question
stringlengths 13
215
| ground_truth
stringlengths 2
3.15k
| context
stringlengths 0
157k
|
|---|---|---|
What is known about diseases associated with mutations in the CHCHD10 gene? | Mutation c.197G>T p.G66V in CHCHD10 is the cause of the lower motor neuron syndrome LOSMoN/SMAJ.
Mutations in the CHCHD10 gene have been identified in a large family with a complex phenotype variably associating frontotemporal dementia (FTD) with amyotrophic lateral sclerosis (ALS), cerebellar ataxia, myopathy, and hearing impairment.
Other findings links CHCHD10 mutations to mitochondrial myopathy. | Mitochondrial DNA instability disorders are responsible for a large clinical
spectrum, among which amyotrophic lateral sclerosis-like symptoms and
frontotemporal dementia are extremely rare. We report a large family with a
late-onset phenotype including motor neuron disease, cognitive decline
resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all
patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative
fibres with combined respiratory chain deficiency and abnormal assembly of
complex V. The multiple mitochondrial DNA deletions found in skeletal muscle
revealed a mitochondrial DNA instability disorder. Patient fibroblasts present
with respiratory chain deficiency, mitochondrial ultrastructural alterations and
fragmentation of the mitochondrial network. Interestingly, expression of
matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not
inhibited in patient fibroblasts. Using whole-exome sequencing we identified a
missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a
coiled-coil helix coiled-coil helix protein, whose function is unknown. We show
that CHCHD10 is a mitochondrial protein located in the intermembrane space and
enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa
cells led to fragmentation of the mitochondrial network and ultrastructural
major abnormalities including loss, disorganization and dilatation of cristae.
The observation of a frontotemporal dementia-amyotrophic lateral sclerosis
phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21
families with pathologically proven frontotemporal dementia-amyotrophic lateral
sclerosis. We identified the same missense p.Ser59Leu mutation in one of these
families. This work opens a novel field to explore the pathogenesis of the
frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by
showing that mitochondrial disease may be at the origin of some of these
phenotypes. Mitochondrial myopathies belong to a larger group of systemic diseases caused by
morphological or biochemical abnormalities of mitochondria. Mitochondrial
disorders can be caused by mutations in either the mitochondrial or nuclear
genome. Only 5% of all mitochondrial disorders are autosomal domit. We
analyzed DNA from members of the previously reported Puerto Rican kindred with
an autosomal domit mitochondrial myopathy (Heimann-Patterson et al. 1997).
Linkage analysis suggested a putative locus on the pericentric region of the
long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we
established chromosome 22 open reading frame 16 (C22orf16) (later designated as
CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal
candidate region. Sequence analysis revealed a double-missense mutation (R15S
and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix
protein of unknown function. These two mutations completely co-segregated with
the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic
(including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10
is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10
protein was confirmed using immunofluorescence in cells expressing either
wild-type or mutant CHCHD10. We found that the expression of the G58R, but not
the R15S, mutation induced mitochondrial fragmentation. Our findings identify a
novel gene causing mitochondrial myopathy, thereby expanding the spectrum of
mitochondrial myopathies caused by nuclear genes. Our findings also suggest a
role for CHCHD10 in the morphologic remodeling of the mitochondria. OBJECTIVE: A study was undertaken to identify the responsible gene defect
underlying late onset spinal motor neuronopathy (LOSMoN/SMAJ; Online Mendelian
Inheritance in Man #615048), an autosomal domit disease mapped to chromosome
22q11.2.
METHODS: The previous genetic linkage approach by microsatellite haplotyping was
continued in new families. A whole genome sequencing was performed to find all
possibly pathogenic mutations in the linked area. The detected variations were
verified by Sanger sequencing.
RESULTS: Six new SMAJ families were identified based on the unique founder
haplotype. A critical recombination in 1 family restricted the linked area to
727kb between markers SHGC-106816 and D22S345. In whole genome sequencing a
previously unknown mutation c.197G>T p.G66V in CHCHD10 was identified. The
mutation was shown to segregate with the disease in 55 patients from 17
families.
INTERPRETATION: Mutation c.197G>T p.G66V in CHCHD10 is the cause of the lower
motor neuron syndrome LOSMoN/SMAJ. During the preparation of this article other
mutations were reported to cause frontotemporal dementia-amyotrophic lateral
sclerosis syndrome, indicating that the CHCHD10 gene is largely important for
the motor and cognitive neuronal systems. |
Which drug should be used as an antidote in benzodiazepine overdose? | Flumazenil should be used in all patients presenting with suspected benzodiazepine overdose. Flumazenil is a potent benzodiazepine receptor antagonist that competitively blocks the central effects of benzodiazepines and reverses behavioral, neurologic, and electrophysiologic effects of benzodiazepine overdose. Clinical efficacy and safety of flumazenil in treatment of benzodiazepine overdose has been confirmed in a number of rigorous clinical trials. In addition, flumazenil is also useful to to reverse benzodiazepine induced sedation and to and to diagnose benzodiazepine overdose. | Anterograde amnesia, possibly accompanied by acute brain syndrome, is a
potential side-effect of certain benzodiazepines, particularly triazolam.
Flumazenil is a benzodiazepine antagonist that is highly effective in reversing
the central nervous system effects of benzodiazepine overdose. We report a case
of triazolam overdose resulting in anterograde amnesia after flumazenil
administration had restored clear consciousness. The defect in memory may have
been due to too little flumazenil being given or failure of memory consolidation
affected by the character of triazolam during the induced lucent period. We feel
that physicians should be aware of the potential occurrence of acute brain
syndrome in patients with benzodiazepine overdose despite treatment with
flumazenil. Flumazenil is a recently discovered pharmacologic antagonist of the CNS effects
of benzodiazepines. It acts by binding CNS benzodiazepine receptors and
competitively blocking benzodiazepine activation of inhibitory GABAergic
synapses. Animal studies and some human studies appear to demonstrate that
flumazenil has weak intrinsic agonist activity; on the other hand, studies are
inconclusive in demonstrating any inverse agonist effects of this agent.
Evidence available suggests that flumazenil is well tolerated in human beings
over a broad range of doses when given either orally or parenterally and does
not produce serious adverse effects. In the setting of isolated benzodiazepine
overdose, flumazenil is capable of completely reversing coma within one to two
minutes, with this effect lasting between one and five hours. Repeat doses can
be given safely to reverse recurrent effects of longer-acting benzodiazepines.
Flumazenil is undergoing further evaluation by the Food and Drug Administration;
should this drug receive approval, it is likely to be used in emergency
departments as well as in a variety of other clinical settings. First, it could
be used to effect rapid reversal of benzodiazepine-induced sedation that has
been administered to facilitate medical, orthopedic, and surgical procedures,
particularly in the event of inadvertent respiratory depression. Second,
flumazenil might have a therapeutic role in the management of patients who have
taken benzodiazepine overdoses. Although most of these patients can be managed
successfully with supportive therapy alone, it is possible that the use of
flumazenil may obviate the need for intubation and respiratory support in such
patients and eliminate the possible adverse effects of even short-term
endotracheal intubation. Finally, flumazenil could have both diagnostic and
therapeutic value in patients with acute alterations of mental status of unknown
etiology, particularly when possible drug overdose is a consideration. Because
flumazenil appears to be specific in its antagonism of benzodiazepine-induced
respiratory and CNS depression, it could be used empirically to confirm or
exclude a role of benzodiazepines in the generation of mental status changes in
the setting of overdose or coma of unknown origin. This in turn might obviate
the need for further expensive (eg, computed tomography) and sometimes invasive
(eg, lumbar puncture) diagnostic modalities. This might be particularly useful
because there is nothing about benzodiazepine-induced coma that clearly
distinguishes it from other causes of coma; thus, there are no signs or symptoms
that may reasonably allow benzodiazepine overdose to be confirmed or eliminated
on clinical grounds. Further studies will continue to define the ultimate use of
this new agent. In a double-blind placebo-controlled prospective clinical trial we studied the
efficacy and safety of the benzodiazepine antagonist, flumazenil. In 23 patients
admitted to the Intensive Care Unit with coma due to overdose with
benzodiazepines or other sedatives, flumazenil i.v. (up to 2 mg or placebo) was
given. In 13 patients given flumazenil the Glasgow Coma Scale (GCS) increased
significantly from 4.9 to 7.8 (p less than 0.05). Six of these 13 patients,
including mainly benzodiazepine mono-intoxications, needed only one series of
injections (up to 1.0 mg flumazenil); the GCS increased thereby from 4.5 to 10.7
within a maximum of 5 min (p less than 0.01). In the remaining 7 patients,
needing two series of injections of flumazenil (up to 2.0 mg), GCS did not rise
significantly and coma was related to intoxications with nonbenzodiazepine
sedatives, flunitrazepam and in one patient, encephalitis. In the 10 patients
receiving placebo, the GCS did not change. A significant increase in the GCS
from 5.5 to 10.8 (p less than 0.001) was, however, observed when flumazenil (up
to 1.0 mg) was given after placebo. In patients with EEG monitoring the changes
in waveform pattern paralleled the clinical response. Effects could be detected
within 1-2 min after flumazenil injection and lasted up to 45 min. There were no
adverse reactions or benzodiazepine withdrawal symptoms. We conclude that
flumazenil is an effective and safe drug in the treatment of benzodiazepine
overdose. The use of flumazenil is of diagnostic value in mixed-drug
intoxications or coma of unknown origin and is of therapeutic importance for
reversal of benzodiazepine intoxications. The clinical efficacy of different doses of the specific benzodiazepine
antagonist flumazenil was studied in a total of 72 patients with benzodiazepine
or ethanol overdose. In a randomized double-blind study, 18 patients (group 1)
and eight patients (group 2) with suspected benzodiazepine overdose received 5
mg (group 1) or 1 mg (group 2) flumazenil or placebo, respectively. The stage of
coma, heart rate, blood pressure and respiratory rate were monitored within the
following 15 min. If no change in the stage of coma was observed, 5 mg (group 1)
or 1 mg (group 2) flumazenil were given, and the stage of coma, heart rate and
blood pressure were again monitored. In a similar way, the effect of 5 and 1 mg
flumazenil was investigated in 13 patients (group 3) and four patients (group 4)
with ethanol intoxication. In an open trial, the clinical efficacy of flumazenil
for the diagnosis of benzodiazepine or ethanol overdose was studied in 29
patients (group 5). In all patients, a toxicological screening confirmed
benzodiazepine or ethanol overdose. None of the patients receiving placebo
showed effects on stage of coma, heart rate, blood pressure or respiratory rate.
Patients with benzodiazepine overdose who received 5 mg flumazenil regained
consciousness about 1-2 min after the end of injection. The effect of 1 mg
flumazenil (group 2) on benzodiazepine-induced coma was less pronounced. In
patients with ethanol overdose (group 3), ethanol-induced coma was reversed
after 5 mg flumazenil more slowly than in patients of group 1. No effect of
flumazenil on ethanol-induced coma was observed in group 4. In group 5,
flumazenil proved to be useful for diagnosing benzodiazepine or ethanol
intoxication. In one patient with coma due to carbamazepine overdose, flumazenil
was also found to be effective. Additionally, a possible analytical interference
of flumazenil and its metabolites with the identification of other
benzodiazepines by a toxicological screening procedure was studied. Even after
an oral dose of 200 mg flumazenil, no interference with immunological
benzodiazepine assays (EMIT, TDX, and RIA) was found. A metabolite and an
artifact of flumazenil could be identified in urine by gas chromatography/mass
spectrometry. For the first time a causal treatment of hepatic encephalopathy may be possible
by the benzodiazepine antagonist flumazenil. In contrast to all other treatments
used so far by flumazenil hepatic encephalopathy improves within minutes.
Flumazenil is the first benzodiazepine antagonist which can be used in humans
and is a well established for treatment of benzodiazepine overdose. For
treatment of hepatic encephalopathy development of a new antagonist with a
longer half-life is desirable. However, ut should be stressed that the current
experience with flumazenil is limited and that the effects of flumazenil on
hepatic encephalopathy is not proven by randomized controlled studies.
Therefore, this drug should only be used in clinical studies. In anaesthesia and in the intensive care unit, benzodiazepines have proven safe
and effective agents for the induction and maintece of sedation for a variety
of therapeutic goals. However, in these contexts, or in benzodiazepine overdose,
it is often desirable to be able to terminate or interrupt sedation without
waiting for the effect of the benzodiazepine to become dissipated by normal
metabolism and excretion. Flumazenil, a 1,4-imidazobenzodiazepine, is a highly
effective, specific benzodiazepine antagonist which is indicated for use when
the effect of a benzodiazepine must be attenuated or terminated at short notice.
It acts by displacing other benzodiazepines from the receptor site by
competitive inhibition. The onset of effect after intravenous administration
occurs within 1 to 3 minutes. The optimal dosage is determined for each patient
by a dose titration procedure and lies in the range 0.2 to 1.0mg in
anaesthesiology, and 0.1 to 2.0mg in intensive care use. Despite its short
elimination half-life of around 1 hour, after general anaesthesia or conscious
to moderate sedation for short procedures, a single dose of flumazenil is
usually sufficient to attain and maintain the desired level of consciousness.
After intoxication with high benzodiazepine doses, the duration of effect of a
single dose of flumazenil is not expected to exceed 1 hour. In such cases, the
period of wakefulness can be prolonged as necessary by repeated low intravenous
doses of flumazenil or by infusion (0.1 mg/hour). Flumazenil is well tolerated
both systemically and locally. The only adverse events seen with greater
frequency after flumazenil compared with placebo were nausea and/or vomiting
after general anaesthesia, although the incidence of actual vomiting was not
significantly different between the 2 groups. Since these effects were virtually
absent in studies of intensive care patients and after sedation for short
procedures, and were not seen in tolerability studies in healthy volunteers
receiving intravenous bolus doses of up to 100mg, there may be a link between
these symptoms and the other agents used in general anaesthesia, some of which
have well-known emetic properties. Thus, flumazenil provides a safe and
effective means of attenuating or reversing the CNS-depressant effects of
benzodiazepines whenever indicated, e.g. following benzodiazepine-induced
general anaesthesia, conscious sedation, or after benzodiazepine overdose,
either alone or in combination with other agents.(ABSTRACT TRUNCATED AT 400
WORDS) Flumazenil (Ro 15-1788) proved to be a very efficacious competitive antagonist
of benzodiazepines that reliably counteracts their pharmacological actions
within 1-2 min as could be demonstrated in clinical and EEG studies. In general,
a total dose of 0.3-0.8 mg will be sufficient in clinical practice, avoiding
side effects like nausea, tremor, sweating, or transient anxiety that could be
observed when higher dosages were administered. Its therapeutic range is very
high as could be demonstrated in experimental animal in which up to 8.000-fold
the clinical dose was administered. The total volume of distribution (Vdes)
amounts to nearly 1.000 ml/kg BW and the total clearance exceeds 1.200 ml/min,
resulting in a biological half-life of less than 60 min. According to the
benzodiazepine dosage and the rapid plasma concentration decline of flumazenil,
in some cases a resedation could be observed. Hence, a careful observation of
the antagonised patient on the ward is mandatory for 1.5-2 h, even if at first
sight the antagonization seemed successful and the patient fully awake and
cooperative. In anaesthesia, indications to administer flumazenil are adverse
drug reactions and prolonged recovery after adequate benzodiazepine dosage. In
intensive care medicine, the antagonist may be used in the treatment of
benzodiazepine overdose as well as in the differential diagnosis of a coma of
unknown origin. Additionally, the antagonist may be administered to interrupt
benzodiazepine sedation e.g. for neurological examination. OBJECTIVE: In the assessment and management of the potentially poisoned patient
with altered consciousness, the most consequential and controversial
interventions occur during the first 5 minutes of care. In this review article,
the risks and benefits of standard diagnostic and therapeutic interventions are
presented to guide clinicians through this critical period of decision making.
DATA SOURCES: Data for discussion were obtained from a search of
English-language publications referenced on MEDLINE for the years 1966 to 1994.
Older literature was included when pertinent. Search terms included poisoning,
overdose, toxicity, naloxone, glucose, thiamine, and flumazenil.
STUDY SELECTION: Only large trials were used for determinations of diagnostic
utility and efficacy. Small trials, case series, and case reports were reviewed
extensively for adverse effects.
DATA EXTRACTION AND SYNTHESIS: Trials were reviewed for overall methodology,
inclusion and exclusion criteria, sources of bias, and outcome.
CONCLUSION: Analysis favors empirical administration of hypertonic dextrose and
thiamine hydrochloride to patients with altered consciousness. Although rapid
reagent test strips can be used to guide this therapy, they are not infallible,
and they fail to recognize clinical hypoglycemia that may occur without
numerical hypoglycemia. Administration of naloxone hydrochloride should be
reserved for patients with signs and symptoms of opioid intoxication. Flumazenil
is best left for reversal of therapeutic conscious sedation and rare select
cases of benzodiazepine overdose. The mechanism of action, pharmacokinetics, and use of flumazenil in
benzodiazepine overdose, as well as in the management of other disease states,
are reviewed. Flumazenil interacts at the central benzodiazepine receptor to
antagonize or reverse the behavioral, neurologic, and electrophysiologic effects
of benzodiazepine agonists and inverse agonists. Flumazenil has been studied for
a variety of indications, including as an antidote to benzodiazepine overdose
and for awakening of comatose patients, reversal of sedation after surgery and
in critically ill patients, and management of hepatic encephalopathy. It
improves the level of consciousness in patients with benzodiazepine overdose;
however, resedation may occur within one to two hours after administration, so
repeated doses or a continuous infusion may be required to maintain therapeutic
efficacy. It appears to be effective in reversing sedation induced by midazolam
or diazepam, and case reports suggest that it is useful in awakening comatose
patients, although its clinical utility is questionable. Flumazenil has proved
useful in reversing conscious sedation in critically ill patients, although
response may be dose dependent. Animal models indicate that flumazenil is of
some benefit in hepatic encephalopathy, but until well-designed clinical trials
are conducted, hepatic encephalopathy must be considered an investigational
indication for flumazenil. Adverse reactions include CNS manifestations,
resedation, cardiovascular effects, seizures, and alterations in intracranial
pressure and cerebral perfusion pressure. Hepatic dysfunction results in a
substantial change in the pharmacokinetic profile of flumazenil; therefore,
dosage adjustment may be necessary in patients with hepatic dysfunction or in
those receiving medications that alter flumazenil metabolism. Flumazenil has
been shown to reverse sedation caused by intoxication with benzodiazepines alone
or benzodiazepines in combination with other agents, but it should not be used
when cyclic antidepressant intoxication is suspected. It may be beneficial after
surgery when benzodiazepines have been used as part of anesthesia and after a
diagnostic or surgical procedure when assessment of CNS function is necessary. Flumazenil, a specific benzodiazepine antagonist, is useful in reversing the
sedation and respiratory depression that often occur when benzodiazepines are
administered to patients undergoing anesthesia or when patients have taken an
intentional benzodiazepine overdose. Judicious use of flumazenil may provide
useful diagnostic information and may obviate the need for mechanical
ventilation and other invasive supportive measures. Although some controversy
exists regarding the possible precipitation of seizure activity in the setting
of mixed tricyclic antidepressant-benzodiazepine overdose, worldwide experience
with flumazenil has validated its safety and efficacy. OBJECTIVES: To assess the efficacy, usefulness, safety, and dosages of
flumazenil required when flumazenil is used in the diagnosis of
benzodiazepine-induced coma (vs. other drug-induced coma), and to reverse or
prevent the recurrence of unconsciousness.
DESIGN: A two-phase study: a controlled, randomized, double-blind study followed
by a prospective, open study.
SETTING: An 800-bed, teaching, university-affiliated hospital.
PATIENTS: Unconscious patients (n = 110) suspected of benzodiazepine overdose,
graded 2 to 4 on the Matthew and Lawson coma scale, were treated with
flumazenil, the specific benzodiazepine receptor antagonist. The first 31
patients were studied in a double-blind fashion, while the rest of the patients
were given flumazenil according to an open protocol. INTERVENTIONS; All patients
received supplemental oxygen; endotracheal intubation was performed, and
synchronized intermittent mandatory ventilation was initiated whenever it was
deemed necessary. A peripheral intravenous cannula was inserted, as were
indwelling arterial and urinary bladder catheters. Blood pressure,
electrocardiogram, respiratory rate, end-tidal CO2, and core temperature were
continuously monitored. The first 31 double-blind patients received either
intravenous flumazenil (to a maximum of 1 mg) or saline, while the rest of the
patients were given flumazenil until either regaining consciousness or a maximum
of 2.5 mg was injected. Patients remaining unconscious among double-blind
patients or those patients relapsing into coma after the first dose were later
treated in the open phase of the study. Treatment continued by boluses or
infusion as long as efficacious.
MEASUREMENTS AND MAIN RESULTS: Fourteen of 17 double-blind, flumazenil-treated
patients woke after a mean of 0.8 +/- 0.3 (SD) mg vs. one of 14 placebo patients
(p < .001). Seventy-five percent of the aggregated controlled and uncontrolled
patients awoke from coma scores of 3.1 +/- 0.6 to 0.4 +/- 0.5 (p < .01) after
the injection of 0.7 +/- 0.3 mg of flumazenil. These patients had high
benzodiazepine serum blood concentrations. Twenty-five percent of the patients
did not regain consciousness. These patients had very high serum concentrations
of nonbenzodiazepine drugs. Sixty percent of the responders who had primarily
ingested benzodiazepines remained awake for 72 +/- 37 mins after flumazenil
administration; 40% relapsed into coma after 18 +/- 7 mins and various central
nervous system depressant drugs were detected in their blood in addition to
benzodiazepines. Seventy-one percent of the patients had ingested tricyclic
antidepressants. Seventy-eight percent of the responders were continually and
efficaciously treated for < or = 8 days. Fourteen (25%) of the intubated
patients were extubated safely while 12 patients, who had shown increased
respiratory insufficiency, resumed satisfactory respiration after flumazenil
injection. Five cases of transient increase in blood pressure and heart rate
were encountered. There were 27 mildly unpleasant "waking" episodes, such as
anxiety, restlessness, and aggression, but no patient had benzodiazepine
withdrawal signs, convulsions, or dysrhythmia, most noticeably absent in
tricyclic antidepressant-intoxicated patients.
CONCLUSIONS: Flumazenil is a valid diagnostic tool for distinguishing pure
benzodiazepine from mixed-drug intoxication or nondrug-induced coma. Flumazenil
is effective in preventing recurrence of benzodiazepine-induced coma.
Respiratory insufficiency is reversed after its administration. Flumazenil is
safe when administered cautiously, even in patients with coma caused by a mixed
overdose of benzodiazepine plus tricyclic antidepressants. Sedation is an important aspect of care for critically ill newborns. Proper
sedation reduces stress during procedures such as mechanical ventilation.
Midazolam, a short-acting benzodiazepine, is widely administered as a sedative
in newborn intensive care units but is not without side effects. Three term
newborns developed myoclonic-like abnormal movements after receiving midazolam.
In one, flumazenil controlled the abnormal movements. Flumazenil is a potent
benzodiazepine antagonist that competitively blocks the central effects of
benzodiazepines. It can reverse the sedative effects of benzodiazepines
occurring after diagnostic or therapeutic procedures or after benzodiazepine
overdose. Flumazenil may be considered in cases of abnormal movements associated
with midazolam. However, further studies are needed to provide guidelines for
the administration of this drug in newborns. Flumazenil is indicated for reversal of sedation from benzodiazepines
administered during therapeutic or diagnostic procedures and during induction or
maintece of general anesthesia, as well as for benzodiazepine overdose. Bolus
doses of flumazenil are usually adequate to achieve reversal; however, when
medical conditions may lead to a prolonged half-life of the benzodiazepine
involved, continuous infusion may be warranted. A 67-year-old man with
chlordiazepoxide toxicity required a 9-day infusion of flumazenil to prevent
resedation and respiratory insufficiency; he initially was admitted to the
hospital for alcohol detoxification. Concomitant medical conditions and the
metabolism characteristics of each benzodiazepine must dictate the agent of
choice. When measures are required to ensure adequate recovery of a patient's
respiratory function and mental awareness, such as in patients with
benzodiazepine toxicity, consideration of continuous-infusion flumazenil is
warranted. The actions of benzodiazepines are due to the potentiation of the neural
inhibition that is mediated by gamma-aminobutyric acid (GABA). Practically all
effects of the benzodiazepines result from their actions on the ionotropic
GABA(A) receptors in the central nervous system. Benzodiazepines do not activate
GABA(A) receptors directly but they require GABA. The main effects of
benzodiazepines are sedation, hypnosis, decreased anxiety, anterograde amnesia,
centrally mediated muscle relaxation and anti-convulsant activity. In addition
to their action on the central nervous system, benzodiazepines have a
dose-dependent ventilatory depressant effect and they also cause a modest
reduction in arterial blood pressure and an increase in heart rate as a result
of a decrease of systemic vascular resistance. The four benzodiazepines, widely
used in clinical anaesthesia, are the agonists midazolam, diazepam and lorazepam
and the antagonist flumazenil. Midazolam, diazepam and flumazenil are
metabolized by cytochrome P450 (CYP) enzymes and by glucuronide conjugation
whereas lorazepam directly undergoes glucuronide conjugation. CYP3A4 is
important in the biotransformation of both midazolam and diazepam. CYP2C19 is
important in the biotransformation of diazepam. Liver and renal dysfunction have
only a minor effect on the pharmacokinetics of lorazepam but they slow down the
elimination of the other benzodiazepines used in clinical anaesthesia. The
duration of action of all benzodiazepines is strongly dependent on the duration
of their administration. Based on clinical studies and computer simulations,
midazolam has the shortest recovery profile followed by lorazepam and diazepam.
Being metabolized by CYP enzymes, midazolam and diazepam have many clinically
significant interactions with inhibitors and inducers of CYP3A4 and 2C19. In
addition to pharmacokinetic interactions, benzodiazepines have synergistic
interactions with other hypnotics and opioids. Midazolam, diazepam and lorazepam
are widely used for sedation and to some extent also for induction and
maintece of anaesthesia. Flumazenil is very useful in reversing
benzodiazepine-induced sedation as well as to diagnose or treat benzodiazepine
overdose. OBJECTIVE: Benzodiazepine (BZD) overdose (OD) continues to cause significant
morbidity and mortality in the UK. Flumazenil is an effective antidote but there
is a risk of seizures, particularly in those who have co-ingested tricyclic
antidepressants. A study was undertaken to examine the frequency of use, safety
and efficacy of flumazenil in the management of BZD OD in the UK.
METHODS: A 2-year retrospective cohort study was performed of all enquiries to
the UK National Poisons Information Service involving BZD OD.
RESULTS: Flumazenil was administered to 80 patients in 4504 BZD-related
enquiries, 68 of whom did not have ventilatory failure or had recognised
contraindications to flumazenil. Factors associated with flumazenil use were
increased age, severe poisoning and ventilatory failure. Co-ingestion of
tricyclic antidepressants and chronic obstructive pulmonary disease did not
influence flumazenil administration. Seizure frequency in patients not treated
with flumazenil was 0.3%. The frequency of prior seizure in flumazenil-treated
patients was 30 times higher (8.8%). Seven patients who had seizures prior to
flumazenil therapy had no recurrence of their seizures. Ventilation or
consciousness improved in 70% of flumazenil-treated patients. Flumazenil
administration was followed by one instance each of agitation and brief seizure.
CONCLUSIONS: Flumazenil is used infrequently in the management of BZD OD in the
UK. It was effective and associated with a low incidence of seizure. These
results compare favourably with the results of published randomised controlled
trials and cohort studies, although previous studies have not reported the use
of flumazenil in such a high-risk population. This study should inform the
continuing review of national guidance on flumazenil therapy. |
Does thyroid hormone signaling affect microRNAs expression in the heart? | YES | The heart responds to diverse forms of stress by hypertrophic growth accompanied
by fibrosis and eventual diminution of contractility, which results from
down-regulation of alpha-myosin heavy chain (alphaMHC) and up-regulation of
betaMHC, the primary contractile proteins of the heart. We found that a
cardiac-specific microRNA (miR-208) encoded by an intron of the alphaMHC gene is
required for cardiomyocyte hypertrophy, fibrosis, and expression of betaMHC in
response to stress and hypothyroidism. Thus, the alphaMHC gene, in addition to
encoding a major cardiac contractile protein, regulates cardiac growth and gene
expression in response to stress and hormonal signaling through miR-208. MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene
expression and play important roles in gene regulation in the heart. To examine
the role of miRNAs in the expression of the two isoforms of the cardiac myosin
heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility,
endogenous miRNAs were downregulated in neonatal rat ventricular myocytes
(NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer,
an essential enzyme for miRNA biosynthesis, and MHC expression levels were
examined. As a result, Dicer siRNA could downregulate endogenous miRNAs
simultaneously and the β-MHC gene but not α-MHC, which implied that specific
miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was
found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC
protein was downregulated by silencing of endogenous miR-27a. Through a
bioinformatics screening using TargetScan, we identified thyroid hormone
receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target
of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation
via thyroid hormone signaling and to be upregulated during the differentiation
of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with
β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC
gene expression by targeting TRβ1 in cardiomyocytes. |
Give examples of next-generation sequencing applications in mutation screening? | Next generation sequencing data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. NGS is introduced to an increasing number of mutation screening applications. An NGS based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence was used for the case of c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2. Another NGS study screened BRCA1 and BRCA2 resulting in overall sensitivity for SOLiD and PGM of 97.8% (95% CI = 94.7 to 100.0) and 98.9% (95% CI = 96.8 to 100.0) respectively. The specificity for the SOLiD platform was high, at 100.0% (95% CI = 99.3 to 100.0). PGM correctly identified all 3 indels, but 68 false-positive indels were also called. Genes known to cause deafness were sequenced by the Illumina NGS platform. Results demonstrated that targeted exons captured by our approach achieved specificity, multiplexicity, uniformity, and depth of coverage suitable for accurate sequencing applications by the NGS systems. Reliable genotype calls for various homozygous and heterozygous mutations were achieved. In the context of von Willebrand disease 43 mutations, including 36 substitutions, 2 intronic splice site mutations, 2 indels, and 3 deletions were screened on the next-generation sequencing instrument. This demonstrated that at least 350 patients and relatives per run can be simultaneously analyzed in a fast, inexpensive manner. The Alport syndrome is caused by mutations in three key genes namely COL4A3, COL4A4 and COL4A5, each of which consists of approximately 50 exons, thus rendering mutations screening a highly time consuming and expensive endeavor. NGS is now being established for the simultaneous, fast and cost-effective detection of all possible variants in these three genes. NGS has also been used screening EGFR, KRAS and BRAF for mutations associated with cancer diagnosis and/or response to several anticancer therapies. NGS has also been used in mutation screening for hereditary spastic paraplegias, X linked leucoencephalopathy, retinitis pigmentosa, inherited urea cycle disorders, as well as the Marfan (MFS), Loeys-Dietz (LDS) and Meckel syndromes. | BACKGROUND: Conventional PCR-based direct sequencing of candidate genes for a
family with X-linked leucoencephalopathy with unknown aetiology failed to
identify any causative mutations.
OBJECTIVE: To carry out exome sequencing of entire transcripts of the whole X
chromosome to investigate a family with X linked leucoencephalopathy.
METHODS AND RESULTS: Next-generation sequencing of all the transcripts of the X
chromosome, after liquid-based genome partitioning, was performed on one of the
two affected male subjects (the proband) and an unaffected male subject (his
brother). A nonsense mutation in MCT8 (c.1102A→T (p.R368X)) was identified in
the proband. Subsequent PCR-based direct sequencing of other family members
confirmed the presence of this mutation, hemizygous in the other affected
brother and heterozygous in the proband's mother and maternal grandmother. MCT8
mutations usually cause abnormal thyroid function in addition to neurological
abnormalities, but this proband had normal thyroid function.
CONCLUSION: Single-lane exome next-generation sequencing is sufficient to fully
analyse all the transcripts of the X chromosome. This method is particularly
suitable for mutation screening of X-linked recessive disorders and can avoid
biases in candidate gene choice. The Marfan (MFS) and Loeys-Dietz (LDS) syndromes are caused by mutations in the
fibrillin-1 (FBN1) and Transforming Growth Factor Beta Receptor 1 and 2 (TGFBR1
and TGFBR2) genes, respectively. With the current conventional mutation
screening technologies, analysis of this set of genes is time consuming and
expensive. We have tailored a cost-effective and reliable mutation discovery
strategy using multiplex PCR followed by Next Generation Sequencing (NGS). In a
first stage, genomic DNA from five MFS or LDS patient samples with previously
identified mutations and/or polymorphisms in FBN1 and TGFBR1 and 2 were analyzed
and revealed all expected variants. In a second stage, we validated the
technique on 87 samples from MFS patients fulfilling the Ghent criteria. This
resulted in the identification of 75 FBN1 mutations, of which 67 were unique.
Subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis of
the remaining negative samples identified four large deletions/insertions.
Finally, Sanger sequencing identified a missense mutation in FBN1 exon 1 that
was not included in the NGS workflow. In total, there was an overall mutation
identification rate of 92%, which is in agreement with data published
previously. We conclude that multiplex PCR of all coding exons of FBN1 and
TGFBR1/2 followed by NGS analysis and MLPA is a robust strategy for time- and
cost-effective identification of mutations. Alport syndrome (ATS) is a hereditary nephropathy often associated with
sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene
cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in
both autosomal recessive and autosomal domit ATS. The conventional mutation
screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and
has relatively high costs because of the absence of hot spots and to the high
number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several
months are usually necessary to complete the diagnosis, especially in cases with
less informative pedigrees. To overcome these limitations, we designed a
next-generation sequencing (NGS) protocol enabling simultaneous detection of all
possible variants in the three genes. We used a method coupling selective
amplification to the 454 Roche DNA sequencing platform (Genome Sequencer
junior). The application of this technology allowed us to identify the second
mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4)
and to reconsider the diagnosis of ATS in a third patient. This study,
therefore, illustrates the successful application of NGS to mutation screening
of Mendelian disorders with locus heterogeneity. BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors
KRAS and BRAF are mutated in several types of cancer, affecting the clinical
response to EGFR inhibitors. Mutations in the EGFR kinase domain predict
sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung
adenocarcinoma, while activating point mutations in KRAS and BRAF confer
resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer.
The development of new generation methods for systematic mutation screening of
these genes will allow more appropriate therapeutic choices.
METHODS: We describe a high resolution melting (HRM) assay for mutation
detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using
formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was
also analysed by massively parallel pyrosequencing of amplicons with the GS
Junior 454 platform.
RESULTS: We tested 120 routine diagnostic specimens from patients with
colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in
41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually
exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D,
7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF
activating mutations. Regarding EGFR, two cases showed exon 19 deletions
(delE746-A750 and delE746-T751insA) and another two substitutions in exon 21
(one showed L858R with the resistance mutation T590M in exon 20, and the other
had P848L mutation). Consistent with earlier reports, our results show that KRAS
and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%,
respectively, while EGFR mutations were detected in 11.1% of the lung cancer
specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM
results and allowed detection and quantitation of KRAS somatic mutations.
CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput
cost-effective screening of oncogene mutations in clinical samples. Rather than
Sanger sequence validation, next-generation sequencing technology results in
more accurate quantitative results in somatic variation and can be achieved at a
higher throughput scale. Genetic analysis of von Willebrand disease by von Willebrand factor gene
sequencing has not yet become routine practice. Nevertheless, the prospects for
molecular diagnosis have changed dramatically in recent years with the unveiling
of next-generation sequencing platforms. With the goal of applying this
technology to von Willebrand disease, we designed a strategy for von Willebrand
factor gene enrichment and multiplexing based on short polymerase chain
reactions. Forty patients were simultaneously analyzed enabling the
identification of 43 mutations, including 36 substitutions, 2 intronic splice
site mutations, 2 indels, and 3 deletions. By pooling patient genomic DNA before
polymerase chain reaction enrichment, indexing samples with barcode tags, and
re-sequencing on the next-generation sequencing instrument, at least 350
patients and relatives per run can be simultaneously analyzed in a fast,
inexpensive manner. This is one of the first reports in which this technology
has been shown to be feasible for large-scale mutation screening by single gene
re-sequencing. Current major barriers for using next-generation sequencing (NGS) technologies
in genetic mutation screening on an epidemiological scale appear to be the high
accuracy demanded by clinical applications and high per-sample cost. How to
achieve high efficiency in enriching targeted disease genes while keeping a low
cost/sample is a key technical hurdle to overcome. We validated a
cDNA-probe-based approach for capturing exons of a group of genes known to cause
deafness. Polymerase chain reaction amplicons were made from cDNA clones of the
targeted genes and used as bait probes in hybridization for capturing human
genomic DNA (gDNA) fragments. The cDNA library containing the clones of targeted
genes provided a readily available, low-cost, and regenerable source for
producing capture probes with standard molecular biology equipment. Captured
gDNA fragments by our method were sequenced by the Illumina NGS platform.
Results demonstrated that targeted exons captured by our approach achieved
specificity, multiplexicity, uniformity, and depth of coverage suitable for
accurate sequencing applications by the NGS systems. Reliable genotype calls for
various homozygous and heterozygous mutations were achieved. The results were
confirmed independently by conventional Sanger sequencing. The method validated
here could be readily expanded to include all-known deafness genes for
applications such as genetic hearing screening in newborns. The high coverage
depth and cost benefits of the cDNA-probe-based exon capture approach may also
facilitate widespread applications in clinical practices beyond screening
mutations in deafness genes. Pathogenic Alu element insertions are rarely reported, whereas their occurrence
is expected to be much higher. Alu containing alleles are usually out-competed
during the PCR process and consequently undetectable with the classical
screening methods. However, with the introduction of the next generation
sequencing (NGS) technology in the diagnostic field, new opportunities are
emerging. NGS data for a particular genomic region can be seen as the summation
of all the individual sequences (reads) obtained for that region and no longer
as the mean of this sum as it is the case for traditional Sanger sequencing.
Because each single read covering that region is expected to be generated from a
different template molecule, the presence of one single mutant read must
theoretically be sufficient to identify the mutation. However, generation and
identification of mutant reads bearing Alu insertions remains challenging and
several wet/dry bench parameters need to be optimized. Hereby we present the
proof of principle of a NGS-based mutation screening procedure allowing the
detection of inherited Alu insertions within any predefined sequence by
investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2. |
List common features of Shapiro syndrome | Shapiro syndrome is a rare entity, comprising a triad of recurrent hypothermia, hyperhidrosis and congenital agenesis of the corpus callosum. Hypermelatoninemia has also been described in a patient with Shapiro syndrome. | We present a patient diagnosed with Shapiro syndrome without corpus callosum
agenesis. A 4-year-old-girl was admitted to the hospital with complaints of
sweating, cooling, and drowsiness that continued during the last week of her
admission. Attacks occurred almost daily, and lasted for about 1 hour. All
laboratory findings, as well as Holter and echocardiography results, were
normal. Cranial magnetic resoce imaging demonstrated an intact corpus
callosum, and electroencephalography obtained during an attack revealed normal
findings. However, technetium 99m-labeled hexamethylpropylene amine oxime brain
single-photon emission computed tomography indicated increased perfusion in the
right thalamus, basal ganglia, and inferior frontal areas during a hypothermic
period. Although oxcarbazepine reduced the frequency of attacks, they were not
halted completely. The patient responded better to carbamazepine. Melatonin, a major photoperiod-dependent hormone, regulates circadian rhythms
and biological rhythms and acts as a prominent sleep promoter. Symptoms related
to hypermelatoninemia have been reported in individuals supplemented with
melatonin. However, spontaneous endogenous hypermelatoninemia has not been
reported previously. A 6-year-old girl previously diagnosed with Shapiro's
syndrome was admitted to our hospital on several occasions during a 1-year
period with complaints of altered consciousness, syncope, hypothermia and
episodes of sweating. The episodes occurred daily and during sleep and lasted
for 1-6 h. During these episodes, she sweated profusely and felt faint and her
skin was pale and cool. Other complaints included recurrent abdominal pain, urge
incontinence and myopia. She was shown to have hypermelatoninemia (>1,000 pg/ml,
normal range 0-150 pg/ml) during these episodes. The duration of her attacks
decreased with phototherapy and she was successfully treated with propranolol.
To our knowledge, this is the first case of hypermelatoninemia without any
detectable organic pathology. We did not determine the exact mechanism of
hypermelatoninemia in this patient; however, it might have been related to
irregular control of pinealocytes by the suprachiasmatic nucleus or related
pathways. Hypermelatoninemia should be considered in patients with spontaneous
periodic hypothermia and hyperhidrosis, and also in patients with Shapiro's
syndrome. Shapiro syndrome and periodic hypothermia have been reported approximately fifty
times in the literature. Shapiro syndrome is defined as the constellation of
periodic hypothermia and hyperhidrosis along with agenesis of the corpus
callosum by Shapiro et al. in 1969. Periodic hypothermia is a more broad
diagnosis with a number of proposed mechanisms; it occurs in patients without
structural brain abnormalities. Hematologic abnormalities beyond iron-deficiency
anemia have not been documented in any of the reported cases of Shapiro syndrome
or periodic hypothermia. Though accidental and therapeutic hypothermia have been
associated with thrombocytopenia, this is, to our knowledge, the first reported
case of periodic intrinsic hypothermia causing bicytopenia. In this report, we
present the case of a patient with Shapiro syndrome who experienced cyclic
bicytopenia mirroring hypothermic episodes. We address the differential
diagnosis of bicytopenia, review the mechanisms proposed for cytopenias related
to hypothermia, and propose possible mechanisms for the finding in this case. Shapiro syndrome is an extremely rare condition consisting the clinical triad of
recurrent hypothermia, hyperhydrosis and agenesis of the corpus callosum. On the
other hand, reverse Shapiro's sydrome is characterized periodic hyperthermia and
agenesis of the corpus callosum. Here, we describe a 3.5-year-old girl with
complete agenesis of corpus callosum presenting with recurrent fever and
vomiting. She also had hypothermia attacks with accompanying diaphoresis. To the
best of our knowledge, there is no described case with episodes of hyperthermia,
hypothermia, and vomiting associated with agenesis of the corpus callosum.
Recurrent vomiting may be a newly defined symptom associated with these
syndromes. |
List mouse models for autism spectrum disorder (ASD). | Numerous mouse models exists for autism spectrum disorder, such as: BTBR T+tf/J (BTBR), maternal immune, activation (MIA) mouse model of gestational poly(IC) exposure, C58/J and ProSAP1/Shank2. | BACKGROUND: Impairments in social processes characterize one of the core
deficits in autism spectrum disorders (ASD) and accumulating evidence suggests
that oxytocin neurotransmission is implicated in mediating social adaptation in
ASD. Using a mouse model, CD38, a transmembrane protein expressed in immune
cells but also in brain, was found to be critical for social behavior via
regulation of oxytocin secretion. This prompted us to both examine CD38
expression in human lymphoblastoid cell lines (LBC) as well as to test
association between SNPs across the CD38 gene and ASD.
METHODS: LBC’s were derived from 44 ASD lines and 40 "unaffected" parents.
Family-based association (UNPHASED) was examined by genotyping 11 tagging SNPs
spanning the CD38 gene identified using HapMap data in 170 trios. An additional
SNP (rs3796863) associated in a study by Munesue et al. with ASD was also
genotyped.
RESULTS: A highly significant reduction in CD38 expression was observed in
immortalized lymphocytes derived from ASD subjects compared to their
"unaffected" parents (F517.2, P50.00024, df51). Haplotype analysis showed
significant association (permutation corrected) between three and seven locus
haplotypes and DSM IV ASD in low functioning (IQ < 70) subjects.
CONCLUSIONS: The current report supports a role for CD38 in conferring risk for
ASD. Notably, our study shows that this gene is not only associated with low
functioning ASD but that CD38 expression is markedly reduced in LBC derived from
ASD subjects compared to "unaffected" parents, strengthening the connection
between oxytocin and ASD. Although twin studies indicate clear genetic bases of autism spectrum disorder
(ASD), the precise mechanisms through which genetic variations causally result
in ASD are poorly understood. Individuals with 3 Mb and nested 1.5 Mb
hemizygosity of the chromosome 22q11.2 represent genetically identifiable cases
of ASD. However, because more than 30 genes are deleted even in the minimal
deletion cases of 22q11.2 deficiency, the individual 22q11.2 gene(s) responsible
for ASD remain elusive. Here, we examined the impact of constitutive
heterozygosity of Tbx1, a 22q11.2 gene, on the behavioral phenotypes of ASD and
characterized the regional and cellular expression of its mRNA and protein in
mice. Congenic Tbx1 heterozygous (HT) mice were impaired in social interaction,
ultrasonic vocalization, memory-based behavioral alternation, working memory and
thigmotaxis, compared with wild-type (WT) mice. These phenotypes were not due to
non-specific alterations in olfactory function, exploratory behavior, motor
movement or anxiety-related behavior. Tbx1 mRNA and protein were ubiquitously
expressed throughout the brains of C57BL/6J mice, but protein expression was
enriched in regions that postnatally retain the capacity of neurogenesis, and in
fact, postnatally proliferating cells expressed Tbx1. In postnatally derived
hippocampal culture cells of C57BL/6J mice, Tbx1 levels were higher during
proliferation than during differentiation, and expressed in neural progenitor
cells, immature and matured neurons and glial cells. Taken together, our data
suggest that Tbx1 is a gene responsible for the phenotypes of 22q11.2
hemizygosity-associated ASD possibly through its role in diverse cell types,
including postnatally and prenatally generated neurons. Potocki-Lupski syndrome (PTLS; MIM #610883), characterized by neurobehavioral
abnormalities, intellectual disability and congenital anomalies, is caused by a
3.7-Mb duplication in 17p11.2. Neurobehavioral studies determined that ∼70-90%
of PTLS subjects tested positive for autism or autism spectrum disorder (ASD).
We previously chromosomally engineered a mouse model for PTLS (Dp(11)17/+) with
a duplication of a 2-Mb genomic interval syntenic to the PTLS region and
identified consistent behavioral abnormalities in this mouse model. We now
report extensive phenotyping with behavioral assays established to evaluate core
and associated autistic-like traits, including tests for social abnormalities,
ultrasonic vocalizations, perseverative and stereotypic behaviors, anxiety,
learning and memory deficits and motor defects. Alterations were identified in
both core and associated ASD-like traits. Rearing this animal model in an
enriched environment mitigated some, and even rescued selected, neurobehavioral
abnormalities, suggesting a role for gene-environment interactions in the
determination of copy number variation-mediated autism severity. Genetic disorders that present with a high incidence of autism spectrum
disorders (ASD) offer tremendous potential both for elucidating the underlying
neurobiology of ASD and identifying therapeutic drugs and/or drug targets. As a
result, clinical trials for genetic disorders associated with ASD are no longer
a hope for the future but rather an exciting reality whose time has come.
Tuberous sclerosis complex (TSC) is one such genetic disorder that presents with
ASD, epilepsy, and intellectual disability. Cell culture and mouse model
experiments have identified the mTOR pathway as a therapeutic target in this
disease. This review summarizes the advantages of using TSC as model of ASD and
the recent advances in the translational and clinical treatment trials in TSC. BTBR T+tf/J (BTBR) inbred mice are frequently used as a model of autism spectrum
disorders (ASD) as they display social deficits and repetitive behaviors that
resemble the symptoms of the human syndrome. Since deficits on tasks that
measure cognitive (executive) control are also reliable phenotypes in ASD, we
wanted to determine whether executive abilities were compromised in the mouse
model. BTBR mice were trained on two visual discrimination paradigms requiring
differing degrees of cognitive control. BTBR mice performed normally on a visual
discrimination reversal where rule switching was relatively automatic, but were
severely impaired on a task-switch paradigm that required the active use of
contextual information to switch between rules in a flexible manner. The present
findings further characterize the behavior of BTBR mice as a model of ASD.
Moreover, the demonstration of both intact and impaired executive functions in
BTBR mice illustrates the importance of developing new cognitive assays for
comprehensive behavioral assessment of mouse models of human brain disorders. Reduced NMDA-receptor (NMDAR) function has been implicated in the
pathophysiology of neuropsychiatric disease, most strongly in schizophrenia but
also recently in autism spectrum disorders (ASD). To determine the direct
contribution of NMDAR dysfunction to disease phenotypes, a mouse model with
constitutively reduced expression of the obligatory NR1 subunit has been
developed and extensively investigated. Adult NR1(neo-/-) mice show multiple
abnormal behaviors, including reduced social interactions, locomotor
hyperactivity, self-injury, deficits in prepulse inhibition (PPI) and sensory
hypersensitivity, among others. Whereas such phenotypes have largely been
interpreted in the context of schizophrenia, these behavioral abnormalities are
rather non-specific and are frequently present across models of diseases
characterized by negative symptom domains. This study investigated auditory
electrophysiological and behavioral paradigms relevant to autism, to determine
whether NMDAR hypofunction may be more consistent with adult ASD-like
phenotypes. Indeed, transgenic mice showed behavioral deficits relevant to all
core ASD symptoms, including decreased social interactions, altered ultrasonic
vocalizations and increased repetitive behaviors. NMDAR disruption recapitulated
clinical endophenotypes including reduced PPI, auditory-evoked response N1
latency delay and reduced gamma synchrony. Auditory electrophysiological
abnormalities more closely resembled those seen in clinical studies of autism
than schizophrenia. These results suggest that NMDAR hypofunction may be
associated with a continuum of neuropsychiatric diseases, including
schizophrenia and autism. Neural synchrony abnormalities suggest an imbalance of
glutamatergic and GABAergic coupling and may provide a target, along with
behavioral phenotypes, for preclinical screening of novel therapeutics. Autism spectrum disorders (ASD) form a common group of neurodevelopmental
disorders appearing to be under polygenic control, but also strongly influenced
by multiple environmental factors. The brain mechanisms responsible for ASD are
not understood and animal models paralleling related emotional and cognitive
impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice
display behaviors consistent with the three diagnostic categories for ASD. They
show impaired social interaction and communication as well as increased
repetitive behaviors. This review covers much of the data available to date on
BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers,
which could both serve as diagnostic tools and help to design effective
treatments for the behavioral symptoms of ASD. Tuberous sclerosis complex (TSC) is a domit tumor suppressor disorder caused
by mutations in either TSC1 or TSC2. TSC causes substantial neuropathology,
often leading to autism spectrum disorders (ASDs) in up to 60% of patients. The
anatomic and neurophysiologic links between these two disorders are not well
understood. We have generated and characterized a novel TSC mouse model with
Purkinje cell specific Tsc2 loss. These Tsc2f/-;Cre mice exhibit progressive
Purkinje cell degeneration. Since loss of Purkinje cells is a well reported
postmortem finding in patients with ASD, we conducted a series of behavior tests
to asses if Tsc2f/-;Cre mice displayed autistic-like deficits. Tsc2f/-;Cre mice
demonstrated increased repetitive behavior as assessed with marble burying
activity. Using the three chambered apparatus to asses social behavior, we found
that Tsc2f/-;Cre mice showed behavioral deficits, exhibiting no preference
between a stranger mouse and an iimate object, or between a novel and a
familiar mouse. We also detected social deficits in Tsc2f/f;Cre mice, suggesting
that Purkinje cell pathology is sufficient to induce ASD-like behavior.
Importantly, social behavior deficits were prevented with rapamycin treatment.
Altogether, these results demonstrate that loss of Tsc2 in Purkinje cells in a
Tsc2-haploinsufficient background leads to autistic-like behavioral deficits.
These studies provide compelling evidence that Purkinje cell loss and/or
dysfunction may be an important link between TSC and ASD as well as a general
anatomic phenomenon that contributes to the ASD phenotype. Mutations that cause intellectual disability (ID) and autism spectrum disorder
(ASD) are commonly found in genes that encode for synaptic proteins. However, it
remains unclear how mutations that disrupt synapse function impact intellectual
ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine
synapses develop prematurely during the early postnatal period. Premature spine
maturation dramatically enhanced excitability in the developing hippocampus,
which corresponded with the emergence of behavioral abnormalities. Inducing
SYNGAP1 mutations after critical developmental windows closed had minimal impact
on spine synapse function, whereas repairing these pathogenic mutations in
adulthood did not improve behavior and cognition. These data demonstrate that
SynGAP protein acts as a critical developmental repressor of neural excitability
that promotes the development of life-long cognitive abilities. We propose that
the pace of dendritic spine synapse maturation in early life is a critical
determit of normal intellectual development. Autism is a neurodevelopmental disorder characterized by social and
communication impairments and repetitive behaviours. The inbred BTBR T+ tf/J
(BTBR) strain, a putative mouse model of autism, exhibits lower social
interactions, higher repetitive self-grooming levels and unusual pattern of
vocalizations as compared to C57BL/6J strain. First aim of the present study was
to evaluate at adolescence (postnatal days 30-35) male BTBR and C57BL/6J
performances in two different tasks involving either investigation of social
cues (same strain partners) or non social ones (iimate objects). In the
social interaction test, BTBR mice showed a reduction of investigation of the
social partner, due to a selective reduction of head sniffing, associated with a
decrease in ultrasonic vocalizations. By contrast, no strain differences were
detected in object investigations. Second aim of the study was to evaluate adult
male BTBR and C57BL/6J performances in a fear conditioning task. Strain
differences were evident during contextual retest: these strain differences
primarily suggested a lack of behavioural flexibility in BTBR mice (i.e.,
realizing the occurrence of changes in the experimental paradigm). Subsequent
electrophysiological analysis in hippocampal slices from adult BTBR and C57BL/6J
mice revealed a significant reduction of Brain Derived Neurotrophic Factor
(BDNF)-induced potentiation of synaptic transmission in BTBR mice. BDNF and
tyrosine kinase B (TrkB) protein levels measured in the hippocampal region were
also lower in BTBR as compared to C57BL/6J mice. These data confirm the presence
of low levels of direct interaction with social stimuli in BTBR mice at
adolescence, in the absence of any strain difference as for investigation of
physical objects. At adulthood in BTBR mice clear signs of behavioural
inflexibility were evident whereas both biochemical and electrophysiological
data point to decreased BDNF signalling (likely due to a reduction in TrkB
levels) in the hippocampus of this mouse strain. BACKGROUND: Autism spectrum disorders (ASDs) are caused by both genetic and
environmental factors. Mitochondria act to connect genes and environment by
regulating gene-encoded metabolic networks according to changes in the chemistry
of the cell and its environment. Mitochondrial ATP and other metabolites are
mitokines-signaling molecules made in mitochondria-that undergo regulated
release from cells to communicate cellular health and danger to neighboring
cells via purinergic signaling. The role of purinergic signaling has not yet
been explored in autism spectrum disorders.
OBJECTIVES AND METHODS: We used the maternal immune activation (MIA) mouse model
of gestational poly(IC) exposure and treatment with the non-selective purinergic
antagonist suramin to test the role of purinergic signaling in C57BL/6J mice.
RESULTS: We found that antipurinergic therapy (APT) corrected 16 multisystem
abnormalities that defined the ASD-like phenotype in this model. These included
correction of the core social deficits and sensorimotor coordination
abnormalities, prevention of cerebellar Purkinje cell loss, correction of the
ultrastructural synaptic dysmorphology, and correction of the hypothermia,
metabolic, mitochondrial, P2Y2 and P2X7 purinergic receptor expression, and
ERK1/2 and CAMKII signal transduction abnormalities.
CONCLUSIONS: Hyperpurinergia is a fundamental and treatable feature of the
multisystem abnormalities in the poly(IC) mouse model of autism spectrum
disorders. Antipurinergic therapy provides a new tool for refining current
concepts of pathogenesis in autism and related spectrum disorders, and
represents a fresh path forward for new drug development. Despite recent advances in understanding the molecular mechanisms of autism
spectrum disorders (ASD), the current treatments for these disorders are mostly
focused on behavioral and educational approaches. The considerable clinical and
molecular heterogeneity of ASD present a significant challenge to the
development of an effective treatment targeting underlying molecular defects.
Deficiency of SHANK family genes causing ASD represent an exciting opportunity
for developing molecular therapies because of strong genetic evidence for SHANK
as causative genes in ASD and the availability of a panel of Shank mutant mouse
models. In this article, we review the literature suggesting the potential for
developing therapies based on molecular characteristics and discuss several
exciting themes that are emerging from studying Shank mutant mice at the
molecular level and in terms of synaptic function. BACKGROUND: Haploinsufficiency of SHANK3, due to either hemizygous gene deletion
(termed 22q13 deletion syndrome or Phelan-McDermid syndrome) or to gene
mutation, accounts for about 0.5% of the cases of autism spectrum disorder (ASD)
and/or developmental delay, and there is evidence for a wider role for SHANK3
and glutamate signaling abnormalities in ASD and related conditions. Therapeutic
approaches that reverse deficits in SHANK3-haploinsufficiency may therefore be
broadly beneficial in ASD and in developmental delay.
FINDINGS: We observed that daily intraperitoneal injections of human
insulin-like growth factor 1 (IGF-1) over a 2-week period reversed deficits in
hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
signaling, long-term potentiation (LTP), and motor performance that we had
previously reported in Shank3-deficient mice. Positive effects were observed
with an IGF-1 peptide derivative as well.
CONCLUSIONS: We observed significant beneficial effects of IGF-1 in a mouse
model of ASD and of developmental delay. Studies in mouse and human neuronal
models of Rett syndrome also show benefits with IGF-1, raising the possibility
that this compound may have benefits broadly in ASD and related conditions, even
with differing molecular etiology. Given the extensive safety data for IGF-1 in
children with short stature due to primary IGF-1 deficiency, IGF-1 is an
attractive candidate for controlled clinical trials in SHANK3-deficiency and in
ASD. Mouse ultrasonic vocalisations have been often used as a paradigm to extrapolate
vocal communication defects observed in patients with autism spectrum disorders
(ASD). The role of these vocalisations as well as their development, structure
and informational content, however, remain largely unknown. In the present
study, we characterised in depth the emission of pup and adult ultrasonic
vocalisations of wild-type mice and their ProSAP1/Shank2(-/-) littermates
lacking a synaptic scaffold protein mutated in ASD. We hypothesised that the
vocal behaviour of ProSAP1/Shank2(-/-) mice not only differs from the vocal
behaviour of their wild-type littermates in a quantitative way, but also
presents more qualitative abnormalities in temporal organisation and acoustic
structure. We first quantified the rate of emission of ultrasonic vocalisations,
and analysed the organisation of vocalisations sequences using Markov models. We
subsequently measured duration and peak frequency characteristics of each
ultrasonic vocalisation, to characterise their acoustic structure. In wild-type
mice, we found a high level of organisation in sequences of ultrasonic
vocalisations, suggesting a communicative function in this complex system. Very
limited significant sex-related variations were detected in their usage and
acoustic structure, even in adult mice. In adult ProSAP1/Shank2(-/-) mice, we
found abnormalities in the call usage and the structure of ultrasonic
vocalisations. Both ProSAP1/Shank2(-/-) male and female mice uttered less
vocalisations with a different call distribution and at lower peak frequency in
comparison with wild-type littermates. This study provides a comprehensive
framework to characterise abnormalities of ultrasonic vocalisations in mice and
confirms that ProSAP1/Shank2(-/-) mice represent a relevant model to study
communication defects. Increasing clinical and experimental evidence links immune and inflammatory
alterations with the pathogenesis of autism spectrum disorders (ASD). Autistic
individuals show signs of neuroinflammation, altered inflammatory responses, and
immune abnormalities throughout life. Mice injected subcutaneously with 600
mg/kg valproic acid (VPA600) at gestational day 12.5 show reduced social
interaction in adulthood (at 8 weeks of age), and they have been proposed as a
mouse model of autism. Here, we show that these adult animals present signs of
chronic glial activation in the hippocampus and the cerebellum. Moreover, when
they are challenged with a peripheral inflammatory stimulus (intraperitoneal
lipopolysaccharides, LPS), VPA600 animals show an exacerbated inflammatory
response. Two hours after LPS injection, VPA600 animals secrete more
corticosterone to the blood than control mice, and show an increase in the
levels of expression of proinflammatory cytokines in the spleen. After LPS
challenge, VPA600 mice also show signs of increased neuroinflammation compared
with control mice: they have more microglial cells in the hippocampus, and they
show higher levels of proinflammatory cytokines in the cerebellum. Our results
provide evidence of basal neuroinflammation and an altered inflammatory response
in the VPA model of autism. We propose that this model can be used to evaluate
the contribution of inflammatory reactivity to autism-related behaviors. These
studies will contribute to elucidate the role of the inflammatory alterations
observed in ASD individuals. Cognitive abilities are impaired in neurodevelopmental disorders, including
autism spectrum disorder (ASD) and schizophrenia. Preclinical models with strong
endophenotypes relevant to cognitive dysfunctions offer a valuable resource for
therapeutic development. However, improved assays to test higher order cognition
are needed. We employed touchscreen technology to design a complex transitive
inference (TI) assay that requires cognitive flexibility and relational
learning. C57BL/6J (B6) mice with good cognitive skills and BTBR T+tf/J (BTBR),
a model of ASD with cognitive deficits, were evaluated in simple and complex
touchscreen assays. Both B6 and BTBR acquired visual discrimination and
reversal. BTBR displayed deficits on components of TI, when 4 stimuli pairs were
interspersed, which required flexible integrated knowledge. BTBR displayed
impairment on the A > E inference, analogous to the A > E deficit in ASD. B6 and
BTBR mice both reached criterion on the B > D comparison, unlike the B > D
impairment in schizophrenia. These results demonstrate that mice are capable of
complex discriminations and higher order tasks using methods and equipment
paralleling those used in humans. Our discovery that a mouse model of ASD
displays a TI deficit similar to humans with ASD supports the use of the
touchscreen technology for complex cognitive tasks in mouse models of
neurodevelopmental disorders. Neurodevelopmental disorders, including autism spectrum disorder (ASD), are
defined by core behavioral impairments; however, subsets of individuals display
a spectrum of gastrointestinal (GI) abnormalities. We demonstrate GI barrier
defects and microbiota alterations in the maternal immune activation (MIA) mouse
model that is known to display features of ASD. Oral treatment of MIA offspring
with the human commensal Bacteroides fragilis corrects gut permeability, alters
microbial composition, and ameliorates defects in communicative, stereotypic,
anxiety-like and sensorimotor behaviors. MIA offspring display an altered serum
metabolomic profile, and B. fragilis modulates levels of several metabolites.
Treating naive mice with a metabolite that is increased by MIA and restored by
B. fragilis causes certain behavioral abnormalities, suggesting that gut
bacterial effects on the host metabolome impact behavior. Taken together, these
findings support a gut-microbiome-brain connection in a mouse model of ASD and
identify a potential probiotic therapy for GI and particular behavioral symptoms
in human neurodevelopmental disorders. |
What is BioASQ? | BIOASQ assesses the ability of systems to semantically index very large numbers of biomedical scientific articles, and to return concise and user-understandable answers to given natural language questions by combining information from biomedical articles and ontologies. | |
Which are the main functions of G3BP1 and G3BP2 proteins? | The main functions of G3BP1 and/or G3BP2 include translation of interferon stimulated mRNAs during dengue virus infection, initiation of assembly of stress granules, regulation of PMP22 mRNA which was found to affect cell proliferation in breast cancer cells, participation in several signaling pathways involved in growth, differentiation and apoptosis in human tumor cells after overexpression, limit viral replication events during Sindbis virus (SINV) infection, and modulation of p53 and MDM2 activity. | Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose
members cause severe human diseases for which there is no specific treatment. To
ascertain host factors important in the replication of the SINV RNA genome, we
generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with
an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes
isolated from cells infected with the tagged virus revealed 29 associated host
proteins. Of these, 10 proteins were associated only at a later time of
infection (12 h), 14 were associated both early and late, and five were isolated
only at the earlier time (6 h postinfection). These results demonstrate the
dynamic nature of the virus-host interaction that occurs over the course of
infection and suggest that different host proteins may be required for the
multiple functions carried out by nsP4. Two related proteins found in
association with nsP4 at both times of infection, GTPase-activating protein (SH3
domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as
associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for
these host factors in limiting SINV replication events. The present study also
identifies 10 host factors associated with nsP4 6 h after infection that were
not found to be associated with nsP2 or nsP3. These factors are candidates for
playing important roles in the RNA replication process. Identifying host factors
essential for replication should lead to new strategies to interrupt alphavirus
replication. Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever
(DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases
for which there are no available therapies or vaccines. The DENV-2
positive-strand RNA genome contains 5' and 3' untranslated regions (UTRs) that
have been shown to form secondary structures required for virus replication and
interaction with host cell proteins. In order to comprehensively identify host
cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using
the DENV-2 5' and 3' UTRs as "bait", combined with quantitative mass
spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2,
Caprin1, and USP10, implicated in P body (PB) and stress granule (SG) function,
and not previously known to bind DENV RNAs. Indirect immunofluorescence
microscopy showed these proteins to colocalize with the DENV replication
complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious
particles and viral RNA in tissue culture supernatants following DENV infection.
DDX6 interacted with DENV RNA in vivo during infection and in vitro this
interaction was mediated by the DB1 and DB2 structures in the 3' UTR, possibly
by formation of a pseudoknot structure. Additional experiments demonstrate
that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind
to the variable region (VR) in the 3' UTR. These results suggest that the
DENV-2 3' UTR is a site for assembly of PB and SG proteins and, for DDX6,
assembly on the 3' UTR is required for DENV replication. Ras-GTPase-activating protein SH3 domain-binding proteins (G3BP) are
overexpressed in various human tumors and participate in several signaling
pathways involved in growth, differentiation and apoptosis. G3BP interact with
RasGAP (Ras-GTPase activating protein) only in growing cells and depend on Ras
activation, and participate in the Ras signal pathway. Therefore, the blockage
and downregulation of G3BP may be a new strategy for cancer therapy. In this
report, we demonstrate that a novel peptide GAP161 blocked the functions of G3BP
and markedly suppressed HCT116 cell growth through the induction of apoptosis.
The peptide bound with G3BP, which interfered with the interaction of G3BP1 with
RasGAP and further suppressed Ras signaling pathways. GAP161 downregulated G3BP1
and G3BP2 proteins. Similarly, the knockdown of G3BP substantially decreased the
proliferation of HCT116 cells and inhibited Ras signal pathways. Furthermore,
the downregulation of G3BP could enhance cisplatin-induced apoptosis and growth
inhibition of HCT116 cells. We also found that GAP161 suppressed the growth of
BALB/c mice bearing colon CT26 tumors and nude mice bearing HCT116 xenografts.
These results suggest that downregulation of G3BP might be useful in cancer
therapy and that GAP161 is a promising new therapeutic agent for cancers. Upon exposure to various environmental stresses such as arsenite, hypoxia, and
heat shock, cells inhibit their translation and apoptosis and then repair
stress-induced alterations, such as DNA damage and the accumulation of misfolded
proteins. These types of stresses induce the formation of cytoplasmic RNA
granules called stress granules (SGs). SGs are storage sites for the many mRNAs
released from disassembled polysomes under these stress conditions and are
essential for the selective translation of stress-inducible genes.
Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a
component of SGs that initiates the assembly of SGs by forming a multimer. In
this study, we examined the role of G3BP2, a close relative of G3BP1, in SG
formation. Although single knockdown of either G3BP1 or G3BP2 in 293T cells
partially reduced the number of SG-positive cells induced by arsenite, the
knockdowns of both genes significantly reduced the number. G3BP2 formed a
homo-multimer and a hetero-multimer with G3BP1. Moreover, like G3BP1, the
overexpression of G3BP2 induced SGs even without stress stimuli. Collectively,
these results suggest that both G3BP1 and G3BP2 play a role in the formation of
SGs in various human cells and thereby recovery from these cellular stresses. BACKGROUND: Regulation of mRNAs is one way to control protein levels and thereby
important cellular processes such as growth, invasion and apoptosis. G3BPs
constitute a family of mRNA-binding proteins, shown to be overexpressed in
several cancer types, including breast, colon and pancreas cancer. G3BP has been
reported to both stabilize and induce degradation of specific mRNAs.
RESULTS: Here, we show that G3BP1, but not G3BP2, supports proliferation of
several breast cancer cell lines. Global gene expression analyses of G3BP1- and
G3BP2-depleted cells indicate that primarily G3BP1, and much less G3BP2,
influences mRNA expression levels. Peripheral myelin protein 22 (PMP22) was one
gene that was significantly influenced by G3BP1 depletion which led to a 2-3
fold increased expression. Depletion of PMP22 resulted in increased
proliferation and the G3BP1-mediated effect on proliferation was not seen upon
PMP22-depletion.
CONCLUSIONS: This indicates a novel role for G3BP1 in the regulation of cell
proliferation in breast cancer cells, perhaps via a regulatory effect on PMP22
expression. Viral RNA-host protein interactions are critical for replication of
flaviviruses, a genus of positive-strand RNA viruses comprising major
vector-borne human pathogens including dengue viruses (DENV). We examined three
conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue
virus (DENV-2) infection and found them to be novel regulators of the interferon
(IFN) response against DENV-2. The three RBPs were required for the accumulation
of the protein products of several interferon stimulated genes (ISGs), and for
efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and
CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was
antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA),
which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to
profound inhibition of ISG mRNA translation. This work describes a new and
unexpected level of regulation for interferon stimulated gene expression and
presents the first mechanism of action for an sfRNA as a molecular sponge of
anti-viral effectors in human cells. |
What is the definitive treatment for low pressure headache? | epidural blood patch | We describe thickening and contrast enhancement of the intracranial
pachymeninges, revealed by MRI in a patient with presumed low-pressure headache
following dural puncture and a blood patch. The clinical and radiological
abnormalities resolved within 2 weeks. OBJECTIVE: This Evidence-Based Case Management article evaluates and grades the
evidence for two anesthesiology-related interventions: prophylaxis after
unintentional meningeal puncture and treatment of spontaneous intracranial
hypotension (SIH).
METHODS: A search was made of relevant English language clinical studies or
reports pertinent to the topic of low-pressure headache, but excluding the
treatment of meningeal puncture headache.
RESULTS: Thirty-seven case reports, case series, and clinical trials were
included to develop the best available evidence-based recommendations for the
prophylaxis of unintentional meningeal puncture and for the treatment of SIH.
CONCLUSION: The highest quality randomized controlled trials suggest that
prophylactic epidural blood patch (EBP) does not reduce the incidence of
headache after unintentional meningeal puncture. The weight of existing
literature supports EBP as an initial treatment of SIH, although its
effectiveness does not approach that seen when EBP is used to treat meningeal
puncture headache. Women presenting with low pressure post-partum headache following neuraxial
techniques are frequently offered an epidural blood patch, despite its inherent
risks. We present two parturients with classical symptoms of low-pressure
headache, who each received neuraxial labour analgesia without a documented
dural puncture with a Tuohy needle. Both parturients were successfully managed
using acupuncture rather than an epidural blood patch. Low pressure headache typically occurs as a complication of dural puncture.
