancy of each antipsychotic drug, using the
average spinal fluid concentration and the correct dissociation constant of the
antipsychotic, thereby revealing that all antipsychotic drugs, including
clozapine, occupy approximately 70-80% of dopamine D2 receptors in the human
striatum, and possibly higher in the limbic regions. As determined by the new
dissociation constants, antipsychotic drugs which elicit Parkinsonism
(trifluperazine, chlorpromazine, raclopride, haloperidol, fluphenazine,
risperidone) bind more tightly than dopamine to D2, while those antipsychotic
drugs which elicit little or no Parkinsonism (melperone, seroquel, perlapine,
clozapine, remoxipride, molindone, sulpiride, olanzapine, sertindole) bind more
loosely than dopamine to D2 receptors. Compared to the tightly bound
antipsychotic drugs, the more loosely bound antipsychotics generally require
higher clinical doses, require fewer days for clinical adjustment, but may
dissociate from the D2 receptor more rapidly and could lead to clinical relapse
somewhat earlier than that found with the traditional tightly bound
antipsychotic drugs. |
What is the function of the enzymes known as dual specificity phoshpatases (DUSPs)? | DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. | The protein tyrosine phosphatase family (PTP) contains a group of
dual-specificity phosphatases (DUSPs) that regulate the activivity of MAP
kinases (MAPKs), which are key effectors in the control of cell growth and
survival in physiological and pathological processes, including cancer. These
phosphatases, named as MKP-DUSPs, include the MAPK phosphatases (MKPs) as well
as a group of small-size atypical DUSPs structurally and functionally related to
the MKPs. MKP-DUSPs, in most of the cases, are direct inactivators of MAPKs by
dephosphorylation of both the Thr and the Tyr regulatory residues at the MAPKs
catalytic loop. In some other cases, MKP-DUSPs regulate the activity of MAPKs
indirectly, acting through upstream MAPK pathways components. The active
involvement of MKP-DUSPs in oncogenesis or resistance to cancer therapies is now
well documented, making the search and validation of MKP-DUSPs inhibitors a
prominent area in clinical cancer research. Here, we review the current
knowledge on the role of MKP-DUSPs in human cancer, the status of the
preclinical development and validation of specific MKP-DUSP inhibitors, and the
potential of MKP-DUSPs as targets for anti-cancer drugs. Dual-specificity protein phosphatases participate in signal transduction
pathways inactivating mitogen-activated protein kinases (MAP kinases). These
signaling pathways are of critical importance in the regulation of numerous
biological processes, including cell proliferation, differentiation and
development. The social ameba Dictyostelium discoideum harbors 14 genes coding
for proteins containing regions very similar to the dual-specificity protein
phosphatase domain. One of these genes, mkpB, additionally codes for a region
similar to the Rhodanase domain, characteristic of animal MAP
kinase-phosphatases, in its N-terminal region. Cells that over-express this gene
show increased protein phosphatase activity. mkpB is expressed in D. discoideum
ameba at growth but it is greatly induced at 12h of multicellular development.
Although it is expressed in all the cells of developmental structures, mkpB mRNA
is enriched in cells with a distribution typical of anterior-like cells. Cells
that express a catalytically inactive mutant of MkpB grow and aggregate like
wild-type cells but show a greatly impaired post-aggregative development. In
addition, the expression of cell-type specific genes is very delayed, indicating
that this protein plays an important role in cell differentiation and
development. Cells expressing the MkpB catalytically inactive mutant show
increased sensitivity to cisplatin, while cells over-expressing wild type MkpB,
or MkpA, proteins or mutated in the MAP kinase erkB gene are more resistant to
this chemotherapeutic drug, as also shown in human tumor cells. Dual specificity phosphatase 1 (DUSP1) dephosphorylates and, hence, regulates
the activity of MAP kinases. The present study investigated the effect of DUSP1
on inflammatory gene expression and on the development of carragee-induced
inflammation. It was found that DUSP1 expression was increased by LPS, and the
down-regulation of DUSP1 by siRNA enhanced the phosphorylation of p38 MAPK,
while JNK phosphorylation was not affected in murine macrophages. LPS-induced
interleukin (IL)-6, tumor-necrosis factor (TNF) and cyclooxygenase-2 (COX2)
expression were enhanced in bone marrow-derived macrophages (BMMs) from
DUSP1(-/-) mice as compared to those from wild-type mice. In addition,
down-regulation of DUSP1 by siRNA enhanced IL-6, TNF and COX2 expression in J774
macrophages, while p38 MAPK inhibitors SB202190 and BIRB 796 inhibited the
expression of those inflammatory factors. In vivo, the intensity of the
carragee-induced paw edema reaction was increased in DUSP1(-/-) mice as
compared to the wild-type animals. In conclusion, DUSP1 is an important negative
regulator of the acute inflammatory response by limiting p38 MAPK, and compounds
which enhance DUSP1 expression or activity may hold a promise as
anti-inflammatory drugs. Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative
regulatory network that acts to shape the duration, magnitude and spatiotemporal
profile of MAP kinase activities in response to both physiological and
pathological stimuli. Individual MKPs may exhibit either exquisite specificity
towards a single mitogen-activated protein kinase (MAPK) isoform or be able to
regulate multiple MAPK pathways in a single cell or tissue. They can act as
negative feedback regulators of MAPK activity, but can also provide mechanisms
of crosstalk between distinct MAPK pathways and between MAPK signalling and
other intracellular signalling modules. In this review, we explore the current
state of knowledge with respect to the regulation of MKP expression levels and
activities, the mechanisms by which individual MKPs recognize and interact with
different MAPK isoforms and their role in the spatiotemporal regulation of MAPK
signalling. Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions
and are key pharmaceutical targets. Regulation of MAPKs is achieved via a
plethora of regulatory proteins including activating MAPKKs and an abundance of
deactivating phosphatases. Although all regulatory proteins use an identical
interaction site on MAPKs, the common docking and hydrophobic pocket, they use
distinct kinase interaction motif (KIM or D-motif) sequences that are present in
linear, peptide-like, or well folded protein domains. It has been recently shown
that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a
unique binding mode to interact with p38α. Here we describe the interaction of
the MAPK binding domain of DUSP16 with p38α and show that despite belonging to
the same dual specificity phosphatase (DUSP) family, its interaction mode
differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an
additional helix, α-helix 4, to further engage p38α. This leads to an additional
interaction surface on p38α. Together, these structural and energetic
differences in p38α engagement highlight the fine-tuning necessary to achieve
MAPK specificity and regulation among multiple regulatory proteins. Several dual-specificity phosphatases (DUSPs) that play key roles in the direct
or indirect inactivation of different MAP kinases (MAPKs) have been implicated
in human cancers over the past decade. This has led to a growing interest in
identifying DUSPs and their specific inhibitors for further testing and
validation as therapeutic targets in human cancers. However, the lack of
understanding of the complex regulatory mechanisms and cross-talks between MAPK
signaling pathways, combined with the fact that DUSPs can act as a double-edged
sword in cancer progression, calls for a more careful and thorough
investigation. Among the various types of brain cancer, glioblastoma multiforme
(GBM) is notorious for its aggressiveness and resistance to current treatment
modalities. This has led to the search for new molecular targets, particularly
those involving various signaling pathways. DUSPs appear to be a promising
target, but much more information on DUSP targets and their effects on GBM is
needed before potential therapies can be developed, tested, and validated. This
review identifies and summarize the specific roles of DUSP1, DUSP4, DUSP6 and
DUSP26 that have been implicated in GBM. SIGNIFICANCE: The dual-specificity phosphatases (DUSPs) constitute a
heterogeneous group of cysteine-based protein tyrosine phosphatases, whose
members exert a pivotal role in cell physiology by dephosphorylation of
phosphoserine, phosphothreonine, and phosphotyrosine residues from proteins, as
well as other non-proteinaceous substrates.
RECENT ADVANCES: A picture is emerging in which a selected group of DUSP enzymes
display overexpression or hyperactivity that is associated with human disease,
especially human cancer, making feasible targeted therapy approaches based on
their inhibition. A panoply of molecular and functional studies on DUSPs have
been performed in the previous years, and drug-discovery efforts are ongoing to
develop specific and efficient DUSP enzyme inhibitors. This review summarizes
the current status on inhibitory compounds targeting DUSPs that belong to the
MAP kinase phosphatases-, small-sized atypical-, and phosphatases of
regenerating liver subfamilies, whose inhibition could be beneficial for the
prevention or mitigation of human disease.
CRITICAL ISSUES: Achieving specificity, potency, and bioavailability are the
major challenges in the discovery of DUSP inhibitors for the clinics. Clinical
validation of compounds or alternative inhibitory strategies of DUSP inhibition
has yet to come.
FUTURE DIRECTIONS: Further work is required to understand the dual role of many
DUSPs in human cancer, their function-structure properties, and to identify
their physiologic substrates. This will help in the implementation of therapies
based on DUSPs inhibition. BACKGROUND: Angiopoietin-1 (Ang-1) promotes survival and migration of
endothelial cells, in part through the activation of mitogen-activated protein
kinase (MAPK) pathways downstream of Tie-2 receptors. Dual-specificity
phosphatases (DUSPs) dephosphorylate phosphotyrosine and
phosphoserine/phosphothreonine residues on target MAPKs. The mechanisms by which
DUSPs modulate MAPK activation in Ang-1/Tie-2 receptor signaling are unknown in
endothelial cells.
METHODS AND RESULTS: Expression of various DUSPs in human umbilical vein
endothelial cells exposed to Ang-1 was measured. The functional roles of DUSPs
in Ang-1-induced regulation of MAPK activation, endothelial cell survival,
migration, differentiation, and permeability were measured using selective siRNA
oligos. Ang-1 differentially induces DUSP1, DUSP4, and DUSP5 in human umbilical
vein endothelial cells through activation of the PI-3 kinase, ERK1/2, p38, and
SAPK/JNK pathways. Lack-of-function siRNA screening revealed that DUSP1
preferentially dephosphorylates p38 protein and is involved in Ang-1-induced
cell migration and differentiation. DUSP4 preferentially dephosphorylates
ERK1/2, p38, and SAPK/JNK proteins and, under conditions of serum deprivation,
is involved in Ang-1-induced cell migration, several antiapoptotic effects, and
differentiation. DUSP5 preferentially dephosphorylates ERK1/2 proteins and is
involved in cell survival and inhibition of permeability.
CONCLUSIONS: DUSP1, DUSP4, and DUSP5 differentially modulate MAPK signaling
pathways downstream of Tie-2 receptors, thus highlighting the importance of
these phosphatases to Ang-1-induced angiogenesis. MAPK activity is negatively regulated by members of the dual specificity
phosphatase (Dusp) family, which differ in expression, substrate specificity,
and subcellular localization. Here, we investigated the function of Dusp16/MKP-7
in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly
expressed in macrophages and dendritic cells following Toll-like receptor
stimulation. A gene trap approach was used to generate Dusp16-deficient mice.
Homozygous Dusp16tp/tp mice developed without gross abnormalities but died
perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently
reconstituted the lymphoid and myeloid compartments with Dusp16-deficient
hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow
progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge
with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a
Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not
dendritic cells, selectively overexpressed a subset of TLR-induced genes,
including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced
activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and
siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and
its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp
macrophages. Altogether, the Dusp16 gene trap mouse model identifies an
essential role in perinatal survival and reveals selective control of
differentiation and cytokine production of myeloid cells by the MAPK phosphatase
Dusp16. Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both
phosphoserine/threonine and phosphotyrosine, play vital roles in immune
activation, brain function and cell-growth signalling. A family-wide structural
library of human DUSPs was constructed based on experimental structure
determination supplemented with homology modelling. The catalytic domain of each
individual DUSP has characteristic features in the active site and in
surface-charge distribution, indicating substrate-interaction specificity. The
active-site loop-to-strand switch occurs in a subtype-specific manner,
indicating that the switch process is necessary for characteristic substrate
interactions in the corresponding DUSPs. A comprehensive analysis of the
activity-inhibition profile and active-site geometry of DUSPs revealed a novel
role of the active-pocket structure in the substrate specificity of DUSPs. A
structure-based analysis of redox responses indicated that the additional
cysteine residues are important for the protection of enzyme activity. The
family-wide structures of DUSPs form a basis for the understanding of
phosphorylation-mediated signal transduction and the development of
therapeutics. |
How is bladder wall thickness measured? | Ultrasound | OBJECTIVE: To determine whether transvaginal ultrasound measurement of bladder
wall thickness can be used as a screening test for detrusor instability in women
with urinary symptoms.
DESIGN: A blinded prospective study.
SETTING: A London teaching hospital.
PARTICIPANTS: One hundred and eight-four symptomatic women presenting to a
urodynamic clinic.
MAIN OUTCOME MEASURE: The detection of detrusor instability by means of
videocystourethrography (VCU) and ambulatory urodynamics in women with a mean
bladder wall thickness of greater than 5 mm measured by transvaginal ultrasound.
RESULTS: One hundred and eight women had a mean bladder wall thickness of
greater than 5 mm. Ninety-four percent (102) of these women had detrusor
instability either when undergoing VCU or ambulatory urodynamics. Seventeen
women had a bladder wall thickness of less than 3.5 mm of whom three were found
to have detrusor instability on VCU.
CONCLUSION: The measurement of a mean bladder wall thickness greater than 5 mm
with transvaginal ultrasound is a sensitive screening method for diagnosing
detrusor instability in symptomatic women without outflow obstruction. PURPOSE: In infancy it has been reported that intravesical pressure is generally
higher in males than in females. We investigated whether there are significant
differences in the sonographic characteristics of the urinary tract in male and
female neonates.
MATERIALS AND METHODS: The study comprised 280 consecutive full-term newborns,
including 146 males and 134 females. Kidneys were assessed for hydronephrosis
and graded according to Society for Fetal Urology guidelines. Kidney
measurements included maximum longitudinal length and largest longitudinal area.
Bladder measurements included bladder dimensions and wall thickness. Bladder
wall thickness was measured only in neonates with an estimated bladder volume of
greater than 10 ml.
RESULTS: Of the 280 neonates 114 had mild renal pelvic dilatation in at least 1
kidney and the incidence in males was significantly higher than in females (53%
versus 27%). Kidney longitudinal length and largest area were significantly
greater in males on each side. Mean bladder volume was essentially equal in the
169 male and female neonates with an estimated bladder volume of greater than 10
ml. but mean bladder wall thickness was significantly greater in males than in
females (1.63 versus 1.38 mm.).
CONCLUSIONS: Our study shows that there are marked differences in sonographic
findings in male and female neonates in regard to renal pelvic dilatation, renal
size and bladder wall thickness. These differences should be considered when
sonography is done for screening for urinary tract anomalies in newborns. OBJECTIVES: To evaluate the effectiveness of transabdominal ultrasonography for
bladder volume and detrusor muscle thickness and to research the relationship of
these measurements with age, height, and body mass index.
METHODS: We reviewed the records of 244 healthy, school-aged children from
February to May 2003. After a complete urologic examination, the weight and
height of all children were measured, and their body mass index was determined.
Transabdominal ultrasonography with a high-frequency probe was performed to
obtain the anterior, posterior, and lateral bladder wall thicknesses.
RESULTS: The mean age of the children was 10.7 +/- 3.6 years (range 7 to 15),
and the mean bladder volume was 256 cm3 (range 78 to 790). The relationship
between bladder volume and age was significant (P = 0.0001, r = 0.568). The mean
anterior, posterior, and lateral detrusor thickness was 1.42 mm (range 0.8 to
2.8), 1.57 mm (range 0.7 to 3.1), and 1.49 mm (range 0.6 to 2.6), respectively.
The relationships between increasing age and the anterior and posterior wall
thicknesses were significant (P <0.05), but the relationship between age and the
lateral wall thickness was not (P >0.05). The relationship between bladder
volume and body mass index was significant (P = 0.0001, r = 0.2959). A strong
positive and significant correlation was found between the anterior (P = 0.0001)
and posterior (P = 0.001) wall thicknesses and body mass index, but the
correlation between the lateral wall thickness and body mass index was not
significant (P = 0.079, r = 0.113).
CONCLUSIONS: Strong, positive correlations were found between the anterior and
posterior wall detrusor thicknesses and increased age and body mass index, but
the same correlations for lateral wall detrusor thickness were not found. PURPOSE: We investigated the correlations between ultrasonographic bladder wall
thickness and urodynamic parameters, and estimated the diagnostic accuracy of
bladder wall thickness for predicting unfavorable urodynamic patterns in
children with myelodysplasia.
MATERIALS AND METHODS: A total of 57 children (median age 5.1 years) with
myelodysplasia were enrolled in the study. All children underwent
ultrasonography to measure bladder wall thickness. Videourodynamic evaluation
was also performed within 3 months of ultrasound assessment. Bladder wall
thickness was compared to urodynamic data. A urodynamic risk of upper urinary
tract deterioration was defined as maximum detrusor pressure greater than 40 cm
H(2)O during filling or at leakage, or sphincter dyssynergia during voiding.
RESULTS: Bladder wall thickness was significantly correlated to detrusor leak
point pressure, maximum amplitude of detrusor overactivity and maximum detrusor
pressure during storage phase. In 16 children who had unfavorable urodynamic
risk patterns the mean bladder wall thickness was 3.9 +/- 1.0 mm, compared to
2.4 +/- 0.7 mm in 41 patients with favorable urodynamic patterns. There was a
significant difference between bladder wall thickness in children with and those
without urodynamic risk factors (p <0.001). For a diagnosis of unfavorable
urodynamic patterns bladder wall thickness greater than 3.3 mm had a positive
predictive value of 85.7%, a negative predictive value of 90.7%, specificity of
75.0% and sensitivity of 95.1%. Receiver operator characteristic analysis
revealed that bladder wall thickness had a high predictive value for unfavorable
urodynamic patterns, with an area under the curve of 0.908.
CONCLUSIONS: Ultrasonographic assessment of bladder wall thickness is a
sensitive screening tool for the diagnosis of urodynamic risk factors for upper
urinary tract deterioration in children with myelodysplasia. OBJECTIVE: To identify measurements of ultrasonography (US)-derived bladder wall
thickness (BWT) and bladder weight in community-dwelling men with presumably
normal bladder function.
SUBJECTS AND METHODS: A total of 100 male volunteers underwent transabdominal US
measurements of BWT and bladder weight, using the BVM 9500 bladder scanner
(Verathon Medical, Bothell, WA, USA), at a variety of bladder filling volumes.
The data were explored for any correlation between measurements of BWT and
US-estimated bladder weight (UEBW) with subject age, height, weight, body mass
index (BMI), International Consultation on Incontinence Questionnaire-Male Lower
Urinary Tract Symptoms (ICIQ M-LUTS) score, International Prostate Symptom Score
(IPSS) and IPSS Quality of Life index (IPSS QoL).
RESULTS: Several statistically significant but weak correlations were observed:
BWT and weight (r = 0.216, P = 0.032); BWT and BMI (r = 0.246, P = 0.014); UEBW
and weight (r = 0.304, P = 0.002); and UEBW and BMI (r = 0.260, P = 0.009).
Bladder filling volume appeared to have a greater effect on BWT than on UEBW,
although this could not be determined accurately. There was a substantial
difference in measurements of BWT and UEBW in the assessment of inter- and
intra-observer reliability testing.
CONCLUSION: Further studies are required to validate automated measurements of
BWT and UEBW and to investigate such measurements in the symptomatic and
asymptomatic male population. |
LY450139 is investigational name of which drug? | LY450139 is investigational name of Semagacestat. It is a γ-secretase inhibitor developed for treatment for Alzheimer's disease. Chemical name of LY450139 is hydroxylvaleryl monobenzocaprolactam. | Alzheimer's disease (AD) represents the most common age-related
neurodegenerative disorder. It is characterized by the invariant accumulation of
the beta-amyloid peptide (Abeta), which mediates synapse loss and cognitive
impairment in AD. Current therapeutic approaches concentrate on reducing Abeta
levels and amyloid plaque load via modifying or inhibiting the generation of
Abeta. Based on in vivo two-photon imaging, we present evidence that side
effects on the level of dendritic spines may counteract the beneficial potential
of these approaches. Two potent gamma-secretase inhibitors (GSIs), DAPT
(N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) and
LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the
density of dendritic spines in wild-type mice. In mice deficient for the amyloid
precursor protein (APP), both GSIs had no effect on dendritic spine density,
demonstrating that gamma-secretase inhibition decreases dendritic spine density
via APP. Independent of the effects of gamma-secretase inhibition, we observed a
twofold higher density of dendritic spines in the cerebral cortex of adult
APP-deficient mice. This observation further supports the notion that APP is
involved in the modulation of dendritic spine density--shown here for the first
time in vivo. INTRODUCTION: LY450139 (semagacestat) inhibits gamma-secretase, a key enzyme for
generation of amyloid beta (Abeta), the peptide deposited in plaques in
Alzheimer disease (AD). Previous data have shown that LY450139 lowers plasma
Abeta, but has no clear effect on Abeta1-40 or Abeta1-42 levels in cerebrospinal
fluid (CSF). By using targeted proteomics techniques, we recently identified
several shorter Abeta isoforms, such as Abeta1-16, that in experimental settings
increase during gamma-secretase inhibitor treatment, and thus may serve as
sensitive biochemical indices of the treatment effect. Here, we test the
hypothesis that these shorter Abeta isoforms may be biomarkers of
gamma-secretase inhibitor treatment in clinical trials.
METHODS: In a phase II clinical trial, 35 individuals with mild to moderate AD
were randomized to placebo (n = 10) or LY450139 (100 mg (n = 15) or 140 mg (n =
10)) and underwent lumbar puncture at baseline and after 14 weeks of treatment.
The CSF Abeta isoform pattern was analyzed with immunoprecipitation combined
with MALDI-TOF mass spectrometry.
RESULTS: The CSF levels of Abeta1-14, Abeta1-15, and Abeta1-16 showed a
dose-dependent increase by 57% and 74%, 21% and 35%, and 30% and 67%,
respectively in the 100-mg and 140-mg treatment groups. Abeta1-40 and Abeta1-42
were unaffected by treatment.
CONCLUSIONS: CSF Abeta1-14, Abeta1-15, and Abeta1-16 increase during
gamma-secretase inhibitor treatment in AD, even at doses that do not affect
Abeta1-42 or Abeta1-40, probably because of increased substrate availability of
the C99 APP stub (APP beta-CTF) induced by gamma-secretase inhibition. These
Abeta isoforms may be novel sensitive biomarkers to monitor the biochemical
effect in clinical trials.
TRIAL REGISTRATION: Clinical Trials.gov NCT00244322. Alzheimer's disease (AD) is associated with deposition of amyloid-β (Aβ) in the
brain, which is reflected by low concentration of the Aβ(1-42) peptide in the
cerebrospinal fluid (CSF). The γ-secretase inhibitor LY450139 (semagacestat)
lowers plasma Aβ(1-40) and Aβ(1-42) in a dose-dependent manner but has no clear
effect on the CSF level of these isoforms. Less is known about the potent
γ-secretase modulator E2012. Using targeted proteomics techniques, we recently
identified several shorter Aβ isoforms in CSF, such as Aβ(1-16), which is
produced by a novel pathway. In a Phase II clinical trial on AD patients,
Aβ(1-14), Aβ(1-15) and Aβ(1-16) increased several-fold during γ-secretase
inhibitor treatment. In the present study, 9 dogs were treated with a single
dose of the γ-secretase modulator E2012, the γ-secretase inhibitor LY450139, or
vehicle with a dosing interval of 1 week. The CSF Aβ isoform pattern was
analyzed by immunoprecipitation combined with MALDI-TOF mass spectrometry. We
show here that Aβ(1-15) and Aβ(1-16) increase while Aβ(1-34) decreases in
response to treatment with the γ-secretase inhibitor LY450139, which is in
agreement with previous studies. The isoform Aβ(1-37) was significantly
increased in a dose-dependent manner in response to treatment with E2012, while
Aβ(1-39), Aβ(1-40) and A(1-42) decreased. The data presented suggests that the
γ-secretase modulator E-2012 alters the cleavage site preference of γ-secretase.