"Spontaneous" low pressure headache is a relatively rare but under-recognised
cause of intractable headache. Clinical suspicion of this condition warrants
imaging of the brain to confirm the diagnosis; spinal imaging may be needed to
identify the site of the leak. Epidural blood patching may be necessary to seal
the leak - CT fluoroscopy may be helpful in delivering the patch directly to the
site of the leak. Surgical intervention may be required in intractable cases. We
describe a patient with spontaneous intracranial hypotension and review the
clinical and radiological features of this syndrome. A 32-year-old woman presented with low pressure headache 3 days after delivery
of her baby. An assessment of postdural puncture headache was made. This was
initially treated with analgesia, caffeine, and fluids for the presumed
cerebrospinal fluid (CSF) leak. The woman was readmitted two days after her
hospital discharge with generalised seizures. A brain scan showed features of
intracranial hypotension, and she was treated for CSF leak using an epidural
blood patch. Her symptoms worsened and three days later, she developed a left
homonymous quadrantanopia. An MRI scan confirmed a right parietal haematoma with
evidence of isolated cortical vein thrombosis (ICVT). |
What is the role of the histidine rich calcium binding protein (HRC) in cardiomyopathy? | The histidine-rich Ca-binding protein (HRC), a 165 kDa sarcoplasmic reticulum (SR) protein, regulates SR Ca cycling during excitation–contraction coupling. HRC mutations or polymorphisms lead to cardiac dysfunction. The Ser96Ala genetic variant of HRC is associated with life-threatening ventricular arrhythmias and sudden death in idiopathic dilated cardiomyopathy (DCM). | OBJECTIVE: The histidine-rich Ca-binding protein (HRC) is a Ca-storage protein
in cardiac sarcoplasmic reticulum. Recent transgenic studies revealed that this
protein inhibits the maximal rates of sarcoplasmic reticulum Ca-transport,
leading to cardiac dysfunction. In view of the role of sarcoplasmic reticulum
Ca-cycling in myocardial ischemia/reperfusion injury, we designed this study to
gain further insight into the role of HRC during ischemia/reperfusion.
METHODS AND RESULTS: The transgenic mouse model with cardiac-specific
overexpression of HRC was utilized and cardiac contractile parameters were
assessed before and after ischemia/reperfusion injury by Langendorff perfusion.
After a 20-min stabilization period, the hearts were subjected to 40 min of
global ischemia, followed by 60 min of reperfusion. We found that although
transgenic (TG) hearts showed depressed cardiac function (25%) compared to wild
types (WTs) at baseline, they exhibited better recovery of left ventricular
developed pressure (86.6+/-2.6% in TGs vs. 58.3+/-4.0% in WTs of pre-ischemic
values, P<0.05) and higher rates of contraction and relaxation after
ischemia/reperfusion than WTs. This improvement was accompanied by smaller
infarcts (23.1+/-1.7% in TGs vs. 41.1+/-2.5% in WTs of infarct region-to-risk
region ratio, P<0.05) and lower creatine kinase release. Notably, the extent of
apoptotic cell death was significantly attenuated, as evidenced by decreased DNA
fragmentation, upregulation of the antiapoptotic protein Bcl-2, and
downregulation of the active caspases (3, 9 and 12) following
ischemia/reperfusion in TG hearts, compared with WTs. Extension of these studies
to an in vivo model of 30-min myocardial ischemia, via coronary artery
occlusion, followed by 24-h reperfusion, showed that the infarct region-to-risk
region ratio was 9+/-0.9% in TGs, compared with 20.4+/-2.9% in WTs (P<0.05).
CONCLUSIONS: Our findings suggest that increased cardiac HRC expression protects
against ischemia/reperfusion injury in the heart, resulting in improved recovery
of function and reduced infarction. AIMS: To investigate whether genetic variants of the histidine-rich calcium
(HRC)-binding protein are associated with idiopathic dilated cardiomyopathy
(DCM) and its progression.
METHODS AND RESULTS: We screened 123 idiopathic DCM patients and 96 healthy
individuals by single-strand conformation polymorphism analysis and direct
sequencing for genetic variants in HRC. Six polymorphisms were detected:
Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (-/GAG),
Asp261del (GAT/-), and an in-frame insertion of 51 amino acids at His321. The
analysis of their frequencies did not reveal any significant correlation with
DCM development. However, the Ser96Ala polymorphism exhibited a statistically
significant correlation with the occurrence of life-threatening ventricular
arrhythmias. During a follow-up of 4.02 +/- 2.4 years, the risk for ventricular
arrhythmias was higher (HR, 9.620; 95% CI, 2.183-42.394; P = 0.003) in the
Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable
Cox regression analysis, the Ser96Ala polymorphism was the only significant
genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI,
0.838-20.967; P = 0.018).
CONCLUSION: The Ser96Ala genetic variant of HRC is associated with
life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an
independent predictor of susceptibility to arrhythmogenesis in the setting of
DCM. |
What is the incidence of sudden cardiac death among young athletes? | the incidence of sudden cardiac death among young athletes ranges from 0.5 to 3 per 100,000 athletes per year . | Sudden cardiac death in a young athlete is a tragic and marking event, even
though the media attention it gets is more important than its incidence
(1-2/100000 per year). The main etiology is hypertrophic cardiomyopathy,
followed by coronary artery anomalies. Sometimes signs of myocarditis are found
at autopsy. The pre-participation examination recommandations ask for oriented
history and physical examination, routine EKG being controversial. A systematic
screening program in Italy has reduced the occurrence of such deaths, which
statistics are non-existant in Switzerland. It would be appropriate to implement
a standardized pre-participation examination, as well as to make sure that the
trainers, teachers and athletes themselves are able to recognize the frequent
warning symptoms. BACKGROUND: The problem of sudden death in the young is currently debated and
measures for prevention are being evaluated worldwide. Information on the
incidence and causes of sudden (cardiac) death in the young (1-40 years) is
essential for the development of these preventive strategies.
METHODS: Incidence estimates and causes of death were determined using death
certificate data of out-of-hospital sudden deaths recorded by Statistics
Netherlands from 1996 to 2006. To define sudden death, International
Classification of Diseases codes were selected based on a systematic review of
the literature assessing the most common causes of sudden death in the young.
RESULTS: The incidence of sudden death was 2.07 (2.06-2.07 per 100 000
person-years). The incidence was higher for men (2.86 per 100 000 person-years)
than for women (1.24 per 100 000 person-years) and increased by age. The
majority of sudden deaths was of cardiac origin. Sudden cardiac death incidence
was 1.62 (1.61-1.62 per 100 000 person-years). In 9% the cause of death remained
unexplained.
CONCLUSION: The incidence of sudden death in the young is 2.07 per 100 000
person-years. Treatable cardiac causes (such as coronary atherosclerosis and
inherited cardiac diseases) are often underlying for the sudden death. This
information is helpful in the development of preventive strategies. The incidence of sudden cardiac death (SCD) among young athletes is estimated to
be 1-3 per 100,000 person years, and may be underestimated. The risk of SCD in
athletes is higher than in non-athletes because of several factors associated
with sports activity that increase the risk in people with an underlying
cardiovascular abnormality. A clear gender difference in the incidence of SCD
exists in young athletes, with the risk in male athletes being up to 9 times
higher than in female athletes. The most common causes of SCD in young athletes
is underlying inherited/congenital cardiac disease, such as cardiomyopathies,
congenital coronary anomalies and ion channelopathies. Blunt chest trauma also
may cause ventricular fibrillation in a structurally normal heart, known as
commotio cordis. Although geographical differences in the causes of SCD in young
athletes have been reported, these disparities are more likely to be related to
the type and implementation of pre-participation screening leading to the
identification of athletes at risk, rather than reflecting a truly different
ethiology. More studies are needed to clarify the role of ethnicity in the
prevalence of diseases known to cause SCD in young athletes. Sudden cardiac death is the leading cause of mortality among young athletes with
an incidence of 1-2 per 100,000 athletes per annum. It is described as 'an event
that is non-traumatic, non-violent, unexpected, and resulting from sudden
cardiac arrest within six hours of previously witnessed normal health'. Most
predisposed athletes have no symptoms and there is no warning for the impending
tragic event. The majority of cases are caused by an underlying structural
cardiac abnormality, most commonly hypertrophic cardiomyopathy. More recently,
the understanding of non-structural causes such as long QT syndrome and Brugada
syndrome has grown and diagnostic criteria have been developed. This review
presents the known aetiologies of sudden cardiac death among athletes and
outlines their identification and management including implications for future
sporting participation as laid out in the consensus documents produced by the
European Society of Cardiology and the 36th Bethesda Conference. |
What is the effect of amitriptyline in the mdx mouse model of Duchenne muscular dystrophy? | Amitriptyline is efficacious in ameliorating muscle inflammation and depressive symptoms in the mdx mouse model of Duchenne muscular dystrophy | Mutations in the structural protein dystrophin underlie muscular dystrophies
characterized by progressive deterioration of muscle function.
Dystrophin-deficient mdx mice are considered a model for Duchenne muscular
dystrophy (DMD). Individuals with DMD are also susceptible to mood disorders,
such as depression and anxiety. Therefore, the study objectives were to
investigate the effects of the tricyclic antidepressant amitriptyline on mood,
learning, central cytokine expression and skeletal muscle inflammation in mdx
mice. Amitriptyline-induced effects (10 mg kg(-1) daily s.c. injections,
25 days) on the behaviour of mdx mice were investigated using the open field
arena and tail suspension tests. The effects of chronic amitriptyline treatment
on inflammatory markers were studied in the muscle and plasma of mdx mice, and
mood-associated monoamine and cytokine concentrations were measured in the
amygdala, hippocampus, prefrontal cortex, striatum, hypothalamus and midbrain.
The mdx mice exhibited increased levels of anxiety and depressive-like behaviour
compared with wild-type mice. Amitriptyline treatment had anxiolytic and
antidepressant effects in mdx mice associated with elevations in serotonin
levels in the amygdala and hippocampus. Inflammation in mdx skeletal muscle
tissue was also reduced following amitriptyline treatment as indicated by
decreased immune cell infiltration of muscle and lower levels of the
pro-inflammatory cytokines tumour necrosis factor-α and interleukin-6 in the
forelimb flexors. Interleukin-6 mRNA expression was remarkably reduced in the
amygdala of mdx mice by chronic amitriptyline treatment. Positive effects of
amitriptyline on mood, in addition to its anti-inflammatory effects in skeletal
muscle, may make it an attractive therapeutic option for individuals with DMD. |
Which post-translational histone modifications are characteristic of facultative heterochromatin? | Nuclear VapB methyltransferase diminishes the establishment of facultative heterochromatin by decreasing histone 3 lysine 9 trimethylation (H3K9me3). Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin. | X inactivation is associated with chromosome-wide establishment of inactive
chromatin. Although this is classically regarded as facultative heterochromatin
that is uniform in nature, the exact distribution of associated epigenetic marks
is not well defined. Here we have analysed histone modifications in human
somatic cells within two selected regions of the X chromosome. Intergenic,
coding and promoter regions are segregated into differentially marked chromatin.
H3K27me3 is most prominent in intergenic and silenced coding regions, but is
associated with some active coding regions as well. Histone H3/H4 acetylation
and H3K4me3 are locally enriched at promoter regions but do not necessarily mark
continuing transcription. Remarkably, H3K9me3 is predomit in coding regions
of active genes, a phenomenon that is not restricted to the X chromosome. These
results argue against the exclusiveness of individual marks to heterochromatin
or euchromatin, but rather suggest that composite patterns of interdependent or
mutually exclusive modifications together signal the gene expression status. The mammalian genome contains numerous regions known as facultative
heterochromatin, which contribute to transcriptional silencing during
development and cell differentiation. We have analyzed the pattern of histone
modifications associated with facultative heterochromatin within the mouse
imprinted Snurf-Snrpn cluster, which is homologous to the human Prader-Willi
syndrome genomic region. We show here that the maternally inherited Snurf-Snrpn
3-Mb region, which is silenced by a potent transcription repressive mechanism,
is uniformly enriched in histone methylation marks usually found in constitutive
heterochromatin, such as H4K20me3, H3K9me3, and H3K79me3. Strikingly, we found
that trimethylated histone H3 at lysine 36 (H3K36me3), which was previously
identified as a hallmark of actively transcribed regions, is deposited onto the
silenced, maternally contributed 3-Mb imprinted region. We show that H3K36me3
deposition within this large heterochromatin domain does not correlate with
transcription events, suggesting the existence of an alternative pathway for the
deposition of this histone modification. In addition, we demonstrate that
H3K36me3 is markedly enriched at the level of pericentromeric heterochromatin in
mouse embryonic stem cells and fibroblasts. This result indicates that H3K36me3
is associated with both facultative and constitutive heterochromatin. Our data
suggest that H3K36me3 function is not restricted to actively transcribed regions
only and may contribute to the composition of heterochromatin, in combination
with other histone modifications. Mature rod photoreceptor cells contain very small nuclei with tightly condensed
heterochromatin. We observed that during mouse rod maturation, the nucleosomal
repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult
retina. At the same time, the total level of linker histone H1 increased
reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a
dramatic increase in H1c. Genetic elimination of the histone H1c gene is
functionally compensated by other histone variants. However, retinas in
H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei,
decreased heterochromatin area, and notable morphological changes suggesting
that the process of chromatin condensation and rod cell structural integrity are
partly impaired. In triple knock-outs, nuclear chromatin exposed several
epigenetic histone modification marks masked in the wild type chromatin.
Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate
that during development linker histone plays a role in establishing the
facultative heterochromatin territory and architecture in the nucleus. During
retina development, the H1c gene and its promoter acquired epigenetic patterns
typical of rod-specific genes. Our data suggest that histone H1c gene expression
is developmentally up-regulated to promote facultative heterochromatin in mature
rod photoreceptors. BACKGROUND: Supernumerary chromosomes have been found in many organisms. In
fungi, these "accessory" or "dispensable" chromosomes are present at different
frequencies in populations and are usually characterized by higher repetitive
DNA content and lower gene density when compared to the core chromosomes. In the
reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete
accessory chromosomes have been found. So far, no functional role has been
assigned to these chromosomes; however, they have existed as separate entities
in the karyotypes of Zymoseptoria species over evolutionary time. In this study,
we addressed what-if anything-distinguishes the chromatin of accessory
chromosomes from core chromosomes. We used chromatin immunoprecipitation
combined with high-throughput sequencing ("ChIP-seq") of DNA associated with the
centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and
ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and
trimethylated H3K27 to determine the relative distribution and proportion of
euchromatin, obligate and facultative heterochromatin, respectively.
RESULTS: Centromeres of the eight accessory chromosomes have the same sequence
composition and structure as centromeres of the 13 core chromosomes and they are
of similar length. Unlike those of most other fungi, Z. tritici centromeres are
not composed entirely of repetitive DNA; some centromeres contain only unique
DNA sequences, and bona fide expressed genes are located in regions enriched
with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici
do not cluster into a single chromocenter during interphase. We found
dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory
chromosomes, consistent with the twofold higher proportion of repetitive DNA and
poorly transcribed genes. In contrast, no single histone modification tested
here correlated with the distribution of centromeric nucleosomes.
CONCLUSIONS: All centromeres are similar in length and composed of a mixture of
unique and repeat DNA, and most contain actively transcribed genes. Centromeres,
subtelomeric regions or telomere repeat length cannot account for the
differences in transfer fidelity between core and accessory chromosomes, but
accessory chromosomes are greatly enriched in nucleosomes with H3K27
trimethylation. Genes on accessory chromosomes appear to be silenced by
trimethylation of H3K9 and H3K27. |
Mutations in which genes have been associated with Aicardi-Goutieres syndrome? | Aicardi-Goutières syndrome (AGS) is an inflammatory disorder caused by mutations in any of six genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR). | Aicardi-Goutieres syndrome (AGS) is a genetic encephalopathy whose clinical
features mimic those of acquired in utero viral infection. AGS exhibits locus
heterogeneity, with mutations identified in genes encoding the 3'-->5'
exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To
define the molecular spectrum of AGS, we performed mutation screening in
patients, from 127 pedigrees, with a clinical diagnosis of the disease.
Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in
31, 3, 47, and 18 families, respectively. In five families, we identified an
RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease
occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no
mutations were found. Null mutations were common in TREX1, although a specific
missense mutation was observed frequently in patients from northern Europe.
Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We
identified an RNASEH2C founder mutation in 13 Pakistani families. We also
collected clinical data from 123 mutation-positive patients. Two clinical
presentations could be delineated: an early-onset neonatal form, highly
reminiscent of congenital infection seen particularly with TREX1 mutations, and
a later-onset presentation, sometimes occurring after several months of normal
development and occasionally associated with remarkably preserved neurological
function, most frequently due to RNASEH2B mutations. Mortality was correlated
with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations
versus 8.0% RNASEH2B mutation-positive patients were known to have died
(P=.001). Our analysis defines the phenotypic spectrum of AGS and suggests a
coherent mutation-screening strategy in this heterogeneous disorder.
Additionally, our data indicate that at least one further AGS-causing gene
remains to be identified. Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been
identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if
the AGS RNaseH2 mutations result in the loss of nuclease activity, the human
wild-type RNaseH2 and four mutant complexes that constitute the majority of
mutations identified in AGS patients have been prepared and tested for
ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2
complexes are intact. Furthermore, the ribonuclease H activities of the mutant
complexes are indistinguishable from the wild-type enzyme with the exception of
the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered
nuclease specificity. These data indicate that the mechanism of RNaseH2
dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity
and points to a more complex mechanism perhaps mediated through altered
interactions with as yet identified nucleic acids or protein partners. TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded
DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and
Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit
domit immune dysfunction phenotypes. Failure to appropriately disassemble
genomic DNA during normal cell death processes could lead to persistent DNA
signals that trigger the innate immune response and autoimmunity. We tested this
concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease
locates 3' termini generated by endonucleases and degrades the nicked DNA
polynucleotide. A competition assay was designed using TREX1 domit mutants
and variants to demonstrate that an intact DNA binding process, coupled with
dysfunctional chemistry in the active sites, explains the domit phenotypes in
TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175
positioned adjacent to the active sites act with the Arg-128 residues positioned
in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for
entry into the active sites. Metal-dependent ssDNA binding in the active sites
of the catalytically inactive domit TREX1 mutants contributes to DNA
retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus,
the domit disease genetics exhibited by the TREX1 D18N, D200N, and D200H
alleles parallel precisely the biochemical properties of these TREX1 dimers
during dsDNA degradation of plasmid and chromatin DNA in vitro. These results
support the concept that failure to degrade genomic dsDNA is a principal pathway
of immune activation in TREX1-mediated autoimmune disease. Aicardi-Goutières syndrome is a genetically determined infantile encephalopathy,
manifesting as progressive microcephaly, psychomotor retardation, and in ∼25% of
patients, death in early childhood. Aicardi-Goutières syndrome is caused by
mutations in any of the genes encoding TREX1, RNASEH2-A, -B, -C and SAMHD1, with
protein dysfunction hypothesized to result in the accumulation of nucleic acids
within the cell, thus triggering an autoinflammatory response with increased
interferon-α production. Astrocytes have been identified as a major source of
interferon-α production in the brains of patients with Aicardi-Goutières
syndrome. Here, we study the effect of interferon-α treatment on astrocytes
derived from immortalized human neural stem cells. Chronic interferon-α
treatment promoted astrocyte activation and a reduction in cell proliferation.
Moreover, chronic exposure resulted in an alteration of genes and proteins
involved in the stability of white matter (ATF4, eIF2Bα, cathepsin D, cystatin
F), an increase of antigen-presenting genes (human leukocyte antigen class I)
and downregulation of pro-angiogenic factors and other cytokines (vascular
endothelial growth factor and IL-1). Interestingly, withdrawal of interferon-α
for 7 days barely reversed these cellular alterations, demonstrating that the
interferon-α mediated effects persist over time. We confirmed our in vitro
findings using brain samples from patients with Aicardi-Goutières syndrome. Our
results support the idea of interferon-α as a key factor in the pathogenesis of
Aicardi-Goutières syndrome relating to the observed leukodystrophy and
microangiopathy. Because of the sustained interferon-α effect, even after
withdrawal, therapeutic targets for Aicardi-Goutières syndrome, and other
interferon-α-mediated encephalopathies, may include downstream interferon-α
signalling cascade effectors rather than interferon-α alone. BACKGROUND: Aicardi-Goutières syndrome (AGS) is an inflammatory disorder caused
by mutations in any of six genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1,
and ADAR). The disease is severe and effective treatments are urgently needed.
We investigated the status of interferon-related biomarkers in patients with AGS
with a view to future use in diagnosis and clinical trials.
METHODS: In this case-control study, samples were collected prospectively from
patients with mutation-proven AGS. The expression of six interferon-stimulated
genes (ISGs) was measured by quantitative PCR, and the median fold change, when
compared with the median of healthy controls, was used to create an interferon
score for each patient. Scores higher than the mean of controls plus two SD
(>2·466) were designated as positive. Additionally, we collated historical data
for interferon activity, measured with a viral cytopathic assay, in CSF and
serum from mutation-positive patients with AGS. We also undertook neutralisation
assays of interferon activity in serum, and looked for the presence of
autoantibodies against a panel of interferon proteins.
FINDINGS: 74 (90%) of 82 patients had a positive interferon score (median 12·90,
IQR 6·14-20·41) compared with two (7%) of 29 controls (median 0·93, IQR
0·57-1·30). Of the eight patients with a negative interferon score, seven had
mutations in RNASEH2B (seven [27%] of all 26 patients with mutations in this
gene). Repeat sampling in 16 patients was consistent for the presence or absence
of an interferon signature on 39 of 41 occasions. Interferon activity (tested in
147 patients) was negatively correlated with age (CSF, r=-0·604; serum,
r=-0·289), and was higher in CSF than in serum in 104 of 136 paired samples.
Neutralisation assays suggested that measurable antiviral activity was related
to interferon α production. We did not record significantly increased
concentrations of autoantibodies to interferon subtypes in patients with AGS, or
an association between the presence of autoantibodies and interferon score or
serum interferon activity.
INTERPRETATION: AGS is consistently associated with an interferon signature,
which is apparently sustained over time and can thus be used to differentiate
patients with AGS from controls. If future studies show that interferon status
is a reactive biomarker, the measurement of an interferon score might prove
useful in the assessment of treatment efficacy in clinical trials.
FUNDING: European Union's Seventh Framework Programme; European Research
Council. Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized
by the presence of nucleic acid- and protein-targeting autoantibodies and an
aberrant type I IFN expression signature. Aicardi-Goutières syndrome (AGS) is an
autosomal-recessive encephalopathy in children that is characterized by
mutations in numerous nucleic acid repair enzymes and elevated IFN levels.
Phenotypically, patients with AGS and SLE share many similarities. Ribonuclease
H2 (RNase H2) is a nucleic acid repair enzyme that removes unwanted
ribonucleotides from DNA. In this issue of the JCI, Günther and colleagues
provide an in-depth investigation of the mechanisms underlying the link between
defective removal of ribonucleotides in AGS and SLE, and these findings will
likely serve as a strong springboard to provide novel therapeutic inroads. |
List bacteria that may be useful in uranium bioremediation. | The main bacteria studied in uranium bioremediation are Geobacteraceae. Other bacteria are:
Firmicutes,
Shewanella oneidensis
Pseudomonas aeruginosa
Anaeromyxobacter dehalogenans
strain Rf4T | Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows
promise as a strategy for immobilizing uranium in uranium-contaminated
subsurface environments. In order to learn more about which microorganisms might
be involved in U(VI) reduction in situ, the changes in the microbial community
when U(VI) reduction was stimulated with the addition of acetate were monitored
in sediments from three different uranium-contaminated sites in the floodplain
of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction
was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of
microorganisms in the family Geobacteraceae, which are known U(VI)- and
Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III)
reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the
16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial
PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA
sequences in control sediments that were not amended with acetate and in which
U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these
Geobacteraceae sequences were most similar to sequences from Desulfuromonas
species, with the remainder being most closely related to Geobacter species.
Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR
and TaqMan analyses indicated that the number of Geobacteraceae sequences
increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III)
reduction in the acetate-amended sediments from the three sites. No increase in
Geobacteraceae sequences was observed in control sediments. In contrast to the
predomice of Geobacteraceae sequences, no sequences related to other known
Fe(III)-reducing microorganisms were detected in sediments. These results
compare favorably with an increasing number of studies which have demonstrated
that Geobacteraceae are important components of the microbial community in a
diversity of subsurface environments in which Fe(III) reduction is an important
process. The combination of these results with the finding that U(VI) reduction
takes place during Fe(III) reduction and prior to sulfate reduction suggests
that Geobacteraceae will be responsible for much of the Fe(III) and U(VI)
reduction during uranium bioremediation in these sediments. Shewanella oneidensis is an important model organism for bioremediation studies
because of its diverse respiratory capabilities, conferred in part by
multicomponent, branched electron transport systems. Here we report the
sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair
circular chromosome with 4,758 predicted protein-encoding open reading frames
(CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first
Shewanella lambda-like phage, providing a potential tool for further genome
engineering. Genome analysis revealed 39 c-type cytochromes, including 32
previously unidentified in S. oneidensis, and a novel periplasmic [Fe]
hydrogenase, which are integral members of the electron transport system. This
genome sequence represents a critical step in the elucidation of the pathways
for reduction (and bioremediation) of pollutants such as uranium (U) and
chromium (Cr), and offers a starting point for defining this organism's complex
electron transport systems and metal ion-reducing capabilities. The proposed research will investigate two microbial communities that are of
direct relevance to Department of Energy interests. One is the microbial
community associated with the in situ bioremediation of uranium-contaminated
groundwater. The second is a microbial community that harvests energy from waste
organic matter in the form of electricity. These studies will address DOE needs
for (1) remediation of metals and radionuclides at DOE sites and (2) the
development of cleaner forms of energy and biomass conversion to energy. Our
previous studies have demonstrated that the microbial communities involved in
uranium bioremediation and energy harvesting are both dominated by
microorganisms in the family Geobacteraceae and that the organisms in this
family are responsible for uranium bioremediation and electron transfer to
electrodes. The initial objectives of this study are to (1) describe the genetic
potential of the Geobacteraceae that predominate in the environments of
interest; (2) identify conserved patterns of gene expression within the
Geobacteraceae family in response to a range of environmental conditions; (3)
begin to identify mechanisms controlling the expression of key genes related to
survival, growth, and activity in subsurface environments and on electrodes; and
(4) use the results from subobjectives 1-3 to develop a conceptual model for
predicting gene expression of Geobacteraceae in the environments of interest.
This will serve as the basis for a subsequent simulation model of the growth and
activity of Geobacteraceae in the subsurface and on electrodes. Although stimulation of dissimilatory metal reduction to promote the reductive
precipitation of uranium has been shown to successfully remove uranium from some
aquifer sediments, the organisms in the family Geobacteraceae that have been
found to be associated with metal reduction in previous studies are not known to
grow at the high salinities found in some uranium-contaminated groundwaters.
Studies with a highly saline uranium-contaminated aquifer sediment demonstrated
that the addition of acetate could stimulate the removal of U(VI) from the
groundwater. This removal was associated with an enrichment in microorganisms
most closely related to Pseudomonas and Desulfosporosinus species. A system for growing Geobacter sulfurreducens under anaerobic conditions in
chemostats was developed in order to study the physiology of this organism under
conditions that might more closely approximate those found in the subsurface
than batch cultures. Geobacter sulfurreducens could be cultured under
acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron
acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was
threefold higher with fumarate as the electron acceptor than with Fe(III),
despite the lower mid-point potential of the fumarate/succinate redox couple.
When growth was limited by availability of fumarate, high steady-state
concentrations were detected, suggesting that fumarate is unlikely to be an
important electron acceptor in sedimentary environments. The half-saturation
constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that
the growth of Geobacter species is likely to be acetate limited in most
subsurface sediments, but that when millimolar quantities of acetate are added
to the subsurface in order to promote the growth of Geobacter for bioremediation
applications, this should be enough to overcome any acetate limitations. When
the availability of electron acceptors, rather than acetate, limited growth, G.
sulfurreducens was less efficient in incorporating acetate into biomass but had
higher respiration rates, a desirable physiological characteristic when adding
acetate to stimulate the activity of Geobacter species during in situ uranium
bioremediation. These results demonstrate that the ability to study the growth
of G. sulfurreducens under steady-state conditions can provide insights into its
physiological characteristics that have relevance for its activity in a
diversity of sedimentary environments. The geochemistry and microbiology of a uranium-contaminated subsurface
environment that had undergone two seasons of acetate addition to stimulate
microbial U(VI) reduction was examined. There were distinct horizontal and
vertical geochemical gradients that could be attributed in large part to the
manner in which acetate was distributed in the aquifer, with more reduction of
Fe(III) and sulfate occurring at greater depths and closer to the point of
acetate injection. Clone libraries of 16S rRNA genes derived from sediments and
groundwater indicated an enrichment of sulfate-reducing bacteria in the order
Desulfobacterales in sediment and groundwater samples. These samples were
collected nearest the injection gallery where microbially reducible Fe(III)
oxides were highly depleted, groundwater sulfate concentrations were low, and
increases in acid volatile sulfide were observed in the sediment. Further
down-gradient, metal-reducing conditions were present as indicated by
intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and
increased abundance of 16S rRNA gene sequences belonging to the dissimilatory
Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI)
reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene
sequences in both groundwater and sediment; however, the sites at which these
maxima occurred were spatially separated within the aquifer. The substantial
microbial and geochemical heterogeneity at this site demonstrates that attempts
should be made to deliver acetate in a more uniform manner and that closely
spaced sampling intervals, horizontally and vertically, in both sediment and
groundwater are necessary in order to obtain a more in-depth understanding of
microbial processes and the relative contribution of attached and planktonic
populations to in situ uranium bioremediation. Limitations on the availability of Fe(III) as an electron acceptor are thought
to play an important role in restricting the growth and activity of Geobacter
species during bioremediation of contaminated subsurface environments, but the
possibility that these organisms might also be limited in the subsurface by the
availability of iron for assimilatory purposes was not previously considered
because copious quantities of Fe(II) are produced as the result of Fe(III)
reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of
a three-gene cluster consisting of homologues of two iron-dependent regulators,
fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II)
uptake protein. This cluster appears to be conserved among members of the
Geobacteraceae and was detected in several environments. Expression of the
fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat
cultures. The number of Geobacteraceae feoB transcripts in groundwater samples
from a site undergoing in situ uranium bioremediation was relatively high until
the concentration of dissolved Fe(II) increased near the end of the field
experiment. These results suggest that, because much of the Fe(II) is
sequestered in solid phases, Geobacter species, which have a high requirement
for iron for iron-sulfur proteins, may be limited by the amount of iron
available for assimilatory purposes. These results demonstrate the ability of
transcript analysis to reveal previously unsuspected aspects of the in situ
physiology of microorganisms in subsurface environments. In order to elucidate the potential mechanisms of U(VI) reduction for the
optimization of bioremediation strategies, the structure-function relationships
of microbial communities were investigated in microcosms of subsurface materials
cocontaminated with radionuclides and nitrate. A polyphasic approach was used to
assess the functional diversity of microbial populations likely to catalyze
electron flow under conditions proposed for in situ uranium bioremediation. The
addition of ethanol and glucose as supplemental electron donors stimulated
microbial nitrate and Fe(III) reduction as the predomit terminal
electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction
overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with
sulfate reduction but preceded Fe(III) reduction in the ethanol treatments.
Phyllosilicate clays were shown to be the major source of Fe(III) for microbial
respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and
Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs
observed in biostimulated microcosms and were affiliated with the genera
Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas.
Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal
were observed in ethanol-amended treatments compared to the results in
glucose-amended treatments. Quantification of citrate synthase (gltA) levels
demonstrated a stimulation of Geobacteraceae activity during metal reduction in
carbon-amended microcosms, with the highest expression observed in the glucose
treatment. Phylogenetic analysis indicated that the active FeRB share high
sequence identity with Geobacteraceae members cultivated from contaminated
subsurface environments. Our results show that the functional diversity of
populations capable of U(VI) reduction is dependent upon the choice of electron
donor. Previous field studies on in situ bioremediation of uranium-contaminated
groundwater in an aquifer in Rifle, Colorado identified two distinct phases
following the addition of acetate to stimulate microbial respiration. In phase
I, Geobacter species are the predomit organisms, Fe(III) is reduced, and
microbial reduction of soluble U(VI) to insoluble U(IV) removes uranium from the
groundwater. In phase II, Fe(III) is depleted, sulfate is reduced, and
sulfate-reducing bacteria predominate. Long-term monitoring revealed an
unexpected third phase during which U(VI) removal continues even after acetate
additions are stopped. All three of these phases were successfully reproduced in
flow-through sediment columns. When sediments from the third phase were heat
sterilized, the capacity for U(VI) removal was lost. In the live sediments U(VI)
removed from the groundwater was recovered as U(VI) in the sediments. This
contrasts to the recovery of U(IV) in sediments resulting from the reduction of
U(VI) to U(IV) during the Fe(III) reduction phase in acetate-amended sediments.
Analysis of 16S rRNA gene sequences in the sediments in which U(VI) was being
adsorbed indicated that members of the Firmicutes were the predomit organisms
whereas no Firmicutes sequences were detected in background sediments which did
not have the capacity to sorb U(VI), suggesting that the U(VI) adsorption might
be due to the presence of these living organisms or at least their intact cell
components. This unexpected enhanced adsorption of U(VI) onto sediments
following the stimulation of microbial growth in the subsurface may potentially
enhance the cost effectiveness of in situ uranium bioremediation. Versaphilic Anaeromyxobacter dehaloges strains implicated in hexavalent
uranium reduction and immobilization are present in the fractured saprolite
subsurface environment at the U.S. Department of Energy Integrated Field-Scale
Subsurface Research Challenge (IFC) site near Oak Ridge, TN. To provide insight
into the in situ distribution of Anaeromyxobacter strains in this system with a
nonuniform groundwater flow, 16S rRNA gene-targeted primers and linear
hybridization (TaqMan) probes were designed for Oak Ridge IFC Anaeromyxobacter
isolates FRC-D1 and FRC-W, along with an Anaeromyxobacter genus-targeted probe
and primer set. Multiplex quantitative real-time PCR (mqPCR) was applied to
samples collected from Oak Ridge IFC site areas 1 and 3, which are not connected
by the primary groundwater flow paths; however, transport between them through
cross-plane fractures is hypothesized. Strain FRC-W accounted for more than 10%
of the total quantifiable Anaeromyxobacter community in area 1 soils, while
strain FRC-D1 was not detected. In FeOOH-amended enrichment cultures derived
from area 1 site materials, strain FRC-D1 accounted for 30 to 90% of the total
Anaeromyxobacter community, demonstrating that this strain was present in situ
in area 1. The area 3 total Anaeromyxobacter abundance exceeded that of area 1
by 3 to 5 orders of magnitude, but neither strain FRC-W- nor FRC-D1-like
sequences were quantifiable in any of the 33 area 3 groundwater or sediment
samples tested. The Anaeromyxobacter community in area 3 increased from <10(5)
cells/g sediment outside the ethanol biostimulation treatment zone to 10(8)
cells/g sediment near the injection well, and 16S rRNA gene clone library
analysis revealed that representatives of a novel phylogenetic cluster dominated
the area 3 Anaeromyxobacter community inside the treatment loop. The combined
applications of genus- and strain-level mqPCR approaches along with clone
libraries provided novel information on patterns of microbial variability within
a bacterial group relevant to uranium bioremediation. Implementation of uranium bioremediation requires methods for monitoring the
membership and activities of the subsurface microbial communities that are
responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a
proteomics-based approach for simultaneously documenting the strain membership
and microbial physiology of the domit Geobacter community members during in
situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three
planktonic Geobacter-dominated samples were obtained from two wells
down-gradient of acetate addition. Over 2,500 proteins from each of these
samples were identified by matching liquid chromatography-tandem mass
spectrometry spectra to peptides predicted from seven isolate Geobacter genomes.
Genome-specific peptides indicate early proliferation of multiple M21 and
Geobacter bemidjiensis-like strains and later possible emergence of M21 and G.
bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout
biostimulation, the proteome is dominated by enzymes that convert acetate to
acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides
matching tricarboxylic acid cycle proteins and ATP synthase subunits were also
detected, indicating the importance of energy generation during the period of
rapid growth following the start of biostimulation. Evolving Geobacter strain
composition may be linked to changes in protein abundance over the course of
biostimulation and may reflect changes in metabolic functioning. Thus,
metagenomics-independent community proteogenomics can be used to diagnose the
status of the subsurface consortia upon which remediation biotechnology relies. Nutrient limitation is an environmental stress that may reduce the effectiveness
of bioremediation strategies, especially when the contamits are organic
compounds or when organic compounds are added to promote microbial activities
such as metal reduction. Genes indicative of phosphate-limitation were
identified by microarray analysis of chemostat cultures of Geobacter
sulfureducens. This analysis revealed that genes in the pst-pho operon, which is
associated with a high-affinity phosphate uptake system in other microorganisms,
had significantly higher transcript abundance under phosphate-limiting
conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR
analysis of pstB and phoU transcript levels in G. sulfurreducens grown in
chemostats demonstrated that the expression of these genes increased when
phosphate was removed from the culture medium. Transcripts of pstB and phoU
within the subsurface Geobacter species predominating during an in situ
uranium-bioremediation field experiment were more abundant than in chemostat
cultures of G. sulfurreducens that were not limited for phosphate. Addition of
phosphate to incubations of subsurface sediments did not stimulate dissimilatory
metal reduction. The added phosphate was rapidly adsorbed onto the sediments.
The results demonstrate that Geobacter species can effectively reduce U(VI) even
when experiencing suboptimal phosphate concentrations and that increasing
phosphate availability with phosphate additions is difficult to achieve because
of the high reactivity of this compound. This transcript-based approach
developed for diagnosing phosphate limitation should be applicable to assessing
the potential need for additional phosphate in other bioremediation processes. Uranium biomineralization by a metal-resistant Pseudomonas aeruginosa strain
isolated from uranium mine waste was characterized for its potential in
bioremediation. Uranium resistance, its cellular localization and chemical
nature of uranium-bacteria interaction were elucidated. Survival and uranium
biomineralization from mine water were investigated using microcosm experiments.
The selected bacterium showed U resistance and accumulation (maximum of 275 mg U
g(-1)cell dry wt.) following incubation in 100 mg U L(-1), pH 4.0, for 6 h.
Transmission electron microscopy and X-ray diffraction analyses revealed that
bioaccumulated uranium was deposited within the cell envelope as needle shaped
U-phosphate compounds that attain crystallinity only at pH 4.0. A synergistic
involvement of deprotonated phosphate and carboxyl moieties in facilitating
bioprecipitation of uranium was evident from FTIR analysis. Based on these
findings we attribute the localized U sequestration by this bacterium as
innocuous complex to its possible mechanism of uranium resistance. Microcosm
data confirmed that the strain can remove soluble uranium (99%) and sequester it
as U oxide and phosphate minerals while maintaining its viability. The study
showed that indigenous bacteria from contaminated site that can survive uranium
and other heavy metal toxicity and sequester soluble uranium as biominerals
could play important role in uranium bioremediation. Monitoring the activity of target microorganisms during stimulated
bioremediation is a key problem for the development of effective remediation
strategies. At the US Department of Energy's Integrated Field Research Challenge
(IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via
subsurface acetate addition leads to precipitation of U(VI) from groundwater as
U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism
that controls flux into the TCA cycle. Here, we utilize shotgun proteomic
methods to demonstrate that the measurement of gltA peptides can be used to
track Geobacter activity and strain evolution during in situ biostimulation.
Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in
U(VI) concentrations during biostimulation, whereas changing patterns of unique
peptide abundances between samples suggested sample-specific strain shifts
within the Geobacter population. Abundances of unique peptides indicated
potential differences at the strain level between Fe(III)-reducing populations
stimulated during in situ biostimulation experiments conducted a year apart at
the Rifle IFRC. These results offer a novel technique for the rapid screening of
large numbers of proteomic samples for Geobacter species and will aid monitoring
of subsurface bioremediation efforts that rely on metal reduction for desired
outcomes. The increasing availability of the genome sequences of microorganisms involved
in important bioremediation processes makes it feasible to consider developing
genome-scale models that can aid in predicting the likely outcome of potential
subsurface bioremediation strategies. Previous studies of the in situ
bioremediation of uranium-contaminated groundwater have demonstrated that
Geobacter species are often the domit members of the groundwater community
during active bioremediation and the primary organisms catalysing U(VI)
reduction. Therefore, a genome-scale, constraint-based model of the metabolism
of Geobacter sulfurreducens was coupled with the reactive transport model
HYDROGEOCHEM in an attempt to model in situ uranium bioremediation. In order to
simplify the modelling, the influence of only three growth factors was
considered: acetate, the electron donor added to stimulate U(VI) reduction;
Fe(III), the electron acceptor primarily supporting growth of Geobacter; and
ammonium, a key nutrient. The constraint-based model predicted that growth
yields of Geobacter varied significantly based on the availability of these
three growth factors and that there are minimum thresholds of acetate and
Fe(III) below which growth and activity are not possible. This contrasts with
typical, empirical microbial models that assume fixed growth yields and the
possibility for complete metabolism of the substrates. The coupled genome-scale
and reactive transport model predicted acetate concentrations and U(VI)
reduction rates in a field trial of in situ uranium bioremediation that were
comparable to the predictions of a calibrated conventional model, but without
the need for empirical calibration, other than specifying the initial biomass of
Geobacter. These results suggest that coupling genome-scale metabolic models
with reactive transport models may be a good approach to developing models that
can be truly predictive, without empirical calibration, for evaluating the
probable response of subsurface microorganisms to possible bioremediation
approaches prior to implementation. To better understand the microbial functional diversity changes with subsurface
redox conditions during in situ uranium bioremediation, key functional genes
were studied with GeoChip, a comprehensive functional gene microarray, in field
experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO).
The results indicated that functional microbial communities altered with a shift
in the domit metabolic process, as documented by hierarchical cluster and
ordination analyses of all detected functional genes. The abundance of dsrAB
genes (dissimilatory sulfite reductase genes) and methane generation-related mcr
genes (methyl coenzyme M reductase coding genes) increased when redox conditions
shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes
detected were primarily from Geobacter sp. and decreased with lower subsurface
redox conditions. Statistical analysis of environmental parameters and
functional genes indicated that acetate, U(VI), and redox potential (E(h)) were
the most significant geochemical variables linked to microbial functional gene
structures, and changes in microbial functional diversity were strongly related
to the domit terminal electron-accepting process following acetate addition.
The study indicates that the microbial functional genes clearly reflect the in
situ redox conditions and the domit microbial processes, which in turn
influence uranium bioreduction. Microbial functional genes thus could be very
useful for tracking microbial community structure and dynamics during
bioremediation. There is increasing interest in harnessing the functional capacities of
indigenous microbial communities to transform and remediate a wide range of
environmental contamits. Information about which community members respond to
stimulation can guide the interpretation and development of remediation
approaches. To comprehensively determine community membership and abundance
patterns among a suite of samples associated with uranium bioremediation
experiments, we employed a high-density microarray (PhyloChip). Samples were
unstimulated, naturally reducing, or collected during Fe(III) (early) and
sulfate reduction (late biostimulation) from an acetate re-amended/amended
aquifer in Rifle, Colorado, and from laboratory experiments using
field-collected materials. Deep community sampling with PhyloChip identified
hundreds-to-thousands of operational taxonomic units (OTUs) present during
amendment, and revealed close similarity among highly enriched taxa from drill
core and groundwater well-deployed column sediment. Overall, phylogenetic data
suggested that stimulated community membership was most affected by a carryover
effect between annual stimulation events. Nevertheless, OTUs within the Fe(III)-
and sulfate-reducing lineages, Desulfuromonadales and Desulfobacterales, were
repeatedly stimulated. Less consistent, co-enriched taxa represented additional
lineages associated with Fe(III) and sulfate reduction (e.g. Desulfovibrionales;
Syntrophobacterales; Peptococcaceae) and autotrophic sulfur oxidation
(Sulfurovum; Campylobacterales). Data implies complex membership among highly
stimulated taxa and, by inference, biogeochemical responses to acetate, a
nonfermentable substrate. Molecular tools that can provide an estimate of the in situ growth rate of
Geobacter species could improve understanding of dissimilatory metal reduction
in a diversity of environments. Whole-genome microarray analyses of a subsurface
isolate of Geobacter uraniireducens, grown under a variety of conditions,
identified a number of genes that are differentially expressed at different
specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC
and rplL, was further evaluated with quantitative reverse transcription-PCR
(qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h.
Transcript abundance of rpsC correlated best (r(2) = 0.90) with specific growth
rates. Therefore, expression patterns of rpsC were used to estimate specific
growth rates of Geobacter species during an in situ uranium bioremediation field
experiment in which acetate was added to the groundwater to promote
dissimilatory metal reduction. Initially, increased availability of acetate in
the groundwater resulted in higher expression of Geobacter rpsC, and the
increase in the number of Geobacter cells estimated with fluorescent in situ
hybridization compared well with specific growth rates estimated from levels of
in situ rpsC expression. However, in later phases, cell number increases were
substantially lower than predicted from rpsC transcript abundance. This change
coincided with a bloom of protozoa and increased attachment of Geobacter species
to solid phases. These results suggest that monitoring rpsC expression may
better reflect the actual rate that Geobacter species are metabolizing and
growing during in situ uranium bioremediation than changes in cell abundance. As a key part of water management at the Ranger Uranium Mine (Northern
Territory, Australia), stockpile (ore and waste) runoff water was applied to
natural woodland on the mine lease in accordance with regulatory requirements.
Consequently, the soil in these Land Application Areas (LAAs) presents a range
of uranium concentrations. Soil samples were collected from LAAs with different
concentrations of uranium and extracts were plated onto LB media containing no
(0 ppm), low (3 ppm), medium (250 ppm), high (600 ppm) and very high (1500 ppm)
uranium concentrations. These concentrations were similar to the range of
measured uranium concentrations in the LAAs soils. Bacteria grew on all plates
except for the very high uranium concentrations, where only fungi were
recovered. Identifications based on bacterial 16S rRNA sequence analysis showed
that the domit cultivable bacteria belonged to the genus Bacillus. Members of
the genera Paenibacillus, Lysinibacillus, Klebsiella, Microbacterium and
Chryseobacterium were also isolated from the LAAs soil samples. Fungi were
identified by sequence analysis of the intergenic spacer region, and members of
the genera Aspergillus, Cryptococcus, Penicillium and Curvularia were domit
on plates with very high uranium concentrations. Members of the Paecilomyces and
Alternaria were also present but in lower numbers. These findings indicate that
fungi can tolerate very high concentrations of uranium and are more resistant
than bacteria. Bacteria and fungi isolated at the Ranger LAAs from soils with
high concentrations of uranium may have uranium binding capability and hence the
potential for uranium bioremediation. High-performance MS instrumentation coupled with improved protein extraction
techniques enables metaproteomics to identify active members of soil and
groundwater microbial communities. Metaproteomics workflows were applied to
study the initial responses (i.e. 4 days post treatment) of the indigenous
aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a
uranium-contaminated site. Members of the Betaproteobacteria (i.e.
Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and
the Firmicutes dominated the biostimulated aquifer community. Proteome
characterization revealed distinct differences between the microbial biomass
collected from groundwater influenced by biostimulation and groundwater
collected upgradient of the EVO injection points. In particular, proteins
involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate
granule formation were prominent following biostimulation. Interestingly, the
atypical NosZ of Dechloromonas spp. was highly abundant, suggesting active
nitrous oxide (N2 O) respiration. c-Type cytochromes were barely detected, as
was citrate synthase, a biomarker for hexavalent uranium reduction activity,
suggesting that uranium reduction has not commenced 4 days post EVO amendment.
Environmental metaproteomics identified microbial community responses to
biostimulation and elucidated active pathways demonstrating the value of this
technique as a monitoring tool and for complementing nucleic acid-based
approaches. |
How much should be the duration of the QT interval in patients with short QT syndrome? | The short-QT syndrome is characterized by QT intervals <300-330 msec | The short QT syndrome constitutes a new clinical entity that is associated with
a high incidence of sudden cardiac death, syncope, and/or atrial fibrillation
even in young patients and newborns. Patients with this congenital electrical
abnormality are characterized by rate-corrected QT intervals<320 ms. Missense
mutations in KCNH2 (HERG) linked to a gain-of-function of the rapidly activating
delayed-rectifier current I(Kr) have been identified in the first two reported
families with familial sudden cardiac death. Recently, two further
gain-of-function mutations in the KCNQ1 gene encoding the alpha-subunit of the
KvLQT1 (I(Ks)) channel and in the KCNJ2 gene encoding the strong inwardly
rectifying channel protein Kir2.1 confirmed a genetically heterogeneous disease.
The possible substrate for the development of ventricular tachyarrhythmias may
be a significant transmural dispersion of the repolarisation due to a
heterogeneous abbreviation of the action potential duration. The implantable
cardioverter defibrillator is the therapy of choice in patients with syncope and
a positive family history of sudden cardiac death. However, ICD therapy in
patients with a short QT syndrome has an increased risk for inappropriate shock
therapies due to possible T wave oversensing. The impact of sotalol, ibutilide,
flecainide, and quinidine on QT prolongation has been evaluated, but only
quinidine effectively suppressed gain-of-function in I(Kr) with prolongation of
the QT interval. In patients with a mutation in HERG, it rendered ventricular
tachycardias/ventricular fibrillation non-inducible and restored the QT
interval/heart rate relationship towards a normal range. It may serve as an
adjunct to ICD therapy or as a possible alternative treatment, especially for
children and newborns. Short QT syndrome (SQTS) is an inheritable primary electrical disease of the
heart, discovered in 1999. It is characterized by an abnormally short QT
interval (<300 ms) and a propensity to atrial fibrillation and sudden cardiac
death (SCD). Like in the case of long QT syndrome there is more than one genetic
mutation that can lead to a short QT interval in the ECG and so far two have
been identified. Shortening of the effective refractory period combined with
increased dispersion of repolarization is the likely substrate for reentry and
life threatening tachyarrhythmias. Only 22 people have been classified as having
SQTS: 15 from the actual measurement of a short QT interval in their ECG and 7
by history, all having died from SCD. It is very likely that several cases,
especially among children, have been overlooked, since the shortness of the QT
interval only becomes apparent at heart rates <80 beats/min. The best form of
treatment is still not known, but prevention of atrial fibrillation has been
accomplished by propafenone, and an implantable cardioverter defibrillator is
recommended for prevention of SCD. Short QT syndrome is an inheritable primary electrical disease of the heart that
was discovered in 1999. The disorder is characterized by an abnormally short QT
interval (<300 ms) and a propensity to atrial fibrillation, sudden cardiac death
or both. As in the case of long QT syndrome, more than one relevant genetic
mutation has been identified that can lead to a short QT interval on
electrocardiography; so far two have been identified. Shortening of the
effective refractory period combined with increased dispersion of repolarization
is the likely substrate for re-entry and life-threatening tachyarrhythmias. Thus
far, 22 people have been classified as having short QT syndrome: 15 from the
actual measurement of a short QT interval on electrocardiograms and 7 by history
after they died from sudden cardiac death. Several cases, especially among
children, have probably been overlooked, since the shortness of the QT interval
becomes apparent only at heart rates less than 80 beats/min. The best form of
treatment is still unknown, but prevention of atrial fibrillation has been
accomplished by propafenone. Implantation of an implantable cardioverter
defibrillator is recommended for prevention of sudden cardiac death. Short QT syndrome is a new genetic disorder associated with familial atrial
fibrillation and/or sudden death or syncope. To date, different mutations in
genes encoding for cardiac ion channels (KCNH2, KCNQ1, and KCNJ2) have been
identified to cause the short QT syndrome. The mutations lead to a gain of
function of the affected current (IKr, IKs, and IK1). The phenotype is
characterized by a shortened QT interval<335 ms after correction for heart rate
at rates<80 beats/min. Furthermore, the QT interval poorly adapts to heart rate.
Patients exhibit shortened atrial and ventricular effective refractory periods
and, in the majority, inducibility of ventricular fibrillation. Death occurs
already in newborns. Therapy of choice seems to be the implantable cardioverter
defibrillator because of the high incidence of sudden death. Pharmacological
treatment has been studied and it could be demonstrated, that some mutant
currents may be insufficiently suppressed by drugs targeted to block the
specific current such as, e.g., sotalol or ibutilide in patients with a mutation
in the IKr-coding gene KCNH2 (HERG). Quinidine proved to be efficient in
prolonging the QT interval and normalizing the effective refractory periods in
some patients. The past 2 decades have witnessed the emergence of many disease states related
to ion-channel disorders, the so-called "channelopathies," usually associated
with structurally normal hearts. The initial emphasis was directed toward the
congenital long QT syndrome and the Brugada syndrome. Recently, the hereditary
short QT syndrome has emerged as yet another rare channelopathy. This autosomal
domit syndrome can afflict infants, children, or young adults; often a
remarkable family background of sudden cardiac death is elucidated. The
electrocardiogram is characterized by a strikingly short QT interval (typically
<320 milliseconds); virtual absence of the ST segment; and tall, peaked,
narrow-based T waves. There is a marked propensity for paroxysmal atrial
fibrillation, and increased risk for sudden cardiac death from ventricular
tachyarrhythmias. At electrophysiology study, short atrial and ventricular
refractory periods are found, with easily inducible atrial fibrillation and
polymorphic ventricular tachycardia with programmed electrical stimulation.
Gain-of-function mutations in 3 genes encoding potassium channels have been
identified, which explain the abbreviated repolarization seen in this condition.
The suggested treatment is an implantable cardioverter-defibrillator, though the
possibilities of inappropriate shocks have caused some concern, especially in
younger patients. The ability of quinidine and disopyramide to prolong the QT
interval has the potential to be effective pharmacological therapy for patients
with short QT syndrome, but awaits additional confirmatory clinical data. |
What is known about potential implication of thyroid hormone receptors in arterial hypertension? | thyroid hormone receptors are implicated in arterial hypertension | Hypertension is more common in hypothyroidic patients than in euthyroid controls
in older age groups. Treatment of the thyroid deficiency alone lowers blood
pressure in most patients. Hemodynamically, cardiac output is reduced and total
peripheral resistance is elevated. The latter probably is secondary to an
increase of sympathetic nervous tone and a relative increase in alpha-adrenergic
response. In hyperthyroidism, elevation of diastolic blood pressure is uncommon.
Systolic hypertension is more common in younger age groups. Treatment of the
hyperthyroidism alone lowers systolic blood pressure in most patients. An
increase in cardiac output and a decrease in total peripheral resistance
accompany the hyperthyroidism. Potentiation of catecholamine action by an excess
of thyroid hormone has been invoked as an explanation, because thyroid hormone
excess is accompanied by increased beta-adrenergic receptors in some tissue,
including heart. Thyroid hormones (THs) enhance MHC alpha gene- and repress MHC beta
gene-transcription in the heart, by interacting with specific nuclear receptors
(TRs), that bind to regulatory sequences localized upstream of basal promoter of
myosin heavy chain (MHC) genes. The overall effects of THs include an increase
in V1- and a decrease in V3-myosin isozyme concentration in the heart. Myosin V1
contains two MHC alpha chains and has a higher ATPase activity than V3 isoform,
which contains two beta chains. Previous studies on papillary muscles of
spontaneously hypertensive rats (SHRs) showed that heart hypertrophy is
accompanied by a shift from alpha to beta MHC accumulation. The present study
was aimed at evaluating whether this event relates to differential expression of
alpha1, alpha2, and beta1 isoforms of TRs. At the ages of 8 and 15 weeks, SHRs
and Harlan Sprague-Dawley control rats were sacrificed under anesthesia and
their hearts were dissected into left and right ventricles, free of atria and
great vessels. The results of Western blot analyses showed that the levels of
the three TR isoforms do not differ significantly between SHRs and control rats
of the same age, either in the left or in the right ventricle. Thus, the
expression of MHC beta in SHR hypertrophic heart does not seem to depend on
changes in TR isoform concentrations. Thyroid hormone action has profound consequences for the heart, ranging from
atrial fibrillation to hemodynamic collapse. It has long been known that the
cardiovascular signs and symptoms seen in thyrotoxicosis resemble those seen in
states of catecholamine excess. However, measured concentrations of serum
catecholamines in patients with thyrotoxicosis are typically normal or even low,
suggesting an increase in the adrenergic responsiveness of the thyrotoxic heart.
In spite of several decades of work, the question of whether thyroid hormone
increases cardiac adrenergic responsiveness is still controversial. In this
brief review, we consider the reasons underlying this controversy, focusing on
the complexity of the adrenergic signaling cascade. BACKGROUND: Thyroid hormones (THs) exert multiple biological roles including
effects on the cardiovascular system (lipid profile, blood pressure (BP) and
cardiac output). The lipid-lowering actions of TH are mediated by the TH
receptor-β whereas the mechanisms explaining the BP variations concomitant with
the thyroid disorders are less understood. As the TH receptor-α (TR-α) has been
associated with many of TH actions on the cardiovascular system in mice models,
we hypothesized that it could be involved in the latter. We thus tested whether
polymorphisms in TR-α (THRA gene) could be associated with BP level variation.
Secondarily, we tested for association with coronary heart disease (CHD) risk.
METHODS: We analyzed the associations between five THRA polymorphisms and (i) BP
level in two population-based studies (MONICA Lille n = 1,155; MONICA Toulouse n
= 1,170) and (ii) the risk of CHD in two case-control studies (Lille CHD n = 558
cases/568 controls; PRIME n = 527 cases/584 controls).
RESULTS: Individuals carrying the rs939348 T allele had higher systolic BP
(~+1.3 mm Hg) than CC individuals in both the MONICA Lille (P = 0.02) and
Toulouse (P = 0.03) studies. The odds ratio (OR) for hypertension was 1.25 (P =
0.02) in the combined sample. Concerning the CHD risk, no significant
association could be detected.
CONCLUSIONS: For the first time, our study showed associations between the THRA
rs939348 polymorphism and systolic BP and the risk of hypertension but not with
CHD, although we admit that the statistical power available to study any
relationship with CHD was very limited. Further larger association studies are
needed to confirm our findings. OBJECTIVE: To review cardiac manifestations in the syndrome of resistance to
thyroid hormone (RTH) and to question the general recommendation that the
thyroid-stimulating hormone (TSH) value be the guide to thyroid hormone
replacement.
METHODS: The syndrome of RTH is caused by mutations in the carboxy-terminal
portion of the β isoform of the thyroid hormone receptor, resulting in variable
clinical manifestations. It is generally recommended that the replacement of
thyroid hormone in patients with RTH be guided by the serum TSH concentration.
The variable responsiveness of tissues to thyroid hormone, however, makes it
difficult to balance the correct replacement dose. We present a case that brings
into question the conventional wisdom about the replacement dose of thyroid
hormone in this scenario, and we review the pertinent literature.
RESULTS: A 54-year-old man with RTH was treated with levothyroxine and
increasing doses of liothyronine sodium as part of an evaluation of RTH. On day
10 of the protocol, he developed atrial fibrillation despite a normal level of
TSH (1.1 mIU/L). Administration of liothyronine was discontinued, and
cardioversion was planned; however, the patient's heart rhythm converted
spontaneously to normal sinus rhythm.
CONCLUSION: Replacement of thyroid hormone in patients with RTH should include
careful monitoring of thyrotoxic cardiac side effects in addition to
consideration of normalization of the TSH level. Pulmonary arterial hypertension (PAH) is a multi-factorial condition and the
underlying pulmonary vascular disease is shaped by the combined action of
genetic, epigenetic and immune-related factors. Whether and how gender, obesity
and the metabolic syndrome modify PAH and associated right heart failure is
under intense investigation. Estrogens may enhance the process of pulmonary
angioproliferation, but may also protect the right ventricle under pressure.
Obesity may affect the pulmonary circulation via interactions with inflammatory
cells and mediators, or via alterations in endocrine signaling. Obesity is a
major risk factor for pulmonary hypertension in patients with elevated pulmonary
venous pressure and preserved LV ejection fraction. Given the overlap between
PAH and autoimmune diseases, hypothyroidism in patients with PAH is commonly
considered a consequence of an autoimmune thyroiditis. In the clinical setting
of hyperthyroidism, severe pulmonary hypertension may develop due to a
hyperdynamic circulation, but a more complex situation presents itself when
hyperthyroidism is associated with PAH. We recently showed in a relevant animal
model of severe PAH that thyroid hormone, via its endothelial cell-proliferative
action, can be permissive and drive angioproliferation. Signaling via the
integrin αvβ3 and FGF receptors may participate in the formation of the lung
vascular lesions in this model of PAH. Whether thyroid hormones in euthyroid PAH
patients play a pathobiologically important role is unknown- as we also do not
know whether the commonly diagnosed hypothyroidism in patients with severe PAH
is cardioprotective. This brief review highlights some recent insights into the
role of metabolic and endocrine disorders in PAH. Thyroid hormone is well known for its profound direct effects on cardiovascular
function and metabolism. Recent evidence, however, suggests that the hormone
also regulates these systems indirectly through the central nervous system.
While some of the molecular mechanisms underlying the hormone's central control
of metabolism have been identified, its actions in the central cardiovascular
control have remained enigmatic. Here, we describe a previously unknown
population of parvalbuminergic neurons in the anterior hypothalamus that
requires thyroid hormone receptor signaling for proper development. Specific
stereotaxic ablation of these cells in the mouse resulted in hypertension and
temperature-dependent tachycardia, indicating a role in the central autonomic
control of blood pressure and heart rate. Moreover, the neurons exhibited
intrinsic temperature sensitivity in patch-clamping experiments, providing a new
connection between cardiovascular function and core temperature. Thus, the data
identify what we believe to be a novel hypothalamic cell population potentially
important for understanding hypertension and indicate developmental
hypothyroidism as an epigenetic risk factor for cardiovascular disorders.
Furthermore, the findings may be beneficial for treatment of the recently
identified patients that have a mutation in thyroid hormone receptor α1. |
What is the mechanism of action of abiraterone? | Abiraterone acts by inhibiting cytochrome P450 17α-hydroxylase (CYP17A1), a critical step in androgen biosynthesis, thus leading to inhibition of androgen biosynthesis. | Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor
(IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in
studies of the role of the 16,17-double bond in its mechanism of action, the
novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and
17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were
synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via
photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate
16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of
6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19
(IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent
(IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation
of the inhibitor with the enzyme. Inhibition following preincubation with these
compounds is not lessened by dialysis for 24 h, implying irreversible binding to
the enzyme. In contrast under these conditions the still potent (IC50 27 nM)
17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h
of dialysis and complete reversal of inhibition after 24 h. This behavior was
also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24.
Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double
bond, the inhibition of the enzymic reaction was not enhanced by preincubation
either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack
this structural feature. The results show that the 16,17-double bond is
necessary for irreversible binding of these pyridyl steroids to cytochrome
P45017alpha. However oxidation to an epoxide is probably not involved since
epoxide 6 was only a moderately potent inhibitor (IC50 260 nM). Prostate cancer is the second leading cause of cancer death in men in the US and
Europe. The treatment of advanced-stage prostate cancer has been androgen
deprivation. Medical castration leads to decreased production of testosterone
and dihydrotestosterone by the testes, but adrenal glands and even prostate
cancer tissue continue to produce androgens, which eventually leads to continued
prostate cancer growth despite castrate level of androgens. This stage is known
as castrate-resistant prostate cancer (CRPC), which continues to be a challenge
to treat. Addition of androgen antagonists to hormonal deprivation has been
successful in lowering the prostate-specific antigen levels further, but has not
actually translated into life-prolonging options. The results of several
contemporary studies have continued to demonstrate activation of the androgen
receptor as being the key factor in the continued growth of prostate cancer.
Blockade of androgen production by nongonadal sources has led to clinical
benefit in this setting. One such agent is abiraterone acetate, which
significantly reduces androgen production by blocking the enzyme, cytochrome
P450 17 alpha-hydroxylase (CYP17). This has provided physicians with another
treatment option for patients with CRPC. The landscape for prostate cancer
treatment has changed with the approval of cabazitaxel, sipuleucel-T and
abiraterone. Here we provide an overview of abiraterone acetate, its mechanism
of action, and its potential place for therapy in CRPC. PURPOSE OF REVIEW: Prior to 2010, docetaxel was the only treatment shown to
prolong survival in metastatic castrate-resistant prostate cancer (CRPC). In the
past 3 years, several therapeutic agents have demonstrated survival improvements
for CRPC after the receipt of prior docetaxel, leading to multiple approvals by
the US Food and Drug Administration.
RECENT FINDINGS: The development of these novel agents, each with a distinct
mechanism of action, is the fruition of sedulous preclinical research and well
designed clinical trials. Cabazitaxel, a next generation taxane, was the first
Food and Drug Administration-approved drug for the postdocetaxel setting. The
recognition of sustained androgen dependence of CRPC has led to the
identification of more potent and selective inhibitors of androgen synthesis and
androgen-receptor signaling, such as abiraterone and enzalutamide, respectively.
Radium-223, an α-emitting radionuclide still under regulatory review, recently
showed a significant survival benefit for CRPC. Finally, sipuleucel-T, a form of
immunotherapy, may benefit a subset of patients in the postdocetaxel setting.
SUMMARY: Post-docetaxel management of CRPC has undergone a dramatic yet welcome
paradigm change in the past 3 years. With multiple life-prolonging agents
available, it now becomes imperative to coordinate how and when these new
therapies should be used and sequenced to achieve optimal patient outcomes. |
The protein neprilysin has an positive effect on Alzheimer disease, how can it be delivered to the brain? | The protein neprilysin can be deliverered to the brain (crossing the blood brain barrier) through: gene tranfer, transgenesis, gene induction, ex-vivo gene therapy, intracardiac (peripheral) administration of viral neprilysin construct, syringe-focused ultrasound device, convection-enhanced delivery and the use of human adipose tissue-derived mesenchymal stem cells that secrete functional neprilysin-bound exosomes | BACKGROUND: Understanding the mechanisms of amyloid-beta protein (Abeta)
production and clearance in the brain has been essential to elucidating the
etiology of Alzheimer disease (AD). Chronically decreasing brain Abeta levels is
an emerging therapeutic approach for AD, but no such disease-modifying agents
have achieved clinical validation. Certain proteases are responsible for the
catabolism of brain Abeta in vivo, and some experimental evidence suggests they
could be used as therapeutic tools to reduce Abeta levels in AD. The objective
of this study was to determine if enhancing the clearance of Abeta in the brain
by ex vivo gene delivery of an Abeta-degrading protease can reduce amyloid
plaque burden.
METHODS AND FINDINGS: We generated a secreted form of the Abeta-degrading
protease neprilysin, which significantly lowers the levels of naturally secreted
Abeta in cell culture. We then used an ex vivo gene delivery approach utilizing
primary fibroblasts to introduce this soluble protease into the brains of
beta-amyloid precursor protein (APP) transgenic mice with advanced plaque
deposition. Brain examination after cell implantation revealed robust clearance
of plaques at the site of engraftment (72% reduction, p = 0.0269), as well as
significant reductions in plaque burden in both the medial and lateral
hippocampus distal to the implantation site (34% reduction, p = 0.0020; and 55%
reduction, p = 0.0081, respectively).
CONCLUSIONS: Ex vivo gene delivery of an Abeta-degrading protease reduces
amyloid plaque burden in transgenic mice expressing human APP. These results
support the use of Abeta-degrading proteases as a means to therapeutically lower
Abeta levels and encourage further exploration of ex vivo gene delivery for the
treatment of Alzheimer disease. Most disease-modifying therapeutic approaches in Alzheimer's disease (AD) aim to
reduce the accumulation of neurotoxic amyloid-beta (Abeta) peptides as a
hallmark of AD pathogenesis. Here we report the in vitro basis for a potential
autologous stem cell-based strategy for widespread delivery of enzymatic
activities against Abeta formation in the brain. We detected the functional
induction of two genes upon neuroectodermal conversion of human adult
mesenchymal stem cells (MSCs), namely F-spondin and neprilysin (CD10), with a
4,992 + or - 697-fold and 692 + or - 226-fold increase of mRNA levels in
converted cells compared to MSCs, respectively (n = 3; P < 0.01). These genes
are known to be involved in the formation and degradation of Abeta peptides,
respectively. Consistently, coincubation of the neuroectodermally converted MSCs
with HEK-293 cells stably expressing amyloid precursor protein (APP) lead to a
significant cell dose-dependent decrease of Abeta peptides. These in vitro
results indicate that MSCs might be useful vehicles for delivering anti-Abeta
activity depicting a causal stem cell-based therapeutic approach to treat AD. Alzheimer's disease (AD), an incurable, progressive neurodegenerative disorder,
is the most common form of dementia. Therapeutic options have been elusive due
to the inability to deliver proteins across the blood-brain barrier (BBB). In
order to improve the therapeutic potential for AD, we utilized a promising new
approach for delivery of proteins across the BBB. We generated a lentivirus
vector expressing the amyloid β-degrading enzyme, neprilysin, fused to the ApoB
transport domain and delivered this by intra-peritoneal injection to amyloid
protein precursor (APP) transgenic model of AD. Treated mice had reduced levels
of Aβ, reduced plaques and increased synaptic density in the CNS. Furthermore,
mice treated with the neprilysin targeting the CNS had a reversal of memory
deficits. Thus, the addition of the ApoB transport domain to the secreted
neprilysin generated a non-invasive therapeutic approach that may be a potential
treatment in patients with AD. Enzymatic degradation contributes to the control of intracerebral amyloid-β (Aβ)
peptide levels. Previous studies have demonstrated the therapeutic potential of
viral vector-mediated neprilysin (NEP) gene therapy in mouse models of
Alzheimer's disease (AD). However, clinical translation of NEP gene therapy is
limited by ethical and practical considerations. In this study we have assessed
the potential of convection-enhanced delivery (CED) as a means of elevating
intracerebral NEP level and activity and degrading endogenous Aβ. We analyzed
the interstitial and perivascular distribution of NEP following CED into rat
striatum. We measured NEP protein level, clearance, activity, and toxicity by
ELISA for NEP and synaptophysin, NEP-specific activity assay, and
immunohistochemistry for NEP, NeuN, glial fibrillary acidic protein and Iba1. We
subsequently performed CED of NEP in normal aged rats and measured endogenous Aβ
by ELISA. CED resulted in widespread distribution of NEP, and a 20-fold
elevation of NEP protein level with preservation of enzyme activity and without
evidence of toxicity. CED in normal, aged rats resulted in a significant
reduction in endogenous Aβ(40) (p = 0.04), despite rapid NEP clearance from the
brain (half-life ~3 h). CED of NEP has therapeutic potential as a dynamically
controllable Aβ(40)-degrading therapeutic strategy for AD. Further studies are
required to determine the longer term effects on Aβ (including Aβ(42)) and on
cognitive function. Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid
peptide (Aβ) in the brain because of an imbalance between Aβ production and
clearance. Neprilysin (NEP) is the most important Aβ-degrading enzyme in the
brain. Thus, researchers have explored virus-mediated NEP gene delivery.