The increase in Aβ(1-37) may inhibit Aβ(1-42) oligomerization and toxicity. The outcomes of the clinical trials of the γ-secretase inhibitor Semagacestat
(LY-450139) and the γ-secretase modulator (GSM) Tarenflurbil were disappointing,
but may not represent the end of the γ-secretase era. γ-Secretase modulators, by
definition, only block the γ-secretase cleavage of amyloid-β protein precursor
(AβPP) to generate the longer, 42-residue amyloid-β (Aβ42) without changing the
production of total Aβ. The first generation GSMs were shown to block Aβ42
generation while increasing Aβ38. The non-steroidal anti-inflammatory drug,
Tarenflurbil, binds to AβPP and shifts the cleavage site from Aβ42 to Aβ38. In
addition, Tarenflurbil does not affect the γ-secretase cleavage of Notch. Even
before the failed clinical trials of Tarenflurbil, second generation GSMs had
emerged, and some of these GSMs interact with presenilin, which carries the
active site of the γ-secretase. While second generation GSMs are
pharmacologically superior to first generation GSMs, in vivo Aβ profiles
(decreased levels of Aβ38, Aβ40, and Aβ42) in animals treated with potent GSMs
are strikingly different from those in cultured cells. Thus, the unique
pharmacologic properties of new GSMs and their mechanisms of action need to be
elucidated in order to avoid the fate of Tarenflurbil. It is critical to
understand how GSMs shift the "end" in vivo, i.e., shifting the γ-secretase
cleavage at the C-terminal end of Aβ. In view of the myriad effects of candidate
GSMs on Aβ production in cells and animals, drug development would benefit from
better definition of the target-GSM interaction and physiological function of
shorter Aβ peptides. CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's
disease transgenic mice. In this study, the effect of an acute or subacute
CHF5074 treatment on in vivo novel object recognition test and on
[³H]Acetylcholine (ACh) and GABA release in pre-plaque (7-month-old) Tg2576 mice
have been compared with those induced by the γ-secretase inhibitor LY450139
(semagacestat). Vehicle-treated Tg2576 mice displayed an impairment of
recognition memory compared with wild-type animals. This impairment was
recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while
LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from
vehicle-treated Tg2576 mice, K⁺-evoked [³H]ACh release was lower than that
measured in wild-type mice. This reduction was absent in transgenic animals
subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was
slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days).
There were no differences between the groups on spontaneous [³H]ACh release as
well as spontaneous and K⁺-evoked GABA release. These results suggest that
CHF5074 has beneficial effects on visual memory and cortical cholinergic
dysfunctions in pre-plaque Tg2576 mice. Together with previous findings, these
data suggest that CHF5074 could be a possible candidate for early Alzheimer's
disease therapeutic regimens. |
Are there any Decision support systems for chronic pain management ? | Yes, there is a variety of decision support systems for chronic pain management. | BACKGROUND: Opioid prescribing for chronic pain is common and controversial, but
recommended clinical practices are followed inconsistently in many clinical
settings. Strategies for increasing adherence to clinical practice guideline
recommendations are needed to increase effectiveness and reduce negative
consequences of opioid prescribing in chronic pain patients.
METHODS: Here we describe the process and outcomes of a project to
operationalize the 2003 VA/DOD Clinical Practice Guideline for Opioid Therapy
for Chronic Non-Cancer Pain into a computerized decision support system (DSS) to
encourage good opioid prescribing practices during primary care visits. We based
the DSS on the existing ATHENA-DSS. We used an iterative process of design,
testing, and revision of the DSS by a diverse team including guideline authors,
medical informatics experts, clinical content experts, and end-users to convert
the written clinical practice guideline into a computable algorithm to generate
patient-specific recommendations for care based upon existing information in the
electronic medical record (EMR), and a set of clinical tools.
RESULTS: The iterative revision process identified numerous and varied problems
with the initially designed system despite diverse expert participation in the
design process. The process of operationalizing the guideline identified areas
in which the guideline was vague, left decisions to clinical judgment, or
required clarification of detail to insure safe clinical implementation. The
revisions led to workable solutions to problems, defined the limits of the DSS
and its utility in clinical practice, improved integration into clinical
workflow, and improved the clarity and accuracy of system recommendations and
tools.
CONCLUSIONS: Use of this iterative process led to development of a
multifunctional DSS that met the approval of the clinical practice guideline
authors, content experts, and clinicians involved in testing. The process and
experiences described provide a model for development of other DSSs that
translate written guidelines into actionable, real-time clinical
recommendations. The purpose of this paper is to describe potential improvements in patient
safety resulting from design decisions in the development of a computerized
decision support system (DSS) for managing opioid therapy for chronic noncancer
pain. ATHENA-DSS is an automated decision support system developed in a
collaboration between Stanford University and the U.S. Department of Veterans
Affairs (VA) to increase guideline-adherent prescribing and to change physician
behavior. Based on data in patients’ computerized medical record and knowledge
of the clinical domain encoded in a knowledge base, the system gives
patient-specific recommendations to primary care providers at the point of care.
ATHENA-Opioid Therapy is based on a previous system, ATHENA-Hypertension, and is
designed to follow the VA/Department of Defense clinical practice guideline for
the management of opioid therapy for chronic noncancer pain. We describe the
rationale for development of decision support system elements and a graphical
user interface to increase patient safety during primary care treatment for
chronic pain. The ATHENA-Opioid Therapy system focuses on reducing patient risk
in four main ways by: (1) identifying patients with comorbidities or concurrent
prescriptions that raise risk for overdose and recommending more conservative
dosing; (2) identifying patients with mental health problems that increase risk
of medication abuse and recommending referral to psychiatric care and close
monitoring; (3) assisting doctors with complex pharmacologic calculations to
reduce the risk of mistakes when initiating, titrating, or switching
medications; and (4) presenting relevant information to clinicians in an
easy-to-use format. We describe a system evaluation plan that we believe is
essential to ensure that deployment of ATHENA-Opioid Therapy leads to
improvements in patient safety and increases in guideline-concordant
prescribing, and we discuss the limitations of this system for patient safety
efforts. BACKGROUND: For many healthcare decisions, multiple alternatives are available
with different combinations of advantages and disadvantages across several
important dimensions. The complexity of current healthcare decisions thus
presents a significant barrier to informed decision making, a key element of
patient-centered care.Interactive decision dashboards were developed to
facilitate decision making in Management, a field marked by similarly
complicated choices. These dashboards utilize data visualization techniques to
reduce the cognitive effort needed to evaluate decision alternatives and a
non-linear flow of information that enables users to review information in a
self-directed fashion. Theoretically, both of these features should facilitate
informed decision making by increasing user engagement with and understanding of
the decision at hand. We sought to determine if the interactive decision
dashboard format can be successfully adapted to create a clinically realistic
prototype patient decision aid suitable for further evaluation and refinement.
METHODS: We created a computerized, interactive clinical decision dashboard and
performed a pilot test of its clinical feasibility and acceptability using a
multi-method analysis. The dashboard summarized information about the
effectiveness, risks of side effects and drug-drug interactions, out-of-pocket
costs, and ease of use of nine analgesic treatment options for knee
osteoarthritis. Outcome evaluations included observations of how study
participants utilized the dashboard, questionnaires to assess usability,
acceptability, and decisional conflict, and an open-ended qualitative analysis.
RESULTS: The study sample consisted of 25 volunteers - 7 men and 18 women - with
an average age of 51 years. The mean time spent interacting with the dashboard
was 4.6 minutes. Mean evaluation scores on scales ranging from 1 (low) to 7
(high) were: mechanical ease of use 6.1, cognitive ease of use 6.2, emotional
difficulty 2.7, decision-aiding effectiveness 5.9, clarification of values 6.5,
reduction in decisional uncertainty 6.1, and provision of decision-related
information 6.0. Qualitative findings were similarly positive.
CONCLUSIONS: Interactive decision dashboards can be adapted for clinical use and
have the potential to foster informed decision making. Additional research is
warranted to more rigorously test the effectiveness and efficiency of patient
decision dashboards for supporting informed decision making and other aspects of
patient-centered care, including shared decision making. Development of clinical decision support systems (CDSs) has tended to focus on
facilitating medication management. An understanding of behavioral medicine
perspectives on the usefulness of a CDS for patient care can expand CDSs to
improve management of chronic disease. The purpose of this study is to explore
feedback from behavioral medicine providers regarding the potential for CDSs to
improve decision-making, care coordination, and guideline adherence in pain
management. Qualitative methods were used to analyze semi-structured interview
responses from behavioral medicine stakeholders following demonstration of an
existing CDS for opioid prescribing, ATHENA-OT. Participants suggested that a
CDS could assist with decision-making by educating providers, providing
recommendations about behavioral therapy, facilitating risk assessment, and
improving referral decisions. They suggested that a CDS could improve care
coordination by facilitating division of workload, improving patient education,
and increasing consideration and knowledge of options in other disciplines.
Clinical decision support systems are promising tools for improving behavioral
medicine care for chronic pain. |
Which gene has been found to be mutant in Lesch-Nyhan Disease patients? | Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). | De novo purine biosynthesis has been studied in lymphocyte cell lines
established from Lesch-Nyhan patients deficient in hypoxanthine-guanine
phosphoribosyltransferase (HGPRT), in in vitro differentiating erythroleukaemic
cell lines cloned from cells charactistic of virus-induced murine leukaemia, and
in mutant hamster cells deficient in amidophosphoribosyltransferase. The
relationship between cellular phosphoribosylpyrophosphate (PP-ribose-P)
metabolism and the activity of the enzymes which catalyse the early steps of de
novo purine biosynthesis has been explored. It was found that hamster cells
deficient in amidophosphoribosyltransferase did not accumulate PP-ribose-P as do
HGPRT-deficient cells. In these model systems, an accelerated rate of de novo
purine biosynthesis tended to be associated with an increase in cellular
PP-ribose-P cotent, but decreases in this rate results from the reduction in the
activity of amidophosphoribosyltransferase. Regulation of ammonia-dependent de
novo purine biosynthesis was similar to that of glutamine-dependent purine
biosynthesis. Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase
(HPRT) gene were identified in a partially HPRT deficient patient with gout and
in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR)
technique coupled with direct sequencing, the nucleotide sequences of the entire
HPRT coding region amplified from the cDNA and also of each exon amplified form
the genomic DNA were analyzed. Three independent point mutations in the coding
region were detected in the partially HPRT deficient patient (Case 1) and in two
Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid
substitutions. The family study of Case 3, utilizing a PvuII restriction site
created in the mutant gene, indicated that the mother was a heterozygote, and a
sister and a fetal brother had inherited the normal HPRT gene from the mother.
In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene
was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at
the 5' end of exon 4 resulted in formation of three different types of abnormal
mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26 bp
of intron 8 instead of the deleted 58 bp at the 5' end of exon 9, because of a
74-bp deletion from intron 8 to exon 9. Lesch-Nyhan syndrome is a rare X-linked recessive disorder of purine metabolism
associated with a virtually complete deficiency of the enzyme
hypoxanthine-guanine phosphoribosyl-transferase (HPRT). The disease is
characterized by hyperuricemia, self-multilation, choreoathetosis, spasticity,
and mental retardation. The abnormalities of purine metabolism are present at
birth and may lead to uric acid crystalluria and stone formation early in life.
Radiographic findings described in Lesch-Nyhan syndrome include faintly
radio-opaque stones on abdominal radiographs or, if renal disease is present,
small kidneys with poor function on intravenous urogram. Radiolucent stones are
usually composed of uric acid; however, several cases of xanthine and
hypoxanthine-containing calculi in Lesch-Nyhan patients receiving allopurinl
therapy have also been described. Oxypurine is the collective name for the
compounds hypoxanthine, xanthine, and uric acid, and all may be radiolucent. We
report a case of Lesch-Nyhan syndrome with presumed renal parenchymal oxypurine
deposition demonstrated readily by ultrasonography but not detected on standard
radiographs or intravenous urograms. The Lesch-Nyhan syndrome is characterized clinically by choreoathetosis,
spasticity, selfmutilation, and mental and growth retardation. Biochemically,
there is a striking reduction of hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) activity in affected individuals. We have examined erythrocytes from 14
patients with the Lesch-Nyhan syndrome for the presence of hypoxanthine-guanine
phosphoribosyltransferase activity and enzyme protein. In contrast to the usual
finding of no detectable hypoxanthine-guanine phosphoribosyltransferase
activity, we have found low levels (0.002-0.79 nmoles/mg protein per hr) of
hypoxanthine-guanine phosphoribosyltransferase activity in erythrocyte lysates
from five of these patients. In three of the five patients, hypoxanthine-guanine
phosphoribosyltransferase activity appeared to be substantially more labile in
vivo than normal using erythrocytes which had been separated according to their
density (age). Immunochemical studies using a monospecific antiserum prepared
from a homogeneous preparation of normal human erythrocyte hypoxanthine-guanine
phosphoribosyltransferase revealed immunoreactive protein (CRM) in hemolysate
from all 14 patients with the Lesch-Nyhan syndrome. The immunoreactive protein
from each patient gave a reaction of complete identity with normal erythrocyte
hypoxanthine-guanine phosphoribosyltransferase and was present in quantities
equal to those observed in normal erythrocytes. In addition, a constant amount
of CRM was found in erythrocytes of increasing density (age) from patients with
the Lesch-Nyhan syndrome despite the decreasing hypoxanthine-guanine
phosphoribosyltransferase activity. These studies confirm previous data which
indicate that the mutations leading to the Lesch-Nyhan syndrome are usually, if
not always on the structural gene coding for hypoxanthine-guanine
phosphoribosyltransferase. In addition, although the mutant proteins appear to
be present in normal amounts, they are often very labile in vivo with respect to
enzymatic activity. These observations suggest that therapy directed at
stabilization or activation of enzyme activity in vivo may be of potential
benefit. We have investigated the molecular basis of hypoxanthine-guanine
phosphoribosyltransferase (HPRT) deficiency in a patient who presented with the
Lesch-Nyhan syndrome. A catalytically incompetent form of HPRT has been isolated
from this patient's erythrocytes and lymphoblasts. This enzyme variant, which we
have termed HPRTKinston, is indistinguishable from the normal enzyme in terms of
its intracellular concentration and maximal velocity, but differs with respect
to its isoelectric point (more basic) and Michaelis constants for both
substrates (markedly elevated). The tryptic peptides of HPRTKinston were mapped
by reverse-phase high pressure liquid chromatography in an attempt to define the
precise abnormality in its primary structure. Sequence analysis of the single
aberrant tryptic peptide in HPRTKinston revealed an aspartic acid to asparagine
amino acid substitution at position 193. Electrophoretic analysis of the CNBr
peptides of HPRTKinston confirmed the location of the proposed mutation. This
amino acid substitution can be explained by a single nucleotide change in the
codon for aspartic acid 193 (GAC leads to AAC). This is the first specific
mutation described at the molecular level in a patient with the Lesch-Nyhan
syndrome. For three patients with the Lesch-Nyhan syndrome the existence of normal amounts
of catalytically inactive hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
protein was demonstrated by using antibodies against the normal enzyme subunits.
The lack of enzyme activity is reverted in virus transformed cells. Individual
revertant cell clones contain different HGPRT enzymes as demonstrated here by
isoelectric focusing. The data strongly support the idea of a structural gene
mutation as the cause of enzyme deficiency in the Lesch-Nyhan syndrome. BACKGROUND: The Lesch-Nyhan syndrome is an X-linked recessive inherited disease
caused by a complete deficiency of hypoxanthine guanine
phosphoribosyl-transferase (HPRT) activity. Many different mutations throughout
the HPRT coding region of Lesch-Nyhan patients have been described, including
single base substitutions, partial or entire gene deletions, gene insertions or
endoduplication of exons. However, study of gene mutation in Chinese patients
has rarely been reported in Taiwan.
METHODS: Polymerase chain reaction (PCR) and nucleotide sequence analysis were
used to identify the location and the nature of the mutation at the HPRT locus
in two brothers affected with Lesch-Nyhan syndrome. The HPRT cDNA, amplified
from total RNA of patient's peripheral blood by reverse transcription-polymerase
chain reaction, was cloned into a pGEM-3Zf(-) vector and then sequenced. Family
study involved initial screening using single-strand conformation polymorphism,
and further confirmation by direct sequencing of the exon encompassing the
mutation.
RESULTS: The mutation identified in these two affected siblings was a single
nucleotide substitution, from cytosine to guanine, in exon 3 of the HPRT coding
region. This transversion putatively caused a single amino acid substitution
from phenylalanine to leucine at codon 74 in the translated protein. This base
change was further confirmed by direct sequencing of both the HPRT cDNA fragment
and the exon 3 of HPRT gene amplified from genomic DNA. The family study
revealed that the patient's mother was a heterozygous carrier, and the mutation
seemed to have occurred de novo in a germinal cell from one of the maternal
grandparents.
CONCLUSIONS: This is the first family study on Chinese patients with Lesch-Nyhan
syndrome identified by molecular analysis in Taiwan. The mutation described
herein is a novel substitution which occurs in a suggested "hotspot" of mutation
(exon 3) of the HPRT gene. The application of molecular analysis of HPRT-gene
allows not only DNA diagnosis by directly detecting the mutant alleles, but also
prenatal diagnosis and carrier identification within individual families
affected by Lesch-Nyhan syndrome. Mutations in the HPRT gene cause a spectrum of diseases that ranges from
hyperuricemia alone to hyperuricemia with profound neurological and behavioral
dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases
in which the germinal HPRT mutation has been characterized, 218 different
mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of
one exon or greater and four (2%) are partial gene duplications. The deletion
breakpoint junctions have been defined for only three of the 34 macro-deletions.
The molecular basis of two of the four duplications has been defined. We report
here the breakpoint junctions for three new deletion mutations, encompassing
exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp),
respectively. The deletion breakpoints were defined by a combination of long
polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA
sequencing. All three deletions are the result of non-homologous recombinations.
A fourth mutation, a duplication of exons 2 and 3, is the result of an
Alu-mediated homologous recombination between identical 19bp sequences in
introns 3 and 1. In toto, two of three germinal HPRT duplication mutations
appear to have been caused by Alu-mediated homologous recombination, while only
one of six deletion mutations appears to have resulted from this type of
recombination mechanism. The other five deletion mutations resulted from
non-homologous recombination. With this admittedly limited number of
characterized macro-mutations, Alu-mediated unequal homologous recombinations
account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of
the total HPRT germinal mutations. Mutations in the X chromosome hypoxanthine-guanine phosphoribosyl transferase
(HPRT) gene are responsible for Lesch-Nyhan syndrome and related diseases in
humans. Because the gene is on the X chromosome, males are affected and females
in the families are at risk of being carriers of the mutation. Because there are
so many different mutations that can cause the disease (218 different mutations
in 271 families), genetic testing for carrier status of females requires
detailed molecular analysis of the familial mutation. This analysis can be
complicated by the unavailability of an affected male for study. In addition,
when the mutation is a deletion (34 reported instances), molecular analysis in
females is difficult because of the two X chromosomes. We have applied a
peripheral blood T lymphocyte cloning assay that uses resistance to the purine
analogue 6-thioguanine (TG) to measure the frequency of cells in females
expressing a mutant HPRT allele to determine mutation carrier status in 123
females in 61 families. In families in which the HPRT mutation was determined
and could be easily analyzed in samples from females, we found a mean (+/- SD)
mutant frequency of 9.7 (+/- 8.7) x 10(-6) in noncarrier females and 2.9 (+/-
3.0) x 10(-2) in carrier females. The frequency in carrier females is less than
the 0.5 expected for nonrandom X inactivation because of in vivo selection
against HPRT mutation-expressing T lymphocytes or stem cells during prenatal
development. The use of this cloning assay allows determination of the carrier
status of females even when the HPRT mutation is not yet known or is difficult
to determine in DNA samples from females. This approach provides a rapid assay
that yields information on carrier status within 10 days of sample receipt. Human embryonic stem (ES) cells are pluripotent cells derived from
blastocyst-stage embryos. It has been suggested that these cells should play a
major role in transplantation medicine and be able to advance our knowledge in
human embryology. We propose that these cells should also play a vital role in
the creation of models of human disorders. This aspect would be most valuable
where animal models failed to faithfully recapitulate the human phenotype.
Lesch-Nyhan disease is caused by a mutation in the HPRT1 gene that triggers an
overproduction of uric acid, causing gout-like symptoms and urinary stones, in
addition to neurological disorders. Due to biochemical differences between
humans and rodents, a mouse lacking the HPRT expression will fail to accumulate
uric acid. In this research we demonstrate a model for Lesch-Nyhan disease by
mutating the HPRT1 gene in human ES cells using homologous recombination. We
have verified the mutation in the HPRT1 allele at the DNA and RNA levels. By
using selection media, we show that HPRT1 activity is abolished in the mutant
cells, and the HPRT1-cells show a higher rate of uric acid accumulation than the
wild-type cells. Therefore, these cells recapitulate to some extent the
characteristics of Lesch-Nyhan syndrome and can help researchers further
investigate this genetic disease and analyze drugs that will prevent the onset
of its symptoms. We therefore suggest that human diseases may be modeled using
human ES cells. Mutations in the gene encoding the purine biosynthetic enzyme
hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable
neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant
development of brain dopamine pathways. In the current study, we have identified
an increased expression of the microRNA miR181a in HPRT-deficient human
dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated
by miR181a are several known to be required for neural development, including
Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these
genes are down-regulated in HPRT-deficient SH-SY5Y cells and that
over-expression of miR181a significantly reduces endogenous expression of these
genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a
3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the
expression of these genes and enhances translation of luciferase constructs
bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate
that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to
be functional partners of Lmx1a and Brn2 are also markedly down-regulated in
SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings
in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by
dysregulation of some of the important pathways that regulate the development of
dopaminergic neurons and dopamine pathways and that this defect is associated
with and possibly due at least partly to aberrant expression of miR181a. Because
aberrant expression of miR181a is not as apparent in HPRT-deficient LND
fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease
remains to be proven. Nevertheless, we propose that these pleiotropic
neurodevelopment effects of miR181a may play a role in the pathogenesis of LND. Lesch-Nyhan disease and its attenuated variants are caused by mutations in the
HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine-guanine
phosphoribosyltransferase. The mutations are heterogeneous, with more than 400
different mutations already documented. Prior efforts to correlate variations in
the clinical phenotype with different mutations have suggested that milder
phenotypes typically are associated with mutants that permit some residual
enzyme function, whereas the most severe phenotype is associated with null
mutants. However, multiple exceptions to this concept have been reported. In the
current studies 44 HPRT1 mutations associated with a wide spectrum of clinical
phenotypes were reconstructed by site-directed mutagenesis, the mutant enzymes
were expressed in vitro and purified, and their kinetic properties were examined
toward their substrates hypoxanthine, guanine, and phosphoribosylpyrophosphate.