However, such strategies may entail unexpected risks, and thus exploration of a
new possibility for NEP delivery is also required. Here, we show that human
adipose tissue-derived mesenchymal stem cells (ADSCs) secrete exosomes carrying
enzymatically active NEP. The NEP-specific activity level of 1 μg protein from
ADSC-derived exosomes was equivalent to that of ~ 0.3 ng of recombit human
NEP. Of note, ADSC-derived exosomes were transferred into N2a cells, and were
suggested to decrease both secreted and intracellular Aβ levels in the N2a
cells. Importantly, these characteristics were more pronounced in ADSCs than
bone marrow-derived mesenchymal stem cells, suggesting the therapeutic relevance
of ADSC-derived exosomes for AD. Accumulation of amyloid-β peptide (Aβ) in the brain is closely associated with
cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of
neprilysin-encoding viral vectors into the hippocampus has been shown to
decrease Aβ in AD-model mice, but more efficient and global delivery is
necessary to treat the broadly distributed burden in AD. Here we developed an
adeno-associated virus (AAV) vector capable of providing neuronal gene
expression throughout the brains after peripheral administration. A single
intracardiac administration of the vector carrying neprilysin gene in AD-model
mice elevated neprilysin activity broadly in the brain, and reduced Aβ
oligomers, with concurrent alleviation of abnormal learning and memory function
and improvement of amyloid burden. The exogenous neprilysin was localized mainly
in endosomes, thereby effectively excluding Aβ oligomers from the brain. AAV
vector-mediated gene transfer may provide a therapeutic strategy for
neurodegenerative diseases, where global transduction of a therapeutic gene into
the brain is necessary. |
Which species of bacteria did the mitochondria originate from? | Biologists agree that the ancestor of mitochondria was an alpha-proteobacterium. Although the Alphaproteobacteria are thought to be the closest relatives of the mitochondrial progenitor, there is dispute as to what its particular sister group is. Accumulating evolutionary data point to a monophyletic origin of mitochondria from the order Rickettsiales. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far. | We have cloned and sequenced the genes encoding cytochrome b (cob) and
cytochrome c oxidase subunit I (cox1) from Rickettsia prowazekii, a member of
the alpha-proteobacteria. The phylogenetic analysis supports the hypothesis that
mitochondria are derived from the alpha-proteobacteria and more specifically
from within the Rickettsiaceae. We have estimated that the common ancestor of
mitochondria and Rickettsiaceae dates back to more than 1500 million years ago. The sequence of an alpha-proteobacterial genome, that of Rickettsia prowazekii,
is a substantial advance in microbial and evolutionary biology. The genome of
this obligately aerobic intracellular parasite is small and is apparently still
undergoing reduction, reflecting gene losses attributable to its intracellular
parasitic lifestyle. Evolutionary analyses of proteins encoded in the genome
contain the strongest phylogenetic evidence to date for the view that
mitochondria descend from alpha-proteobacteria. Although both Rickettsia and
mitochondrial genomes are highly reduced, it appears that genome reduction in
these lineages has occurred independently. Rickettsia's genome encodes an
ATP-generating machinery that is strikingly similar to that of aerobic
mitochondria. But it does not encode homologues for the ATP-producing pathways
of anaerobic mitochondria or hydrogenosomes, leaving an important issue
regarding the origin and nature of the ancestral mitochondrial symbiont
unresolved. The availability of complete genome sequence data from both bacteria and
eukaryotes provides information about the contribution of bacterial genes to the
origin and evolution of mitochondria. Phylogenetic analyses based on genes
located in the mitochondrial genome indicate that these genes originated from
within the alpha-proteobacteria. A number of ancestral bacterial genes have also
been transferred from the mitochondrial to the nuclear genome, as evidenced by
the presence of orthologous genes in the mitochondrial genome in some species
and in the nuclear genome of other species. However, a multitude of
mitochondrial proteins encoded in the nucleus display no homology to bacterial
proteins, indicating that these originated within the eukaryotic cell subsequent
to the acquisition of the endosymbiont. An analysis of the expression patterns
of yeast nuclear genes coding for mitochondrial proteins has shown that genes
predicted to be of eukaryotic origin are mainly translated on polysomes that are
free in the cytosol whereas those of putative bacterial origin are translated on
polysomes attached to the mitochondrion. The strong relationship with
alpha-proteobacterial genes observed for some mitochondrial genes, combined with
the lack of such a relationship for others, indicates that the modern
mitochondrial proteome is the product of both reductive and expansive processes. Although mitochondria derive from alpha-proteobacteria, many proteins acting in
this organelle did not originate from bacteria. In particular, phylogenetic
evidence indicates that RNA polymerase, DNA polymerase and DNA primase--with
homologues encoded by T3/T7-like bacteriophages--have replaced the ancestral
proteins of bacterial origin. To date, there was no clear explanation for this
puzzling observation. Bacterial genomics has now revealed the presence of
cryptic prophages that are related to T3/T7 in several genomes of
proteobacteria. We propose that such a prophage was present in the ancestral
alpha-proteobacterium at the origin of mitochondria and that RNA polymerase, DNA
polymerase and DNA primase encoded by this prophage replaced the original
bacterial enzymes to function in mitochondria. Another T3/T7 viral-like RNA
polymerase is functional in the chloroplast, indicating that a strong selection
pressure has favored replacement of some cellular proteins by viral proteins in
organelle evolution. Mitochondria originated by permanent enslavement of purple non-sulphur bacteria.
These endosymbionts became organelles through the origin of complex
protein-import machinery and insertion into their inner membranes of protein
carriers for extracting energy for the host. A chicken-and-egg problem exists:
selective advantages for evolving import machinery were absent until inner
membrane carriers were present, but this very machinery is now required for
carrier insertion. I argue here that this problem was probably circumvented by
conversion of the symbiont protein-export machinery into protein-import
machinery, in three phases. I suggest that the first carrier entered the
periplasmic space via pre-existing beta-barrel proteins in the bacterial outer
membrane that later became Tom40, and inserted into the inner membrane probably
helped by a pre-existing inner membrane protein, thereby immediately providing
the protoeukaryote host with photosynthesate. This would have created a powerful
selective advantage for evolving more efficient carrier import by inserting
Tom70 receptors. Massive gene transfer to the nucleus inevitably occurred by
mutation pressure. Finally, pressure from harmful, non-selected gene transfer to
the nucleus probably caused evolution of the presequence mechanism, and
photosynthesis was lost. Biologists agree that the ancestor of mitochondria was an alpha-proteobacterium.
But there is no consensus as to what constitutes an alpha-proteobacterial gene.
Is it a gene found in all or several alpha-proteobacteria, or in only one? Here,
we examine the proportion of alpha-proteobacterial genes in
alpha-proteobacterial genomes by means of sequence comparisons. We find that
each alpha-proteobacterium harbours a particular collection of genes and that,
depending upon the lineage examined, between 97 and 33% are
alpha-proteobacterial by the nearest-neighbour criterion. Our findings bear upon
attempts to reconstruct the mitochondrial ancestor and upon inferences
concerning the collection of genes that the mitochondrial ancestor possessed at
the time that it became an endosymbiont. Recently, α-proteobacteria have been shown to possess virus-like gene transfer
agents that facilitate high frequency gene transfer in natural environments
between distantly related lineages. This system could have driven the genomic
integration of the mitochondrial progenitor and its proto-eukaryote host and
contributed to the evolutionary mosaic of genes seen in modern-day prokaryotic
and eukaryotic genomes. |
Which disease is linked to mutations within BRAG1? | Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). | |
Which is the main calcium pump of the sarcoplasmic reticulum? | Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is the pump crucial for calcium homeostasis. SERCA is a membrane protein that belongs to the family of P-type ion translocating ATPases and pumps free cytosolic calcium into intracellular stores. | This review mainly focuses on the structure, function of the sarco(endo)plasmic
reticulum calcium pump (SERCA) and its role in genesis of arrhythmias. SERCA is
a membrane protein that belongs to the family of P-type ion translocating
ATPases and pumps free cytosolic calcium into intracellular stores. Active
transport of Ca2+ is achieved, according to the E1-E2 model, changing of SERCA
structure by Ca2+. The affinity of Ca2+ -binding sites varies from high (E1) to
low (E2). Three different SERCA genes were identified-SERCA1, SERCA2, and
SERCA3. SERCA is mainly represented by the SERCA2a isoform in the heart. In
heart muscle, during systole, depolarization triggers the release of Ca2+ from
the sarcoplasmic reticulum (SR) and starts contraction. During diastole, muscle
relaxation occurs as Ca2+ is again removed from cytosol, predomitly by
accumulation into SR via the action of SERCA2a. The main regulator of SERCA2a is
phospholamban and another regulator proteolipid of SERCA is sarcolipin. There
are a lot of studies on the effect of decreased and/or increased SERCA activity
in genesis of arrhythmia. Actually both decrease and increase of SERCA activity
in the heart result in some pathological mechanisms such as heart failure and
arrhythmia. Endoplasmic reticulum calcium homeostasis is involved in a multitude of
signaling, as well as "house-keeping" functions that control cell growth,
differentiation or apoptosis in every human/eukaryotic cell. Calcium is actively
accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium
transport ATPases (SERCA enzymes). SERCA-dependent calcium transport is the only
calcium uptake mechanism in this organelle, and therefore the regulation of
SERCA function by the cell constitutes a key mechanism to adjust calcium
homeostasis in the endoplasmic reticulum depending on the cell type and its
state of differentiation. The direct pharmacological modulation of SERCA
activity affects cell differentiation and survival. SERCA expression levels can
undergo significant changes during cell differentiation or tumorigenesis,
leading to modified endoplasmic reticulum calcium storage. In several cell types
such as cells of hematopoietic origin or various epithelial cells, two SERCA
genes (SERCA2 and SERCA3) are simultaneously expressed. Expression levels of
SERCA3, a lower calcium affinity calcium pump are highly variable. In several
cell systems SERCA3 expression is selectively induced during differentiation,
whereas during tumorigenesis and blastic transformation SERCA3 expression is
decreased. These observations point at the existence of a cross-talk, via the
regulation of SERCA3 levels, between endoplasmic reticulum calcium homeostasis
and the control of cell differentiation, and show that endoplasmic reticulum
calcium homeostasis itself can undergo remodeling during differentiation. The
investigation of the anomalies of endoplasmic reticulum differentiation in tumor
and leukemia cells may be useful for a better understanding of the contribution
of calcium signaling to the establishment of maligt phenotypes. The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays
a central role in cardiac contraction, shows decreased expression in heart
failure (HF). Increasing SERCA2a expression in HF models improves cardiac
function. We used direct cardiac delivery of adeno-associated virus encoding
human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety,
efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received
left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued
postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were
analyzed for hSERCA2a expression (Western blot) and immune responses (histology
and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were
analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and
immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks
and then declined (to ~50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end
systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05)
whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a
developed neutralizing antibodies (titer ≥1:120) and cardiac cellular
infiltration. Immunosuppression dramatically reduced immune responses (reduced
inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a
expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a
expression and improves cardiac function. However, the hSERCA2a protein level is
reduced by host immune responses. Immunosuppression alleviates immune responses
and sustains transgene expression, and may be an important adjuvant for clinical
gene therapy trials. The sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) is a membrane-bound pump that
utilizes ATP to drive calcium ions from the myocyte cytosol against the higher
calcium concentration in the sarcoplasmic reticulum. Conformational transitions
associated with Ca²⁺-binding are important to its catalytic function. We have
identified collective motions that partition SERCA crystallographic structures
into multiple catalytically-distinct states using principal component analysis.
Using Brownian dynamics simulations, we demonstrate the important contribution
of surface-exposed, polar residues in the diffusional encounter of Ca²⁺.
Molecular dynamics simulations indicate the role of Glu309 gating in binding
Ca²⁺, as well as subsequent changes in the dynamics of SERCA's cytosolic
domains. Together these data provide structural and dynamical insights into a
multistep process involving Ca²⁺ binding and catalytic transitions. Although pharmacologic therapies have provided gains in reducing the mortality
of heart failure, the rising incidence of the disease requires new approaches to
combat its health burden. Twenty-five years ago, abnormal calcium cycling was
identified as a characteristic of failing human myocardium. Sarcoplasmic
reticulum calcium ATPase (SERCA2a), the sarcoplasmic reticulum calcium pump, was
found to be a key factor in the alteration of calcium cycling. With the
advancement of gene vectors, SERCA2a emerged as an attractive clinical target
for gene delivery purposes. Using adeno-associated virus constructs, SERCA2a
upregulation has been found to improve myocardial function in animal models. The
clinical benefits of overexpressing SERCA2a have been demonstrated in the phase
I study Calcium Upregulation by Percutaneous Administration of Gene Therapy in
Cardiac Disease (CUPID). This study has demonstrated that a persistent
expression of the transgene SERCA2a is associated with a significant improvement
in associated biochemical alterations and clinical symptoms of heart failure. In
the coming years, additional targets will likely emerge that are amenable to
genetic manipulations along with the development of more advanced vector systems
with safer delivery approaches. Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is the pump crucial for calcium
homeostasis and its impairment results in pathologies such as myopathy, heart
failure or diabetes. Modulation of SERCA activity may represent an approach to
the therapy of diseases with SERCA impairment involvment. Quercetin is flavonoid
known to modulate SERCA activity. We examined the effect of nine novel quercetin
derivatives on the activity of the pump. We found that
5-morpholinohydroxypoxyquercetin, di(prenylferuoyl)quercetin,
di(diacetylcaffeoyl)-mono-(monoacetylcaffeoyl)quercetin and
monoacetylferuloylquercetin stimulated the activity of SERCA. On the contrary,
monochloropivaloylquercetin, tri(chloropivaloyl)quercetin, pentaacetylquercetin,
tri(trimethylgalloyl)quercetin and diquercetin inhibited the activity of the
pump. To identify compounds with a potential to protect SERCA against free
radicals, we assessed the free radical scavenging activity of quercetin
derivatives. We also related lipophilicity, an index of the ability to
incorporate into the membrane of sarcoplasmic reticulum, to the modulatury
effect of quercetin derivatives on SERCA activity. In addition to its ability to
stimulate SERCA, di(prenylferuloyl)quercetin showed excellent radical scavenging
activity. Adipose tissue is central to the regulation of lipid metabolism.
Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most
severe lipodystrophy diseases, is caused by mutation of the Seipin gene. Seipin
plays an important role in adipocyte differentiation and lipid homeostasis, but
its exact molecular functions are still unknown. Here, we show that Seipin
physically interacts with the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA)
in both Drosophila and man. SERCA, an endoplasmic reticulum (ER) calcium pump,
is solely responsible for transporting cytosolic calcium into the ER lumen. Like
dSeipin, dSERCA cell-autonomously promotes lipid storage in Drosophila fat
cells. dSeipin affects dSERCA activity and modulates intracellular calcium
homeostasis. Adipose tissue-specific knockdown of the ER-to-cytosol calcium
release channel ryanodine receptor (RyR) partially restores fat storage in
dSeipin mutants. Our results reveal that Seipin promotes adipose tissue fat
storage by regulating intracellular calcium homeostasis. |
What is the inheritance pattern of Hunter disease or mucopolysaccharidosis II? | X- linked recessive | Profound iduronate sulfatase deficiency, characteristic of the Hunter syndrome,
has been found in cultured fibroblasts, serum, lymphocytes, and tissues of two
clinically affected girls. The patients are karyotypically normal and have
normal fathers; cloning of the mothers' fibroblasts did not reveal the mosaicism
expected of carriers of an X-linked disease. Homozygosity for a previously
unsuspected autosomal recessive gene for iduronate sulfatase is considered the
most likely explanation, although heterozygosity for the X-linked gene and
subsequent selection cannot be completely excluded. A female child of healthy, unrelated parents presented at 12 months of age with
a history of moderately severe developmental delay, macrocephaly, dysmorphic
facies, hypotonia, hepatosplenomegaly, mild generalized dysostosis multiplex,
mucopolysacchariduria (dermatan and heparan sulfates), and Alder-Reilly bodies
in peripheral blood leukocytes. Iduronate sulfatase activity in plasma was
markedly depressed: 0.11 units/ml/h (normal, 1.75 +/- 0.56, N = 6). Analyses of
arylsulfatases A, B, and C, heparan N-sulfatase, alpha-mannosidase,
beta-mannosidase, beta-glucuronidase, beta-hexosaminidase, beta-galactosidase,
and alpha-fucosidase activities in plasma, leukocytes, and/or cultured skin
fibroblasts were all normal. Urinary sulfatide excretion was also within normal
limits. Karyotypes of peripheral blood leukocytes and cultured skin fibroblasts
were normal. Serum iduronate sulfatase activities in the parents were in the
normal range (father, 1.63 units/ml/h; mother, 1.25 units/ml/h). The results of
analyses of restriction fragment length polymorphisms (RFLP) of DNA from
cultured skin fibroblasts with the use of probes for loci extending from Xpter
to Xq28 showed X chromosome heterozygosity and confirmed the paternal origin of
one of the X chromosomes. Studies on sulfur-35 uptake in mixed fibroblast
cultures showed cross-correction of [35S]-glycosaminoglycan accumulation between
cells from the patient and normal cells or cells from a patient with Hurler
disease; however, there was no cross-correction between cells from the patient
and those from boys affected with classical Hunter disease. This represents only
the second confirmed case of Hunter disease reported in a karyotypically normal
girl. A 2.5-year-old girl who presented with abdominal distension, hepatomegaly,
coarse facies, hirsutism and contraction deformities was investigated for
mucopolysaccharidoses. Urinary excretion showed increased total
glycosaminoglycans (105 mg/mmol creatinine; normal for age 9-20 mg/mmol) with
marked increases of dermatan and heparan sulphates. A number of lysosomal enzyme
activities were measured on leucocytes, serum and cultured fibroblasts. Normal
or high activities were found for alpha-iduronidase,
N-acetylgalactosamine-6-sulphatase, beta-galactosidase, arylsulphatase B and
beta-glucuronidase. However a marked deficiency of iduronate sulphate sulphatase
activity was observed, consistent with a diagnosis of Hunter's disease.
Activities were reduced to less than 2% of mean control values in the patient's
leucocytes, serum and cultured fibroblasts. Normal activities were measured in
samples from the father and younger sister but a partial deficiency (43% of
control serum) was found in the mother. Chromosome studies on the patient
revealed a partial deletion of the long arm of one X-chromosome, most probably
of band Xq25, which was not inherited from either parent. Studies using BrdU
indicated that the deleted X chromosome was consistently late replicating, and
as a result the Hunter gene was fully expressed on the other X chromosome. Hunter disease, Mucopolysaccharidosis type II, is an X-linked recessive
lysosomal storage disorder caused by a deficiency in iduronate sulfatase
activity. We studied at molecular level a Neapolitan family with the disease. We
report, in patient, the delta 139 mutation on the third exon of the gene, on
female family members, the DNA analysis that allowed to assess or exclude their
carrier status and on fetal DNA from a pregcy of patient's mother, a prenatal
diagnosis that resulted negative. Hunter syndrome is an X-linked recessive disorder. Determination of
heterozygotes is of vital importance in genetic counselling. We describe the DNA
linkage analysis in 6 Hunter syndrome families and compare it to previous
results based on a serum assay for IDS activity. Our results confirm the
reliability of the serum assay. The serum test correctly detected 11/12 of the
1st degree relatives tested by the serum assay (6/7 carriers and 5/5
non-carriers). The only case with an apparent false negative result in the serum
test was a daughter of a "probable heterozygote" whose serum test was also
negative. We suggest that in this family the mother represented a case of
germinal mosaicism and her daughter, based on the serum test, was not a carrier.
If our interpretation is correct, then the apparent false negative results were
correct. It is concluded that in families where the mutation is not known and
DNA analysis is not possible due to the lack of informative RFLPs or due to the
lack of DNA samples on key individuals, as well as in sporadic cases, the serum
test should be applied as an alternative option for heterozygote detection. Most lysosomal diseases (LD) are inherited as autosomal recessive traits, but
two important conditions have X-linked inheritance: Fabry disease and
Mucopolysaccharidosis II (MPS II). These two diseases show a very different
pattern regarding expression on heterozygotes, which does not seem to be
explained by the X-inactivation mechanism only. While MPS II heterozygotes are
asymptomatic in most instances, in Fabry disease most of female carriers show
some disease manifestation, which is sometimes severe. It is known that there is
a major difference among X-linked diseases depending on the cell autonomy of the
gene product involved and, therefore, on the occurrence of cross-correction.
Since lysosomal enzymes are usually secreted and uptaken by neighbor cells, the
different findings between MPS II and Fabry disease heterozygotes can also be
due to different efficiency of cross-correction (higher in MPS II and lower in
Fabry disease). In this paper, we review these two X-linked LD in order to
discuss the mechanisms that could explain the different rates of penetrance and
expressivity observed in the heterozygotes; this could be helpful to better
understand the expression of X-linked traits. |
Do ephrins play a role in brain cancer? | Eph receptors and ephrin ligands are involved in the development of the central nervous system. Their expression is often reported to be up-regulated in brain tumours and they may be considered molecular markers for the diagnosis of invasive and metastatic tumours. However, there are also reports describing the down-regulation of the Eph/ephrin family in brain cancer. | BACKGROUND: Eph receptors and their ligands, the ephrins, represent a large
class of cell-cell communication molecules with well-defined developmental
functions. Their role in healthy adult tissues and in human disease is still
largely unknown, although diverse roles in carcinogenesis have been postulated.
METHODS: We established a set of fluorescent PCR probes and primers for the
definition of individual gene expression profiles of 12 different Eph receptors
and 8 ephrins in 13 different healthy tissues. The mRNA expression profiles were
studied in human lung, colorectal, kidney, liver, and brain cancers.
RESULTS: The family of Eph receptors/ephrins was widely expressed in adult
tissues with organ-site-specific patterns: EphB6 was highest in the thymus,
compatible with an involvement in T-cell maturation. Brain and testis shared a
unique pattern with EphA6, EphA8, and EphB1 being the most prominent. EphA7 had
a high abundance in the kidney vasculature. Ephrin-A3 was up-regulated 26-fold
in lung cancer, and EphB2 was up-regulated 9-fold in hepatocellular carcinoma.
EphA8 was down-regulated in colon cancer, and EphA1/EphA8 was down-regulated in
glioblastomas.
CONCLUSION: Eph/Ephrin genes are widely expressed in all adult organs with
certain organ-site-specific patterns. Because their function in adult tissues
remains unknown, further analysis of their role in disease may disclose new
insights beyond their well-defined meaning in development. Eph receptor tyrosine kinases and their ligands, ephrins, mediate
neurodevelopmental processes such as boundary formation, axon guidance,
vasculogenesis, and cell migration. We determined the expression profiles of the
Eph family members in five glioma cell lines under migrating and nonmigrating
conditions. EphB2 mRNA was overexpressed in all five during migration
(1.2-2.8-fold). We found abundant EphB2 protein as well as strong
phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2
localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc
chimera stimulated migration and invasion of U87, whereas treatment with a
blocking EphB2 antibody significantly inhibited migration and invasion. Forced
expression of EphB2 in U251 cells stimulated cell migration and invasion and
diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251
stably transfected with EphB2 showed more scattered and more pronounced invasive
growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2
expression was higher in glioblastomas than in low-grade astrocytomas or normal
brain; patterns of phosphorylated EphB2 matched the expression levels. Laser
capture microdissection of invading glioblastoma cells revealed elevated EphB2
mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry
demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases)
and not in normal brain. This is the first demonstration that migrating
glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and
invasion are promoted by activation of EphB2 or inhibited by blocking EphB2.
Dysregulation of EphB2 expression or function may underlie glioma invasion. BACKGROUND & OBJECTIVE: EphrinB2 is a novel angiogenic factor. EphrinB2 and its
receptor EphB4 express in several kinds of tumor cells,and correlate with
tumorigenesis and neoangiogenesis. This study was designed to explore the
characteristics of EphB4 and EphrinB2 protein expression in astrocytomas.
METHODS: Double labelling immunofluorescence was used to detect co-expression of
EphB4/EphrinB2 with glial fibrilary acid protein (GFAP)or CD34 protein in 35
fresh glioma specimens, and 2 kinds of human glioma cell lines (CHG-5,and
SHG-44).
RESULTS: EphB4/EphrinB2 and CD34 proteins co-expressed in some tumor stromal
microvessels, and mainly localizing in endothelial cells. Co-expression of
EphB4/EphrinB2 and GFAP proteins was also noticed in tumor cells,and 2 glioma
cell lines. In poorly-differentiated SHG-44 cells, the average fluorescence
intensity of EphB4 was 72.48+/-33.78,and that of EphrinB2 was 96.80+/-36.98,
both higher than those in well-differentiated CHG-5 cells (56.7+/-21.7, and
53.6+/-18.8). But the green fluorescence intensity of GFAP in SHG-44 cells was
22.3+/-15.3, while in CHG-5 cells was 47.5+/-16.7.
CONCLUSION: Expressions of EphB4 and EphrinB2 proteins may be related to
differentiation degree of tumor cells. We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma
multiforme (GBM), a maligt neoplasm of glial cells, and normal brain. We also
initially examined the functional importance of the interaction between EphA2
and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1
in human GBM and normal brain were examined using Western blotting,
immunofluorescence, and immunohistochemistry. A functional role for EphA2 was
investigated by assessing the activation status of the receptor and the effect
of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells.
We found EphA2 to be elevated in approximately 90% of GBM specimens and cell
lines but not in normal brain, whereas ephrinA1 was present at consistently low
levels in both GBM and normal brain. EphA2 was activated and phosphorylated by
ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent
inhibitory effect on the anchorage-independent growth and invasiveness of GBM
cells highly overexpressing EphA2, which was not seen in cells expressing low
levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM
and expressed differentially with respect to its ligand, ephrinA1, which may
reflect on the oncogenic processes of maligt glioma cells. EphA2 seems to be
functionally important in GBM cells and thus may play an important role in GBM
pathogenesis. Hence, EphA2 represents a new marker and novel target for the
development of molecular therapeutics against GBM. Eph receptor tyrosine kinases are involved in nervous system development. Eph
ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors,
the mutual activation of which causes repulsive effects in reciprocally
contacting cells. Previously, we showed that overexpression of EphB2 in glioma
cells increases cell invasion. Here, expression profiles of ephrin-B family
members were determined in four glioma cell lines and in invading glioblastoma
cells collected by laser capture microdissection. Ephrin-B3 mRNA was
up-regulated in migrating cells of four of four glioma cell lines (1.3- to
1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2-
to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87,
T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant
with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251,
SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type
cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed
significant morphologic change and decreased invasion in vitro and ex vivo.
Depletion of endogenous ephrin-B3 expression abrogated the increase of migration
and invasion induced by EphB2/Fc, indicating increased invasion is dependent on
ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed
that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA
negated the increased invasion by addition of EphB2/Fc. In human glioma
specimens, ephrin-B3 expression and phosphorylation correlated with increasing
tumor grade. Immunohistochemistry revealed robust staining for phosphorylated
ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that
ephrin-B3 expression and signaling through Rac1 are critically important to
glioma invasion. Eph receptors, the largest receptor tyrosine kinases, and their ephrin ligands
play important roles in axon guidance and cell migration during development of
the nervous system. Recently, these molecules are also found involved in
tumorigenesis of different kinds of cancers. In this study, we demonstrated that
expression of ephrin-A1 was dramatically down-regulated in glioma cell lines and
in primary gliomas compared to the matched normal tissues. Forced expression of
ephrin-A1 attenuated cell migration, cell proliferation, and
adhesion-independent growth in human glioma U251 cells. EphA2, a receptor for
ephrin-A1 and an oncoprotein, was greatly decreased in ephrin-A1-transfected
glioma cells. Overexpression of ephrin-A1 stimulated activation of EphA2 by
phosphorylation and led to its degradation. Furthermore, focal adhesion kinase
(FAK), a known downstream molecule of EphA2, was also down-regulated in the
ephrin-A1 transfected cells. These results suggested that ephrin-A1 serves as a
critical negative regulator in the tumorigenesis of gliomas by down-regulating
EphA2 and FAK, which may provide potential valuable targets for therapeutic
intervention. We have previously shown that the EphA2 receptor tyrosine kinase is
overexpressed in glioblastoma multiforme (GBM) and represents a novel,
attractive therapeutic target for the treatment of brain tumors. Here, we have
developed an EphA2-targeted agent, ephrinA1-PE38QQR, a novel cytotoxin composed
of ephrinA1, a ligand for EphA2, and PE38QQR, a mutated form of Pseudomonas
aeruginosa exotoxin A. EphrinA1-PE38QQR showed potent and dose-dependent killing
of GBM cells overexpressing the EphA2 receptor in cell viability and clonogenic
survival assays, with an average IC(50) of approximately 10(-11) mol/L. The
conjugate was also highly effective in killing breast and prostate cancer cells
overexpressing EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, as
it was neutralized by an excess of EphA2 ligands. Moreover, normal human
endothelial cells and breast cancer cells that do not overexpress EphA2, as well
as GBM cells that have down-regulated EphA2, were not susceptible to the
cytotoxin. EphrinA1-PE38QQR-mediated cytotoxicity induced caspase-dependent
apoptosis, which was, however, not responsible for cell death in response to the
conjugate. In addition, the conjugate elicited no changes in the activity of
survival pathways such as phosphoinositide 3-kinase, measured by AKT
phosphorylation. This is the first attempt to create a cytotoxic therapy using
any of the ephrin ligands of either class (A or B) conjugated to a bacterial
toxin. EphrinA1-PE38QQR is very potent and specific, produces cell death that is
caspase independent, and forms the basis for the further development of
clinically applicable EphA2-targeted cytotoxins. BACKGROUND: Maligt gliomas are lethal cancers, highly dependent on
angiogenesis and treatment options and prognosis still remain poor for patients
with recurrent glioblastoma multiforme (GBM). Ephs and ephrins have many
well-defined functions during embryonic development of central nervous system
such as axon mapping, neural crest cell migration, hindbrain segmentation and
synapse formation as well as physiological and abnormal angiogenesis.
Accumulating evidence indicates that Eph and ephrins are frequently
overexpressed in different tumor types including GBM. However, their role in
tumorigenesis remains controversial, as both tumor growth promoter and
suppressor potential have been ascribed to Eph and ephrins while the function of
EphA7 in GBM pathogenesis remains largely unknown.
METHODS: In this study, we investigated the immunohistochemical expression of
EphA7 in a series of 32 primary and recurrent GBM and correlated it with
clinical pathological parameters and patient outcome. In addition, intratumor
microvascular density (MVD) was quantified by immunostaining for endothelial
cell marker von Willebrand factor (vWF).
RESULTS: Overexpression of EphA7 protein was predictive of the adverse outcome
in GBM patients, independent of MVD expression (p = 0.02). Moreover, high
density of MVD as well as higher EphA7 expression predicted the disease outcome
more accurately than EphA7 variable alone (p = 0.01). There was no correlation
between MVD and overall survival or recurrence-free survival (p > 0.05).
However, a statistically significant correlation between lower MVD and tumor
recurrence was observed (p = 0.003).
CONCLUSION: The immunohistochemical assessment of tissue EphA7 provides
important prognostic information in GBM and would justify its use as surrogate
marker to screen patients for tyrosine kinase inhibitor therapy. The ephrinA1 ligand exerts antioncogenic effects in tumor cells through
activation and downregulation of the EphA2 receptor and has been described as a
membrane-anchored protein requiring clustering for function. However, while
investigating the ephrinA1/EphA2 system in the pathobiology of glioblastoma
multiforme (GBM), we uncovered that ephrinA1 is released from GBM and breast
adenocarcinoma cells as a soluble, monomeric protein and is a functional form of
the ligand in this state. Conditioned media containing a soluble monomer of
ephrinA1 caused EphA2 internalization and downregulation, dramatic alteration of
cell morphology and suppression of the Ras-MAPK pathway. Moreover, soluble
monomeric ephrinA1 was functional in a physiological context, eliciting collapse
of embryonic neuronal growth cones. We also found that ephrinA1 is cleaved from
the plasma membrane of GBM cells, an event which involves the action of a
metalloprotease. Thus, the ephrinA1 ligand can, indeed, function as a soluble
monomer and may act in a paracrine manner on the EphA2 receptor without the need
for juxtacrine interactions. These findings have important implications for
further deciphering the function of these proteins in pathology and physiology,
as well as for the design of ephrinA1-based EphA2-targeted antitumor
therapeutics. The Eph receptor tyrosine kinases and ephrin ligands have been studied
extensively for their roles in developmental processes. In recent years, Eph
receptors and ephrins have been found to be integral players in cancer formation
and progression. Among these are EphA2 and ephrinA1, which are involved in the
development and maintece of many different types of solid tumors. The
function of EphA2 and ephrinA1 in tumorigenesis and tumor progression is complex
and seems to be dependent on cell type and microenvironment. These variables
affect the expression of the EphA2 and ephrinA1 proteins, the pathways through
which they induce signaling, and the functional consequences of that signaling
on the behavior of tumor cells and tumor-associated cells. This review will
specifically focus on the roles that EphA2 and ephrinA1 play in the different
cell types that contribute to the maligcy of solid tumors, with emphasis on
the opportunities for therapeutic targeting. Eph receptors, the largest subfamily of receptor tyrosine kinases, and their
ephrin ligands play important roles in nervous system development. Recently,
they have been implicated in tumorigenesis of different cancers. In this study,
we showed that the expression of ephrinA5 was dramatically downregulated in
primary gliomas compared with normal tissues. Forced expression of ephrinA5
reduced tumorigenicity of human glioma U373 cells. Epidermal growth factor
receptor (EGFR), which frequently acts as an oncoprotein in glioma, was greatly
decreased in ephrinA5-transfected glioma cells, and the two molecules exhibited
a mutually exclusive expression pattern in primary glioma samples. We found that
ephrinA5 enhanced c-Cbl binding to EGFR, thus promoted ubiquitylation and
degradation of the receptor. Either ephrinA5-Fc or EphA2-Fc treatment simulating
bidirectional signaling of Eph/ephrin system resulted in EGFR decrease. This
study discovered that ephrinA5 acted as a tumor suppressor in glioma, and its
negative regulation of EGFR contributed to the suppressive effects. In addition
to identifying a novel mechanism underlying tumor suppressor activity of
ephrinA5, we also showed cross-talk between different receptor tyrosine kinase
families in glioma. These findings may improve therapeutic strategies for
glioma. To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM)
cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM
specimens using laser capture microdissection. Isolated RNA underwent whole
human genome expression profiling to identify differentially expressed genes.
Pathway enrichment analysis highlighted the bidirectional receptor/ligand
tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the
invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a
clinically annotated expression data set of 195 brain biopsy specimens. Levels
of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in
normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not
ephrin-B1, expression levels were significantly associated with short term
survival in maligt astrocytomas (n = 97, p = 0.016). In human brain tumor
specimens, the production and phosphorylation of ephrin-B2 were high in GBM.
Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells
but not in normal brain. A highly invasive glioma cell line, U87, expressed high
levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment
with EphB2/Fc chimera further enhanced migration and invasion of U87 cells,
whereas treatment with an ephrin-B2 blocking antibody significantly slowed
migration and invasion. Forced expression of ephrin-B2 in the U251 cell line
stimulated migration and invasion in vitro and ex vivo, concomitant with
tyrosine phosphorylation of ephrin-B2. These results demonstrate that high
expression of ephrin-B2 is a strong predictor of short-term survival and that
ephrin-B2 plays a critical role in glioma invasion rendering this signaling
pathway as a potential therapeutic target. EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2
receptor, which is overexpressed in glioblastoma (GBM), among other cancers.
Activation of the receptor by ephrinA1 leads to a suppression of oncogenic
properties of GBM cells. We documented that a monomeric functional form of
ephrinA1 is released from cancer cells and thus explored the mechanism of
ephrinA1 release and the primary protein sequence. We demonstrate here that
multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably
MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs
at three positions near the C terminus, producing three forms ending in
valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174
to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released
by proteolysis, underlining the necessity of amino acids 175 to 181 for release
from the membrane. Furthermore, recombit ephrinA1 ending at residue 175
retains activity toward the EphA2 receptor. These findings suggest a mechanism
of release and provide evidence for the existence of several forms of monomeric
ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175
in fusions or conjugates with other molecules in order to prevent likely
proteolysis within physiological and pathobiological environments. Eph/ephrin signaling has been implicated in various types of key
cancer-enhancing processes, like migration, proliferation, and angiogenesis. In
medulloblastoma, invading tumor cells characteristically lead to early
recurrence and a decreased prognosis. Based on kinase-activity profiling data
published recently, we hypothesized a key role for the Eph/ephrin signaling
system in medulloblastoma invasion. In primary medulloblastoma samples, a
significantly higher expression of EphB2 and the ligand ephrin-B1 was observed
compared with normal cerebellum. Furthermore, medulloblastoma cell lines showed
high expression of EphA2, EphB2, and EphB4. Stimulation of medulloblastoma cells
with ephrin-B1 resulted in a marked decrease in in vitro cell adhesion and an
increase in the invasion capacity of cells expressing high levels of EphB2. The
cell lines that showed an ephrin-B1-induced phenotype possessed increased levels
of phosphorylated EphB2 and, to a lesser extent, EphB4 after stimulation.
Knockdown of EphB2 expression by short hairpin RNA completely abolished ephrin
ligand-induced effects on adhesion and migration. Analysis of signal
transduction identified p38, Erk, and mTOR as downstream signaling mediators
potentially inducing the ephrin-B1 phenotype. In conclusion, the observed
deregulation of Eph/ephrin expression in medulloblastoma enhances the invasive
phenotype, suggesting a potential role in local tumor cell invasion and the
formation of metastases. |
What is the effect of the absence of Saccharomyces cerevisiae Rrm3p? | The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers. These replication defects were associated with activation of the intra-S phase checkpoint. Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures. | The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While
replication of most of the yeast genome was not dependent upon Rrm3p, in its
absence, replication forks paused and often broke at an estimated 1400 discrete
sites, including tRNA genes, centromeres, inactive replication origins, and
transcriptional silencers. These replication defects were associated with
activation of the intra-S phase checkpoint. Activation of the checkpoint was
critical for viability of rrm3Delta cells, especially at low temperatures. Each
site whose replication was affected by Rrm3p is assembled into a nonnucleosomal
protein-DNA complex. At tRNA genes and the silent mating type loci, disruption
of these complexes eliminated dependence upon Rrm3p. These data indicate that
the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes,
naturally occurring impediments that are encountered in each S phase. Termination of replication forks at the natural termini of the rDNA of
Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by
the interaction of the Fob1p replication terminator protein with the tandem Ter
sites located in the nontranscribed spacers. Here we show, by both 2D gel
analyses and chromatin immunoprecipitations (ChIP), that there exists a second
level of global control mediated by the intra-S-phase checkpoint protein complex
of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity
of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks
presumably by the transient displacement of the Ter-bound Fob1p. Consistent with
this mechanism, very few replication forks were arrested at the natural
replication termini in the absence of the two checkpoint proteins. In the
absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at
the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes
restored fork arrest by removing both the fork-releasing and fork-protection
activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in
the MRC1 checkpoint pathway were not involved in this global control. This
observation suggests that Tof1p-Csm3p function differently from MRC1 and the
other above-mentioned genes. This mechanism is not restricted to the natural Ter
sites but was also observed at fork arrest caused by the meeting of a
replication fork with transcription approaching from the opposite direction. How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal
conditions and in the presence of genotoxic stress is less understood. We
demonstrate that the inefficient mtDNA replication process of mutant yeast cells
lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA
helicase RRM3. The rescue effect is likely due to the increase in the
deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In
contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of
genotoxic stress is not suppressed if RRM3 is lacking suggesting that this
phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found
to stimulate the incorporation of dNTPs into newly synthesised mtDNA of
gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA
helicase in mitochondria affects mtDNA replication directly. Possible roles of
Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the
temporarily displacement of tightly bound mtDNA-binding proteins, or the
stabilization of the mitochondrial replication complex during mtDNA replication. The DNA helicase Rrm3 promotes replication fork progression through >1000
discrete genomic regions and represses the cDNA-mediated mobility of the Ty1
retrotransposon. We explored the connection between DNA replication and Ty1
retromobility by investigating the basis of increased retromobility in an rrm3
mutant. Even though Ty1 cDNA levels are increased in the absence of RRM3,
neither the level nor target-site specificity of cDNA integration was altered.
Instead, cDNA was incorporated into the genome by a Rad52-dependent mechanism
that did not involve gene conversion of genomic Ty1 sequences. In rrm3 isolates,
incorporated cDNA was often present in tandem arrays. Multimeric cDNA arrays
probably arise during chromosomal break repair, since their appearance was
strongly correlated with the formation of gross chromosomal rearrangements.
Moreover, Ty1 multimers were invariantly located on rearranged chromosomes, when
present. Overexpression of a cellular RNase H, which degrades RNA in an RNA:DNA
hybrid, completely suppressed the increase in Ty1 multimer formation in an rrm3
mutant. We propose that RNA:DNA hybrid regions within nascent retrotransposition
events block replication in an rrm3 mutant, leading to chromosome breaks within
Ty1 sequences. Multiple extragenomic Ty1 cDNA molecules are then used as donors
in recombinational repair of the break before it is healed. Replication forks face multiple obstacles that slow their progression. By
two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes,
and this pausing is greatly increased in the absence of the Rrm3 helicase. We
used a genome-wide approach to identify 96 sites of very high DNA polymerase
binding in wild-type cells. Most of these binding sites were not previously
identified pause sites. Rather, the most highly represented genomic category
among high DNA polymerase binding sites was the open reading frames (ORFs) of
highly transcribed RNA polymerase II genes. Twice as many pause sites were
identified in rrm3 compared with wild-type cells, as pausing in this strain
occurred at both highly transcribed RNA polymerase II genes and the previously
identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II
genes are a class of natural pause sites that are not exacerbated in rrm3 cells. |
Do all archaea possess multiple origins of DNA replication? | Origins of DNA replication differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Though most archaea replicate their chromosomes using multiple origins, there are also certain archaea that possess a single origin of DNA replication (such as Pyrococcus abyssi and some archaea belonging in the hyperthermophilic order of Themococcales). | Eukaryotic chromosomes possess multiple origins of replication, whereas
bacterial chromosomes are replicated from a single origin. The archaeon
Pyrococcus abyssi also appears to have a single origin, suggesting a common rule
for prokaryotes. However, in the current work, we describe the identification of
two active origins of replication in the single chromosome of the
hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify
conserved sequence motifs within the origins that are recognized by a family of
three Sulfolobus proteins that are homologous to the eukaryotic initiator
proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by
distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an
analysis of the levels of the three Orc1/Cdc6 proteins in different growth
phases and cell cycle stages, lead us to propose a model for the roles for these
proteins in modulating origin activity. Until recently, the only archaeon for which a bona fide origin of replication
was reported was Pyrococcus abyssi, where a single origin was identified.
Although several in silico analyses have suggested that some archaeal species
might contain more than one origin, this has only been demonstrated recently.
Two studies have shown that multiple origins of replication function in two
archaeal species. One study identified two origins of replication in the
archaeon Sulfolobus solfataricus, whereas a second study used a different
technique to show that both S. solfataricus and Sulfolobus acidocaldarius have
three functional origins. These are the first reports of archaea having multiple
origins. This finding has implications for research on the mechanisms of DNA
replication and evolution. Genome replication is a crucial and essential process for the continuity of
life.In all organisms it starts at a specific region of the genome known as
origin of replication (Ori) site. The number of Ori sites varies in prokaryotes
and eukaryotes. Replication starts at a single Ori site in bacteria, but in
eukaryotes multiple Ori sites are used for fast copying across all chromosomes.
The situation becomes complex in archaea, where some groups have single and
others have multiple origins of replication. Themococcales, are a
hyperthermophilic order of archaea. They are anaerobes and heterotrophs-peptide
fermenters, sulphate reducers, methanogens being some of the examples of
metabolic types. In this paper we have applied a combination of multiple in
silico approaches - Z curve, the cell division cycle (cdc6) gene location and
location of consensus origin recognition box (ORB) sequences for location of
origin of replication in Thermococcus onnurineus, Thermococcus gammatolerans and
other Themococcales and compared the results to that of the well-documented case
of Pyrococcus abyssi. The motivation behind this study is to find the number of
Ori sites based on the data available for members of this order. Results from
this in silico analysis show that the Themococcales have a single origin of
replication. DNA replication initiates at defined sites called origins, which serve as
binding sites for initiator proteins that recruit the replicative machinery.
Origins differ in number and structure across the three domains of life and
their properties determine the dynamics of chromosome replication. Bacteria and
some archaea replicate from single origins, whereas most archaea and all
eukaryotes replicate using multiple origins. Initiation mechanisms that rely on
homologous recombination operate in some viruses. Here we show that such
mechanisms also operate in archaea. We use deep sequencing to study replication
in Haloferax volcanii and identify four chromosomal origins of differing
activity. Deletion of individual origins results in perturbed replication
dynamics and reduced growth. However, a strain lacking all origins has no
apparent defects and grows significantly faster than wild type. Origin-less
cells initiate replication at dispersed sites rather than at discrete origins
and have an absolute requirement for the recombinase RadA, unlike strains
lacking individual origins. Our results demonstrate that homologous
recombination alone can efficiently initiate the replication of an entire
cellular genome. This raises the question of what purpose replication origins
serve and why they have evolved. DNA replication initiation, which starts at specific chromosomal site (known as
replication origins), is the key regulatory stage of chromosome replication.
Archaea, the third domain of life, use a single or multiple origin(s) to
initiate replication of their circular chromosomes. The basic structure of
replication origins is conserved among archaea, typically including an AT-rich
unwinding region flanked by several conserved repeats (origin recognition box,
ORB) that are located adjacent to a replication initiator gene. Both the ORB
sequence and the adjacent initiator gene are considerably diverse among
different replication origins, while in silico and genetic analyses have
indicated the specificity between the initiator genes and their cognate origins.
These replicator-initiator pairings are reminiscent of the oriC-dnaA system in
bacteria, and a model for the negative regulation of origin activity by a
downstream cluster of ORB elements has been recently proposed in haloarchaea.
Moreover, comparative genomic analyses have revealed that the mosaics of
replicator-initiator pairings in archaeal chromosomes originated from the
integration of extrachromosomal elements. This review summarizes the research
progress in understanding of archaeal replication origins with particular focus
on the utilization, control and evolution of multiple replication origins in
haloarchaea. Precise DNA replication is critical for the maintece of genetic integrity in
all organisms. In all three domains of life, DNA replication starts at a
specialized locus, termed as the replication origin, oriC or ORI, and its
identification is vital to understanding the complex replication process. In
bacteria and eukaryotes, replication initiates from single and multiple origins,
respectively, while archaea can adopt either of the two modes. The Z-curve
method has been successfully used to identify replication origins in genomes of
various species, including multiple oriCs in some archaea. Based on the Z-curve
method and comparative genomics analysis, we have developed a web-based system,
Ori-Finder, for finding oriCs in bacterial genomes with high accuracy. Predicted
oriC regions in bacterial genomes are organized into an online database, DoriC.
Recently, archaeal oriC regions identified by both in vivo and in silico methods
have also been included in the database. Here, we summarize the recent advances
of in silico prediction of oriCs in bacterial and archaeal genomes using the
Z-curve based method. DNA replication is one of the most basic processes in all three domains of
cellular life. With the advent of the post-genomic era, the increasing number of
complete archaeal genomes has created an opportunity for exploration of the
molecular mechanisms for initiating cellular DNA replication by in vivo
experiments as well as in silico analysis. However, the location of replication
origins (oriCs) in many sequenced archaeal genomes remains unknown. We present a
web-based tool Ori-Finder 2 to predict oriCs in the archaeal genomes
automatically, based on the integrated method comprising the analysis of base
composition asymmetry using the Z-curve method, the distribution of origin
recognition boxes identified by FIMO tool, and the occurrence of genes
frequently close to oriCs. The web server is also able to analyze the
unotated genome sequences by integrating with gene prediction pipelines and
BLAST software for gene identification and function annotation. The result of
the predicted oriCs is displayed as an HTML table, which offers an intuitive way
to browse the result in graphical and tabular form. The software presented here
is accurate for the genomes with single oriC, but it does not necessarily find
all the origins of replication for the genomes with multiple oriCs. Ori-Finder 2
aims to become a useful platform for the identification and analysis of oriCs in
the archaeal genomes, which would provide insight into the replication
mechanisms in archaea. The web server is freely available at
http://tubic.tju.edu.cn/Ori-Finder2/. |
What is the ubiquitin proteome? | The ubiquitin proteome is the entire set ubiquitinated proteins and of their respective ubiquitination sites. | The successful use of proteasome inhibitors in clinical trials revealed the
potential of the Ubiquitin Proteasome System for drug development. Protein
remodeling through ubiquitylation is known to regulate the stability and
activity of essential cellular factors through largely uncharacterized
mechanisms. Here, we used Tandem repeated Ubiquitin Binding Entities (TUBEs)
under non-denaturing conditions followed by mass spectrometry analysis to study
global ubiquitylation events that may lead to the identification of potential
drug targets. Using this approach we identified 643 proteins including known and
unknown ubiquitin targets from human breast adenocarcinoma MCF7 cells treated
with Adriamycin. Coherent with a global cellular response to this genotoxic
insult, cellular factors identified are involved in protein synthesis, cellular
transport, RNA post-transcriptional modification and signaling pathways
regulating early stress responses. This includes components of large
macromolecular complexes such as subunits and regulators of the proteasome,
supporting the use of this method to characterize networks of molecular
interactions coordinated by ubiquitylation. Further in vitro and in silico
analysis confirmed that 84% of the total proteins identified here, are
ubiquitylated. More importantly the enrichment of known biomarkers and targets
for drug development, underlined the potential of this approach for the
identification of this clinically relevant information. This article is part of
a Special Issue entitled: Proteomics: The clinical link. Protein ubiquitination plays an important role in the regulation of many
cellular processes, including protein degradation, cell cycle regulation,
apoptosis, and DNA repair. To study the ubiquitin proteome we have established
an immunoaffinity purification method for the proteomic analysis of endogenously
ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated
factors was achieved using the FK2 antibody bound to protein G-beaded agarose,
which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass
spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the
identification of 296 FK2-specific proteins in both experiments. The isolation
of ubiquitinated and ubiquitination-related proteins was confirmed by pathway
analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation
enrichment). Additionally, comparing the proteins that specifically came down in
the FK2 IP with databases of ubiquitinated proteins showed that a high
percentage of proteins in our enriched fraction was indeed ubiquitinated.
Finally, assessment of protein-protein interactions revealed that significantly
more FK2-specific proteins were residing in protein complexes than in random
protein sets. This method, which is capable of isolating both endogenously
ubiquitinated proteins and their interacting proteins, can be widely used for
unraveling ubiquitin-mediated protein regulation in various cell systems and
tissues when comparing different cellular states. Ubiquitination is a posttranslational modification of proteins that involves the
covalent attachment of ubiquitin, either as a single moiety or as polymers. This
process controls almost every cellular metabolic pathway through a variety of
combinations of linkages. Mass spectrometry now allows high throughput
approaches for the identification of the thousands of ubiquitinated proteins and
of their ubiquitination sites. Despite major technological improvements in mass
spectrometry in terms of sensitivity, resolution and acquisition speed, the use
of efficient purification methods of ubiquitinated proteins prior to mass
spectrometry analysis is critical to achieve an efficient characterization of
the ubiquitome. This critical step is achieved using different approaches that
possess advantages and pitfalls. Here, we discuss the limits that can be
encountered when deciphering the ubiquitome. This article is part of a Directed
Issue entitled: Molecular basis of muscle wasting. |
Which major signaling pathways are regulated by RIP1? | necroptosis
apoptosis
pro-survival/inflammation NF-κB activation | Virus-induced apoptosis is thought to be the primary mechanism of cell death
following reovirus infection. Induction of cell death following reovirus
infection is initiated by the incoming viral capsid proteins during cell entry
and occurs via NF-κB-dependent activation of classical apoptotic pathways.
Prototype reovirus strain T3D displays a higher cell-killing potential than
strain T1L. To investigate how signaling pathways initiated by T3D and T1L
differ, we methodically analyzed cell death pathways activated by these two
viruses in L929 cells. We found that T3D activates NF-κB, initiator caspases,
and effector caspases to a significantly greater extent than T1L. Surprisingly,
blockade of NF-κB or caspases did not affect T3D-induced cell death. Cell death
following T3D infection resulted in a reduction in cellular ATP levels and was
sensitive to inhibition of the kinase activity of receptor interacting protein 1
(RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on
the dispensability of caspases, a requirement for RIP1 kinase function, and the
physiological status of infected cells, we conclude that reovirus can also
induce an alternate, necrotic form of cell death described as necroptosis. We
also found that induction of necroptosis requires synthesis of viral RNA or
proteins, a step distinct from that necessary for the induction of apoptosis.
Thus, our studies reveal that two different events in the reovirus replication
cycle can injure host cells by distinct mechanisms.
IMPORTANCE: Virus-induced cell death is a determit of pathogenesis. Mammalian
reovirus is a versatile experimental model for identifying viral and host
intermediaries that contribute to cell death and for examining how these factors
influence viral disease. In this study, we identified that in addition to
apoptosis, a regulated form of cell death, reovirus is capable of inducing an
alternate form of controlled cell death known as necroptosis. Death by this
pathway perturbs the integrity of host membranes and likely triggers
inflammation. We also found that apoptosis and necroptosis following viral
infection are activated by distinct mechanisms. Our results suggest that host
cells can detect different stages of viral infection and attempt to limit viral
replication through different forms of cellular suicide. While these death
responses may aid in curbing viral spread, they can also exacerbate tissue
injury and disease following infection. Programmed cell death is a basic cellular process that is critical to
maintaining tissue homeostasis. In contrast to apoptosis, necrosis was
previously regarded as an unregulated and uncontrollable process. However, as
research has progressed, necrosis, also known as necroptosis or programmed
necrosis, is drawing increasing attention, not least becasu of its possible
impications for cancer research. Necroptosis exhibits a unique signaling pathway
that requires the involvement of receptor interaction protein kinases 1 and 3
(RIP1 and RIP3), mixed lineage kinase domain-like (MLKL), and phosphoglycerate
mutase 5 (PGAM5) and can be specifically inhibited by necrostatins. Not only
does necroptosis serve as a backup cell death program when apoptosis is
inhibited, but it is now recognized to play a pivotal role in regulating various
physiological processes and the pathogenesis of a variety of human diseases such
as ischemic brain injury, immune system disorders and cancer. The control of
necroptosis by various defined trigger factors and signaling pathways now offers
the opportunity to target this cellular process for therapeutic purposes. The
purpose of this paper is to review current findings concerning the connections
between various trigger factors and the RIP1/RIP3 signaling pathway as it
relates to necroptosis. The NLRP3 inflammasome functions as a crucial component of the innate immune
system in recognizing viral infection, but the mechanism by which viruses
activate this inflammasome remains unclear. Here we found that inhibition of the
serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA
virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus
initiated assembly of the RIP1-RIP3 complex, which promoted activation of the
GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage
and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove
the NLRP3 inflammasome independently of MLKL, an essential downstream effector
of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for
the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3
inflammasome and establish a direct link between inflammation and cell-death
signaling pathways. RIP1 is an adaptor kinase originally identified as being able to associate with
TNFR1 and Fas, and is later shown to be involved in signaling induced by TLRs.
Major signaling pathways regulated by RIP1 include necroptosis, apoptosis, and
pro-survival/inflammation NF-κB activation. Previous studies show that RIP1
deficiency has no effect on mouse embryogenesis, but blocks postnatal
development. This phenotype could not readily be explained, since mice lacking
TNFR1, Fas, or TLRs show no apparent developmental defect. Certain types of
RIP1-deficient cells are hypersensitive to TNF-induced apoptosis. However, in
our previous study, deletion of the apoptotic adaptor protein, FADD, provides
marginal improvement of postnatal development of rip1 (-/-) mice. Remarkably,
the current data shows that haploid insufficiency of RIP3, a known mediator of
necroptosis, allowed survival of rip1 (-/-) fadd (-/-) mice beyond weaning age,
although the resulting rip1(-/-)fadd(-/-) rip3(+/-) mice were significant
smaller in size and weight. Moreover, complete absence of RIP3 further improved
postnatal development of the resulting rip1 (-/-) fadd (-/-) rip3 (-/-) mice,
which display normal size and weight. In such triple knockout (TKO) mice,
lymphocytes underwent normal development, but progressively accumulated as mice
age. This lymphoproliferative (lpr) disease in TKO mice is, however, less severe
than that of fadd(-/-)rip3 (-/-) double knockout mice. In total, the data show
that the postnatal developmental defect in rip1 (-/-) mice is due in part to
FADD-mediated apoptosis as well as RIP3-dependent necroptosis. Moreover, the
function of RIP1 contributes to development of lpr diseases. |
What is the effect induced by sympathetic nervous system on pupil size? | Pupil size is determined by the interaction of the parasympathetic and the sympathetic nervous system. The sympathetic nervous system acts either directly on the dilator muscle (peripherally) or centrally by inhibiting the Edinger-Westphal nucleus. Thus, the sympathetic nervous system mediates pupillary dilatation. | Pupil is richly innervated by autonomic nerves. Therefore, its size or movement
well reflects the autonomic nervous function. By analyzing dynamics of pupil
movement for light stimulus followed by infrared video-pupillography, the
abnormality of autonomic nervous system can be expressed in a quantitative
manner. Clinical applications of pupillography and pharmacological examination
to Horner syndrome, Adie syndrome, diabetes mellitus, pupillary involvement of
Behçet disease, pupil of brain death and patients with chemical sensitivity have
been described. Segmental spasm of iris in Horner and Aide syndrome, especially,
nasal side of iridoplegia was seen in early stage of Aide syndrome from
pupillary dynamics to light stimulus. An involvement of sympathetic nerve
subserving iris dilator muscle was confirmed from pupillography, drug reaction
to topically applied l-epinephrine in the diabetics. An involvement of
cholinergic nerve which subserve the iris sphincter muscle was seen in Behçet
disease when there was no manifest eye involvement. Mild mydriasis in brain
death patients was seen under denervation of central nervous system. Dysfunction
of sympathetic nerve from pupillary light reflex in chemically sensitive
patients for cholinated hydrocarbon pesticides. This study tested the hypothesis that the Type A behavior pattern and its
components are associated with increased activity of the sympathetic nervous
system. To do this, we employed two new markers of sympathetic tone--pupil size
and platelet catecholamine content. Thirty-three healthy males were blindly
rated for Type A behavior and verbal stylistic components by Structured
Interview. Adrenergic tone to the pupils was assessed from pupil diameter
recorded by infrared television pupillometry, and pupil alpha-adrenergic
sensitivity was estimated from mydriatic response to ophthalmic phenylephrine.
Platelet epinephrine and norepinephrine contents were assayed because these
levels are associated with chronic, long-term circulating catecholamine levels.
The results showed Type As had consistently larger pupil diameters than Type Bs
(p = 0.03, 0.03, and 0.01). There was no difference in pupillary response to
phenylephrine instillation between groups. Platelet epinephrine content was
greater in Type A subjects (chi 2 = 4.25, p less than 0.04, t = 1.6, p = 0.06).
Only the component of explosiveness was modestly associated with resting pupil
size, and no components significantly predicted platelet catecholamines. We
conclude that the results of these two new markers of autonomic activity,
namely, pupil size and platelet epinephrine, suggest that Type A behavior is
characterized by increased sympathetic nervous system tone although we cannot
rule out the possibility of decreased activity of the parasympathetic nervous
system. Psychosomatic elements, behavior, and sympathetic overactivity are presumed to
underlie many cardiovascular diseases; the most investigated conditions are
borderline hypertension and type A/B behavior. Many patients with borderline
hypertension show signs of hyperkinetic circulation, that is, elevated heart
rate and cardiac index. This elevation is neurogenic, since it can be abolished
with a combination of beta-adrenergic and parasympathetic blockade. Response to
such stepwise blockade shows a larger beta-adrenergic and a lower
parasympathetic change in cardiac tone, characteristic of the functional
organization of the medullary integrative centers of cardiovascular control. The
most likely cause for this abnormal integration is psychosomatic. These patients
may be expected to respond to treatment with behavioral and/or cardioselective
beta-adrenergic blocking agents. There is little doubt that type A behavior is a
predictor of coronary heart disease. The description of the pathophysiology of
these transient but characteristically excessive cardiovascular episodes is
difficult, however. Type A individuals do not always hyperreact in the same way;
the response can be limited to heart rate, blood pressure, or occasionally
exaggerated urinary or plasma catecholamine responses. In our laboratory we
concentrated on steady markers of type A behavior. Type A subjects had
reproducibly larger pupil size--indicative of increased sympathetic
arousal--higher heart rate and diastolic blood pressure, and significantly
elevated platelet epinephrine values when challenged by a mental task. Treatment
with behavioral methods, beta-blocking agents, or both might be useful for
prevention of coronary heart disease in type A individuals. Because onset of autonomic neural dysfunction in the diabetic syndrome has not
been well established, sensitive and quantitative measures of autonomic nervous
system (ANS) function were made in 19 non-insulin-dependent (NIDD) and 14
insulin-dependent (IDD) recent-onset diabetic subjects. The known duration of
diabetes mellitus in the NIDD subjects was less than or equal to 12 mo. The
duration in the IDD subjects was less than or equal to 24 mo. RR-variation
during beta adrenergic blockade (an index of an ANS reflex involving the cardiac
parasympathetic nervous system [PNS] pathway) was smaller than that of control
subjects in both NIDD (P less than 0.001) and IDD subjects (P less than 0.01).
This PNS abnormality was not likely to be due to volume depletion since acute
volume depletion induced by furosemide in six normal subjects (1608 +/- 105 ml,
mean +/- SEM) did not change RR-variation. Dark-adapted pupil size after topical
PNS blockade (an index of iris sympathetic nervous system [SNS] activity) was
also smaller in both groups of diabetic subjects (NIDD, P less than 0.01; IDD, P
less than 0.05). Pupillary latency time (an index of an ANS reflex involving
iris PNS pathway) was prolonged in the NIDD subjects (P less than 0.005) but was
not significantly altered in the IDD subjects. Thus, it would appear that the
ANS is impaired soon after the diagnosis of diabetes mellitus. We hypothesize
that early impairment of the ANS is common in IDD and NIDD subjects. This
finding is consistent with the hypothesis that abnormal carbohydrate metabolism
is an important factor in the etiology of diabetic autonomic neuropathy. To assess the relationship between aging and autonomic nervous system function,
cardiovascular and pupillary autonomic nervous system reflexes were measured in
subgroups of 103 normal male subjects ranging in age from 19 to 82 years (mean
age = 39 years). Both the plasma norepinephrine level, a measure of
cardiovascular sympathetic nervous system activity, and the mean arterial blood
pressure increased with age (r = 0.68 and 0.67, respectively, both p less than
0.001). In contrast, the plasma epinephrine level, a measure of adrenomedullary
sympathetic nervous system activity, was unrelated to age (r = 0.08, p = NS).
Respiratory variation of heart rate during beta-adrenergic blockade, an index of
cardiac parasympathetic nervous system activity, was reduced in older subjects
(r = -0.54, p less than 0.001). Thus, there was evidence of an age-related
increase of cardiovascular sympathetic nervous system activity and a reduction
of cardiac parasympathetic nervous system activity. These findings are
consistent with the hypothesis that there is sympathetic nervous system and
parasympathetic nervous system compensation of cardiovascular function in
response to an age-related decrease in baroreceptor sensitivity. However,
dark-adapted pupil size during parasympathetic nervous system blockade, an index
of iris sympathetic nervous system activity, declined with age (r = -0.81, p
less than 0.001). The latency time for the pupillary response to a light
stimulus, an index of iris parasympathetic nervous system activity, was
prolonged in older subjects (r = 0.58, p less than 0.001). Thus, both
sympathetic nervous system and parasympathetic nervous system inputs to the iris
were diminished in older subjects, findings consistent with the generalized
decrease of peripheral somatic nerve function that has been reported with aging
in man. It is concluded that autonomic nervous system function also declines
with aging, but that other age-related changes such as a decline of baroreceptor
sensitivity may lead to compensatory autonomic nervous system response, which
could mask underlying functional defects. The vasoconstrictor supplies to different tissues show distinct patterns of
ongoing and reflex activity, indicating that they are driven by distinct central
pathways. Vasomotor tone depends heavily on connections from the brainstem, so
class-specific vasomotor drives have been sought amongst the sympathetic
premotor neurons which provide those connections. Premotor neurons of the
rostral ventrolateral medulla (subretrofacial nucleus) provide most descending
vasomotor drive. Together, they drive the sympathetic supplies to heart, blood
vessels and adrenal, but not 'non-cardiovascular' sympathetic responses
(sweating, pupil dilatation, piloerection, etc.). Individually, they provide
preferential or selective drives to particular classes of 'cardiovascular'
sympathetic outflow. Subretrofacial neurons are arranged topographically,
forming a neural map of the functional class (target tissue), not the body
region, of the driven outflows. It is still unknown whether other premotor cell
groups are organised this way. Nor are the premotor pathways to
'non-cardiovascular' sympathetic nerves yet well-defined. BACKGROUND: Estimation of the mu-agonist opioid effect in anesthetized and
paralyzed patients is often imprecise and can be obscured by concomitant
administration of drugs that affect the sympathetic nervous system, such as
beta-adrenergic blocking agents. As an alternative to hemodynamic measures of
opioid effect, the authors tested the hypothesis that the pupillary light reflex
or pupillary reflex dilation correlated with alfentanil concentrations during
isoflurane anesthesia.
METHODS: Six volunteers were anesthetized on 4 days with 0.8% isoflurane.
Alfentanil was administered intravenously to target total plasma concentrations
of 0, 25, 50, and 100 ng/ml. A 5-s tetanic electrical stimulus was applied to
the skin. Pupil size and the pupillary light reflex were recorded before and
after alfentanil administration, and before and for 8 min after the stimulus.
RESULTS: Alfentanil exponentially impaired reflex pupillary dilation, decreasing
the maximum response amplitude from 5 mm at 0 ng/ml, to 2.3 mm at 25 ng/ml, to
1.0 mm at 50 ng/ml, and finally to 0.2 mm at 100 ng/ml. In contrast, only the
highest concentration of alfentanil depressed the dilation of the pupil in the
first 2 s after the stimulus. Alfentanil administration had no effect on the
pupillary light reflex.
CONCLUSIONS: Dilation of the pupil in response to a noxious stimulus is a
measure of opioid effect in isoflurane-anesthetized volunteers. In contrast, the
pupillary light reflex is unaffected by alfentanil during isoflurane anesthesia.
These data suggest that stimulus-induced pupillary dilation may be used to
evaluate the analgesic component of a combined volatile and opioid anesthetic. Vaginocervical mechanostimulation (VS) was shown previously to release oxytocin
within the spinal cord and to induce pupillary dilatation. In the present study,
(a) injection of oxytocin directly to the spinal cord (10 or 25 microg
intrathecally [i.t.] in 5 microl saline) induced pupillary dilatation when
observed 1 min after the end of the injection and (b) injection of an oxytocin
receptor antagonist ([d(CH2)5-Tyr (Me)2-Orn8]-Vasotocin [OTA]; 25 microg i.t. in
5 microl saline) significantly attenuated the pupillary dilatation response to
VS, when VS was applied 3 min after the end of the injection. Since activation
of autonomic sympathetic preganglionic neurons in the thoracic spinal cord
produces pupillary dilatation, we propose that oxytocin is a central nervous
system neurotransmitter that stimulates these neurons directly, or perhaps
indirectly, and thus is a mediator of VS-produced pupillary dilatation. BACKGROUND: The mechanism of reflex pupillary dilation was investigated in eight
patients who were declared brain dead after rupture of intracranial vascular
malformations and in eight awake volunteers. The authors hypothesized that the
reflex was primarily a spinal sympathetic reflex that would be blocked by
topical application of the alpha1-adrenergic blocking agent dapiprazole and that
it would be present in organ donors with intact spinal reflexes and no history
of hypoxia.
METHODS: In volunteers, pupil size was measured with an infrared pupillometer
while brief painful electric stimuli were delivered to the finger. Pain was
assessed with a visual analog scale and adjusted with each volunteer to equal 3
on a visual analog scale of 0-10. Subjects were studied before and after topical
application of the alpha1-adrenergic antagonist dapiprazole. In organ donors,
the authors measured pupil size after high-intensity tetanic electric
stimulation and in dapiprazole-blocked and -unblocked pupils after surgically
induced nociception.
RESULTS: In volunteers, the pupil dilated 0.43 +/- 0.23 mm after nociceptive
stimuli. Dapiprazole eyedrops blocked this dilation, confirming that the reflex
in awake humans is primarily a sympathetic reflex. Baseline diameters were 5.7
+/- 0.5 mm before dapiprazole and 4.1 +/- 0.9 mm after dapiprazole. In organ
donors, a tetanic electric current failed to dilate the pupil, whereas the skin
incision dilated the pupil 0.4 +/- 0.4 mm, but this dilation was not blocked by
dapiprazole.
CONCLUSION: The authors conclude that pupillary reflex dilation, as it is
clinically performed in awake subjects by stimulating somatic nociceptors, is a
sympathetic reflex. Because it is not present in organ donors, the neural
pathway must require a supraspinal component for completion. |
Is p100 the precursor protein molecule of the NF-kappaB transcription factor subunit p50? | No, the precursor molecule for NF-kappaB p50 is p105 and not p100. Nfkb2 encodes two members of the NF-kappa B/Rel family of proteins: p52 and p100. The p100 polypeptide has been proposed to serve as a precursor of p52 (and not of p50), which corresponds to the N-terminal half of p100. NF-kappaB functions as a hetero- or homo-dimer which can be formed from five NF-kappaB subunits, NF-kappaB1 (p50 and its precursor p105), NF-kappaB2 (p52 and its precursor p100), RelA (p65), RelB and c-Rel. | The p50 subunit of NF-kappa B is derived from the amino terminus of a 105
kilodalton precursor. The p105 carboxyl terminus, which contains ankyrin-like
repeats, a feature of I kappa B molecules, regulates the cytoplasmic retention
of p105 and inhibits DNA binding by the precursor. Here, we describe an I kappa
B protein identical to the carboxyl-terminal region of p105. Probes spanning the
COOH terminus but not the rel homology domain of p105 hybridize to a distinct
2.6-kilobase mRNA expressed in a wide range of murine tissues. The nucleotide
sequence of complementary DNA clones for this transcript, in vitro translation,
and immune precipitation of metabolically labeled cell lysates establish that it
encodes a 70 kilodalton protein that corresponds to the COOH-terminal 607 amino
acids of p105. p70 suppresses p65 and p75c-rel mediated transactivation of
reporter genes under the control of NF-kappa B elements and in vitro can prevent
DNA binding of p50 and p75c-rel homodimers to NF-kappa B sites. The ability of
p70 to stably associate with p49 and p65 in vitro, but not inhibit DNA binding
by these proteins, suggests that the specific inhibitory properties of this I
kappa B may reflect its relative affinity for different rel targets. p70
phosphorylated by protein kinase A fails to inhibit DNA binding by p50 or the
c-rel protein, and sequencing of radiolabeled p70 tryptic phosphopeptides
establishes that protein kinase A phosphorylates serine residue 576 of p70. This
finding suggests that the inhibitory activity of p70 can be regulated by
signaling via the adenylate cyclase pathway. Proteolytic processing of the p105 precursor (NF-kappa B1) generates the p50
subunit of NF-kappa B. To address the function of p105 precursor, we have
generated embryonic stem (ES) cells which lack the C-terminal inhibiting
ankyrin-containing domain of p105 (I kappa B gamma) but contain a functional p50
by homologous recombination. Two forms of p50 were found in ES cells lacking
p105. While one was a p50 molecule with the expected size of 415 amino acids,
the other was an isoform of 358 amino acids generated by alternative splicing.
This isoform lacked the C-terminal 62 amino acids of p50 including the putative
nuclear localization signal (NLS), but included five unrelated amino acids. This
p50 molecule lacking the NLS behaved similar to wild type p50. Importantly, the
p50 isoform-RelA complex interacted efficiently with I kappa B alpha, implying
that the NLS of p50 is not essential for these functions. Also, p50 isoform
dimers were present in the nucleus. Loss of p105 and the augment of total p50
produced an increase in the nuclear kappa B-binding activity of p50-RelA and p50
dimers. In wild type ES cells, a significant proportion of total p50 is
associated with p105. Therefore, the mutation introduced in NF-kappa B1
eliminates both the processing to generate p50 and the trapping of p50 by p105.
In contrast to p50, only a small fraction of RelA is associated with p105. These
findings indicate that p105 plays a unique role in controlling p50 dimer
activity. p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the
source of the NF-kappaB p50 subunit upon processing. p105 can form various
heterodimers with other NF-kappaB subunits, including its own processing
product, p50, and these complexes are signal responsive. Signaling through the
IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer
formation, involving p105 phosphorylation at a C-terminal destruction box. We
show here that IKKbeta phosphorylation of p105 is direct and does not require
kinases downstream of IKK. p105 contains an IKK docking site located in a death
domain, which is separate from the substrate site. The substrate residues were
identified as serines 923 and 927, the latter of which was previously assumed to
be a threonine. S927 is part of a conserved DSGPsi motif and is functionally
most critical. The region containing both serines is homologous to the
N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon
phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate
recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination
and complete degradation by the proteasome. However, processing of p105 is
independent of IKK signaling. In line with this and as a physiologically
relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105
and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B
cells lacking IKKgamma, processing was unaffected, but LPS-induced p105
degradation was abolished. Thus, a functional endogenous IKK complex is required
for signal-induced p105 degradation but not for processing. IkappaB kinase (IKK) is a key mediator of NF-kappaB activation induced by
various immunological signals. In T cells and most other cell types, the primary
target of IKK is a labile inhibitor of NF-kappaB, IkappaBalpha, which is
responsible for the canonical NF-kappaB activation. Here, we show that in T
cells infected with the human T-cell leukemia virus (HTLV), IKKalpha is targeted
to a novel signaling pathway that mediates processing of the nfkappab2 precursor
protein p100, resulting in active production of the NF-kappaB subunit, p52. This
pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears
to act by physically recruiting IKKalpha to p100, triggering
phosphorylation-dependent ubiquitylation and processing of p100. These findings
suggest a novel mechanism by which Tax modulates the NF-kappaB signaling
pathway. Targeted disruption of the Rel/NF-kappaB family members NF-kappaB2, encoding
p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid
tissues. Here, we report that development of Peyer's patch (PP)-organizing
centers is impaired in both NF-kappaB2- and RelB-deficient animals. IL-7-induced
expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP
development, is not impaired in RelB-deficient embryos. LTbeta receptor
(LTbetaR)-deficient mice also lack PPs, and we demonstrate that LTbetaR
signaling induces p52-RelB and classical p50-RelA heterodimers, while tumor
necrosis factor (TNF) activates only RelA. LTbetaR-induced binding of p52-RelB
requires the degradation of the inhibitory p52 precursor, p100, which is
mediated by the NF-kappaB-inducing kinase (NIK) and the IkappaB kinase (IKK)
complex subunit IKKalpha, but not IKKbeta or IKKgamma. Activation of RelA
requires all three IKK subunits, but is independent of NIK. Finally, we show
that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA
binding via the C-terminus of p100. Our data indicate an important role of
p52-RelB heterodimers in lymphoid organ development downstream of LTbetaR, NIK
and IKKalpha. Processing of the NF-kappaB2 precursor protein p100 to generate p52 is an
important step of NF-kappaB regulation. This proteolytic event is tightly
regulated by sequences located at the C-terminal portion of p100. Constitutive
processing of p100 occurs in certain lymphoma cells due to the loss of its
C-terminal regulatory domain, although the underlying mechanisms remain unknown.
We show here that the constitutive processing of C-terminal truncation mutants
of p100 is associated with their active nuclear translocation. Deletion of the
C-terminal death domain of p100 triggers a low, but significant, level of
nuclear translocation and processing. Disruption of the ankyrin-repeat domain of
p100 further enhances its nuclear shuttling activity, which is again associated
with elevated level of processing. More importantly, mutation of the nuclear
localization signal (NLS) of p100 abolishes its processing, and this defect can
be rescued by fusion of a heterologous NLS to the amino- or carboxyl-terminus of
the p100 mutant. These results suggest that nuclear shuttling is a mechanism
regulating the processing of NF-kappaB2/p100. Casper/c-FLIP is a caspase-8-related molecule critically involved in regulation
of death receptor-induced apoptosis. It has been shown that Casper can either
promote or antagonize apoptosis and can activate the transcription factor
NF-kappaB. The exact functions of Casper are controversial. To further
understand how Casper signals, we searched Casper-interacting proteins by yeast
two-hybrid screening. This effort identified NF-kappaB1 (p105), an atypical
IkappaB molecule and the precursor of NF-kappaB subunit p50.
Co-immunoprecipitation experiments indicated that Casper interacted with p105 in
293 cells and this interaction was mediated through the C-terminal IkappaB-like
domain (IkappaBgamma). Overexpression of p105 and IkappaBgamma inhibited
Casper-induced NF-kappaB activation and potentiated Casper-induced apoptosis.
Furthermore, Casper and its C-terminal caspase-like domain inhibited p105
processing into p50. Our findings suggest that p105 is involved in
Casper-mediated regulation of apoptosis and NF-kappaB activation. Processing of NF-kappa B2 precursor protein p100 to generate p52 is tightly
regulated. However, this proteolytic event could be actively induced by the
NF-kappa B-inducing kinase and the human T-cell leukemia virus-encoded
oncoprotein Tax or be constitutively turned on due to the loss of the C-terminal
portion of p100. Whereas NF-kappa B-inducing kinase-mediated p100 processing
requires beta-transducin repeat-containing protein, constitutive processing of
p100 is independent of this protein. On the other hand, Tax-induced processing
of p100 appears to be both beta-transducin repeat-containing protein-dependent
and -independent. We show here that, besides the C-terminal sequences, multiple
functional regions, including the two alpha-helices, dimerization domain,
nuclear localization sequence, and glycine-rich region, located in the N
terminus of p100, also play important roles in both constitutive and inducible
processing, suggesting a common mechanism for p100 processing. We further
demonstrate that with the help of the C-terminal death domain and I kappa B
kinase alpha-targeting serines, the C-terminal ankyrin-repeat domain of p100
strongly interacts with its N-terminal dimerization domain and nuclear
localization sequence, thereby bringing the C- and N-terminal sequences together
to form a three-dimensional domain. This presumptive domain is not only
responsible for suppression of constitutive processing but also required for
inducible processing of p100. Taken together, these studies highlight the
mechanism by which the different sequences within p100 work in concert to
regulate its processing and shed light on the mechanisms of how p100 processing
is tightly and delicately controlled. Processing of NF-kappaB2 precursor protein p100 to generate p52 is tightly
controlled, which is important for proper function of NF-kappaB. Accordingly,
constitutive processing of p100, caused by the loss of its C-terminal processing
inhibitory domain due to nfkappab2 gene rearrangements, is associated with the
development of various lymphomas and leukemia. In contrast to the physiological
processing of p100 triggered by NF-kappaB-inducing kinase (NIK) and its
downstream kinase, IkappaB kinase alpha (IKKalpha), which requires the E3
ligase, beta-transducin repeat-containing protein (beta-TrCP), and occurs only
in the cytoplasm, the constitutive processing of p100 is independent of
beta-TrCP but rather is regulated by the nuclear shuttling of p100. Here, we
show that constitutive processing of p100 also requires IKKalpha, but not
IKKbeta (IkappaB kinase beta) or IKKgamma (IkappaB kinase gamma). It seems that
NIK is also dispensable for this pathogenic processing of p100. These results
demonstrate a general role of IKKalpha in p100 processing under both
physiological and pathogenic conditions. Additionally, we find that IKKalpha is
not required for the nuclear translocation of p100. Thus, these results also
indicate that p100 nuclear translocation is not sufficient for the constitutive
processing of p100. The mammalian Rel/NF-kappaB family of transcription factors, including RelA,
c-Rel, RelB, NF-kappaB1 (p50 and its precursor p105), and NF-kappaB2 (p52 and
its precursor p100), plays a central role in the immune system by regulating
several processes ranging from the development and survival of lymphocytes and
lymphoid organs to the control of immune responses and maligt transformation.
The five members of the NF-kappaB family are normally kept inactive in the
cytoplasm by interaction with inhibitors called IkappaBs or the unprocessed
forms of NF-kappaB1 and NF-kappaB2. A wide variety of signals emanating from
antigen receptors, pattern-recognition receptors, receptors for the members of
TNF and IL-1 cytokine families, and others induce differential activation of
NF-kappaB heterodimers. Although work over the past two decades has shed
significant light on the regulation of NF-kappaB transcription factors and their
functions, much progress has been made in the past two years revealing new
insights into the regulation and functions of NF-kappaB. This recent progress is
covered in this review. Proteolytic processing of the nuclear factor (NF)-kappaB2 precursor protein p100
generates the active NF-kappaB2 subunit p52, which in turn transcriptionally
up-regulates p100 expression. p100 also functions as an IkappaB molecule capable
of repressing p52 activity. The biological significance of this negative
feedback control loop has yet to be demonstrated in vivo. Here we show that mice
deficient in p100 but with constitutive expression of p52 in lymphocytes
developed fatal lung inflammation characterized by diffuse alveolar damage with
marked peribronchial fibrosis. In contrast, their littermates with only p100
deficiency or constitutive expression of p52 in lymphocytes developed mild lung
inflammation with perivascular lymphocyte infiltration and had a normal life
span. The fatal lung inflammation is associated with high-level induction of
interferon-gamma and its inducible inflammatory chemokines, suggesting the
involvement of a T-helper-1 immune response. These findings demonstrate the
physiological relevance of the NF-kappaB2 p100 precursor protein in limiting the
potentially detrimental effects of constitutive NF-kappaB2 signaling in
lymphocytes. We previously reported that alymphoplasia (aly/aly) mice, which have a natural
loss-of-function mutation in the Nik gene, which encodes a kinase essential for
the processing of p100 to p52 in the alternative nuclear factor-κB (NF-κB)
pathway, show mild osteopetrosis with an increase in several parameters of bone
formation: bone formation rate, mineral apposition rate, and osteoblast number.
We therefore investigated the molecular mechanisms triggered by the alternative
NF-κB pathway in the regulation of osteoblast differentiation using primary
osteoblasts (POB) prepared from aly/aly mice. Alkaline phosphatase (ALP)
activity and mineralization induced by the presence of β-glycerophosphate and
ascorbic acid were enhanced in POB from aly/aly compared with wild-type (WT)
mice. Furthermore, osteoblastic differentiation induced by bone morphogenetic
protein 2 (BMP2), as shown by ALP activity, mRNA expression of osteocalcin, Id1,
Osterix and Runx2, and Sma- and Mad-related protein (Smad)1/5/8 phosphorylation,
was also enhanced in POB from aly/aly mice. The ectopic bone formation in vivo
that was induced by BMP2 was enhanced in aly/aly mice compared with controls.
Transfection of a mutant form of p100, p100ΔGRR, which cannot be processed to
p52, stimulated ALP activity and Smad phosphorylation. In contrast to p100ΔGRR,
overexpression of p52 inhibited these events. Both BMP2-induced ALP activity and
Smad phosphorylation were reduced in POB from p100-deficient mice, which carry a
homozygous deletion of the COOH-terminal ankyrin repeats of p100 but still
express functional p52 protein. p52 and p100ΔGRR interacted with a BMP receptor,
ALK2, in overexpressed COS7 cells and changed the ALK2 protein levels in
opposite directions: p52 reduced ALK2 and p100 increased it. Thus, the
alternative the NF-κB pathway via the processing of p52 from p100 negatively
regulates osteoblastic differentiation and bone formation by modifying BMP
activity. |
What is the definition and the biological role of epithelial-mesenchymal transition (EMT) | Epithelial-mesenchymal transition (EMT) is a complex process in which epithelial cells acquire the characteristics of invasive mesenchymal cells. EMT has been implicated in cancer progression and metastasis as well as the formation of many tissues and organs during development. Epithelial cells undergoing EMT lose cell-cell adhesion structures and polarity, and rearrange their cytoskeletons. Several oncogenic pathways such as transforming growth factor (TGF) -β, Wnt, and Notch signaling pathways, have been shown to induce EMT. The epithelial-mesenchymal transition (EMT) is a fundamental process governing morphogenesis in multicellular organisms. This process is also reactivated in a variety of diseases including fibrosis and in the progression of carcinoma. | Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism
by which cells change phenotype during carcinogenesis, as well as tumor
metastasis. Whether EMT is involved in cancer metastasis has a specific,
practical impact on the field of circulating tumor cells (CTCs). Since the
generally accepted definition of a CTC includes the expression of epithelial
surface markers, such as EpCAM, if a cancer cell loses its epithelial surface
markers (which is suggested in EMT), it will not be separated and/or identified
as a CTC. We have developed, and previously reported on the use of, a purely
negative enrichment technology enriching for CTCs in the blood of squamous cell
carcinoma of the head and neck (SCCHN). This methodology does not depend on the
expression of surface epithelial markers. Using this technology, our initial
data on SCCHN patient blood indicates that the presence of CTCs correlates with
worse disease-free survival. Since our enrichment is not dependent on epithelial
markers, we have initiated investigation of the presence of mesenchymal markers
in these CTC cells to include analysis of: vimentin, epidermal growth factor
receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have
demonstrated not only presumed CTCs that express and/or contain: a nucleus,
cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells
that contain all of the aforementioned proteins except cytokeratins, suggesting
that the cells have undergone the EMT process. We suggest that our negative
depletion enrichment methodology provides a more objective approach in
identifying and evaluating CTCs, as opposed to positive selection approaches, as
it is not subjective to a selection bias and can be tailored to accommodate a
variety of cytoplasmic and surface markers which can be evaluated to identify a
multitude of phenotypic patterns within CTCs from individual patients, including
so-called EMT as presented here. Epithelial-mesenchymal transition (EMT) is a biological process that drives
polarized, immotile epithelial cells to undergo multiple biochemical changes to
acquire a mesenchymal cell phenotype. The characteristic features of EMT are
cell apolarity, loss of cellular adhesion, reduced expression of E-cadherin and
increased migratory capacity, as well as invasiveness. EMT is a physiological
process that is essential for normal embryonic development. Additionally,
abnormal activation of EMT contributes to some human pathologies such as tissue
fibrosis, cancer cell invasion and metastasis. In both situations, the basic
molecular mechanisms are similar, but lead to different effects depending on
cell type and biological conditions of the environment. TGF-β is a
multifunctional cytokine that controls proliferation, differentiation and other
functions in many cell types. It has been found that neoplastic development
converts TGF-β into an oncogenic cytokine. It activates various molecular
processes, which are engaged in EMT initiation. All that makes TGF-β a key
regulator of EMT. Connective tissue growth factor (CTGF) is involved in human cancer development
and progression. Epithelial to mesenchymal transition (EMT) plays an important
role in many biological processes. In this study, we wished to investigate the
role of CTGF in EMT of peritoneal mesothelial cells and the effects of CTGF on
adhesion of gastric cancer cells to mesothelial cells. Human peritoneal
mesothelial cells (HPMCs) were cultured with TGF-β1 or various concentrations of
CTGF for different time. The EMT process was monitored by morphology. Real-time
RT-PCR and Western blot were used to evaluate the expression of vimentin, α-SMA
, E-cadherin and β-catenin. RNA interference was used to achieve selective and
specific knockdown of CTGF. We demonstrated that CTGF induced EMT of mesothelial
cells in a dose- and time-dependent manner. HPMCs were exposed to TGF-β1 also
underwent EMT which was associated with the induction of CTGF expression.
Transfection with CTGF siRNA was able to reverse the EMT partially after
treatment of TGF-β1. Moreover, the induced EMT of HPMCs was associated with an
increased adhesion of gastric cancer cells to mesothelial cells. These findings
suggest that CTGF is not only an important mediator but a potent activator of
EMT in peritoneal mesothelial cells, which in turn promotes gastric cancer cell
adhesion to peritoneum. Epithelial-mesenchymal transition (EMT) is a complex process in which epithelial
cells acquire the characteristics of invasive mesenchymal cells. EMT has been
implicated in cancer progression and metastasis as well as the formation of many
tissues and organs during development. Epithelial cells undergoing EMT lose
cell-cell adhesion structures and polarity, and rearrange their cytoskeletons.
Several oncogenic pathways such as transforming growth factor (TGF) -β, Wnt, and
Notch signaling pathways, have been shown to induce EMT. These pathways have
activated transcription factors including Snail, Slug, and the ZEB family which
work as transcriptional repressors of E-cadherin, thereby making epithelial
cells motile and resistant to apoptosis. Mounting evidence shows that EMT is
associated with cell invasion and tumor progression.In this review, we summarize
the characteristic features of EMT, pathways leading to EMT, and the role of EMT
in cell invasion. Three topics are addressed in this review: (1) Definition of
EMT, (2) Signaling pathways leading to EMT, (3) Role of EMT in cell invasion.
Understanding the role of EMT in cell invasion will provide valuable information
for establishing strategies to develop anti-metastatic therapeutics which
modulate maligt cellular processes mediated by EMT. |
Is there a difference in the rate between gene fusion and gene fission? | Yes. Several studies have estimated that gene fusion and fission are relatively rare events and the gene fusion/fission rate is approximately between 2 and 6. A conflicting case has been discovered in an analysis of plant genomes, where in Oryza sativa the opposite trend was observed. | During evolution genes can produce more complex proteins by gene fusion or less
complex proteins by gene fission. Considering proteins from 131 completely
sequenced genomes from all three kingdoms of life, we identified 2869 groups of
multi-domain proteins as a single protein in certain organisms and as two or
more smaller proteins with equivalent domain architectures in other organisms.
We found that fusion events are approximately four times more common than
fission events, and we established that, in most cases, any particular fusion or
fission event only occurred once during the course of evolution. Modular rearrangements play an important role in protein evolution. Functional
modules, often tantamount to structural domains or smaller fragments, are in
many cases well conserved but reoccur in a different order and across many
protein families. The underlying genetic mechanisms are gene duplication,
fusion, and loss of sequence fragments. As a consequence, the sequential order
of domains can be inverted, leading to what is known as circularly permutated
proteins. Using a recently developed algorithm, we have identified a large
number of such rearrangements and analyzed their evolutionary history. We
searched for examples which have arisen by one of the three postulated
mechanisms: independent fusion/fission, "duplication/deletion," and
plasmid-mediated "cut and paste." We conclude that all three mechanisms can be
observed, with the independent fusion/fission being the most frequent. This can
be partly attributed to highly mobile domains. Duplication/deletion has been
found in modular proteins such as peptide synthases. Most proteins comprise one or several domains. New domain architectures can be
created by combining previously existing domains. The elementary events that
create new domain architectures may be categorized into three classes, namely
domain(s) insertion or deletion (indel), exchange and repetition. Using
'DomainTeam', a tool dedicated to the search for microsyntenies of domains, we
quantified the relative contribution of these events. This tool allowed us to
collect homologous bacterial genes encoding proteins that have obviously evolved
by modular assembly of domains. We show that indels are the most frequent
elementary events and that they occur in most cases at either the N- or
C-terminus of the proteins. As revealed by the genomic neighbourhood/context of
the corresponding genes, we show that a substantial number of these terminal
indels are the consequence of gene fusions/fissions. We provide evidence showing
that the contribution of gene fusion/fission to the evolution of multi-domain
bacterial proteins is lower-bounded by 27% and upper-bounded by 64%. We conclude
that gene fusion/fission is a major contributor to the evolution of multi-domain
bacterial proteins. Two-component systems (TCSs) are common signal transduction systems, typically
comprising paired histidine protein kinase (HK) and response regulator (RR)
proteins. In many examples, it appears RR and HK genes have fused, producing a
"hybrid kinase " We have characterized a set of prokaryotic genes encoding RRs,
HKs, and hybrid kinases, enabling characterization of gene fusion and fission.
Primary factors correlating with fusion rates are the presence of transmembrane
helices in HKs and the presence of DNA-binding domains in RRs, features that
require correct (and separate) spatial location. In the absence of such
features, there is a relative abundance of fused genes. The order of paired HK
and RR genes and the nucleotide distance between encoded domains also correlate
with apparent gene fusion rates. We propose that localization requirements and
relative positioning of encoded domains within TCS genes affect the function
(and therefore retention) of hybrid kinases resulting from gene fusion. Protein domains are compact evolutionary units of structure and function that
usually combine in proteins to produce complex domain arrangements. In order to
study their evolution, we reconstructed genome-based phylogenetic trees of
architectures from a census of domain structure and organization conducted at
protein fold and fold-superfamily levels in hundreds of fully sequenced genomes.
These trees defined timelines of architectural discovery and revealed remarkable
evolutionary patterns, including the explosive appearance of domain combinations
during the rise of organismal lineages, the domice of domain fusion processes
throughout evolution, and the late appearance of a new class of multifunctional
modules in Eukarya by fission of domain combinations. Our study provides a
detailed account of the history and diversification of a molecular interactome
and shows how the interplay of domain fusions and fissions defines an
evolutionary mechanics of domain organization that is fundamentally responsible
for the complexity of the protein world. Although the possibility of gene evolution by domain rearrangements has long
been appreciated, current methods for reconstructing and systematically
analyzing gene family evolution are limited to events such as duplication, loss,
and sometimes, horizontal transfer. However, within the Drosophila clade, we
find domain rearrangements occur in 35.9% of gene families, and thus, any
comprehensive study of gene evolution in these species will need to account for
such events. Here, we present a new computational model and algorithm for
reconstructing gene evolution at the domain level. We develop a method for
detecting homologous domains between genes and present a phylogenetic algorithm
for reconstructing maximum parsimony evolutionary histories that include domain
generation, duplication, loss, merge (fusion), and split (fission) events. Using
this method, we find that genes involved in fusion and fission are enriched in
signaling and development, suggesting that domain rearrangements and reuse may
be crucial in these processes. We also find that fusion is more abundant than
fission, and that fusion and fission events occur predomitly alongside
duplication, with 92.5% and 34.3% of fusion and fission events retaining
ancestral architectures in the duplicated copies. We provide a catalog of ∼9,000
genes that undergo domain rearrangement across nine sequenced species, along
with possible mechanisms for their formation. These results dramatically expand
on evolution at the subgene level and offer several insights into how new genes
and functions arise between species. Modularity is a hallmark of molecular evolution. Whether considering gene
regulation, the components of metabolic pathways or signaling cascades, the
ability to reuse autonomous modules in different molecular contexts can expedite
evolutionary innovation. Similarly, protein domains are the modules of proteins,
and modular domain rearrangements can create diversity with seemingly few
operations in turn allowing for swift changes to an organism's functional
repertoire. Here, we assess the patterns and functional effects of modular
rearrangements at high resolution. Using a well resolved and diverse group of
pancrustaceans, we illustrate arrangement diversity within closely related
organisms, estimate arrangement turnover frequency and establish, for the first
time, branch-specific rate estimates for fusion, fission, domain addition and
terminal loss. Our results show that roughly 16 new arrangements arise per
million years and that between 64% and 81% of these can be explained by simple,
single-step modular rearrangement events. We find evidence that the frequencies
of fission and terminal deletion events increase over time, and that modular
rearrangements impact all levels of the cellular signaling apparatus and thus
may have strong adaptive potential. Novel arrangements that cannot be explained
by simple modular rearrangements contain a significant amount of repeat domains
that occur in complex patterns which we term "supra-repeats". Furthermore, these
arrangements are significantly longer than those with a single-step
rearrangement solution, suggesting that such arrangements may result from
multi-step events. In summary, our analysis provides an integrated view and
initial quantification of the patterns and functional impact of modular protein
evolution in a well resolved phylogenetic tree. This article is part of a
Special Issue entitled: The emerging dynamic view of proteins: Protein
plasticity in allostery, evolution and self-assembly. |
What type of cancers and inherited diseases have been associated to mutations in the Notch pathway? | So far, mutations in Notch and other components of its signaling pathway have been implicated in an array of human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL, Alagille Syndrome, Spondylocostal Dysostosis), but more pathologies are likely to be associated with Notch in the future due to its network complexity. | Alagille syndrome is an autosomal domit disorder characterized by abnormal
development of liver, heart, skeleton, eye, face and, less frequently, kidney.
Analyses of many patients with cytogenetic deletions or rearrangements have
mapped the gene to chromosome 20p12, although deletions are found in a
relatively small proportion of patients (< 7%). We have mapped the human Jagged1
gene (JAG1), encoding a ligand for the developmentally important Notch
transmembrane receptor, to the Alagille syndrome critical region within 20p12.
The Notch intercellular signalling pathway has been shown to mediate cell fate
decisions during development in invertebrates and vertebrates. We demonstrate
four distinct coding mutations in JAG1 from four Alagille syndrome families,
providing evidence that it is the causal gene for Alagille syndrome. All four
mutations lie within conserved regions of the gene and cause translational
frameshifts, resulting in gross alterations of the protein product Patients with
cytogenetically detectable deletions including JAG1 have Alagille syndrome,
supporting the hypothesis that haploinsufficiency for this gene is one of the
mechanisms causing the Alagille syndrome phenotype. Alagille syndrome (AGS) is a domitly inherited disorder characterized by
liver disease in combination with heart, skeletal, ocular, facial, renal, and
pancreatic abnormalities. We have recently demonstrated that Jagged1 (JAG1) is
the AGS gene. JAG1 encodes a ligand in the Notch intercellular signaling
pathway. AGS is the first developmental disorder to be associated with this
pathway and the first human disorder caused by a Notch ligand. We have screened
54 AGS probands and family members to determine the frequency of mutations in
JAG1. Three patients (6%) had deletions of the entire gene. Of the remaining 51
patients, 35 (69%) had mutations within JAG1, identified by SSCP analysis. Of
the 35 identified intragenic mutations, all were unique, with the exceptions of
a 5-bp deletion in exon 16, seen in two unrelated patients, and a C insertion at
base 1618 in exon 9, also seen in two unrelated patients. The 35 intragenic
mutations included 9 nonsense mutations (26%); 2 missense mutations (6%); 11
small deletions (31%), 8 small insertions (23%), and 1 complex rearrangement
(3%), all leading to frameshifts; and 4 splice-site mutations (11%). The
mutations are spread across the coding sequence of the gene within the
evolutionarily conserved motifs of the JAG1 protein. There is no phenotypic
difference between patients with deletions of the entire JAG1 gene and those
with intragenic mutations, which suggests that one mechanism involved in AGS is
haploinsufficiency. The two missense mutations occur at the same amino acid
residue. The mechanism by which these missense mutations lead to the disease is
not yet understood; however, they suggest that mechanisms other than
haploinsufficiency may result in the AGS phenotype. Alagille syndrome (AGS) is an autosomal domit disorder caused by mutations in
Jagged1 (JAG1), a ligand in the evolutionarily conserved Notch signaling
pathway. Previous studies have demonstrated that a wide spectrum of JAG1
mutations result in AGS. These include total gene deletions, protein truncating,
splicing and missense mutations which are distributed across the coding region
of the gene. Here we present results of JAG1 mutation screening by SSCP and FISH
in 105 patients with AGS. For these studies, new primers were designed for 12
exons. Mutations were identified in 63/105 patients (60%). The spectrum of the
JAG1 mutations presented here is consistent with previously reported results.
Eighty three percent (52/63) of the mutations were protein truncating, 11%
(7/63) were missense, 2% (1/63) were splice site, and 5% (3/63) were total gene
deletions demonstrable by FISH. Six of the missense mutations are novel. As has
been reported previously, there is no apparent relationship between genotype and
clinical phenotype. Alagille syndrome (AGS) is an autosomal domit disorder characterized by five
major symptoms: cholestasis, vertebral deformity, heart malformations, ocular
defects and peculiar facial appearance. The previously described Jagged1 (JAG1)
gene on chromosome 20p12 has been identified as being responsible for AGS. JAG1
encodes a transmembrane protein acting as ligand for the evolutionarily
conserved Notch signaling pathway. Here we report 36 novel mutations in the JAG1
gene. We identified 12 novel deletions, 4 insertions, 8 missense, 7 nonsense and
5 splice site mutations. All mutations map to the sequence encoding the
extracellular part of the Jagged1 protein. The mutations spread over the entire
gene with slightly increased rates in exons 2 to 6 and exon 23 and 24. Eight
novel missense mutations map to the Delta-Serrate-Lag2 (DSL) domain and adjacent
sequences which are important for ligand-receptor interaction. Inheritance was
determined in 27 families. Sixteen mutations (55%) were de novo and eleven
mutations (45%) were transmitted. Altogether 226 different JAG1 mutations have
been described in association with AGS, including our novel 36 mutations. AGS
variants are spread over the entire gene with only a few mutations in exon 26. A
relatively high number of mutations are clustered in exons 2 to 6. This sequence
region shows high interspecies conservation and encodes the Notch
receptor-binding region (DSL domain). The Notch signaling pathway is involved in a wide variety of highly conserved
developmental processes in mammals. Importantly, mutations of the Notch protein
and components of its signaling pathway have been implicated in an array of
human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL,
Alagille Syndrome, Spondylocostal Dysostosis). In mammals, Notch becomes
activated upon binding of its extracellular domain to ligands (Delta and
Jagged/Serrate) that are present on the surface of apposed cells. The
extracellular domain of Notch contains up to 36 tandem Epidermal Growth
Factor-like (EGF) repeats. Many of these EGF repeats are modified at
evolutionarily-conserved consensus sites by an unusual form of O-glycosylation
called O-fucose. Work from several groups indicates that O-fucosylation plays an
important role in ligand mediated Notch signaling. Recent evidence also suggests
that the enzyme responsible for addition of O-fucose to Notch, protein
O-fucosyltransferase-1 (POFUT1), may serve a quality control function in the
endoplasmic reticulum. Additionally, some of the O-fucose moieties are further
elongated by the action of members of the Fringe family of
beta-1,3-N-acetylglucosaminyltransferases. The alteration in O-fucose saccharide
structure caused by Fringe modulates the response of Notch to its ligands. Thus,
glycosylation serves an important role in regulating Notch activity. This review
focuses on the role of glycosylation in the normal functioning of the Notch
pathway. As well, potential roles for glycosylation in Notch-related human
diseases, and possible roles for therapeutic targeting of POFUT1 and Fringe in
Notch-related human diseases, are discussed. Spondylocostal dysostosis (SCD) is an inherited disorder that is characterized
by the presence of extensive hemivertebrae, truncal shortening and abnormally
aligned ribs. It arises during embryonic development by a disruption of
formation of somites (the precursor tissue of the vertebrae, ribs and associated
tendons and muscles). Previously, three genes causing a subset of autosomal
recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300),
MESP2 (SCDO2: MIM 608681) and LFNG (SCDO3: MIM609813). These genes are all
important components of the Notch signaling pathway, which has multiple roles in
development and disease. Here we have used autozygosity mapping to identify a
mutation in a fourth Notch pathway gene, Hairy-and-Enhancer-of-Split-7 (HES7),
in an autosomal recessive SCD family. HES7 encodes a bHLH-Orange domain
transcriptional repressor protein that is both a direct target of the Notch
signaling pathway, and part of a negative feedback mechanism required to
attenuate Notch signaling. A missense mutation was identified in the DNA-binding
domain of the HES7 protein. Functional analysis revealed that the mutant HES7
was not able to repress gene expression by DNA binding or protein
heterodimerization. This is the first report of mutation in the human HES7 gene,
and provides further evidence for the importance of the Notch signaling pathway
in the correct patterning of the axial skeleton. PURPOSE: The molecular pathogenesis of pediatric ependymoma remains unclear. Our
study was designed to identify genetic changes implicated in ependymoma
progression.
PATIENTS AND METHODS: We characterized 59 ependymoma samples (33 at diagnosis
and 26 at relapse) using array-comparative genomic hybridization (aCGH).
Specific chromosomal imbalances were confirmed by fluorescent in situ
hybridization, and candidate genes were assessed by real-time quantitative
polymerase chain reaction (qPCR), immunohistochemistry, sequencing, and in vitro
functional studies.
RESULTS: aCGH analysis revealed a significant increase in genomic imbalances on
relapse compared with diagnosis, such as gain of 9qter and 1q (54% v 21% and 12%
v 0%, respectively) and loss of 6q (27% v 6%). Supervised tumor classification
showed that gain of 9qter was associated with tumor recurrence, age older than 3
years, and posterior fossa location. Using a candidate-gene strategy, we found
an overexpression of two potential oncogenes at the locus 9qter: Tenascin-C and
Notch1. Moreover, Notch pathway analysis (qPCR) revealed overexpression of Notch
ligands, receptors, and target genes (Hes-1, Hey2, and c-Myc), and
downregulation of Notch repressor Fbxw7. We confirmed by immunohistochemistry
the overexpression of Tenascin-C and Hes-1. We detected Notch1 missense
mutations in 8.3% of the tumors (only in the posterior fossa location and in
case of 9q33-34 gain). Furthermore, inhibition of Notch pathway with a
gamma-secretase inhibitor impaired the growth of ependymoma stem cell cultures.
CONCLUSION: The activation of the Notch pathway and Tenascin-C seem to be
important events in ependymoma progression and may represent future targets for
therapy. We report, to our knowledge for the first time, recurrent oncogenic
mutations in pediatric posterior fossa ependymomas. Spondylocostal dysostosis (SCD) is an inherited disorder with abnormal vertebral
segmentation that results in extensive hemivertebrae, truncal shortening and
abnormally aligned ribs. It arises during embryonic development by a disruption
of formation of somites (the precursor tissue of the vertebrae, ribs and
associated tendons and muscles). Four genes causing a subset of autosomal
recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300),
MESP2 (SCDO2: MIM 608681), LFNG (SCDO3: MIM609813) and HES7 (SCDO4). These genes
are all essential components of the Notch signalling pathway, which has multiple
roles in development and disease. Previously, only a single SCD-causative
missense mutation was described in HES7. In this study, we have identified two
new missense mutations in the HES7 gene in a single family, with only
individuals carrying both mutant alleles being affected by SCD. In vitro
functional analysis revealed that one of the mutant HES7 proteins was unable to
repress gene expression by DNA binding or protein heterodimerization. Notch signalling is a central regulator of differentiation in a variety of
organisms and tissue types. Its activity is controlled by the multi-subunit
γ-secretase (γSE) complex. Although Notch signalling can play both oncogenic and
tumour-suppressor roles in solid tumours, in the haematopoietic system it is
exclusively oncogenic, notably in T-cell acute lymphoblastic leukaemia, a
disease characterized by Notch1-activating mutations. Here we identify novel
somatic-inactivating Notch pathway mutations in a fraction of patients with
chronic myelomonocytic leukaemia (CMML). Inactivation of Notch signalling in
mouse haematopoietic stem cells (HSCs) results in an aberrant accumulation of
granulocyte/monocyte progenitors (GMPs), extramedullary haematopoieisis and the
induction of CMML-like disease. Transcriptome analysis revealed that Notch
signalling regulates an extensive myelomonocytic-specific gene signature,
through the direct suppression of gene transcription by the Notch target Hes1.
Our studies identify a novel role for Notch signalling during early
haematopoietic stem cell differentiation and suggest that the Notch pathway can
play both tumour-promoting and -suppressive roles within the same tissue. JAGGED1 mutations cause Alagille syndrome, comprising a constellation of
clinical findings, including biliary, cardiac and craniofacial anomalies.
Jagged1, a ligand in the Notch signaling pathway, has been extensively studied
during biliary and cardiac development. However, the role of JAGGED1 during
craniofacial development is poorly understood. Patients with Alagille syndrome
have midface hypoplasia giving them a characteristic 'inverted V' facial
appearance. This study design determines the requirement of Jagged1 in the
cranial neural crest (CNC) cells, which encompass the majority of mesenchyme
present during craniofacial development. Furthermore, with this approach, we
identify the autonomous and non-autonomous requirement of Jagged1 in a cell
lineage-specific approach during midface development. Deleting Jagged1 in the
CNC using Wnt1-cre; Jag1 Flox/Flox recapitulated the midfacial hypoplasia
phenotype of Alagille syndrome. The Wnt1-cre; Jag1 Flox/Flox mice die at
postnatal day 30 due to inability to masticate owing to jaw misalignment and
poor occlusion. The etiology of midfacial hypoplasia in the Wnt1-cre; Jag1
Flox/Flox mice was a consequence of reduced cellular proliferation in the
midface, aberrant vasculogenesis with decreased productive vessel branching and
reduced extracellular matrix by hyaluronic acid staining, all of which are
associated with midface anomalies and aberrant craniofacial growth. Deletion of
Notch1 from the CNC using Wnt1-cre; Notch1 F/F mice did not recapitulate the
midface hypoplasia of Alagille syndrome. These data demonstrate the requirement
of Jagged1, but not Notch1, within the midfacial CNC population during
development. Future studies will investigate the mechanism in which Jagged1 acts
in a cell autonomous and cell non-autonomous manner. Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is
characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting
overexpression of cyclin D1 (CCND1). Our current knowledge of this disease
encompasses frequent secondary cytogenetic aberrations and the recurrent
mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these
findings insufficiently explain the biologic underpinnings of MCL. Here, we
performed whole transcriptome sequencing on a discovery cohort of 18 primary
tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a
finding that we confirmed in an extension cohort of 108 clinical samples and 8
cell lines. In total, 12% of clinical samples and 20% of cell lines harbored
somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and
predomitly consisted of truncating mutations or small frame-shifting indels.
NOTCH1 mutations were associated with poor overall survival (P = .003).
Furthermore, we showed that inhibition of the NOTCH pathway reduced
proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have
identified recurrent NOTCH1 mutations that provide the preclinical rationale for
therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL. Alagille syndrome (ALGS, OMIM #118450) is an autosomal domit disorder that
affects multiple organ systems including the liver, heart, eyes, vertebrae, and
face. ALGS is caused by mutations in one of two genes in the Notch Signaling
Pathway, Jagged1 (JAG1) or NOTCH2. In this study, analysis of 21 Vietnamese ALGS
individuals led to the identification of 19 different mutations (18 JAG1 and 1
NOTCH2), 17 of which are novel, including the third reported NOTCH2 mutation in
Alagille Syndrome. The spectrum of JAG1 mutations in the Vietnamese patients is
similar to that previously reported, including nine frameshift, three missense,
two splice site, one nonsense, two whole gene, and one partial gene deletion.
The missense mutations are all likely to be disease causing, as two are loss of
cysteines (C22R and C78G) and the third creates a cryptic splice site in exon 9
(G386R). No correlation between genotype and phenotype was observed. Assessment
of clinical phenotype revealed that skeletal manifestations occur with a higher
frequency than in previously reported Alagille cohorts. Facial features were
difficult to assess and a Vietnamese pediatric gastroenterologist was only able
to identify the facial phenotype in 61% of the cohort. To assess the agreement
among North American dysmorphologists at detecting the presence of ALGS facial
features in the Vietnamese patients, 37 clinical dysmorphologists evaluated a
photographic panel of 20 Vietnamese children with and without ALGS. The
dysmorphologists were unable to identify the individuals with ALGS in the
majority of cases, suggesting that evaluation of facial features should not be
used in the diagnosis of ALGS in this population. This is the first report of
mutations and phenotypic spectrum of ALGS in a Vietnamese population. Splenic marginal zone lymphoma (SMZL) is a B cell maligcy of unknown
pathogenesis, and thus an orphan of targeted therapies. By integrating
whole-exome sequencing and copy-number analysis, we show that the SMZL exome
carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene
required for marginal-zone (MZ) B cell development, represent the most frequent
lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted
to cause impaired degradation of the NOTCH2 protein by eliminating the
C-terminal PEST domain, which is required for proteasomal recruitment. Among
indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted
to SMZL, thus representing a potential diagnostic marker for this lymphoma type.
In addition to NOTCH2, other modulators or members of the NOTCH pathway are
recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and
DTX1. We also noted mutations in other signaling pathways normally involved in
MZ B cell development, suggesting that deregulation of MZ B cell development
pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have
direct implications for the treatment of SMZL patients, given the availability
of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this
disease. BACKGROUND & AIMS: The Notch signaling pathway is activated in leukemia and
solid tumors (such as lung cancer), but little is known about its role in liver
cancer.
METHODS: The intracellular domain of Notch was conditionally expressed in
hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This
was achieved through Cre expression under the control of an albumin and
α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain
[NICD]). We used comparative functional genomics to integrate transcriptome data
from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A
Notch gene signature was generated using the nearest template prediction method.
RESULTS: AFP-NICD mice developed HCC with 100% penetrance when they were 12
months old. Activation of Notch signaling correlated with activation of 3
promoters of insulin-like growth factor 2; these processes appeared to
contribute to hepatocarcinogenesis. Comparative functional genomic analysis
identified a signature of Notch activation in 30% of HCC samples from patients.
These samples had altered expression in Notch pathway genes and activation of
insulin-like growth factor signaling, despite a low frequency of mutations in
regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver
cancer cells with the Notch activation signature using γ-secretase inhibitors or
by expressing a domit negative form of mastermind-like 1 reduced their
proliferation in vitro.
CONCLUSIONS: Notch signaling is activated in human HCC samples and promotes
formation of liver tumors in mice. The Notch signature is a biomarker of
response to Notch inhibition in vitro. The Notch signaling pathway is an important contributor to the development and
homeostasis of the cardiovascular system. Not surprisingly, mutations in Notch
receptors and ligands have been linked to a variety of hereditary diseases that
impact both the heart and the vasculature. In particular, mutations in the gene
encoding the human Notch ligand jagged 1 result in a multisystem autosomal
domit disorder called Alagille syndrome, which includes tetralogy of Fallot
among its more severe cardiac pathologies. Jagged 1 is expressed throughout the
developing embryo, particularly in endothelial cells. Here, we demonstrate that
endothelial-specific deletion of Jag1 leads to cardiovascular defects in both
embryonic and adult mice that are reminiscent of those in Alagille syndrome.
Mutant mice display right ventricular hypertrophy, overriding aorta, ventricular
septal defects, coronary vessel abnormalities and valve defects. Examination of
mid-gestational embryos revealed that the loss of Jag1, similar to the loss of
Notch1, disrupts endothelial-to-mesenchymal transition during endocardial
cushion formation. Furthermore, adult mutant mice exhibit cardiac valve
calcifications associated with abnormal matrix remodeling and induction of bone
morphogenesis. This work shows that the endothelium is responsible for the wide
spectrum of cardiac phenotypes displayed in Alagille Syndrome and it
demonstrates a crucial role for Jag1 in valve morphogenesis. Notch signaling pathway activation is known to contribute to the pathogenesis of
a spectrum of human maligcies, including T cell leukemia. However, recent
studies have implicated the Notch pathway as a tumor suppressor in
myeloproliferative neoplasms and several solid tumors. Here we report a novel
tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and
demonstrate that Notch pathway activation could represent a therapeutic strategy
in this disease. We show that Notch signaling is silenced in human AML samples,
as well as in AML-initiating cells in an animal model of the disease. In vivo
activation of Notch signaling using genetic Notch gain of function models or in
vitro using synthetic Notch ligand induces rapid cell cycle arrest,
differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate
that Notch inactivation cooperates in vivo with loss of the myeloid tumor
suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor
suppressor role for Notch signaling in AML and elucidate the potential
therapeutic use of Notch receptor agonists in the treatment of this devastating
leukemia. |
Can cognitive behavioral therapy improve fatigue in cancer patients? | Yes, it has been documented that cognitive behavioral therapy reduces fatigue symptom severity in cancer patients. In addition, cognitive behavioral therapy has been also shown to improve mood and overall quality of life, and it can be successfully delivered through a variety of treatment modalities in patients with cancer. | PURPOSE: Persistent fatigue is a long-term adverse effect experienced by 30% to
40% of patients cured of cancer. The main objective of this randomized
controlled trial was to show the effectiveness of cognitive behavior therapy
(CBT) especially designed for fatigue in cancer survivors.
PATIENTS AND METHODS: A total of 112 cancer survivors with somatically
unexplained fatigue were allocated randomly to immediate cognitive behavior
therapy or to a waiting list condition for therapy. Both conditions were
assessed two times, at baseline and 6 months later. The primary outcome
variables were fatigue severity (Checklist Individual Strength) and functional
impairment (Sickness Impact Profile). Data were analyzed by intention to treat.
RESULTS: Analyses were based on 50 patients in the intervention condition and 48
patients in the waiting list condition. Patients in the intervention condition
reported a significantly greater decrease than patients in the waiting list
condition in fatigue severity (difference, 13.3; 95% CI, 8.6 to 18.1) and in
functional impairment (difference, 383.2; 95% CI, 197.1 to 569.2). Clinically
significant improvement for the CBT group compared with the waiting list group
was seen in fatigue severity (54% v 4% of the patients, respectively) and in
functional impairment (50% v 18% of the patients, respectively).
CONCLUSION: Cognitive behavior therapy has a clinically relevant effect in
reducing fatigue and functional impairments in cancer survivors. This case report describes an effort to control two primary side-effects of
breast cancer radiotherapy (fatigue and skin discomfort) that used a combination
of cognitive-behavioral therapy with hypnosis (CBTH). Two patients, matched on
demographic and medical variables (marital status, employment status, number of
children, cancer diagnosis, surgical history, radiation dose), were compared:
one who received a CBTH intervention and one who received standard care. Results
were consistent with the view that CBTH was effective in managing fatigue and
skin discomfort, and increasing relaxation. OBJECTIVES: The purpose of this work was to assess the long-term outcome of
adolescents with chronic fatigue syndrome who received cognitive behavioral
therapy and to determine the predictive value of fatigue severity and physical
impairments of the adolescent and the fatigue severity of the mother at baseline
for the outcome of the treatment at follow-up.
PATIENTS AND METHODS: Sixty-six adolescent patients with chronic fatigue
syndrome who previously participated in a randomized, controlled trial that
showed that cognitive behavioral therapy was more effective than a waiting-list
condition in reducing fatigue and improving physical functioning were contacted
for a follow-up assessment. Fifty participants of the follow-up study had
received cognitive behavioral therapy for chronic fatigue syndrome (32 formed
the cognitive behavioral therapy group in the original trial, and 18 patients
received cognitive behavioral therapy after the waiting period). The remaining
16 patients had refused cognitive behavioral therapy after the waiting period.
The main outcome measures were fatigue severity (Checklist Individual Strength),
physical functioning (Short-Form General Health Survey), and school attendance.
RESULTS: Data were complete for 61 patients at follow-up (cognitive behavioral
therapy group: 47 patients; no-treatment group: 14 patients). The mean follow-up
time was 2.1 years. There was no significant change in fatigue severity between
posttreatment and follow-up in the cognitive behavioral therapy group. There was
a significant further increase in physical functioning and school attendance
(10% increase). The adolescents in the cognitive behavioral therapy group were
significantly less fatigued and significantly less functionally impaired and had
higher school attendance at follow-up than those in the no-treatment group.
Fatigue severity of the mother was a significant predictor of treatment outcome.
CONCLUSIONS: The positive effects of cognitive behavioral therapy in adolescents
with chronic fatigue syndrome are sustained after cognitive behavioral therapy.
Higher fatigue severity of the mother predicts lower treatment outcome in
adolescent patients. BACKGROUND: To determine whether sleep quality and fatigue associated with
breast cancer adjuvant chemotherapy treatments can be improved with behavioral
therapy (BT) [Individualized Sleep Promotion Plan (ISPP)] including modified
stimulus control, modified sleep restriction, relaxation therapy, and sleep
hygiene.
METHODS: Randomized-controlled trial based on Piper Integrated Fatigue Model,
219 stages I-IIIA breast cancer patients. Prior to the initial chemotherapy
treatment, BT participants developed an ISPP plan that was regularly reinforced
and revised. Controls received healthy eating information and attention.
Pittsburgh Sleep Quality Index (PSQI), daily diary, actigraph, and Piper Fatigue
Scale (PFS) data were collected 2 days prior, during the 7 days after each
treatment, and 30 days after the last treatment. Repeated measures analysis of
variance was used.
RESULTS: Prior to chemotherapy, participants reported mild fatigue and fairly
poor sleep quality. All variables changed over time. A group by time interaction
was found for sleep quality (PSQI) improving in the BT group. Diary revealed
group differences on number of awakenings, minutes awake after sleep onset, and
sleep efficiency. Fatigue (PFS) was similar between groups.
CONCLUSIONS: The BT group showed improved sleep quality over time and better
sleep (diary). Perceptions of improved sleep quality over time are not
consistently associated with diary or actigraph, or result in lower fatigue. OBJECTIVE: The study purpose was to test the effectiveness of a psychological
intervention combining cognitive-behavioral therapy and hypnosis (CBTH) to treat
radiotherapy-related fatigue.
DESIGN: Women (n = 42) scheduled for breast cancer radiotherapy were randomly
assigned to receive standard medical care (SMC) (n = 20) or a CBTH intervention
(n = 22) in addition to SMC. Participants assigned to receive CBTH met
individually with a clinical psychologist. CBTH participants received training
in hypnosis and CBT. Participants assigned to the SMC control condition did not
meet with a study psychologist.
MAIN OUTCOME MEASURES: Fatigue was measured on a weekly basis by using the
fatigue subscale of the Functional Assessment of Chronic Illness Therapy (FACIT)
and daily using visual analogue scales.
RESULTS: Multilevel modeling indicated that for weekly FACIT fatigue data, there
was a significant effect of the CBTH intervention on the rate of change in
fatigue (p < .05), such that on average, CBTH participants' fatigue did not
increase over the course of treatment, whereas control group participants'
fatigue increased linearly. Daily data corroborated the analyses of weekly data.
CONCLUSION: The results suggest that CBTH is an effective means for controlling
and potentially preventing fatigue in breast cancer radiotherapy patients. BACKGROUND: Research suggests that cancer rehabilitation reduces fatigue in
survivors of cancer. To date, it is unclear what type of rehabilitation is most
beneficial.
OBJECTIVE: This randomized controlled trial compared the effect on
cancer-related fatigue of physical training combined with cognitive behavioral
therapy with physical training alone and with no intervention.
DESIGN: In this multicenter randomized controlled trial, 147 survivors of cancer
were randomly assigned to a group that received physical training combined with
cognitive-behavioral therapy (PT+CBT group, n=76) or to a group that received
physical training alone (PT group, n=71). In addition, a nonintervention control
group (WLC group) consisting of 62 survivors of cancer who were on the waiting
lists of rehabilitation centers elsewhere was included.
SETTING: The study was conducted at 4 rehabilitation centers in the Netherlands.
PATIENTS: All patients were survivors of cancer.
INTERVENTION: Physical training consisting of 2 hours of individual training and
group sports took place twice weekly, and cognitive-behavioral therapy took
place once weekly for 2 hours.
MEASUREMENTS: Fatigue was assessed with the Multidimensional Fatigue Inventory
before and immediately after intervention (12 weeks after enrollment). The WLC
group completed questionnaires at the same time points.
RESULTS: Baseline fatigue did not differ significantly among the 3 groups. Over
time, levels of fatigue significantly decreased in all domains in all groups,
except in mental fatigue in the WLC group. Analyses of variance of
postintervention fatigue showed statistically significant group effects on
general fatigue, on physical and mental fatigue, and on reduced activation but
not on reduced motivation. Compared with the WLC group, the PT group reported
significantly greater decline in 4 domains of fatigue, whereas the PT+CBT group
reported significantly greater decline in physical fatigue only. No significant
differences in decline in fatigue were found between the PT+CBT and PT groups.
CONCLUSIONS: Physical training combined with cognitive-behavioral therapy and
physical training alone had significant and beneficial effects on fatigue
compared with no intervention. Physical training was equally effective as or
more effective than physical training combined with cognitive-behavioral therapy
in reducing cancer-related fatigue, suggesting that cognitive-behavioral therapy
did not have additional beneficial effects beyond the benefits of physical
training. Cancer-related fatigue (CRF) is a disabling and distressing symptom that is
highly prevalent across the cancer continuum from a patient's diagnosis and
treatment through survivorship and end of life. It has a multifactorial etiology
and significant individual variability in its clinical expression, determits,
and sequelae. Despite the significance of CRF, it is often underdiagnosed, and
management is frequently suboptimal. This review synthesizes the state of the
science concerning the features, possible mechanisms, and predictors of CRF;
offers recommendations for the evaluation of CRF; and appraises the strength of
the evidence for a wide range of pharmacologic and nonpharmacologic
interventions to prevent and manage fatigue during and after cancer and its
treatment. There is evidence from methodologically rigorous controlled trials
that exercise, psycho-educational interventions, and cognitive-behavioral
therapy for insomnia are effective in the treatment of CRF, and a wide range of
pharmacologic and nonpharmacologic interventions has shown initial promise in
single-arm pilot studies with small, heterogeneous samples. Rigorously designed
and adequately powered randomized trials are warranted to (1) determine the
effectiveness of promising approaches and (2) identify the interventions that
are most effective in treating CRF in specific subpopulations (eg, stem cell
transplant recipients, older adults, patients with lung or colorectal cancers,
survivors, and those at the end of life). Studies to elucidate the biologic
expression profiles of CRF, to explicate the mechanisms through which particular
interventions impact CRF, and to identify the mediators and moderators of
fatigue outcomes will ultimately permit individually tailored approaches for the
treatment of CRF. Dyspnea, nausea and vomiting, anorexia, fatigue, and sleep disturbances are
common and distressing in advanced cancer. We updated previous systematic
reviews of how these symptoms can be alleviated with targeted literature
searches. The approach to these symptoms requires comprehensive symptom
assessment; treating underlying causes when benefits exceed risks; prioritizing
treatment, as patients usually have many symptoms; and addressing psychosocial
and spiritual distress. For dyspnea, evidence supports systemic opioids and
nonpharmacological treatments such as a fan. The strongest evidence supports
metoclopramide for cancer-related nausea and octreotide for bowel obstruction.
For anorexia, enteral or parenteral nutrition is indicated with obstruction and
expected prognosis of at least 6 weeks. Evidence supports several drugs for
appetite affecting quality of life. For fatigue, evidence supports psychosocial
interventions and methylphenidate. For insomnia, evidence supports
cognitive-behavioral therapy in cancer; no sleep agents have superior
effectiveness. BACKGROUND: Two interventions for fatigue were given during curative cancer
treatment. The aim of this multicenter randomized controlled trial (RCT) with
three conditions was to demonstrate the efficacy and to determine the
contribution of physical activity.
METHODS: Recruited from seven hospitals, 220 patients with various maligcies
participated in a RCT. The brief nursing intervention (BNI) consisted of two
1-hour sessions, 3 months apart, given by 12 trained nurses, focusing only on
physical activity. Cognitive behavior therapy (CBT) consisted of up to ten
1-hour sessions, within 6 months, provided by two therapists, focusing on
physical activity and psychosocial elements. The control group received only
usual care (UC). Assessments took place before and at least 2 months after
cancer treatment, when patients had recovered from acute fatigue. Fatigue was
the primary outcome. Efficacy was tested using analyses of covariance. A
nonparametric bootstrap approach was used to test whether the effect on fatigue
was mediated by physical activity.
RESULTS: The CBT group was significantly less fatigued than the UC group.
Between the BNI and the UC groups, no significant difference was found in
fatigue. The mediation hypothesis was rejected.
DISCUSSION: CBT given during curative cancer treatment proved to be an effective
intervention to reduce fatigue at least 2 months after cancer treatment. The BNI
was not effective. Contrary to what was expected, physical activity did not
mediate the effect of CBT on fatigue. Thus, the reduction in fatigue elicited by
CBT was realized without a lasting increase in physical activity. BACKGROUND: Radiotherapy can have multiple adverse effects, including patient
complaints of persistent fatigue and low quality of life. Cognitive-behavior
therapy (CBT) has alleviated fatigue and improved QOL of cancer patients;
however, little is known about the effects of nurse-led CBT on breast cancer
patients undergoing radiotherapy.
OBJECTIVE: The purpose of this study was to analyze the effects of a nurse-led
CBT program on fatigue and QOL of patients with breast cancer undergoing
radiotherapy.
METHODS: This study was performed using a quasi-experimental pretest/posttest
design, with a nonequivalent control group. The participants were patients with
breast cancer (N = 71: experimental group = 35, control group = 36) undergoing
radiotherapy at P University Hospital in Korea. The experimental group received
a 6-week intervention program that included cognitive restructuring, education
about the disease and medical treatment, relaxation therapy, and rehabilitation
exercise.
RESULTS: After the 6-week intervention, the level of fatigue increased in
patients in both groups. However, the increase in the experimental group was
lower than that in the control group. Quality of life of the experimental group
was significantly higher than that of the control group.
CONCLUSIONS: The nurse-led CBT seemed to control fatigue level and improve QOL.
Therefore, the use of nurse-led CBT for patients with breast cancer undergoing
radiotherapy must be promoted.
IMPLICATIONS FOR PRACTICE: Active involvement of experienced nurses in the
counseling program should be considered. In addition, further research must be
conducted into the implementation of the nurse-led cognitive-behavioral
intervention to a broader spectrum of patients. OBJECTIVE: Insomnia is a common complaint among cancer survivors. Fortunately,
cognitive-behavioral therapy for insomnia (CBT-I) has been shown to be an
effective treatment in this population. However, it is rarely implemented given
its limited availability. To address this barrier, we examined the ability of an
easily accessible online CBT-I program to improve insomnia symptoms in cancer
survivors.
METHODS: Twenty-eight cancer survivors with insomnia were randomly assigned to
either an Internet insomnia intervention (n = 14) or to a waitlist control group
(n = 14). The online program, Sleep Healthy Using The Internet, delivers the
primary components of CBT-I (sleep restriction, stimulus control, cognitive
restructuring, sleep hygiene, and relapse prevention). Pre- and post-assessment
data were collected via online questionnaires and daily sleep diaries.
RESULTS: Participants in the Internet group showed significant improvements at
post-assessment compared with those in the control group in overall insomnia
severity (F(1,26) = 22.8; p<0.001), sleep efficiency (F(1,24) = 11.45; P =
0.002), sleep onset latency (F(1,24) = 5.18; P = 0.03), soundness of sleep
(F(1,24) = 9.34; P = 0.005), restored feeling upon awakening (F(1,24) = 11.95; P
= 0.002), and general fatigue (F(1,26) = 13.88; P = 0.001). Although other group
× time interactions were not significant, overall adjusted effect sizes for all
sleep variables as well as for fatigue, depression, anxiety, and quality of life
ranged from small to large.
CONCLUSIONS: CBT-I delivered through an interactive, individually tailored
Internet intervention may be a viable treatment option for cancer survivors
experiencing insomnia. PURPOSE: The objective of this study was to test the efficacy of
cognitive-behavioral therapy plus hypnosis (CBTH) to control fatigue in patients
with breast cancer undergoing radiotherapy. We hypothesized that patients in the
CBTH group receiving radiotherapy would have lower levels of fatigue than
patients in an attention control group.
PATIENTS AND METHODS: Patients (n = 200) were randomly assigned to either the
CBTH (n = 100; mean age, 55.59 years) or attention control (n = 100; mean age,
55.97 years) group. Fatigue was measured at four time points (baseline, end of
radiotherapy, 4 weeks, and 6 months after radiotherapy). Fatigue was measured
using the Functional Assessment of Chronic Illness Therapy (FACIT) -Fatigue
subscale and Visual Analog Scales (VASs; Fatigue and Muscle Weakness).
RESULTS: The CBTH group had significantly lower levels of fatigue (FACIT) at the
end of radiotherapy (z, 6.73; P < .001), 4-week follow-up (z, 6.98; P < .001),
and 6-month follow-up (z, 7.99; P < .001) assessments. Fatigue VAS scores were
significantly lower in the CBTH group at the end of treatment (z, 5.81; P <
.001) and at the 6-month follow-up (z, 4.56; P < .001), but not at the 4-week
follow-up (P < .07). Muscle Weakness VAS scores were significantly lower in the
CBTH group at the end of treatment (z, 9.30; P < .001) and at the 6-month
follow-up (z, 3.10; P < .02), but not at the 4-week follow-up (P < .13).
CONCLUSION: The results support CBTH as an evidence-based intervention to
control fatigue in patients undergoing radiotherapy for breast cancer. CBTH is
noninvasive, has no adverse effects, and its beneficial effects persist long
after the last intervention session. CBTH seems to be a candidate for future
dissemination and implementation. OBJECTIVE: Fatigue is the most frequently occurring and distressing symptom in
patients with advanced cancer, caused by multiple factors. Neither a specific
histological diagnosis of maligcy nor the type of anticancer treatment seem
to be strongly related to fatigue, which support the idea that other factors may
play a role. This study investigated to what extent the model of
fatigue-perpetuating factors that is known for cancer survivors was applicable
for patients with advanced cancer.
METHODS: Patients on active treatment for various incurable cancers were asked
to complete the Checklist Individual Strength, subscale fatigue severity and
physical activity, the Acceptance scale of the Illness Cognition Questionnaire,
the Hospital Anxiety and Depression Scale, the Fatigue Catastrophizing Scale,
the Symptom Checklist subscale sleep, and the van Sonderen Social Support
List-Discrepancies.
RESULTS: The questionnaires were filled in by 137 patients. Inappropriate
coping, fear of progression, fatigue catastrophizing, discrepancies in social
support, depressive mood, self reported physical activity, and sleeping problems
were all related to fatigue severity in univariate analyses, of which the latter
two were significant in a multivariate linear regression analysis.
CONCLUSION: This study tested fatigue-perpetuating factors known to be of
relevance in cancer survivors, for their relation with fatigue severity in
palliative patients. We demonstrated that these factors were also relevant for
patients on palliative treatment. On the basis of our results, we suggest
clinicians confronted with palliative patients with serious fatigue to address
sleeping problems and promote physical activity. In case of persistent fatigue,
personalized cognitive behavioral therapy can be considered. PURPOSE/OBJECTIVES: To examine the effect of cognitive-behavioral therapy for
insomnia (CBTI) on sleep improvement, daytime symptoms, and quality of life
(QOL) in breast cancer survivors (BCSs) after cancer treatment.
DESIGN: A prospective, longitudinal, randomized, controlled trial.
SETTING: Oncology clinics, breast cancer support groups, and communities in
Colorado.
SAMPLE: 56 middle-aged BCSs with chronic insomnia.
METHODS: Women were randomly assigned to CBTI or behavioral placebo treatment
(BPT) and completed measures of sleep, QOL, functioning, fatigue, and mood at
baseline, postintervention, and at three- and six-month follow-ups.
MAIN RESEARCH VARIABLES: Sleep outcomes (e.g., sleep efficiency, sleep latency,
total sleep time, wake after sleep onset, number of nightly awakenings);
secondary variables included sleep medication use, insomnia severity, QOL,
physical function, cognitive function, fatigue, depression, anxiety, and sleep
attitudes or knowledge.
FINDINGS: Sleep efficiency and latency improved more in the CBTI group than the
BPT group; this difference was maintained during follow-up. Women in the CBTI
group had less subjective insomnia, greater improvements in physical and
cognitive functioning, positive sleep attitudes, and increased sleep hygiene
knowledge. No group differences in improvement were noted relative to QOL,
fatigue, or mood.
CONCLUSIONS: Nurse-delivered CBTI appears to be beneficial for BCSs' sleep
latency/efficiency, insomnia severity, functioning, sleep knowledge, and
attitudes more than active placebo, with sustained benefit over time.
IMPLICATIONS FOR NURSING: Oncology nurses are in a unique position to identify
insomnia in cancer survivors. When sleep disturbances become chronic, nurses
need to make recommendations and referrals. Individuals with cancer are disproportionately affected by sleep disturbance and
insomnia relative to the general population. These problems can be a consequence
of the psychological, behavioral, and physical effects of a cancer diagnosis and
treatment. Insomnia often persists for years and, when combined with already
high levels of cancer-related distress, may place cancer survivors at a higher
risk of future physical and mental health problems and poorer quality of life.
The recommended first-line treatment for insomnia is cognitive behavioral
therapy for insomnia (CBT-I), a non-pharmacological treatment that incorporates
cognitive and behavior-change techniques and targets dysfunctional attitudes,
beliefs, and habits involving sleep. This article presents a comprehensive
review of the literature examining the efficacy of CBT-I on sleep and
psychological outcomes in cancer patients and survivors. The search revealed 12
studies (four uncontrolled, eight controlled) that evaluated the effects of
CBT-I in cancer patients or survivors. Results suggest that CBT-I is associated
with statistically and clinically significant improvements in subjective sleep
outcomes in patients with cancer. CBT-I may also improve mood, fatigue, and
overall quality of life, and can be successfully delivered through a variety of
treatment modalities, making it possible to reach a broader range of patients
who may not have access to more traditional programs. Future research in this
area should focus on the translation of evidence into clinical practice in order
to increase awareness and access to effective insomnia treatment in cancer care. STUDY OBJECTIVE: To assess the short-term efficacy of a video-based cognitive
behavioral therapy for insomnia (CBT-I) as compared to a professionally
administered CBT-I and to a no-treatment group.
DESIGN: Randomized controlled trial.
SETTING: Radio-oncology department of a public hospital affiliated with
Université Laval (CHU de Québec).
PARTICIPANTS: Two hundred forty-two women with breast cancer who had received
radiation therapy in the past 18 mo and who had insomnia symptoms or were using
hypnotic medications were randomized to: (1) professionally administered CBT-I
(PCBT-I; n = 81); (2) video-based CBT-I (VCBT-I; n = 80); and (3) no treatment
(CTL; n = 81).
INTERVENTIONS: PCBT-I composed of six weekly, individual sessions of
approximately 50 min; VCBT-I composed of a 60-min animated video + six booklets.
MEASUREMENT AND RESULTS: Insomnia Severity Index (ISI) total score and sleep
parameters derived from a daily sleep diary and actigraphy, collected at
pretreatment and posttreatment. PCBT-I and VCBT-I were associated with
significantly greater sleep improvements, assessed subjectively, as compared to
CTL. However, relative to VCBT-I, PCBT-I was associated with significantly
greater improvements of insomnia severity, early morning awakenings, depression,
fatigue, and dysfunctional beliefs about sleep. The remission rates of insomnia
(ISI < 8) were significantly greater in PCBT-I as compared to VCBT-I (71.3%
versus 44.3%, P < 0.005).
CONCLUSIONS: A self-administered cognitive behavioral therapy for insomnia
(CBT-I) using a video format appears to be a valuable treatment option, but
face-to-face sessions remain the optimal format for administering CBT-I
efficaciously in patients with breast cancer. Self-help interventions for
insomnia may constitute an appropriate entry level as part of a stepped care
model.
TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00674830.
CITATION: Savard J, Ivers H, Savard MH, Morin CM. Is a video-based cognitive
behavioral therapy for insomnia as efficacious as a professionally administered
treatment in breast cancer? Results of a randomized controlled trial. INTRODUCTION: Many breast cancer patients and survivors experience pain and
emotional stress related to their disease, its diagnostic procedures, or
treatment. Hypnosis has long been used for the treatment of such symptoms. The
aim of this review was to systematically assess the effectiveness of hypnosis in
women with breast cancer, breast cancer survivors, and in women undergoing
diagnostic breast biopsy.
METHODS: PubMed, Scopus, the Cochrane Library, PsycINFO, and CAMBASE were
screened through February 2014 for randomized controlled trials (RCTs) of
hypnosis in women with breast cancer or undergoing diagnostic breast biopsy.
RCTs on postmenopausal women without a history of breast cancer were also
eligible. Primary outcomes were pain, distress, fatigue, nausea/vomiting, and
hot flashes. Safety was defined as secondary outcome measure. Risk of bias was
assessed by 2 reviewers independently using the Cochrane Risk of Bias Tool.
RESULTS: Thirteen RCTs with 1357 patients were included. In women undergoing
diagnostic breast biopsy (3 RCTs), hypnosis positively influenced pain and
distress; 1 RCT on breast cancer surgery found effects of hypnosis on pain,
distress, fatigue, and nausea. For women undergoing radiotherapy (3 RCTs),
hypnosis combined with cognitive-behavioral therapy improved distress and
fatigue. In 3 RCTs on women with and without a history of breast cancer
experiencing hot flashes, hypnosis improved hot flashes and distress. Three RCTs
on women with metastatic breast cancer found effects on pain and distress.
CONCLUSIONS: This systematic review found sparse but promising evidence for the
effectiveness of hypnosis in breast cancer care. While more research is needed
to underpin these results, hypnosis can be considered as an ancillary
intervention in the management of breast cancer-related symptoms. Cancer-related fatigue is a common yet underappreciated problem with a
significant impact on functional ability and quality of life. Practice
guidelines mandate that all cancer patients and survivors be screened for
cancer-related fatigue (CRF) at regular intervals. Comorbidities that could
contribute to fatigue should be treated, and patients with moderate to severe
fatigue should undergo a comprehensive evaluation. Nonpharmacologic
interventions are important tools to combat CRF and should be incorporated into
routine practice. Physical activity, educational interventions, and
cognitive-behavioral therapy have the most supportive data and can be
recommended to patients with confidence. From a practical standpoint, general
education on CRF is something that most care providers can readily offer
patients as part of routine care. Other interventions that appear promising but
are as yet lacking convincing evidence include mindfulness-based stress
reduction, yoga, and acupuncture. Reiki, Qigong, hypnosis, and music therapy may
be worthy of further investigation. |
Is protein Fbw7 a SCF type of E3 ubiquitin ligase? | Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members.The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7) facilitates polyubiquitination and subsequent degradation of various proteins such as Notch, cyclin E, c-Myc and c-Jun. | FBW7 (F-box and WD repeat domain-containing 7) is the substrate recognition
component of an evolutionary conserved SCF (complex of SKP1, CUL1 and F-box
protein)-type ubiquitin ligase. SCF(FBW7) degrades several proto-oncogenes that
function in cellular growth and division pathways, including MYC, cyclin E,
Notch and JUN. FBW7 is also a tumour suppressor, the regulatory network of which
is perturbed in many human maligcies. Numerous cancer-associated mutations in
FBW7 and its substrates have been identified, and loss of FBW7 function causes
chromosomal instability and tumorigenesis. This Review focuses on structural and
functional aspects of FBW7 and its role in the development of cancer. The SCF(Fbw7) ubiquitin ligase complex plays important roles in cell growth,
survival, and differentiation via the ubiquitin-proteasome-mediated regulation
of protein stability. Fbw7 (also known as Fbxw7, Sel-10, hCdc4, or hAgo), a
substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, facilitates the
degradation of several proto-oncogene products by the proteasome. Given that
mutations in Fbw7 are found in various types of human cancers, Fbw7 is
considered to be a potent tumor suppressor. In the present study, we show that
E1A, an oncogene product derived from adenovirus, interferes with the activity
of the SCF(Fbw7) ubiquitin ligase. E1A interacted with SCF(Fbw7) and attenuated
the ubiquitylation of its target proteins in vivo. Furthermore, using in vitro
purified SCF(Fbw7) component proteins, we found that E1A directly bound to
Roc1/Rbx1 and CUL1 and that E1A inhibited the ubiquitin ligase activity of the
Roc1/Rbx1-CUL1 complex but not that of another RING-type ubiquitin ligase, Mdm2.