The results provide strong evidence for a correlation between disease severity
and residual catalytic activity of the enzyme (k(cat)) toward each of its
substrates as well as several mechanisms that result in exceptions to this
correlation. There was no correlation between disease severity and the affinity
of the enzyme for its substrates (K(m)). These studies provide a valuable model
for understanding general principles of genotype-phenotype correlations in human
disease, as the mechanisms involved are applicable to many other disorders. Lesch-Nyhan disease (LND) is caused by deficiency of hypoxanthine guanine
phosphoribosyltransferase (HPRT). The aim of the present study is to
characterize the molecular deficiency of a clinical diagnosed Chinese patient
with attenuated variant of LND. The coding region and the intron-exon boundaries
of HPRT1 gene were sequenced by standard methods, and HPRT activity was assayed
by HPLC method. Structure analysis was performed to estimate the consequence of
the mutant of HPRT1 gene. A new mutation c.245T>G (p.Ile82Ser) was identified in
this patient, and heterozygous mutation was found in the patient's mother. The
activity of HPRT in the patient was completely undetectable. Structure study
indicates that the mutation of p.Ile82Ser may lead to loss of hydrophobic side
chain and disrupt its normal conformation of HPRT protein. It is helpful for
diagnosis of LND that sequencing analysis of HPRT1 gene is performed in male
infant and juvenile with hyperuricaemia and neurologic dysfunction in Chinese. We describe a family of seven boys affected by Lesch-Nyhan disease with various
phenotypes. Further investigations revealed a mutation c.203T>C in the gene
encoding HGprt of all members, with substitution of leucine to proline at
residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety
of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared
by site-directed mutagenesis and the kinetics of the enzyme revealed that the
catalytic activity of the mutant was reduced, in association with marked
reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km
for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with
guanine as substrate with associated reduced catalytic potential. Molecular
modeling confirmed that the most prominent defect was the dramatically reduced
affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a
strong impact on PRPP binding and on stability of the active conformation. This
suggests that factors other than HGprt activity per se may influence the
phenotype of Lesch-Nyhan patients. |
Which is the methyl donor of histone methyltransferases? | The major methyl donor of histone methyltransferases (HMTs) is S-adenosyl-L–methionine (SAM, AdoMet). | Protein methylase III (S-adenosylmethionine:proteinlysine methyltransferase; ED
2.1.1.25) and protein methylase I (S-adenosylmethionine:protein-arginine
methyltransferase; EC 2.1.1.23) activities were examined in isolated nuclei and
cytosol fraction, respectively, from various hepatomas with different growth
rates. The enzyme activities of both enzymes paralleled the rates of tumor
growth in fast- and moderately growing hepatomas. The parallelism was more
evident with protein methylase I than with protein methylase III. While protein
methylase III activity was elevated in the fast- to moderately growing
hepatomas, the enzyme that is responsible for demethylating proteins,
epsilon-alkyllysinase (epsilon-slkyl-L-lysine:oxygen oxidoreductase; EC
1.5.3.4), had an inverse relationship to the rate of tumor growth, thus
suggesting a possible physiological antagonism. When isolated rat liver nuclei
were methylated in vitro with S-adenosyl-L-[methyl-14 C]methionine as methyl
donor, H2SO4-insoluble protein and histones had almost equal amounts of
methyl-14 C incorporated. However, amino acid analysis revealed that methylated
arginines are the predomit form of radioactivity in the H2SO4-insoluble
protein (product of protein methylase I), while methylated lysines are the major
methylated amino acids in the histones (product of protein methylase III).
Furthermore, the hydrolysate of the H2SO4-insoluble protein showed four unknown
radioactivity peaks on the amino acid analyzer in addition to the known
methylated arginine and lysine derivatives. A protein methylase III responsible for specifically methylating the cytochrome
c in Neurospora crassa was partially characterized by using unmethylated horse
heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine
as the methyl donor. An analysis of the distribution of [14C]methyl groups in
the peptides obtained by chymotrypsin digestion of the enzymically methylated
cytochrome c showed that all of the radioactivity could be recovered within a
single peak after chromatography. This indicates that the enzyme methylates a
specific amino acid sequence within cytochrome c. On hydrolysis of the
radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine,
epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The
enzyme can easily be extracted from the N. crassa mycelial pads and was purified
approx. 30-fold. Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet),
and methionine (Met) by a unique reaction and, like other organisms, use SMM as
a methyl donor for Met synthesis from homocysteine (Hcy). These reactions
comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate
the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by
homology and authenticated by complementing an Escherichia coli yagD mutant and
by detecting HMT activity in complemented cells. Gel blot analyses indicate that
these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced
polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack
obvious targeting sequences, and are 55% identical to each other. The
recombit enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or
(S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both
enzymes also use either methyl donor in vivo because both restore the ability to
utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly
inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to
the control of flux through the HMT reaction and the SMM cycle. Plant HMT is
known to transfer the pro-R methyl group of SMM. This enabled us to use
recombit AtHMT-1 to establish that the other enzyme of the SMM cycle,
AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These
opposing stereoselectivities suggest a way to measure in vivo flux through the
SMM cycle. Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and
S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met
S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from
homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT
together have been proposed to constitute a futile SMM cycle that stops the free
Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis
and maize have one MMT gene, and at least three HMT genes that belong to two
anciently diverged classes and encode enzymes with distinct properties and
expression patterns. SMM, and presumably its cycle, must therefore have
originated before dicot and monocot lineages separated. Arabidopsis leaves,
roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met
to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the
plant. This appears to be a general feature of angiosperms, as digital gene
expression profiles show that MMT and HMT are co-expressed in leaves, roots and
reproductive tissues of maize and other species. An in silico model of the SMM
cycle in mature Arabidopsis leaves was developed from radiotracer kinetic
measurements and pool size data. This model indicates that the SMM cycle
consumes half the AdoMet produced, and suggests that the cycle serves to stop
accumulation of AdoMet, rather than to prevent depletion of free Met. Because
plants lack the negative feedback loops that regulate AdoMet pool size in other
eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve
short-term control of AdoMet level. Histone methyltransferase (HMT)(1) class enzymes that methylate lysine residues
of histones or proteins contain a conserved catalytic core termed the SET
domain, which shares sequence homology with an independently described sequence
motif, the PR domain. Intact PR or SET sequence is required for tumor
suppression functions, but it remains unclear whether it is histone
methyltransferase activity that underlies tumor suppression. We now show that
tumor suppressor RIZ1 (PRDM2) methylates histone H3 on lysine 9, and this
activity is reduced by mutations in the PR domain found in human cancers. Also,
S-adenosylhomocysteine or methyl donor deficiency inhibits RIZ1 and other H3
lysine 9 methylation activities. These results support the hypothesis that H3
lysine 9 methylation activities of a PR/SET domain have tumor suppression
functions and may underlie carcinogenesis associated with dietary methyl donor
deficiency. Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The
elongated structure of the conserved core of yeast Dot1p contains an N-terminal
helical domain and a seven-stranded catalytic domain that harbors the binding
site for the methyl-donor and an active site pocket sided with conserved
hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended
conformation distinct from the folded conformation observed in structures of SET
domain histone lysine methyltransferases. A catalytic asparagine (Asn479),
located at the bottom of the active site pocket, suggests a mechanism similar to
that employed for amino methylation in DNA and protein glutamine methylation.
The acidic, concave cleft between the two domains contains two basic residue
binding pockets that could accommodate the outwardly protruding basic side
chains around Lys79 of histone H3 on the disk-like nucleosome surface.
Biochemical studies suggest that recombit Dot1 proteins are active on
recombit nucleosomes, free of any modifications. Evidence indicates that failure of nuclear transfer (NT) embryos to develop
normally can be attributed, at least partially, to the use of a differentiated
cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst
production and development to term of cloned embryos may differ between
population doublings (PDs) of the same cell line as a consequence of changes in
DNA methylation and histone acetylation patterns during in vitro culture. The
objective of this study was to determine gene expression patterns of the
chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG
binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in
addition, to measuring levels of DNA methylation and histone acetylation of
bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were
established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1,
methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45,
and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global
levels of methylated DNA and acetylated histone were determined by incubation of
fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody,
respectively. Cells were labeled with a second antibody, counter-stained with
propidium iodide and analyzed by flow cytometry. These data demonstrate that
chromatin remodeling protein mRNAs involved in epigenetic modifications are
altered during in vitro culture. Methylated DNA and acetylated histone patterns
of in vitro cells change with time in culture. Subsequent use of these cells for
NT will provide insight as to how these epigenetic modifications affect
reprogramming. Multiple protein arginine methyltransferases are involved in transcriptional
activation of nuclear receptors. Coactivator-associated arginine
methyltransferase 1 (CARM1)-mediated histone methylation has been shown to
activate nuclear receptor-dependent transcription; however, little is known
about the regulation of its enzymatic activity. Here, we report that the
methyltransferase activity of CARM1 is negatively regulated through
phosphorylation at a conserved serine residue. When the serine residue is
mutated to glutamic acid, which mimics the phosphorylated serine residue, the
mutant CARM1 exhibits diminished ability to bind the methyl donor
adenosylmethionine and diminished histone methylation activity. Moreover, such
mutation leads to the inhibition of CARM1 transactivation of estrogen
receptor-dependent transcription. Our results provide an example for the
regulation of protein arginine methyltransferase activity by phosphorylation. As
CARM1 is a potent transcriptional coactivator of estrogen receptor, our results
suggest that phosphorylation of CARM1 serves as a unique mechanism for
inactivating CARM1-regulated estrogen-dependent gene expression. We previously showed that S-adenosylmethionine (SAMe) and its metabolite
methylthioadenosine (MTA) blocked lipopolysaccharide (LPS)-induced tumor
necrosis factor alpha (TNFalpha) expression in RAW (murine macrophage cell line)
and Kupffer cells at the transcriptional level without affecting nuclear factor
kappa B nuclear binding. However, the exact molecular mechanism or mechanisms of
the inhibitory effect were unclear. While SAMe is a methyl donor, MTA is an
inhibitor of methylation. SAMe can convert to MTA spontaneously, so the effect
of exogenous SAMe may be mediated by MTA. The aim of our current work is to
examine whether the mechanism of SAMe and MTA's inhibitory effect on
proinflammatory mediators might involve modulation of histone methylation. In
RAW cells, we found that LPS induced TNFalpha expression by both transcriptional
and posttranscriptional mechanisms. SAMe and MTA treatment inhibited the
LPS-induced increase in gene transcription. Using the chromatin
immunoprecipitation assay, we found that LPS increased the binding of
trimethylated histone 3 lysine 4 (H3K4) to the TNFalpha promoter, and this was
completely blocked by either SAMe or MTA pretreatment. Similar effects were
observed with LPS-mediated induction of inducible nitric oxide synthase (iNOS).
LPS increased the binding of histone methyltransferases Set1 and
myeloid/lymphoid leukemia to these promoters, which was unaffected by SAMe or
MTA. The effects of MTA in RAW cells were confirmed in vivo in LPS-treated mice.
Exogenous SAMe is unstable and converts spontaneously to MTA, which is stable
and cell-permeant. Treatment with SAMe doubled intracellular MTA and
S-adenosylhomocysteine (SAH) levels. SAH also inhibited H3K4 binding to TNFalpha
and iNOS promoters.
CONCLUSION: The mechanism of SAMe's pharmacologic inhibitory effect on
proinflammatory mediators is mainly mediated by MTA and SAH at the level of
histone methylation. Polycomb gene silencing requires histone methyltransferase activity of Polycomb
repressive complex 2 (PRC2), which methylates lysine 27 of histone H3.
Information on how PRC2 works is limited by lack of structural data on the
catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant
alleles that alter its SET domain. Here we analyze missense alleles of
Drosophila E(z), selected for molecular study because of their domit genetic
effects. Four missense alleles identify key E(Z) SET domain residues, and a
fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes
in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation
disrupts PRC2 histone methyltransferase. Based on known SET domain structures,
the mutations likely affect either the lysine-substrate binding pocket, the
binding site for the adenosylmethionine methyl donor, or a critical tyrosine
predicted to interact with the substrate lysine epsilon-amino group. In
contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and
trimethylation capacity. Deletion analysis also reveals a functional requirement
for a conserved E(Z) domain N-terminal to CXC and SET. These results identify
critical SET domain residues needed for PRC2 enzyme function, and they also
emphasize functional inputs from outside the SET domain. Metnase is a human SET and transposase domain protein that methylates histone H3
and promotes DNA double-strand break repair. We now show that Metnase physically
interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key
chromosome decatenating enzyme. Metnase promotes progression through
decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193
and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of
kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase
decatenated kDNA more rapidly than those without Metnase, and neutralizing
anti-sera against Metnase reversed that enhancement of decatenation. Metnase
automethylates at K485, and the presence of a methyl donor blocked the
enhancement of Topo IIalpha decatenation by Metnase, implying an internal
regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and
this activity is repressed by automethylation. These results suggest that cancer
cells could subvert Metnase to mediate clinically relevant resistance to Topo
IIalpha inhibitors. The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain
SET histone methyltransferases that are implicated in human cancer progression.
Here we report an analysis of the crystal structure of the full length human
SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl
donor cofactor. The structure revealed an overall compact architecture in which
the "split-SET" domain adopts a canonical SET domain fold and closely assembles
with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle
similar to that observed for the mouse SMYD1 structure. Together, these
structurally interlocked domains impose a highly confined binding pocket for
histone substrates, suggesting a regulated mechanism for its enzymatic activity.
Our mutational and biochemical analyses confirm regulatory roles of the unique
structural elements both inside and outside the core SET domain and establish a
previously undetected preference for trimethylation of H4K20. Protein lysine methyltransferases are important regulators of epigenetic
signaling. These enzymes catalyze the transfer of donor methyl groups from
S-adenosylmethionine to specific acceptor lysines on histones, leading to
changes in chromatin structure and transcriptional regulation. These enzymes
also methylate nonhistone protein substrates, revealing an additional mechanism
to regulate cellular physiology. The oncogenic protein SMYD2 represses the
functional activities of the tumor suppressor proteins p53 and Rb, making it an
attractive drug target. Here we report the discovery of AZ505, a potent and
selective inhibitor of SMYD2 that was identified from a high throughput chemical
screen. We also present the crystal structures of SMYD2 with p53 substrate and
product peptides, and notably, in complex with AZ505. This substrate competitive
inhibitor is bound in the peptide binding groove of SMYD2. These results have
implications for the development of SMYD2 inhibitors, and indicate the potential
for developing novel therapies targeting this target class. vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2
(PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation
on gene transcription, as both catalyze histone H3K27 trimethylation. To control
the enzymatic activity of vSET in vivo with an engineered
S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried
out structure-guided design, synthesis, and characterization of orthogonal vSET
methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy. The filamentous fungus Aspergillus nidulans carries a single gene for the
S-adenosylmethionine (SAM) synthetase SasA, whereas many other organisms possess
multiple SAM synthetases. The conserved enzyme catalyzes the reaction of
methionine and ATP to the ubiquitous methyl group donor SAM. SAM is the main
methyl group donor for methyltransferases to modify DNA, RNA, protein,
metabolites, or phospholipid target substrates. We show here that the single A.
nidulans SAM synthetase encoding gene sasA is essential. Overexpression of sasA,
encoding a predomitly cytoplasmic protein, led to impaired development
including only small sterile fruiting bodies which are surrounded by unusually
pigmented auxiliary Hülle cells. Hülle cells are the only fungal cell type which
does not contain significant amounts of SasA. Sterigmatocystin production is
altered when sasA is overexpressed, suggesting defects in coordination of
development and secondary metabolism. SasA interacts with various metabolic
proteins including methionine or mitochondrial metabolic enzymes as well as
proteins involved in fungal morphogenesis. SasA interaction to histone-2B might
reflect a putative epigenetic link to gene expression. Our data suggest a
distinct role of SasA in coordinating fungal secondary metabolism and
development. Certain lysine residues on histone tails could be methylated by protein lysine
methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl
donor. Since the methylation states of the target lysines play a fundamental
role in the regulation of chromatin structure and gene expression, it is
important to study the property of PKMTs that allows a specific number of methyl
groups (one, two or three) to be added (termed as product specificity). It has
been shown that the product specificity of PKMTs may be controlled in part by
the existence of specific residues at the active site. One of the best examples
is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular
mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are
performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants
for understanding the energetic origin of the product specificity and the
reasons for the change of product specificity as a result of single-residue
mutations at the Phe/Tyr switch as well as other positions. The free energy
barriers of the methyl transfer processes calculated from our simulations are
consistent with experimental data, supporting the suggestion that the relative
free energy barriers may determine, at least in part, the product specificity of
PKMTs. The changes of the free energy barriers as a result of the mutations are
also discussed based on the structural information obtained from the
simulations. The results suggest that the space and active-site interactions
around the ε-amino group of the target lysine available for methyl addition
appear to among the key structural factors in controlling the product
specificity and activity of PKMTs. Protein lysine methylation occurs extensively in the Crenarchaeota, a major
kingdom in the Archaea. However, the enzymes responsible for this type of
posttranslational modification have not been found. Here we report the
identification and characterization of the first crenarchaeal protein lysine
methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon
Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to
selected lysine residues in a substrate protein using S-adenosyl-l-methionine
(SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved
among crenarchaea, and distantly related homologs also exist in Bacteria and
Eukarya. aKMT was active over a wide range of temperatures, from ~25 to 90 °C,
with an optimal temperature at ~60 to 70 °C. Amino acid residues Y9 and T12 at
the N terminus appear to be the key residues in the putative active site of
aKMT, as indicated by sequence conservation and site-directed mutagenesis.
Although aKMT was identified based on its methylating activity on Cren7, the
crenarchaeal chromatin protein, it exhibited broad substrate specificity and was
capable of methylating a number of recombit Sulfolobus proteins overproduced
in Escherichia coli. The finding of aKMT will help elucidate mechanisms
underlining extensive protein lysine methylation and the functional significance
of posttranslational protein methylation in crenarchaea. Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the
Polycomb repressive complex 2 (PRC2), which represses gene expression by
methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and
differentiation during embryonic development. Recently, hot-spot mutations of
Ezh2 were identified in diffused large B-cell lymphomas and follicular
lymphomas. To investigate if tumor growth is dependent on the enzymatic activity
of Ezh2, we developed a potent and selective small molecule inhibitor, EI1,
which inhibits the enzymatic activity of Ezh2 through direct binding to the
enzyme and competing with the methyl group donor S-Adenosyl methionine.
EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation
of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large
B-cell lymphomas cells carrying the Y641 mutations results in decreased
proliferation, cell cycle arrest, and apoptosis. These results provide strong
validation of Ezh2 as a potential therapeutic target for the treatment of
cancer. |
How are human accelerated regions (HAR) defined? | Human accelerated regions (HAR) are defined as previously slowly evolving regions of the genome that have evolved most quickly along the human lineage. These represent genomic regions that are conserved among vertebrates but have accumulated substitutions on the human lineage at an accelerated rate. | The developmental and evolutionary mechanisms behind the emergence of
human-specific brain features remain largely unknown. However, the recent
ability to compare our genome to that of our closest relative, the chimpanzee,
provides new avenues to link genetic and phenotypic changes in the evolution of
the human brain. We devised a ranking of regions in the human genome that show
significant evolutionary acceleration. Here we report that the most dramatic of
these 'human accelerated regions', HAR1, is part of a novel RNA gene (HAR1F)
that is expressed specifically in Cajal-Retzius neurons in the developing human
neocortex from 7 to 19 gestational weeks, a crucial period for cortical neuron
specification and migration. HAR1F is co-expressed with reelin, a product of
Cajal-Retzius neurons that is of fundamental importance in specifying the
six-layer structure of the human cortex. HAR1 and the other human accelerated
regions provide new candidates in the search for uniquely human biology. Regions of the genome that have been the target of positive selection
specifically along the human lineage are of special importance in human biology.
We used high throughput sequencing combined with methods to enrich human genomic
samples for particular targets to obtain the sequence of 22 chromosomal samples
at high depth in 40 kb neighborhoods of 49 previously identified 100-400 bp
elements that show evidence for human accelerated evolution. In addition to
selection, the pattern of nucleotide substitutions in several of these elements
suggested an historical bias favoring the conversion of weak (A or T) alleles
into strong (G or C) alleles. Here we found strong evidence in the derived
allele frequency spectra of many of these 40 kb regions for ongoing
weak-to-strong fixation bias. Comparison of the nucleotide composition at
polymorphic loci to the composition at sites of fixed substitutions additionally
reveals the signature of historical weak-to-strong fixation bias in a subset of
these regions. Most of the regions with evidence for historical bias do not also
have signatures of ongoing bias, suggesting that the evolutionary forces
generating weak-to-strong bias are not constant over time. To investigate the
role of selection in shaping these regions, we analyzed the spatial pattern of
polymorphism in our samples. We found no significant evidence for selective
sweeps, possibly because the signal of such sweeps has decayed beyond the power
of our tests to detect them. Together, these results do not rule out functional
roles for the observed changes in these regions-indeed there is good evidence
that the first two are functional elements in humans-but they suggest that a
fixation process (such as biased gene conversion) that is biased at the
nucleotide level, but is otherwise selectively neutral, could be an important
evolutionary force at play in them, both historically and at present. GC-biased gene conversion (gBGC) is a recombination-associated evolutionary
process that accelerates the fixation of guanine or cytosine alleles, regardless
of their effects on fitness. gBGC can increase the overall rate of
substitutions, a hallmark of positive selection. Many fast-evolving genes and
noncoding sequences in the human genome have GC-biased substitution patterns,
suggesting that gBGC-in contrast to adaptive processes-may have driven the human
changes in these sequences. To investigate this hypothesis, we developed a
substitution model for DNA sequence evolution that quantifies the nonlinear
interacting effects of selection and gBGC on substitution rates and patterns.
Based on this model, we used a series of lineage-specific likelihood ratio tests
to evaluate sequence alignments for evidence of changes in mode of selection,
action of gBGC, or both. With a false positive rate of less than 5% for
individual tests, we found that the majority (76%) of previously identified
human accelerated regions are best explained without gBGC, whereas a substantial
minority (19%) are best explained by the action of gBGC alone. Further, more
than half (55%) have substitution rates that significantly exceed local
estimates of the neutral rate, suggesting that these regions may have been
shaped by positive selection rather than by relaxation of constraint. By
distinguishing the effects of gBGC, relaxation of constraint, and positive
selection we provide an integrated analysis of the evolutionary forces that
shaped the fastest evolving regions of the human genome, which facilitates the
design of targeted functional studies of adaptation in humans. |
Carbapenemase-producing gram-negative bacteria is a major health concern because their resistance to antibiotics.
List the most frequent carbapenemases found in Enterobacteriaceae. | The most frequent carbapenemases in Enterobacteriaceae are OXA-48, KPC, VIM, NDM, IMP, SME, NMC, GES, IMI and MBL. | Emergence and dissemination of Enterobacteriaceae isolates harboring
carbapenemases in various geographic regions represents a significant threat to
the management of nosocomial infections. Enterobacteriaceae isolates from the
SENTRY Antimicrobial Surveillance Program (2000-2004) demonstrating decreased
susceptibility to imipenem and meropenem (minimum inhibitory concentration
[MIC], > or =2 mg/L) were evaluated for the production of
metallo-beta-lactamases and serine carbapenemases using disk approximation and
polymerase chain reaction (PCR) tests. Carbapenemase-producing strains were
epidemiologically typed by automated riboprinting and pulsed-field gel
electrophoresis (PFGE) to establish clonality. Among 37,557 Enterobacteriaceae
(5 genus groups) evaluated, 119 (0.32%) had increased carbapenem MIC values, and
a carbapenemase was identified in 51 (42.9%) of these strains. KPC-2 and KPC-3
were the most frequently occurring carbapenemases (24 isolates, 20.2%) in the
United States and were detected in Klebsiella spp, Citrobacter spp.,
Enterobacter spp., and Serratia marcescens strains isolated in New York,
Arkansas, and Virginia. SME-2-producing S. marcescens were isolated in the New
York City area, Texas, and Ohio, while NMC-A was found in one E. cloacae strain
from New York. In contrast, metallo-beta-lactamases were prevalent in Europe.
IMP-1-producing E. cloacae (11 isolates) were detected in Turkey, while
VIM-1-producing strains were found in Italy (Enterobacter spp.) and Greece
(Klebsiella pneumoniae). Clonal dissemination of carbapenemase-producing strains
was observed in several medical centers on both continents. The occurrence of
carbapenemases in various Enterobacteriaceae remains rare but appears to be
spreading geographically (not in Latin America), mainly with
metallo-beta-lactamases being found in Mediterranean Europe and KPC enzymes in
the New York City area. Strains of Klebsiella pneumoniae that produce one of three possible
carbapenemases--KPC--have recently been identified with increasing frequency
among isolates recovered from patients residing along the East Coast of the
United States, particularly within the New York City metropolitan region. These
strains have exhibited resistance to multiple antibiotic classes, including
carbapenem agents. We report a case of nosocomial pneumonia and empyema caused
by a KPC-producing isolate of K. pneumoniae at a large midwestern U.S. tertiary
care facility in which the patient was treated with tigecycline. Although the
pneumonia was treated successfully, the empyema recurred in association with a
treatment-emergent tigecycline minimum inhibitory concentration (MIC) increase
from 0.75 to 2 microg/ml. Clinicians should be aware of the potential occurrence
of this treatment-emergent MIC increase, especially in the setting of sustained
tigecycline therapy. In addition, the emergence of carbapenem-resistant
Enterobacteriaceae reinforces the importance of antibiotic stewardship and
strict infection control practices. ChromID extended-spectrum beta-lactamase (ESBL) culture medium is routinely used
for screening ESBL producers. This medium was tested for detecting
carbapenemase-producing Enterobacteriaceae isolates from a collection of
reference strains and compared to the CHROMagar KPC culture medium previously
evaluated for detecting KPC-producing isolates. Producers of IMP-, VIM-, and
KPC-type carbapenemases with high levels of resistance to cephalosporins and to
carbapenems were detected at 1x10(1) CFU/ml. The OXA-48 producers were not
detected on ChromID ESBL medium unless coexpressing ESBLs, whereas
carbapenemase-producing isolates with MICs of <4 microg/ml were not detected on
CHROMagar KPC medium. We assessed the ability of three commercial systems to infer carbapenem
resistance mechanisms in 39 carbapenemase-producing and 16 other
carbapenem-resistant Enterobacteriaceae. The sensitivity/specificity values for
"flagging" a likely carbapenemase were 100%/0% (BD Phoenix), 82 to 85%/6 to 19%
(MicroScan), and 74%/38% (Vitek 2), respectively. OXA-48 producers were poorly
detected, but all systems reliably detected isolates with KPC and most with
metallo-carbapenemases. The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion
carbapenem breakpoints for the detection of carbapenemase-producing Klebsiella
pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20)
carbapenemases were characterized by disk diffusion (Oxoid) and Etest
(bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux,
five different cards). Extended-spectrum β-lactamase (ESBL) testing was
performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson)
and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints,
respectively, 11 and seven of the strains were susceptible to imipenem, 12 and
eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological
cut-off (ECOFF) values for meropenem and ertapenem identified all carbapenemase
producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase
producers were detected with EUCAST disk diffusion breakpoints for ertapenem and
meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion
breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem)
isolates as susceptible. When cards with a single carbapenem were used,
detection failures with VITEK2 were four for imipenem, none for meropenem and
one for ertapenem. Cards containing all three carbapenems had one to two
failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM
producers were positive. With VITEK2, no VIM producers and between none and
seven KPC producers were ESBL-positive. All carbapenemase producers were
detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for
ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem
detected all carbapenemase producers. VITEK2 had between none and four failures
in detecting carbapenemase producers, depending on the antibiotic card. Matrix-assisted laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial
resistance, especially that conferred by β-lactamases. Here we describe a
modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay.
The modified method was validated on 108 carbapenemase-producing members of the
Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35
carbapenem-resistant enterobacteria producing no carbapenemase. The detection of
carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective
method for microbiological laboratories. The spread of carbapenemase-producing gram-negative bacteria is one of the major
challenges of the present. Since 2009, the National Reference Laboratory for
gram-negative nosocomial pathogens has observed the molecular epidemiology of
carbapenemases in Germany. In 2011, 1,454 referred bacterial isolates were
tested for the presence of carbapenemases. Carbapenemase was found in 34.4% of
Enterobacteriaceae isolates, in 19.9% of Pseudomonas aeruginosa isolates and in
96.3% of Acinetobacter baumannii isolates. The most frequent carbapenemases in
Enterobacteriaceae were OXA-48, KPC and VIM-1; in P. aeruginosa it was VIM-2 and
in A. baumannii OXA-23. |
How are GRBs (Genomic Regulatory Blocks) defined? | Genomic regulatory blocks (GRBs) are chromosomal regions spanned by highly conserved non-coding elements (HCNEs), most of which serve as regulatory inputs of one target gene in the region. The target genes are most often transcription factors involved in embryonic development and differentiation. GRBs often contain extensive gene deserts, as well as additional 'bystander' genes intertwined with HCNEs but whose expression and function are unrelated to those of the target gene. | We report evidence for a mechanism for the maintece of long-range conserved
synteny across vertebrate genomes. We found the largest mammal-teleost conserved
chromosomal segments to be spanned by highly conserved noncoding elements
(HCNEs), their developmental regulatory target genes, and phylogenetically and
functionally unrelated "bystander" genes. Bystander genes are not specifically
under the control of the regulatory elements that drive the target genes and are
expressed in patterns that are different from those of the target genes.
Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2,
rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the
expression patterns of these genes even if located inside or beyond bystander
genes, suggesting that the regulatory domain of a developmental regulatory gene
can extend into and beyond adjacent transcriptional units. We termed these
chromosomal segments genomic regulatory blocks (GRBs). After whole genome
duplication in teleosts, GRBs, including HCNEs and target genes, were often
maintained in both copies, while bystander genes were typically lost from one
GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy
GRBs of higher vertebrates intact. We show that loss of bystander genes and
other mutational events suffered by duplicated GRBs in teleost genomes permits
target gene identification and HCNE/target gene assignment. These findings
explain the absence of evolutionary breakpoints from large vertebrate
chromosomal segments and will aid in the recognition of position effect
mutations within human GRBs. Insect genomes contain larger blocks of conserved gene order (microsynteny) than
would be expected under a random breakage model of chromosome evolution. We
present evidence that microsynteny has been retained to keep large arrays of
highly conserved noncoding elements (HCNEs) intact. These arrays span key
developmental regulatory genes, forming genomic regulatory blocks (GRBs). We
recently described GRBs in vertebrates, where most HCNEs function as enhancers
and HCNE arrays specify complex expression programs of their target genes. Here
we present a comparison of five Drosophila genomes showing that HCNE density
peaks centrally in large synteny blocks containing multiple genes. Besides
developmental regulators that are likely targets of HCNE enhancers, HCNE arrays
often span unrelated neighboring genes. We describe differences in core
promoters between the target genes and the unrelated genes that offer an
explanation for the differences in their responsiveness to enhancers. We show
examples of a striking correspondence between boundaries of synteny blocks, HCNE
arrays, and Polycomb binding regions, confirming that the synteny blocks
correspond to regulatory domains. Although few noncoding elements are highly
conserved between Drosophila and the malaria mosquito Anopheles gambiae, we find
that A. gambiae regions orthologous to Drosophila GRBs contain an equivalent
distribution of noncoding elements highly conserved in the yellow fever mosquito
Aëdes aegypti and coincide with regions of ancient microsynteny between
Drosophila and mosquitoes. The structural and functional equivalence between
insect and vertebrate GRBs marks them as an ancient feature of metazoan genomes
and as a key to future studies of development and gene regulation. A large-scale enhancer detection screen was performed in the zebrafish using a
retroviral vector carrying a basal promoter and a fluorescent protein reporter
cassette. Analysis of insertional hotspots uncovered areas around developmental
regulatory genes in which an insertion results in the same global expression
pattern, irrespective of exact position. These areas coincide with vertebrate
chromosomal segments containing identical gene order; a phenomenon known as
conserved synteny and thought to be a vestige of evolution. Genomic comparative
studies have found large numbers of highly conserved noncoding elements (HCNEs)
spanning these and other loci. HCNEs are thought to act as transcriptional
enhancers based on the finding that many of those that have been tested direct
tissue specific expression in transient or transgenic assays. Although gene
order in hox and other gene clusters has long been known to be conserved because
of shared regulatory sequences or overlapping transcriptional units, the
chromosomal areas found through insertional hotspots contain only one or a few
developmental regulatory genes as well as phylogenetically unrelated genes. We
have termed these regions genomic regulatory blocks (GRBs), and show that they
underlie the phenomenon of conserved synteny through all sequenced vertebrate
genomes. After teleost whole genome duplication, a subset of GRBs were retained
in two copies, underwent degenerative changes compared with tetrapod loci that
exist as single copy, and that therefore can be viewed as representing the
ancestral form. We discuss these findings in light of evolution of vertebrate
chromosomal architecture and the identification of human disease mutations. Despite a recent explosion in the production of vertebrate genome sequence data
and large-scale efforts to completely annotate the human genome, we still have
scant knowledge of the principles that built vertebrate genomes in evolution,
and of genome architecture and its functional significance. We review approaches
using bioinformatics, zebrafish transgenesis, and recent findings in the
molecular basis of gene regulation and tie these in with mechanisms for the
maintece of long-range conserved synteny across all vertebrate genomes.
Specifically, we discuss the recently discovered genomic regulatory blocks which
we argue are principal units of vertebrate genome evolution and serve as the
foundations onto which evolutionary innovations are built through sequence
evolution and insertion of new cis-regulatory elements. We subsequently discuss
how these arrangements relate to common human heritable diseases and their
significance in disease causality. 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. Using a comparative genomics approach to reconstruct the fate of genomic
regulatory blocks (GRBs) and identify exonic remts that have survived the
disappearance of their host genes after whole-genome duplication (WGD) in
teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs)
with predicted target genes. These elements demonstrate evolutionary separation
of overlapping protein-coding and regulatory information after WGD in teleosts.
We present evidence that the corresponding mammalian exons are still under both
coding and non-coding selection pressure, are more conserved than other protein
coding exons in the host gene and several control sets, and share key
characteristics with highly conserved non-coding elements in the same regions.
Their dual function is corroborated by existing experimental data. Additionally,
we show examples of human exon remts stemming from the vertebrate 2R WGD. Our
findings suggest that long-range cis-regulatory inputs for developmental genes
are not limited to non-coding regions, but can also overlap the coding sequence
of unrelated genes. Thus, exonic regulatory elements in GRBs might be
functionally equivalent to those in non-coding regions, calling for a
re-evaluation of the sequence space in which to look for long-range regulatory
elements and experimentally test their activity. Genome-wide association studies identified noncoding SNPs associated with type 2
diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE
and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881-885;
Steinthorsdottir V, et al. (2007) Nat. Genet 39:770-775; Frayling TM, et al.
(2007) Science 316:889-894]. We show that these LD blocks contain highly
conserved noncoding elements and overlap with the genomic regulatory blocks of
the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly
conserved noncoding elements in LD with the risk SNPs drive expression in
endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have
recently been implicated in pancreas development and the regulation of insulin
secretion, but IRX3 had no prior association with pancreatic function or
development. Knockdown of its orthologue in zebrafish, irx3a, increased the
number of pancreatic ghrelin-producing epsilon cells and decreased the number of
insulin-producing beta-cells and glucagon-producing alpha-cells, thereby
suggesting a direct link of pancreatic IRX3 function to both obesity and type 2
diabetes. Developmental genes are regulated by complex, distantly located cis-regulatory
modules (CRMs), often forming genomic regulatory blocks (GRBs) that are
conserved among vertebrates and among insects. We have investigated GRBs
associated with Iroquois homeobox genes in 39 metazoans. Despite 600 million
years of independent evolution, Iroquois genes are linked to
ankyrin-repeat-containing Sowah genes in nearly all studied bilaterians. We show
that Iroquois-specific CRMs populate the Sowah locus, suggesting that regulatory
constraints underlie the maintece of the Iroquois-Sowah syntenic block.
Surprisingly, tetrapod Sowah orthologs are intronless and not associated with
Iroquois; however, teleost and elephant shark data demonstrate that this is a
derived feature, and that many Iroquois-CRMs were ancestrally located within
Sowah introns. Retroposition, gene, and genome duplication have allowed
selective elimination of Sowah exons from the Iroquois regulatory landscape
while keeping associated CRMs, resulting in large associated gene deserts. These
results highlight the importance of CRMs in imposing constraints to genome
architecture, even across large phylogenetic distances, and of gene
duplication-mediated genetic redundancy to disentangle these constraints,
increasing genomic plasticity. The order of genes in eukaryotic genomes has generally been assumed to be
neutral, since gene order is largely scrambled over evolutionary time. Only a
handful of exceptional examples are known, typically involving deeply conserved
clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we
report the first systematic survey of microsynteny conservation across
metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of
unrelated genes that have remained tightly physically linked in diverse lineages
across over 600 million years of evolution. Integrating sequence conservation,
gene expression data, gene function, epigenetic marks, and other genomic
features, we provide extensive evidence that many conserved ancient linkages
involve (1) the coordinated transcription of neighboring genes, or (2) genomic
regulatory blocks (GRBs) in which transcriptional enhancers controlling
developmental genes are contained within nearby bystander genes. In addition, we
generated ChIP-seq data for key histone modifications in zebrafish embryos,
which provided further evidence of putative GRBs in embryonic development.
Finally, using chromosome conformation capture (3C) assays and stable transgenic
experiments, we demonstrate that enhancers within bystander genes drive the
expression of genes such as Otx and Islet, critical regulators of central
nervous system development across bilaterians. These results suggest that
ancient genomic functional associations are far more common than previously
thought-involving ∼12% of the ancestral bilaterian genome-and that
cis-regulatory constraints are crucial in determining metazoan genome
architecture. MOTIVATION: Genomic context analysis, also known as phylogenetic profiling, is
widely used to infer functional interactions between proteins but rarely applied
to non-coding cis-regulatory DNA elements. We were wondering whether this
approach could provide insights about utlraconserved non-coding elements
(UCNEs). These elements are organized as large clusters, so-called gene
regulatory blocks (GRBs) around key developmental genes. Their molecular
functions and the reasons for their high degree of conservation remain
enigmatic.
RESULTS: In a special setting of genomic context analysis, we analyzed the fate
of GRBs after a whole-genome duplication event in five fish genomes. We found
that in most cases all UCNEs were retained together as a single block, whereas
the corresponding target genes were often retained in two copies, one completely
devoid of UCNEs. This 'winner-takes-all' pattern suggests that UCNEs of a GRB
function in a highly cooperative manner. We propose that the multitude of
interactions between UCNEs is the reason for their extreme sequence
conservation.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online and at http://ccg.vital-it.ch/ucne/ |
What is the gene frequently mutated in Multiple endocrine neoplasia 2 (MEN2) and Hisrchsprung disease? | The Ret gene may have gain of mutation functions in MEN2 cancer as well as loss of function mutations in Hirschprung disease. | BACKGROUND AND OBJECTIVES: Medullary thyroid carcinoma (MTC) occurs both
sporadically and in the autosomal domitly inherited multiple endocrine
neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and
a new mutation familial case is important for future clinical management of both
the patient and family. The susceptibility gene for MEN 2 is the RET
proto-oncogene. Systematic analysis for germline mutations of the RET
proto-oncogene was performed in a series of 67 patients with apparently sporadic
MTC to determine whether they were true sporadic cases or unsuspected de novo
MEN 2 cases.
DESIGN AND PATIENTS: Sixty-seven unselected patients with sporadic MTC were
randomly ascertained from clinic patients from four centres. The diagnosis of
MTC was confirmed by histopathology. Germline DNA was extracted from peripheral
blood leucocytes or from paraffin-embedded tissue and subsequently used for
polymerase chain reaction amplification.
MEASUREMENTS: Polymerase chain reaction based RET mutation analysis was
performed by direct double-stranded cycle sequencing of exons 10, 11, 13 and 16,
within which the majority of MEN2 mutations have been shown to occur.
RESULTS: In this series, there was one proven case of germline mutation in RET
codon 620, which previously has been shown to be responsible for MEN 2, thus
indicating heritable disease. No germline mutation in codon 918, typical of MEN
2B, was found.
CONCLUSIONS: A figure of 1.5% germline mutations in 67 apparently sporadic MTC
is lower than the incidence of familial disease reported in previous series
involving clinical and biochemical screening. The presence of a germline
mutation in the RET proto-oncogene in a patient with MTC indicates heritable
disease. The absence of germline RET exon 10, 11, 13 or 16 mutation appears to
rule out MEN 2A to a high probability, although the presence of a familial form
of MTC other than classical MEN 2A cannot be excluded conclusively. The clinical association between multiple endocrine neoplasia type 2 (MEN2) and
Hirschsprung disease (HSCR) is infrequent. Germline mutations of the ret
protooncogene are the underlying cause of the MEN2 syndromes and a proportion of
cases of HSCR. In this report, we describe a new kindred in which the MEN2 and
HSCR phenotypes are associated with a single C620S point mutation at one of the
cysteine codons of the extracellular domain of the ret protooncogene. We also
speculate about the role of a silent mutation in exon 2 of this same gene
(A45A), present in a homozygous state in the patient with both MEN2A and HSCR.
To investigate the contribution of GDNF to the phenotype observed in this
kindred, we scanned the coding region of GDNF in the patient with MEN2/HSCR, but
no mutation was found. PURPOSE: The RET proto-oncogene is involved in neural crest disorders.
Activating germline mutations in the RET proto-oncogene cause the development of
familial medullary thyroid carcinoma (FMTC) or medullary thyroid carcinoma (MTC)
as a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Inactivating
germline mutations in the RET proto-oncogene are detected in Hirschsprung's
disease (HSCR). Only in a very small number of families are these 2 diseases
expressed together.
METHODS: This study presents a novel Czech kindred with FMTC-HSCR phenotype. Two
family members (mother and daughter) were tested for RET germline mutations in
exons 10, 11, 13, 14, 15, and 16.
RESULTS: Direct fluorescent sequencing of genomic DNA revealed a heterozygous
mutation in the RET proto-oncogene in exon 10 at codon C609Y in both persons
tested. This family was reclassified, thanks to genetic screening from the
apparently sporadic MTC-HSCR to FMTC-HSCR.
CONCLUSION: The germline mutation was detected because of the systematic genetic
screening of the RET proto-oncogene, which is useful for genetic counseling of
potential risk of HSCR and MTC in other family members. This family could be
added to the small worldwide cohort of families with MEN2A/FMTC-HSCR. OBJECTIVE: Medullary thyroid carcinoma (MTC) occurs both sporadically and in the
context of autosomal domitly inherited multiple endocrine neoplasia type 2
(MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC),
which are caused by activating germline mutations in the RET proto-oncogene. The
aim of this study was to characterize the RET mutational spectrum in MEN2
families and apparently sporadic MTC (AS-MTC) cases originating from the central
region of Portugal.
SUBJECTS AND METHODS: We studied a total of 82 individuals (64 affected and 18
family members), comprising five MEN2 families (four MEN2A and one MEN2B), as
well as 53 AS-MTC cases. RET germline mutations were screened using PCR-DNA
sequencing, SSCP and RFLP. The haplotypes associated with recurrent mutations
were determined by fragment analysis of microsatellite markers, and by RFLP, in
the case of intragenic polymorphisms.
RESULTS: Frequency of the Cys611Tyr (TGC-TAC) mutation was significantly
increased in this region of Portugal, due to the fact that three apparently
unrelated MEN2A/FMTC families, out of the five in which mutations were
identified, harboured this specific mutation. Haplotype analysis revealed that a
common haplotype was shared between two of these three families. We have also
characterized a novel RET mutation, Arg886Trp, located in the tyrosine kinase
domain, which was found in an AS-MTC case.
CONCLUSIONS: There are regional specificities in the relative frequency of RET
mutations, which are consistent with a cluster-like distribution of specific
disease-causing mutations, as a result of the inheritance of a shared haplotype.
These data, along with the finding of a novel RET mutation (Arg886Trp), have
important implications towards facilitating and improving the molecular
diagnosis of hereditary MTC on a regional basis. Multiple endocrine neoplasia type 2 (MEN2) is an inherited, autosomal-domit
disorder caused by deleterious mutations within the RET protooncogene. MEN2 RET
mutations are mainly heterozygous, missense sequence changes found in RET exons
10, 11, and 13-16. Our group has developed the publicly available, searchable
MEN2 RET database to aid in genotype/phenotype correlations, using Human Genome
Variation Society recommendations for sequence variation nomenclature and
database content. The MEN2 RET database catalogs all RET sequence variation
relevant to the MEN2 syndromes, with associated clinical information. Each
database entry lists a RET sequence variation's location within the RET gene,
genotype, pathogenicity classification, MEN2 phenotype, first literature
reference, and comments (which may contain information on other clinical
features, complex genotypes, and additional literature references). The MEN2
phenotype definitions were derived from the International RET Mutation
Consortium guidelines for classification of MEN2 disease phenotypes. Although
nearly all of the 132 RET sequence variation entries initially cataloged in the
database were from literature reports, novel sequence variation and updated
phenotypic information for any existing database entry can be submitted
electronically on the database website. The database website also contains links
to selected MEN2 literature reviews, gene and protein information, and RET
reference sequences. The MEN2 RET database
(www.arup.utah.edu/database/MEN2/MEN2_welcome.php) will serve as a repository
for MEN2-associated RET sequence variation and reference for RET genotype/MEN2
phenotype correlations. Activating germline RET mutations are presented in patients with familial
medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia (MEN) types
2A and 2B, whereas inactivating germline mutations in patients with
Hirschsprung's disease (HSCR). The aim of this study was to evaluate
genotype-phenotype correlations of the frequently discussed Tyr791Phe mutation
in exon 13 of the RET proto-oncogene. Screening of three groups of patients was
performed (276 families with medullary thyroid carcinoma (MTC), 122 families
with HSCR, and 29 patients with pheochromocytoma). We found this mutation in 3
families with apparently sporadic MTC, 3 families with FMTC/MEN2, 1 patient with
pheochromocytoma, and 3 families with HSCR. All gene mutation carriers have a
silent polymorphism Leu769Leu in exon 13. In three families second germline
mutations were detected: Cys620Phe (exon 10) in MEN2A family, Met918Thr (exon
16) in MEN2B family, and Ser649Leu (exon 11) in HSCR patient. Detection of the
Tyr791Phe mutation in MEN2/MTC and also in HSCR families leads to the question
whether this mutation has a dual character (gain-of-function as well as
loss-of-function). A rare case of maligt pheochromocytoma in a patient with
the Tyr791Phe mutation is presented. This study shows various clinical
characteristics of the frequently discussed Tyr791Phe mutation. BACKGROUND: RET germline mutations predispose to the development of inherited
cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Several variants of
the RET proto-oncogene including G691S and S904S have been suggested to act as
genetic modifiers at the age of onset ofMEN2.
AIM: The aim of this study is to characterize clinically and molecularly 7
Cypriot patients with familial medullary thyroid carcinoma (FMTC) and 1 with
MEN2A and also to determine the allelic frequencies of the RET variants G691S
and S904S.
SUBJECTS AND METHODS: Seven probands from FMTC families and 1 from MEN2A were
screened for the presence of RET mutations and the G691S and S904S variants.
Additionally, 226 healthy Cypriots, who served as controls were analysed in an
attempt to compare the frequencies of G691S and S904S RET variants to those
observed in the 8 patients.
RESULTS: The clinical diagnosis of the probands was based on clinical
presentation and supported with biochemical findings. The germline C618R
mutation of exon 10 was identified in all 8 probands and in 15 relatives from 7
different families. No significant difference in the G691S/S904S variants allele
frequencies between patients (4/16 or 25%) and controls (124/452 or 27.4%) was
found.
CONCLUSIONS: Mutational screening of the RET gene identified a common mutation
(C618R) in all 8 (7 FMTC and 1 MEN2A) unrelated Cypriot patients which may be
explained by a founder effect. Additionally, no association of the G691S/S904S
variants was linked with the disease. The RET proto-oncogene (REarranged during Transfection; RET) plays an important
role in the causation of many thyroid tumours. Germline RET proto-oncogene
missense mutations have been clearly linked to medullary thyroid carcinoma (MTC)
and the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2A,
MEN2B).
METHODS: We investigated a cohort of MEN2-related patients referred to Tygerberg
Hospital, W Cape (2003-2009). The study cohort was divided into three groups
based on pathology (viz. MEN/MTC, phaeochromocytoma, and a miscellaneous group
of MEN pathologies). Families with identified high-risk factors were recalled.