Ectopically expressed E1A interacted with cellular endogenous Roc1/Rbx1 and CUL1
and decelerated the degradation of several protooncogene products that were
degraded by SCF(Fbw7) ubiquitin ligase. Moreover, after wild-type adenovirus
infection, adenovirus-derived E1A interacted with endogenous Roc1/Rbx1 and
decelerated degradation of the endogenous target protein of SCF(Fbw7). These
observations demonstrated that E1A perturbs protein turnover regulated by
SCF(Fbw7) through the inhibition of SCF(Fbw7) ubiquitin ligase. Our findings may
help to explain the mechanism whereby adenovirus infection induces unregulated
proliferation. The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago,
and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase
complex acts as a tumor suppressor in several tissues and targets multiple
transcriptional activators and protooncogenes for ubiquitin-mediated
degradation. To understand Fbxw7 function in the murine intestine, in this
study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre
(Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of
the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and
induced development of adenomas at 9-10 mo of age. In the context of APC
(adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7
accelerated intestinal tumorigenesis and death and promoted accumulation of
β-catenin in adenomas at late but not early time points. At early time points,
Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression
promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may
also influence cell proliferation. DEK accumulation and altered TPM RNA splicing
were also detected in FBXW7 mutant human colorectal tumor tissues. Given their
reduced lifespan and increased incidence of intestinal tumors,
Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug
screening. The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a
substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7)
facilitates polyubiquitination and subsequent degradation of various proteins
such as Notch, cyclin E, c-Myc and c-Jun. Fbw7 is highly expressed in the
nervous system and controls neural stem cell differentiation and apoptosis via
Notch and c-Jun during embryonic development (Hoeck et al., 2010). Fbw7 deletion
in the neural lineage is perinatal lethal and thus prohibits studying the role
of Fbw7 in the adult nervous system. fbw7 mRNA is highly expressed in the
postnatal brain and to gain insights into the function of Fbw7 in postnatal
neurogenesis we analysed Fbw7 function in the cerebellum. We generated
conditional Fbw7-knockout mice (fbw7(∆Cb)) by inactivating Fbw7 specifically in
the cerebellar anlage. This resulted in decreased cerebellar size, reduced
Purkinje cell number and defects in axonal arborisation. Moreover,
Fbw7-deficient cerebella showed supranumeral fissures and aberrant progenitor
cell migration. Protein levels of the Fbw7 substrates Notch1 and N-terminally
phosphorylated c-Jun were upregulated in fbw7(∆Cb) mice. Concomitant deletion of
c-Jun, and also the junAA knock-in mutation which specifically abrogates c-Jun
N-terminal phosphorylation, rescued Purkinje cell numbers and arborisation in
the fbw7(∆Cb) background. Taken together these data demonstrate that Fbw7 is
essential during cerebellar development, and identify N-terminally
phosphorylated c-Jun as an important substrate of SCF(Fbw7) during neurogenesis. Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type
E3 ligase complex and a well-characterized tumor suppressor that targets
numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has
been frequently found in various types of human cancers; however, little is
known about the upstream signaling pathway(s) governing Fbw7 stability and
cellular functions. Here we report that Fbw7 protein destruction and tumor
suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1
interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7
self-ubiquitination and protein degradation by disrupting Fbw7 dimerization.
Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's
ability to inhibit proliferation and transformation. By contrast, depletion of
Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently
reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated
inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug
target for anticancer therapy. F-box and WD repeat domain-containing 7 (FBW7), the substrate-binding subunit of
E3 ubiquitin ligase SCF(FBW7) (a complex of SKP1, cullin-1 and FBW7), plays
important roles in various physiological and pathological processes. Although
FBW7 is required for vascular development, its function in the endothelium
remains to be investigated. In this study, we show that FBW7 is an important
regulator of endothelial functions, including angiogenesis, leukocyte adhesion
and the endothelial barrier integrity. Using RNA interference, we found that the
depletion of FBW7 markedly impairs angiogenesis in vitro and in vivo. We
identified the zinc finger transcription factor Krüppel-like factor 2 (KLF2) as
a physiological target of FBW7 in endothelial cells. Knockdown of FBW7
expression resulted in the accumulation of endogenous KLF2 protein in
endothelial cells. FBW7-mediated KLF2 destruction was shown to depend on the
phosphorylation of KLF2 via glycogen synthase kinase-3 (GSK3) at two conserved
phosphodegrons. Mutating these phosphodegron motifs abolished the FBW7-mediated
degradation and ubiquitination of KLF2. The siRNA-mediated knockdown of FBW7
showed that KLF2 is an essential target of FBW7 in the regulation of endothelial
functions. Moreover, FBW7-mediated KLF2 degradation was shown to be critical for
angiogenesis in teratomas and in zebrafish development. Taken together, our
study suggests a role for FBW7 in the processes of endothelial cell migration,
angiogenesis, inflammation and barrier integrity, and provides novel insights
into the regulation of KLF2 stability in vivo. FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates
degradation of an array of different target proteins. The Fbw7 locus comprises
three different isoforms, each with its own promoter and each suspected to have
a distinct set of substrates. Most FBW7 targets have important functions in
developmental processes and oncogenesis, including Notch proteins, which are
functionally important substrates of SCF(Fbw7). Notch signalling controls a
plethora of cell differentiation decisions in a wide range of species. A
prominent role of this signalling pathway is that of mediating lateral
inhibition, a process where exchange of signals that repress Notch ligand
production amplifies initial differences in Notch activation levels between
neighbouring cells, resulting in unequal cell differentiation decisions. Here we
show that the downstream Notch signalling effector HES5 directly represses
transcription of the E3 ligase Fbw7β, thereby directly bearing on the process of
lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for
Notch degradation causing impaired intestinal progenitor cell and neural stem
cell differentiation. Notably, concomitant inactivation of Hes5 rescued both
phenotypes and restored normal stem cell differentiation potential. In silico
modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies
Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch
signalling is an essential mechanism that is coupled to and required for the
correct specification of cell fates induced by lateral inhibition. |
Is there increased incidence of incontinence in athletes? | There is a very high prevalence of urinary incontinence in women athletes. | OBJECTIVE: To determine the prevalence of the symptom of urinary incontinence
during athletic endeavors among a group of nulliparous, elite college varsity
female athletes.
METHODS: All women currently participating in varsity athletics at a large state
university were asked to fill out a questionnaire about the occurrence of
urinary incontinence while participating in their sport and during activities of
daily life. One hundred forty-four of 156 eligible women (92%) responded.
RESULTS: The mean age was 19.9 years, and all women were nulliparous. Overall,
40 athletes (28%) reported urine loss while participating in their sport. The
proportions in different sports were: gymnastics 67%, basketball 66%, tennis
50%, field hockey 42%, track 29%, swimming 10%, volleyball 9%, softball 6%, and
golf 0%. Two-thirds of the women who noted urine loss during athletics were
incontinent more often than rarely. There were no statistically significant
relations between incontinence and amenorrhea, weight, hormonal therapy, or
duration of athletic activity. Activities most likely to provoke incontinence
included jumping, high-impact landings, and running. Forty percent and 17% of
the women first noted incontinence during their sport while in high school and
junior high school, respectively.
CONCLUSIONS: Incontinence during physical stresses is common in young, highly
fit, nulliparous women. This suggests that there is a continence threshold
which, when exceeded, can result in urine loss, even in the absence of known
risk factors for incontinence. More than 6 million women now compete in strenous exercis, worldwide. The sports
participation is a sage experience for the teenage athlete. But intense exercise
had been reported to delay menarche when the sports activity was begun before
puberty. Is has been shown to increase beta-endorphins and catecholamine in
women. Menstrual cyclicity is influenced by prior and concomitant exercise
intensity and/or duration in long-distance runners, gymnasts, ballet dancers,
fencers, rowers. Altered menstrual cyclicity can approach 70% in strenuously
exercising women. Menstrual dysfunction has been associated with significant
weight loss, decreased body fat, previous history od menstrual dysfunction,
stress and intensity of athletic training. A complication of amenorrhea is the
loss of bone mineral content and bone density. Urinary incontinence during
physical stresses is common in young nulliparous women. Some female athletes to
increase their sports performance artificially take illegal steroid substances.
Perineo-sphincter exercise must be started very early during the post-partum,
before the start of sports. Most of the athletes of all ages resulted to prefer
the oral contraceptive method. Over the past 30 years, the number of women participating in organized sports
has grown dramatically. Several forms of menstrual irregularities have been
described in the female athlete: primary and secondary amenorrhoea,
oligomenorrhoea, short luteal phases and anovulation. The incidence of menstrual
irregularities is much higher in activities where a thin body is required for
better performance. The hormonal pattern seen in these athletes is a
hypothalamic amenorrhoea profile. There appears to be a decrease in
gonadotrophin-releasing hormone (GnRH) pulses from the hypothalamus, which in
turn decreases the pulsatile secretion of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) and shuts down stimulation of ovary.
Recently, another type of amenorrhoea has been described in swimmers which is
characterized by mild hyperandrogenism. Athletes with low weight are at risk of
developing the female athletic triad, which includes amenorrhoea, osteoporosis
and disordered eating. Athletes with this triad are susceptible to stress
fractures. Other issues include the pregt athlete. Intensive exercise during
pregcy can cause bradycardia. Safe limits of aerobic exercise in pregcy
depend on previous exercise habits. Infertility, which may develop with
exercise, is probably reversible with reduction of exercise or weight gain. High
impact sports activities may produce urinary incontinence. Oestrogen replacement
therapy is often prescribed in amenorrhoeic athletes, but bone loss may not be
completely reversible. PURPOSE: The purposes of the present study were to examine the prevalence of
stress and urge incontinence in female elite athletes and controls, assess a
possible association between urge and stress incontinence and eating disorders,
and assess a possible association between stress and urge incontinence and
menstrual irregularity.
METHODS: This was a two-stage study including a screening part by questionnaire
and a clinical interview. Questions included in the questionnaire were related
to training history, menstrual history, eating behavior, and stress and urge
incontinence. DSM-IV criteria were used in the clinical part of the study to
diagnose eating disorders in those classified as being at risk. The total
population of female elite athletes, defined as one representing the national
team for junior or senior (N = 660), and age-matched nonathlete controls (N =
765) aged 15-39 were asked to answer the questionnaire. The response rate was
87% and 75% for athletes and controls, respectively.
RESULTS: Four percent of the athletes and 33% of the controls had delivered (P <
0.05). There was no significant difference in prevalence of stress urinary
incontinence (SUI) in the athletes and controls, 41% and 39%, respectively. No
significant difference was observed between sport groups. No difference in the
frequency of urge incontinence was found when athletes (16%) and controls (19%)
were compared. Twenty percent of the elite athletes and 9% of the controls met
the DSM-IV criteria for eating disorder. The prevalence of SUI (49.5%) and urge
incontinence (20%) in eating disordered athletes was significantly higher than
in healthy athletes, 38.8% (P = 0.003) and 15% (P = 0.048). No difference in
prevalence was observed when eating disordered nonathletes (39%) and healthy
nonathletes (39%) were compared (P = 0.426).
CONCLUSION: There is a high prevalence of stress and urge incontinence in female
elite athletes. The frequency of SUI and urge incontinence was significantly
higher in eating disordered athletes compared with healthy athletes. The aim of this study was, to determine the frequency of urinary loss in elite
women athletes and dancers. Elite athletes in eight different sports, including
ballet, filled in an evaluated questionnaire about urinary incontinence while
participating in their sport/dancing and during daily life activities. A total
of 291 women with a mean age of 22.8 years completed the questionnaire,
providing a response rate of 73.9%. Overall, 151 women (51.9%) had experienced
urine loss, 125 (43%) while participating in their sport and 123 (42%) during
daily life. The proportion of urinary leakage in the different sports was:
gymnastics 56%, ballet 43%, aerobics 40%, badminton 31%, volleyball 30%,
athletics 25%, handball 21% and basketball 17%. During sport 44% had experienced
leakage a few times, 46.4% now and then, and 9.6% frequently. During daily life
the figures were: 61.7% a few times, 37.4% now and then, and 0.8% frequently. Of
those who leaked during sport, 95.2% experienced urine loss while training
versus only 51.2% during competition (P<0.001). The activity most likely to
provoke leakage was jumping. Sixty per cent (91/151) occasionally wore pads or
panty shields because of urine loss. Urinary leakage is common among elite
athletes and dancers, particularly during training, but also during daily life
activities. Urinary incontinence is defined as "the complaint of any involuntary leakage of
urine" and is a common problem in the female population with prevalence rates
varying between 10% and 55% in 15- to 64-year-old women. The most frequent form
of urinary incontinence in women is stress urinary incontinence, defined as
"involuntary leakage on effort or exertion, or on sneezing or coughing". The aim
of this article is to systematically review the literature on urinary
incontinence and participation in sport and fitness activities with a special
emphasis on prevalence and treatment in female elite athletes. Stress urinary
incontinence is a barrier to women's participation in sport and fitness
activities and, therefore, it may be a threat to women's health, self-esteem and
well-being. The prevalence during sports among young, nulliparous elite athletes
varies between 0% (golf) and 80% (trampolinists). The highest prevalence is
found in sports involving high impact activities such as gymnastics, track and
field, and some ball games. A 'stiff' and strong pelvic floor positioned at an
optimal level inside the pelvis may be a crucial factor in counteracting the
increases in abdominal pressure occurring during high-impact activities. There
are no randomised controlled trials or reports on the effect of any treatment
for stress urinary incontinence in female elite athletes. However, strength
training of the pelvic floor muscles has been shown to be effective in treating
stress urinary incontinence in parous females in the general population. In
randomised controlled trials, reported cure rates, defined as <2g of leakage on
pad tests, varied between 44% and 69%. Pelvic floor muscle training has no
serious adverse effects and has been recommended as first-line treatment in the
general population. Use of preventive devices such as vaginal tampons or
pessaries can prevent leakage during high impact physical activity. The pelvic
floor muscles need to be much stronger in elite athletes than in other women.
There is a need for more basic research on pelvic floor muscle function during
physical activity and the effect of pelvic floor muscle training in female elite
athletes. Urinary incontinence is a common problem in women and may significantly impair
their quality of life. Although women often report stress urinary incontinence
during exercise, current data indicates that most types of exercise are not a
risk factor for the development of urinary incontinence. However, certain
extreme high-impact sports such as parachute jumping may cause pelvic organ
support defects that result in stress urinary incontinence. Eating disorders
also increase the risk of urinary incontinence in athletes. Overall, women
should be encouraged to pursue physical activity that will benefit their general
health without the risk of development of urinary incontinence later in life.
Women athletes should be counseled about the increased risk of urinary
incontinence with ultra high-impact sports and eating disorders. OBJECTIVE: 1) To assess the prevalence of stress urinary incontinence (SUI) and
urge urinary incontinence (UTI) in elite women athletes versus the general
female population, and 2) to analyze the conditions of occurrence of urine loss
in search of etiological clues in elite athletes. DECISION: An anonymous
self-questionnaire was collected transversally from women aged 18 to 35 years.
The exposed group was composed of elite female athletes; the non-exposed group
was made up of women in the same age range accepting to answer the
questionnaire.
RESULTS: A total of 157 answers from elite athletes and 426 from control
subjects were available for analysis. Urinary incontinence prevalence was 28%
for athletes and 9.8% for control subjects (p = .001). There was no significant
difference in the relative prevalence of SUI between the athletes and control
subjects. Athletes reported urine loss more frequently during the second part of
the training session (p < 0.0003), and the second part of competition (p <
0.05). Urinary incontinence prevalence was 9.87% in physically-active control
subjects versus 9.84% in sedentary control subjects (NS). Even a small quantity
of urine loss was felt to be embarrassing. Most incontinent women did not dare
to speak of their condition to anybody.
CONCLUSIONS: There is a very high prevalence of urinary incontinence in women
athletes. Detailed studies of the patho-physiology of this problem are necessary
to formulate preventive recommendations. Research has demonstrated that young female athletes participating in
high-impact sports may be at higher risk for urinary incontinence. Using a
modified Bristol Female Lower Urinary Tract Symptoms Questionnaire, a group of
young adult female athletes was surveyed in Central Illinois to identify the
prevalence of stress incontinence and assess education needs. Results indicated
that more than 25% of those completing surveys experienced incontinence and that
more than 90% had never told anyone about their problem and had no knowledge of
preventive measures; 16% reported incontinence negatively impacted their quality
of life. BACKGROUND: To determine the prevalence of urinary incontinence in female
long-distance runners and to compare it with the presence or not of eating
disorders. Methods - A total of 37 women have completed the International
Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and the short
version of the Eating Attitudes Test (EAT-26). A one-hour pad test was performed
to determine urine loss. Mean values of continuous variables were compared using
an independent sample t-test or the Mann-Whitney U test.
RESULTS: 23 athletes (62.2%) reported urine loss. The mean of the ICIQ-SF was
4.03 +/- 5.06. There was a significant relation between the 1-hour pad test
(p=0.02) and eating disorders (p=0.03).
CONCLUSIONS: There was urinary incontinence in female long-distance runners and
a correlation with eating disorders. Coaches should improve their knowledge
about this problem and establish cooperation with a multidisciplinary team. Stress urinary incontinence (SUI) occurs due to anatomic and/or neurologic
factors involving connective tissues, muscles and nerves. Although SUI is more
common in post-menopausal and multiparous women, studies have also shown a high
prevalence of SUI in young, physically fit female athletes. With a goal toward
dynamic subject-specific mechanical characterization of the interaction between
anatomical structures during physical activities that elicit SUI in females
during physical or daily activities, a computer aided design (CAD)-based
computer model of the female pelvis has been developed to test the feasibility
of the computer modeling approach in understanding the measurable differences
between stress-continent and stress-incontinent women. In the present study, a
fluid-structure interaction analysis was conducted by using the finite element
(FE) analysis technique based on the CAD-based computer model of the female
pelvis to investigate the urine leakage in females during jumping. To the best
of our knowledge, this is the first application of a fluid-structure interaction
FE analysis approach in understanding the mechanisms of SUI in females. Through
a series of computer simulations, the effects of varying impact forces
determined by jumping height and bladder volume were investigated. The dynamic
computer simulation results revealed that jumping heights have a significant
influence on the volume of urine leakage caused by the landing impact of
jumping. Bladder volume did not have a significant influence on leakage when the
jumping heights were smaller than 1 ft, which indicates that normal walking
(corresponds to a jumping height smaller than 0.1 ft) is not the primary cause
of urine leakage for healthy females. The computer simulation results also
showed that the deformation difference between the anterior and posterior
portion of the female pelvis causes opening of the urethra and resultant urine
leakage. The present study demonstrates the feasibility of using a computer
modeling approach to study female SUI during physical and daily activities. A relationship between sport or fitness activities and urinary incontinence (UI)
previously has been described in women. We report our preliminary experience
with the use of a complete pelvic floor rehabilitation program in three female
athletes affected by UI. The athletes were submitted to a combined pelvic floor
rehabilitation program, including biofeedback, functional electrical
stimulation, pelvic floor muscle exercises, and vaginal cones. After the
scheduled rehabilitation scheme, none of the patients reported incontinence, nor
referred to urine leakage during sport or during daily life. We therefore
conclude that UI that affects female agonistic athletes may be effectively
treated with this combined approach. INTRODUCTION AND HYPOTHESIS: To assess the prevalence of lower urinary tract
symptoms (LUTS) and incontinence in female athletes and to determine the
etiological factors.
METHODS: An anonymous self-questionnaire was collected from 623 casual female
athletes aged 18 to 56 years, who were involved in 12 different sports. The
surveys were distributed by hand to the athletes, during their sports fitness
tests, in a sports center. We investigated the relationship between urinary
disorders and factors such as age, body mass index (BMI), parity, duration of
physical exercise, and type of sport.
RESULTS: The prevalence of LUTS was 54.7%, and 30% for urinary incontinence.
Changes in urinary frequency were detected in 91 (14.6%) women. Prevalence of
dysuria was 13.3%, urinary straining was present in 173 (27.8%) athletes,
whereas urinary urgency had an estimated prevalence of 37.2% with 232 athletes
suffering from this disorder. Urgency was very common in volleyball players, as
was dysuria among hockey and basketball players, whereas straining mainly
affected aerobic participants and cyclists. Long training hours and competitive
practices were correlated with the onset of LUTS. High-impact sports were more
frequently associated with incontinence, while low-impact sports with LUTS. The
sport with the main number of incontinent people was football. Urge incontinence
affected a lot of athletes, mainly cyclists and football players. Stress
incontinence was more frequent in hockey and volleyball players.
CONCLUSIONS: LUTS and incontinence are prevalent in female athletes. In many
cases, the disorders were present only during sports activities. In this sample,
the presence of urinary disorders did not seem to be a barrier during sports or
exercise. BACKGROUND: Regular physical activity usually confers health benefits, but
high-level sport may induce harmful outcomes, such as pelvic floor dysfunction.
Urinary incontinence (UI) was previously documented, but few data are available
about anal incontinence (AI) in female athletes. Our aim was to determine the
role of high-level sport practice on AI in a young, healthy female population.
METHODS: In this cross-sectional study, we included women aged 18-40 years.
Self-administered questionnaires were delivered to each female volunteer. Two
groups were defined: (1) intensive sport (IS) group: high-level sport (>8 hours
weekly), and (2) nonintensive sport (NIS) group: all other subjects.
RESULTS: Of the 393 women enrolled, 169 were in the IS group and 224 were in the
NIS group. Women of the IS group were significantly younger than the others
(21.74±4.28 vs. 24.87±5.61 years, p<0.001) and had less births (0.07±0.31 vs.
0.20±0.62, p=0.005). The prevalence of AI was statistically higher in the IS
group than in the NIS group (14.8% vs. 4.9%, p=0.001), as was UI (33.1% vs.
18.3%, p=0.001). Multivariate analysis showed that IS practice (odds ratio [OR]
2.99, 95% confidence interval [CI] 1.29-6.87, p=0.010) and body mass index (BMI)
(OR 1.14, 95% CI 1.01-1.28, p=0.033) were significantly linked to AI when taking
into account major confounding factors (age and births). In the IS group, AI was
mainly represented by loss of flatus in 84%.
CONCLUSIONS: High-level sport appears to be a significant independent risk
factor for AI in healthy young women. These results suggest that preventive
measures, such as pelvic floor muscle training, may be proposed for this young
population. OBJECTIVE: To assess the prevalence of urinary incontinence (UI) in a group of
female athletes and to explore its impact on their lives.
METHODS: In total, 106 female athletics, basketball, and indoor football
athletes were recruited. Data were collected through a questionnaire and a focus
group and analyzed via Pearson's χ(2) test, 2-way analysis of variance, and
thematic analysis.
RESULTS: Urinary incontinence was experienced by 41.5% of the athletes. Its
prevalence across the 3 types of sport was similar and was not affected by age.
However, athletes who experienced UI had a lower body weight (P = 0.011) and a
lower body mass index (P = 0.035). Most (95.5%) athletes had never discussed
their condition with a health professional. In the focus group, all athletes
described preventive urination to avoid urine leakage. It was mentioned that UI
affected their performance and made them feel uncomfortable and frustrated.
CONCLUSION: Urinary incontinence is a prevalent condition among athletes that is
not openly discussed. However, it causes regular changes in their physical
performance and daily life. More research is needed to increase awareness of UI
and to design interventions. OBJECTIVE: To evaluate the pressure of the pelvic floor muscles in female
athletes and the associated signs and symptoms of stress urinary incontinence.
DESIGN: A prospective observational study.
SETTING: An academic institution, primary level of clinical care.
PARTICIPANTS: Forty women between 18 and 30 years of age divided into 4 groups:
10 volleyball players, 10 handball players, 10 basketball players, and 10
nonathletes.
METHODS: The measurement of intracavity pressure was performed with use of a
perineometer. The volunteers were instructed to perform 3 maximum isometric
contractions of the perineum, held for 4 seconds. Data regarding specific
training and urinary symptoms were collected through a questionnaire.
MAIN OUTCOME MEASUREMENTS: Statistical analysis was performed by analysis of
variance, with a significance level of 5%. The Spearman correlation was used to
verify the degree of association between variables related to training, urinary
symptoms, and perineal pressure.
RESULTS: The average (standard deviation) perineal pressure for nonathletes was
6.73 ± 1.91 mm Hg. The average perineal pressure for handball players was 5.55 ±
1.43 mm Hg; for volleyball players, 4.36 ± 1.43 mm Hg; and for basketball
players, 3.65 ± 1.35 mm Hg. Statistically significant differences were found in
the perineal pressure of volleyball (P = .009) and basketball players (P = .039)
compared with nonathletes. The number of games per year, strength training, and
on-court workout correlated significantly with perineal pressure (Spearman
correlation coefficient [Rs] of -0.512 for the 3 variables). Urine leakage
through effort and nocturia correlated moderately with perineal pressure (Rs of
-0.51 and -0.54, respectively). A strong correlation was found between urinary
frequency and perineal pressure (Rs of -0.85).
CONCLUSIONS: Analysis of these data suggests that perineal pressure is decreased
in female athletes compared with nonathlete women. A lower perineal pressure
correlates with increased symptoms of urinary incontinence and pelvic floor
dysfunction. INTRODUCTION AND HYPOTHESIS: The prevalence of female stress urinary
incontinence is high, and young adults are also affected, including athletes,
especially those involved in "high-impact" sports. To date there have been
almost no studies testing pelvic floor muscle (PFM) activity during dynamic
functional whole body movements. The aim of this study was the description and
reliability test of PFM activity and time variables during running.
METHODS: A prospective cross-sectional study including ten healthy female
subjects was designed with the focus on the intra-session test-retest
reliability of PFM activity and time variables during running derived from
electromyography (EMG) and accelerometry.
RESULTS: Thirteen variables were identified based on ten steps of each subject:
Six EMG variables showed good reliability (ICC 0.906-0.942) and seven time
variables did not show good reliability (ICC 0.113-0.731). Time variables (e.g.
time difference between heel strike and maximal acceleration of vaginal
accelerator) showed low reliability. However, relevant PFM EMG variables during
running (e.g., pre-activation, minimal and maximal activity) could be identified
and showed good reliability.
CONCLUSION: Further adaptations regarding measurement methods should be tested
to gain better control of the kinetics and kinematics of the EMG probe and
accelerometers. To our knowledge this is the first study to test the reliability
of PFM activity and time variables during dynamic functional whole body
movements. More knowledge of PFM activity and time variables may help to provide
a deeper insight into physical strain with high force impacts and important
functional reflexive contraction patterns of PFM to maintain or to restore
continence. |
What is the inheritance pattern of Apert syndrome? | The Apert syndrome is a disorder of autosomal dominant inheritance. | This report presents the first example of male transmission of Apert
acrocephalosyndactyly syndrome. Female transmission has been reported in the
five previous well-documented cases of domit inheritance of the syndrome. We report two observations of antenatal diagnosis of Apert syndrome. This
uncommon genetic disorder suggest an autosomal domit inheritance, but almost
all cases described are sporadic; the responsible gene is yet not located.
Ultrasonographic detection is difficult, based on the following signs:
brachycephalic skull (unusually detected), flat facial profile with a nasal
bridge depression, tall appearance of the forehead (inconstant), total bilateral
and symmetrical syndactylies of the hands and feet. At last we present our
arguments for medical abortion, when this disorder is detected. A fourteen years old girl showed the classic signs of acrocephalosyndactyly I:
dysostosis craniofacialis with hypertelorism, exophthalmus, strabism, amblyopia
and cleft palate as well as syndactyly of the fingers and toes. The feet showed
on both side a 6 cm long horny band. Since the twelfth year of life, she had
suffered from papulo-pustular acne with many comedomes. Her menstruation started
one year later. Intellectual development was normal. At time of her birth, her
father was 54 years old, and her mother 36 years old. Two elder siblings are
healthy. The inheritance of acrocephalosyndactyly I is usually autosomal
domit, but sporadic cases are frequent. The Apert syndrome is characterized by craniosynostosis and syndactyly of hands
and feet. Although most cases are sporadic, an autosomal domit mode of
inheritance is well documented. Two mutations in the FGFR2 gene (Ser252Trp and
Pro253Arg) account for most of the cases. We report a patient with a rare form
of Apert syndrome with polydactyly. The proposita has turribrachycephaly.
complete syndactyly of 2nd to 5th digits ("mitten hands" and cutaneous fusion of
all toes). The X-rays revealed craniosynostosis of the coronal suture and
preaxial polydactyly of hands and feet with distal bony fusion. Molecular
analysis found a C755G transversion (Ser252Trp) in the FGFR2 gene. Only eight
patients with Apert syndrome and preaxial polydactyly have been reported and
this is the first case in which molecular diagnosis is available. On the basis
of the molecular findings in this patient, polydactyly should be considered part
of the spectrum of abnormalities in the Apert syndrome. This assertion would
establish the need for a new molecular classification of the
acrocephalopolysyndactylies. INTRODUÇÃO: A síndrome de Apert é um distúrbio raro de herança autossômica
domite causado por mutações no lócus 10q26 do gene FGFR2; pacientes com esta
síndrome apresentam sindactilia severa, exoftalmia, hiperteleorbitismo e
hipoplasia da face média com má oclusão de Classe III, além de alterações
sistêmicas. A maior parte dos estudos disponíveis sobre a síndrome de Apert
aborda o aspecto genético ou manejo cirúrgico, com pouca ênfase nos aspectos
bucais.
OBJETIVO: investigar os achados bucais, incluindo anomalias dentárias, irrupção
ectópica dos primeiros molares permanentes superiores e alterações de tecido
mole, em indivíduos com síndrome de Apert.
MATERIAL E MÉTODOS: exame clínico e radiográfico de nove pacientes com síndrome
de Apert, de 6 a 15 anos de idade, não anteriormente submetidos a tratamento
ortodôntico ou ortognático.
RESULTADOS: anomalias dentárias estavam presentes em todos os pacientes, com uma
a oito anomalias por indivíduo. As anomalias mais freqüentes foram agenesia
dentária, principalmente afetando os caninos superiores, e opacidades de esmalte
(44,4% para ambos). Foi observada irrupção ectópica dos primeiros molares
superiores em 33,3% dos pacientes; foram observados aumentos volumétricos
laterais da mucosa palatina em 88,8% dos pacientes.
CONCLUSÕES: a ocorrência de aumentos volumétricos laterais típicos da mucosa
palatina concorda com a literatura. A alta prevalência de anomalias dentárias e
irrupção ectópica pode sugerir uma possível relação etiológica com a síndrome. |
Which is the most widely used model for the study of multiple sclerosis (MS)? | Experimental autoimmune encephalomyelitis (EAE) is a classical, conventional and widely recognized animal model for studying multiple sclerosis (MS). EAE is the best available model for the inflammatory processes that occur in MS, and for the disease process. A less commonly used model is that of Theiler's murine encephalomyelitis virus (TMEV). | We examined the therapeutic effect of 15-DOS in the two models of acute and
chronic relapsing EAE in Lewis rats. In the first model adult rats developed an
acute severe EAE and by day 16 all animals died. Lewis rats treated with 15-DOS
showed a delayed and reduced onset of clinical symptoms and mortality was
prevented. In the second model Lewis rats (aged animals) developed a chronic
relapsing EAE with up to three relapses. The second and third episodes were both
milder and shorter in duration. All animals treated with 15-DOS survived the
delayed first attack and developed no further relapses. Two questions were posed at the beginning of this article. Is EAE a good model
for MS? And, is MS an autoimmune disease? The first question is easier to
address than the second. EAE is the best available model for the inflammatory
processes that occur in MS, and for the disease process. The latter depends
somewhat on study of chronic relapsing EAE, rather than early or mono-episodic
EAE, which, although of great immunological interest, is of less relevance to
the established disease that presents as MS. The second question asks whether MS
fulfills Koch's postulates as an autoimmune disease. MS has all the hallmarks of
an inflammatory disease of the CNS. The question then is whether the
inflammation is autoimmune. The evidence presented shows a considerable
autoimmune component to MS inflammation, raising the subsidiary question of
whether autoimmune reactivity induces MS. This remains uswerable for the
present, and it should be kept in mind that the same question also would be
uswerable by observation of EAE. The major postulate therefore remains
unfulfilled. Diagnosis of MS as an autoimmune disease requires definitive
identification of the autoantigen; otherwise, the possibility remains open that
this is a disease resulting from the inadvertent activation and dysregulation of
immune processes in the CNS that, themselves, are not directed at that organ. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central
nervous system (CNS). Most patients undergo an initial relapsing-remitting
(RR-MS) course that transforms into a relentless neurodegenerative disorder,
termed secondary progressive (SP)-MS. Reversible inflammation and demyelination
account readily for the pattern of RR-MS but provide an unsatisfactory
explanation for irrevocable decline in SP-MS. Axon loss is thought to be
responsible for progressive, non-remitting neurological disability during SP-MS.
There is considerable potential for neuroprotective therapies in MS, but their
application awaits animal models in which axonal loss correlates with permanent
neurological disability. In this report, we describe quantitative
immunohistochemical methods that correlate inflammation and axonal loss with
neurological disability in chronic-relapsing experimental autoimmune
encephalomyelitis (EAE). At first attack, CNS inflammation, but not axon loss,
correlated with the degree of neurological disability. In contrast, fixed
neurological impairment in chronic EAE correlated with axon loss that, in turn,
correlated with the number of symptomatic attacks. As proposed for MS, these
observations imply a causal relationship between inflammation, axon loss, and
irreversible neurological disability. This chronic-relapsing EAE model provides
an excellent platform for 2 critical objectives: investigating mechanisms of
axon loss and evaluating efficacy of neuroprotective therapies. To assess neurological impairments quantitatively in an animal model of multiple
sclerosis (MS), we have used a targeted model of experimental autoimmune
encephalomyelitis (EAE), which leads to the formation of anatomically defined
lesions in the spinal cord. Deficits in the hindlimb locomotion are therefore
well defined and highly reproducible, in contrast to the situation in
generalized EAE with disseminated lesions. Behavioral tests for hindlimb
sensorimotor functions, originally established for traumatic spinal cord injury,
revealed temporary or persistent deficits in open field locomotion, the grid
walk, the narrow beam and the measurement of the foot exorotation angle. Such
refined behavioral testing in EAE will be crucial for the analysis of new
therapeutic approaches for MS that seek to improve or prevent neurological
impairment. Many individuals with multiple sclerosis (MS) experience clinically significant
pain, yet the underlying neural mechanisms for MS pain are not understood.
Experimental autoimmune encephalomyelitis (EAE) is a well-studied disease in
rodents that mimics many clinical and pathological features of MS, including
central nervous system inflammation and demyelination. To determine whether EAE
is an appropriate model for MS-related pain, nociceptive responses in both male
and female SJL mice were measured before and after immunization with myelin
proteolipid protein peptide 139-151 (PLP(139-151)) in complete Freund's adjuvant
to induce 'active' EAE. To determine if changes in nociception were due to
direct effects of encephalitogenic T cells, nociceptive responses in female SJL
mice were measured following the transfer of activated, PLP(139-151) specific T
cells to induce 'passive' EAE. Both forepaw and tail withdrawal latencies to a
radiant heat stimulus were measured. In both active and passive EAE, there was
an initial increase in tail withdrawal latency (hypoalgesia) that peaked several
days prior to the peak in motor deficits during the acute disease phase. During
the chronic disease phase, tail withdrawal latencies decreased and were
significantly faster than control latencies for up to 38 days post-immunization.
This hyperalgesia was seen in both sexes and in both active and passive EAE
models. Forepaw withdrawal latencies remained within 1-2 s of baseline latencies
for the entire testing period, indicating that the hypoalgesia and hyperalgesia
were most pronounced in clinically affected body regions. These results suggest
that both active and passive EAE are useful models of MS-related pain. In multiple sclerosis (MS), inflammation in the central nervous system (CNS)
leads to damage of axons and myelin. Early during the clinical course, patients
can compensate this damage, but little is known about the changes that underlie
this improvement of neurological function. To study axonal changes that may
contribute to recovery, we made use of an animal model of MS, which allows us to
target inflammatory lesions to the corticospinal tract (CST), a major descending
motor pathway. We demonstrate that axons remodel at multiple levels in response
to a single neuroinflammatory lesion as follows: (a) surrounding the lesion,
local interneurons show regenerative sprouting; (b) above the lesion, descending
CST axons extend new collaterals that establish a "detour" circuit to the lumbar
target area, whereas below the lesion, spared CST axons increase their terminal
branching; and (c) in the motor cortex, the distribution of projection neurons
is remodeled, and new neurons are recruited to the cortical motor pool.
Behavioral tests directly show the importance of these changes for recovery.
This paper provides evidence for a highly plastic response of the motor system
to a single neuroinflammatory lesion. This framework will help to understand the
endogenous repair capacity of the CNS and to develop therapeutic strategies to
support it. Both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis
(EAE), its animal model, involve inflammatory attack on central nervous system
(CNS) white matter, leading to demyelination and axonal damage. Changes in
astrocytic morphology and function are also prominent features of MS and EAE.
Resting astrocytes form a network that is interconnected through gap junctions,
composed mainly of connexin43 (Cx43) protein. Although astrocytic gap junctional
connectivity is known to be altered in many CNS pathologies, little is known
about Cx43 expression in inflammatory demyelinating disease. Therefore, we
evaluated the expression of Cx43 in spinal cords of EAE mice compared with
healthy controls. Lumbar ventral white matter areas were heavily infiltrated
with CD11beta-immunoreactive monocytes, and within these infiltrated regions
loss of Cx43 immunoreactivity was evident. These regions also showed axonal
dystrophy, demonstrated by the abnormally dephosphorylated heavy-chain
neurofilament proteins. Astrocytes in these Cx43-depleted lesions were strongly
glial fibrillary acidic protein reactive. Significant loss (38%) of Cx43 protein
in EAE mouse at the lumbar portion of spinal cords was confirmed by Western blot
analysis. Decreased Cx43 transcript level was also observed on cDNA microarray
analysis. In addition to changes in Cx43 expression, numerous other genes were
altered, including those encoding adhesion and extracellular matrix proteins.
Our data support the notion that, in addition to damage of myelinating glia,
altered astrocyte connectivity is a prominent feature of inflammatory
demyelination. The tyrosine kinase receptor RON and its ligand, macrophage stimulating protein
(MSP), exert inhibitory effects on systemic innate immunity, but their CNS
expression and impact on human neuroinflammatory diseases are unknown were RON
and MSP present in human brain perivascular macrophages and microglia, but RON
mRNA and protein abundance in the CNS were diminished in both MS patients and
the MS animal model, experimental autoimmune encephalomyelitis (EAE). Treatment
of differentiated human monocytoid cells with MSP resulted in significant
reduction of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and MMP-9
mRNA levels, whereas minimal effects were observed in human astrocytes. After
induction of EAE, RON knockout and heterozygote animals exhibited significantly
increased CNS proinflammatory gene (TNF-alpha, MMP-12) expression compared with
wild-type littermate controls, although IL-4 levels were suppressed in both
RON-deficient groups. Neurological disease in RON-deficient animals showed a
more rapid onset with overall worsened severity, together with exacerbated
demyelination, axonal injury, and neuroinflammation after EAE induction. The
proto-oncogene, c-Cbl, which modulates ubiquitylation of RON, was increased in
glia in both MS brains and EAE spinal cords. Thus, the MSP-RON pathway
represents a novel regulatory mechanism within the CNS by which innate immunity
and its pathogenic effects could be targeted for future therapeutic
interventions. Intravenous immunoglobulin (IVIG) is an established treatment of immune-mediated
demyelinating neuropathy. Since IVIG possesses multiple immunomodulatory and
anti-inflammatory properties, IVIG therapy may represent a way of interfering
with the disease process in multiple sclerosis (MS). In the MS animal model
experimental autoimmune encephalomyelitis (EAE), infusions of IVIG significantly
reduced disease symptoms as well as the underlying CNS pathology. IVIG was only
effective in EAE when administered in a prophylactic treatment protocol, since
IVIG infusions during the established phase of EAE did not alter the disease
course or the degree of inflammation found in the central nervous system. IVIG
also has the potential to act through myelin repair mechanisms as evidenced by
work done in the Theilers murine encephalomyelitis virus model of demyelination.
Together these observations have led to certain expectations for IVIG as a
treatment for MS, and have resulted in various clinical trials. Several
controlled trials report beneficial effects of IVIG on relapse rate, new MRI
lesions, and disease progression in relapsing-remitting MS, while a
remyelinating effect of IVIG has not been documented. IVIG is, therefore,
presently regarded as a second-line therapy of MS. Impaired remyelination due to degeneration of both postmitotic oligodendrocytes
and oligodendrocyte progenitors (OPs) is the major hallmark of inflammatory
demyelination in multiple sclerosis (MS) lesions and experimental autoimmune
encephalomyelitis (EAE). Here, we have demonstrated the potential of lovastatin,
a HMG-CoA reductase inhibitor, for the restoration of impaired remyelination
mediated through enhanced survival and differentiation of OPs in the spinal cord
of treated EAE animals. Lovastatin treatment significantly increased the level
of myelin lipids in the spinal cord of treated EAE animals, coinciding with the
attenuation of disease severity and inflammatory demyelination as compared with
untreated EAE animals. The increased expression of myelin proteins and
transcription factors associated with differentiating oligodendrocytes along
with the increase in number of NG2+/BrdU- and NG2+/BrdU+ cells, and the
expression of proliferating OP-specific proteins, demonstrated the restoration
of remyelination in the spinal cord of lovastatin-treated EAE animals.
Corresponding to this, in vitro studies further corroborated the increased
survival and differentiation of OPs in lovastatin-treated activated mixed glial
cells suggesting that lovastatin protects against the degeneration of OPs and
enhances their differentiation through induction of a pro-remyelinating
environment in the spinal cord of treated EAE animals. Together, these data
demonstrate that lovastatin has the potential to augment remyelination in MS
lesions and other neuroinflammatory diseases. The pathogenic events in multiple sclerosis (MS) that result in immune cell
infiltration, multifocal demyelination and axonal loss have been focused by the
strong impact of the classical MS model experimental autoimmune
encephalomyelitis (EAE) towards the hypothesis that MS is an entirely T
cell-mediated disease. Although conspicuous humoral immune responses have been
known since Kabal's seminal finding of elevated immunoglobulins (Igs) in the
cerebrospinal fluid (CSF), only in the past few years evidence derived from
recent studies of the MS lesion of anti-myelin antibodies (Abs) in patients with
early MS and of MS animal models has led to a renewed interest in the role for B
cells, plasma cells and their products in the pathogenesis of MS. This review
surveys the actual data concerning the role of the humoral immune system in MS
and EAE and explains potential modes of action and long-time persistence in the
inflamed brain tissue as a B cell-supportive microenvironment in MS. These
mechanisms include the modulation of antigen presentation and costimulation to T
cells, increased myelin opsonisation und recruitment of inflammatory cells to
the CNS, but also immunoregulatory influences on the remyelination by
anti-myelin B cells and Abs. So, affecting the humoral immune system in MS would
be a reasonable therapeutic option. Inflammatory diseases of the CNS, such as MS and its animal model EAE, are
characterized by infiltration of activated lymphocytes and phagocytes into the
CNS. Within the CNS, activation of resident cells initiates an inflammatory
cascade, leading to tissue destruction, demyelination, and neurologic deficit.
TLRs recognize microbes and are pivotal mediators of innate immunity. Within the
CNS, augmented TLR expression during EAE is observed, even in the absence of any
apparent microbial involvement. To determine the functional relevance of this
phenomenon during sterile autoimmunity, we studied the role of different TLRs as
well as their common signaling adaptor MyD88 in the development of EAE. We found
that MyD88 mice were completely EAE resistant. Surprisingly, this protection is
partly due to engagement of the CpG receptor TLR9. Restricting the MyD88 or TLR9
mutation to host radio-resistant cells, including the cells within the CNS,
revealed that engagement of radio-resistant cells modulated the disease course
and histopathological changes. Our data clearly demonstrate that both TLR9 and
MyD88 are essential modulators of the autoimmune process during the effector
phase of disease and suggest that endogenous "danger signals" modulate the
disease pathogenesis. Although the central nervous system (CNS) is thought to be immunoprivileged,
under special circumstances it can produce antibody. Antibody production within
the CNS, called intrathecal antibody production (ITAbP), is a prominent feature
of neurological infections and inflammatory diseases, and is thought to possibly
contribute to disease in multiple sclerosis (MS), but it has not been
extensively studied. We investigated ITAbP in a viral model of MS. ELISpot,
real-time RT-PCR for IgG mRNA in CNS tissue, and CSF analysis were used to
assess ITAbP in two types of SJL mice infected with one of two strains of
Theiler's murine encephalomyelitis virus (TMEV). The amplitude of ITAbP
increased during the first 4 months of infection. TMEV viral load remained high
during the course of the infection, which likely was the main stimulus for
ITAbP, since within samples of infected CNS tissues, levels of IgG gene
expression were highly correlated with viral RNA levels, and a large percentage
of intrathecally produced antibody was directed against TMEV. This study
provides the first extensive analysis of ITAbP in TMEV infection, and
demonstrates that, in this animal model of MS, antibody production within the
CNS is likely driven by the presence of the causative pathogen. Multiple sclerosis (MS) is the leading cause of neurological disability in young
adults, affecting some two million people worldwide. Traditionally, MS has been
considered a chronic, inflammatory disorder of the central white matter in which
ensuing demyelination results in physical disability [Frohman EM, Racke MK,
Raine CS (2006) N Engl J Med 354:942-955]. More recently, MS has become
increasingly viewed as a neurodegenerative disorder in which neuronal loss,
axonal injury, and atrophy of the CNS lead to permanent neurological and
clinical disability. Although axonal pathology and loss in MS has been
recognized for >100 years, very little is known about the underlying molecular
mechanisms. Progressive axonal loss in MS may stem from a cascade of ionic
imbalances initiated by inflammation, leading to mitochondrial dysfunction and
energetic deficits that result in mitochondrial and cellular Ca2+ overload. In a
murine disease model, experimental autoimmune encephalomyelitis (EAE) mice
lacking cyclophilin D (CyPD), a key regulator of the mitochondrial permeability
transition pore (PTP), developed EAE, but unlike WT mice, they partially
recovered. Examination of the spinal cords of CyPD-knockout mice revealed a
striking preservation of axons, despite a similar extent of inflammation.
Furthermore, neurons prepared from CyPD-knockout animals were resistant to
reactive oxygen and nitrogen species thought to mediate axonal damage in EAE and
MS, and brain mitochondria lacking CyPD sequestered substantially higher levels
of Ca2+. Our results directly implicate pathological activation of the
mitochondrial PTP in the axonal damage occurring during MS and identify CyPD, as
well as the PTP, as a potential target for MS neuroprotective therapies. Although pain was previously not considered an important element of multiple
sclerosis (MS), recent evidence indicates that over 50% of MS patients suffer
from chronic pain. In the present study, we utilized the Theiler's murine
encephalomyelitis virus (TMEV) model of MS to examine whether changes in
nociception occur during disease progression and to investigate whether sex
influences the development of nociception or disease-associated neurological
symptoms. Using the rotarod assay, TMEV infected male mice displayed increased
neurological deficits when compared to TMEV infected female mice, which mimics
what is observed in human MS. While both male and female TMEV infected mice
exhibited thermal hyperalgesia and mechanical allodynia, female mice developed
mechanical allodynia at a faster rate and displayed significantly more
mechanical allodynia than male mice. Since neuropathic symptoms have been
described in MS patients, we quantified sensory nerve fibers in the epidermis of
TMEV-infected and non-infected mice to determine if there were alterations in
epidermal nerve density. There was a significantly higher density of PGP9.5 and
CGRP-immunoreactive axons in the epidermis of TMEV-infected mice versus
controls. Collectively these results indicate that the TMEV model is well suited
to study the mechanisms of MS-induced nociception and suggest that alterations
in peripheral nerve innervation may contribute to MS pain. Experimental autoimmune encephalomyelitis (EAE), a widely recognized animal
model of multiple sclerosis (MS), is highly useful for studying inflammation,
demyelination, and neurodegeneration in the central nervous system (CNS). EAE
exhibits many similarities with MS, which is a chronic inflammatory disease
affecting CNS white matter in humans. Various studies have indicated that EAE is
a particularly useful animal model for understanding both the mechanisms of
immune-mediated CNS pathology and also the progressive clinical course of MS.
Demyelination and axonal dysfunction have previously been shown in MS and EAE
but current evidences indicate that axonal damage and neuron death also occur,
demonstrating that these diseases harbor a neurodegenerative component. Recent
studies also have shown that the activation of calpain and caspase pathways
contribute to the apoptotic death of oligodendrocytes and neurons, promoting the
pathological events leading to neurological deficits. Apoptosis is involved in
the disease-regulating as well as in the disease-promoting processes in EAE.
This review discusses the major involvement of calpain and caspase pathways in
causing demyelination and neurodegeneration in EAE animals. BACKGROUND: Multiple sclerosis (MS) is an immune mediated demyelinating disease
of the central nervous system (CNS). A potential new therapeutic approach for MS
is cell transplantation which may promote remyelination and suppress the
inflammatory process.
METHODS: We transplanted human embryonic stem cells (hESC)-derived early
multipotent neural precursors (NPs) into the brain ventricles of mice induced
with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We
studied the effect of the transplanted NPs on the functional and pathological
manifestations of the disease.
RESULTS: Transplanted hESC-derived NPs significantly reduced the clinical signs
of EAE. Histological examination showed migration of the transplanted NPs to the
host white matter, however, differentiation to mature oligodendrocytes and
remyelination were negligible. Time course analysis of the evolution and
progression of CNS inflammation and tissue injury showed an attenuation of the
inflammatory process in transplanted animals, which was correlated with the
reduction of both axonal damage and demyelination. Co-culture experiments showed
that hESC-derived NPs inhibited the activation and proliferation of lymph
node-derived T cells in response to nonspecific polyclonal stimuli.
CONCLUSIONS: The therapeutic effect of transplantation was not related to graft
or host remyelination but was mediated by an immunosuppressive neuroprotective
mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may
serve as the first step towards further developments of hESC for cell therapy in
MS. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the
central nervous system (CNS). Recent studies suggest that, beside focal lesions,
diffuse inflammatory and degenerative processes take place throughout the MS
brain. Especially, molecular alterations in the so-called normal appearing white
matter suggest the induction of neuroprotective mechanisms against oxidative
stress preserving cellular homeostasis and function. In this study we
investigated whether in an animal model for MS, namely in experimental
autoimmune encephalomyelitis (EAE), similar changes occur. We isolated normal
appearing white and grey matter from the corpus callosum and the above lying
cerebral cortex from DA rats with rMOG-induced EAE and carried out a gene
expression analysis. Examination of corpus callosum revealed only minor changes
in EAE rats. In contrast, we identified a number of gene expression alterations
in the cerebral cortex even though morphological and cellular alterations were
not evident. One of the most striking observations was the downregulation of
genes involved in mitochondrial function as well as a whole set of genes coding
for different glutamate receptors. Our data imply that molecular alterations are
present in neurons far distant to inflammatory demyelinating lesions. These
alterations might reflect degenerative processes induced by lesion-mediated
axonal injury in the spinal cord. Our results indicate that the MOG-induced EAE
in DA rats is a valuable model to analyze neuronal alterations due to axonal
impairment in an acute phase of a MS-like disease, and could be used for
development of neuroprotective strategies. Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying
multiple sclerosis (MS). Calpain has been implicated in many inflammatory and
neurodegenerative events that lead to disability in EAE and MS. Thus, treating
EAE animals with calpain inhibitors may block these events and ameliorate
disability. To test this hypothesis, acute EAE Lewis rats were treated dose
dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain
activity, gliosis, loss of myelin, and axonal damage were attenuated by
calpeptin therapy, leading to improved clinical scores. Neuronal and
oligodendrocyte death were also decreased, with down-regulation of proapoptotic
proteins, suggesting that decreases in cell death were due to decreases in the
expression or activity of proapoptotic proteins. These results indicate that
calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS. The pathological and radiological hallmarks of multiple sclerosis (MS) include
multiple demyelinated lesions disseminated throughout the white matter of the
central nervous system (CNS). More recently, the cerebral cortex has been shown
to be affected in MS, but the elucidation of events causing cortical
demyelination has been hampered by the lack of animal models reflecting such
human cortical pathology. In this report, we have described the presence of
cortical gray matter and callosal white matter demyelinating lesions in the
chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS.
Similar to the pathological lesions of MS patients, EAE lesions have been
classified as type I-leukocortical, type II-intracortical and type III-subpial.
All of these lesions had varying degrees of demyelination, inflammatory cells
and reactive astrocytes. Similar to MS, cortical layers during EAE showed
demyelination, microglia activation, synaptic protein alterations and apoptotic
cells. In addition, the callosal white matter during EAE had many inflammatory
demyelinating lesions and axon degeneration. Functional electrophysiological
conduction analysis showed deficits in both myelinated and unmyelinated callosal
axons during early and late EAE. The chronic EAE mouse model has features that
mimic cortical and callosal pathology of MS, and can be potentially used to
screen agents to prevent these features of disease. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous
system (CNS) characterized by inflammation, but also degenerative changes.
Besides neurological deficits, the rate of affective disorders such as
depression and anxiety is at least six fold increased. Many aspects of MS can be
mimicked in the animal model of myelin oligodendrocyte glycoprotein experimental
autoimmune encephalomyelitis (MOG-EAE). Here we investigate behavioral changes
in C57BL/6 mice suffering from mild MOG-EAE. In the later phase of the disease,
mice were subjected to behavioral tests including the light-dark-box (LD Box),
the acoustic startle response (SR) with a pre-pulse inhibition protocol as well
as the learned helplessness (LH) paradigm. Behavioral data were correlated with
the motor performance in an open field and rotarod test (RR). In the RR and open
field, there was no significant difference in the motor performance between
controls and mice suffering from mild MOG-EAE. Yet EAE mice displayed an
increased anxiety-like behavior with a 23% reduction of the time spent in the
bright compartment of the LD Box as well as an increased SR. In the LH paradigm,
mice suffering from MOG-EAE were twice as much prone to depressive-like
behavior. These changes correlate with an increase of hippocampal tissue tumor
necrosis factor alpha levels and neuronal loss in the hippocampus. Modulation of
monoaminergic transmission by chronic application of the antidepressant
amitriptyline resulted in a decreased startle reaction and increased hippocampal
norepinephrine levels. These data imply that chronic inflammation in the CNS may
impact on emotional responses in rodent models of anxiety. |
What is the usual HER-2 status in breast cancer associated with Li-Fraumeni syndrome? | In up to two thirds of breast cancer patients associated with Li-Fraumeni syndrome, the HER-2 status was found to be positive. | BACKGROUND: Germline TP53 mutations predispose to early onset breast cancer in
women and are associated with Li-Fraumeni syndrome. Published data on the
pathological characteristics of breast cancer among women with TP53 mutations is
limited.
METHODS: We retrospectively reviewed the clinical records of women who underwent
genetic testing for suspected germline TP53 mutations and who were diagnosed
with breast cancer between 2000 and 2011. The pathological characteristics of
the breast tumors from patients testing positive for a mutation (cases) were
compared with those testing negative (controls).
RESULTS: Patients who tested positive for germlineTP53 mutations (n = 30) were
compared with controls (n = 79). Human epidermal growth factor receptor 2 (HER2)
amplification and/or overexpression was found in 67% of the tumors from the
cases, compared with 25% for the controls (P = .0001). Among patients with a
mutation, 70% had estrogen receptor- and/or progesterone receptor-positive
tumors, compared with 68% in the control group (P = .87). After adjusting for
age at breast cancer diagnosis, having a HER2-positive tumor increased the odds
of testing positive for a germline TP53 mutation (odds ratio, 6.9; 95%
confidence interval, 2.6-18.2). For each yearly increment in age at breast
cancer diagnosis, there was decreased likelihood of having a TP53 mutation of 5%
(odds ratio, 0.95; 95% confidence interval0.91-0.99).
CONCLUSIONS: This study suggests an association between germline TP53 mutations
and early onset HER2-positive breast cancer. If confirmed in a larger cohort,
these results could guide genetic testing strategies, lead to chemoprevention
trials incorporating HER2-targeted therapies, and elucidate some of the
molecular pathways involved in breast cancer. |
What is the treatment of triiodothyronine toxicosis? | Treatment of T3 toxicosis is a complex medical problem because not well responsive to the various options. Usual treatment includes antithyroid drugs such as propyltiouracil, radioactive iodine or beta blockers like propanol; surgery may be also necessary in some cases. | Triiodothyronine (T3) toxicity has been well documented in adults, but only
isolated cases have been reported in children. Two girls presented with firm
goitres and overt hyperthyroidism. In each patient, total serum thyroxine (T4)
values by competitive protein binding were normal, however total T3 values by
radioimmunoassay were elevated. One patient had Graves' disease, the second
patient had Hashimoto's disease which has been only infrequently associated with
T3 toxicity in adults. Both patients responded to therapy with propylthiouracil.
The mechanisms by which T3 is preferentially secreted in thyrotoxic states in
man are poorly understood, but iodine deficiency and poor iodination of
thyroglobulin may be important factors. Serum thyroid hormone concentrations were measured before and during 6 months
treatment with propranolol (160 mg/day) in eight patients with T3
(triiodothyronine) toxicosis. Serum total T3 concentrations showed a significant
(p less than 0.01) and sustained fall to approximately 80% of pre-treatment
values. Six of the patients, however, remained clinically and biochemically
hyperthyroid and our data do not support the use of propranolol as sole therapy
in T3 toxicosis. PURPOSE: To develop a strategy to identify cases of endogenous subclinical
hyperthyroidism and free triiodothyronine (free T3) thyrotoxicosis in otherwise
healthy ambulatory patients.
PATIENTS AND METHODS: In a retrospective study we reviewed the records of
ambulatory patients who had thyroid stimulating hormone (TSH) levels determined
between October 1, 1991 and August 31, 1992. Each patient also had a
simultaneous free thyroxine (free T4) measurement. Patients were excluded from
consideration if they had active, concurrent non-thyroidal illness, psychiatric
disease, known hypothalamic/pituitary lesions, were under treatment for hyper-
or hypothyroidism, were on drugs known to affect TSH levels, or were pregt.
Patients without exclusions were diagnosed with free T3 toxicosis if they had:
(1) a markedly subnormal TSH level (less than or equal to 0.1 mU/L), (2) a
normal free T4, (3) a normal total T3, (4) evidence of a primary thyroid
abnormality (e.g., autonomous function on a thyroid scan), and (5) an elevated
free T3 level by tracer equilibrium dialysis. Patients meeting conditions 1-4,
but with normal free T3 levels, were considered to have subclinical
hyperthyroidism.
RESULTS: One thousand twenty-five patients had TSH and simultaneous free T4
determinations, and 148 of these had markedly subnormal TSH but normal free T4
levels. Three patients met the criteria for free T3 toxicosis and three had
subclinical hyperthyroidism. All six patients had either multinodular glands or
a single nodule on thyroid exam. Four patients were treated with radioactive
iodine or surgery, resulting in reversal of the TSH suppression in three cases.
CONCLUSION: Apparently healthy ambulatory patients with subnormal TSH levels
should be worked up with measurements of free T4 and total T3. If these are
normal, a T3 level (by tracer equilibrium dialysis) be obtained to distinguish
subclinical hyperthyroidism from overt free T3 toxicosis. A thyroid scan and
radioiodine uptake measurement can be obtained to substantiate the diagnosis.
Some patients with these conditions will benefit from treatment. Appropriate methimazole dosing for initial treatment of childhood Graves'
disease is uncertain. A retrospective chart review was performed on 5 to 17
year-old children treated for Graves' disease. Patients were divided into two
groups depending on initial methimazole dosing: low-dose and high-dose regimens
using <0.5 mg/kg/day and >0.5 mg/kg/day, respectively. The low-dose regimen was
effective in 5/12 (42%) of patients and the high-dose regimen was effective in
27/33 (82%) of patients (p = 0.016). There was also a statistically significant
dose/time interaction for levels of free thyroxine (T4) (p = 0.025). During
treatment, 63.3% of diagnosable samples showed unambiguous hyperthyroidism or
triiodothyronine (T3) toxicosis, 16.7% elevated free T3 with normal free T4 and
T3 levels, indicating borderline hyperthyroidism, and 20% showed
thyroid-stimulating hormone (TSH) suppression with normal or low levels of free
T4 and free T3, indicating delayed recovery of pituitary TSH secretion. Free T3
levels combined with concurrent TSH levels permit differentiation of mild
hyperthyroidism from delayed pituitary recovery. |
Are there enhancer RNAs (eRNAs)? | Yes. Active enhancers are transcribed, producing a class of noncoding RNAs called enhancer RNAs (eRNAs). eRNAs are distinct from long noncoding RNAs (lncRNAs), but these two species of noncoding RNAs may share a similar role in the activation of mRNA transcription. | We used genome-wide sequencing methods to study stimulus-dependent enhancer
function in mouse cortical neurons. We identified approximately 12,000 neuronal
activity-regulated enhancers that are bound by the general transcriptional
co-activator CBP in an activity-dependent manner. A function of CBP at enhancers
may be to recruit RNA polymerase II (RNAPII), as we also observed
activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at
enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs)
within enhancer domains defined by the presence of histone H3 monomethylated at
lysine 4. The level of eRNA expression at neuronal enhancers positively
correlates with the level of messenger RNA synthesis at nearby genes, suggesting
that eRNA synthesis occurs specifically at enhancers that are actively engaged
in promoting mRNA synthesis. These findings reveal that a widespread mechanism
of enhancer activation involves RNAPII binding and eRNA synthesis. The functional importance of gene enhancers in regulated gene expression is well
established. In addition to widespread transcription of long non-coding RNAs
(lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers,
and are thus referred to as enhancer RNAs (eRNAs). However, it has remained
unclear whether these eRNAs are functional or merely a reflection of enhancer
activation. Here we report that in human breast cancer cells 17β-oestradiol
(E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA
transcription on enhancers adjacent to E2-upregulated coding genes. These
induced eRNAs, as functional transcripts, seem to exert important roles for the
observed ligand-dependent induction of target coding genes, increasing the
strength of specific enhancer-promoter looping initiated by ER-α binding.
Cohesin, present on many ER-α-regulated enhancers even before ligand treatment,
apparently contributes to E2-dependent gene activation, at least in part by
stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate
that eRNAs are likely to have important functions in many regulated programs of
gene transcription. Author information:
(1)1] Division of Gene Regulation, The Netherlands Cancer Institute, Plesmanlaan
121, 1066 CX, Amsterdam, The Netherlands [2] Doctoral Programme in Biomedicine
and Experimental Biology, Centre for Neuroscience and Cell Biology, Erasmus MC,
Rotterdam University, The Netherlands. Enhancers are cis-acting elements capable of regulating transcription in a
distance and orientation-independent manner. A subset of enhancers are occupied
by RNA polymerase II (RNAP II) and transcribed to produce long non-coding RNAs
termed eRNAs. We thoroughly investigated the association between eRNA
productivity and various chromatin marks and transcriptional regulators in mouse
embryonic stem cells (ESCs) through an integrative approach. We found that
eRNA-producing enhancers exhibited elevated levels of the active mark H3K27Ac,
decreased DNA methylation, and enrichment for the DNA hydroxylase Tet1. Many
eRNA-producing enhancers have recently been characterized as "super-enhancers,"
suggesting an important role in the maintece of pluripotency. Using
experimental methods, we focally investigated a well-characterized enhancer
linked to the Nanog locus and confirmed its exclusive eRNA productivity in ESCs.
We further demonstrate that the binding of Sall4 and Tet family proteins were
required for eRNA productivity at this locus. Collectively, we demonstrate that
Tet1 binding and DNA hypomethylation are hallmarks of eRNA production. Following reports by ENCyclopedia Of DNA Elements (ENCODE; GENCODE) Consortium
and others, it is now fairly evident that the majority (70-80%) of the mammalian
genome has the potential to be transcribed into non-protein-coding RNAs
(ncRNAs). Critical to our understanding of genetic processes is the mechanism by
which ncRNAs exert their roles. Accordingly, ncRNAs are shown to regulate the
expression of protein-coding loci (i.e., genes) at the transcriptional as well
as post-transcriptional stages. We recently reported on a widespread
transcription at the DNA enhancer elements in myogenic cells. In our study, we
found certain enhancer RNAs (eRNAs) regulate chromatin accessibility of the
transcriptional machinery at loci encoding master regulators of myogenesis
(i.e., MyoD/MyoG), thus suggesting their significance and site-specific impact
in cellular programming. Here, we examine recent discoveries pertinent to the
proposed role(s) of eRNAs in regulating gene expression. We will highlight
consistencies, discuss confounding observations, and consider a lack of critical
information in a way to prioritize future objectives. Author information:
(1)Department of Cellular and Molecular Medicine, University of California, San
Diego, 9500 Gilman Drive, La Jolla, CA, USA.
(2)Howard Hughes Medical Institute, Department of Medicine, University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA.
(3)Howard Hughes Medical Institute, Department of Medicine, University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA. Electronic address:
[email protected].
(4)Department of Cellular and Molecular Medicine, University of California, San
Diego, 9500 Gilman Drive, La Jolla, CA, USA; Howard Hughes Medical Institute,
Department of Medicine, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA, USA. Electronic address: [email protected]. Recent studies have revealed that active enhancers are transcribed, producing a
class of noncoding RNAs called enhancer RNAs (eRNAs). eRNAs are distinct from
long noncoding RNAs (lncRNAs), but these two species of noncoding RNAs may share
a similar role in the activation of mRNA transcription. Emerging studies,
showing that eRNAs function in controlling mRNA transcription, challenge the
idea that enhancers are merely sites of transcription factor assembly. Instead,
communication between promoters and enhancers can be bidirectional with
promoters required to activate enhancer transcription. Reciprocally, eRNAs may
then facilitate enhancer-promoter interaction or activate promoter-driven
transcription. |
Which are the known human transmembrane nucleoporins? | Transmembrane nucleoporins (NUPs) are integral membrane components of the eukaryotic nuclear pore, playing an important role in the Nuclear Pore Complex (NPC) assembly. Even though the NPC is a conserved feature of all eukaryotes, different lineages possess some distinct transmembrane NUP subunits. Currently, four human transmembrane NUPs have been characterized, namely: NDC1 (also known as TMEM48 or NET3 or hNDC1), POM121 (also known as Nup121), GP210 (also known as Nuclear pore membrane glycoprotein 210 or Nuclear envelope pore membrane protein POM 210, POM210 or Nup210) and TMEM33 (or DB83). | Patients with primary biliary cirrhosis (PBC) frequently have autoantibodies
against a 210-kD integral glycoprotein of the nuclear envelope pore membrane.
This protein, termed gp210, has a 1,783-amino acid amino-terminal domain located
in the perinuclear space, a 20-amino acid transmembrane segment, and a 58-amino
acid cytoplasmic carboxy-terminal tail. We now demonstrate that autoantibodies
from 25 patients with PBC that recognize gp210 react with the cytoplasmic
carboxy-terminal tail while none react with unmodified linear epitopes in the
amino-terminal domain. The epitope(s) recognized by autoantibodies from all 25
patients is contained within a stretch of 15 amino acids. The recognized amino
acid sequence is homologous to the protein products of the Escherichia coli mutY
gene and Salmonella typhimurium mutB gene with an exact identity of six
consecutive amino acids, suggesting that anti-gp210 antibodies may arise by
molecular mimicry of bacterial antigenic determits. Patients with primary biliary cirrhosis frequently develop autoantibodies
directed to gp210, a major glycoprotein of the nuclear pore complex. This
protein contains a large glycosylated cisternal domain, a single transmembrane
segment, and a short cytoplasmic tail. It has been previously shown that
autoantibodies from primary biliary cirrhosis patients exclusively react with
the cytoplasmic tail. We demonstrate that autoantibodies against gp210 recognize
at least two different epitopes. 4 out of 12 anti-gp210 positive sera reacted
with the fragment consisting of the cytoplasmic tail, and 8 sera targeted a
novel epitope located within the large glycosylated lumenal domain. Moreover,
our data prove that carbohydrate moieties are an essential part of this novel
epitope. We propose, therefore, that future screening assays should be performed
with antigens possessing both epitopes to detect all sera with anti-gp210
specificity. The membrane-spanning glycoprotein gp210 is a major component of the nuclear
pore complex. This nucleoporin contains a large cisternal N-terminal domain, a
short C-terminal cytoplasmic tail, and a single transmembrane segment. We show
here that dimers of native gp210 can be isolated from cell extracts by
immunoprecipitation, and from purified rat liver nuclear envelopes by velocity
sedimentation and gel filtration. Cross-linking of proteins in isolated
membranes prior to solubilization dramatically increases the proportion of
dimers. The dimers are SDS-resistant, as previously observed for some integral
membrane proteins of cis-Golgi and plasma membrane proteins, including
glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration
and denaturing electrophoresis, but unlike the dimers are dissociated by
reduction and heating in the presence of SDS. We propose that gp210 is organized
into the pore membrane as a large array of gp210 dimers that may constitute a
luminal submembranous protein skeleton. RNA undergoing nuclear export first encounters the basket of the nuclear pore.
Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their
molecular partners within the pore are largely unknown. Because the mechanism of
RNA export will be in question as long as significant vertebrate pore proteins
remain undiscovered, we set out to find their partners. Fragments of Nup98 and
Nup153 were used for pulldown experiments from Xenopus egg extracts, which
contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each
bound the same four large proteins. Purification and sequence analysis revealed
that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two
mapped to ORFs of unknown function. The genes encoding the novel proteins were
cloned, and antibodies were produced. Immunofluorescence reveals them to be new
nucleoporins, designated Nup160 and Nup133, which are accessible on the basket
side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a
complex in Xenopus egg extracts and in assembled pores, now termed the Nup160
complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be
a member of the Nup160 complex. We have mapped the sites that are required for
binding the Nup160 subcomplex, and have found that in Nup98, the binding site is
used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153
to the nuclear pore. With transfection and in vivo transport assays, we find
that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not
protein import or export. These results demonstrate that two novel vertebrate
nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but
themselves play a role in mRNA export. We tested the hypothesis that gp210, an integral membrane protein of nuclear
pore complexes (NPCs), mediates nuclear pore formation. Gp210 has a large
lumenal domain and small COOH-terminal tail exposed to the cytoplasm. We studied
the exposed tail. We added recombit tail polypeptides to Xenopus nuclear
assembly extracts, or inhibited endogenous gp210 tails using anti-tail
antibodies. Both strategies had no effect on the formation of fused flattened
nuclear membranes, but blocked NPC assembly and nuclear growth. Inhibited nuclei
accumulated gp210 and some nucleoporin p62, but failed to incorporate
nup214/CAN, nup153, or nup98 and were defective for nuclear import of lamin B3.