Serum calcitonin levels were monitored where indicated. DNA was extracted from
whole blood by standard techniques and polymerase chain reaction (PCR) products
screened for RET gene variations by heteroduplex single-strand duplication
techniques (heteroduplex single-strand conformation polymorphism analysis) being
validated with automated sequencing techniques showing conformational variants
in acrylamide gel.
RESULTS: We screened 40 persons, male/female ratio 1:1.5. Three ethnic groups
were represented (white (12), black (11) and mixed race (17)). Nine were index
MTC cases, 5 phaeochromocytoma, 3 Hirschsprung's disease-MEN associations and 2
miscellaneous (1 neuroblastoma, 1 intestinal neuronal dysplasia), while 1 fell
into the MEN2B category. The remaining 19 were unaffected relatives screened for
carrier status, among whom afamilial recurrence was observed in 7. On genetic
testing, an RET point mutation at the high-risk 634 cysteine allele was
identified in 11 cases. A further cysteine radical mutation at the 620 position
was related to MEN2 in 3 families plus 1 other family referred from elsewhere.
Other less-recognised gene variations were detected throughout the RET gene in
70% of cases and included the 691 position on codon 11 (11 cases); the 432
position (4 cases, 1 homozygous) intronic mutations on exon 4 (1 case); and an
IVS19-37G/C and a D1017N variation in exon 19 in 2 MEN families. Fifteen MTC
patients have had thyroidectomies, of which 2 were prophylactic (C-cell
hyperplasia; early occult MTC). A further 3 are awaiting prophylactic surgery.
CONCLUSION: RET gene mutation carries a risk of MEN2 and MTC in all ethnic
groups in South Africa. Prophylactic surgery may prevent MTC, so genetic
screening is important to identify and treat high-risk patients. To describe the coexistence of mutations of both the multiple endocrine
neoplasia type 1 (MEN1) and type 2 (MEN2) genes in a large Italian family and
evaluate if it could be associated with more aggressive clinical manifestations
of the two syndromes. Blood samples were obtained for genetic and biochemical
analyses. The RET gene exons (8, 10, 11, 13, 14, 15, 16, 18) and the MEN1 coding
regions, including the exon-intron boundaries, were amplified by PCR and
directly sequenced. We identified two germline mutations in the proband: the
first one, K666M, located at the exon 11 of RET proto-oncogene and the second
one, IVS4+1G>T, located in the MEN1 gene. The functional characterization of
IVS4+1G>T variation, located in the splicing donor site of exon 4 of MEN1 gene,
caused the in-frame junction of exon 3 to exon 5, thus obtaining a shorter
protein. The same proband's germline mutations were found in 16 relatives out of
21 screened subjects: 8 carried IVS4+1G>T, 4 RET K666M, and 4 both the
mutations. This is the second report in literature of coexistence in the same
family of germline mutations of both RET proto-oncogene and MEN1 gene. The
simultaneous presence of the two mutations was not apparently associated with
more aggressive diseases, since at last follow-up all patients appeared to be
disease-free or well compensated by medical therapy; finally, no one exhibited
metastatic diseases. BACKGROUND: Screening medullary thyroid carcinomas (MTCs) for rearranged during
transfection (RET) mutations becomes increasingly important for clinical
assessment of the disease. The role of mutations in other genes including RAS
(i.e. HRAS, KRAS, and NRAS), v-raf murine sarcoma viral oncogene homolog B1
(BRAF), v-akt murine thymoma viral oncogene homolog 1 (AKT1), and CTNNB1
(β-catenin) is unknown or not fully explored yet for this disease.
MATERIALS AND METHODS: Formalin-fixed and paraffin-embedded (FFPE) material was
the primary source for screening 13 sporadic and inherited MTCs and matched
non-tumor specimens. Multiplex PCR was included in the PCR protocol. Sequence
analysis encompassed mutational hotspot regions in RET exons 5, 8, 10, 11, and
13 to 16; HRAS exons 1 and 2; KRAS exons 1 and 2; NRAS exons 1 and 2; BRAF exon
15; AKT1 exon 2, and CTNNB1 exon 3.
RESULTS: We identified RET mutations in seven of 13 MTCs: five RET-positive
cases revealed a mutation in exon 16 (M918T) and two a mutation in exon 10
(C618S and C620S). In four of the RET-positive cases, the mutation was
inherited, out of which three were reportedly associated with a multiple
endocrine neoplasia type 2 (MEN2) syndrome, i.e. MEN2A (C618S), MEN2A/familial
MTC (FMTC) (C620S), and MEN2B (M918T). These cases reflect the known MEN2
genotype-phenotype correlation. Three of the five stage IVc MTCs were inherited
RET-positive cases. Mutational screening in HRAS, KRAS, NRAS, BRAF, AKT1, and
CTNNB1 disclosed one sporadic RET-negative MTC (stage III) with mutation in HRAS
codon 13 (G13R).
CONCLUSION: Our study supports the clinical relevance of screening MTC patients
for RET mutations. The role of RAS mutations, in particular HRAS mutations, in
sporadic RET-negative MTC has not been fully explored yet. Mutations in BRAF,
AKT1, and CTNNB1 are likely not to play a role in MTC. BACKGROUND: Genetic screening for germline mutations in the RET proto-oncogene
has been extensively exploited worldwide to optimize the diagnostic and clinical
management of multiple endocrine neoplasia type 2 (MEN2) patients and their
relatives. However, a distinct lag period exists not only in the recognition but
also in the medical treatment of patients with MEN2. Here we present a
comprehensive genetic and clinical analysis of MEN2 among Chinese families
followed from 1975 to 2011. Our series comprises 36 index cases and 134
relatives from 11 independent families.
METHODS: Genetic diagnosis was performed in all participants by direct
sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients
with varying cervical neck dissection procedures. Patients with pheochromocytoma
(PHEO) underwent specific surgery. Demographic, clinical profiles, mutation
types, tumor histopathologic features, and follow-up records were systematically
analyzed.
RESULTS: The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13),
p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were
confirmed in 31 index cases and then identified in 32 at-risk relatives
(mutation carriers), with MEN2A as the most common clinical subtype. The overall
penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients
underwent thyroidectomy, and there was a significant lowering of their mean age
at thyroidectomy and the tumor diameter of the mutation carriers that were
detected and operated on compared with the index cases (age at first surgery:
29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was
also a decrease in the TNM staging and the proportion of patients who underwent
inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001;
inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS)
increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid
carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the
study period.
CONCLUSIONS: Our results further substantiate that gene scanning of all relevant
RET exons is a powerful tool in the management of MEN2 patients, especially in
asymptomatic carriers, and has led to earlier diagnosis and more complete
initial treatment of patients with MEN2 in China. |
Which are the clinical characteristics of isolated Non-compaction cardiomyopathy? | The clinical characteristics of isolated Non-compaction cardiomyopathy are excessively thickened endocardial layer with deep intertrabecular recesses (with ratio of non-compacted to compacted myocardium >2), heart failure, syncope, ventricular arrhythmias, stroke, pulmonary hypertension, complete left branch conductive block, sick sinus syndrome and paroxysmal supraventricular tachycardia | Isolated ventricular non-compaction is a rare congenital cardiomyopathy,
manifested morphologically as prominent myocardial trabeculations and deep
inter-trabecular recesses that communicate with the ventricular cavity. Heart
failure is the most common presenting condition. Other manifestations include
arrhythmia and cardioembolic events. This report is illustrative of isolated
ventricular non-compaction in a 78-year-old woman. The diagnosis was made when
she presented with ventricular tachycardia many years after a stroke. She
subsequently underwent implantation of a cardioverter-defibrillator. This report
documents an uncommon presentation of this disease entity in the oldest patient
at presentation as yet reported in the literature. AIMS: Non-compaction of the left ventricle (LVNC) is a disorder of
endomyocardial morphogenesis that results in multiple trabeculations in the left
ventricular myocardium. The current literature suggests that LVNC in adults is
rare and associated with a poor prognosis. Given that the disorder is present at
birth and that several studies have reported asymptomatic familial disease in
some patients, we hypothesized that there is a long pre-clinical phase of the
disease. The aim of this study was to define the prognosis and familial
incidence of LVNC.
METHODS AND RESULTS: This study cohort comprised 45 patients (mean age at
diagnosis 37 years) consecutively identified at a referral centre for
cardiomyopathy over a 10-year period. Twenty-eight patients (62%) had dyspnoea
at presentation; 41 (91%) an abnormal ECG; and 30 (66%) left ventricular
dilatation and impaired systolic function. Nine patients (20%) had non-sustained
ventricular tachycardia on 24 h Holter monitoring. Mean survival from death or
transplantation was 97% at 46 months. There were three thromboembolic events in
two patients (4%). On systematic family screening, 8 of 32 (25%) asymptomatic
relatives had a range of echocardiographic abnormalities, including LVNC, LVNC
with impaired systolic function, and left ventricular enlargement without LVNC.
CONCLUSION: This study demonstrates that LVNC is associated with a better
prognosis than previously reported. In patients with familial disease, relatives
may have features consistent with dilated cardiomyopathy rather than LVNC. Isolated non-compaction of the ventricular myocardium (INVM), also known as left
ventricular hypertrabeculation or spongy myocardium, belongs to the
"unclassified" cardiomyopathies according to the World Health Organization. The
main characteristic of this entity is a prominent trabeculation of the left
ventricle with deep intertrabecular recesses communicating with the ventricular
cavity. The pathomechanism of INVM is thought to be an arrest in cardiac
myogenesis with persistence of embryonic myocardial morphology. The most
frequent clinical manifestations include congestive heart failure, ventricular
arrhythmias and systemic thromboembolic events. The therapy of INVM comprises
standard medical therapy for heart failure. Non-compaction of the left ventricle or spongy myocardium is a rare congenital
cardiomyopathy which is characterized by impaired endomyocardial morphogenesis,
hypertrophy of left ventricular myocardium with prominent trabeculation, and
deep inter-trabecular recesses. According to WHO classification (1995) this
disease belongs to unclassified cardiomyopathies. Main clinical signs of
ventricular non-compaction are heart failure, ventricular arrhythmias, systemic
and pulmonary embolism. Echocardiography which is a basic method of diagnosis
can reveal double layer structure of thickened left ventricular wall and
multiple prominent trabeculation with wide inter-trabecular spaces. Predomit
localizations of pathological process are region of cardiac apex, inferior and
lateral left ventricular walls. Treatment of patients with ventricular
non-compaction concentrates on elimination of its main clinical symptoms. BACKGROUND: Isolated ventricular non-compaction (IVNC) is a rare disorder
characterized by prominent trabecular meshwork and deep recesses. We
retrospectively assessed the clinical characteristics and natural course of IVNC
in adults diagnosed at our hospital.
METHODS AND RESULTS: Sixty-seven adult patients (44 male, mean age 41 +/- 18
years) with the diagnosis of IVNC were evaluated in this retrospective cohort.
Its prevalence was found to be .14%. Forty-seven patients (70%) had class I/II
functional capacity. Fifty-seven patients (85%) had electrocardiographic
abnormalities, and the most common one was left ventricular (LV) hypertrophy
(25%). LV systolic function was depressed in 44 patients (66%), with a median
ejection fraction (EF) of 35% (range: 20%-48%) at diagnosis. Multiple regression
analysis revealed that age at initial presentation, the total number of affected
segments, and the ratio of non-compaction/compaction (NC/C) were the independent
predictors of LV systolic dysfunction. Familial occurrence of IVNC was 33%.
During a mean follow-up of 30 months (range: 9-50 months), major complications
including ventricular tachycardia, heart failure requiring hospitalization, and
cerebrovascular events were observed in 36%, 34%, and 9% of the patients,
respectively. Ten patients (15%) with IVNC died in this study. LVEF at initial
presentation and functional capacity at last visit were found to be independent
predictors of mortality.
CONCLUSION: This study suggests that IVNC is a form of cardiomyopathy with
higher prevalence and relatively better prognosis than previously reported. Age
at initial presentation, ratio of NC/C, and number of affected segments seem to
be major determits of LV systolic dysfunction, while initial LVEF and last
functional capacity predict mortality in this cohort. INTRODUCTION: Isolated left ventricular non-compaction is a recently described
form of cardiomyopathy that is associated with a significant risk of
life-threatening arrhythmia and thromboembolic complications.
CASE PRESENTATION: We report the presentation, diagnosis and management of
isolated left ventricular non-compaction in a 54-year-old Caucasian woman
presenting with progressive symptoms of heart failure.
CONCLUSION: Advances in diagnostic imaging have undoubtedly led to an increase
in the detection of isolated left ventricular non-compaction. Diagnosing and
differentiating this uncommon condition from other forms of cardiomyopathy are
important as treatment and prognosis may differ significantly. Our current
understanding of isolated left ventricular non-compaction, including diagnostic
criteria, management and prognosis, is discussed. Isolated left ventricular non-compaction is a rare, morphologically distinct
primary genetic cardiomyopathy, which was first described in children, but is
becoming increasingly recognised in adults. The most common clinical
manifestations include heart failure, arrhythmias and embolic events, and in
children it may be associated with facial dysmorphisms and Wolff-Parkinson-White
syndrome. Misdiagnosis may occur through lack of awareness of the condition, but
with increased vigilance detailed cardiac imaging can reliably differentiate
isolated left ventricular non-compaction from other pathological causes of left
ventricular dysfunction. This is important because the condition may be
associated with significant morbidity and mortality, and genetic counselling and
echocardiographic screening of first-degree relatives is recommended to detect
asymptomatic cases. BACKGROUND: It is apparent that despite lack of family history, patients with
the morphological characteristics of left ventricular non-compaction develop
arrhythmias, thrombo-embolism and left ventricular dysfunction.
METHODS: Forty two patients, aged 48.7 +/- 2.3 yrs (mean +/- SEM) underwent
cardiovascular magnetic resoce (CMR) for the quantification of left
ventricular volumes and extent of non-compacted (NC) myocardium. The latter was
quantified using planimetry on the two-chamber long axis LV view (NC area). The
patients included those referred specifically for CMR to investigate suspected
cardiomyopathy, and as such is represents a selected group of patients.
RESULTS: At presentation, 50% had dyspnoea, 19% chest pain, 14% palpitations and
5% stroke. Pulmonary embolism had occurred in 7% and brachial artery embolism in
2%. The ECG was abnormal in 81% and atrial fibrillation occurred in 29%.
Transthoracic echocardiograms showed features of NC in only 10%. On CMR,
patients who presented with dyspnoea had greater left ventricular volumes (both
p < 0.0001) and a lower left ventricular ejection fraction (LVEF) (p < 0.0001)
than age-matched, healthy controls. In patients without dyspnoea (n = 21), NC
area correlated positively with end-diastolic volume (r = 0.52, p = 0.0184) and
end-systolic volume (r = 0.56, p = 0.0095), and negatively with EF (r = -0.72, p
= 0.0001).
CONCLUSION: Left ventricular non-compaction is associated with dysrrhythmias,
thromboembolic events, chest pain and LV dysfunction. The inverse correlation
between NC area and EF suggests that NC contributes to left ventricular
dysfunction. HISTORY AND CLINICAL FINDINGS: A 55-year-old woman was referred to our hospital
with signs of cerebral ischaemia i. e. dysarthria and weakness of the buccal
branch of the facial nerve. Additionally the patient reported symptoms of heart
failure NYHA class II. Six months earlier the patient also had visual
disturbances. Magnetic resoce imaging (MRI) had shown ischaemic lesions.
INVESTIGATIONS: A recent MRI confirmed the suspected diagnosis of ischaemia in
the territory supplied by the left middle cerebral artery. The echocardiography
was characterized by a reduced left ventricular ejection fraction (25 %) due to
isolated ventricular non-compaction (IVNC).
TREATMENT AND COURSE: The patient was treated with a combination therapy
including ACE-inhibitors and diuretics. An oral anticoagulation was recommended
as secondary prophylaxis. At the time of discharge the patient had no residual
neurological deficits.
CONCLUSION: Isolated ventricular non-compaction is a rare type of
cardiomyopathy. Possible manifestations include systemic embolic events,
arrhythmias and heart failure. Echocardiography is the investigation of choice
in identifying characteristic changes. BACKGROUND: Isolated non-compaction cardiomyopathy (NCCM) was first described in
1984. This disorder, a primary genetic cardiomyopathy, is now attracting
increased attention.
METHOD: The current state of the epidemiology, pathogenesis, pathophysiology,
clinical features, diagnosis, treatment, and prognosis of NCCM are discussed on
the basis of a review of selected literature as well as the authors' personal
experience.
RESULTS: The pathogenesis of NCCM is thought to involve a genetically determined
disturbance of the myocardial compaction process during fetal endomyocardial
morphogenesis. It is not accompanied by any other cardiac anomalies.
Echocardiography is the diagnostic method of choice. The diagnosis is based on
the following echocardiographic criteria: the presence of at least 4 prominent
trabeculations and deep intertrabecular recesses, blood flow from the
ventricular cavity into the intertrabecular recesses, and a typical bilaminar
structure of the affected portion of the left ventricular myocardium. NCCM can
also be diagnosed with magnetic resoce imaging of the heart. The clinical
severity of NCCM is variable; its manifestations include heart failure,
thromboembolic events, and arrhythmias. The treatment is symptom-based. Patients
with symptomatic NCCM have a poor prognosis.
CONCLUSION: NCCM is a type of cardiomyopathy that was first described 25 years
ago. Its molecular genetic basis is not yet fully clear, and the same is true of
its diagnosis, treatment, and prognosis. Further study of these matters is
needed. Isolated left ventricular non-compaction (LVNC) is a rare genetic form of
cardiomyopathy (CM) characterized by prominent left ventricular wall
trabeculation and intertrabecular recesses communicating with the ventricular
cavity. Clinical signs are variable, ranging from lack of symptoms to severe
manifestations including heart failure, sustained ventricular arrhythmias,
cardioembolism and sudden death. The diagnosis of LVNC is frequently missed, due
to limited awareness in the medical community. Contemporary diagnostic
sensitivity has been enhanced by the introduction of specific morphologic
criteria by high resolution echocardiography and cardiac magnetic resoce. As
a consequence, LVNC has been diagnosed more frequently in association with other
disorders such as congenital heart disease or genetic CM. The clinical relevance
of regional non-compaction in the context of other cardiac diseases is still
uncertain. Recent evidence points to an overlapping genetic background
encompassing LVNC, hypertrophic and dilated CM, suggesting a continuum of
disease associated with sarcomere protein gene mutations. This concept may prove
relevant to the understanding of common pathogenetic mechanisms of CM and offer
novel research opportunities. AIMS: The clinical features, prognosis, and even definition of left ventricular
non-compaction (LVNC) are still the subject of much debate. The aim of this
registry was to describe the clinical, echocardiographic, and prognostic
features of LVNC in France. The main endpoint was to assess clinical and
echocardiographic predictors of adverse outcome, defined as death or heart
transplantation.
METHODS AND RESULTS: Between 2004 and 2006, 154 suspected cases of LNVC were
identified from a nationwide survey in France. The diagnosis of LVNC was
confirmed in 105 cases by echocardiographic evaluation in a core laboratory.
Clinical and echocardiographic data for the 105 cases of LVNC are presented.
Left ventricular non-compaction was first detected from heart failure symptoms
in 45 patients, rhythm disorders in 12, and familial screening in 8. Left
ventricular ejection fraction (LVEF) was < 30% in 46% of patients, but ≥ 50% in
16%. The latter had less symptoms of severe heart failure (11 vs. 54%, P =
0.001), but similar extension of the NC zone. During 2.33 ± 1.47 years of
follow-up, several complications occurred, including severe heart failure in 33
patients, transplantation in 9, ventricular arrhythmia in 7, embolic events in
9, and death in 12. Factors associated with death or heart transplantation were
NYHA 3 or 4 (HR = 6.69; P = 0.0007), high LV filling pressures (HR = 7.59; P =
0.001), LVEF (HR = 0.93; P = 0.006), and hospitalization for heart failure (HR =
13.55; P < 0.0001).
CONCLUSION: In this large reported series of LVNC, we observed that: (i) Left
ventricular non-compaction was detected by familial screening in asymptomatic
patients in 8% of cases. (ii) Left ventricular non-compaction was frequently
over-diagnosed by echocardiography. (iii) Patients identified as LVNC presented
with a high risk of severe complications, transplantation or death and needed
close follow-up. Left ventricular hypertrophy is a non-specific physiological or maladaptive
cardiac response to a large array of stimuli mediated by exercise and numerous
cardiac and systemic diseases. The precise characterization and quantification
of left ventricular hypertrophy may allow a more timely diagnosis of the
underlying condition. The clinical reference standard to assess left ventricular
hypertrophy is echocardiography, but a comprehensive description of how to
approach this frequent finding in clinical practice is lacking. The current
review systematically describes the typical echocardiographic patterns of
important types of cardiac hypertrophy using both established and advanced
imaging modalities. In hypertrophic obstructive cardiomyopathy a markedly
reduced regional systolic function is found in the prominent thickened septum,
whereas in essential arterial hypertension a typical concentric left ventricular
hypertrophy with a less prominent basal septal bulge is present. The
echocardiographic characteristics of cardiac amyloidosis are ventricular
hypertrophy with sparkling granular myocardial texture and a small epicardial
effusion. In addition, the strain rate curve for longitudinal function shows a
typically reduced function which reaches maximum already in early systole. The
typical feature of Friedreich cardiomyopathy is concentric left ventricular
hypertrophy and sparkling granular texture with preserved regional systolic
function. In Fabry cardiomyopathy a prominent papillary muscle is presented and
a typical strain rate curve can be extracted from the basal lateral wall,
indicating replacement fibrosis. Prominent hypertrabecularisation (ratio of
non-compacted to compacted myocardium >2) in the apical and mid left ventricular
segments is typical for non-compaction cardiomyopathy. Knowledge of these
typical echocardiographic features enables the cardiologist to distinguish
between the different hypertrophic entities, thus paving the way to early
diagnosis. BACKGROUND: Non compaction cardiomyopathy is a rare disorder caused by the
arrest of myocardial compaction during embryogenesis, leading to a non compacted
endocardial layer with marked hypertrabeculation and deep recesses.
AIM: To report the clinical and echocardiographic characteristics of a series of
15 adult patients with non-compaction cardiomyopathy.
PATIENTS AND METHODS: We included a total of 15 patients aged 52 ± 17 years (40%
males) diagnosed at our echocardiography laboratory between January 2001 and
July 2010.
RESULTS: The form of presentation was heart failure in 53% of subjects, syncope
in 20%o, ventricular arrhythmias in 13%o and stroke in 7%>. Left ventricular
end-diastolic diameter was 66 ± 11 mm and estimated ejection fraction was 27 ±
10%>. Apical and/or mid-ventricular segments of the left ventricle were involved
in all the cases. Pulmonary hypertension was present in 40%o. The average
follow-up was 19 months and no patient died during this period. Sixty seven
percent of the patients had manifestations of heart failure, 27%o presented
sustained ventricular arrhythmias and 20%> had atrial fibrillation or flutter,
whereas 13%o had cerebral embolic events. An automated internal cardioverter
defibrillator was implanted in 47%o of patients.
CONCLUSIONS: Non-compaction cardiomyopathy is associated with high
cardiovascular morbidity. The diagnosis is made in advanced stages of the
disease, with significant dilation and ventricular dysfunction. Isolated noncompaction of the left ventricle (LV) is a rare disorder, classified
as a primary genetic cardiomyopathy by the American Heart Association. The
European Society of Cardiology Working Group on Myocardial and Pericardial
Diseases classified LV noncompaction as an unclassified cardiomyopathy. LV
noncompaction cardiomyopathy characterized by the following features: 1) an
altered myocardial wall with prominent trabeculae and deep intertrabecular
recesses resulting in thickened myocardium with two layers, consisting of
compacted and noncompacted myocardium and 2) continuity between the left
ventricular cavity and the deep intertrabecular recesses, which are filled with
blood from the ventricular cavity, without evidence of communication with the
epicardial coronary artery system. Features of LV noncompaction can overlap with
dilated cardiomyopathy, hypertrophic cardiomyopathy (especially the apical
variant), and restrictive cardiomyopathy. The phenotypic expression can vary
considerably within the same family. The LV noncompaction can rarely occur as a
transient phenomenon during myocarditis. We present the case of a 23-year-old
patient, admitted to our Department for cardiac evaluation because of ECG
changes and cardiac enlargement revealed at thoracic radiography. She had a
history of chronic toxoplasmosis. An echocardiography was performed revealing
left ventricular enlargement with severe systolic and diastolic dysfunction,
diffuse hypokinesia and signs of isolated left ventricular non-compaction. Under
these circumstances, we have considered the presence of isolated left
ventricular non-compaction. A cardiac Magnetic Resoce Imaging was performed
and it sustained the diagnosis. The alternative cause of isolated left
ventricular noncompaction (prominent trabeculation due to myocardial
toxoplasmosis) was considered improbable. |
What is STARR-seq? | STARR-seq is a method to directly and quantitatively assess enhancer activity for millions of candidates from arbitrary sources of DNA, which enables screens across entire genomes. When applied to the Drosophila genome, STARR-seq identifies thousands of cell type-specific enhancers across a broad continuum of strengths, links differential gene expression to differences in enhancer activity, and creates a genome-wide quantitative enhancer map. This map reveals the highly complex regulation of transcription, with several independent enhancers for both developmental regulators and ubiquitously expressed genes. STARR-seq can be used to identify and quantify enhancer activity in other eukaryotes, including humans. | Steroid hormones act as important developmental switches, and their nuclear
receptors regulate many genes. However, few hormone-dependent enhancers have
been characterized, and important aspects of their sequence architecture,
cell-type-specific activating and repressing functions, or the regulatory roles
of their chromatin structure have remained unclear. We used STARR-seq, a
recently developed enhancer-screening assay, and ecdysone signaling in two
different Drosophila cell types to derive genome-wide hormone-dependent
enhancer-activity maps. We demonstrate that enhancer activation depends on
cis-regulatory motif combinations that differ between cell types and can predict
cell-type-specific ecdysone targeting. Activated enhancers are often not
accessible prior to induction. Enhancer repression following hormone treatment
seems independent of receptor motifs and receptor binding to the enhancer, as we
show using ChIP-seq, but appears to rely on motifs for other factors, including
Eip74. Our strategy is applicable to study signal-dependent enhancers for
different pathways and across organisms. |
In which genomic positions is the histone variant macroH2A enriched? | macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. The transcribed regions of most active genes are depleted of macroH2A, often in sharply localized domains that show depletion of 4-fold or more relative to bulk mouse liver chromatin. This repressor activity of marcroH2A is further supported by the substantial and relatively uniform macroH2A1 enrichment along the inactive X chromosome, which averages 4-fold. In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome | One of several features acquired by chromatin of the inactive X chromosome (Xi)
is enrichment for the core histone H2A variant macroH2A within a distinct
nuclear structure referred to as a macrochromatin body (MCB). In addition to
localizing to the MCB, macroH2A accumulates at a perinuclear structure centered
at the centrosome. To better understand the association of macroH2A1 with the
centrosome and the formation of an MCB, we investigated the distribution of
macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which
associates with the Xi throughout interphase, the appearance of an MCB is
predomitly a feature of S phase. Although the MCB dissipates during late S
phase and G2 before reforming in late G1, macroH2A1 remains associated during
mitosis with specific regions of the Xi, including at the X inactivation center.
This association yields a distinct macroH2A banding pattern that overlaps with
the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24.
The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of
the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is
likely part of a degradation pathway, a mechanism common to a variety of other
chromatin proteins. Female mammalian cells inactivate transcription from one of their X chromosomes
to equalize gene expression of X-linked genes between males and females.
Inactivation is a multistep process that involves a large non-coding RNA termed
XIST, a variety of epigenetic modifications of chromatin, and alterations in
protein composition such as enrichment of the histone variant macroH2A. We show
here that inactive X chromosomes are also enriched in a well-characterized
protein component of the nuclear scaffold, SAF-A. This protein has been
implicated in chromatin organization, owing to its high specificity for
scaffold-associated region (SAR)-DNA, in transcriptional regulation, e.g. of
hormone-regulated genes, owing to its functional interaction with steroid
receptors, and in RNA processing, owing to its interaction with RNA and
heterogeneous nuclear ribonucleoprotein (hnRNP) particles. After near complete
removal of DNA and associated chromatin proteins such as macroH2A, SAF-A remains
with the "nuclear matrix", still highlighting the former position of inactive X
chromosomes. Interestingly, the enrichment of SAF-A in the inactive X chromosome
depends on the RNA binding domain of the protein, the RGG box, raising the
possibility that interaction of SAF-A with XIST RNA may contribute to the
silencing of X-linked genes by local changes in nuclear architecture. Histone variants replace the core histones in a substantial fraction of
nucleosomes, affecting chromatin structure and impacting chromatin-templated
processes. In many instances incorporation of histone variants results in
formation of specialized regions of chromatin. Proper localization of histone
variants to distinct regions of the genome is critical for their function, yet
how this specific localization is achieved remains unclear. macroH2A1 is
enriched on the inactive X chromosome in female mammalian cells, where it
functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like
histone domain and a large, globular C-terminal macro domain that is not present
in other histone proteins. The histone domain of macroH2A1 is alone sufficient
to direct enrichment on the inactive X chromosome when expressed in female
cells, indicating that sequences important for correct localization lie in this
domain. Here we investigate whether divergent sequences of the H2A variant
macroH2A1 contribute to its correct localization. We mapped the regions of the
macroH2A1 histone domain that are sufficient for localization to the inactive X
chromosome using chimeras between H2A and the histone domain of macroH2A1.
Multiple short sequences dispersed along the macroH2A1 histone domain
individually supported enrichment on the inactive X chromosome when introduced
into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are
buried in the crystal structure of the macroH2A1 containing nucleosome,
suggesting that they may contribute to recognition by macroH2A1/H2B deposition
factors. We studied the enrichment and distribution of the histone variant mH2A1 in the
condensed inactive X (Xi) chromosome. By using highly specific antibodies
against mH2A1 and stable HEK 293 cell lines expressing either green fluorescent
protein (GFP)-mH2A1 or GFP-H2A, we found that the Xi chromosome contains
approximately 1.5-fold more mH2A1 than the autosomes. To determine the in vivo
distribution of mH2A1 along the X chromosome, we used a native chromatin
immunoprecipitation-on-chip technique. DNA isolated from
mH2A1-immunoprecipitated nucleosomes from either male or female mouse liver were
hybridized to tiling microarrays covering 5 kb around most promoters or the
entire X chromosome. The data show that mH2A1 is uniformly distributed across
the entire Xi chromosome. Interestingly, a stronger mH2A1 enrichment along the
pseudoautosomal X chromosome region was observed in both sexes. Our results
indicate a potential role for macroH2A in large-scale chromosome structure and
genome stability. |
Does amiodarone affect thyroid hormone receptors in the myocardium? | Yes | Desethylamiodarone (DEA) is the major metabolite of amiodarone and has similar
electrophysiologic effects with prolongation of the repolarization that is
reversed by thyroid hormone (T3). Some of the electrophysiologic effects are
probably due to antagonism of T3 at the receptor level. Such effects of T3 are
mediated by modulation of gene transcription. The aim of this study was to
investigate whether cycloheximide (Cy), an inhibitor of protein synthesis, and
actinomycin D (ActD), a RNA-synthesis inhibitor, block DEA-induced prolongation
of the repolarization and whether DEA takes part in the autoregulation of the
nuclear thyroid hormone-receptor subtypes (ThR). Corrected monophasic action
potentials (MAPc) and QTc were measured in Langendorff-perfused guinea pig
hearts for 1 h. The hearts were continuously perfused with (a) vehicle, (b) 7.5
microM Cy, (c) 5 microM DEA, (d) 5 microM DEA + 7.5 microM Cy, (e) 1 microM T3,
(f) 5 microM DEA + 1 microM T3, (g) 1.5 microM ActD, and (h) ActD + DEA. A
potassium channel blocker with class III antiarrhythmic effects, 0.5 microM
almokalant, was used as a control, separately and together with Cy. Western blot
analysis for the ThR subtypes alpha, beta1, and beta2 was performed on vehicle-
and DEA-treated hearts. DEA increased MAPc by 19% (p < 0.0005) and QTc by 18% (p
< 0.0005). There was no effect on MAPc or QTc when Cy, ActD, or T3 was added
with DEA. Almokalant increased MAPc by 14% (p < 0.005) and QTc by 13% (p <
0.0005). When Cy was present, almokalant still induced a similar prolongation of
MAPc by 14% (p < 0.005) and QTc by 17% (p < 0.0005). Western blot analysis
revealed no change in the expression of the ThR protein. In conclusion, the
prolongation of the cardiac repolarization by DEA, but not almokalant, can be
totally blocked by Cy and ActD. This indicates that the class III action of DEA
is at least in part dependent on transcription rather than a direct effect on
cell-membrane channels or receptors. The action of DEA could be reversed by T3,
indicating an antagonism between DEA and T3. These results suggest a new
antiarrhythmic mechanism dependent on gene expression. Amiodarone, a powerful antiarrhythmic drug, may exert its effect by antagonism
of the thyroid hormone, probably at the receptor level. The aim of this study
was to investigate whether amiodarone affects the levels of thyroid hormone
receptor (TR) messenger RNA (mRNA) subtypes in mouse hearts. Mice were treated
with 10, 25, and 50 mg/kg body weight (BW) amiodarone or vehicle
(propyleneglycol) intraperitoneally, daily for 14 days. The heart rate
dose-dependently decreased in the 25 mg/kg BW (p < 0.05) and 50 mg/kg BW (p <
0.005) amiodarone-treated mice compared with control. Serum T3 levels were
significantly decreased by 25% (4.2 +/- 0.7 pM) in the 50 mg/kg BW amiodarone
group in comparison to control (5.6 +/- 1.4 pM; p < 0.05). The serum T4 levels
were 1.3 times higher in 50 mg/kg BW amiodarone-treated mice (13.2 +/-1.6 pM)
compared with the control (10.3 +/- 1.3 pM; p < 0.005). Determination of
TRalpha1, alpha2, beta1, and beta2 mRNA in the heart were performed by reverse
transcriptase-polymerase chain reaction (RT-PCR)/enzyme-linked immunosorbent
assay (ELISA). Both in treated and untreated mice, TRalpha2 mRNA had the highest
density in mouse heart, whereas TRbeta2 mRNA had the lowest density. Amiodarone
dose-dependently downregulated the levels of TRalpha1 and beta1 mRNA in
comparison to the control. There were, however, no differences in the TRalpha2
and TRbeta2 mRNA levels in the mice heart treated with different doses of
amiodarone in comparison with the control group. In conclusion, this study shows
that amiodarone subtype selectively downregulates the TR mRNA levels in mouse
myocardium in a dose-dependent manner. These results support a thyroid
hormone-dependent action of amiodarone. |
From which sequence does the Alu repeat originate from? | The presence of Alu-like structural motifs supports the hypothesis of the monophyletic origin of Alu and B1 repeats, i.e., from a common 7SL RNA-derived retroposing monomeric element, The origin of Alu subfamilies in human populations may be related to evolution of chromosome Y. | We have determined sequences of PCR-amplified B1 elements from hamster and rat
(Myomorpha), chipmunk (Sciuromorpha), and guinea pig (Caviomorpha). Between
three and six B1 subfamilies were found in these species. In the phylogenetic
analysis B1 sequences of hamster, mouse, and rat clustered separately from those
of chipmunk and those of guinea pig. This is consistent with an independent
evolution of B1 elements in separate rodent lineages. We exclude the possibility
of convergent mutations to explain certain diagnostic characters within the
modern B1 quasi-dimers and view these elements as mosaic structures assembling
preexisting mutations. Furthermore, the presence of Alu-like structural motifs
supports the hypothesis of the monophyletic origin of Alu and B1 repeats, i.e.,
from a common 7SL RNA-derived retroposing monomeric element. Alu sequences are frequently encountered during study of human genomic nucleic
acid and form a major component of repetitive DNA. This review describes the
origin of Alu sequences and their subsequent amplification and evolution into
distinct subfamilies. In recent years a number of different functional roles for
Alu sequences have been described. The multiple influences of Alu sequences on
RNA polymerase II-mediated gene expression and the presence of Alu sequences in
RNA polymerase III-generated transcripts are discussed. We analyze minisatellites derived from Alu fragments corresponding approximately
to the first 44 bases of human Alu consensus sequences from different
subfamilies. The origin of Alu-derived minisatellites appears to have been
mediated by short flanking repeats, as first proposed by Haber and Louis [Haber,
J.E., Louis, E.J., 1998. Minisatellite origins in yeast and humans. Genomics 48,
132-135.]. We also present evidence for base substitutions and deletions
introduced to minisatellites by gene conversion with partially similar but
unrelated flanking regions. Segments flanked by short direct repeats are
relatively common in different regions of Alu and other repetitive sequences.
Our analysis shows that they can be effectively used in comparative studies of
the overall sequence context which may contribute to instability of DNA segments
flanked by short direct repeats. |
Abnormality in which vertebral region is important in the Bertolotti's syndrome? | Lumbosacral vertebral region is implicated in the Bertolotti's syndrome. Lumbosacral transitional vertebra is an anatomical variation of the fifth lumbar vertebra in which an enlarged transverse process can form a joint or fusion with the sacrum or ilium. Patients often complain of intractable sciatica that arises from impingement of the nerve root extraforaminally by compression caused by the enlarged transverse process. | Bertolotti's syndrome refers to the association of back pain with lumbosacral
transitional vertebrae. Such vertebrae were observed in 140 of 2,000 adults with
back pain over a 4-year period of study. Each patient had radiographic
evaluation of the lumbar spine by plain films as well as a sectional imaging
modality (magnetic resoce [MR] or computed tomography [CT]). The overall
incidence of structural pathology (eg, spinal stenosis and disc protrusion)
detected by CT or MR was not apparently higher in patients with transitional
vertebrae, but the distribution of these lesions was significantly different.
Disc bulge or herniation, when it occurred, was nearly nine times more common at
the interspace immediately above the transitional vertebra than at any other
level. Spinal stenosis and nerve root canal stenosis were more common at or near
the interspace above the transitional vertebra than at any other level.
Degenerative change at the articulation between the transverse process of the
transitional vertebra and the pelvis was an uncommon occurrence; when seen there
was no significant correlation with the reported side of pain. It is postulated
that hypermobility and altered stresses become concentrated in the spine at the
level immediately above a lumbar transitional vertebra. Accelerated disc and
facet joint degeneration at this level may then result. We surgically treated 16 patients with Bertolotti's syndrome (chronic,
persistent low back pain and radiographically diagnosed transitional lumbar
vertebra). Eight had posterolateral fusion and another eight resection of the
transitional articulation. Thirteen patients had in addition to the chronic low
back pain, suffered from repeated episodes or chronic sciatica. In six cases
with resection treatment, local injections were administered at the transitional
articulation before deciding for resection of the transitional joint; each
patient reported transient relief of pain, while this preoperative test did not
correlate with successful outcome of treatment. Six patients had to be treated
with second operations. Ten of the 16 operatively treated patients showed
improvement of the low back pain, and this result was similar in the group
treated with fusion and in that treated with resection. Seven had no low back
pain at follow-up, and the improvement according to the Oswestry pain scale was
similar in the two groups, and statistically significant. Eleven patients still
had persisting episodes of sciatica (versus 13 preoperatively). The average
disability according to the Oswestry total disability scale was 30%,
corresponding with moderate outcome, and both operatively treated groups did
equally well. At follow-up the first disc above the fused segments was found to
be degenerated in seven out of eight cases, and in the group treated with
resection the first disc above the transitional vertebra was degenerated in five
cases.(ABSTRACT TRUNCATED AT 250 WORDS) STUDY DESIGN: Case report of surgically treated mechanical low back pain from
the facet joint contralateral to a unilateral anomalous lumbosacral articulation
(Bertolotti's syndrome).
OBJECTIVES: To describe the clinical presentation, diagnostic evaluation, and
management of facet-related low back pain in a 17-year-old cheerleader and its
successful surgical treatment with resection of a contralateral anomalous
articulation.
SUMMARY OF BACKGROUND DATA: Lumbosacral transitional vertebrae are common in the
general population. Bertolotti's syndrome is mechanical low back pain associated
with these transitional segments. Little is known about the pathophysiology and
mechanics of these vertebral segments and their propensity to be pain
generators. Treatment of this syndrome is controversial, and surgical
intervention has been infrequently reported.
METHOD: A retrospective chart analysis and radiographic review were performed.
RESULTS: Repeated fluoroscopically guided injections implicated a symptomatic
L6-S1 facet joint contralateral to an anomalous lumbosacral articulation.
Eventually, a successful surgical outcome was achieved with resection of the
anomalous articulation.
CONCLUSION: Clinicians should consider the possibility that mechanical low back
pain may occur from a facet contralateral to a unilateral anomalous lumbosacral
articulation, even in a young patient. Although reports of surgical treatment of
Bertolotti's syndrome are infrequent, resection of the anomalous articulation
provided excellent results in this patient, presumably because of reduced
stresses on the symptomatic facet. Bertolotti's syndrome is characterised by anomalous enlargement of the
transverse process(es) of the most caudal lumbar vertebra which may articulate
or fuse with the sacrum or ilium and cause isolated L4/5 disc disease. We
analysed the elective MR scans of the lumbosacral spine of 769 consecutive
patients with low back pain taken between July 2003 and November 2004. Of these
568 showed disc degeneration. Bertolotti's syndrome was present in 35 patients
with a mean age of 32.7 years (15 to 60). This was a younger age than that of
patients with multiple disc degeneration, single-level disease and isolated disc
degeneration at the L4/5 level (p </= 0.05). The overall incidence of
Bertolotti's syndrome in our study was 4.6% (35 of 769). It was present in 11.4%
(20 patients) of the under-30 age group. Our findings suggest that Bertolotti's
syndrome must form part of a list of differential diagnoses in the investigation
of low back pain in young people. STUDY DESIGN: A case report and literature review is presented.
OBJECTIVE: To review relevant data for the management of Bertolotti's syndrome
and to determine whether the transverse process-ilium articulation may be a pain
generator.
BACKGROUND: Bertolotti's syndrome is associated with axial low back pain
secondary to arthritic changes; the pain generator in the disorder is unclear.
METHODS: We present a case report of symptomatic Bertolotti's syndrome managed
with intra-articular steroid injections.
RESULTS: A patient with Bertolotti's syndrome had significant relief of axial
pain after steroid injection of the ilium-transverse process articulation.
CONCLUSIONS: Steroid therapy may be a non-surgical alternative for the treatment
of symptomatic Bertolotti's syndrome. OBJECTIVE: Bertolotti's syndrome is a spine disorder characterized by the
occurrence of a congenital lumbar transverse mega-apophysis in a transitional
vertebral body that usually articulates with the sacrum or the iliac bone. It
has been considered a possible cause of low back pain.
METHOD: We analyzed the cases of Bertolotti's syndrome that failed clinical
treatment and reviewed the literature concerning this subject.
RESULTS: Five patients in our series had severe low back pain due to the
neo-articulation and two of them were successfully submitted to surgical
resection of the transverse mega-apophysis. Taking into account the clinical and
surgical experience acquired with these cases, we propose a
diagnostic-therapeutic algorithm.
CONCLUSION: There is still no consensus about the most appropriate therapy for
Bertolotti's syndrome. In patients in whom the mega-apophysis itself may be the
source of back pain, surgical resection may be a safe and effective procedure. INTRODUCTION: Lumbosacral transitional vertebra is an anatomical variation of
the fifth lumbar vertebra in which an enlarged transverse process can form a
joint or fusion with the sacrum or ilium. The association of that variant with
low back pain and the change in the biomechanical properties of the lumbar spine
is called Bertolotti's syndrome.
CASE PRESENTATION: We report a case of a 40-year-old male patient with chronic
low back pain extending to the left buttock, just above the ipsilateral
sacroiliac joint. Radiographic investigation revealed an anomalous enlargement
of the left transverse process of the fifth lumbar vertebra forming a
pseudarthrosis with the infrajacent ala of the sacrum.
CONCLUSION: In young patients with back pain the possibility of Bertolotti's
syndrome should always be taken in account. OBJECTIVE: Describe the clinical presentation, diagnostic evaluation, and
successful treatment of a case of symptomatic unilateral lumbosacral junction
pseudarticulation using a novel radiofrequency nerve ablation technique.
CASE: A 56-year-old female patient who had suffered with low back and right
upper buttock pain for 16 years experienced incomplete relief with L4/5 facet
joint radiofrequency ablation. She was found to have an elongated right L5
transverse process that articulated with the sacral ala (Bertolotti's syndrome).
Fluoroscopically guided local anesthetic/corticosteroid injection into the
pseudarthrosis eliminated her residual right buttock pain for the duration of
the local anesthetic only. Complete pain relief was achieved by injecting local
anesthetic circumferentially around the posterior pseudarthrosis articular
margin. Accordingly, bipolar radiofrequency strip thermal lesions were created
at the same locations. Complete pain relief and full restoration of function was
achieved for 16 months postprocedure.
CONCLUSION: This case report describes a novel radiofrequency technique for
treating symptomatic lumbosacral junction pseudarticulation that warrants
further evaluation. Patients with Bertolotti's syndrome have characteristic lumbosacral anomalies
and often have severe sciatica. We describe a patient with this syndrome in whom
standard decompression of the affected nerve root failed, but endoscopic
lumbosacral extraforaminal decompression relieved the symptoms. We suggest that
the intractable sciatica in this syndrome could arise from impingement of the
nerve root extraforaminally by compression caused by the enlarged transverse
process. BACKGROUND AND INTRODUCTION: In 1991, a deceased human male was found frozen in
a glacier pool in the Italian Alps in north west Italy, and is now carefully
preserved in the South Tyrol Museum of Archaeology, in Bolzano, Italy. The
bodily tissues of the 5,300 year old male (colloquially referred to as the
Iceman or Ötzi) were well preserved despite damage related to freezing, and
glacial movement. Associated articles of well-preserved clothing, tools, weapons
and other devices were also present and have been studied in detail. Clinical
examination and imaging investigations have also shown that the Icemen had
experienced possible illnesses in his lifetime and had identifiable areas of
arthritis and musculoskeletal injury. This report includes some key observations
on the musculoskeletal state of Ötzi and reference to the involvement of tattoo
markings. Some aspects about the aetiology of his abnormalities and inflammatory
arthritis are considered along with possible treatments that he might have
employed.
METHODS AND RESULTS: We (WFK and MK) undertook a clinical musculoskeletal
examination of the Iceman, details of which with available photographs and
radiographic imaging pertaining to the musculoskeletal findings of the Iceman
are reported here. The skin of the Iceman has numerous linear carbon tattoos,
which are not of a decorative type. These have been presumed to possibly be
"medicinal" tattoos administered for therapeutic reasons and may have been used
in acupuncture-like treatment of pain. Spinal imaging identified areas of spinal
damage and our observations have provided clues as to possible sites of spinal
initiated pain and hence sites for administration of the "medicinal" tattoos. We
observed body areas of the Iceman, in which imaging demonstrated arthritis and
other forms of long-term musculoskeletal damage, but which do not have adjacent
or corresponding "medicinal" tattoos. We contend that the back and leg
"medicinal" tattoos correspond directly to sites of chronic right knee and right
ankle pain, and left thoracolumbar pain. They also correspond to lower lumbar
and sciatic referred radicular pain which may have a contributory cause related
to the presence of a transitional lumbar 5 vertebra. Using recent published data
(Keller et al. in Nature Commun 3:698, 2012. doi: 10.1038/ncomms1701 ) of the
genome structure of the Iceman, we suggest some potential causes of the
osteoarthritis or inflammatory joint injury may relate to presence of coronary
heart disease (CHD) and Lyme disease (Borrelia burgdorferi) infection. We
speculate on possible medical applications of natural products for
self-medication.