Scanning and transmission EM revealed a lack of "closely apposed" inner and
outer membranes, and the accumulation of novel arrested structures including
"mini-pores." We conclude that gp210 has early roles in nuclear pore formation,
and that pore dilation is mediated by gp210 and its tail-binding partner(s). We
propose that membrane fusion and pore dilation are coupled, acting as a
mechanism to control nuclear pore size. gp210 is a major constituent of the nuclear pore complex (NPC) with possible
structural and regulatory roles. It interacts with components of the NPC via its
C-terminal domain (CTD), which follows a transmembrane domain and a massive (
approximately 200 kDa) N-terminal region that resides in the lumen of the
perinuclear space. Here, we report the solution structure of the human gp210 CTD
as determined by various spectroscopic techniques. In water, the CTD adopts an
extended, largely unordered conformation, which contains a significant amount of
left-handed polyproline type II (PII) helical structure. The conformation of the
CTD is altered by high pH, charged detergents, and the hydrogen bond-promoting
reagent trifluoroethanol (TFE), which decrease the PII fraction of the fragment.
TFE also induces a conformational change in a region containing an SPXX motif
whose serine becomes specifically phosphorylated during mitosis. We propose that
PII elements in the CTD may play a role in its interaction with the NPC and may
serve as recognition sites for regulatory proteins bearing WW or other, unknown
PII-binding motifs. Resident integral proteins of the inner nuclear membrane (INM) are synthesized
as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and
are transported to the INM throughout interphase using an unknown trafficking
mechanism. To study this transport, we developed a live cell assay that measures
the movement of transmembrane reporters from the ER to the INM by
rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with
small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM.
However, increasing the size of either domain by 47 kD strongly inhibited
movement. Reduced temperature and ATP depletion also inhibited movement, which
is characteristic of membrane fusion mechanisms, but pharmacological inhibition
of vesicular trafficking had no effect. Because reporter accumulation at the INM
was inhibited by antibodies to the nuclear pore membrane protein gp210, our
results support a model wherein transport of integral proteins to the INM
involves lateral diffusion in the lipid bilayer around the nuclear pore
membrane, coupled with active restructuring of the nuclear pore complex. BACKGROUND: Glycoprotein 210 (GP210) is a transmembrane component of the nuclear
pore complex of metazoans, with a short carboxyterminus protruding towards the
cytoplasm. Its function is unknown, but it is considered to be a major
structural component of metazoan nuclear pores. Yet, our previous findings
showed pronounced differences in expression levels in embryonic mouse tissues
and cell lines. In order to identify factors regulating GP210, the genomic
organization of human GP210 was analyzed in silico.
RESULTS: The human gene was mapped to chromosome 3 and consists of 40 exons
spread over 102 kb. The deduced 1887 amino acid showed a high degree of
alignment homology to previously reported orthologues. Experimentally we defined
two transcription initiation sites, 18 and 29 bp upstream of the ATG start
codon. The promoter region is characterized by a CpG island and several
consensus binding motifs for gene regulatory transcription factors, including
clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc
finger protein (WT1). In addition, distal to the translation start we found a
(GT)n repetitive sequence, an element known for its ability to bind WT1.
Homologies for these motifs could be identified in the corresponding mouse
genomic region. However, experimental tetracycline dependent induction of WT1 in
SAOS osteosarcoma cells did not influence GP210 transcription.
CONCLUSION: Although mouse GP210 was identified as an early response gene during
induced metanephric kidney development, and WT1 binding sites were identified in
the promoter region of the human GP210 gene, experimental modulation of WT1
expression did not influence expression of GP210. Therefore, WT1 is probably not
regulating GP210 expression. Instead, we suggest that the identified Sp binding
sites are involved. Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the
nuclear envelope (NE), through which exchange of molecules between the nucleus
and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In
particular, almost nothing is known about how NPCs are anchored in the NE. Here,
we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between
yeast and metazoans. We show by RNA interference (RNAi) and biochemical
depletion that NDC1 plays an important role in NPC and NE assembly in vivo and
in vitro. RNAi experiments suggest a functional link between NDC1 and the
soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with
Nup53 in vitro. This suggests that NDC1 function involves forming a link between
the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the
membrane. The nuclear pore complex (NPC) is a large channel that spans the two lipid
bilayers of the nuclear envelope and mediates transport events between the
cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins,
and the role of these proteins in NPC function and assembly remains poorly
understood. We investigate the function of the three integral membrane
nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and
transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the
correct localization of nuclear transport cargoes and of components of the NPC.
However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p,
as cells lacking both of these proteins show enhanced NPC disruption. Electron
microscopy studies reveal that the absence of Ndc1p and Pom152p results in
aberrant pores that have enlarged diameters and lack proteinaceous material,
leading to an increased diffusion between the cytoplasm and the nucleus. POM121 and gp210 were, until this point, the only known membrane-integral
nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for
nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying
study (Stavru et al.), we provided evidence that NPCs can exist independently of
POM121 and gp210, and we predicted that vertebrate NPCs contain additional
membrane-integral constituents. We identify such an additional membrane protein
in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast
Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments,
and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa
cells interferes with the assembly of phenylalanine-glycine repeat Nups into
NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC
defects and very high larval and embryonic mortality. However, it is not
ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated.
This indicates that none of the membrane-integral Nups is universally essential
for NPC assembly, and suggests that NPC biogenesis is an extremely
fault-tolerant process. So far, POM121 and gp210 are the only known anchoring sites of vertebrate
nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope
(NE) and, thus, are excellent candidates for initiating the NPC assembly
process. Indeed, we demonstrate that POM121 can recruit several nucleoporins,
such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a
nucleation site for the assembly of NPC substructures. Nonetheless, we observed
functional NPCs and intact NEs in severely POM121-depleted cells. Double
knockdowns of gp210 and POM121 in HeLa cells, as well as depletion of POM121
from human fibroblasts, which do not express gp210, further suggest that NPCs
can assemble or at least persist in a POM121- and gp210-free form. This points
to extensive redundancies in protein-protein interactions within NPCs and
suggests that vertebrate NPCs contain additional membrane-integral nucleoporins
for anchorage within the lipid bilayer of the NE. In Stavru et al., we describe
such an additional transmembrane nucleoporin as the metazoan orthologue of yeast
Ndc1p. The nuclear pore complex is the predomit structure in the nuclear envelope
that spans the double nuclear membranes of all eukaryotes. Yeasts have one
additional organelle that is also embedded in the nuclear envelope: the spindle
pole body, which functions as the microtubule organizing center. The only
protein known to localize to and be important in the assembly of both of these
yeast structures is the integral membrane protein, Ndc1p. However, no homologues
of Ndc1p had been characterized in metazoa. Here, we identify and analyze NDC1
homologues that are conserved throughout evolution. We show that the overall
topology of these homologues is conserved. Each contains six transmembrane
segments in its N-terminal half and has a large soluble C-terminal half of
approximately 300 amino acids. Charge distribution analysis infers that the N-
and C-termini are exposed to the cytoplasm. Limited proteolysis of yeast Ndc1p
in cellular membranes confirms the orientation of its C-terminus. Although it is
not known whether vertebrate NDC1 protein localizes to nuclear pores like its
yeast counterpart, the human homologue contains three FG repeats in the
C-terminus, a feature of many nuclear pore proteins. Moreover, a small region
containing mutations that affect assembly of the nuclear pore in yeast is highly
conserved throughout evolution. Lastly, we bring together data from another
study to demonstrate that the human homologue of NDC1 is the known inner nuclear
membrane protein, NET3. NDC1 is a transmembrane nucleoporin that is required for NPC assembly and
nucleocytoplasmic transport. We show here that NDC1 directly interacts with the
nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome,
and that this interaction is required for targeting of ALADIN to nuclear pore
complexes (NPCs). Furthermore, we show that NDC1 is required for selective
nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN
to NPCs is essential for selective nuclear protein import, and that abrogation
of the interaction between ALADIN and NDC1 may be important for the development
of triple-A syndrome. The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the
assembly of high-complexity macromolecular structures. So far, only three
integral pore membrane proteins are known to function redundantly in NPC
anchoring within the nuclear envelope. Here, we describe the identification and
functional characterization of Pom33, a novel transmembrane protein dynamically
associated with budding yeast NPCs. Pom33 becomes critical for yeast viability
in the absence of a functional Nup84 complex or Ndc1 interaction network, which
are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover,
POM33 loss of function impairs NPC distribution, a readout for a subset of genes
required for pore biogenesis, including members of the Nup84 complex and RTN1.
Consistently, we show that Pom33 is required for normal NPC density in the
daughter nucleus and for proper NPC biogenesis and/or stability in the absence
of Nup170. We hypothesize that, by modifying or stabilizing the nuclear
envelope-NPC interface, Pom33 may contribute to proper distribution and/or
efficient assembly of nuclear pores. RanGTP mediates nuclear import and mitotic spindle assembly by dissociating
import receptors from nuclear localization signal (NLS) bearing proteins. We
investigated the interplay between import receptors and the transmembrane
nucleoporin Pom121. We found that Pom121 interacts with importin alpha/beta and
a group of nucleoporins in an NLS-dependent manner. In vivo, replacement of
Pom121 with an NLS mutant version resulted in defective nuclear transport,
induction of aberrant cytoplasmic membrane stacks and decreased cell viability.
We propose that the NLS sites of Pom121 affect its function in NPC assembly both
by influencing nucleoporin interactions and pore membrane structure. The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a
crucial process for various cellular activities. The NPC comprises ~30
nucleoporins and is well characterized in vertebrates and yeast. However, only
eight plant nucleoporins have been identified, and little information is
available about the complete molecular structure of plant NPCs. In this study,
an interactive proteomic approach was used to identify Arabidopsis thaliana
nucleoporins. A series of five cycles of interactive proteomic analysis was
performed using green fluorescent protein (GFP)-tagged nucleoporins. The
identified nucleoporins were then cloned and subcellular localization analyses
were performed. We found that the plant NPC contains at least 30 nucleoporins,
22 of which had not been previously annotated. Surprisingly, plant nucleoporins
shared a similar domain organization to their vertebrate (human) and yeast
(Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins
exhibited higher sequence homology to vertebrate nucleoporins than to yeast
nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358],
Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN
OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a
nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis
failed to identify a vertebrate homolog for this protein. Interestingly,
Nup136-GFP showed greater mobility on the nuclear envelope than did other
nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant
development. These findings provide valuable new information about plant NPC
structure and function. The nuclear envelope contains >100 transmembrane proteins that continuously
exchange with the endoplasmic reticulum and move within the nuclear membranes.
To better understand the organization and dynamics of this system, we compared
the trafficking of 15 integral nuclear envelope proteins using FRAP. A
surprising 30-fold range of mobilities was observed. The dynamic behavior of
several of these proteins was also analyzed after depletion of ATP and/or Ran,
two functions implicated in endoplasmic reticulum-inner nuclear membrane
translocation. This revealed that ATP- and Ran-dependent translocation
mechanisms are distinct and not used by all inner nuclear membrane proteins. The
Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin
Nup35, which is consistent with use of the nuclear pore complex peripheral
channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP
recovery times, and this also depends on Nup35. Modeling of three proteins that
were unaffected by either ATP or Ran depletion indicates that the wide range in
mobilities could be explained by differences in binding affinities in the inner
nuclear membrane. Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope
(NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of
macromolecules between these compartments. Most NPC proteins do not contain
integral membrane domains and thus it is largely unclear how NPCs are embedded
and anchored in the NE. Here, we show that the evolutionary conserved nuclear
pore protein Nup53 binds independently of other proteins to membranes, a
property that is crucial for NPC assembly and conserved between yeast and
vertebrates. The vertebrate protein comprises two membrane binding sites, of
which the C-terminal domain has membrane deforming capabilities, and is
specifically required for de novo NPC assembly and insertion into the intact NE
during interphase. Dimerization of Nup53 contributes to its membrane interaction
and is crucial for its function in NPC assembly. |
Which E3 ubiquitin ligase mediates the ubiquitination and degradation of the interferon receptor type 1 (IFNAR1)? | Ubiquitination and degradation of the IFNAR1 (interferon alpha receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCFbeta-Trcp (Skp1-Cullin1-F-box protein beta transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. | Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon
alpha receptor 1) subunit of the type I interferon (IFN) receptor is mediated by
the SCFbeta-Trcp (Skp1-Cullin1-F-box protein beta transducin repeat-containing
protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition,
stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we
characterize the determits of IFNAR1 ubiquitination and degradation. We found
that the integrity of two Ser residues at positions 535 and 539 within the
specific destruction motif present in the cytoplasmic tail of IFNAR1 is
essential for the ability of IFNAR1 to recruit beta-Trcp as well as to undergo
efficient ubiquitination and degradation. Using an antibody that specifically
recognizes IFNAR1 phosphorylated on Ser535 we found that IFNAR1 is
phosphorylated on this residue in cells. This phosphorylation is promoted by
treatment of cells with IFNalpha. Although the cytoplasmic tail of IFNAR1
contains seven Lys residues that could function as potential ubiquitin acceptor
sites, we found that only three (Lys501, Lys525, and Lys526), all located
proximal to the destruction motif, are essential for ubiquitination and
degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was
dependent neither on its binding to beta-Trcp nor IFNAR1 ubiquitination. We
discuss the complexities and specifics of the ubiquitination and degradation of
IFNAR1, which is a beta-Trcp substrate that undergoes degradation via a
lysosomal pathway. The type I IFNR (interferon receptor) is a heterodimer composed of two
transmembrane chains, IFNAR1 (interferon-alpha receptor 1 subunit) and IFNAR2,
which are associated with the tyrosine kinases Tyk2 and Jak1 (Janus kinase 1)
respectively. Ligand-induced down-regulation of the type I IFNR is a major
mechanism of negative regulation of cellular signalling and involves the
internalization and lysosomal degradation of IFNAR1. IFNalpha promotes the
phosphorylation of IFNAR1 on Ser535, followed by recruitment of the E3 ubiquitin
ligase, beta-TrCP2 (beta-transducin repeats-containing protein 2),
ubiquitination of IFNAR1 and proteolysis. The non-catalytic role of Tyk2 in
sustaining the steady-state IFNAR1 level at the plasma membrane is well
documented; however, little is known about the function of Tyk2 in the steps
that precede and succeed serine phosphorylation and ubiquitination of IFNAR1 in
response to ligand binding. In the present study, we show that catalytic
activation of Tyk2 is not essential for IFNAR1 internalization, but is required
for ligand-induced IFNAR1 serine phosphorylation, ubiquitination and efficient
lysosomal proteolysis. Interferon alpha (IFNalpha) is widely used in treatment of maligt melanoma
patients. This cytokine acts on cells by engaging Type I IFN receptor consisting
of two subunits, (IFNAR1 and IFNAR2) followed by activation of Janus kinases
(Jak). Levels of IFNAR1 (regulated via degradation mediated by the betaTrcp E3
ubiquitin ligase) and IFNalpha signaling were reduced in 1205Lu melanoma cell
line that harbors activated BRAF and exhibits high levels of betaTrcp ubiquitin
ligase. Expression of stabilized IFNAR1 in melanoma cells decreased their
tumorigenicity. Furthermore, RNAi-mediated BRAF knockdown and pharmacologic
inhibition of either Raf or MEK1 decreased levels of betaTrcp and stabilized
IFNAR1. However, despite causing stabilization of IFNAR1, Raf inhibitor BAY
43-9006 interfered with cellular responses to IFNalpha most likely due to its
ability to directly inhibit Jak activity. We discuss the implications of this
result for combination therapy with BAY 43-9006 and IFNalpha in melanoma
patients. Phosphorylation-dependent ubiquitination and ensuing down-regulation and
lysosomal degradation of the interferon alpha/beta receptor chain 1 (IFNAR1) of
the receptor for Type I interferons play important roles in limiting the
cellular responses to these cytokines. These events could be stimulated either
by the ligands (in a Janus kinase-dependent manner) or by unfolded protein
response (UPR) inducers including viral infection (in a manner dependent on the
activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and
-independent pathways converge on phosphorylation of Ser(535) within the IFNAR1
degron leading to recruitment of beta-Trcp E3 ubiquitin ligase and concomitant
ubiquitination and degradation. Casein kinase 1 alpha (CK1 alpha) was shown to
directly phosphorylate Ser(535) within the ligand-independent pathway. Yet given
the constitutive activity of CK1 alpha, it remained unclear how this pathway is
stimulated by UPR. Here we report that induction of UPR promotes the
phosphorylation of a proximal residue, Ser(532), in a pancreatic endoplasmic
reticulum kinase-dependent manner. This serine serves as a priming site that
promotes subsequent phosphorylation of IFNAR1 within its degron by CK1 alpha.
These events play an important role in regulating ubiquitination and degradation
of IFNAR1 as well as the extent of Type I interferon signaling. Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells,
regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor
growth. This cytokine is often used to treat cancers and chronic viral
infections. The extent of cellular responses to IFN-α is limited by the
IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1
(IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by
the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron
phosphorylation, which is induced by the ligand. Here we report identification
of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible
phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor.
Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates
its interaction with IFNAR1. Expression and kinase activity of PKD2 are required
for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis
and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or
knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and
increases the efficacy of its antiviral effects. The mechanisms of the
ligand-inducible elimination of IFNAR1 are discussed, along with the potential
medical significance of this regulation. |
Are conserved noncoding elements associated with the evolution of animal body plans? | Yes. Cis-regulatory inputs identified by CNEs arose during the "re-wiring" of regulatory interactions that occurred during early animal evolution. Consequently, different animal groups, with different core GRNs, contain alternative sets of CNEs. Due to the subsequent stability of animal body plans, these core regulatory sequences have been evolving in parallel under strong purifying selection in different animal groups. | |
Which genes were found to be methylated in bladder cancer cells? | In bladder cancer, hepaCAM (hepatocyte cell adhesion molecule), RARβ(2), APC, TPEF (transmembrane protein containing epidermal growth factor and follistatin domain), RASSF1A, p14(ARF) and p16 genes were found to be methylated. These methylation events were demostrated to associate with downregulation of gene expression. | Methylation of CpG sites in the control regions of tumor suppressor genes may be
an important mechanism for their heritable, yet reversible, transcriptional
inactivation. These changes in methylation may impair the proper expression
and/or function of cell cycle regulatory genes and confer a selective growth
advantage to affected cells. Detailed methylation analysis using genomic
bisulfite sequencing was performed on a series of subclones of a bladder cancer
cell line in which a hypermethylated p16 gene had been reactivated by transient
treatment with 5-aza-2'-deoxycytidine. Methylation of the CpG island in the
promoter of the p16 gene in human bladder cancer cells did not stop the
formation of a transcript initiated 20 kb upstream by the p19 promoter but did
prevent the expression of a p16 transcript. Furthermore, we show that reactivant
clones that expressed p16 at varying levels contained heterogeneous methylation
patterns, suggesting that p16 expression can occur even in the presence of a
relatively heavily methylated coding region. We also present the first
functional evidence that methylation of only a small number of CpG sites can
significantly down-regulate p16 promoter activity, thus providing support for
the model of progressive inactivation of this tumor suppressor gene by DNA
methylation. The role of promoter methylation in the process of cancer cell metastasis has,
however, not yet been studied. Recently, methylation of the TPEF (transmembrane
protein containing epidermal growth factor and follistatin domain) gene was
reported in human colon, gastric, and bladder cancer cells. Using the Methylight
assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n
= 47), matched normal colon mucosa, as well as in the liver metastasis of 24
patients with colorectal cancer, and compared to the methylation status of the
TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in
primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of
21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was
restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were
also frequently methylated in primary colorectal cancers (P < .02) compared to
the normal colon mucosa. Interestingly, promoter methylation was significantly
more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high
degree of methylation of the TPEF gene was also observed in liver metastasis (19
of 24). In summary, we observed frequent TPEF methylation in primary colorectal
cancers and liver metastases, indicating that epigenetic alterations are not
only present in the early phases of carcinogenesis, but are also common in
metastatic lesions. The high frequency of TPEF methylation in this series of
colorectal cancers underscores the importance of epigenetic changes as targets
for the development of molecular tests for cancer diagnosis. AIM: We aimed to investigate the mechanisms of hepaCAM inactivation in
transitional cell carcinoma of the bladder through the analysis of hepaCAM exon
2 methylation.
METHODS: The methylation of hepaCAM exon 2 and the expression of hepaCAM were
determined by methylation-specific restriction PCR assay and RT-PCR in bladder
cancer cells (T24, BIU-87) as well as in 55 paired bladder cancer specimens. The
methylated bladder cancer cells were treated with 5-Aza- 2'-deoxycytidine
(5-Aza-CdR), a demethylating agent. MTT was used to detect the proliferation of
T24 and BIU-87 cells.
RESULTS: The proliferation of T24 and BIU-87 cells was suppressed by treatment
with different concentrations of 5-Aza-CdR; the expression of hepaCAM was absent
in T24 and BIU-87 cells, and we found that exon 2 of hepaCAM was methylated in
the 2 cells. hepaCAM mRNA was re-expressed and the methylation status of hepaCAM
exon 2 was reversed after treatment with 5-Aza-CdR. The expression of hepaCAM
mRNA in bladder cancer tissues was significantly lower than that in adjacent
tissues. The methylation rate of hepaCAM exon 2 was significantly higher in
bladder cancer tissues than in adjacent tissues. The methylation of hepaCAM exon
2 was related to hepaCAM expression in bladder cancer tissues.
CONCLUSIONS: Downregulation of hepaCAM expression plays an important role in the
tumorigenesis and development of bladder cancer. DNA methylation may be
important for downregulation of hepaCAM expression in bladder cancer. BACKGROUND: Bladder cancer cells illustrate major disruptions in their DNA
methylation patterns as compared with normal ones. Authors aimed to identify
epigenetic molecular markers in urine for early detection of bladder cancer.
MATERIALS AND METHODS: We retrospectively analyzed the methylation status of
RARβ(2) and APC genes in urine samples from 210 bladder cancer patients, 61
patients with benign urological diseases, and 49 healthy volunteers by using
methylation-specific PCR.
RESULTS: Methylated RARβ(2) and APC were significantly higher in bladder cancer
patients (62.8%, 59.5%) than benign (16.4%, 5%) but not detected in healthy
volunteers (0%) at (P < 0.0001). Both methylated genes showed no significant
difference among clinicopathologic factors; however, they were detected in all
grades and stages. Among the 128 patients with bilharzial bladder cancer, 94
(73.4%) showed methylated RARβ(2) and 86 (67.2%) showed methylated APC.
Homoplasmic methylation pattern of both genes were only detected in bilharzial
bladder cancer cases. Both sensitivities and specificities of the methylated
genes for bladder cancer detection were superior to urine cytology and when
altogether combined, the sensitivities improved to (91.8%), (93.5%), (91.9%),
and (80.9%) in detection of: bladder cancer, non-muscle invasive bladder cancer,
low-grade tumors, and bilharzial associated bladder cancer, respectively.
CONCLUSION: Thus, methylated RARβ(2) and APC genes might be valuable urinary
molecular markers for early detection of bilharzial and nonbilharzial bladder
cancer. BACKGROUND: Epigenetic regulation such as aberrant hypermethylation of CpG
islands in promoter plays a key role in tumorigenesis. 5-Aza-2'-deoxycytidine
(5-aza-CdR) which is a potent inhibitor of DNA methylation can reverse the
abnormal hypermethylation of the silenced tumor suppressor genes (TSGs). It has
been reported that hepatocyte cell adhesion molecule (hepaCAM) acts as a tumor
suppressor gene and expression of its mRNA and protein were down-regulated in
bladder cancer. Over-expression of hepaCAM can inhibit cancer growth and arrest
renal cancer cells at G0/G1 phase. In this study, we investigated the
methylation status of hepaCAM gene, as well as the influence of 5-aza-CdR on
expression of hepaCAM gene in bladder cancer cells.
METHODS: CpG islands in hepaCAM promoter and methprimers were predicted and
designed using bioinformatics program. Methylation status of hepaCAM promoter
was evaluated in bladder cancer tissues and two cell lines (T24 and BIU-87) by
Methylation-specific PCR; Western blot and Immunofluorescence were used to
detect expression of hepaCAM protein after 5-aza-CdR treatment; Flow cytometry
assay was performed to determine effectiveness of 5-aza-CdR on cell cycle
profile.
RESULTS: CpG island in promoter of hepaCAM gene was hyper-methylated both in
bladder carcinoma tissues and cell lines (T24 and BIU-87). Otherwise, aberrant
methylation of its promoter was associated with its decreased expression.
Hypermethylation of hepaCAM gene was reversed and expression of its mRNA and
protein were re-activated in two cell lines by DNA methyltransferases inhibitor
5-aza-CdR. Flow cytometry assay demonstrated that 5-aza-CdR can inhibit growth
of cancer cells by arresting cancer cells at G0/G1 phase.
CONCLUSION: Abnormal hypermethylation in CpG island of hepaCAM promoter is
involved in absence of hepaCAM gene expression when bladder cancer occurs.
Re-activation of hepaCAM gene by 5-aza-CdR can inhibit growth of cancer cells
and arrest cells at G0/G1 phase. |
Are DNA methylation maps applicable to the diagnosis of non-small-cell lung carcinomas? | Yes. | BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a complex maligcy that
owing to its heterogeneity and poor prognosis poses many challenges to
diagnosis, prognosis and patient treatment. DNA methylation is an important
mechanism of epigenetic regulation involved in normal development and cancer. It
is a very stable and specific modification and therefore in principle a very
suitable marker for epigenetic phenotyping of tumors. Here we present a
genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues,
where we combine MethylCap and next generation sequencing (MethylCap-seq) to
provide comprehensive DNA methylation maps of the tumor and paired lung samples.
The MethylCap-seq data were validated by bisulfite sequencing and
methyl-specific polymerase chain reaction of selected regions.
RESULTS: Analysis of the MethylCap-seq data revealed a strong positive
correlation between replicate experiments and between paired tumor/lung samples.
We identified 57 differentially methylated regions (DMRs) present in all NSCLC
tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to
any particular functional category of genes, the hypermethylated DMRs were
strongly associated with genes encoding transcriptional regulators. Furthermore,
subtelomeric regions and satellite repeats were hypomethylated in the NSCLC
samples. We also identified DMRs that were specific to two of the major subtypes
of NSCLC, adenocarcinomas and squamous cell carcinomas.
CONCLUSIONS: Collectively, we provide a resource containing genome-wide DNA
methylation maps of NSCLC and their paired lung tissues, and comprehensive lists
of known and novel DMRs and associated genes in NSCLC. INTRODUCTION: DNA methylation is part of the epigenetic regulatory mechanism
present in all normal cells. It is tissue-specific and stably maintained
throughout development, but often abnormally changed in cancer. Non-small-cell
lung carcinoma (NSCLC) is the most deadly type of cancer, involving different
tumor subtypes. This heterogeneity is a challenge for correct diagnosis and
patient treatment. The stability and specificity make of DNA methylation a very
suitable marker for epigenetic phenotyping of tumors.
METHODS: To identify candidate markers for use in NSCLC diagnosis, we used
genomewide DNA methylation maps that we had previously generated by MethylCap
and next-generation sequencing and listed the most significant differentially
methylated regions (DMRs). The 25 DMRs with highest significance in their
methylation scores were selected. The methylation status of these DMRs was
investigated in 61 tumors and matching control lung tissues by
methylation-specific polymerase chain reaction.
RESULTS: We found 12 novel DMRs that showed significant differences between
tumor and control lung tissues. We also identified three novel DMRs for each of
the two most common NSCLC subtypes, adenocarcinomas and squamous cell
carcinomas. We propose a panel of five DMRs, composed of novel and known markers
that exhibit high specificity and sensitivity to distinguish tumors from control
lung tissues.
CONCLUSION: Novel markers will aid the development of a highly specific
epigenetic panel for accurate identification and subtyping of NSCLC tumors. |
Which domain of TIA-1 is necessary for stress granule assembly? | TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. | TIA-1 is an RNA binding protein that promotes the assembly of stress granules
(SGs), discrete cytoplasmic inclusions into which stalled translation initiation
complexes are dynamically recruited in cells subjected to environmental stress.
The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related
domain (PRD). Truncation mutants lacking the PRD domain do not induce
spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD
forms aggregates that are recruited to SGs in low-level-expressing cells but
prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits
many characteristics of prions: concentration-dependent aggregation that is
inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to
protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of
HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with
the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly,
confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic
fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although
they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha
in response to arsenite. Our results reveal that prion-like aggregation of TIA-1
regulates SG formation downstream of eIF2alpha phosphorylation in response to
stress. Stress granules aid cell survival in response to environmental stressors by
acting as sites of translational repression. We report an uticipated link
between stress granules and the serine/threonine kinase RSK2. In stressed breast
cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding
protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The
RSK2 N-terminal kinase domain controls the direct interaction with the
prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in
response to stress. Mitogen releases RSK2 from the stress granules and permits
its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal
domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear
localization of RSK2 is sufficient to enhance proliferation through induction of
cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a
pivotal factor linking the stress response to survival and proliferation. Tia1/Pub1 is a stress granule component carrying a Q/N-rich prion domain. We
provide direct evidence that Tia1 forms a prion in yeast. Moreover, Tia1/Pub1
acts cooperatively with release factor Sup35/eRF3 to establish a two-protein
self-propagating state. This two-protein prion driven by the Q/N-rich prion
domains of Sup35 and Tia1/Pub1 can be visualized as distinctive line structures
along tubulin cytoskeleton. Furthermore, we find that tubulin-associated complex
containing Pub1 and Sup35 oligomers normally exists in yeast, and its assembly
depends on prion domains of Pub1 and Sup35. This Sup35/Pub1 complex, which also
contains TUB1 mRNA and components of translation machinery, is important for the
integrity of the tubulin cytoskeleton: PUB1 disruption and Sup35 depletion from
the complex lead to cytoskeletal defects. We propose that the complex is
implicated in protein synthesis at the site of microtubule assembly. Thus our
study identifies the role for prion domains in the assembly of multiprotein
complexes. |
Is the protein product of the cylindromatosis gene (CYLD) a deubiquitinating enzyme? | CYLD is a tumour-suppressor gene that is mutated in a benign skin tumour syndrome called cylindromatosis. The CYLD gene product is a deubiquitinating enzyme that was shown to regulate cell proliferation, cell survival and inflammatory responses, mainly through inhibiting NF-kappaB signalling. | Familial cylindromatosis is an autosomal domit predisposition to tumours of
skin appendages called cylindromas. Familial cylindromatosis is caused by
mutations in a gene encoding the CYLD protein of previously unknown function.
Here we show that CYLD is a deubiquitinating enzyme that negatively regulates
activation of the transcription factor NF-kappaB by specific tumour-necrosis
factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD
correlates with tumorigenesis. CYLD inhibits activation of NF-kappaB by the TNFR
family members CD40, XEDAR and EDAR in a manner that depends on the
deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated
interference augments both basal and CD40-mediated activation of NF-kappaB. The
inhibition of NF-kappaB activation by CYLD is mediated, at least in part, by the
deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a
lesser extent, TRAF6. These results indicate that CYLD is a negative regulator
of the cytokine-mediated activation of NF-kappaB that is required for
appropriate cellular homeostasis of skin appendages. Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients
with familial cylindromatosis, a condition characterized by numerous benign
adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here
we show that the development of B cells, T cells, and myeloid cells was
unaffected in CYLD-deficient mice, but that the activation of these cells with
mediators of innate and adaptive immunity resulted in enhanced NF-kappaB and JNK
activity associated with increased TNF receptor-associated factor 2 (TRAF2) and
NF-kappaB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were
more susceptible to induced colonic inflammation and showed a dramatic increase
in the incidence of tumors compared with controls in a colitis-associated cancer
model. These results suggest that CYLD limits inflammation and tumorigenesis by
regulating ubiquitination in vivo. Deubiquitinating enzymes (DUB) form a family of cysteine proteases that digests
ubiquitin chains and reverses the process of protein ubiquitination. Despite the
identification of a large number of DUBs, their physiological functions remain
poorly defined. Here we provide genetic evidence that CYLD, a recently
identified DUB, plays a crucial role in regulating the peripheral development
and activation of B cells. Disruption of the CYLD gene in mice results in B cell
hyperplasia and lymphoid organ enlargement. The CYLD-deficient B cells display
surface markers indicative of spontaneous activation and are hyperproliferative
upon in vitro stimulation. When challenged with antigens, the CYLD(-/-) mice
develop exacerbated lymphoid organ abnormalities and abnormal B cell responses.
Although the loss of CYLD has only a minor effect on B cell development in bone
marrow, this genetic deficiency disrupts the balance of peripheral B cell
populations with a significant increase in marginal zone B cells. In keeping
with these functional abnormalities, the CYLD(-/-) B cells exhibit constitutive
activation of the transcription factor NF-kappaB due to spontaneous activation
of IkappaB kinase beta and degradation of the NF-kappaB inhibitor IkappaBalpha.
These findings demonstrate a critical role for CYLD in regulating the basal
activity of NF-kappaB and maintaining the naive phenotype and proper activation
of B cells. Mutations in the cylindromatosis (CYLD) gene cause benign tumors of skin
appendages, referred to as cylindromas. The CYLD gene encodes a deubiquitinating
enzyme that removes Lys-63-linked ubiquitin chains from I kappa B kinase
signaling components and thereby inhibits NF-kappaB pathway activation. The
dysregulation of NF-kappaB activity has been proposed to promote cell
transformation in part by increasing apoptosis resistance, but it is not clear
whether this is CYLD's only or predomit tumor-suppressing function. Here, we
show that CYLD is also required for timely entry into mitosis. Consistent with a
cell-cycle regulatory function, CYLD localizes to microtubules in interphase and
the midbody during telophase, and its protein levels decrease as cells exit from
mitosis. We identified the protein kinase Plk1 as a potential target of CYLD in
the regulation of mitotic entry, based on their physical interaction and similar
loss-of-function and overexpression phenotypes. Our findings raise the
possibility that, as with other genes regulating tumorigenesis, CYLD has not
only tumor-suppressing (apoptosis regulation) but also tumor-promoting
activities (enhancer of mitotic entry). We propose that this additional function
of CYLD could provide an explanation for the benign nature of most cylindroma
lesions. CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis.
Despite biochemical and cell culture studies, the physiological functions of
CYLD in animal development and tumorigenesis remain poorly understood. To
address these questions, we generated Drosophila CYLD (dCYLD) mutant and
transgenic flies expressing wild-type and mutant dCYLD proteins. Here we show
that dCYLD is essential for JNK-dependent oxidative stress resistance and normal
lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death
through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of
the JNKK kinase dTAK1. We show that dCYLD encodes a deubiquitinating enzyme that
deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic
degradation. These data provide a molecular mechanism for the tumor suppressor
function of this evolutionary conserved molecule by indicating that dCYLD plays
a critical role in modulating TNF-JNK-mediated cell death. Data from several studies suggest that the ubiquitin-proteasome system may play
a role in the progression of atherosclerosis. Here, we examined the potential
role of the deubiquitinating enzyme CYLD (cylindromatosis), mutation of which
has been reported to cause familial cylindromatosis. Northern blot analysis
revealed expression of CYLD mRNA in the aorta, as well as in cultured human
aortic endothelial cells (ECs) and vascular smooth muscle cells. Treatment with
recombit tumor necrosis factor (TNF)-alpha significantly increased CYLD
expression in ECs and vascular smooth muscle cells. Immunostaining showed CYLD
expression in atherosclerotic lesions from human carotid arteries and
up-regulation of CYLD expression in the neointima of rat carotid arteries after
balloon injury. Overexpression of CYLD in ECs resulted in inhibition of
TNF-alpha-induced nuclear factor-kappaB activity through deubiquitination of
TNFR-associated factor 2 (TRAF2), whereas overexpression of catalytically
inactive CYLD had no effect. CYLD overexpression also inhibited expression of
cyclin D1 and activation of the E2F pathway through deubiquitination of the
upstream molecule Bcl-3 and inhibition of its translocation into the nucleus.
Overexpressed CYLD also significantly inhibited cell viability. Furthermore,
overexpression of CYLD in rat balloon-injured carotid artery attenuated
neointimal formation through inactivation of nuclear factor-kappaB and E2F. In
conclusion, these data demonstrate that the deubiquitinating enzyme CYLD may
inhibit inflammation and proliferation in vascular cells and may represent a
novel target for the treatment or prevention of atherosclerosis. Molecular studies of cylindromas, which arise from the eccrine or apocrine cells
of the skin, have demonstrated frequent alterations at chromosome 16q12-13,
recently found to house the cylindromatosis (CYLD) gene. CYLD, a tumor
suppressor gene, has deubiquitinating enzyme activity and inhibits the
activation of transcription factor NF-kappaB. Loss of the deubiquitinating
activity of CYLD is correlated with tumorigenesis. It has been reported that the
expression of CYLD is observed in various organs. We demonstrated previously
that human salivary gland tumor (SGT) cell line, HSG spontaneously expresses
CYLD and also found that adenoid cystic carcinoma (ACC) arising from the hard
palate was distinctly positive for CYLD, immunohistochemically. However, it is
unclear whether loss of CYLD is associated with development of SGTs. This study
examined CYLD function in SGT cells and attempted to clarify whether CYLD is
associated with development of SGTs. The expression of CYLD and NF-kappaB mRNAs
in HSG cells was increased by TNF-alpha. Translocation of NF-kappaB protein from
the cytoplasm to the nucleus in HSG cells peaked at 30 min after TNF-alpha
stimulation, then decreased at 60 min, whereas that of CYLD protein increased
gradually in a time-dependent manner. Luciferase reporter assay indicated that
TNF-alpha induced a 5-fold increase of NF-kappaB-dependent transcription at 4 h,
which was further enhanced by knockdown of CYLD using RNA interference. Taken
together, these data demonstrated that the levels of both CYLD and NF-kappaB
mRNAs accumulated in HSG cells during 24 h after TNF-alpha stimulation, although
the NF-kappaB activity in the cells was at least negatively regulated by CYLD.
Immunohistochemical examinations revealed that there are several correlations
between the expression of CYLD and NF-kappaB-related factors in 17 cases of ACC
tissues. These findings suggest that loss of CYLD is associated with development
of SGTs. Bacteraemic pneumonia is a common cause of sepsis in critically ill patients
today and is characterized by dysregulation of inflammation. The genetic factors
predisposing to bacteraemic pneumonia are not yet fully understood. Innate
immunity is pivotal for host defence against invading bacteria, and nuclear
factor-kappa B (NF-kappaB) is central to bacteria-induced inflammation and
immune responses. The deubiquitinating enzyme CYLD has been identified as a key
negative regulator for NF-kappaB. In the present study, we investigated the role
of CYLD in innate immune response in Escherichia coli pneumonia. Upon E. coli
inoculation, Cyld(-/-) mice were hypersusceptible to E. coli pneumonia with
higher mortality. Innate immune response to E. coli was enhanced in Cyld(-/-)
cells and mice. Cyld(-/-) cells exhibited enhanced NF-kappaB activation upon E.
coli inoculation, and the enhanced NF-kappaB activation by E. coli was abolished
by perturbing IkappaB kinase (IKK) signalling. Furthermore, IKK inhibitor
rescued Cyld(-/-) mice from lethal infection during E. coli pneumonia along with
reduced inflammation. Taken together, these data showed that CYLD acts as a
crucial negative regulator for E. coli pneumonia by negatively regulating
NF-kappaB. These findings provide novel insight into the regulation of
bacteraemic pneumonia and related diseases and may help develop novel
therapeutic strategies for these diseases. Cyld encodes a 956-amino acid deubiquitinating enzyme (CYLD), which is a
negative regulator of nuclear factor kappaB and mitogen-activated protein kinase
pathways. Mutations that truncate and inactivate the carboxyl-terminal
deubiquitinating domain of CYLD underlie the development of skin appendage
tumors in humans, whereas down-regulation of Cyld expression has been associated
with the development of various types of human maligcies including lung
cancer. To establish an animal model of human CYLD inactivation and characterize
the biological role of CYLD in vivo, we generated mice carrying a homozygous
deletion of Cyld exon 9 (Cyld(Delta 9/Delta 9) mice) using a conditional
approach. Deletion of exon 9 would cause a carboxyl-terminal truncation of CYLD
and inactivation of its deubiquitinating activity. In accordance with previous
studies, fibroblasts from Cyld(Delta 9/Delta 9) embryos had hyperactive nuclear
factor kappaB and c-Jun kinase pathways compared with control fibroblasts.
Cyld(Delta 9/Delta 9) newborn mice were smaller than wild-type littermates with
a short and kinky tail and no major developmental defects. However, Cyld(Delta
9/Delta 9) mice died shortly after birth from apparent respiratory dysfunction.
Histological examination of E18.5 Cyld(Delta 9/Delta 9) lungs demonstrated an
immature phenotype characterized by hyperplasic mesenchyme but apparently normal
epithelial, smooth muscle. and endothelial structures. Our study identifies an
important role of CYLD in lung maturation, which may underlie the development of
many cases of lung cancer. The mechanism by which Wnt receptors transduce signals to activate downstream
beta-catenin-mediated target gene transcription remains incompletely understood
but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and
activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the
deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor
gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling.
Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation
of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive
Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD.
At the molecular level, CYLD interacts with and regulates K63-linked
ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX
domain in CYLD-deficient cells positively links to the signaling activity of
Dvl. Together, our results argue that loss of CYLD instigates tumor growth in
human cylindromatosis through a mechanism in which hyperubiquitination of
polymerized Dvl drives enhancement of Wnt responses. The cylindromatosis tumor suppressor gene (Cyld) encodes a deubiquitinating
enzyme (CYLD) with immunoregulatory function. In this study, we evaluated the
role of Cyld in T cell ontogeny by generating a mouse (Cyld(Delta9)) with a
thymocyte-restricted Cyld mutation that causes a C-terminal truncation of the
protein and reciprocates catalytically inactive human mutations. Mutant mice had
dramatically reduced single positive thymocytes and a substantial loss of
peripheral T cells. The analyses of polyclonal and TCR-restricted thymocyte
populations possessing the mutation revealed a significant block in positive
selection and an increased occurrence of apoptosis at the double-positive stage.
Interestingly, in the context of MHC class I and II restricted TCR transgenes,
lack of functional CYLD caused massive deletion of thymocytes that would have
been positively selected, which is consistent with an impairment of positive
selection. Biochemical analysis revealed that Cyld(Delta9) thymocytes exhibit
abnormally elevated basal activity of NF-kappaB and JNK. Most importantly,
inactivation of NF-kappaB essential modulator fully restored the NF-kappaB
activity of Cyld(Delta9) thymocytes to physiologic levels and rescued their
developmental and survival defect. This study identifies a fundamental role for
functional CYLD in establishing the proper threshold of activation for thymocyte
selection by a mechanism dependent on NF-kappaB essential modulator. The cylindromatosis gene (CYLD) was identified as a tumor suppressor gene, which
is mutated in familial cylindromatosis (Brooke-Spiegler syndrome), an
autosomal-domit predisposition to multiple tumors of the skin appendages.
CYLD is a deubiquitinating enzyme acting as a negative regulator of the nuclear
factor κB (NF-κB) signaling pathway by removing lysine-63-linked polyubiquitin
chains from NF-κB activating proteins. In order to investigate the role of CYLD
in apoptotic signaling in human hepatocellular carcinoma (HCC) cells, we first
studied the expression levels of CYLD in HCC tissues. CYLD expression was lower
in HCC both at protein and mRNA levels compared to the surrounding non-maligt
tissue. In order to further study the role of CYLD in the apoptotic sensitivity
of HCC cells, CYLD was specifically down-regulated in HCC cell lines via RNA
interference. The specific down-regulation of CYLD resulted in increased
resistance towards treatment with doxorubicin, 5-fluorouracil and cisplatin. In
addition, the down-regulation of CYLD in HCC cells decreased the sensitivity
towards tumor necrosis factor-α-induced apoptosis. The CYLD knockdown also led
to the degradation of the NF-κB inhibitor, IκB-α, resulting in enhanced NF-κB
activity in HCC cells. Finally, we found that CYLD expression was triggered by
the multikinase inhibitor, sorafenib, by the inhibition of Raf-1, as well as by
the blockage of the pro-survival kinases, MEK (U0126) and the epidermal growth
factor receptor (AG1478). In summary, we show that CYLD is down-regulated in
human HCC and is involved in the apoptotic resistance of HCC cells. Our data
identify the reconstitution of CYLD expression as an attractive approach for
overcoming resistance to treatment in HCC. The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that
has been implicated in various aspects of adaptive and innate immune responses.
Nevertheless, the role of CYLD in the function of specific types of immune cells
remains elusive. In this report we have used conditional gene targeting in mice
to address the role of the deubiquitinating activity of CYLD in the
myelomonocytic lineage. Truncation of the deubiquitinating domain of CYLD
specifically in myelomonocytic cells impaired the development of lethal
LPS-induced endotoxic shock and the accumulation of thioglycollate-elicited
peritoneal macrophages. Our data establish CYLD as a regulator of
monocyte-macrophage activation in response to inflammatory stimuli and identify
it as a potential target for therapeutic intervention in relevant inflammatory
disorders in humans. Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively
regulates various signaling pathways by removing the lysine 63-linked
polyubiquitin chains from several specific substrates. Loss of CYLD in different
types of tumors leads to either cell survival or proliferation. In this study we
demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation
rate of these cells compared to CYLD+/+ in a serum concentration dependent
manner without affecting cell survival. The reduced proliferation rate in
CYLD+/+ in the presence of serum was due to the binding of serum response factor
(SRF) to the serum response element identified in the CYLD promoter for the
up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD
promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity.
Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression
level of CYLD and increased cell proliferation. These results show that SRF acts
as a positive regulator of CYLD expression, which in turn reduces the mitogenic
activation of serum for aberrant proliferation of MEF cells. CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK
signaling. Here, we show that CYLD knock-out mice have markedly increased
numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in
the thymus. In vitro stimulation of CYLD-deficient naive T cells with
anti-CD3/28 in the presence of TGF-β led to a marked increase in the number of
Foxp3-expressing T cells when compared with stimulated naive control CD4(+)
cells. Under endogenous conditions, CYLD formed a complex with Smad7 that
facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues.
Moreover, this site-specific ubiquitination of Smad7 was required for activation
of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38
activity in primary T cells impaired Treg differentiation. Together, our results
show that CYLD regulates TGF-β signaling function in T cells and the development
of Tregs through deubiquitination of Smad7. |
What is the characteristic domain of histone methyltransferases? | SET (suppressor of variegation, enhancer of zest and trithorax) domain | The organization of chromatin into higher-order structures influences chromosome
function and epigenetic gene regulation. Higher-order chromatin has been
proposed to be nucleated by the covalent modification of histone tails and the
subsequent establishment of chromosomal subdomains by non-histone modifier
factors. Here we show that human SUV39H1 and murine Suv39h1--mammalian
homologues of Drosophila Su(var)3-9 and of Schizosaccharomyces pombe
clr4--encode histone H3-specific methyltransferases that selectively methylate
lysine 9 of the amino terminus of histone H3 in vitro. We mapped the catalytic
motif to the evolutionarily conserved SET domain, which requires adjacent
cysteine-rich regions to confer histone methyltransferase activity. Methylation
of lysine 9 interferes with phosphorylation of serine 10, but is also influenced
by pre-existing modifications in the amino terminus of H3. In vivo, deregulated
SUV39H1 or disrupted Suv39h activity modulate H3 serine 10 phosphorylation in
native chromatin and induce aberrant mitotic divisions. Our data reveal a
functional interdependence of site-specific H3 tail modifications and suggest a
dynamic mechanism for the regulation of higher-order chromatin. Methylation of lysine residues in the N-terminal tails of histones is thought to
represent an important component of the mechanism that regulates chromatin
structure. The evolutionarily conserved SET domain occurs in most proteins known
to possess histone lysine methyltransferase activity. We present here the
crystal structure of a large fragment of human SET7/9 that contains a N-terminal
beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies
two residues in the C terminus of the protein that appear essential for
catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the
cofactor AdoMet binds to this domain and present biochemical data supporting the
role of invariant residues in catalysis, binding of AdoMet, and interactions
with the peptide substrate. The SET domain contains the catalytic center of lysine methyltransferases that
target the N-terminal tails of histones and regulate chromatin function. Here we
report the structure of the SET7/9 protein in the absence and presence of its
cofactor product, S-adenosyl-L-homocysteine (AdoHcy). A knot within the SET
domain helps form the methyltransferase active site, where AdoHcy binds and
lysine methylation is likely to occur. A structure-guided comparison of
sequences within the SET protein family suggests that the knot substructure and
active site environment are conserved features of the SET domain. Covalent modifications of histone tails play important roles in gene
transcription and silencing. We recently identified an ERG ( ets -related
gene)-associated protein with a SET (suppressor of variegation, enhancer of zest
and trithorax) domain (ESET) that was found to have the activity of a histone
H3-specific methyltransferase. In the present study, we investigated the
interaction of ESET with other chromatin remodelling factors. We show that ESET
histone methyltransferase associates with histone deacetylase 1 (HDAC1) and
HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A
and mSin3B. Deletion analysis of ESET reveals that an N-terminal region
containing a tudor domain is responsible for interaction with mSin3A/B and
association with HDAC1/2, and that truncation of ESET enhances its binding to
mSin3. When bound to a promoter, ESET represses the transcription of a
downstream luciferase reporter gene. This repression by ESET is independent of
its histone methyltransferase activity, but correlates with its binding to the
mSin3 co-repressors. In addition, the repression can be partially reversed by
treatment with the HDAC inhibitor trichostatin A. Taken together, these data
suggest that ESET histone methyltransferase can form a large, multi-protein
complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in
multiple pathways of transcriptional repression. The methylation of lysine residues of histones plays a pivotal role in the
regulation of chromatin structure and gene expression. Here, we report two
crystal structures of SET7/9, a histone methyltransferase (HMTase) that
transfers methyl groups to Lys4 of histone H3, in complex with
S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A resolution. The
structures reveal an active site consisting of: (i) a binding pocket between the
SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation
binds; (ii) a narrow substrate-specific channel that only unmethylated lysine
residues can access; and (iii) a catalytic tyrosine residue. The methyl group of
AdoMet is directed to the narrow channel where a substrate lysine enters from
the opposite side. We demonstrate that SET7/9 can transfer two but not three
methyl groups to unmodified Lys4 of H3 without substrate dissociation. The
unusual features of the SET domain-containing HMTase discriminate between the
un- and methylated lysine substrate, and the methylation sites for the histone
H3 tail. Dot1 is an evolutionarily conserved histone methyltransferase that methylates
lysine-79 of histone H3 in the core domain. Unlike other histone
methyltransferases, Dot1 does not contain a SET domain, and it specifically
methylates nucleosomal histone H3. We have solved a 2.5 A resolution structure
of the catalytic domain of human Dot1, hDOT1L, in complex with
S-adenosyl-L-methionine (SAM). The structure reveals a unique organization of a
mainly alpha-helical N-terminal domain and a central open alpha/beta structure,
an active site consisting of a SAM binding pocket, and a potential lysine
binding channel. We also show that a flexible, positively charged region at the
C terminus of the catalytic domain is critical for nucleosome binding and
enzymatic activity. These structural and biochemical analyses, combined with
molecular modeling, provide mechanistic insights into the catalytic mechanism
and nucleosomal specificity of Dot1 proteins. In this issue of Molecular Cell, Zhang et al. report the structure of a ternary
complex between the SET domain histone methyltransferase DIM-5, its cofactor,
and a histone H3 peptide. The insight gained from analysis of a key amino acid
provides an exciting opportunity to dissect the possible functional meaning of
mono-, di-, and trimethylation of histone lysine residues in vivo that will
complement existing approaches in the quest to crack the histone methylation
code. 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. The SET domain, first identified within and named after proteins encoded by
three Drosophila genes [Su(var)3-9, E(z), and Trithorax], is recognized as a
signature motif for histone methyltransferases that are involved in epigenetic
processes. The SUV39H family of SET domain proteins methylate specifically the
residue lysine 9 of histone H3, creating a code for gene silencing. This family
of proteins contain at their C termini a unique catalytic domain consisting of
pre-SET, SET, and post-SET domains. Sequence homology-based searches identified
15 Arabidopsis, 14 maize, and 12 rice proteins that can be assigned to the
SUV39H family. These high numbers in plants are in marked contrast to the
situation in animals, in which each species appears to contain only two to three
proteins of this family. Our phylogenetic analyses revealed that plant proteins
can be classified into seven orthology groups. Representative members of each
group can be found in single plant species, suggesting that different group
members are evolutionarily conserved to perform specific functions. Methylation of lysine residues of histones is an important epigenetic mark that
correlates with functionally distinct regions of chromatin. We present here the
crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as
Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is
exclusively a mono-methylase and is therefore responsible for a signaling role
quite distinct from that established by other enzymes that target this histone
residue. We provide evidence from NMR for the C-flanking domains of SET proteins
becoming ordered upon addition of AdoMet cofactor and develop a model for the
catalytic cycle of these enzymes. The crystal structure reveals the basis of the
specificity of the enzyme for H4-K20 because a histidine residue within the
substrate, close to the target lysine, is required for completion of the active
site. We also show how a highly variable component of the SET domain is
responsible for many of the enzymes' interactions with its target histone
peptide and probably also how this part of the structure ensures that Pr-Set7 is
nucleosome specific. Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in
chromatin is associated with transcriptionally silenced genes and is mediated by
histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9
methyltransferase that possesses characteristic SET domain and ANK repeats. In
this paper, we have used a series of green fluorescent protein-tagged deletion
constructs to identify two nuclear localization signals (NLS), the first NLS
embedded between amino acids 24 and 109 and the second between amino acids 394
and 401 of murine G9a. Our data show that both long and short G9a isoforms were
capable of entering the nucleus to methylate chromatin. Full-length or
N-terminus-deleted G9a isoforms were also catalytically active enzymes that
methylated recombit H3 or synthetic peptides representing the N-terminus tail
of H3. In vitro methylation reactions using N-terminus tail peptides resulted in
tri-methylation of K9 that remained processive, even in G9a enzymes that lacked
an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di-
and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa
cells resulted in reduction of global H3-K9 di- and tri-methylation. A
recombit deletion mutant enzyme fused with maltose-binding protein
(MBP-G9aDelta634) was used for steady-state kinetic analysis with various
substrates and was compared with full-length G9a (G9aFL). Turnover numbers of
MBP-G9aDelta634 for various substrates was approximately 3-fold less compared
with G9aFL, while their Michaelis constants (K(m)) for recombit H3 were
similar. The K(AdoMet)m for MBP-G9aDelta634 was approximately 2.3-2.65 microM
with various substrates. Catalytic efficiencies (kcat/K(m)) for both
MBP-G9aDelta634 and G9aFL were similar, suggesting that the N-terminus is not
essential for catalysis. Furthermore, mutation of conserved amino acids R1097A,
W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic
region, resulted in catalytically impaired enzymes, thereby confirming the
involvement of the C-terminus of G9a in catalysis. Thus, distinct domains
modulate nuclear targeting and catalytic functions of G9a. The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors
is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes,
and related HMTases control germ line development in worms, flowering in plants,
and X inactivation in mammals. The fly complex contains a catalytic SET domain
subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The
four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show
that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We
also show that loss of ESC disrupts global methylation of histone H3-lysine 27
in fly embryos. Subunit mutations identify domains required for catalytic
activity and/or binding to specific partners. We describe missense mutations in
surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS
domain of SU(Z)12, which each disrupt HMTase activity but preserve complex
assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce
active HMTase. We also find that a recombit worm complex containing the E(Z)
homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an
ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian
PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase
activity from a distinct partner set. Epigenetic modifications of histones regulate gene expression and chromatin
structure. Here we show that Meisetz (meiosis-induced factor containing a PR/SET
domain and zinc-finger motif) is a histone methyltransferase that is important
for the progression of early meiotic prophase. Meisetz transcripts are detected
only in germ cells entering meiotic prophase in female fetal gonads and in
postnatal testis. Notably, Meisetz has catalytic activity for trimethylation,
but not mono- or dimethylation, of lysine 4 of histone H3, and a transactivation
activity that depends on its methylation activity. Mice in which the Meisetz
gene is disrupted show sterility in both sexes due to severe impairment of the
double-stranded break repair pathway, deficient pairing of homologous
chromosomes and impaired sex body formation. In Meisetz-deficient testis,
trimethylation of lysine 4 of histone H3 is attenuated and meiotic gene
transcription is altered. These findings indicate that meiosis-specific
epigenetic events in mammals are crucial for proper meiotic progression. Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation
and chromatin organization. SUV39H1 methylates H3K9 at the pericentric
heterochromatin region and participates in the maintece of genome stability.
In this study, a recombit purified SUV39H1 is used for substrate specificity
and steady-state kinetic analysis with peptides representing the un- or
dimethylated lysine 9 histone H3 tail or full-length human recombit H3 (rH3).
Recombit SUV39H1 methylated its substrate via a nonprocessive mechanism.
Binding of either peptide or AdoMet first to the enzyme made a catalytically
competent binary complex. Product inhibition studies with SUV39H1 showed that
S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine
and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide
was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of
AdoMet, suggesting a random mechanism in a bi-bi reaction for recombit
SUV39H1 in which either substrate can bind to the enzyme first and either
product can release first. The turnover numbers (k(cat)) for the H3 tail peptide
and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value
of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The
Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower
compared to that of the unmethylated peptide. The Michaelis constants for AdoMet
(K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and
rH3, respectively. A reduction in the level of methylation was observed at high
concentrations of rH3, implying substrate inhibition. Deletion of the
chromodomain or point mutation of the conserved amino acids, W64A or W67A, of
SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET
domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for
catalysis. SET domain genes have been identified in numbers of bacterial genomes based on
similarity to SET domains of eukaryotic histone methyltransferases. Herein, a
Chlamydophila pneumoniae SET domain gene was clarified to be coincidently
expressed with hctA and hctB genes encoding chlamydial histone H1-like proteins,
Hc1 and Hc2, respectively. The SET domain protein (cpnSET) is localized in
chlamydial cells and interacts with Hc1 and Hc2 through the C-terminal SET
domain. As expected from conservation of catalytic sites in cpnSET, it functions
as a protein methyltransferase to murine histone H3 and Hc1. However, little is
known about protein methylation in the molecular pathogenesis of chlamydial
infection. cpnSET may play an important role in chlamydial cell maturation due
to modification of chlamydial histone H1-like proteins. Processive versus distributive methyl group transfer was assessed for pea
Rubisco large subunit methyltransferase, a SET domain protein lysine
methyltransferase catalyzing the formation of trimethyllysine-14 in the large
subunit of Rubisco. Catalytically competent complexes between an immobilized
form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were
used to demonstrate enzyme release that was co-incident with and dependent on
formation of trimethyllysine. Catalytic rate constants determined for formation
of trimethyllysine were considerably lower ( approximately 10-fold) than rate
constants determined for total radiolabel incorporation from
[3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under
catalytic conditions favoring monomethyllysine indicated a random or ordered
reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid
ping-pong mechanism. These results were compared with double-reciprocal velocity
plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and
SpCLR4 (a dimethyltransferase) and suggest a predictive ability of
double-reciprocal velocity plots for single versus multiple methyl group
transfers by SET domain protein lysine methyltransferases. A model is proposed
for SET domain protein lysine methyltransferases in which initial binding of
polypeptide substrate and S-adenosylmethionine is random, with polypeptide
binding followed by deprotonation of the epsilon-amine of the target lysyl
residue and subsequent methylation. Following methyl group transfer,
S-adenosylhomocysteine and monomethylated polypeptide dissociate from
monomethyltransferases, but di- and trimethyltransferases begin a successive and
catalytically obligatory deprotonation of enzyme-bound methylated lysyl
intermediates, which along with binding and release of S-adenosylmethionine and
S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction
mechanism. We previously identified a mammalian Set1A complex analogous to the yeast
Set1/COMPASS histone H3-Lys4 methyltransferase complex (Lee, J.-H., and Skalnik,
D. G. (2005) J. Biol. Chem. 280, 41725-41731). Data base analysis indicates that
human Set1A protein shares 39% identity with an uncharacterized SET domain
protein, KIAA1076, hereafter denoted Set1B. Immunoprecipitation and mass
spectrometry reveal that Set1B associates with a approximately 450 kDa complex
that contains all five non-catalytic components of the Set1A complex, including
CFP1, Rbbp5, Ash2, Wdr5, and Wdr82. These data reveal two human protein
complexes that differ only in the identity of the catalytic histone
methyltransferase. In vitro assays demonstrate that the Set1B complex is a
histone methyltransferase that produces trimethylated histone H3 at Lys(4). Both
Set1A and Set1B are widely expressed. Inducible expression of the carboxyl
terminus of either Set1A or Set1B decreases steady-state levels of both
endogenous Set1A and Set1B protein, but does not alter the expression of the
non-catalytic components of the Set1 complexes. A 123-amino acid fragment
upstream of the Set1A SET domain is necessary for interaction with CFP1, Ash2,
Rbbp5, and Wdr5. This protein domain is also required to mediate feedback
inhibition of Set1A and Set1B expression, which is a consequence of reduced
Set1A and Set1B stability when not associated with the methyltransferase
complex. Confocal microscopy reveals that Set1A and Set1B each localize to a
largely non-overlapping set of euchromatic nuclear speckles, suggesting that
Set1A and Set1B each bind to a unique set of target genes and thus make
non-redundant contributions to the epigenetic control of chromatin structure and
gene expression. Methylation of certain lysine residues in the N-terminal tails of core histone
proteins in nucleosome is of fundamental importance in the regulation of
chromatin structure and gene expression. Such histone modification is catalyzed
by protein lysine methyltransferases (PKMTs). PKMTs contain a conserved SET
domain in almost all of the cases and may transfer one to three methyl groups
from S-adenosyl-L-methionine (AdoMet) to the epsilon-amino group of the target
lysine residue. Here, quantum mechanical/molecular mechanical molecular dynamics
and free-energy simulations are performed on human PKMT SET7/9 and its mutants
to understand two outstanding questions for the reaction catalyzed by PKMTs: the
mechanism for deprotonation of positively charged methyl lysine (lysine) and
origin of product specificity. The results of the simulations suggest that
Tyr-335 (an absolute conserved residue in PKMTs) may play the role as the
general base for the deprotonation after dissociation of AdoHcy
(S-adenosyl-L-homocysteine) and before binding of AdoMet. It is shown that
conformational changes could bring Y335 to the target methyl lysine (lysine) for
proton abstraction. This mechanism provides an explanation why methyl transfers
could be catalyzed by PKMTs processively. The free-energy profiles for methyl
transfers are reported and analyzed for wild type and certain mutants (Y305F and
Y335F) and the active-site interactions that are of importance for the enzyme's
function are discussed. The results of the simulations provide important
insights into the catalytic process and lead to a better understanding of
experimental observations concerning the origin of product specificity for
PKMTs. 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. The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an
essential role in early development and hematopoiesis. The biological function
of MLL1 is mediated by the histone H3K4 methyltransferase activity of the
carboxyl-terminal SET domain. We have determined the crystal structure of the
MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3
peptide. This structure indicates that, in order to form a well-ordered active
site, a highly variable but essential component of the SET domain must be
repositioned. To test this idea, we compared the effect of the addition of MLL
complex members on methyltransferase activity and show that both RbBP5 and Ash2L
but not Wdr5 stimulate activity. Additionally, we have determined the effect of
posttranslational modifications on histone H3 residues downstream and upstream
from the target lysine and provide a structural explanation for why H3T3
phosphorylation and H3K9 acetylation regulate activity. SET domain methyltransferases deposit methyl marks on specific histone tail
lysine residues and play a major role in epigenetic regulation of gene
transcription. We solved the structures of the catalytic domains of GLP, G9a,
Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in
their apo conformation or in complex with the methyl donating cofactor, and
peptide substrates. We analyzed the structural determits for methylation
state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show
that the I-SET domain acts as a rigid docking platform, while induced-fit of the
Post-SET domain is necessary to achieve a catalytically competent conformation.
We also propose a model where long-range electrostatics bring enzyme and histone
substrate together, while the presence of an arginine upstream of the target
lysine is critical for binding and specificity.
ENHANCED VERSION: This article can also be viewed as an enhanced version in
which the text of the article is integrated with interactive 3D representations
and animated transitions. Please note that a web plugin is required to access
this enhanced functionality. Instructions for the installation and use of the
web plugin are available in Text S1. NSD3/WHSC1L1 histone methyltransferase gene aberrations are observed in leukemia
and in breast and lung carcinomas, suggesting that NSD3 is implicated in
carcinogenesis. In this study we examined in human breast cancer cells the NSD3L
isoform which contains the catalytic histone methyltransferase SET-domain. siRNA
directed depletion of NSD3L followed by genome-wide microarray analysis
identified NSD3L regulated genes which could be functionally linked to cellular
signaling pathways such as cell growth, cell cycle, cell motility,
transcription, and apoptosis. Notably up-regulated genes are the cell cycle
regulators E2F2 and Arl2. In accordance with a function of NSD3L in cell cycle
regulation NSD3L depletion resulted in an increase in the number of cells in the
S and G2/M cell cycle phases. Moreover, NSD3L depletion increased the
invasiveness of MDA-MB-231 breast cancer cells indicating that NSD3L normally
restrain cellular metastatic potential. Together the presented data indicates
that NSD3L is a candidate tumor suppressor. Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with
transcription activation and is mainly catalyzed by the MLL/SET1 family histone
methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the
presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic
subunit containing a SET domain. Unlike most other histone lysine
methyltransferases, all four proteins are required for efficient H3 K4
methylation. Despite extensive efforts, mechanisms for how three core components
regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a
heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity.
This activity requires the highly conserved SPRY domain of Ash2L and a short
peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are
capable of binding to S-adenosyl-L- [methyl-(3)H] methionine in the MLL1 core
complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4
methylation also compromise SAM binding by Ash2L. Taken together, our results
show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall
catalysis of MLL1 mediated H3 K4 methylation. The results we describe here
provide mechanistic insights for unique regulation of the MLL1 methyltransferase
activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct
contacts with the substrates and contribute to the formation of a joint
catalytic center. Given the shared core configuration among all MLL/SET1 family
HMTs, it will be interesting to test whether the mechanism we describe here can
be generalized to other MLL/SET1 family members in the future. A survey of the human genome was performed to understand the constituency of
protein methyltransferases (both protein arginine and lysine methyltransferases)
and the relatedness of their catalytic domains. We identified 51 protein lysine
methyltransferase proteins based on similarity to the canonical Drosophila
Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of
telomeric silencing-1-like, a known protein lysine methyltransferase, did not
fit within the protein lysine methyltransferase family, but did group with the
protein arginine methyltransferases, along with 44 other proteins, including the
METTL and NOP2/Sun domain family proteins. We show that a representative METTL,
METTL11A, demonstrates catalytic activity as a histone methyltransferase. We
also solved the co-crystal structures of disruptor of telomeric silencing-1-like
with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site.
The conformation of both ligands is virtually identical to that found in known
protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins
and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste
(E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have
developed biochemical assays for 11 members of the protein methyltransferase
target class and have profiled the affinity of three ligands for these enzymes:
the common methyl-donating substrate S-adenosylmethionine; the common reaction
product S-adenosylhomocysteine; and the natural product sinefungin. The affinity
of each of these ligands is mapped onto the family trees of the protein lysine
methyltransferases and protein arginine methyltransferases to reveal patterns of
ligand recognition by these enzymes. The ESET (also called SETDB1) protein contains an N-terminal tudor domain that
mediates protein-protein interactions and a C-terminal SET domain that catalyzes
methylation of histone H3 at lysine 9. We report here that ESET protein is
transiently upregulated in prehypertrophic chondrocytes in newborn mice. To
investigate the in vivo effects of ESET on chondrocyte differentiation, we
generated conditional knockout mice to specifically eliminate the catalytic SET
domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene
caused acceleration of chondrocyte hypertrophy in both embryos and young
animals, depleting chondrocytes that are otherwise available to form epiphyseal
plates for endochondral bone growth. ESET-deficient mice are thus characterized
by defective long bone growth and trabecular bone formation. To understand the
underlying mechanism for ESET regulation of chondrocytes, we carried out
co-expression experiments and found that ESET associates with histone
deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting
transcription factor. Repression of Runx2-mediated gene transactivation by ESET
is dependent on its H3-K9 methyltransferase activity as well as its associated
histone deacetylase activity. In addition, knockout of ESET is associated with
repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes.
Together, these results provide clear evidence that ESET controls hypertrophic
differentiation of growth plate chondrocytes and endochondral ossification
during embryogenesis and postnatal development. |
What is smFISH? | smFISH (Single-molecule fluorescence in situ hybridization) allows for quantitative imaging of single RNA molecules. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells. | Author information:
(1)1] Verna &Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, Texas, USA. [2] Center for the Physics of
Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
[3] Center for Theoretical Biological Physics, Rice University, Houston, Texas,
USA.
(2)1] Verna &Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, Texas, USA. [2] Center for Theoretical
Biological Physics, Rice University, Houston, Texas, USA.
(3)Integrative Molecular and Biomedical Sciences Graduate Program, Baylor
College of Medicine, Houston, Texas, USA.
(4)Verna &Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, Houston, Texas, USA. |
Which are the bioinformatics tools for gene structure prediction?
| The in silico prediction of the complete structure of genes is one of the main challenges of bioinformatics. A critical part in the gene structure prediction is to identify the boundaries between exons and introns (i.e. splice sites) in the coding region. Several advanced bioinformatics tools have been developed for the precise delineation of a given gene structure: WPSS, SCGPred, TICO, GLIMMER, MetWAMer, WebScipio, GeneSeqer, SplicePredictor, DGSplicer, Transcript Assembly Program (TAP), GeneBuilder, SeqHelp, HSPL, RNASPL, HEXON, CDSB, HBR, FGENE and FGENEH for human genes. | Here we describe a tool to analyze molecular sequences utilizing the internet
and existing computational resources for molecular biology. The computer program
SeqHelp organizes information from database searches, gene structure prediction,
and other information to generate multiply aligned, hypertext-linked reports to
allow for fast analysis of molecular sequences. The efficient and economical
strategy in this program can be employed to study molecular sequences for gene
cloning, mutation analysis, and identical sequence search projects. MOTIVATION: Prediction of gene structure in newly sequenced DNA becomes very
important in large genome sequencing projects. This problem is complicated due
to the exon-intron structure of eukaryotic genes and because gene expression is
regulated by many different short nucleotide domains. In order to be able to
analyse the full gene structure in different organisms, it is necessary to
combine information about potential functional signals (promoter region, splice
sites, start and stop codons, 3' untranslated region) together with the
statistical properties of coding sequences (coding potential), information about
homologous proteins, ESTs and repeated elements.
RESULTS: We have developed the GeneBuilder system which is based on prediction
of functional signals and coding regions by different approaches in combination
with similarity searches in proteins and EST databases. The potential gene
structure models are obtained by using a dynamic programming method. The program
permits the use of several parameters for gene structure prediction and
refinement. During gene model construction, selecting different exon homology
levels with a protein sequence selected from a list of homologous proteins can
improve the accuracy of the gene structure prediction. In the case of low
homology, GeneBuilder is still able to predict the gene structure. The
GeneBuilder system has been tested by using the standard set (Burset and Guigo,
Genomics, 34, 353-367, 1996) and the performances are: 0.89 sensitivity and 0.91
specificity at the nucleotide level. The total correlation coefficient is 0.88.