CONCLUSIONS: These observations highlight several diagnostic features of
musculoskeletal conditions in the Iceman with the possibility that tattoos may
have been used for diagnosis or location of his painful states. The origins of
his musculoskeletal conditions are unclear but there are indications that Lyme
disease and CHD may have been factors. The associations or use of natural
products may give insights into their applications at the time of the life of
the Iceman. BACKGROUND: Bertolotti's syndrome (BS), a form of lumbago in lumbosacral
transitional vertebrae, is an important cause of low back pain in young
patients. The purpose of this study was to assess the etiology of low back pain
and the efficacy of treatment offered to patients with BS.
METHODS: All patients of BS Castellvi type1a during a period of 6 months were
enrolled in the study. The patients underwent interventional pain procedures for
diagnosis and pain relief. Response to the therapy was assessed based on VAS and
ODI scores. A 50% decrease in VAS score or a VAS score less than 3 would be
considered adequate pain relief.
RESULTS: All 20 patients diagnosed with BS during the 6-month observation period
had scoliosis. Common causes of back pain were the ipsilateral L5-S1 facet
joint, neoarticulation, the SI joint, and disc degeneration. Responses to
various interventions for pain relief were different and inconsistent from
patient to patient. In particular, responses to interventions for neoarticular
pain were generally poor.
CONCLUSIONS: Pain in patients with BS does not usually respond to interventional
pain treatment. A very dynamic treatment approach must be pursued while managing
BS patients, and the treatment plan must be individualized at various stages in
order to obtain satisfactory pain relief. |
What does iBAQ stand for in proteomic analysis? | iBAQ stands for intensity-based absolute quantification. | There is a great interest in reliable ways to obtain absolute protein abundances
at a proteome-wide scale. To this end, label-free LC-MS/MS quantification
methods have been proposed where all identified proteins are assigned an
estimated abundance. Several variants of this quantification approach have been
presented, based on either the number of spectral counts per protein or MS1 peak
intensities. Equipped with several datasets representing real biological
environments, containing a high number of accurately quantified reference
proteins, we evaluate five popular low-cost and easily implemented
quantification methods (Absolute Protein Expression, Exponentially Modified
Protein Abundance Index, Intensity-Based Absolute Quantification Index, Top3,
and MeanInt). Our results demonstrate considerably improved abundance estimates
upon implementing accurately quantified reference proteins; that is, using
spiked in stable isotope labeled standard peptides or a standard protein mix, to
generate a properly calibrated quantification model. We show that only the Top3
method is directly proportional to protein abundance over the full
quantification range and is the preferred method in the absence of reference
protein measurements. Additionally, we demonstrate that spectral count based
quantification methods are associated with higher errors than MS1 peak intensity
based methods. Furthermore, we investigate the impact of miscleaved, modified,
and shared peptides as well as protein size and the number of employed reference
proteins on quantification accuracy. |
Which is the main target of the anti-arrhythmic activity of flecainide? | Flecainide is a class 1c antiarrhythmic that acts by blocking sodium channels and is used mainly in the treatment of supraventricular arrhythmias. | AIMS: Ca(2+) waves are thought to be important in the aetiology of ventricular
tachyarrhythmias. There have been conflicting results regarding whether
flecainide reduces Ca(2+) waves in isolated cardiomyocytes. We sought to confirm
whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate
the mechanism.
METHODS AND RESULTS: We imaged spontaneous sarcoplasmic reticulum (SR) Ca(2+)
release events in healthy adult rat cardiomyocytes. Variation in stimulation
frequency was used to produce Ca(2+) sparks or waves. Spark frequency, wave
frequency, and wave velocity were reduced by flecainide in the absence of a
reduction of SR Ca(2+) content. Inhibition of I(Na) via alternative
pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced
similar changes. To assess the contribution of I(Na) to spark and wave
production, voltage clamping was used to activate contraction from holding
potentials of -80 or -40 mV. This confirmed that reducing Na(+) influx during
myocyte stimulation is sufficient to reduce waves and that flecainide only
causes Ca(2+) wave reduction when I(Na) is active. It was found that
Na(+)/Ca(2+)-exchanger (NCX)-mediated Ca(2+) efflux was significantly enhanced
by flecainide and that the effects of flecainide on wave frequency could be
reversed by reducing [Na(+)](o), suggesting an important downstream role for NCX
function.
CONCLUSION: Flecainide reduces spark and wave frequency in the intact rat
cardiomyocyte at therapeutically relevant concentrations but the mechanism
involves I(Na) reduction rather than direct ryanodine receptor (RyR2)
inhibition. Reduced I(Na) results in increased Ca(2+) efflux via NCX across the
sarcolemma, reducing Ca(2+) concentration in the vicinity of the RyR2. Flecainide is recommended as a first-line antiarrhythmic drug to maintain normal
sinus rhythm in patients with atrial fibrillation (AF) who have structurally
normal hearts or hypertension without left ventricular hypertrophy. Flecainide
is a sodium channel blocker with minimal effects expected on ventricular
repolarization. We describe the case of a 32-year-old man with a structurally
normal heart and persistent AF who was started on diltiazem and flecainide 50 mg
twice/day approximately a year prior to presentation. Due to persistent and
bothersome symptoms, his dose was increased to 150 mg twice/day, which was
associated with a progressive lengthening of his corrected QT interval. On the
day of presentation, he underwent an exercise test as part of his job
requirements. While running, he felt lightheaded and experienced a syncopal
event and cardiac arrest. An automated external defibrillator was available that
displayed polymorphic ventricular tachycardia. The patient was successfully
resuscitated. Although rare, this case suggests that flecainide can induce QT
prolongation leading to torsades de pointes. Clinicians should be aware and
consider periodic evaluations with electrocardiograms. Mechanical stretch has been shown to provoke arrhythmia. We wanted to analyze
ventricular arrhythmia induced by local left ventricular stretch in order to
find out, where arrhythmias originate and whether they can be prevented
pharmacologically. Isolated rabbit hearts (Langendorff technique) were submitted
to increased left ventricular stretch at the left wall by insertion of an
additional intraventricular balloon and adjusting the end-diastolic pressure
(EDP) to 25 mmHg for 10 min followed by 20 min recovery at normal EDP of 5-8
mmHg. Activation and repolarization processes were investigated by ventricular
256 electrode epicardial mapping. The hearts were treated during the whole
procedure either with vehicle, 0.5 μM flecainide (sodium channel blocker) or 100
μM streptomycin (here used as stretch-activated ion-channel blocker). In
addition, we performed a series of experiments, in which we enhanced EDP to 30
mmHg (global stretch instead of local stretch) by inflating the left ventricular
pressure balloon (strain, 0.148 ± 0.034). Each series was performed with n = 6.
Stretch resulted in local strain of 25% at the left wall together with a local
slowing of the activation process at the left wall, in a change in the
activation pattern, and in ventricular arrhythmia. Coronary flow was not
affected. Ventricular arrhythmias originated from the border between the
stretched area and the non-stretched region. Flecainide and streptomycin reduced
the prolongation of the activation process at the stretched left wall and
mitigated the difference in total activation time between left and front wall
but only partially prevented arrhythmia. In the additional global stretch
experiments relative coronary flow and the other parameters remained unchanged,
in particular TAT. Thus, in contrast to the local stretch series, there was no
difference in the change in TAT between left and front wall. Only rare single
ventricular extrasystoles (<1/min; originating from LV (front and left wall)
i.e. from within the stretched region) were seen during stretch (but not at the
beginning) and during recovery. Local left ventricular stretch can elicit
ventricular arrhythmias. Local slowing of electrical activation seems involved
so that the difference in total activation time of the stretched free left wall
and the non-stretched increased. It remains unclear whether flecainide, a Class I antiarrhythmic drug, improves
left ventricular pressure gradient (LVPG) or symptoms in patients with
obstructive hypertrophic cardiomyopathy (HCM). Our study evaluated the long-term
efficacy of flecainide, compared to disopyramide, when administered orally, on
LVPG and symptoms in obstructive HCM patients. Among 164 obstructive HCM
patients, 15 were administered oral flecainide therapy and 33 administered oral
disopyramide therapy. LVPG declined from 79.8 ± 36.6 to 39.2 ± 36.7 mmHg
(p = 0.003) after flecainide therapy and from 74.5 ± 26.4 to 31.4 ± 24.8 mmHg
(p < 0.001) after disopyramide therapy. The percent reduction in LVPG was
-47.9 ± 43.2 % in patients treated with flecainide, comparable to the results
for those treated with disopyramide (-57.1 ± 33.0 %; p = 0.425). We found no
significant differences in improvement in NYHA functional class between patients
treated with flecainide and those treated with disopyramide (p = 0.331).
Patients treated with flecainide exhibited no significant adverse side effects,
and there was no need for myectomy or alcohol septal ablation to reduce LVPG and
symptoms. Improvements in LVPG and symptoms were similar in patients treated
with flecainide and patients treated with disopyramide, suggesting that
flecainide is a potentially useful alternative for symptomatic obstructive HCM
patients, particularly those with disopyramide-induced vagolytic side effects,
narrow angle glaucoma, or prostatic hyperplasia and pre-existing urination
difficulties. Our data must be viewed with caution, however, in view of the
small number of study patients. Flecainide therapy will require further proof of
safety before it can be routinely recommended in patients with symptomatic
obstructive HCM. |
Does thyroid hormone affect cardiac remodeling? | TH affects cardiac remodeling | The present study investigated whether changes in thyroid hormone (TH)
signalling can occur after acute myocardial infarction (AMI) with possible
physiological consequences on myocardial performance. TH may regulate several
genes encoding important structural and regulatory proteins particularly through
the TR alpha 1 receptor which is predomit in the myocardium. AMI was induced
in rats by ligating the left coronary artery while sham-operated animals served
as controls. This resulted in impaired cardiac function in AMI animals after 2
and 13 weeks accompanied by a shift in myosin isoforms expression towards a
fetal phenotype in the non-infarcted area. Cardiac hypertrophy was evident in
AMI hearts after 13 weeks but not at 2 weeks. This response was associated with
a differential pattern of TH changes at 2 and 13 weeks; T(3) and T(4) levels in
plasma were not changed at 2 weeks but T(3) was significantly lower and T(4)
remained unchanged at 13 weeks. A twofold increase in TR alpha 1 expression was
observed after 13 weeks in the non-infarcted area, P<0.05 versus sham operated,
while TR alpha 1 expression remained unchanged at 2 weeks. A 2.2-fold decrease
in TR beta 1 expression was found in the non-infarcted area at 13 weeks, P<0.05,
while no change in TR beta 1 expression was seen at 2 weeks. Parallel studies
with neonatal cardiomyocytes showed that phenylephrine (PE) administration
resulted in 4.5-fold increase in the expression of TR alpha 1 and 1.6-fold
decrease in TR beta 1 expression versus untreated, P<0.05. In conclusion,
cardiac dysfunction which occurs at late stages after AMI is associated with
increased expression of TR alpha 1 receptor and lower circulating
tri-iodothyronine levels. Thus, apo-TR alpha 1 receptor state may prevail
contributing to cardiac fetal phenotype. Furthermore, down-regulation of TR beta
1 also contributes to fetal phenotypic changes. alpha1-adrenergic signalling is,
at least in part, involved in this response. Thyroid hormone (TH) is critical for tissue differentiation at early stages of
development, induces physiological hypertrophy and regulates the expression of
important contractile proteins such as myosin heavy chain (MHC) isoform and
calcium cycling proteins. Furthermore, TH seems to control the response to
stress by regulating important cardioprotective molecules such as heat shock
proteins (HSPs). Thus, the present study investigated whether TH administration
immediately after acute myocardial infarction can favourably remodel the
post-infarcted myocardium. Acute myocardial infarction was induced in rats by
coronary artery ligation (AMI, n=10), while SHAM-operated animals served as
controls (SHAM, n = 8). TH was administered for 13 weeks (AMI-THYR, n = 9).
Cardiac contractile function and left ventricular (LV) chamber remodelling was
assessed by serial echocardiography and in Langendorff heart preparations. AMI
significantly reduced LV ejection fraction (EF%); 30.0 (s.e.m, 2.3) Vs. 73.8
(1.8) in SHAM, P < 0.05. In addition, +dp/dt and -dp/dt (in mmHg/s) were 4,051
(343) and 2,333 (118) respectively for SHAM Vs. 2,102 (290) and 1,368 (181) for
AMI, P < 0.05. With TH treatment, EF% was increased to 49.5 (2.7) in AMI-THYR, P
< 0.05, while +dp/dt and -dp/dt (in mmHg/s) were 3,708 (231) and 2,035 (95) for
AMI-THYR, P < 0.05 Vs. AMI. A marked elevation of the expression of beta-MHC and
a reduced ratio of SERCA/Phospholamban were found in viable myocardium of AMI
hearts, which was prevented by TH. Furthermore, heat shock protein 70 myocardial
content was decreased in AMI hearts and was significantly increased after TH
treatment. An ellipsoidal reshaping of LV chamber was observed with TH; cardiac
sphericity index, (ratio of long/short axis, SI), was 1.98 (0.03) for SHAM, 1.52
(0.05) for AMI and 1.72(0.02) for AMI-THYR, P < 0.05. In conclusion, long-term
TH administration immediately after AMI results in sustained improvement of
cardiac haemodynamics. Thyroid hormone has various effects on the cardiovascular system and its effects
on cardiac contractility, heart rhythm and vascular function has long been
recognized. However, new evidence is emerged on the importance of thyroid
hormone in the response of the myocardium to ischaemic stress and cardiac
remodelling following myocardial infarction. Based on this new information, this
review highlights the role of thyroid hormone in myocardial ischaemia and
cardiac remodelling, the possible underlying mechanisms and the potential
therapeutic implications. Thyroid hormone or analogs may prove new therapeutic
agents for treating ischaemic heart disease. Thyroid hormone receptor alpha1 (TRalpha1) is predomitly expressed in the
myocardium but its biological function under physiological or pathological
conditions remains largely unknown. The present study investigated possible
interactions between alpha1 adrenergic and thyroid hormone signaling at the
level of TRalpha1, potential underlying mechanisms and physiological
consequences, as well as the role of TRalpha1 in cell differentiation. This may
be of physiological relevance since both thyroid hormone and adrenergic
signalling are implicated in the pathophysiology of cardiac remodelling.
Neonatal cardiomyocytes obtained from newborn rats (2-3 days) were exposed to
phenylephrine (PE, an alpha1 adrenergic agonist) for 5 days, in the absence or
excess of T3 in the culture medium. PE, in the absence of T3, resulted in 5.0
fold increase in TRalpha1 expression in nucleus and 2.0 fold decrease in
TRalpha1 expression in cytosol, P<0.05. As a result, a fetal pattern of myosin
isoform expression with marked expression of beta-MHC was observed in PE treated
vs the untreated cells, P<0.05. PD98059 (an ERK signalling inhibitor) abrogated
this response. In the presence of T3 in the culture medium, TRalpha1 expression
was increased 1.6 fold in nucleus and 2.0 fold in cytosol in PE-T3 vs PE treated
cells, P<0.05, and the fetal pattern of myosin isoform expression was prevented.
Parallel studies with H9c2 myoblasts showed that reduction of T3 binding to
TRalpha1 receptor delayed cardiac myoblasts differentiation without affecting
proliferation. In conclusion, in neonatal cardiomyocytes, nuclear TRalpha1 is
overexpressed after prolonged activation of the alpha1- adrenergic signalling by
PE. This response seems to be an ERK kinase dependent process. Over-expression
of TRalpha1 may lead to fetal cardiac phenotype in the absence of thyroid
hormone availability. Furthermore, TRalpha1 seems to be critical in cardiac
myoblast differentiation. Viable myocardium undergoes several changes in the course of cardiac remodeling
following myocardial infarction aiming to adapt the heart to the hemodynamic
compromise. This response is characterized by reactivation of the fetal
transcriptional program and results in cardiac dysfunction. Changes in thyroid
hormone (TH)-TH receptors (TRs) axis occur in the course of post-infarction
cardiac remodeling and seem to contribute to cardiac fetal phenotype. TH can
"rebuild" the post-infarcted heart by preventing the fetal-like pattern of
contractile proteins expression, normalizing wall tension, and optimizing
cardiac chamber geometry. This effect seems to be attributed to TH pleiotropic
cellular actions; TH promotes tissue growth and differentiation and favorably
remodels cardiac cell while increases cellular survival upon stress. TH may
constitute a new therapeutic option for mending the ischemic myocardium. Thyroid hormone (TH) is critical in cardiac cell differentiation (regulating
contractile proteins and cell geometry) and this effect could be potentially
exploited therapeutically in reversing the process of de-differentiation which
underlies postischemic cardiac remodeling. Acute myocardial infarction was
induced in male Wistar rats by ligating left coronary artery (AMI, n=8), while
sham operated animals served as control (SHAM, n=8). 13 weeks after AMI, TH was
administered in a group of animals for 4 weeks (AMI-THYR, n=9). TH significantly
increased beta-MHC and decreased alpha-MHC expression in the myocardium. This
response was accompanied by changes in cardiac geometry: sphericity index, (SI,
long to short axis ratio) was found to be 1.95 (SEM, 0.02) in SHAM, 1.51(0.03)
in AMI and 1.64(0.03) in AMI-THYR, p<0.05. As a consequence, cardiac function
was significantly improved: left ventricular ejection fraction (EF%) was 74.5%
(SEM, 2.8) in SHAM vs 29.5% (2.1) in AMI, and 40.0% in AMI-THYR, p<0.05.
Furthermore, +dp/dt and -dp/dt were 4250 (127) and 2278 (55) in SHAM vs
2737(233) and 1508 (95) in AMI vs 3866 (310) and 2137(111) in AMI -THYR,
respectively, p<0.05. TH treatment partially reverses cardiac dysfunction in
rats with old myocardial infarction by favorably changing cardiac chamber
geometry and expression of myosin isoforms. Thyroid hormone, unlike current
treatments, appears to be a paradigm of therapeutic intervention which aims at
restoring cardiac geometry and may prove new effective treatment for heart
failure. Thyroid hormone (TH), apart from its "classical" actions on cardiac
contractility and heart rhythm, appears to regulate various intracellular
signalling pathways related to response to stress and cardiac remodelling. There
is now accumulating experimental and clinical evidence showing a beneficial
effect of TH on limiting myocardial ischaemic injury, preventing/reversing post
infarction cardiac remodelling and improving cardiac hemodynamics. Thyroid
analogs have already been developed and may allow TH use in clinical practice.
However, the efficacy of TH in the treatment of cardiac diseases is now awaiting
to be tested in large clinical trials. Chronic ischemia or pressure overload decreases thyroid hormone (TH) signaling
and activates the fetal gene program in the heart. While these features are of
physiologic importance in the developing heart, their respective roles in the
postnatal heart are debated. Administration of TH can prevent the changes of the
fetal gene program and rebuild the heart after an "index event" such as
ischemia. TH affects cardiac remodeling by limiting reperfusion injury, and, at
later states, by inducing distinct changes in cardiac chamber geometry in a
time-dependent manner. Furthermore, administration of TH can convert pathologic
to physiologic hypertrophy. These effects are the result of favorable cellular
remodeling. While preliminary clinical studies provide encouraging results, the
potential and efficacy of TH in the treatment of heart disease still await
evaluation in large clinical trials. It has been previously shown that regulators of physiological growth such as
thyroid hormone (TH) can favorably remodel the post ischaemic myocardium. Here,
we further explored whether this effect can be preserved in the presence of
co-morbidities such as diabetes which accelerates cardiac remodeling and
increases mortality after myocardial infarction. Acute myocardial infarction
(AMI) was induced by left coronary ligation in rats with type I diabetes (DM)
induced by streptozotocin administration (STZ; 35 mg/kg; i.p.) while
sham-operated animals served as controls (SHAM). AMI resulted in distinct
changes in cardiac function and geometry; EF% was significantly decreased in
DM-AMI [37.9 ± 2.0 vs. 74.5 ± 2.1 in DM-SHAM]. Systolic and diastolic chamber
dimensions were increased without concomitant increase in wall thickness and
thus, wall tension index [WTI, the ratio of (Left Ventricular Internal Diameter
at diastole)/2*(Posterior Wall thickness)], an index of wall stress, was found
to be significantly increased in DM-AMI; 2.27 ± 0.08 versus 1.70 ± 0.05.
2D-Strain echocardiographic analysis showed reduced systolic radial strain in
all segments, indicating increased loss of cardiac myocytes in the infarct
related area and less compensatory hypertrophy in the viable segments. This
response was accompanied by a marked decrease in the expression of TRα1 and TRβ1
receptors in the diabetic myocardium without changes in circulating T3 and T4.
Accordingly, the expression of TH target genes related to cardiac contractility
was altered; β-MHC and PKCα were significantly increased. TH (L-T4 and L-T3)
administration prevented these changes and resulted in increased EF%, normal
wall stress and increased systolic radial strain in all myocardial segments.
Acute myocardial infarction in diabetic rats results in TH receptor
down-regulation with important physiological consequences. TH treatment prevents
this response and improves cardiac hemodynamics. Pressure overload-induced cardiac hypertrophy results in a pathological type of
hypertrophy with activation of signaling cascades like the extracellular
signal-regulated kinase (ERK) pathway, which promotes negative cardiac
remodeling and decreased contractile function. In contrast, thyroid hormone
mediates a physiological type of hypertrophy resulting in enhanced contractile
function. In addition, thyroid hormone action is diminished in pressure
overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone
status modulates ERK activity and that administration of thyroid hormone could
alter the activity of this kinase in cardiac hypertrophy induced by pressure
overload. ERK is activated by phosphorylation; accordingly, we investigated
phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice.
In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied
hearts from transverse aortic-constricted (TAC) mice was investigated. Results
showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid
mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice
presented a greater than fourfold increase of p-ERK compared with control mice.