AVAILABILITY: The GeneBuilder system is implemented as a part of the WebGene a
the URL: http://www.itba.mi. cnr.it/webgene and TRADAT (TRAncription Database
and Analysis Tools) launcher URL: http://www.itba.mi.cnr.it/tradat. Gene identification in genomic DNA from eukaryotes is complicated by the vast
combinatorial possibilities of potential exon assemblies. If the gene encodes a
protein that is closely related to known proteins, gene identification is aided
by matching similarity of potential translation products to those target
proteins. The genomic DNA and protein sequences can be aligned directly by
scoring the implied residues of in-frame nucleotide triplets against the protein
residues in conventional ways, while allowing for long gaps in the alignment
corresponding to introns in the genomic DNA. We describe a novel method for such
spliced alignment. The method derives an optimal alignment based on scoring for
both sequence similarity of the predicted gene product to the protein sequence
and intrinsic splice site strength of the predicted introns. Application of the
method to a representative set of 50 known genes from Arabidopsis thaliana
showed significant improvement in prediction accuracy compared to previous
spliced alignment methods. The method is also more accurate than ab initio gene
prediction methods, provided sufficiently close target proteins are available.
In view of the fast growth of public sequence repositories, we argue that close
targets will be available for the majority of novel genes, making spliced
alignment an excellent practical tool for high-throughput automated genome
annotation. With the availability of a nearly complete sequence of the human genome,
aligning expressed sequence tags (EST) to the genomic sequence has become a
practical and powerful strategy for gene prediction. Elucidating gene structure
is a complex problem requiring the identification of splice junctions, gene
boundaries, and alternative splicing variants. We have developed a software
tool, Transcript Assembly Program (TAP), to delineate gene structures using
genomically aligned EST sequences. TAP assembles the joint gene structure of the
entire genomic region from individual splice junction pairs, using a novel
algorithm that uses the EST-encoded connectivity and redundancy information to
sort out the complex alternative splicing patterns. A method called
polyadenylation site scan (PASS) has been developed to detect poly-A sites in
the genome. TAP uses these predictions to identify gene boundaries by segmenting
the joint gene structure at polyadenylated terminal exons. Reconstructing 1007
known transcripts, TAP scored a sensitivity (Sn) of 60% and a specificity (Sp)
of 92% at the exon level. The gene boundary identification process was found to
be accurate 78% of the time. also reports alternative splicing patterns in EST
alignments. An analysis of alternative splicing in 1124 genic regions suggested
that more than half of human genes undergo alternative splicing. Surprisingly,
we saw an absolute majority of the detected alternative splicing events affect
the coding region. Furthermore, the evolutionary conservation of alternative
splicing between human and mouse was analyzed using an EST-based approach. (See
http://stl.wustl.edu/~zkan/TAP/) MOTIVATION: Accurate gene structure annotation is a challenging computational
problem in genomics. The best results are achieved with spliced alignment of
full-length cDNAs or multiple expressed sequence tags (ESTs) with sufficient
overlap to cover the entire gene. For most species, cDNA and EST collections are
far from comprehensive. We sought to overcome this bottleneck by exploring the
possibility of using combined EST resources from fairly diverged species that
still share a common gene space. Previous spliced alignment tools were found
inadequate for this task because they rely on very high sequence similarity
between the ESTs and the genomic DNA.
RESULTS: We have developed a computer program, GeneSeqer, which is capable of
aligning thousands of ESTs with a long genomic sequence in a reasonable amount
of time. The algorithm is uniquely designed to tolerate a high percentage of
mismatches and insertions or deletions in the EST relative to the genomic
template. This feature allows use of non-cognate ESTs for gene structure
prediction, including ESTs derived from duplicated genes and homologous genes
from related species. The increased gene prediction sensitivity results in part
from novel splice site prediction models that are also available as a
stand-alone splice site prediction tool. We assessed GeneSeqer performance
relative to a standard Arabidopsis thaliana gene set and demonstrate its utility
for plant genome annotation. In particular, we propose that this method provides
a timely tool for the annotation of the rice genome, using abundant ESTs from
other cereals and plants.
AVAILABILITY: The source code is available for download at
http://bioinformatics.iastate.edu/bioinformatics2go/gs/download.html. Web
servers for Arabidopsis and other plant species are accessible at
http://www.plantgdb.org/cgi-bin/AtGeneSeqer.cgi and
http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi, respectively. For non-plant
species, use http://bioinformatics.iastate.edu/cgi-bin/gs.cgi. The splice site
prediction tool (SplicePredictor) is distributed with the GeneSeqer code. A
SplicePredictor web server is available at
http://bioinformatics.iastate.edu/cgi-bin/sp.cgi MOTIVATION: Owing to the complete sequencing of human and many other genomes,
huge amounts of DNA sequence data have been accumulated. In bioinformatics, an
important issue is how to predict the complete structure of genes from the
genomic DNA sequence, especially the human genome. A crucial part in the gene
structure prediction is to determine the precise exon-intron boundaries, i.e.
the splice sites, in the coding region.
RESULTS: We have developed a dependency graph model to fully capture the
intrinsic interdependency between base positions in a splice site. The
establishment of dependency between two position is based on a chi2-test from
known sample data. To facilitate statistical inference, we have expanded the
dependency graph (which is usually a graph with cycles that make probabilistic
reasoning very difficult, if not impossible) into a Bayesian network (which is a
directed acyclic graph that facilitates statistical reasoning). When compared
with the existing models such as weight matrix model, weight array model,
maximal dependence decomposition, Cai et al.'s tree model as well as the
less-studied second-order and third-order Markov chain models, the expanded
Bayesian networks from our dependency graph models perform the best in nearly
all the cases studied.
AVAILABILITY: Software (a program called DGSplicer) and datasets used are
available at http://csrl.ee.nthu.edu.tw/bioinf/
CONTACT: [email protected]. Genomic projects heavily depend on genome annotations and are limited by the
current deficiencies in the published predictions of gene structure and
function. It follows that, improved annotation will allow better data mining of
genomes, and more secure planning and design of experiments. The purpose of the
GeneFarm project is to obtain homogeneous, reliable, documented and traceable
annotations for Arabidopsis nuclear genes and gene products, and to enter them
into an added-value database. This re-annotation project is being performed
exhaustively on every member of each gene family. Performing a family-wide
annotation makes the task easier and more efficient than a gene-by-gene approach
since many features obtained for one gene can be extrapolated to some or all the
other genes of a family. A complete annotation procedure based on the most
efficient prediction tools available is being used by 16 partner laboratories,
each contributing annotated families from its field of expertise. A database,
named GeneFarm, and an associated user-friendly interface to query the
annotations have been developed. More than 3000 genes distributed over 300
families have been annotated and are available at
http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with
the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data
into the protein knowledgebase Swiss-Prot. BACKGROUND: The moss Physcomitrella patens is an emerging plant model system due
to its high rate of homologous recombination, haploidy, simple body plan,
physiological properties as well as phylogenetic position. Available EST data
was clustered and assembled, and provided the basis for a genome-wide analysis
of protein encoding genes.
RESULTS: We have clustered and assembled Physcomitrella patens EST and CDS data
in order to represent the transcriptome of this non-seed plant. Clustering of
the publicly available data and subsequent prediction resulted in a total of
19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have
a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts
are not present in seed plants but can be found in other kingdoms. These
potential "retained genes" might have been lost during seed plant evolution.
Functional annotation of these genes reveals unequal distribution among
taxonomic groups and intriguing putative functions such as cytotoxicity and
nucleic acid repair. Whereas introns in the moss are larger on average than in
the seed plant Arabidopsis thaliana, position and amount of introns are
approximately the same. Contrary to Arabidopsis, where CDS contain on average
44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss
orthologs of Arabidopsis genes show a significant drift of codon fraction usage,
towards the seed plant. While averaged codon bias is the same in Physcomitrella
and Arabidopsis, the distribution pattern is different, with 15% of moss genes
being unbiased. Species-specific, sensitive and selective splice site prediction
for Physcomitrella has been developed using a dataset of 368 donor and acceptor
sites, utilizing a support vector machine. The prediction accuracy is better
than those achieved with tools trained on Arabidopsis data.
CONCLUSION: Analysis of the moss transcriptome displays differences in gene
structure, codon and splice site usage in comparison with the seed plant
Arabidopsis. Putative retained genes exhibit possible functions that might
explain the peculiar physiological properties of mosses. Both the transcriptome
representation (including a BLAST and retrieval service) and splice site
prediction have been made available on http://www.cosmoss.org, setting the basis
for assembly and annotation of the Physcomitrella genome, of which draft shotgun
sequences will become available in 2005. MOTIVATION: The vast majority of introns in protein-coding genes of higher
eukaryotes have a GT dinucleotide at their 5'-terminus and an AG dinucleotide at
their 3' end. About 1-2% of introns are non-canonical, with the most abundant
subtype of non-canonical introns being characterized by GC and AG dinucleotides
at their 5'- and 3'-termini, respectively. Most current gene prediction
software, whether based on ab initio or spliced alignment approaches, does not
include explicit models for non-canonical introns or may exclude their
prediction altogether. With present amounts of genome and transcript data, it is
now possible to apply statistical methodology to non-canonical splice site
prediction. We pursued one such approach and describe the training and
implementation of GC-donor splice site models for Arabidopsis and rice, with the
goal of exploring whether specific modeling of non-canonical introns can enhance
gene structure prediction accuracy.
RESULTS: Our results indicate that the incorporation of non-canonical splice
site models yields dramatic improvements in annotating genes containing GC-AG
and AT-AC non-canonical introns. Comparison of models shows differences between
monocot and dicot species, but also suggests GC intron-specific biases
independent of taxonomic clade. We also present evidence that GC-AG introns
occur preferentially in genes with atypically high exon counts.
AVAILABILITY: Source code for the updated versions of GeneSeqer and
SplicePredictor (distributed with the GeneSeqer code) isavailable at
http://bioinformatics.iastate.edu/bioinformatics2go/gs/download.html. Web
servers for Arabidopsis, rice and other plant species are accessible at
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/AtGDBgs.cgi,
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/OsGDBgs.cgi and
http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/PlantGDBgs.cgi, respectively. A
SplicePredictor web server is available at
http://bioinformatics.iastate.edu/cgi-bin/sp.cgi. Software to generate training
data and parameterizations for Bayesian splice site models is available at
http://gremlin1.gdcb.iastate.edu/~volker/SB05B/BSSM4GSQ/ The frequency distribution of mutation-induced aberrant 3' splice sites (3'ss)
in exons and introns is more complex than for 5' splice sites, largely owing to
sequence constraints upstream of intron/exon boundaries. As a result, prediction
of their localization remains a challenging task. Here, nucleotide sequences of
previously reported 218 aberrant 3'ss activated by disease-causing mutations in
131 human genes were compared with their authentic counterparts using currently
available splice site prediction tools. Each tested algorithm distinguished
authentic 3'ss from cryptic sites more effectively than from de novo sites. The
best discrimination between aberrant and authentic 3'ss was achieved by the
maximum entropy model. Almost one half of aberrant 3'ss was activated by
AG-creating mutations and approximately 95% of the newly created AGs were
selected in vivo. The overall nucleotide structure upstream of aberrant 3'ss was
characterized by higher purine content than for authentic sites, particularly in
position -3, that may be compensated by more stringent requirements for positive
and negative nucleotide signatures centred around position -11. A newly
developed online database of aberrant 3'ss will facilitate identification of
splicing mutations in a gene or phenotype of interest and future optimization of
splice site prediction tools. BACKGROUND: Obtaining the gene structure for a given protein encoding gene is an
important step in many analyses. A software suited for this task should be
readily accessible, accurate, easy to handle and should provide the user with a
coherent representation of the most probable gene structure. It should be
rigorous enough to optimise features on the level of single bases and at the
same time flexible enough to allow for cross-species searches.
RESULTS: WebScipio, a web interface to the Scipio software, allows a user to
obtain the corresponding coding sequence structure of a here given a query
protein sequence that belongs to an already assembled eukaryotic genome. The
resulting gene structure is presented in various human readable formats like a
schematic representation, and a detailed alignment of the query and the target
sequence highlighting any discrepancies. WebScipio can also be used to identify
and characterise the gene structures of homologs in related organisms. In
addition, it offers a web service for integration with other programs.
CONCLUSION: WebScipio is a tool that allows users to get a high-quality gene
structure prediction from a protein query. It offers more than 250 eukaryotic
genomes that can be searched and produces predictions that are close to what can
be achieved by manual annotation, for in-species and cross-species searches
alike. WebScipio is freely accessible at http://www.webscipio.org. BACKGROUND: Translation initiation site (TIS) identification is an important
aspect of the gene annotation process, requisite for the accurate delineation of
protein sequences from transcript data. We have developed the MetWAMer package
for TIS prediction in eukaryotic open reading frames of non-viral origin.
MetWAMer can be used as a stand-alone, third-party tool for post-processing gene
structure annotations generated by external computational programs and/or
pipelines, or directly integrated into gene structure prediction software
implementations.
RESULTS: MetWAMer currently implements five distinct methods for TIS prediction,
the most accurate of which is a routine that combines weighted, signal-based
translation initiation site scores and the contrast in coding potential of
sequences flanking TISs using a perceptron. Also, our program implements
clustering capabilities through use of the k-medoids algorithm, thereby enabling
cluster-specific TIS parameter utilization. In practice, our static weight array
matrix-based indexing method for parameter set lookup can be used with good
results in data sets exhibiting moderate levels of 5'-complete coverage.
CONCLUSION: We demonstrate that improvements in statistically-based models for
TIS prediction can be achieved by taking the class of each potential
start-methionine into account pending certain testing conditions, and that our
perceptron-based model is suitable for the TIS identification task. MetWAMer
represents a well-documented, extensible, and freely available software system
that can be readily re-trained for differing target applications and/or extended
with existing and novel TIS prediction methods, to support further research
efforts in this area. Predicting protein-coding genes still remains a significant challenge. Although
a variety of computational programs that use commonly machine learning methods
have emerged, the accuracy of predictions remains a low level when implementing
in large genomic sequences. Moreover, computational gene finding in newly
sequenced genomes is especially a difficult task due to the absence of a
training set of abundant validated genes. Here we present a new gene-finding
program, SCGPred, to improve the accuracy of prediction by combining multiple
sources of evidence. SCGPred can perform both supervised method in previously
well-studied genomes and unsupervised one in novel genomes. By testing with
datasets composed of large DNA sequences from human and a novel genome of
Ustilago maydi, SCG-Pred gains a significant improvement in comparison to the
popular ab initio gene predictors. We also demonstrate that SCGPred can
significantly improve prediction in novel genomes by combining several foreign
gene finders with similarity alignments, which is superior to other unsupervised
methods. Therefore, SCG-Pred can serve as an alternative gene-finding tool for
newly sequenced eukaryotic genomes. The program is freely available at
http://bio.scu.edu.cn/SCGPred/. In the bioinformatics field, many computer algorithmic and data mining
technologies have been developed for gene prediction, protein-protein
interaction analysis, sequence analysis, and protein folding predictions, to
name a few. This kind of research has branched off from the genomics field,
creating the transcriptomics, proteomics, metabolomics, and glycomics research
areas in the postgenomic age. In the glycomics field, given the complexity of
glycan structures with their branches of monosaccharides in various
conformations, new data mining and algorithmic methods have been developed in an
attempt to gain a better understanding of glycans. However, these methods have
not all been implemented as tools such that the glycobiology community may
utilize them in their research. Thus, we have developed RINGS (Resource for
INformatics of Glycomes at Soka) as a freely available Web resource for
glycobiologists to analyze their data using the latest data mining and
algorithmic techniques. It provides a number of tools including a 2D glycan
drawing and querying interface called DrawRINGS, a Glycan Pathway Predictor
(GPP) tool for dynamically computing the N-glycan biosynthesis pathway from a
given glycan structure, and data mining tools Glycan Miner Tool and Profile
PSTMM. These tools and other utilities provided by RINGS will be described. The
URL for RINGS is http://rings.t.soka.ac.jp/. The prediction of the complete structure of genes is one of the very important
tasks of bioinformatics, especially in eukaryotes. A crucial part in the gene
structure prediction is to determine the splice sites in the coding region.
Identification of splice sites depends on the precise recognition of the
boundaries between exons and introns of a given DNA sequence. This problem can
be formulated as a classification of sequence elements into 'exon-intron' (EI),
'intron-exon' (IE) or 'None' (N) boundary classes. In this study we propose a
new Weighted Position Specific Scoring Method (WPSSM) to recognize splice sites
which uses a position-specific scoring matrix constructed by nucleotide base
frequencies. A genetic algorithm is used in order to tune the weight and
threshold parameters of the positions on. This method consists of two phases:
learning phase and identification phase. The proposed WPSS method poses
efficient results compared with the performance of many methods proposed in the
literature. Computational experiments are performed on the DNA sequence datasets
from 'UCI Repository of machine learning databases'. |
Which is the major phytoalexin in alfalfa (Medicago sativa L.)? | The major phytoalexin in alfalfa (Medicago sativa L.) is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Medicarpin is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. | The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR,
11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in
medicarpin biosynthesis, is responsible for introducing one of two chiral
centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1)
which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin
stereospecifically to (3R)-vestitone, as would be predicted for IFR from
alfalfa. The calculated molecular weight of the polypeptide (35,400) derived
from the 954 bp open reading frame compares favorably to estimated Mrs
determined for IFR proteins purified from other legumes. The transcript (1.4 kb)
is highly induced in elicited alfalfa cell cultures. The kinetics of induction
are consistent with the appearance of IFR activity, the accumulation of
medicarpin, and the observed induction of other enzymes in the pathway. Low
levels of IFR transcripts were found in healthy plant parts (roots and nodules)
which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded
by a single gene in alfalfa. The cloning of IFR opens up the possibility of
genetic manipulation of phytoalexin biosynthesis in alfalfa by altering
isoflavonoid stereochemistry. Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the
isoflavonoid branch of phenylpropanoid metabolism. One of the final steps of
medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol,
is catalyzed by vestitone reductase. A 1245-bp cDNA clone which encodes
vestitone reductase was identified utilizing internal amino acid sequence of
purified vestitone reductase. When expressed in Escherichia coli, the cloned
enzyme exhibits strict substrate stereospecificity for (3R)-vestitone, as was
observed for vestitone reductase purified from alfalfa. The calculated molecular
weight of the protein (35,918) is similar to that of purified vestitone
reductase from alfalfa (38 kDa by SDS-PAGE). The levels of vestitone reductase
transcript (1.35 kb) greatly increase within 2 h of elicitor addition to alfalfa
cell suspension cultures, preceding the rapid increases in vestitione reductase
enzyme activity and medicarpin biosynthesis. In healthy alfalfa plants, the
highest levels of transcripts were detected in roots and root nodules,
consistent with the synthesis of medicarpin and its conjugate in these tissues.
The cloning of the vestitone reductase gene provides a specific tool for the
study and manipulation of pterocarpan biosynthesis in legumes. Medicarpin, the major phytoalexin in alfalfa, is synthesized via the
isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9
position of medicarpin is generally accepted to arise via the methylation of the
4' position (B-ring) of daidzein. Surprisingly, the
isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes
involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of
daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing
internal amino acid sequence information from purified alfalfa IOMT, we have
isolated three full-length IOMT cDNA clones. A search of the protein databases
revealed sequence similarities to O-methyltransferases from various sources. The
highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain
3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT
expressed in Escherichia coli was similar to that of purified IOMT from alfalfa
cell cultures (41 kDa by SDS-PAGE). The recombit enzyme catalyzed the
O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also
methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple
IOMT genes, and closely related sequences are present in the genomes of chickpea
and cowpea, species that also produce B-ring methylated isoflavonoids in vivo.
Northern blot analysis indicated that IOMT transcripts are rapidly induced
following elicitation, prior to the increase in IOMT activity and medicarpin
accumulation. The possible role of the isoflavone 7-OMT in the synthesis of
formononetin in vivo is discussed. Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high
concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum
within 24 hours after exposure to an elicitor preparation from cell walls of the
phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by
increases in the extractable activities of the isoflavonoid biosynthetic enzymes
l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme
A-ligase, chalcone synthase, chalcone isomerase, and isoflavone
O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine
ammonia-lyase activity but did not induce medicarpin accumulation, whereas
reduced glutathione was totally inactive as an elicitor in this system. The
fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes,
caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did
not induce appreciable increases in the activities of the hydrolytic enzymes
chitinase and 1,3-beta-d-glucanase. The results are discussed in relation to the
activation of isoflavonoid biosynthesis in other legumes and the development of
the alfalfa cell culture system as a model for studying the enzymology and
molecular biology of plant defense expression. The isoflavonoid conjugates medicarpin-3-O-glucoside-6''-O-malonate (MGM),
afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6''-O-malonate (AGM)
were isolated and characterized from cell suspension cultures of alfalfa
(Medicago sativa L.), where they were the major constitutive secondary
metabolites. They were also found in alfalfa roots but not in other parts of the
plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured
cells treated with elicitor from Colletotrichum lindemuthianum, and this was
subsequently accompanied by an increase in the levels of MGM. In contrast, net
accumulation of afrormosin conjugates was not affected by elicitor treatment.
Labeling studies with [(14)C]phenylalanine indicated that afrormosin conjugates
were the major de novo synthesized isoflavonoid products in unelicited cells.
During elicitation, [(14)C]phenylalanine was incorporated predomitly into
medicarpin, although a significant proportion of the newly synthesized
medicarpin was also conjugated. Treatment of (14)C-labeled, elicited cells with
l-alpha-aminooxy-beta-phenylpropionic acid, a potent inhibitor of PAL activity
in vivo, resulted in the initial appearance of labeled medicarpin of very low
specific activity, suggesting that the phytoalexin could be released from a
preformed conjugate under these conditions. Our data draw attention to the
involvement of isoflavone hydroxylases during the constitutive and
elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa
cell cultures. In order to determine why the activated methyl cycle is up-regulated in plants
undergoing defence responses to fungal pathogens we have monitored the
utilisation of methyl groups derived from methionine in cell-suspension cultures
of alfalfa (Medicago sativa L.) treated for various times with fungal elicitor,
by carrying out a parallel labelling study with [(35)S]methionine and
[methyl-(3)H]methionine. The distribution of the two radiolabels among the
medium, soluble cellular components and cell wall was then determined. In the
absence of elicitor the utilisation of the two radiolabels was similar. However,
in the presence of the elicitor the total incorporation of radioactivity from
[methyl-(3)H]methionine into metabolites was far greater than from
[(35)S]methionine, indicating that the methyl label had been utilised in
methylation reactions. Elicitor treatment resulted in up to a sixfold increase
in the use of (3)H-methyl groups in the methylation of hydrophobic metabolites.
In the period 0-24 h after elicitor treatment, increased methylation was
directed largely into the synthesis of the isoflavonoid phytoalexin medicarpin
and related metabolites. Newly synthesized phytoalexins were exported into the
medium, while a significant proportion of the medicarpin accumulating in the
cell in the early stages of elicitation was derived from the hydrolysis of its
respective conjugate. Elicitor treatment also modified the incorporation of
(3)H-methyl groups into the cell wall. Between 0 and 24 h after elicitor
treatment the methylation of pectin in the cell wall declined. After 24 h,
pectin methylation recovered and was associated with an increase in the
methylation of other wall-bound polysaccharide components. Since no other major
metabolic sink for the increased methylation was determined we conclude that the
increased activity of the activated methyl cycle during defence interactions in
alfalfa is required to support phytoalexin synthesis and cell wall
modifications. |
What is the correlation between SPARC expression and growth inhibition in human cancer? | Secreted protein acidic and rich in cysteine (SPARC) is a multi-faceted protein-modulating cell-cell and cell-matrix interactions. SPARC seems to act as a tumour suppressor, as it has been found that loss of SPARC accelerates the development of certain types of cancer, whereas its expression impairs tumor growth. However it has also been associated with a aggressive phenotypes of some tumours. The role of SPARC may depend on its subcellular localization. | SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a
matricellular protein with multiple effects on cell behaviour. In vitro, its
major known functions are anti-adhesive and anti-proliferative, and it is
associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in
many cancers, including breast cancer, and the effects of SPARC seem to be cell
type-specific. To study the effects of SPARC on breast cancer, we transfected
SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On
inducible system. By western analysis, we found low background levels in the
MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC
protein expression after doxycycline treatment in SPARC transfected clones X5,
X21, X24 and X75. Induction of SPARC expression did not affect cell morphology
or adhesiveness to collagens type I and IV, but it slowed the rate of
proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed
the progression to S phase. Doxycycline induction of SPARC also slowed the rate
of monolayer wound closure in the cultured wound healing assay. Thymidine
inhibition of proliferation abrogated this effect, confirming that it was due to
anti-proliferation rather than inhibition of migration. Consistent with this, we
were unable to detect any differences in migration and Matrigel outgrowth
analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory
to human breast cancer cell proliferation, and does not stimulate migration, in
contrast to its stimulatory effects reported for melanoma (proliferation and
migration) and glioma (migration) cells. Similar growth repression by SPARC has
been reported for ovarian cancer cells, and this may be a common feature among
carcinomas. Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular
glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC
has been implicated as a tumor suppressor in humans, its function in normal or
maligt hematopoiesis has not previously been studied. We found that the
leukemic cells of AML patients with MLL gene rearrangements express low to
undetectable amounts of SPARC whereas normal hematopoietic progenitors and most
AML patients express this gene. SPARC RNA and protein levels were also low or
undetectable in AML cell lines with MLL translocations. Consistent with its
tumor suppressive effects in various solid tumor models, exogenous SPARC protein
selectively reduced the growth of cell lines with MLL rearrangements by
inhibiting cell cycle progression from G1 to S phase. The lack of SPARC
expression in MLL-rearranged cell lines was associated with dense promoter
methylation. However, we found no evidence of methylation-based silencing of
SPARC in primary patient samples. Our results suggest that low or absent SPARC
expression is a consistent feature of AML cells with MLL rearrangements and that
SPARC may function as a tumor suppressor in this subset of patients. A potential
role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further
investigation. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third
leading cause of cancer-related death worldwide. Current treatments are
extremely disappointing. SPARC (Secreted protein, acidic and rich in cysteine)
is a matricellular glycoprotein with differential expression in several tumors,
including HCC, which significance remains unclear. We infected HCC cells (HepG2,
Hep3B and Huh7) with an adenovirus expressing SPARC (AdsSPARC) to examine the
role of SPARC expression on HCC cells and its effect on tumor aggressiveness.
The in vitro HCC cells substrate-dependent proliferation and cell cycle profile
were unaffected; however, SPARC overexpression reduced HCC proliferation when
cells were grown in spheroids. A mild induction of cellular apoptosis was
observed upon SPARC overexpression. SPARC overexpression resulted in spheroid
growth inhibition in vitro while no effects were found when recombit SPARC
was exogenously applied. Moreover, the clonogenic and migratory capabilities
were largely decreased in SPARC-overexpressing HCC cells, altogether suggesting
a less aggressive HCC cell phenotype. Consistently, AdsSPARC-transduced cells
showed increased E-cadherin expression and a concomitant decrease in N-cadherin
expression. Furthermore, SPARC overexpression was found to reduce HCC cell
viability in response to 5-FU-based chemotherapy in vitro, partially through
induction of apoptosis. In vivo experiments revealed that SPARC overexpression
in HCC cells inhibited their tumorigenic capacity and increased animal survival
through a mechanism that partially involves host macrophages. Our data suggest
that SPARC overexpression in HCC cells results in a reduced tumorigenicity
partially through the induction of mesenchymal-to-epithelial transition (MET).
These evidences point to SPARC as a novel target for HCC treatment. BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC), a
matricellular glycoprotein, modulates cellular interaction with the
extracellular matrix and is capable of altering the growth of various cancers.
We therefore sought to determine the effect of SPARC expression on
medulloblastoma tumour growth and angiogenesis.
METHODS: To this extent, we selected three SPARC full-length cDNA overexpressed
clones (Daoy-SP). Consequences of SPARC overexpression were studied in terms of
cell growth, angiogenesis using co-culture assay in vitro, dorsal skin-fold
chamber assay in vivo, PCR Array for human angiogenic genes, as well as western
blotting for angiogenic molecules and tumour growth, in an orthotopic tumour
model.
RESULTS: The SPARC protein and mRNA levels were increased by approximately
three-fold in Daoy-SP cells compared with parental (Daoy-P) and vector (Daoy-EV)
controls. Daoy-SP clones reduced tumour cell-induced angiogenesis in vitro and
in vivo, and formed small tumours with fewer blood vessels when compared with
controls. Matrix metalloprotease-9 (MMP-9) and vascular endothelial growth
factor (VEGF) expression were decreased in Daoy-SP clones. Further, inhibition
of MMP-9 expression caused SPARC-mediated inhibition of angiogenesis and tumour
growth as MMP-9 rescued SPARC-mediated anti-angiogenic effect in vitro and
tumour growth inhibition in vivo.
CONCLUSION: Overexpression of SPARC decreases angiogenesis, which leads to
decreased tumour growth. Further, the role of MMP-9 could be attributed to the
anti-angiogenic effect of SPARC. The overexpression of secreted protein acidic and rich in cysteine (SPARC) is
associated with increased aggressiveness and poor prognosis in maligt
melanoma. Its roles and underlying mechanisms on melanoma cell growth, however,
are not fully clarified. To validate the potential of SPARC as a therapeutic
target, we examined the effect of the knockdown of SPARC with SPARC-specific
siRNA on the growth of human melanoma cell lines. SPARC siRNAs exerted a potent
knockdown effect. Silencing of SPARC resulted in growth inhibition with G(1)
arrest accompanied by accumulation of p21, a G(1) cyclin-dependent kinase
inhibitor, in MeWo and CRL1579 cells. Moreover, the induction of p53 was
observed in MeWo cells, but not in CRL1579 cells. Conditioned media containing
SPARC from MeWo cells could not restore the growth of SPARC-silenced MeWo cells.
This result suggests that intracellular SPARC, but not secreted SPARC, is
involved in cell proliferation. In addition, silencing of SPARC induced
apoptosis in MeWo and CRL1579 cells. Furthermore, when MeWo cells in which SPARC
expression was transiently knocked down by SPARC siRNA were implanted in nude
mice, the tumor growth was suppressed. Our findings suggest that SPARC
contributes to cell growth and could be a potential target molecule for melanoma
therapy. Krüppel-Like Factor 4 (KLF4) functions as a tumor suppressor in some cancers,
but its molecular mechanism is not clear. Our recent study also showed that the
expression of KLF4 is dramatically reduced in primary lung cancer tissues. To
investigate the possible role of KLF4 in lung cancer, we stably transfected KLF4
into cells from lung cancer cell lines H322 and A549 to determine the cells'
invasion ability. Our results showed that ectopic expression of KLF4 extensively
suppressed lung cancer cell invasion in Matrigel. This effect was independent of
KLF4-mediated p21 up-regulation because ectopic expression of p21 had minimal
effect on cell invasion. Our analysis of the expression of 12 genes associated
with cell invasion in parental, vector-transfected, and KLF4-transfected cells
showed that ectopic expression of KLF4 resulted in extensively repressed
expression of secreted protein acidic and rich in cysteine (SPARC), an
extracellular matrix protein that plays a role in tumor development and
metastasis. Knockdown of SPARC expression in H322 and A549 cells led to
suppression of cell invasion, comparable to that observed in KLF4-transfected
cells. Moreover, retrovirus-mediated restoration of SPARC expression in
KLF4-transfected cells abrogated KLF4-induced anti-invasion activity. Together,
our results indicate that KLF4 inhibits lung cancer cell invasion by suppressing
SPARC gene expression. We investigated whether Ukrain modulates the maligt phenotype of clear cell
renal cell carcinoma (ccRCC) cells Caki-1, Caki-2, and ACHN treated with four
doses (5, 10, 20, and 40 μmol/l) for 24 and 48 h. The epithelial-to-mesenchymal
transition markers E-cadherin, β-catenin, and vimentin were analyzed by
immunofluorescence as well as actin and tubulin; matrix metalloproteinase-2 and
matrix metalloproteinase-9 activity was analyzed by SDS-zymography,
intracellular and secreted SPARC levels by western blot, and cell cycle by flow
cytometry. Ukrain did not induce E-cadherin/β-catenin immunoreactivity at the
cell-cell boundary, although it determined the actin cortical expression in
Caki-2 and ACHN, and did not affect vimentin organization; however, in some
Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent
with its downregulation. Matrix metalloproteinase-2 and matrix
metalloproteinase-9 activity was significantly downregulated 48 h after 20
μmol/l Ukrain administration. At this time point, Ukrain significantly decreased
migration and invasion, and downregulated SPARC levels in cell supernatants at
all doses in Caki-2, and at 20 μmol/l in Caki-1 and ACHN cells. Concomitantly,
SPARC was upregulated in all ccRCC cells, suggesting that Ukrain could also
affect cell proliferation by cell cycle inhibition, as supported by the cell
cycle analysis, as SPARC also acts as a cell cycle inhibitor. Our results
suggest that Ukrain may switch the epithelial-to-mesenchymal transition-related
phenotype of ccRCC cells, and targets the two major aspects involved in RCC
progression, such as tumor invasion/microenvironment remodeling and cell
proliferation. Dynamic cell interaction with ECM components has profound influence in cancer
progression. SPARC is a component of the ECM, impairs the proliferation of
different cell types and modulates tumor cell aggressive features. We previously
reported that SPARC expression significantly impairs medulloblastoma tumor
growth in vivo. In this study, we demonstrate that expression of SPARC inhibits
medulloblastoma cell proliferation. MTT assay indicated a dose-dependent
reduction in tumor cell proliferation in adenoviral mediated expression of SPARC
full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis
showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell
cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC
induced G2/M cell cycle arrest was mediated through inhibition of the
Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression.
Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705;
constitutively active STAT3 expression reversed SPARC induced G2/M arrest.
Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth
and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections
from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3
and increased immunoreactivity for p21 compared to tumor section from mice
treated with mock and Ad-DsRed. Taken together our studies further reveal that
STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells.
These new findings provide a molecular basis for the mechanistic understanding
of the effects of SPARC on medulloblastoma tumor cell proliferation. Epigenetic aberrations and a CpG island methylator phenotype are associated with
poor outcome in children with neuroblastoma (NB). Previously, we have shown that
valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, exerts antitumor
effects in an NB xenograft model. However, the underlying antitumor molecular
mechanisms are largely unknown. In this study, we examined the role of HDAC in
cell proliferation, cell cycle progression, gene expression patterns, and
epigenome in NB. Cell proliferation, cell cycle progression, caspase activity,
RNA and protein expression, quantitative methylation, and global DNA methylation
were examined in NBL-W-N and LA1-55n NB cell lines. Our studies showed that
inhibition of HDAC decreased NB proliferation, and induced caspase activity and
G1 growth arrest. Expression patterns of cancer-related genes were modulated by
VPA. The expression of THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 was
upregulated, and that of MYCN and TIG1 was downregulated. HDAC inhibition
decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC
increased acetylation of histone 4 and overall DNA methylation levels. Our
studies showed that inhibition of HDAC blocked cell proliferation and cell cycle
progression in relation to alteration in cancer-related genes, increased overall
DNA methylation, and decreased methylation of tumor suppressor genes. Further
studies examining the antitumor effects of VPA in NB are warranted. |
Which syndrome is associated with mutations in the LYST gene? | Mutations in LYST, a gene encoding a putative lysosomal trafficking protein, cause Chédiak-Higashi syndrome (CHS), an autosomal recessive disorder typically characterized by infantile-onset hemophagocytic syndrome and immunodeficiency, and oculocutaneous albinism. A small number of reports of rare, attenuated forms of CHS exist, with affected individuals exhibiting progressive neurodegenerative disease beginning in early adulthood with cognitive decline, parkinsonism, features of spinocerebellar degeneration, and peripheral neuropathy, as well as subtle pigmentary abnormalities and subclinical or absent immune dysfunction. | Vesicular transport to and from the lysosome and late endosome is defective in
patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS
and bg cells have giant, perinuclear vesicles with characteristics of late
endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and
bg lysosomes also exhibit compartmental missorting of proteins, such as
elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was
identified by direct complementary DNA selection from a yeast artificial
chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical
region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg
mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The
homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a
frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS
patient. Thus bg mice and human CHS patients have homologous disorders
associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal
prenylation motif and multiple potential phosphorylation sites. Lyst protein is
predicted to form extended helical domains, and has a region of sequence similar
to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating
cellular signal response coupling. During the recent cloning of the mouse Lyst gene we developed both a
high-resolution genetic map and a complete YAC and BAC contig of the Lyst
critical region on mouse Chromosome 13. We also report the mapping of the human
homologue of the mouse Lyst gene (LYST) to 1q43. These data are consistent with
LYST being the gene for the human Chediak-Higashi Syndrome and strengthen the
synteny relationship between MMU13 and human 1q43. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, also known as CD152) has
been shown to play a major role in the regulation of T cell activation. Its
membrane expression is highly regulated by endocytosis and trafficking through
the secretory lysosome pathway. Chediak-Higashi syndrome (CHS) is an inherited
disorder caused by mutations in the lysosomal trafficking regulator gene, LYST.
It results in defective membrane targeting of the proteins present in secretory
lysosomes, and it is associated with a variety of features, including a
lymphoproliferative syndrome with hemophagocytosis. The murine equivalent of
CHS, beige mice, present similar characteristics but do not develop the
lymphoproliferative syndrome. We show herein that CTLA-4 is present in enlarged,
abnormal vesicles in CHS T cells and is not properly expressed at the cell
surface after T cell activation, whereas its surface expression is not impaired.
It is therefore proposed that the defective surface expression of CTLA-4 by CHS
T cells is involved in the generation of lymphoproliferative disease. This
observation may provide insight into the role of CTLA-4 in humans. Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder in which
an immune deficiency occurs in association with pigmentation abnormalities. Most
patients who do not undergo bone marrow transplantation die of a
lymphoproliferative syndrome, though some patients with CHS have a relatively
milder clinical course of the disease. The large size of the LYST gene,
defective in CHS, has made it difficult to screen for mutations in a large
number of patients. Only 8 mutations have been identified so far, and all lead
to a truncated LYST protein. We conducted protein truncation tests on this gene
in 8 patients with CHS. Different LYST mutations were identified in all subjects
through this approach, strengthening the observation of a high frequency of
truncated LYST proteins as the genetic cause of CHS. Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene
encoding a protein named LYST in humans ("lysosomal trafficking regulator") or
Beige in mice. A prominent feature of this disease is the accumulation of
enlarged lysosome-related granules in a variety of cells. The genome of
Dictyostelium discoideum contains six genes encoding proteins that are related
to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA
(large volume sphere), results in profound defects in cytokinesis. To better
understand the function of this family of proteins in membrane trafficking, we
have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of
all these, only lvsA and lvsB mutants displayed interesting phenotypes in our
assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal
looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein
most similar to LYST/Beige, resulted in the formation of enlarged vesicles that
by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis,
phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also,
the rates of processing and the efficiency of targeting of lysosomal
alpha-mannosidase were normal, although lvsB mutants inefficiently retained
alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally,
results of pulse-chase experiments indicated that an increase in fusion rates
accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB
acts as a negative regulator of fusion. Our results support the notion that
LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis. BACKGROUND: Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency
disease characterized by giant lysosomes and impaired leukocyte degranulation.
CHS results from mutations in the lysosomal trafficking regulator (LYST) gene,
which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this
study was to identify proteins that interact with LYST as a first step in
understanding how LYST modulates lysosomal exocytosis.
MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding
domain of LYST, were used as baits to screen five human cDNA libraries by a
yeast two-hybrid method, modified to allow screening in the activation and the
binding domain, three selectable markers, and more stringent confirmation
procedures. Five of the interactions were confirmed by an in vitro binding
assay.
RESULTS: Twenty-one proteins that interact with LYST were identified in yeast
two-hybrid screens. Four interactions, confirmed directly, were with proteins
important in vesicular transport and signal transduction (the SNARE-complex
protein HRS, 14-3-3, and casein kinase II).
CONCLUSIONS: On the basis of protein interactions, LYST appears to function as
an adapter protein that may juxtapose proteins that mediate intracellular
membrane fusion reactions. The pathologic manifestations observed in CHS
patients and in mice with the homologous mutation beige suggest that
understanding the role of LYST may be relevant to the treatment of not only CHS
but also of diseases such as asthma, urticaria, and lupus, as well as to the
molecular dissection of the CHS-associated cancer predisposition. Two spontaneous mutant beige rats, with phenotypes resembling human Chediak-
Higashi syndrome (CHS), were found independently in two inbred strains. Both
beige mutations were identified to be recessive alleles in the Lyst locus on rat
chromosome 17 and the alleles were denoted Lyst(bg) and Lyst(bg-Kyo). As it is
almost impossible to discriminate these mutations phenotypically, we developed
an allele-specific genotyping method for the Lyst gene. The nested PCR
amplification was followed by restriction fragment length polymorphism (RFLP)
analysis. By this method, we could discriminate the mutant Lyst(bg),
Lyst(bg-Kyo) alleles, and the normal Lyst allele, easily and accurately. Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease
characterized by variable degrees of oculocutaneous albinism, recurrent
infections, and a mild bleeding tendency, with late neurologic dysfunction. Most
patients also undergo an accelerated phase of lymphohistiocytosis and die at an
early age unless they receive an allogeneic hematopoietic stem cell transplant
(SCT). Mutations in the CHS1 (LYST) gene result in CHS. Here, we describe an
adopted infant who is compound heterozygous for two novel CHS1 gene mutations,
both of which are predicted to result in truncated proteins. The two mutations
are a nonsense mutation (c.1540 C>T, CGA>TGA, R514X) in exon 5 and a one base
pair deletion (del c.9893T, F3298fsX3304) in exon 43, coding for part of the
CHS1 protein's BEACH domain. These two newly described mutations are expected to
give rise to a severe phenotype and, indeed, the patient had absolutely no
cytotoxicity by natural killer cells or cytotoxic lymphocytes prior to his
allogeneic SCT. The murine beige mutant phenotype and the human Chediak-Higashi syndrome are
caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene
and the human CHS gene, respectively. In this report we have analyzed a novel
murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU
mutation screen and named grey because of the grey coat color of affected mice.
The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive
fashion. Melanosomes of melanocytes associated with hair follicles and the
choroid layer of the eye, as well as melanosomes in the neural tube-derived
pigment epithelium of the retina, were larger and irregularly shaped in
homozygous mutants compared with those of wild-type controls. Secretory vesicles
in dermal mast cells of the mutant skin were enlarged as well. Test crosses with
beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes
(Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either
homozygous parent, demonstrating that the ENU mutation was an allele of the
murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in
Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation
and the loss of the following 77 amino acids encoded by exon 25 but leave the
C-terminal end of the protein intact. Analysis of the genomic Lyst locus around
exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C
transition point mutation. Western blot analysis suggests that the Lyst(bg-grey)
mutation causes instability of the LYST protein. Because the phenotype of
Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect
to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation
defines a critical region for the stability of the murine LYST protein. Disturbance of intracellular trafficking plays a major role in several
neurodegenerative disorders including Alzheimer or Parkinson's disease. The
Chediak-Higashi syndrome (CHS), a life-threatening autosomal recessive disease
with frequent mutations in the LYST gene, and its animal model, the beige mouse,
are both characterized by lysosomal defects with accumulation of giant
lysosomes. Clinically they manifest as hypopigmentation, abnormal bleeding and
increased susceptibility to infection with various degrees of involvement of the
nervous system. In the course of a recessive N-ethyl-N-nitrosurea (ENU)
mutagenesis screen, we identified the first murine missense mutation in the
lysosomal trafficking regulator gene (Lyst(Ing3618)) located at a highly
conserved position in the WD40 protein domain. Nearly all described human Lyst
alleles lead to protein truncation and fatal childhood CHS. Only four different
missense mutations have been reported in patients with adolescent or adult forms
of CHS involving the nervous system. Interestingly, the Lyst(Ing3618) model
presents with a predomit neurodegenerative phenotype with progressive
degeneration and loss of Purkinje cells and lacks severe impairment of the
immune system. Therefore, the Lyst(Ing3618 )allele could represent a new model
for adult CHS with neurological impairment. It could also provide an important
tool to elucidate the role of neuronal lysosomal trafficking in the
pathophysiology of neurodegeneration. PURPOSE OF REVIEW: Chediak-Higashi syndrome, a rare autosomal recessive
disorder, was described over 50 years ago. Patients show hypopigmentation,
recurrent infections, mild coagulation defects and varying neurologic problems.
Treatment is bone marrow transplant, which is effective in treating the
hematologic and immune defects, however the neurologic problems persist. The
CHS1/LYST gene was identified over 10 years ago and homologous CHS1/LYST genes
are present in all eukaryotes. This review will discuss the advances made in
understanding the clinical aspects of the syndrome and the function of
CHS1/LYST/Beige.
RECENT FINDINGS: Clinical reports of Chediak-Higashi syndrome have identified
mutations throughout the CHS1/LYST gene. The nature of the mutation can be a
predictor of the severity of the disease. Over the past decade the CHS1/LYST
family of proteins has been analyzed using model organisms, two-hybrid analysis,
overexpression phenotypes and domit negatives. These studies suggest that the
CHS1/LYST protein is involved in either vesicle fusion or fission.
SUMMARY: Although CHS is a rare disease, the Chediak-like family of proteins is
providing insight into the regulation of vesicle trafficking. Understanding the
basic mechanisms that govern vesicle trafficking will provide essential
information regarding how loss of CHS1/LYST affects hematologic, immunologic and
neurologic processes. Background. Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive
disorder characterized by oculocutaneous albinism, immunodeficiency,
coagulopathy and late-onset, progressive neurological dysfunction. It also has
an "accelerated phase" characterized by hemophagocytic lymphohistiocytosis
(HLH). The disease is caused by mutations in the CHS1/LYST gene located on
chromosome 1, which affects lysosome morphology and function. We report the case
of an African-American child with CHS in Case. This 16-month old
African-American girl presented with fever and lethargy. The proband had pale
skin compared to her parents, with light brown eyes, silvery hair and massive
hepatosplenomegaly. Her laboratory evaluation was remarkable for pancytopenia,
high serum ferritin and an elevated LDH. Bone marrow aspirate revealed large
inclusions in granulocytes and erythrophagocytosis consistent with HLH. Genetic
evaluation revealed two novel nonsense mutations in the CHS1 gene: c.3622C > T
(p.Q1208X) and c.11002G > T (p.E3668X). Conclusions. Our patient is one of the
few cases of CHS reported in the African American population. We identified 2
nonsense mutations in the CHS1 gene, the first mutation analysis published of an
African-American child with Chediak-Higashi Syndrome. These two mutations
predict a severe phenotype and thus identification of these mutations has an
important clinical significance in CHS. Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune disorder
characterized by uncontrolled lymphocyte- and macrophage-activation. The
resulting hypercytokinemia and cell infiltration of organs lead to the clinical
and laboratory features of HLH. Viral infections and other triggers can induce
both, inherited and acquired forms of HLH. Disease-causing mutations in the
genes encoding perforin (PRF1, FHL2), munc13-4 (UNC13D, FHL3), syntaxin 11
(STX11, FHL4), and munc18-2 (UNC18-2/STXBP2, FHL5) have been previously
identified in Familial Hemophagocyic Lymphohistiocytosis (FHL), whereas mutation
in RAB27A and LYST account for Griscelli syndome type 2 and Chediak-Higashi
syndrome, respectively. These genes all encode proteins which are involved in
the cytotoxic activity of lymphocytes. The inability of activated cytotoxic
cells to clear antigen-presenting targets results in sustained immune
stimulation, likely accounting for the unremitting polyclonal CD8 T-cell
activation and hyperimmune reaction which characterizes FHL. Treatment of HLH
consists of elimination of the trigger and immunosuppressive treatment in order
to induce remission from the uncontrolled inflammation. Allogeneic hematopoietic
stem cell transplantation can be indicated in the inherited forms of HLH. LYST is a large cytosolic protein that influences the biogenesis of
lysosome-related organelles, and mutation of the encoding gene, LYST, can cause
Chediak-Higashi syndrome. Recently, Lyst-mutant mice were recognized to also
exhibit an iris disease resembling exfoliation syndrome, a common cause of
glaucoma in humans. Here, Lyst-mutant iris phenotypes were used in a search for
genes that influence Lyst pathways. In a candidate gene-driven approach, albino
Lyst-mutant mice homozygous for a mutation in Tyr, whose product is key to
melanin synthesis within melanosomes, exhibited complete rescue of Lyst-mutant
iris phenotypes. In a genetic background-driven approach using a DBA/2J strain
of congenic mice, an interval containing Tyrp1 enhanced Lyst-dependent iris
phenotypes. Thus, both experimental approaches implicated the melanosome, an
organelle that is a potential source of oxidative stress, as contributing to the
disease phenotype. Confirming an association with oxidative damage, Lyst
mutation resulted in genetic context-sensitive changes in iris lipid
hydroperoxide levels, being lowest in albino and highest in DBA/2J mice.
Surprisingly, the DBA/2J genetic background also exposed a late-onset
neurodegenerative phenotype involving cerebellar Purkinje-cell degeneration.
These results identify an association between oxidative damage to lipid
membranes and the severity of Lyst-mutant phenotypes, revealing a new mechanism
that contributes to pathophysiology involving LYST. One of the colors of mink is Aleutian (aa)-a specific gun-metal gray
pigmentation of the fur-commonly used in combination with other color loci to
generate popular colors such as Violet (aammpp) and Sapphire (aapp). The
Aleutian color allele is a manifestation of mink Chédiak-Higashi syndrome (CHS),
which has been described in humans and several other species. As with forms of
CHS in other species, we report that the mink CHS is linked to the lysosomal
trafficking regulator ( LYST ) gene. Furthermore, we have identified a base
deletion (c.9468delC) in exon 40 of LYST, which causes a frameshift and
virtually terminates the LYST product prematurely (p.Leu3156Phefs*37). We
investigated the blood parameters of three wild-type mink and three CHS mink. No
difference in the platelet number between the two groups was observed, but an
accumulation of platelets between the groups appears different when collagen is
used as a coagulant. Microscopic analysis of peripheral blood indicates giant
inclusions in the neutrophils of the Aleutian mink types. Molecular findings at
the LYST locus enable the development of genetic tests for analyzing the color
selection in American mink. BACKGROUND: Mutations in LYST, a gene encoding a putative lysosomal trafficking
protein, cause Chédiak-Higashi syndrome (CHS), an autosomal recessive disorder
typically characterized by infantile-onset hemophagocytic syndrome and
immunodeficiency, and oculocutaneous albinism. A small number of reports of
rare, attenuated forms of CHS exist, with affected individuals exhibiting
progressive neurodegenerative disease beginning in early adulthood with
cognitive decline, parkinsonism, features of spinocerebellar degeneration, and
peripheral neuropathy, as well as subtle pigmentary abnormalities and
subclinical or absent immune dysfunction.
METHODS: In a consanguineous Pakistani kindred with clinical phenotypes
consistent with attenuated CHS, we performed SNP array-based homozygosity
mapping and whole gene sequencing of LYST.
RESULTS: We identified three individuals homozygous for a novel six base pair
in-frame deletion in LYST (c.9827_9832ATACAA), predicting the loss of asparagine
and threonine residues from the LYST transcript (p.Asn3276_Thr3277del), and
segregating with the phenotype in this family.
CONCLUSIONS: We further characterize the neurologic features of the attenuated
form of CHS, and discuss pathophysiologic mechanisms underlying the
neurodegenerative components of CHS. Attenuated CHS is phenotypically
heterogenous and should be considered when young adults develop
neurodegenerative disease and have pigmentary abnormalities. We briefly discuss
surveillance and management of patients with CHS-related neurodegeneration. BACKGROUND: Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive
disorder characterized by immunodeficiency, neurological dysfunction, and
oculocutaneous albinism. Recently, several clinical CHS phenotypes have been
reported. Here, we report results of a nationwide survey performed to clarify
clinical characteristics and outcomes of CHS patients in Japan.
METHODS: Questionnaires were sent to 287 institutions to collect data regarding
CHS patients diagnosed between 2000 and 2010, including results of lysosomal
trafficking regulator (LYST) gene analysis. Cytotoxicity and degranulation
activity of cytotoxic T lymphocytes were analyzed in available patient samples.
RESULTS: A total of 15 patients diagnosed with CHS were eligible for enrollment
in this study. Of these, 10 (67%) had recurrent bacterial infections, five (33%)
developed life-threatening hemophagocytic lymphohistiocytosis (HLH), and one
patient had complicated maligt lymphoma. Hematopoietic stem cell
transplantation (HSCT) was performed for six patients including three with HLH,
and 10 of the enrolled patients have survived at the time of this writing. LYST
analysis was performed for 10 patients; seven different mutations were detected
in seven patients, whereas no mutation was identified in three patients.
Cytotoxicity and degranulation activity were impaired in patients with and
without LYST mutation.
DISCUSSION: Results of this survey indicate that one or two patients with CHS
were newly diagnosed each year in Japan. The incidence of HLH was not as high as
expected. Mutations of genes other than LYST were suspected in some cases. We
conclude that determining indication for HSCT for CHS patients should be based
on genetic and cytotoxic analysis. BACKGROUND: Chediak-Higashi syndrome (CHS) is a rare, autosomal, recessive
lysosomal disorder with hematological and immunologic abnormalities; however,
stem-cell transplantation from a matched or related donor may be curative. Many
mutations of the CHS1/LYST gene have been reported to date. We report a novel
nonsense mutation of the CHS1/LYST gene in 3 Omani patients.
METHODS AND RESULTS: Three patients from 2 different families presented with
clinical and laboratory features of CHS and a history of death of a previous
sibling because of a severe illness, suggestive of the accelerated phase of CHS.
Giant granules were present in the myeloid cell lines. Before the stem-cell
transplant, the first patient underwent gene sequencing of all exons of the
lysosome trafficking regulator (CHS1/LYST) gene and revealed a nonsense mutation
in exon 5 (c.925C>T, p.R309X). Subsequently, upon presentation, the second and
third patients' direct gene sequencing of exon 5 revealed the same mutation.
CONCLUSIONS: We report a nonsense mutation in exon 5 (c.925C>T, p.R309X). This
supports the allelic heterogeneity of CHS and is in line with most reported
mutation types that lead to a truncated protein. Identification of the mutation
type will facilitate timely diagnosis, management, and family counseling for
those with affected children in Oman. BACKGROUND: Autosomal-recessive hereditary spastic paraplegias (AR-HSP) consist
of a genetically diverse group of neurodegenerative diseases characterised by
pyramidal tracts dysfunction. The causative genes for many types of AR-HSP
remain elusive. We tried to identify the gene mutation for AR-HSP with
cerebellar ataxia and neuropathy.
METHODS: This study included two patients in a Japanese family with their
parents who are first cousins. Neurological examination and gene analysis were
conducted in the two patients and two normal family members. We undertook
genome-wide linkage analysis employing single nucleotide polymorphism arrays
using the two patients' DNAs and exome sequencing using one patient's sample.
RESULTS: We detected a homozygous missense mutation (c.4189T>G, p.F1397V) in the
lysosomal trafficking regulator (LYST) gene, which is described as the causative
gene for Chédiak-Higashi syndrome (CHS). CHS is a rare autosomal-recessive
syndrome characterised by hypopigmentation, severe immune deficiency, a bleeding
tendency and progressive neurological dysfunction. This mutation was
co-segregated with the disease in the family and was located at well-conserved
amino acid. This LYST mutation was not found in 200 Japanese control DNAs.
Microscopic observation of peripheral blood in the two patients disclosed large
peroxidase-positive granules in both patients' granulocytes, although they had
no symptoms of immune deficiency or bleeding tendency.
CONCLUSIONS: We diagnosed these patients as having adult CHS presenting spastic
paraplegia with cerebellar ataxia and neuropathy. The clinical spectrum of CHS
is broader than previously recognised. Adult CHS must be considered in the
differential diagnosis of AR-HSP. Peripheral neuropathy (PN) has been reported in idiopathic and hereditary forms
of parkinsonism, but the pathogenic mechanisms are unclear and likely
heterogeneous. Levodopa-induced vitamin B12 deficiency has been discussed as a
causal factor of PN in idiopathic Parkinson's disease, but peripheral nervous
system involvement might also be a consequence of the underlying
neurodegenerative process. Occurrence of PN with parkinsonism has been
associated with a panel of mitochondrial cytopathies, more frequently related to
a nuclear gene defect and mainly polymerase gamma (POLG1) gene. Parkin (PARK2)
gene mutations are responsible for juvenile parkinsonism, and possible
peripheral nervous system involvement has been reported. Rarely, an association
of parkinsonism with PN may be encountered in other neurodegenerative diseases
such as fragile X-associated tremor and ataxia syndrome related to premutation
CGG repeat expansion in the fragile X mental retardation (FMR1) gene,
Machado-Joseph disease related to an abnormal CAG repeat expansion in ataxin-3
(ATXN3) gene, Kufor-Rakeb syndrome caused by mutations in ATP13A2 gene, or in
hereditary systemic disorders such as Gaucher disease due to mutations in the
β-glucocerebrosidase (GBA) gene and Chediak-Higashi syndrome due to LYST gene
mutations. This article reviews conditions in which PN may coexist with
parkinsonism. |
Has the presence of delayed enhancement been documented in athletes performing strenuous exercise? | There are contrasting literature data on the presence of delayed enhancement, as a sign of myocardial fibrosis, in healthy athletes. More studies are necessary to define the presence, incidence and severity, as well clinical and prognostic meaning, of delayed enhancement magnetic resonance in healthy athletes. | PURPOSE: To prospectively analyze the myocardial distribution of late gadolinium
enhancement (LGE) with delayed-enhancement cardiac magnetic resoce (MR)
imaging, to compare the prevalence of this distribution in nonprofessional male
marathon runners with that in asymptomatic control subjects, and to examine the
prognostic role of LGE.
MATERIALS AND METHODS: Institutional review board and ethics committee approval
were obtained for this study, and all subjects provided written informed
consent. Two-dimensional inversion-recovery segmented k-space gradient-echo MR
sequences were performed after administration of a gadolinium-containing
contrast agent in 102 ostensibly healthy male runners aged 50-72 years who had
completed at least five marathons during the past 3 years and in 102 age-matched
control subjects. Predomitly subendocardial regions of LGE typical of
myocardial infarction (hereafter, coronary artery disease [CAD] pattern) were
distinguished from a predomitly midmyocardial patchy pattern of LGE
(hereafter, non-CAD pattern). Marathon runners with LGE underwent repeat cardiac
MR imaging and additional adenosine perfusion imaging. Runners were followed up
for a mean of 21 months +/- 3 (standard deviation) after initial presentation.
The chi(2), Fisher exact, and McNemar exact tests were used for comparisons.
Event-free survival rates were estimated with the Kaplan-Meier method, and
overall group differences were evaluated with log-rank statistics.
RESULTS: Of the 102 runners, five had a CAD pattern of LGE, and seven had a
non-CAD pattern of LGE. The CAD pattern of LGE was located in the territory of
the left anterior descending coronary artery more frequently than was the
non-CAD pattern (P = .0027, Fisher exact test). The prevalence of LGE in runners
was higher than that in age-matched control subjects (12% vs 4%; P = .077,
McNemar exact test). The event-free survival rate was lower in runners with
myocardial LGE than in those without myocardial LGE (P < .0001, log-rank test).
CONCLUSION: Ostensibly healthy marathon runners have an unexpectedly high rate
of myocardial LGE, and this may have diagnostic and prognostic relevance. Although previous studies including endurance athletes after marathon running
have demonstrated biochemical evidence of cardiac injury and have correlated
these findings with echocardiographic evidence of cardiac dysfunction,
particularly of the right ventricle, a study of marathon athletes incorporating
biomarkers, echocardiography, and cardiac magnetic resoce (CMR) imaging has
not been performed to date. The aim of this study was to demonstrate the cardiac
changes associated with participation in a marathon using serial cardiac
biomarkers, echocardiography, and CMR imaging. Fourteen participants (mean age
33 +/- 6 years, 8 men) completed the full marathon. Myoglobin, creatine kinase,
and troponin T were elevated in all athletes after the race. There was a strong
linear correlation between right ventricular (RV) fractional area change as
assessed by echocardiography and the RV ejection fraction as assessed by CMR
imaging (r = 0.96) after the marathon. RV function, using echocardiography,
transiently decreased from before to after the race (RV fractional area change
43 +/- 4% vs 33 +/- 5%, p <0.05). There were also postrace changes in left
ventricular and RV diastolic filling. Although RV systolic changes were
transient, left ventricular and RV diastolic abnormalities persisted up to 1
week after the marathon. No evidence of delayed enhancement of the left
ventricular myocardium was found on CMR imaging, suggesting that the increase in
cardiac biomarkers after the marathon may not have be due to myocardial
necrosis. In conclusion, RV systolic dysfunction transiently occurs after a
marathon and has been validated for the first time by CMR imaging. The increase
in cardiac troponin after marathon running is likely due to the cytosolic
release of the biomarker, not to the true breakdown of the myocyte, as confirmed
by delayed enhancement CMR imaging. AIMS: Endurance training may be associated with arrhythmogenic cardiac
remodelling of the right ventricle (RV). We examined whether myocardial
dysfunction following intense endurance exercise affects the RV more than the
left ventricle (LV) and whether cumulative exposure to endurance competition
influences cardiac remodelling (including fibrosis) in well-trained athletes.
METHODS AND RESULTS: Forty athletes were studied at baseline, immediately
following an endurance race (3-11 h duration) and 1-week post-race. Evaluation
included cardiac troponin (cTnI), B-type natriuretic peptide, and
echocardiography [including three-dimensional volumes, ejection fraction (EF),
and systolic strain rate]. Delayed gadolinium enhancement (DGE) on cardiac
magnetic resoce imaging (CMR) was assessed as a marker of myocardial
fibrosis. Relative to baseline, RV volumes increased and all functional measures
decreased post-race, whereas LV volumes reduced and function was preserved.
B-type natriuretic peptide (13.1 ± 14.0 vs. 25.4 ± 21.4 ng/L, P = 0.003) and
cTnI (0.01 ± .03 vs. 0.14 ± .17 μg/L, P < 0.0001) increased post-race and
correlated with reductions in RVEF (r = 0.52, P = 0.001 and r = 0.49, P = 0.002,
respectively), but not LVEF. Right ventricular ejection fraction decreased with
increasing race duration (r = -0.501, P < 0.0001) and VO(2)max (r = -0.359, P =
0.011). Right ventricular function mostly recovered by 1 week. On CMR, DGE
localized to the interventricular septum was identified in 5 of 39 athletes who
had greater cumulative exercise exposure and lower RVEF (47.1 ± 5.9 vs. 51.1 ±
3.7%, P = 0.042) than those with normal CMR.
CONCLUSION: Intense endurance exercise causes acute dysfunction of the RV, but
not the LV. Although short-term recovery appears complete, chronic structural
changes and reduced RV function are evident in some of the most practiced
athletes, the long-term clinical significance of which warrants further study. Sudden cardiac death in young athletes is rare but tragic. The cardiology
community is faced with the challenge of providing a sensible strategy for the
prevention of SCD while simultaneously reaffirming that the benefits of regular
exercise far outweigh potential risks. At present, there is a broad range of
screening recommendations dependent upon country, sporting discipline, and
competition level. While much recent debate has focused on the efficacy of
screening with electrocardiography, a number of sporting bodies also mandate the
inclusion of exercise testing and echocardiography in screening protocols.
Cardiac magnetic resoce imaging, coronary calcium scoring and computed
tomography coronary angiography have also been promoted as potentially valuable
screening tools for competitive athletes. This review will examine the
controversial topic of utilizing cardiac imaging for athlete pre-participation
screening. Specifically, the limitations of screening for relatively rare
disorders using imaging tools with uncertain or imperfect accuracy will be
addressed. Current evidence suggests that the accuracy of all cardiac imaging
modalities is insufficient to justify their use as primary screening modalities
in athletes. Atypical findings such as marked cardiac dilation, reduced
deformation, or small patches of delayed gadolinium enhancement may be commonly
encountered in well-trained athletes, but, at present, the prognostic
significance of such findings is unknown. Resulting uncertainty for the
clinician and athlete has the potential for psychological stress, further
testing, and unnecessary exclusions from competition. However, these concerns
must not be confused with the extremely useful applications of cardiac imaging
for the assessment of athletes with symptoms, an abnormal electrocardiogram or a
positive family history. As modern imaging further enhances our understanding of
the spectrum of athlete's heart, its role may expand from the assessment of
athletes with suspected disease to being part of comprehensive pre-participation
screening in apparently healthy athletes. OBJECTIVE: To prospectively document musculoskeletal magnetic resoce imaging
(MRI) use and how it affects diagnosis, playing status, and treatment of
Division I university athletes. We hypothesized that MRI often has little or no
effect on the diagnosis or treatment plan.
DESIGN: Cross-sectional study.
SETTING: Division I university sports medicine program.
PATIENTS: Division I university varsity athletes.
INTERVENTIONS: Data were collected of musculoskeletal MRI use in varsity student
athletes for 2 full academic years from 2010 to 2012 at a National Collegiate
Athletic Association Division I institution.
MAIN OUTCOME MEASURES: Timing of the injury, first physician visit, and MRI and
pre- and post-MRI diagnosis, playing status, and treatment (surgical vs
nonsurgical).
RESULTS: Eighty-six MRIs were obtained during the 2 years studied. Average age
was 19.9 (18-23) years. Forty-five percent of injuries occurred during
competition season, 34% occurred preseason, and 21% occurred postseason. There
was a change in diagnosis in 13 athletes (15.1%, 1 led to surgery performed
after completion of the season), and there was a change in participation status
in 8 athletes (9.3%, 5 increased and 3 decreased). Treatment plan changed in 1
athlete (1.2%). No athlete required surgery immediately after an MRI that was
not already being planned. Every athlete treated nonsurgically pre-MRI was able
to finish their season.
CONCLUSIONS: Magnetic resoce imaging was obtained in 14% of athletes and did
not demonstrate a clear benefit over history, examination, and radiographs.
Magnetic resoce imaging did change diagnosis in 15% of cases, though it did
not appreciably change the playing status or treatment plan. |
How does dronedarone affect thyroid hormone signaling in the heart? | Dronedarone via its metabolite debutyldronedarone acts as a TRalpha(1)-selective inhibitor and selectively mimicks hypothyroidism.
Dronedarone decreases TRalpha 1 and beta 1 expression by about 50% in the right atrium (RA) while in the left ventricle, only TRbeta1 is found to be decreased. | The present study investigated the effects of dronedarone and amiodarone on
plasma thyroid hormones and the possible consequences on the response of the
heart to ischemia. Amiodarone (30 mg/kg/day per os) or dronedarone (30 mg/kg/day
per os) were administered for 2 weeks in normal and thyroxine-treated animals
(25 microg/100 g body weight od sc, for 2 weeks), while animals without
amiodarone and dronedarone served as controls. Isolated rat hearts were perfused
in a Langendorff mode and subjected to 20 and 30 min of zero-flow global
ischemia followed by 45 min of reperfusion. Functional changes were assessed by
measuring left ventricular developed pressure (LVDP) under resting conditions
and in response to ischemia-reperfusion, LVDP%, as well as the severity of
ischemic contracture. Amiodarone resulted in increased T4, T4/T3 and rT3,
whereas dronedarone did not alter the thyroid hormone profile in normal animals.
In thyroxine-treated animals, amiodarone increased T4/T3 ratio but T4, T3 and
rT3 levels were not altered. Basal functional parameters and ischemic
contracture did not change by amiodarone and/or dronedarone neither in normal
nor in thyroxine-treated hearts. In normal hearts, postischemic functional
recovery, LVDP%, was not altered by amiodarone or dronedarone administration.
LVDP% was statistically higher in thyroxine-treated hearts than in normal and
this beneficial effect was not abolished by amiodarone or dronedarone treatment. Dronedarone (Dron), without iodine, was developed as an alternative to the
iodine-containing antiarrhythmic drug amiodarone (AM). AM acts, via its major
metabolite desethylamiodarone, in vitro and in vivo as a thyroid hormone
receptor alpha(1) (TRalpha(1)) and TRbeta(1) antagonist. Here we investigate
whether Dron and/or its metabolite debutyldronedarone inhibit T(3) binding to
TRalpha(1) and TRbeta(1) in vitro and whether dronedarone behaves similarly to
amiodarone in vivo. In vitro, Dron had a inhibitory effect of 14% on the binding
of T(3) to TRalpha(1), but not on TRbeta(1). Desethylamiodarone inhibited T(3)
binding to TRalpha(1) and TRbeta(1) equally. Debutyldronedarone inhibited T(3)
binding to TRalpha(1) by 77%, but to TRbeta(1) by only 25%. In vivo, AM
increased plasma TSH and rT(3), and decreased T(3). Dron decreased T(4) and
T(3), rT(3) did not change, and TSH fell slightly. Plasma total cholesterol was
increased by AM, but remained unchanged in Dron-treated animals.
TRbeta(1)-dependent liver low density lipoprotein receptor protein and type 1
deiodinase activities decreased in AM-treated, but not in Dron-treated, animals.
TRalpha(1)-mediated lengthening of the QTc interval was present in both AM- and
Dron-treated animals. The in vitro and in vivo findings suggest that dronedarone
via its metabolite debutyldronedarone acts as a TRalpha(1)-selective inhibitor. Hypothyroid heart displays a phenotype of cardioprotection against ischemia and
this study investigated whether administration of dronedarone, an
amiodarone-like compound that has been shown to preferentially antagonize
thyroid hormone binding to thyroid hormone receptor alpha1 (TRalpha1), results
in a similar effect. Dronedarone was given in Wistar rats (90 mg/kg, once daily
(od) for 2 weeks) (DRON), while untreated animals served as controls (CONT).
Hypothyroidism (HYPO) was induced by propylthiouracil administration. Isolated
rat hearts were perfused in Langendorff mode and subjected to 20 minutes of
zero-flow global ischemia (I) followed by 45 minutes of reperfusion (R). 3,5,3'
Triiodothyronine remained unchanged while body weight and food intake were
reduced. alpha-Myosin heavy chain (alpha-MHC) decreased in DRON while
beta-myosin heavy chain (beta-MHC) and sarcoplasmic reticulum Ca2+ adenosine
triphosphatase (ATPase) expression (SERCA) was similar to CONT. In HYPO,
alpha-MHC and SERCA were decreased while beta-MHC was increased. Myocardial
glycogen content was increased in both DRON and HYPO. In DRON, resting heart
rate and contractility were reduced and ischemic contracture was significantly
suppressed while postischemic left ventricular end-diastolic pressure and
lactate dehydrogenase release (IU/L min) after I/R were significantly decreased.