Interestingly, T3 administration dramatically canceled TAC-induced ERK
phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK
pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3
treatment inhibited this effect of pressure overload and further decreased
p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid
hormone receptor-α in cultured cardiomyocytes potentiated the inhibitory effect
of T3 on ERK phosphorylation. We concluded that thyroid hormone has an
inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice
with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent
mechanism. Similarities in cardiac gene expression in hypothyroidism and left ventricular
(LV) pathological remodeling after myocardial infarction (MI) suggest a role for
impaired cardiac thyroid hormone (TH) signaling in the development of heart
failure. Increased ventricular activity of the TH-degrading enzyme type 3
deiodinase (D3) is recognized as a potential cause. In the present study, we
investigated the cardiac expression and activity of D3 over an 8-wk period after
MI in C57Bl/6J mice. Pathological remodeling of the noninfarcted part of the LV
was evident from cardiomyocyte hypertrophy, interstitial fibrosis, and
impairment of contractility. These changes were maximal and stable from the
first week onward, as was the degree of LV dilation. A strong induction of D3
activity was found, which was similarly stable for the period examined. Plasma
T(4) levels were transiently decreased at 1 wk after MI, but T(3) levels
remained normal. The high D3 activity was associated with increased D3 mRNA
expression at 1 but not at 4 and 8 wk after MI. Immunohistochemistry localized
D3 protein to cardiomyocytes. In vivo measurement of TH-dependent transcription
activity in cardiomyocytes using a luciferase reporter assay indicated a 48%
decrease in post-MI mice relative to sham-operated animals, and this was
associated with a 50% decrease in LV tissue T(3) concentration. In conclusion,
pathological ventricular remodeling after MI in the mouse leads to high and
stable induction of D3 activity in cardiomyocytes and a local hypothyroid
condition. In the post-reperfusion era, molecular and genetic mechanisms of
cardioprotection and regeneration represent new therapeutic challenges to limit
infarct size and minimize post-ischemic remodeling after acute myocardial
infarction (AMI). Activation of cell survival mechanisms can be promoted by the
administration of external drugs, stimulation of internal mechanisms, and
genetic manipulation to delete or replace pathological genes or enhance gene
expression. Among internal cardiovascular regulatory mechanisms, thyroid
hormones (THs) may play a fundamental role. TH has a critical role in
cardiovascular development and homeostasis in both physiological and
pathological conditions. In experimental AMI, TH has been shown to affect
cardiac contractility, left ventricular (LV) function, and remodeling. Several
experimental studies have clearly shown that THs participate in the regulation
of molecular mechanisms of angiogenesis, cardioprotection, cardiac metabolism,
and ultimately myocyte regeneration, changes that can reverse left ventricular
remodeling by favorably improving myocyte shape and geometry of LV cavity, thus
improving systolic and diastolic performance. This review is focused on the role
of thyroid on AMI evolution and on the potential novel option of thyroid-related
treatment of AMI. Type 1 diabetes (TOD) increases the risk of coronary artery disease and
myocardial infarction and is characterized by baseline cardiac dysfunction. We
investigated the influence of TOD in post-infarct remodeling (REM) and the role
of thyroid hormone (TH) signaling in this response. Acute myocardial infarction
(AMI) was induced in rats with type I diabetes (TOD) and in non diabetic rats
(NTOD-AMI), sham-operated rats serving as controls (SHAM). AMI resulted in
tissue hypothyroidism due to significant downregulation of the TH receptors TRa1
and TRbeta1 in the TOD myocardium, while no change in plasma T3 or T4 was
observed This response was associated with increased expression of beta-MHC and
distinct changes in cardiac function and geometry: EF % was decreased in TOD-AMI
as compared to NTOD-AMI. Systolic and diastolic chamber dimensions were
increased, with no concomitant increase in wall thickness. Thus, WTI (the ratio
of LVIDd/2 x posterior wall thickness), an index of wall stress, was
significantly increased in TOD-AMI. The absence of wall thickening in TOD-AMI
hearts was associated with changes in stretch-induced kinase hypertrophic
signaling: phosporylated (p) ERK and p-p38 MAPK levels were not changed in
TOD-AMI in comparison with non infarcted hearts (TOD-SHAM) and NTOD-A MI hearts.
TH administration after AMI prevented tissue hypothyroidism and resulted in
decreased beta-MHC expression, increased wall thickening and normalized
wallstress, while stretch-induced p38 MAPK activation was increased. We conclude
that diabetes exacerbates post-ischemic cardiac remodeling and that tissue
hypothyroidism may be involved in this response. Thyroid hormone receptor α1 (TRα1) is shown to be critical for the maturation of
cardiomyocytes and for the cellular response to stress. TRα1 is altered during
post ischemic cardiac remodeling but the physiological significance of this
response is not fully understood. Thus, the present study explored the potential
consequences of selective pharmacological inhibition of TRα1 on the mechanical
performance of the post-infarcted heart. Acute myocardial infarction was induced
in mice (AMI), while sham operated animals served as controls (SHAM). A group of
mice was treated with debutyl-dronedarone (DBD), a selective TRα1 inhibitor
(AMI-DBD). AMI resulted in low T3 levels in plasma and in down-regulation of
TRα1 and TRβ1 expression. Left ventricular ejection fraction (LVEF%) was
significantly reduced in AMI [33 (SEM 2.1) vs 79(2.5) in SHAM, p < 0.05] and was
further declined in AMI-DBD [22(1.1) vs 33(2.1), respectively, p < 0.05].
Cardiac mass was increased in AMI but not in AMI-DBD hearts, resulting in
significant increase in wall tension index. This increase in wall stress was
accompanied by marked activation of p38 MAPK, a kinase that is sensitive to
mechanical stretch and exerts negative inotropic effect. Furthermore, AMI
resulted in β-myosin heavy chain overexpression and reduction in the ratio of
SR(Ca)ATPase to phospholamban (PLB). The latter further declined in AMI-DBD
mainly due to increased expression of PLB. AMI induces downregulation of thyroid
hormone signaling and pharmacological inhibition of TRα1 further depresses
post-ischemic cardiac function. p38 MAPK and PLB may, at least in part, be
involved in this response. |
What is the effect of ROS on cyclin B1? | Reactive oxygen species (ROS) production is able to cause growth arrest at the G2-M checkpoint of the cell cycle, partly by deregulation of Cyclin B1 expression. | This study is the first to investigate the anticancer effect of plumbagin in
human melanoma A375.S2 cells. Plumbagin exhibited effective cell growth
inhibition by inducing cancer cells to undergo S-G2/M phase arrest and
apoptosis. Further investigation revealed that plumbagin's inhibition of cell
growth was also evident in a nude mice model. Blockade of cell cycle was
associated with increased levels of p21, and reduced amounts of cyclin B1,
cyclin A, Cdc2, and Cdc25C. Plumbagin also enhanced the levels of inactivated
phosphorylated Cdc2 and Cdc25C. Plumbagin triggered the mitochondrial apoptotic
pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9
activation. We also found the generation of ROS is a critical mediator in
plumbagin-induced cell growth inhibition. Plumbagin increased the activation of
apoptosis signal-regulating kinase 1, JNK and extracellular signal-regulated
kinase 1/2 (ERK1/2), but not p38. In addition, antioxidants vitamin C and
catalase significantly decreased plumbagin-mediated c-Jun N-terminal kinase
(JNK) activation and apoptosis. Moreover, blocking ERK and JNK by specific
inhibitors suppressed plumbagin-triggered mitochondrial apoptotic pathway. Taken
together, these results imply a critical role for ROS and JNK in the plumbagin's
anticancer activity. Emodin was isolated from Rheum palmatum L. and exhibits an anticancer effect on
human cancer cell lines, however, the molecular mechanisms of emodin-mediated
apoptosis in human tongue cancer cells have not been fully investigated. In this
study, treatment of human tongue cancer SCC-4 cells with various concentrations
of emodin led to G2/M arrest through promoted p21 and Chk2 expression but
inhibited cyclin B1 and cdc2; it also induced apoptosis through the pronounced
release of cytochrome c from mitochondria and activations of caspase-9 and
caspase-3. These events were accompanied by the generation of reactive oxygen
species (ROS), disruption of mitochondrial membrane potential (delta psi(m)) and
a decrease in the ratio of mitochondrial Bcl-2 and Bax content; emodin also
promoted the levels of GADD153 and GRP78. The free radical scavenger
N-acetylcysteine and caspase inhibitors markedly blocked emodin-induced
apoptosis. Taken together, these findings suggest that emodin mediated oxidative
injury (DNA damage) based on ROS production and ER stress based on the levels of
GADD153 and GRP78 that acts as an early and upstream change in the cell death
cascade to caspase- and mitochondria-dependent signaling pathways, triggers
mitochondrial dysfunction from Bcl-2 and Bax modulation, mitochondrial
cytochrome c release and caspase activation, consequently leading to apoptosis
in SCC-4 cells. BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and
propionic acid, are metabolic by-products generated by periodontal microflora
such as Porphyromonas gingivalis, and contribute to the pathogenesis of
periodontitis. However, the effects of butyrate on the biological activities of
gingival fibroblasts (GFs) are not well elucidated.
MATERIAL AND METHODS: Human GFs were exposed to various concentrations of
butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were
counted. Cell cycle distribution of GFs was analyzed by propidium
iodide-staining flow cytometry. Cellular reactive oxygen species (ROS)
production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF).
Total RNA and protein lysates were isolated and subjected to RT-PCR using
specific primers or to western blotting using specific antibodies, respectively.
RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the
number of viable cells. This event was associated with an induction of G0/G1 and
G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked
apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm)
inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the
levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR
and western blotting, respectively. This toxic effect of butyrate was associated
with the production of ROS.
CONCLUSION: These results suggest that butyrate generated by periodontal
pathogens may be involved in the pathogenesis of periodontal diseases via the
induction of ROS production and the impairment of cell growth, cell cycle
progression and expression of cell cycle-related genes in GFs. These events are
important in the initiation and prolongation of inflammatory processes in
periodontal diseases. An elevated rate of glucose consumption and the dependency on aerobic glycolysis
for ATP generation have long been observed in cancer cells, a phenomenon known
as the Warburg effect. the altered energy metabolism in cancer cells provides an
attractive opportunity for developing novel cancer therapeutic strategies.
Lactate dehydrogenase (LDH), which catalyzes the transformation of pyruvate to
lactate, plays a vital role in the process of glycolysis. It has been reported
that the level of LDH-A expression is increased both in head and neck cancer
cells and in the blood serum of nasopharyngeal carcinoma (NPC) patients, and is
associated with poor prognosis. However, the effect of LDH-A inhibition on NPC
cells remains unknown. Here, in the present study, we found that oxamate, a
classical inhibitor of LDH-A, suppressed cell proliferation in a dose- and
time-dependent manner both in CNE-1 and CNE-2 cells, two NPC cancer cell lines.
LDH inhibition by oxamate induced G2/M cell cycle arrest via downregulation of
the CDK1/cyclin B1 pathway and promoted apoptosis through enhancement of
mitochondrial ROS generation. N-acetylcysteine, a specific scavenger of ROS,
significantly blocked the growth inhibition effect induced by oxamate. We also
identified that oxamate increased sensitivity to ionizing radiation in the two
NPC cancer cell lines. Furthermore, we verified similar results in tumor
xenograft models. collectively, these results suggest that LDH-A may serve as a
promising therapeutic target for NPC treatment. Magnolol, a small-molecule hydroxylated biphenol, isolated from the root and
stem bark of Magnolia officinalis, has been shown to possess antiproliferative
effect on various cancer cell lines. In the current study, we found that
magnolol potently inhibited proliferation and induced apoptosis in MCF-7 human
breast cancer cells. Further mechanistic studies revealed that induction of
apoptosis is associated with cell cycle arrest at G2/M phase, increased
generation of reactive oxygen species (ROS), reduced mitochondrial membrane
potential (MMP), release of cytochrome c (Cyto c) and apoptosis inducing factor
(AIF) from mitochondria to cytosol, upregulation of Bax, p21 and p53, and
down-regulation of Bcl-2, cyclin B1 and cyclin-dependent kinase 1 (CDK1). Our
findings indicated that magnolol induced apoptosis in MCF-7 cells via the
intrinsic pathway with release of AIF from mitochondrial and G2/M phase arrest
pathway. Therefore, magnolol might be a potential lead compound in the therapy
of breast cancer. The present study was undertaken to determine whether sulforaphane-derived
reactive oxygen species (ROS) might cause growth arrest and apoptosis in human
bladder cancer 5637 cells. Our results show that the reduced viability of 5637
cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The
sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1
and phosphorylation of Cdk1, as well as a concomitant increased complex between
cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the
activation of caspase-8 and -9, the initiators caspases of the extrinsic and
intrinsic apoptotic pathways, respectively, and activation of effector caspase-3
and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase
activation inhibited apoptosis and abrogated growth inhibition in
sulforaphane-treated 5637 cells. This study further investigated the roles of
ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and
the maximum level of ROS accumulation was observed 3h after sulforaphane
treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated
sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these
results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637
cells via a ROS-dependent pathway. |
Are there any HCV replication inhibitors available? | Chronic hepatitis C virus (HCV) infection is a worldwide health problem causing serious complications, such as liver cirrhosis and hepatoma. Small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) have been reported to suppress gene expression significantly. HCV seems a suitable candidate for targets of siRNAs, as HCV is a positive single-strand RNA virus and replicates in the cytoplasm. Based on results, nowadays there are few HCV replication inhibitors such as GS-563253, PSI-6130, NA-808, BMS-790052, GS-9132 and BMS-788329. | Persistent hepatitis C virus (HCV) infection is a leading cause of chronic
hepatitis, cirrhosis, and hepatocellular carcinoma and the major indication for
liver transplantation in adults. Current standard of care treatment (SOC) with
pegylated-interferon-α 2 and ribavirin (RBV) has a limited efficacy and is
associated with significant side effects frequently associated with poor
compliance or treatment discontinuation, requiring specialized and frequent
monitoring. To overcome the limited efficacy of SOC, more than 50 direct-acting
antiviral agents (DAA) designed to target viral-encoded proteins essential in
the HCV life cycle are currently under development. The rapid selection of
resistant mutants associated with the quasispecies nature of HCV with high
mutation and replication rates is one of the main challenges for the new HCV
therapies. Predictive host and viral factors together with combination of DAAs
with or without IFN and/or RBV need to be accurately evaluated to design the
most effective individualized treatment strategy within the shortest time
interval and with minimum side effects. In this study, we established a flow cytometry live cell-based assay that
permits the screening of hepatitis C virus (HCV) inhibitors. Specifically, we
created a stable cell line, which harbors a subgenomic replicon encoding an
NS5A-YFP fusion protein. This system allows direct measurement of YFP
fluorescence in live hepatoma cells in which the HCV replicon replicates. We
demonstrated that this stable fluorescent system permits the rapid and sensitive
quantification of HCV replication inhibition by direct-acting antiviral agents
(DAA) including protease and NS5A inhibitors and host-targeting antiviral agents
(HTA) including cyclophilin inhibitors. This flow cytometry-based live cell
assay is well suited for multiple applications such as the evaluation of HCV
replication as well as antiviral drug screening. BACKGROUND: Hepatitis C virus (HCV) NS5A replication complex inhibitors (RCIs)
have been shown to exhibit picomolar antiviral activity against genotype 1 (GT1)
in vitro. This has translated into rapid and robust declines in HCV RNA in GT1
patients. Less is known about the susceptibility of other genotypes such as GT3
to inhibition by NS5A RCIs.
OBJECTIVES: To detect and phenotype naturally occurring HCVGT3 NS5A
polymorphisms against two NS5A RCIs (daclatasvir [DCV] and GS-5885) currently in
clinical development.
STUDY DESIGN: The NS5A region from 96 HCV GT3 treatment-naive patients spanning
North America, Europe and Australia was determined.
RESULTS: Phylogenetic analysis revealed a broad distribution with no significant
geographic clustering. GT1 DCV resistance-associated variants (RAVs) were
observed in GT3 subjects; variants (and their frequencies) included 28M/V (1%),
30A/K/S/T/V (10%), 31L/M (1%), E92A (1%) and Y93H (8.3%). A consensus sequence
was used to generate a JFH1/3a-NS5A hybrid replicon and employed to assess
susceptibility to NS5A RCIs. Against JFH1/3a-NS5A, DCV was more potent (EC(50) =
0.52 nM) than GS-5885 (EC(50) = 141 nM). DCV sensitivity was increased against
JFH1/3a-NS5A-M28V (EC50 = 0.006 nM), A30V (EC(50) = 0.012 nM), and E92A (EC(50)
= 0.004 nM) while the NS5A-A30K and -Y93H variants exhibited reduced sensitivity
to DCV (EC50 values of 23 nM and 1120 nM, respectively) and to GS-5885 (EC50
values of 1770 nM and 4300 nM, respectively).
CONCLUSIONS: Substitutions conferring resistance to NS5A RCIs pre-existed in
treatment-naive patients infected with HCV GT3. The effectiveness of these NS5A
RCIs to exert efficacy in the clinic may depend on which inhibitor is used in
combination with other antivirals. Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a
series of new monodentate molecules were designed. The synthesis of 36 new
non-dimeric NS5A inhibitors is reported along with their ability to block HCV
replication in an HCV 1b replicon system. Among them compound 5a showed
picomolar range activity along with an excellent selectivity index (SI >
90,000). Treatment of hepatitis C patients with direct-acting antiviral drugs involves
the combination of multiple small-molecule inhibitors of distinctive mechanisms
of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific
for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon
cells and was active in genotype 1 HCV-infected patients in a proof-of-concept
clinical trial (1). Here, we describe a potential mechanism of action (MoA)
wherein ACH-806 alters viral replication complex (RC) composition and function.
We found that ACH-806 did not affect HCV polyprotein translation and processing,
the early events of the formation of HCV RC. Instead, ACH-806 triggered the
formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in
replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production
was negatively regulated by NS3, and its appearance in turn was associated with
reductions in NS3 and, especially, NS4A content in RCs due to their accelerated
degradation. A previously described resistance substitution near the N terminus
of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A
conferred by ACH-806 treatment. Taken together, we show that the compositional
changes in viral RCs are associated with the antiviral activity of ACH-806.
Small molecules, including ACH-806, with this novel MoA hold promise for further
development and provide unique tools for clarifying the functions of NS4A in HCV
replication. Hepatitis C virus (HCV) NS3/4A protease is an important and attractive target
for anti-HCV drug development and discovery. Vaniprevir (phase III clinical
trials) and MK-5172 (phase II clinical trials) are two potent antiviral
compounds that target NS3/4A protease. However, the emergence of resistance to
these two inhibitors reduced the effectiveness of vaniprevir and MK-5172 against
viral replication. Among the drug resistance mutations, three single-site
mutations at residues Arg155, Ala156, and Asp168 in NS3/4A protease are
especially important due to their resistance to nearly all inhibitors in
clinical development. A detailed understanding of drug resistance mechanism to
vaniprevir and MK-5172 is therefore very crucial for the design of novel potent
agents targeting viral variants. In this work, molecular dynamics (MD)
simulation, binding free energy calculation, free energy decomposition, residue
interaction network (RIN), and substrate envelope analysis were used to study
the detailed drug resistance mechanism of the three mutants R155K, A156T, and
D168A to vaniprevir and MK-5172. MD simulation was used to investigate the
binding mode for these two inhibitors to wild-type and resistant mutants of HCV
NS3/4A protease. Binding free energy calculation and free energy decomposition
analysis reveal that drug resistance mutations reduced the interactions between
the active site residues and substituent in the P2 to P4 linker of vaniprevir
and MK-5172. Furthermore, RIN and substrate envelope analysis indicate that the
studied mutations of the residues are located outside the substrate (4B5A)
binding site and selectively decrease the affinity of inhibitors but not the
activity of the enzyme and consequently help NS3/4A protease escape from the
effect of the inhibitors without influencing the affinity of substrate binding.
These findings can provide useful information for understanding the drug
resistance mechanism against vaniprevir and MK-5172. The results can also
provide some potential clues for further design of novel inhibitors that are
less susceptible to drug resistance. BACKGROUND & AIMS: Host cell lipid rafts form a scaffold required for
replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs)
produce sphingolipids, which are essential components of the lipid rafts that
associate with HCV nonstructural proteins. Prevention of the de novo synthesis
of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and
thereby inhibits HCV replication. We investigated the ability of the SPT
inhibitor NA808 to prevent HCV replication in cells and mice.
METHODS: We tested the ability of NA808 to inhibit SPT's enzymatic activity in
FLR3-1 replicon cells. We used a replicon system to select for HCV variants that
became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory
concentration, after 14 rounds of cell passage. We assessed the ability of NA808
or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in
mice with humanized livers (transplanted with human hepatocytes). NA808 was
injected intravenously, with or without pegylated interferon alfa-2a and HCV
polymerase and/or protease inhibitors.
RESULTS: NA808 prevented HCV replication via noncompetitive inhibition of SPT;
no resistance mutations developed. NA808 prevented replication of all HCV
genotypes tested in mice with humanized livers. Intravenous NA808 significantly
reduced viral load in the mice and had synergistic effects with pegylated
interferon alfa-2a and HCV polymerase and protease inhibitors.
CONCLUSIONS: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a,
1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It
might be developed for treatment of HCV infection or used in combination with
pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Treatment of Hepatitis C Virus (HCV) infection is rapidly evolving with the
introduction of direct acting antiviral agents (DAA). HCV NS5A replication
complex inhibitors, exemplified by Daclatasvir (BMS-790052), represent a new
class of DAA. The exceptional in vitro potency (EC50 values at pM to low nM
range) and broad genotype coverage of NS5A inhibitors have translated to robust
anti-HCV effects in infected patients, making NS5A inhibitors an essential
component of effective HCV DAA combination therapies. On the basis of
drug-induced resistance substitutions and computer modeling, NS5A inhibitors
most likely act at the N-terminus of NS5A (domain I). Mechanism of inhibition
studies to elucidate the exquisite potency of these inhibitors have generated
several working models. OBJECTIVES: Direct-acting antiviral agents (DAAs) against hepatitis C virus
(HCV) have recently been developed and are ultimately hoped to replace
interferon-based therapy. However, DAA monotherapy results in rapid emergence of
resistant strains and DAAs must be used in combinations that present a high
genetic barrier to resistance, although viral kinetics of multidrug-resistant
strains remain poorly characterized. The aim of this study is to track the
emergence and fitness of resistance using combinations of telaprevir and NS5A or
NS5B inhibitors with genotype 1b clones.
METHODS: HCV-infected chimeric mice were treated with DAAs, and resistance was
monitored using direct and ultra-deep sequencing.
RESULTS: Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor)
reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A
telaprevir resistance mutation resulted in poor response to telaprevir
monotherapy but showed significant HCV reduction when telaprevir was combined
with BMS-788329. However, a BMS-788329-resistant strain emerged at low
frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly
resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir
resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H
mutations resulted in poor response to combination therapy and development of
telaprevir resistance. Although HCV RNA became undetectable soon after the
beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B
inhibitor), rebound with emergence of resistance against all three drugs
occurred. Triple resistance also occurred following infection with the
NS3V36A/NS5AL31V/NS5AY93H triple mutation.
CONCLUSIONS: Resistant strains easily develop from cloned virus strains.
Sequential use of DAAs should be avoided to prevent emergence of
multidrug-resistant strains. BACKGROUND: Resistance to mericitabine (prodrug of HCV NS5B polymerase inhibitor
PSI-6130) is rare and conferred by the NS5B S282T mutation.
METHODS: Serum HCV RNA from patients who experienced viral breakthrough, partial
response, or nonresponse in 2 clinical trials in which patients received
mericitabine plus peginterferon alfa-2a (40KD)/ribavirin were analyzed by
population and clonal sequence analysis as well as phenotypic assay for
assessment of in vivo mericitabine resistance.
RESULTS: Among 405 patients treated with mericitabine plus peginterferon
alfa-2a/ribavirin in PROPEL and JUMP-C, virologic breakthrough or nonresponse
were not observed; 12 patients experienced a partial response. The NS5B S282T
resistance mutation was not observed in any patient. A number of
treatment-associated NS5B changes were observed and characterized. A novel
double mutant (L159F/L320F) with impaired replication capacity was detected in
one HCV genotype 1b-infected patient. Introduction of double mutant L159F/L320F
into genotype 1a (H77) and 1b (Con-1) replicons, respectively, increased the
EC50 for mericitabine by 3.1- and 5.5-fold and the EC90 by 3.1- and 8.9-fold.
The double mutant also decreased susceptibility to sofosbuvir (GS-7977) and
GS-938 but not setrobuvir, relative to wild-type.
CONCLUSIONS: A novel and replication-deficient double mutation (L159F/L320F)
confers low-level resistance to mericitabine and cross-resistance to both
sofosbuvir and GS-938.
CLINICAL TRIALS REGISTRATION: NCT00869661, NCT01057667. One of the most challenging goals of hepatitis C virus (HCV) research is to
develop well-tolerated regimens with high cure rates across a variety of patient
populations. Such a regimen will likely require a combination of at least two
distinct direct-acting antivirals (DAAs). Combining two or more DAAs with
different resistance profiles increases the number of mutations required for
viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently
reported that the combination of two distinct classes of HCV inhibitors, entry
inhibitors and replication inhibitors, prolonged reductions in extracellular HCV
in persistently infected cells. We therefore sought to identify new inhibitors
targeting aspects of the HCV replication cycle other than RNA replication. We
report here the discovery of the first small-molecule HCV infectivity inhibitor,
GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a
substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs
with low-omolar 50% effective concentrations. It was identified through a
high-throughput screen and subsequent chemical optimization. HCV II-1 only
permits the production and release of noninfectious HCV particles from cells.
Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1
resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal
fusion, suggesting that it either locks the viral envelope in its prefusion
state or promotes a viral envelope conformation change incapable of fusion.
Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors
that prolong viral suppression by HCV replication inhibitors in persistently
infected cell cultures. |
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