In conclusion, dronedarone treatment results in cardioprotection by selectively
mimicking hypothyroidism. This is accompanied by a reduction in body weight
because of the suppression of food intake. TRs might prove novel pharmacologic
targets for the treatment of cardiovascular illnesses. We have previously shown that acute thyroid hormone treatment could limit
reperfusion injury and increase post-ischemic recovery of function. In the
present study, we further explore potential initiating mechanisms of this
response. Thus, isolated rat hearts were subjected to 30 min zero-flow global
ischemia (I) followed by 60-min reperfusion (R). Reperfusion injury was assessed
by post-ischemic recovery of left ventricular developed pressure (LVDP%) and LDH
release. T3 at a dose of 60 nM which had no effect on contractile function of
non-ischemic myocardium, significantly increased LVDP% [48% (2.9) vs. 30.2%
(3.3) for untreated group, P < 0.05] and reduced LDH release [8.3 (0.3) vs. 10
(0.42) for untreated group, P < 0.05] when administered at R. T4 (60 and 400 nM)
had no effect on contractile function either in non-ischemic or ischemic
myocardium. Administration of debutyl-dronedarone (DBD), a TRα1 antagonist
abolished the T3-limiting effect on reperfusion injury: Thus, co-administration
of T3 and DBD resulted in significantly lower LVDP%, [23% (4.7) vs. 48% (2.9)
for T3 group, P < 0.05] and higher LDH release [9.9 (0.3) vs. 8.3 (0.3), for T3
group, P < 0.05]. In conclusion, acute T3 and not T4 treatment will be able to
protect against reperfusion injury. T3 can exert this beneficial effect on
ischemic myocardium at a dose that has no effects on non-ischemic myocardium.
Acute T3-limiting effect on reperfusion injury is mediated, at least in part,
via TRα1 receptor. |
Name the factors required for selenoprotein synthesis in eukaryotes | eFSec, SBP2, SECp43, PSTK, Sec synthase (Sec S, SLA/LP), SPS2 (SelD), tRNASec, SECIS element, (L30), SPS1 | The patufet gene encodes the Drosophila melanogaster homologue of
selenophosphate synthetase, an enzyme required for selenoprotein synthesis, and
appears to have a role in cell proliferation. In this paper we analyse the
expression pattern of patufet during the development of imaginal discs and brain
as well as the function of this gene in relation to cell proliferation.
Wild-type organisms showed a highly dynamic pattern of ptuf mRNA expression
during larval and pupal development. Co-localization analysis of ptuf mRNA
expression and BrdU incorporation showed high levels of ptuf mRNA in dividing
cells and low or undetectable levels in non-dividing cells. In addition,
[(75)Se] incorporation revealed a major selenoprotein band of 42 kDa. Mutant
organisms showed no selenoprotein synthesis, lower levels of cell proliferation,
a higher proportion of cells arrested in G(2) as seen by cyclin B labeling and
increased levels of reactive oxygen species (ROS). Because most selenoproteins
identified so far are antioxidants, the role of ptuf in cell proliferation
through the control of the cellular redox balance is discussed. The synthesis of eukaryotic selenoproteins involves the recoding of an internal
UGA codon as a site for selenocysteine incorporation. This recoding event is
directed by a selenocysteine insertion sequence in the 3'-untranslated region.
Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have
investigated how the rates of translational termination and selenocysteine
incorporation relate to cis-acting elements in the mRNA as well as to
trans-acting factors in the cytoplasm. We used cis-elements from the
phospholipid glutathione peroxidase gene as the basis for this work because of
its relatively high efficiency of selenocysteine incorporation. The last two
codons preceding the UGA were found to exert a far greater influence on
selenocysteine incorporation than nucleotides downstream of it. The efficiency
of selenocysteine incorporation was generally much less than 100% but could be
partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The
combination of two or three UGA codons in one reading frame led to a dramatic
reduction in the yield of full-length protein. It is therefore unlikely that
multiple incorporations of selenocysteine are processive with respect to the
mode of action of the ribosomal complex binding to the UGA site. These
observations are discussed in terms of the mechanism of selenoprotein synthesis
and its ability to compete with termination at UGA codons. 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. Selenoprotein synthesis is conserved from bacteria to man. It involves the
differential decoding of the UGA stop codon as selenocysteine. The proteomes of
both prokaryotes and eukaryotes, with the exception of yeast, contain only few
selenoproteins. This low number is explained by a counterselection of readily
oxidized selenocysteine after the introduction of oxygen into the atmosphere and
the need to conserve selenoenzymes that control redox homeostasis of cells. Lack
of selenoprotein synthesis in vertebrates impairs the oxidative stress defence
and causes lethality. Here we show that Drosophila mutants that lack the
translation elongation factor SelB/eEFsec fail to decode the UGA codon as
selenocysteine, but they are viable and fertile. Oxidative stress responses and
the lifespan of these flies are not affected. Protecting cells from oxidative
stress can therefore not account for the selection pressure that conserves
selenoprotein biosynthesis during the course of evolution. Selenium is biologically active through the functions of selenoproteins that
contain the amino acid selenocysteine. This amino acid is translated in response
to in-frame UGA codons in mRNAs that include a SECIS element in its 3'
untranslated region, and this process requires a unique tRNA, referred to as
tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as
a termination signal, is a candidate restriction point for the regulation of
selenoprotein synthesis by selenium. A specialized reporter construct was used
that permits the evaluation of SECIS-directed UGA translation to examine
mechanisms of the regulation of selenoprotein translation. Using SECIS elements
from five different selenoprotein mRNAs, UGA translation was quantified in
response to selenium supplementation and alterations in tRNA([Ser]Sec) levels
and isoform distributions. Although each of the evaluated SECIS elements
exhibited differences in their baseline activities, each was stimulated to a
similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited
by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using
a domit-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be
limiting for UGA translation under conditions of high selenoprotein mRNA in both
a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data
indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during
selenoprotein translation indicate that it is this isoform that is
translationally active and that selenium-induced tRNA methylation is a mechanism
of regulation of the synthesis of selenoproteins. Selenoproteins are central controllers of cellular redox homeostasis.
Incorporation of selenocysteine (Sec) into selenoproteins employs a unique
mechanism to decode the UGA stop codon. The process requires the Sec insertion
sequence (SECIS) element, tRNASec, and protein factors including the SECIS
binding protein 2 (SBP2). Here, we report the characterization of motifs within
SBP2 that regulate its subcellular localization and function. We show that SBP2
shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear
localization signal and nuclear export signal motifs and that its nuclear export
is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation
of SBP2 via oxidation of cysteine residues within a redox-sensitive
cysteine-rich domain. These modifications are efficiently reversed in vitro by
human thioredoxin and glutaredoxin, suggesting that these antioxidant systems
might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines
using small interfering RNA results in a decrease in Sec incorporation,
providing direct evidence for its requirement for selenoprotein synthesis.
Furthermore, Sec incorporation is reduced substantially after treatment of cells
with agents that cause oxidative stress, suggesting that nuclear sequestration
of SBP2 under such conditions may represent a mechanism to regulate the
expression of selenoproteins. Selenophosphate synthetase (SelD) generates the selenium donor for
selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes
is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as
a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1,
synthesized selenophosphate from selenide, whereas SPS1 may utilize a different
substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain
unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells
using small interfering RNA and found that selenoprotein biosynthesis was
severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2
into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not,
indicating that SPS1 cannot complement SPS2 function. These in vivo studies
indicate that SPS2 is essential for generating the selenium donor for
selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more
specialized, non-essential role in selenoprotein metabolism. Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA
Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the
cis-acting SECIS RNA hairpin in the 3'UTR of selenoprotein mRNAs, and
trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central
to the mechanism. SBP2 has been so far functionally characterized only in rats
and humans. In this work, we report the characterization of the Drosophila
melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same
selenoprotein synthesis-promoting capabilities as the mammalian counterpart.
However, a major difference resides in the SECIS recognition pattern: while
human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar
affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we
report the identification of a K (lysine)-rich domain in all SBP2s, essential
for SECIS and 60S ribosomal subunit binding, differing from the
well-characterized L7Ae RNA-binding domain. Swapping only five amino acids
between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding
properties to the proteins, thus unveiling an important sequence for form 1
binding. The micronutrient selenium is found in proteins as selenocysteine (Sec), the
21st amino acid cotranslationally inserted in response to a UGA codon. In vitro
studies in archaea and mouse showed that Sec-tRNA(Sec) formation is a 3-step
process starting with serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS),
phosphorylation of serine to form phosphoserine (Sep)-tRNA(Sec) by
phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion to Sec-tRNA(Sec) by
Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic
selenoprotein synthesis has been lacking. Here, we present an analysis of
Sec-tRNA(Sec) formation in the parasitic protozoon Trypanosoma brucei in vivo.
Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis,
demonstrating the essentiality of both enzymes for Sec-tRNA(Sec) formation.
Growth of the 2 knockout strains was not impaired; thus, unlike mammals,
trypanosomes do not require selenoproteins for viability. Analysis of
conditional RNAi strains showed that SerRS, selenophosphate synthase, and the
Sec-specific elongation factor, EFSec, are also essential for selenoprotein
synthesis. These results with T. brucei imply that eukaryotes have a single
pathway of Sec-tRNA(Sec) synthesis that requires Sep-tRNA(Sec) as an
intermediate. 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. |
What are the functions of the ESCRT machinery? | The endosomal sorting complexes required for transport (ESCRT) are needed for three distinct cellular functions in higher eukaryotes: (i) Multivesicular body formation for the degradation of transmembrane proteins in lysosomes, (ii) midbody abscission during cytokinesis and (iii) retroviral budding. | The ESCRT (endosomal sorting complex required for transport) machinery is known
to sort ubiquitinated transmembrane proteins into vesicles that bud into the
lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are
ubiquitinated they are excluded from the intraluminal vesicles and recycle back
to the cytoplasm for further rounds of sorting. To obtain insights into the
rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of
artificial ESCRT-like protein fusions. These studies showed that lowering
ESCRT-binding affinity converts a protein from behaving like ESCRT machinery
into cargo of the MVB pathway, highlighting the close relationship between
machinery and the cargoes they sort. Furthermore, our findings give insights
into the targeting of soluble proteins into the MVB pathway and show that
binding to any of the ESCRTs can mediate ubiquitin-independent MVB sorting. HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the
endosomal sorting complex required for transport (ESCRT) machinery to enable
particle release. However, little is known about the temporal and spatial
organization of ESCRT protein recruitment. Using multiple-color live-cell total
internal reflection fluorescence microscopy, we observed that the ESCRT-I
protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly,
whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited
sequentially, once Gag assembly is completed. Chmp4b, a protein that is required
to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before
the ATPase Vps4A. Using two-color superresolution imaging, we observed that the
ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the
periphery of the nascent virions, with the Tsg101 assemblages positioned closer
to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are
consistent with the notion that the ESCRT machinery is recruited transiently to
the neck of the assembling particle and is thus present at the appropriate time
and place to mediate fission between the nascent virus and the plasma membrane. Protein turnover can be achieved via the lysosome/vacuole and the autophagic
degradation pathways. Evidence has accumulated revealing that efficient
autophagic degradation requires functional endosomal sorting complex required
for transport (ESCRT) machinery. However, the interplay between the ESCRT
machinery and the autophagy regulator remains unclear. Here, we show that FYVE
domain protein required for endosomal sorting 1 (FREE1), a recently identified
plant-specific ESCRT component essential for multivesicular body (MVB)
biogenesis and plant growth, plays roles both in vacuolar protein transport and
autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds
and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with
a unique plant autophagy regulator SH3 domain-containing protein2 and associates
with the PI3K complex, to regulate the autophagic degradation in plants. Thus,
FREE1 plays multiple functional roles in vacuolar protein trafficking and
organelle biogenesis as well as in autophagic degradation via a previously
unidentified regulatory mechanism of cross-talk between the ESCRT machinery and
autophagy process. Disassembly of the endosomal sorting complex required for transport (ESCRT)
machinery from biological membranes is a critical final step in cellular
processes that require the ESCRT function. This reaction is catalyzed by VPS4,
an AAA-ATPase whose activity is tightly regulated by a host of proteins,
including LIP5 and the ESCRT-III proteins. Here, we present structural and
functional analyses of molecular interactions between human VPS4, LIP5, and the
ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for
LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly
inhibits the stimulation. Both of these observations are distinct from what was
previously described for homologous yeast proteins. The crystal structure of
LIP5NTD in complex with the MIT (microtubule-interacting and
transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B,
was determined at 1 Å resolution. It reveals an ESCRT-III binding induced
moderate conformational change in LIP5NTD, which results from insertion of a
conserved CHMP5 tyrosine residue (Tyr(182)) at the core of LIP5NTD structure.
Mutation of Tyr(182) partially relieves the inhibition displayed by CHMP5.
Together, these results suggest a novel mechanism of VPS4 regulation in
metazoans, where CHMP5 functions as a negative allosteric switch to control
LIP5-mediated stimulation of VPS4. Endosomal sorting complexes required for transport (ESCRTs) are involved in the
formation of multivesicular bodies and sorting of targeted proteins to the yeast
vacuole. The deletion of seven genes encoding components of the ESCRT machinery
render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2
concentrations as well as to low pH in media. In this work, we focused on
intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT
mutants exhibited an altered pHin level compared to the wild type under standard
growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more
significant drop in pHin levels in these mutants than in the wild type,
suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition
of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells
lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this
protein in the maintece of proper pHin homeostasis when cells need to cope
with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in
the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the
pHin levels of cells, which demonstrates a genetic interaction between VCX1 and
studied ESCRT genes. During telophase, the nuclear envelope (NE) reforms around daughter nuclei to
ensure proper segregation of nuclear and cytoplasmic contents. NE reformation
requires the coating of chromatin by membrane derived from the endoplasmic
reticulum, and a subsequent annular fusion step to ensure that the formed
envelope is sealed. How annular fusion is accomplished is unknown, but it is
thought to involve the p97 AAA-ATPase complex and bears a topological
equivalence to the membrane fusion event that occurs during the abscission phase
of cytokinesis. Here we show that the endosomal sorting complex required for
transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the
forming NE in human cells, and is necessary for proper post-mitotic
nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged
multivesicular body protein 2A (CHMP2A) is directed to the forming NE through
binding to CHMP4B, and provides an activity essential for NE reformation.
Localization also requires the p97 complex member ubiquitin fusion and
degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery
in cell division and demonstrate a conservation of the machineries involved in
topologically equivalent mitotic membrane remodelling events. |
Which genes/proteins have been found to inhibit cyclin dependent kinase 4 (CDK4)? | The p15(ink4b) and p16(ink4a) CDK4 inhibitor genes map within the chromosome band 9p21 region deleted frequently in various cancers.The Cdk4 inhibitor p18(Ink4c) is a tumor suppressor. Recent studies of Cyclin D1/Cdk4 have proposed that p21(Waf1/Cip1/Sdi1) plays a key role as a potent Cdk4 inhibitor. p27KIP1 is also a cdk4 ihibitor. | The cyclin-dependent kinase 4 (cdk4) inhibitor (p16INK4/MTS1/CDKN2) gene has
been recently identified as a putative tumor suppressor gene because of the high
frequency of homozygous deletion observed in numerous human tumor cell lines,
including leukemias. However, results obtained from uncultured tumor samples
have led to discussion of the relevance of these findings. Using reverse
transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis, we
have investigated p16INK4A gene at both RNA and genomic levels in various types
of leukemias: acute myeloid leukemia (AML) (n = 23); acute lymphocytic leukemia
(ALL) (n = 22) and B cell chronic lymphoproliferative disorders (CLPD) (n = 33).
p16INK4A mRNA expression was not found in only 1/20 AML and 2/23 CLPD samples.
Conversely, p16INK4A mRNA was not detected in 5/17 ALL cases, and intensity of
PCR products were barely detectable in seven additional cases, possibly related
to the contamination by normal cells in some cases. By Southern blotting, a
homozygous deletion of p16INK4A gene was found in 6/17 ALL cases (35%) among
which 4/6 were negative or weakly positive by RT-PCR assay. None of the five AML
and 20 CLL samples studied had p16INK4A deletion. Sequence analysis of p16INK4A
exon 2 did not show point mutation in two of these cases lacking mRNA
expression. Our data provide further evidence that among hematological
maligcies, ALL are the most likely to be associated with p16INK4A
inactivation, mainly by homozygous gene deletion. Since most hematological
maligcies-except ALL-are infrequently associated with p16INK4A and
retinoblastoma (Rb) gene alteration it seems worthwhile to explore cdk4 and cdk6
expression to determine whether or not the disruption of the
p16INK4A/Rb/cdk4/cdk6 regulatory loop might play a role in their pathogenesis. The p15 and p16 CDK4 inhibitor genes map within the chromosome band 9p21 region
deleted frequently in maligt mesothelioma and other cancers. p16 has been
implicated recently as a potential target of 9p21 deletions in mesothelioma, but
the role of this gene is uncertain because deletions have been detected more
often in established cell lines than in primary tumor specimens. We determined
p15 and p16 copy number by fluorescence in situ hybridization with a P1 contig
in 50 primary mesotheliomas. Codeletion of p15 and p16 was found in 72% of
mesotheliomas, including all cases with spindle-cell components (n = 21) and
total deletion of p15 and p16 was found in several mesotheliomas that lacked
cytogenetic deletion of the chromosome 9 short arm. Point mutations were not
found, however, in exon 2 of retained p15 and p16 alleles from seven
mesotheliomas. These findings demonstrate that p15, p16 and/or a closely
neighboring gene, are the targets of frequent chromosome 9p deletion in primary
maligt mesothelioma. A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific
antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes
(CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous
cultured melanoma cells and metastasis tissue, but not in the patient's
lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was
part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4
inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in
one additional melanoma among 28 melanomas analyzed. These results suggest that
mutation of CDK4 can create a tumor-specific antigen and can disrupt the
cell-cycle regulation exerted by the tumor suppressor p16INK4a. Cell cycle dependent phosphorylation of the RB tumor suppressor protein is
mediated by a family of G1 cyclin dependent kinases (cdks) and cyclins including
the activated cdk4:cyclin D complex. The identification of a cdk4 inhibitor,
p16INK4, as a target for mutations in cultured tumor lines and primary tumors
suggested that RB activity may be affected in these cells. We have examined 88
lung cancer lines for p16INK4 protein expression and have observed a striking
inverse correlation between the presence of p16INK4 and wildtype RB. We
demonstrated that only 6/55 (11%) of small cell lung cancer (SCLC) samples had
absent p16INK4 protein, and all 6 belonged to the rare subset of SCLC with
wildtype RB expression. Conversely of 48 SCLC samples with absent or mutant RB,
all showed detectable levels of p16INK4 protein. In contrast, we observed that
23/33 (70%) of non-SCLC samples had loss of p16INK4. Twenty-two of 26 non-SCLC
lines with wildtype RB had absent p16INK4 while 6 of 7 non-SCLC lines with
absent or mutant RB had detectable p16INK4. The inverse correlation of RB and
p16INK4 expression and the absence of p16INK4 inactivation in RB (-/-) SCLC
lines (0/48) confirms a common p16INK4/RB growth suppressor pathway in human
cancers and provides evidence that p16INK4, and not an adjacent gene on
chromosome 9p, is a specific target for mutational events. In the present study, we analyzed human ovarian carcinoma cell lines for
abnormalities in the tumor suppressor gene Rb (retinoblastoma) and in
cyclin-dependent kinase 4 (CDK4) inhibitor genes (p16INK4 and p15INK4B) using
molecular biology techniques. For the Rb gene, in all six cell lines (PA-1,
Caov-3 and -4, OVCAR-3, SK-OV-3, and Kuramochi), Rb gene abnormality was not
detected using Southern blotting. In the Caov-3 cell line transcripts were not
detectable by either Northern blot or polymerase chain reaction. Sequence
analysis of the entire coding region of the Rb gene revealed point mutations
(AAC to GAC) resulting in codon 123 (Asn to Asp) changes in the Caov-4 cell
line. In the PA-1 cell line both wild-type Rb and mutant-type Rb (codon 798: CGG
to TGG) were expressed, and in the OVCAR-3 cell line both wild-type Rb and
mutant-type Rb (codon 704: ATG to GTG) were expressed. In four of six human
ovarian carcinoma cell lines Rb gene abnormality was detected. For the p16INK4
and p15INK4B genes, only the SK-OV-3 cell line had abnormalities. There was a
gene rearrangement or minor deletion of the p16INK4 gene in the SK-OV-3 cell
line, while the p15INK4B gene was deleted in this cell line. In the SK-OV-3 cell
line no mRNAs of p16INK4 and p15INK4B were expressed. At the point of Rb gene
inactivation, we can explain five cell lines of six: four cell lines had
abnormalities in the Rb gene itself, which is another mechanism by which the Rb
gene is inactivated, while one cell line (SK-OV-3) had abnormalities in CDK4
inhibitor genes, another of the inactivation mechanisms of the Rb gene. These
data suggest that abnormalities of Rb and CDK4 inhibitor genes (p16INK4,
p15INK4B) may be involved in human ovarian carcinogenesis. Although p27(Kip1) has been considered a general inhibitor of G1 and S phase
cyclin-dependent kinases, we report that the interaction of p27 with two such
kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells
containing a p27 inducible system, a 6-fold increase over the basal p27 level
completely inhibited Cdk2 and cell cycle progression. In contrast, the same or a
larger increase in p27 levels did not inhibit Cdk4 or its homologue Cdk6,
despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed
in vitro was essentially inactive, whereas a p27-cyclin D2-Cdk4 complex was
active as a retinoblastoma kinase and served as a substrate for the
Cdk-activating kinase Cak. High concentrations of p27 inhibited cyclin D2-Cdk4,
apparently by conversion of active complexes into inactive ones by the binding
of additional p27 molecules. In contrast to their differential interaction,
cyclin A-Cdk2 and cyclin D2-Cdk4 were similarly inhibited by bound
p21(Cip1/Waf1). Roles of cyclin A-Cdk2 as a p27 target and cyclin D2-Cdk4 as a
p27 reservoir may result from the differential ability of bound p27 to inhibit
the kinase subunit in these complexes. Members of the INK4 family of cyclin-dependent kinase (CDK) inhibitors
specifically bind and inhibit the G1-specific CDK molecules CDK4 and CDK6. One
of the INK4 molecules, p16, is also known as multiple tumor suppressor and has
been found to be mutated or deleted in various tumors and cell lines. We have
previously identified p18 as a member of the INK4 family. To determine the
molecular basis for the inhibitory function of p18, we introduced 11 missense
mutations of conserved residues that were identified in p16 of cancer cell lines
into p18. The effects of these mutations on the ability of p18 to bind and
inhibit CDK4 and CDK6 or to inhibit cell growth were determined. Our results
indicate that the third ankyrin repeat and the NH2-terminal portion of the
fourth repeat constitute the essential element necessary for the ability of p18
to bind and inhibit CDK4 and CDK6. Apart from this core interaction element, p18
seems to use additional, distinct residues to differentially bind and inhibit
CDK4 and CDK6, accounting for the known penchant of p18 to preferentially
interact with CDK6. Protein-protein interactions usually involve a large number of residues; thus it
is difficult to elucidate functional and structural roles of specific residues
located in the interface. This problem is particularly challenging for ankyrin
repeat proteins (ARs), which consist of linear arrays of small repeating units
and play critical roles in almost every life process via protein-protein
interactions, because the residues involved are discontinuously dispersed in
both the ARs and their partners. Our previous studies showed that while both
specific CDK4 inhibitor p16INK4A (P16) and gankyrin bind to cyclin-dependent
kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of
CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is
oncogenic, the structural basis of these contrasting properties was unknown.
Here we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase
activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated
with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring
about a moderate decrease in the stability of gankyrin. Further structural and
biophysical analyses suggest that the substitution of Ile79 with Asp leads to
local conformational changes in loops I-III of gankyrin. Taken together, our
results allow the dissection of the "protein-protein binding" and "CDK4
inhibition" functions of P16, show that the difference between tumor suppressing
and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a
single residue, and provide structural insight to the contrasting biological
functions of the two AR proteins. The retinoblastoma (RB) tumor suppressor pathway is likely important in
primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of
children born with RB mutations develop brain PNETs, commonly in the pineal
gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and
Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of
sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic
events cooperating with RB pathway disruption in brain PNET, we generated a
transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1
enhanced pinealocyte proliferation, causing pineal gland enlargement. However,
proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb
phosphorylation despite continued expression of the transgene, and the pineal
cells showed heterochromatin foci suggestive of a senescent-like state. In the
absence of the p53 tumor suppressor, cell proliferation continued, resulting in
pineal PNET that limited mouse survival to approximately 4 months.
Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal
glands independently of p53, and transgenic mice that lacked Ink4c developed
invasive PNET, although at an older age than those lacking p53. Analogous to our
mouse model, we found that children with heritable RB often had asymptomatic
pineal gland enlargement that only rarely progressed to PNET. Our finding that
the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET
suggests that pharmacologic interventions to inhibit Cdk4 activity may be a
useful chemoprevention or therapeutic strategy in cancer driven by primary RB
pathway disruption. |
What is the role of TRH in hypertension? | TRH gene overexpression induces hypertension in normal rats and spontaneously hypertensive rats have central TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. TRH antisense treatment reduces hypertension.
central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity.
the pressor effect of intravenous TRH is mediated primarily by a stimulation of alpha-adrenergic receptors. Activation of cardiac beta-adrenoceptors seems to contribute to the blood pressure increasing effect of intravenous TRH. Ang II system is involved in the TRH cardiovascular effects.
Polymorphisms in TRH (thyrotropin-releasing hormone) are significantly associated with both blood pressure variation and hypertension.
TRH may mediate the central leptin-induced hypertension effect
A parallel increase in the density of brain TRH receptors with elevation of blood pressure has been shown and suggests that brain TRH receptors may play an important role in the pathophysiology of hypertension. TRH Receptor gene participates in the etiopathogenesis of essential hypertension. | Centrally administered thyrotropin-releasing hormone exerts a well documented
hypertensive effect. In this study, the possible physiological role of
thyrotropin-releasing hormone in the central cardiovascular regulation was
evaluated in spontaneously hypertensive rats receiving long-term (8-14 days)
intracerebroventricular infusion of a heterologous antiserum to
thyrotropin-releasing hormone. The effect of this passive immunization on the
blood pressure was monitored from conscious animals by the tail-cuff method.
Thyrotropin-releasing hormone antiserum significantly decreased the systolic
arterial pressure in adult rats with established hypertension. No alterations in
serum thyroid hormone status were observed indicating that the antihypertensive
effect of immunological blockade of thyrotropin-releasing hormone was not due to
changes in the serum thyroid hormone levels. These results provide evidence for
a role of endogenous brain thyrotropin-releasing hormone in the maintece of
hypertension in spontaneously hypertensive rats. The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to
brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and
normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound
specifically to rat brain membranes at a single high affinity site. The density
(Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR
rats compared to WKY rats. The apparent dissociation constants (Kd values) for
the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the
various brain regions revealed that the density of [3H]MeTRH was highest in the
hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex
and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex,
hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in
pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To
assess the significance of increased binding sites for [3H]MeTRH in some brain
regions of SHR rats, the binding studies were carried out during normotensive
and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old
SHR rats there was neither an increase in blood pressure nor any increase in
[3H]MeTRH binding in the hypothalamus and striatum as compared to age matched
WKY rats. With the development of elevated blood pressure at 6 weeks, an
increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in
comparison to the tissues from WKY rats was observed. The results provide, for
the first time, evidence for a parallel increase in the density of brain TRH
receptors with elevation of blood pressure, and suggest that brain TRH receptors
may play an important role in the pathophysiology of hypertension. Thyrotropin-releasing hormone (TRH) plays an important role in central
cardiovascular regulation through the activation of different neurotransmitter
systems at distinct extrahypothalamic sites. To study possible alterations in
the TRH system in the hypertensive state, we measured TRH concentration in
cerebrospinal fluid and TRH content of the preoptic area in spontaneously
hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In
addition, we also measured the density of the TRH receptor in this area by a
rapid filtration technique using [3H]methyl-TRH. We found a significant increase
in both the TRH content (634 +/- 61 versus 350 +/- 26 pg/mg protein, SHR versus
WKY; P < .01, n = 5) and density of TRH receptors without changes in affinity
(Bmax, 5.0 +/- 0.1 versus 3.3 +/- 0.1 fmol/mg protein, P < .01, n = 4). An
increase in TRH concentration was also found in the cerebrospinal fluid of SHR
(30 +/- 3 versus 21 +/- 2 pg/mL, P < .01, n = 5), suggesting increased TRH
release in the central nervous system. Northern blot analysis indicated a
threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR.
A polyclonal antibody raised against TRH injected peripherally or
intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY.
In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was
effective in inhibiting serum angiotensin-converting enzyme activity by more
than 50%, decreased arterial blood pressure and preoptic area TRH content of
SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to
produce a similar change.(ABSTRACT TRUNCATED AT 250 WORDS) We report in the present study the effect of intrathecal treatment with
antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH)
receptor mRNA on the pressor response to intrathecal administration of TRH and
on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously
hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester
antisense or mismatch oligonucleotides to TRH receptor mRNA (100 micrograms per
day) were injected intrathecally for 3 days. Twenty-four hours after the last
injection, the magnitude of the pressor response to intrathecal TRH (10
micrograms) was significantly smaller in the antisense-treated group (n = 7)
compared with mismatch-treated controls (n = 7) (change in mean arterial
pressure, +20.3 +/- 3.0 versus +32.6 +/- 2.5 mm Hg, P < .01). No differences
were observed in the pressor responses to injection of N-methyl-D-aspartic acid.
Resting arterial blood pressure was unaffected by antisense treatment in WKY
rats. In separate experiments, 16-week-old male SHR were treated with antisense
(n = 7) or mismatch (n = 6) oligonucleotides for 3 days. Mean resting arterial
blood pressure was significantly reduced by treatment with antisense
oligonucleotides (from 157 +/- 4.8 to 119 +/- 8.8 mm Hg, P < .01), but no
significant changes were observed in mismatch-treated animals. Our results
suggest that the expression of TRH receptors in spinal sympathetic preganglionic
neurons can be selectively reduced by intrathecal treatment with antisense
oligonucleotides and that TRH projections to sympathetic preganglionic neurons
play an important role in the elevation of arterial blood pressure in SHR. 1. The purpose of this study was to examine the contribution of the
sympatho-adrenomedullary system to the blood pressure response to an intravenous
bolus of thyrotropin-releasing hormone (TRH) in conscious medullectomized and
sham-operated rats. 2. The peak pressor effect of 0.5 mg TRH was significantly
increased in rats having no adrenal medulla (+24.2 +/- 1.6 mmHg, mean +/-
s.e.m., P < 0.01) as compared to sham-operated animals (+12.2 +/- 3.0 mmHg). 3.
Blockade of alpha-adrenergic receptors with phentolamine abolished the pressor
effect of TRH in control rats (+2.1 +/- 1.9 mmHg) but did not attenuate the
blood pressure response of medullectomized rats (+21.5 +/- 4.7 mmHg). In
contrast, beta-blockade with propranolol blunted the blood pressure
responsiveness of rats subjected to adrenal medullectomy (+12.4 +/- 2.6 mmHg)
but did not modify the effect of TRH in sham-operated controls (+10.9 +/- 2.9
mmHg). 4. The direct in vitro effect of TRH on isolated mesenteric rat arteries
was also evaluated. TRH did not induce contractions of isolated arteries. 5.
These results suggest that in rats with intact adrenals, the pressor effect of
intravenous TRH is mediated primarily by a stimulation of alpha-adrenergic
receptors. Adrenal medullectomy appears to enhance the blood pressure response
to intravenous TRH. Activation of cardiac beta-adrenoceptors seems to contribute
to the blood pressure increasing effect of intravenous TRH in medullectomized
animals. Thyrotropin-releasing hormone (TRH) plays an important role in central
cardiovascular regulation. Recently, we described that the TRH precursor gene
overexpression induces hypertension in the normal rat. In addition, we published
that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH
hyperactivity with increased TRH synthesis and release and an elevated TRH
receptor number. In the present study, we report that intracerebroventricular
antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH
precursor gene significantly diminished up to 72 hours and in a dose-dependent
manner the increased diencephalic TRH content, whereas normalized systolic blood
pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats.
Although basal thyrotropin was higher in SHR compared with WKY rats and this
difference disappeared after antisense treatment, no differences were observed
in plasma T4 or T3 between strains with or without AS treatment, indicating that
the effect of the AS on SABP was independent of the thyroid status. Because the
encephalic renin-angiotensin system seems to be crucial in the development
and/or maintece of hypertension in SHR, we investigated the effect of
antisense inhibition of TRH on that system and found that TRH antisense
treatment significantly diminished the elevated diencephalic angiotensin II (Ang
II) content in the SHR without any effect in control animals, suggesting that
the Ang II system is involved in the TRH cardiovascular effects. To summarize,
the central TRH system seems to be involved in the etiopathogenesis of
hypertension in this model of essential hypertension. In essential hypertension, a polygenic and multifactorial syndrome, several
genes interact with the environment to produce high blood pressure.
Thyrotropin-releasing hormone (TRH) plays an important role in central
cardiovascular regulation. We have described that TRH overexpression induces
hypertension in a normal rat, which was reversed by TRH antisense treatment.
This treatment also reduces the central TRH hyperactivity in spontaneously
hypertensive rats and normalizes blood pressure. Human TRH receptor (TRHR)
belongs to the G protein-coupled seven-transmembrane domain receptor
superfamily. Mutations of these receptors may result in constitutive activation.
As it has been demonstrated that hypertensive patients have a blunted TSH
response to TRH injection, suggesting a defect in the TRHR, we postulate that
the TRHR gene is involved in human hypertension. We studied 2 independent
populations from different geographic regions of our country: a sample of adult
subjects from a referral clinic and a population-based sample of high school
students. In search of molecular variants of TRHR, we disclosed that a
polymorphic TG dinucleotide repeat (STR) at -68 bp and a novel single nucleotide
polymorphism, a G-->C conversion at -221 located in the promoter of the TRHR are
associated with essential hypertension. As STRs detected in gene promoters are
potential Z-DNA-forming sequences and seem to affect gene expression, we studied
the potentially different transcriptional activity of these TRHR promoter
variants and found that the S/-221C allele has a higher affinity than does the
L/G-221 allele to nuclear protein factor(s). Our findings support the hypothesis
that the TRHR gene participates in the etiopathogenesis of essential
hypertension. Leptin, an adipocyte-released hormone, modifies food intake and energy
expenditure regulating hypothalamic-pituitary-thyroid axis function. We
previously reported that thyrotropin-releasing hormone (TRH) precursor gene
overexpression induces hypertension in the normal rat and that spontaneously
hypertensive rats have central TRH hyperactivity with increased TRH synthesis
and release and an elevated TRH receptor number. In both models,
intracerebroventricular antisense (AS) treatment against the TRH precursor
produced a dose-dependent reduction of the increased diencephalic TRH content
while normalizing high arterial blood pressure. In this article, we report that
male Wistar rats that were made hypertensive by intracerebroventricular
injection of a eucaryotic expression plasmid containing the pre-TRH cDNA showed
decreased leptin plasma levels and that pre-TRH AS treatment reversed this
phenomenon. In addition, male and female spontaneously hypertensive rats showed
lower levels of circulating leptin than did sex-matched Wistar-Kyoto control
rats. This difference also was abated by the pre-TRH AS treatment. Conversely,
20 microg ICV leptin induced a long-lasting pressor effect (18 +/- 5 mm Hg, n=6,
P<0.01, >60 minutes) that was not observed in pre-TRH AS pretreated rats (2 +/-
3 mm Hg, n=6) but persisted in rats used as controls that were treated with
inverted oligonucleotide (20 +/- 6 mm Hg, n=4, P<0.01). These data suggest that
in rats with TRH-induced hypertension, leptin is decreased, inducing
compensatory adiposity. We propose that because leptin produces central TRH
synthesis and release, obesity may induce hypertension through TRH system
activation and that the TRH-leptin interaction may thus contribute to the strong
association between hypertension and obesity. We recently showed that diencephalic TRH may mediate the central leptin-induced
pressor effect. Here, to study the role of TRH in obesity-induced hypertension
(OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male
Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP)
increased in HFD animals. Plasma leptin was correlated with peritoneal adipose
tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and
small interfering (si)RNA against the prepro-TRH. Antisense significantly
decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment.
Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk.
Conversely, vehicle, an inverted antisense sequence and siRNA against green
fluorescence protein, produced no changes. SABP decrease seems to be owing to an
inhibition of the obesity-enhanced sympathetic outflow but not to an alteration
in thyroid status. Using a simple OIH model we demonstrated, for the first time,
that central TRH participates in the hypertension induced by body weight gain
probably through its well-known action on sympathetic activity. Thus the
TRH-leptin interaction may contribute to the strong association between
hypertension and obesity. |
Is triadin involved in cardiac function? | Yes, triadin is involved in the regulation of cardiac excitation-contraction coupling. | OBJECTIVE: Ca2+ release from the cardiac junctional sarcoplasmic reticulum (SR)
is regulated by a complex of proteins, including the ryanodine receptor (RyR),
calsequestrin (CSQ), junctin (JCN), and triadin 1 (TRD). Moreover, triadin 1
appears to anchor calsequestrin to the ryanodine receptor.
METHODS: To determine whether triadin 1 overexpression alters
excitation-contraction coupling, we examined the effects of cardiac-specific
overexpression of triadin 1 on SR Ca2+ handling and contractility in transgenic
(TG) compared to wild-type (WT) mice.
RESULTS: The overexpression of triadin 1 was associated with an enhanced SR Ca2+
load, reflected by a 22% higher amplitude of caffeine-induced Ca2+ transients.
The decline of Ca2+ transients during caffeine exposure was prolonged by 57%.
The detection of resting spontaneous SR Ca2+ release events (Ca2+ sparks)
revealed an increased amplitude (by 16%), decline (by 47%), and width (by 47%)
in TG. This was associated with a redistribution of Ca2+ spark amplitudes from
one population to two populations. Measurement of cardiac function by
echocardiography and left ventricular (LV) catheterization revealed a decreased
cardiac contractility in vivo. The impaired response to beta-adrenergic receptor
(beta-AR) stimulation in TG hearts was associated with an increased protein
expression of beta-AR kinase 1. In addition, the increase of the L-type Ca2+
peak current and the increase of phospholamban (PLB) phosphorylation at Thr17
were reduced under beta-AR stimulation.
CONCLUSION: Taken together, our data suggest that triadin 1 overexpression
results in a complex modulation of SR Ca2+ handling, which may contribute, at
least in part, to the depressed basal contractility and the blunted response to
beta-adrenergic agonists in TG mice. Triadin is involved in the regulation of cardiac excitation-contraction
coupling. However, the extent of its contribution to the regulation of
sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression
of triadin in single-transgenic mice was associated with the downregulation of
its homologous protein, junctin. In the present study, this problem was
circumvented by cross-breeding of mice with heart-directed overexpression of
triadin and junctin (JxT). This resulted in a stable approximately threefold
expression of total triadin but unchanged junctin protein. Transgenic mice
exhibited cardiac hypertrophy and structural abnormalities of myofibrils.
Measurement of cardiac function by echocardiography and edge detection in
myocytes revealed an impaired relaxation in JxT mice. The stimulation of
beta-adrenergic receptors resulted in a depressed contractility and an impaired
relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum
stimulation frequency (5 Hz) was associated with both a lower shortening and
relengthening in isolated myocytes of JxT mice. The contractile effects in JxT
myocytes were paralleled by similar changes of the intracellular Ca
concentration ([Ca](i)) peak amplitude and Ca transient decay kinetics at basal
conditions, under administration of isoproterenol, and with high-frequency
stimulation. Finally, we found a higher caffeine-induced [Ca](i) peak amplitude
in JxT myocytes. Our data show that the stable expression of triadin,
independent of junctin expression, resulted in cardiac hypertrophy, prolonged
basal relaxation, a depressed response to beta-adrenergic agonists, and altered
Ca transients. Thus the maintece of triadin expression is essential for
normal SR Ca cycling and contractile function. |
Which disorders are associated to mutated Hepcidin (HAMP)? | Juvenile hemochromatosis (JH) is the most severe form of heriditary hemochromatosis, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). | Most hereditary hemochromatosis (HH) patients are homozygous for the C282Y
mutation of the HFE gene. Nevertheless, penetrance of the disease is very
variable. In some patients, penetrance can be mediated by concomitant mutations
in other iron master genes. We evaluated the clinical impact of hepcidin (HAMP)
and hemojuvelin mutations in a cohort of 100 Spanish patients homozygous for the
C282Y mutation of the HFE gene. HAMP and hemojuvelin mutations were evaluated in
all patients by bidirectional direct cycle sequencing. Phenotype-genotype
interactions were evaluated. A heterozygous mutation of the HAMP gene (G71D) was
found in only one out of 100 cases. Following, we performed a study of several
members of that family, and we observed several members had a digenic
inheritance of the C282Y mutation of the HFE gene and the G71D mutation of the
HAMP gene. This mutation in the HAMP gene did not modify the phenotype of the
individuals who were homozygous for the C282Y mutation. One other patient
presented a new polymorphism in the hemojuvelin gene, without consequences in
iron load or clinical course of the disease. In conclusion, HAMP and hemojuvelin
mutations are rare among Spanish HH patients, and their impact in this
population is not significant. Hereditary hemochromatosis is an iron overload disorder that can lead to the
impairment of multiple organs and is caused by mutations in one or more
different genes. Type 1 hemochromatosis is the most common form of the disease
and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the
most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin
(HAMP). The autosomal domit form of the disease, type 4, is due to mutations
in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary
hemochromatosis is commonly found in populations of European origin. By
contrast, hemochromatosis in Asia is rare and less well understood and can be
masked by the presence of iron deficiency and secondary iron overload from
thalassemia. Here, we provide a comprehensive report of hemochromatosis in a
group of patients of Asian origin. We have identified novel mutations in HJV,
HAMP, and SLC40A1 in countries not normally associated with hereditary
hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family
studies show a high degree of consanguinity, highlighting the increased risk of
iron overload in many countries of the developing world and in countries in
which there are large immigrant populations from these regions. Porphyria cutanea tarda (PCT) is a vesiculobulIous disorder often associated
with estrogens, hepatitis C virus (HCV), alcoholism, hereditary hemochromatosis
(HH), and human immunodeficiency virus. Hepcidin, a peptide hormone produced by
the liver, has been associated with iron metabolism in 3 common precipitating
factors for PCT: HCV, HH, and alcohol consumption. We present the case of a
patient with erosions and noninflammatory bullae on his hands and forearms who
received a diagnosis of PCT. On further examination, the patient was found to be
positive for 3 precipitating factors: HCV, an HH gene mutation, and alcohol use.
For patients with PCT, it is important to perform phenotypic screening for HCV
and HH. Targeting hepcidin with replacement therapy to decrease iron may be a
treatment of not only HCV, HH, and alcoholic cirrhosis, but also PCT. Author information:
(1)University Hospital of Rennes, French reference center for rare iron overload
diseases of genetic origin, Rennes, France; University of Rennes1, Inserm UMR
991, 35000 Rennes, France; University Hospital of Rennes, Liver disease
department, Rennes, France. Electronic address:
[email protected].
(2)University Hospital of Rennes, French reference center for rare iron overload
diseases of genetic origin, Rennes, France; University Hospital of Rennes, Liver
disease department, Rennes, France.
(3)University Hospital of Rennes, French reference center for rare iron overload
diseases of genetic origin, Rennes, France; University Hospital of Rennes,
Molecular Genetics Department, Rennes, France.
(4)University Hospital of Rennes, French reference center for rare iron overload
diseases of genetic origin, Rennes, France; University of Rennes1, Inserm UMR
991, 35000 Rennes, France.
(5)University Hospital of Rennes, French reference center for rare iron overload
diseases of genetic origin, Rennes, France; University of Rennes1, Inserm UMR
991, 35000 Rennes, France; University Hospital of Rennes, Liver disease
department, Rennes, France. |
Which protein is the main inhibitor of protein phosphatase 1 (PP1)? | Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. | Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The
protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes'
radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a
threonyl residue; this phosphorylation was necessary for inhibition of protein
phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with
rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and
cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of
protein phosphatase-1. By the following biochemical and immunochemical criteria,
bovine adipose tissue inhibitor-1 was found to be very similar and possibly
identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1:
molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine
residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed
by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer
electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic
peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody
recognition by immunoprecipitation and immunoblotting; effect of cyanogen
bromide cleavage on protein phosphatase inhibitor activity. Based on these
results we conclude that bovine brain and adipose tissue contain an identical
phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct
from that of skeletal muscle (inhibitor-1). Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major
eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas
phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on
Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase
(A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase
that phosphorylates Ser(67) had not been identified and the physiological role
of Ser(67) phosphorylation was unknown. In this study we detected a high level
of kinase activity in brain extract when a glutathione S-transferase (GST)
fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was
used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein
kinase (NCLK) in the brain extract could not be separated from each other by a
series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated
with anti-NCLK antibody from kinase-active column fractions. Purified
NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of
I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed
mutagenesis determined that NCLK phosphorylates Ser(67) of I-1.
NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values
approximately 9.5 and 13. 8 nM, respectively. When compared, A
kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than
NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1
kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1. Inhibitor-1 (I-1) is a selective inhibitor of protein phosphatase-1 (PP1) and
regulates several PP1-dependent signaling pathways, including cardiac
contractility and regulation of learning and memory. The human I-1 gene has been
spliced to generate two alternative mRNAs, termed I-1alpha and I-1beta, encoding
polypeptides that differ from I-1 in their C-terminal sequences. Reverse
transcription-PCR established that I-1alpha and I-1beta mRNAs are expressed in a
developmental and tissue-specific manner. Functional analysis of I-1 in a
Saccharomyces cerevisiae strain dependent on human I-1 for viability established
that a novel domain encompassing amino acids 77-110 is necessary for PP1
inhibition in yeast. Expression of human I-1 in S. cerevisiae with a partial
loss-of-function eukaryotic initiation factor-2alpha (eIF2alpha) kinase (Gcn2p)
mutation permitted growth during amino acid starvation, consistent with the
inhibition of Glc7p/PP1, the yeast eIF2alpha phosphatase. In contrast, human
I-1alpha, which lacks amino acids 83-134, and I-1 with C-terminal deletions were
significantly less effective in promoting yeast growth under starvation
conditions. These data suggest that C-terminal sequences of I-1 enhance
regulation of the eukaryotic eIF2alpha phosphatase. In vitro studies established
that C-terminal sequences, deleted in both I-1alpha and I-1beta, enhance PP1
binding and inhibition. Expression of full-length and C-terminally truncated I-1
in HEK293T cells established the importance of the I-1 C terminus in transducing
cAMP signals that promote eIF2alpha phosphorylation. This study demonstrates
that multiple domains in I-1 target cellular PP1 complexes and establishes I-1
as a cellular regulator of eIF2alpha phosphorylation. Transgenesis based on organ specific gene expression has provided the basis to
elucidate the functional role of proteins for the past 15 years. Using this
technology, we showed that inhibition of the protein phosphatase 1, by its
constitutively active inhibitor-1, significantly increases cardiac contractility
and calcium handling. To uncover protein changes accompanying the chronic
increases in cardiac function of these transgenic hearts, we analyzed the
cardiac proteome. Interestingly, we found significant increases in the levels of
6 proteins involved in metabolism, calcium binding and scavenging of
oxido-reductive stress. These proteins were identified as: hydroxyacyl CoA
dehydrogenase II, alpha subunit of the mitochondrial proton ATPase,
peroxiredoxin 2, a novel EF-hand containing protein-2, annexin 5, and a
previously uncharacterized cDNA. Thus, long-term cardiac specific overexpression
of the protein phosphatase 1 inhibitor-1 and the associated increases in cardiac
contractility appear to herald changes in a rather small number of proteins,
which may reflect important compensatory adaptations in a hyperdynamic heart
[corrected] The type 1 protein phosphatase (PP1) is a critical negative regulator of Ca(2+)
cycling and contractility in the cardiomyocyte. In particular, it mediates
restoration of cardiac function to basal levels, after beta-adrenergic
stimulation, by dephosphorylating key phospho-proteins. PP1 is a holoenzyme
comprised of its catalytic and auxiliary subunits. These regulatory proteins
dictate PP1's subcellular localization, substrate specificity and activity.
Amongst them, inhibitor-1 is of particular importance since it has been
implicated as an integrator of multiple neurohormonal pathways, which finely
regulate PP1 activity, at the level of the sarcoplasmic reticulum (SR). In fact,
perturbations in the regulation of PP1 by inhibitor-1 have been implicated in
the pathogenesis of heart failure, suggesting that inhibitor-1-based therapeutic
interventions may ameliorate cardiac dysfunction and remodeling in the failing
heart. This review will discuss the current views on the role of inhibitor-1 in
cardiac physiology, its possible contribution to cardiac disease and its
potential as a novel therapeutic strategy. Cardiac sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) promotes Ca(2+)
uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a
activity. We found a distinct dephosphorylation of PLB at Thr(17) and Ser(16)
after 20-30min of ischemia produced by coronary artery occlusion in rats. The
aim of the study was to investigate how PLB is dephosphorylated in ischemia and
to determine whether PLB dephosphorylation causes myocardial hypercontraction
and calpain activation through Ca(2+) overload in reperfusion. Protein
inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the
predomit PLB phosphatase in heart. A Ca(2+)-dependent phosphatase calcineurin
may also induce PLB dephosphorylation. Ischemia for 30min induced PKC-α
translocation, resulting in inactivation of I-1 through PKC-α-dependent
phosphorylation at Ser(67). The PP1 activation following I-1 inactivation was
thought to induce PLB dephosphorylation in ischemia. Ischemia for 30min
activated calcineurin, and pre-treatment with a calcineurin inhibitor,
cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at
Ser(67), and PLB dephosphorylation in ischemia. Reperfusion for 5min following
30min of ischemia induced spreading of contraction bands (CBs) and proteolysis
of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of
PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These
results reveal a novel pathway via which ischemia induces calcineurin-dependent
activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation
at Ser(67), and PP1-dependent PLB dephosphorylation. The pathway contributes to
the spreading of CBs and calpain activation through Ca(2+) overload in early
reperfusion. |
What is the role of NETs in systemic lupus erythematosus? | Neutrophil extracellular traps (NETs) are released via a novel form of cell death called NETosis. NETs, consisting of a chromatin meshwork decorated with antimicrobial peptides, play an important role in the innate response to microbial infections. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in these patients low-density granulocytes, a phenotype that correlates with disease activity. NETs are composed of secreted chromatin that may act as a source of autoantigens typical for SLE. NETs can directly damage tissues - including the endothelium - with implications for lupus nephritis and accelerated atherosclerosis. | Systemic lupus erythematosus (SLE) is a systemic autoimmune disease
characterized by a breakdown of tolerance to nuclear antigens and the
development of immune complexes. Genomic approaches have shown that human SLE
leukocytes homogeneously express type I interferon (IFN)-induced and
neutrophil-related transcripts. Increased production and/or bioavailability of
IFN-α and associated alterations in dendritic cell (DC) homeostasis have been
linked to lupus pathogenesis. Although neutrophils have long been shown to be
associated with lupus, their potential role in disease pathogenesis remains
elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I
IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies,
releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as
large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake
and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs
activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like
receptor 9)-dependent manner. Our results reveal an unsuspected role for
neutrophils in SLE pathogenesis and identify a novel link between nucleic
acid-recognizing antibodies and type I IFN production in this disease. Ongoing inflammation including activation of the complement system is a hallmark
of systemic lupus erythematosus (SLE). Antimicrobial neutrophil extracellular
traps (NETs) are composed of secreted chromatin that may act as a source of
autoantigens typical for SLE. In this study, we investigated how complement
interacts with NETs and how NET degradation is affected by complement in SLE
patients. We found that sera from a subset of patients with active SLE had a
reduced ability to degrade in vitro-generated NETs, which was mostly restored
when these patients were in remission. Patients that failed to degrade NETs had
a more active disease and they also displayed lower levels of complement
proteins C4 and C3 in blood. We discovered that NETs activated complement in
vitro and that deposited C1q inhibited NET degradation including a direct
inhibition of DNase-I by C1q. Complement deposition on NETs may facilitate
autoantibody production, and indeed, Abs against NETs and NET epitopes were more
pronounced in patients with impaired ability to degrade NETs. NET-bound
autoantibodies inhibited degradation but also further increased C1q deposition,
potentially exacerbating the disease. Thus, NETs are a potent complement
activator, and this interaction may play an important role in SLE. Targeting
complement with inhibitors or by removing complement activators such as NETs
could be beneficial for patients with SLE. PURPOSE OF REVIEW: Historically, researchers have focused on the role of
adaptive immunity in lupus pathogenesis; recently, however, there has been
renewed interest in the contributions of a prototypical innate immune cell - the
neutrophil.
RECENT FINDINGS: Neutrophil extracellular traps (NETs) are released via a novel
form of cell death called NETosis. NETs, consisting of a chromatin meshwork
decorated with antimicrobial peptides, play an important role in the innate
response to microbial infections. Some lupus patients do not clear NETs
normally, a phenotype that correlates with disease activity. Further, lupus
neutrophils - and, in particular, an aberrant subset called low-density
granulocytes - have an increased propensity to undergo NETosis. Both interferon
alpha (IFNα) and immune complexes are potential triggers of enhanced NETosis in
lupus patients.
SUMMARY: NETs are a potent stimulus for IFNα release by plasmacytoid dendritic
cells, and, as such, may play an important role in propagation of the lupus
phenotype. NETs can also directly damage tissues - including the endothelium -
with implications for lupus nephritis and accelerated atherosclerosis. Whether
aberrant NETosis is sufficient to trigger systemic lupus erythematosus, and
whether inhibition of NETosis can ameliorate clinical manifestations of lupus,
are open questions, and will be exciting topics of future research. Neutrophil extracellular traps (NETs) are characterized by the presence of
extracellular DNA fibers studded with antimicrobial proteins, including
myeloperoxidase (MPO). Although NETs play an important role in the innate immune
system, the scattered extracellular enzymes, such as MPO, pose risks to the
host. Therefore, NETs are strictly regulated by DNase I in the serum, which
prevents them from persisting. Recent studies have demonstrated that
dysregulation of NETs could be involved in the pathogenesis of autoimmune
diseases, including systemic lupus erythematosus. In this review, we interpret
the association of disordered NETs with autoimmune diseases, especially
propylthiouracil-induced MPO-ANCA-associated vasculitis. Neutrophils are the most abundant leukocytes in circulation and represent one of
the first lines of defense against invading pathogens. Neutrophils possess a
vast arsenal of antimicrobial proteins, which can be released from the cell by a
death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like
structures consisting of decondensed chromatin decorated with granular and
cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have
been implicated in the organ damage of autoimmune diseases, such as systemic
lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs
may also represent an important source of modified autoantigens in SLE and SVV.
Here, we review the autoimmune diseases linked to NETosis, with a focus on how
modified proteins externalized on NETs may trigger loss of immune tolerance and
promote organ damage. Neutrophil extracellular traps (NETs) represent an important defense mechanism
against microorganisms. Clearance of NETs is impaired in a subset of patients
with systemic lupus erythematosus, and NETosis is increased in neutrophils and,
particularly, in low-density granulocytes derived from lupus patients. NETs are
toxic to the endothelium, expose immunostimulatory molecules, activate
plasmacytoid dendritic cells, and may participate in organ damage through
incompletely characterized pathways. To better understand the role of NETs in
fostering dysregulated inflammation, we examined inflammasome activation in
response to NETs or to LL-37, an antibacterial protein externalized on NETs.
Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome,
in both human and murine macrophages, resulting in release of active IL-1β and
IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7
receptor-mediated potassium efflux. NET and LL-37-mediated activation of the
inflammasome is enhanced in macrophages derived from lupus patients. In turn,
IL-18 is able to stimulate NETosis in human neutrophils. These results suggest
that enhanced formation of NETs in lupus patients can lead to increased
inflammasome activation in adjacent macrophages. This leads to release of
inflammatory cytokines that further stimulate NETosis, resulting in a
feed-forward inflammatory loop that could potentially lead to disease flares
and/or organ damage. INTRODUCTION: The ability to degrade neutrophil extracellular traps (NETs) is
reduced in a subset of patients with systemic lupus erythematosus (SLE). NETs
consist of chromatin covered with antimicrobial enzymes and are normally
degraded by DNase-I, an enzyme which is known to have reduced activity in SLE.
Decreased ability to degrade NETs is associated with disease activity. In the
current study we investigated how the ability of serum from SLE patients to
degrade NETs varies during the course of SLE as well as what impact this may
have for the clinical phenotype of SLE.
METHODS: Serum from 69 patients with SLE, included in a prospective study, was
taken every 60 days for a median of 784 days. The ability of serum to degrade
NETs was determined and associated with clinical parameters occurring before and
at the time of sampling, as well as after sampling by using conditional logistic
regression.
RESULTS: As many as 41% of all patients in the study showed decreased ability to
degrade NETs at least once, but with a median of 20% of all time points.
Decreased degradation was associated with manifestations of glomerulonephritis
as well as low complement levels and elevated levels of antibodies directed
against histones and DNA. Furthermore, the odds ratio for the patient to develop
alopecia and fever after an episode of decreased NETs degradation was increased
by four to five times compared to normal.
CONCLUSIONS: Decreased degradation of NETs is associated with clinical
manifestations in SLE and may contribute to disease pathogenesis. Potential
therapeutics restoring the ability to degrade NETs could be beneficial for
certain patients with SLE. Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis
of systemic Lupus erythematosus (SLE), since netting neutrophils release
potentially immunogenic autoantigens including histones, LL37, human neutrophil
peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic
cells resulting in aggravation of inflammation and disease. How suppression of
NET formation can be targeted for treatment has not been reported yet. Signal
Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively
expressed on phagocytes. We recently identified SIRL-1 as a negative regulator
of human neutrophil function. Here, we determine whether ligation of SIRL-1
prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils
from SLE patients with mild to moderate disease activity and healthy donors were
freshly isolated. NET release was assessed spontaneously or after exposure to
anti-neutrophil antibodies or plasma obtained from SLE patients. The formation
of NETs was determined by microscopic evaluation using DNA dyes and
immunostaining of NET components, as well as by live cell imaging. We show that
SLE neutrophils spontaneously release NETs. NET formation is enhanced by
stimulation with antibodies against LL37. Inhibition of nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents
NET release in response to these antibodies. Signaling via the inhibitory
receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies.
Both spontaneous and anti-neutrophil antibody-induced NET formation is
suppressed by engagement of SIRL-1. Furthermore, NET release by healthy
neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1
engagement can dampen spontaneous and anti-neutrophil antibody-induced NET
formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity.
Together, these findings reveal a regulatory role for SIRL-1 in NET formation,
potentially providing a novel therapeutic target to break the pathogenic loop in
SLE. OBJECTIVES: A decreased ability to degrade neutrophil extracellular traps (NETs)
is seen in a subgroup of patients with systemic lupus erythematosus (SLE) and
correlates with the presence of autoantibodies. Antiphospholipid syndrome (APS)
can develop secondary to SLE or as a primary disease. In the current study we
investigated the ability of sera from patients with APS to degrade NETs. The
presence of antibodies against NETs and neutrophil remts were also determined
in the same patients.
METHODS: In the study, 106 patients with APS (73 primary and 33 secondary), 76
patients with systemic sclerosis (SSc) and 77 healthy donors as control samples
were included. NETs generated from neutrophils isolated from healthy volunteers
were incubated with patient sera, followed by measurement of degraded NETs or
deposited IgG.
RESULTS: Sera of APS patients had a decreased ability to degrade NETs compared
to healthy controls, with no difference between primary and secondary APS. Sera
from SSc patients did not differ significantly from healthy controls in the
ability to degrade NETs. A decreased degradation of NETs correlated weakly to
increased levels of antibodies against NETs/neutrophil remts in patients with
primary APS, but stronger in patients with secondary APS.
CONCLUSIONS: The ability to degrade NETs is decreased in a subgroup of patients
with APS and is associated with antibodies against NETs and specific clinical
manifestations in those patients. RATIONALE: The structural and functional integrity of the endothelium is crucial
in maintaining vascular homeostasis and preventing atherosclerosis. Patients
with systemic lupus erythematosus (SLE) have an increased risk of developing
endothelial dysfunction and premature cardiovascular disease. Neutrophil
extracellular trap (NET) formation is increased in SLE and has been proposed to
contribute to endothelial damage, but the mechanism remains unclear.
OBJECTIVE: To determine the mechanism by which enhanced NET formation by
low-density granulocytes (LDGs) in SLE contributes to endothelial damage and
disrupts the endothelium.
RESULTS: The putative role of NET-externalised matrix metalloproteinases (MMPs)
in altering the functional integrity of the endothelium was examined. MMP-9
externalised by lupus LDGs during NET formation specifically impaired murine
aortic endothelium-dependent vasorelaxation and induced endothelial cell
apoptosis. Endothelial dysfunction correlated with the activation of endothelial
MMP-2 by MMP-9 present in NETs, while inhibition of MMP-2 activation restored
endothelium-dependent function and decreased NET-induced vascular cytotoxicity.
Moreover, immunogenic complexes composed of MMP-9 and anti-MMP-9 were identified
in SLE sera. These complexes, as well as anti-MMP-9 autoantibodies, induced
NETosis and enhanced MMP-9 activity.
CONCLUSIONS: These observations implicate activation of endothelial MMP-2 by
MMP-9 contained in NETs as an important player in endothelial dysfunction, and
MMP-9 as a novel self-antigen in SLE. These results further support that
aberrant NET formation plays pathogenic roles in SLE. OBJECTIVE: Oxidative stress and oxidized high-density lipoprotein (HDL) are
implicated as risk factors for cardiovascular disease (CVD) in systemic lupus
erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE
remains unclear. Neutrophil extracellular traps (NETs), the levels of which are
elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase
(MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized
that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC).
METHODS: Plasma MPO levels and CEC activity were examined in controls and lupus
patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from
reactive nitrogen species) were quantified in human HDL. Multivariable linear
models were used to estimate and test differences between groups. HDL was
exposed to NETs from control and lupus neutrophils in the presence or absence of
MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was
quantified after NET inhibition in vivo.
RESULTS: SLE patients displayed higher MPO levels and diminished CEC compared to
controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than
control HDL, with site-specific oxidation signatures on apolipoprotein A-I.
Experiments with human and murine NETs confirmed that chlorination was mediated
by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the
NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation. CQ
inhibited NET formation in vitro.
CONCLUSION: Active NOS, NOX, and MPO within NETs significantly modify HDL,
rendering the lipoprotein proatherogenic. Since NET formation is enhanced in
SLE, these findings support a novel role for NET-derived lipoprotein oxidation
in SLE-associated CVD and identify additional proatherogenic roles of
neutrophils and putative protective roles of antimalarials in autoimmunity. OBJECTIVE: The abnormal formation and insufficient clearance of neutrophil
extracellular traps (NETs) has been reported to be involved in the pathogenesis
of lupus nephritis (LN). The abnormal regulation of NETs may contribute to
increases in the levels of circulating cell-free DNA (cfDNA). The present study
tested the hypothesis that elevated plasma cfDNA levels are related to LN.
METHODS: Fifty-four systemic lupus erythematosus (SLE) patients and 43 control
subjects were included in this study. The cfDNA concentrations were measured
using the Picogreen Kit, the low-density granulocyte (LDG) percentage in
peripheral blood mononuclear cells (PBMCs) was tested using a flow cytometer and
the DNase I activity was measured according to the radial enzyme-diffusion
method.
RESULTS: The mean cfDNA concentration in the SLE group was 236.66±40.09 ng/mL,
which was significantly higher than that observed in the healthy control group
(187.96±40.55 ng/mL, p<0.0001). Meanwhile, the mean cfDNA concentration in the
patients with LN was significantly higher than that observed in the patients
without LN (247.27±46.79 ng/mL vs. 213.56±31.34 ng/mL, p=0.0094), and the mean
cfDNA concentration in the patients with active LN was significantly higher than
that observed in the patients with inactive LN (254.22±50.16 ng/mL vs.
215.93±29.10 ng/mL, p=0.0349). In the SLE group, the cfDNA concentration was to
positively correlate with the quantitative 24-hour urinary protein (r=0.350,
p=0.013), LDG (r=0.6361, p=0.0019) and neutrophil (r=0.5990, p<0.0001) levels
and inversely correlate with the albumin level (r=-0.500, p<0.0001) and
endogenous creatinine clearance rate (r=-0.354, p=0.044). Compared to that
observed in the control group, the SLE group exhibited a significantly increased
percentage of LDGs in PBMCs and a significantly decreased DNase I activity.
CONCLUSION: Our data indicate that elevated plasma cfDNA concentrations may be
associated with active LN and partially attributed to the abnormal regulation of
NETs in SLE patients, thus suggesting that NETs constitute an intrinsic link
between cfDNA and active LN. |
Which protein is required for Argonaute 2 recruitment to stress granules and P-bodies? | Hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and P-bodies. | Argonaute proteins are effectors of RNA interference that function in the
context of cytoplasmic ribonucleoprotein complexes to regulate gene expression.
Processing bodies (PBs) and stress granules (SGs) are the two main types of
ribonucleoprotein complexes with which Argonautes are associated. Targeting of
Argonautes to these structures seems to be regulated by different factors. In
the present study, we show that heat-shock protein (Hsp) 90 activity is required
for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological
inhibition of Hsp90 was associated with reduced microRNA- and short interfering
RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding
protein (TRBP) associates with PBs or SGs, but interestingly, protein activator
of the double-stranded RNA-activated protein kinase (PACT), another Dicer
cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs
were not dependent upon PACT (or TRBP) expression. Together, our data suggest
that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose
that Ago2 and PACT form a complex that functions at the level of SGs. Previously, we found that treatment of cells with the Hsp90 inhibitor
geldanamycin (GA) leads to a substantial reduction in the number of processing
bodies (P-bodies), and also alters the size and subcellular localization of
stress granules. These findings imply that the chaperone activity of Hsp90 is
involved in the formation of P-bodies and stress granules. To verify these
observations, we examined whether another Hsp90 inhibitor radicicol (RA)
affected P-bodies and stress granules. Treatment with RA reduced the level of
the Hsp90 client protein Argonaute 2 and the number of P-bodies. Although stress
granules still assembled in RA-treated cells upon heat shock, they were smaller
and more dispersed in the cytoplasm than those in untreated cells. Furthermore
eIF4E and eIF4E-transporter were dissociated selectively from stress granules in
RA-treated cells. These observations were comparable to those obtained upon
treatment with GA in our previous work. Thus, we conclude that abrogation of the
chaperone activity of Hsp90 affects P-body formation and the integrity of stress
granules. |
Is Mammaprint approved by the United States Food and Drug Administration? | Yes, Mammaprint has been approved by the US Food and Drug Administration. | How molecular biology can improve clinical management: the MammaPrint
experience. In the past 5 years, a number of commercialized multigene prognostic and
predictive tests have entered the complex and expanding landscape of breast
cancer companion diagnostics. These tests have used a variety of formats ranging
from the familiar slide-based assays of immunohistochemistry and fluorescence in
situ hybridization to the nonmorphology-driven molecular platforms of
quantitative multiplex real-time polymerase chain reaction and genomic
microarray profiling. In this review, 14 multigene assays are evaluated as to
their scientific validation, current clinical utility, regulatory approval
status, and estimated cost-benefit ratio. Emphasis is placed on two tests:
oncotype DX and MammaPrint. Current evidence indicates that the oncotype DX test
has the advantages of earlier commercial launch, wide acceptance for payment by
third-party payors in the U.S., ease of use of formalin-fixed paraffin-embedded
tissues, recent listing by the American Society of Clinical Oncology Breast
Cancer Tumor Markers Update Committee as recommended for use, continuous scoring
system algorithm, ability to serve as both a prognostic test and predictive test
for certain hormonal and chemotherapeutic agents, demonstrated
cost-effectiveness in one published study, and a high accrual rate for the
prospective validation clinical trial (Trial Assigning Individualized Options
for Treatment). The MammaPrint assay has the advantages of a 510(k) clearance by
the U.S. Food and Drug Administration, a larger gene number, which may enhance
further utility, and a potentially wider patient eligibility, including lymph
node-positive, estrogen receptor (ER)-negative, and younger patients being
accrued into the prospective trial (Microarray in Node-Negative Disease May
Avoid Chemotherapy). A number of other assays have specific predictive goals
that are most often focused on the efficacy of tamoxifen in ER-positive
patients, such as the two-gene ratio test and the cytochrome P450 CYP2D6
genotyping assay. BACKGROUND: Numerous studies have used microarrays to identify gene signatures
for predicting cancer patient clinical outcome and responses to chemotherapy.
However, the potential impact of gene expression profiling in cancer diagnosis,
prognosis and development of personalized treatment may not be fully exploited
due to the lack of consensus gene signatures and poor understanding of the
underlying molecular mechanisms.
METHODS: We developed a novel approach to derive gene signatures for breast
cancer prognosis in the context of known biological pathways. Using unsupervised
methods, cancer patients were separated into distinct groups based on gene
expression patterns in one of the following pathways: apoptosis, cell cycle,
angiogenesis, metastasis, p53, DNA repair, and several receptor-mediated
signaling pathways including chemokines, EGF, FGF, HIF, MAP kinase, JAK and
NF-kappaB. The survival probabilities were then compared between the patient
groups to determine if differential gene expression in a specific pathway is
correlated with differential survival.
RESULTS: Our results revealed expression of cell cycle genes is strongly
predictive of breast cancer outcomes. We further confirmed this observation by
building a cell cycle gene signature model using supervised methods. Validated
in multiple independent datasets, the cell cycle gene signature is a more
accurate predictor for breast cancer clinical outcome than the previously
identified Amsterdam 70-gene signature that has been developed into a FDA
approved clinical test MammaPrint.
CONCLUSION: Taken together, the gene expression signature model we developed
from well defined pathways is not only a consistently powerful prognosticator
but also mechanistically linked to cancer biology. Our approach provides an
alternative to the current methodology of identifying gene expression markers
for cancer prognosis and drug responses using the whole genome gene expression
data. A series of multigene classifiers, prognostic and predictive tests have recently
been introduced as potentially useful adjuncts for the management of recently
diagnosed breast cancer patients. These tests have used both slide-based methods
including immunohistochemistry and fluorescence in situ hybridization and
nonmorphology driven molecular platforms including quantitative multiplex real
time polymerase chain reaction and genomic microarray profiling. In this review,
a series of partially and completely commercialized multigene assays are
compared with the standard breast cancer clinico-pathologic variables and
biomarkers and evaluated as to the level of their scientific validation, current
clinical utility, regulatory approval status, and estimated cost-benefit. A
comparison of the Oncotype Dx and MammaPrint assays indicates that the Oncotype
Dx test has the advantages of an earlier commercial launch in the US, wide
acceptance for payment by third party payors, the ease of use of formalin fixed
paraffin embedded tissues, a recommendation as ready for use by the American
Society of Clinical Oncology Breast Cancer Tumor Markers Update Committee, a
continuous rather than dichotomous algorithm, inclusion of both estrogen
receptor (ER) and human epidermal growth factor receptor 2 in the mRNA profile,
an ability to serve as both a prognostic and predictive test for certain
hormonal and chemotherapeutic agents, demonstrated cost-effectiveness in 1
published study, and a high accrual rate for the prospective validation clinical
trial (Trial Assigning Individualized Options for Treatment Rx). The MammaPrint
assay has the advantages of a 510(k) clearance by the US Food and Drug
Administration, a larger gene number which may enhance further utility, and the
potentially wider patient eligibility including lymph node-positive,
ER-negative, and younger patients being accrued into the prospective trial (the
Microarray in Node-negative Disease may Avoid ChemoTherapy). A number of other
assays have specific predictive goals most often focused on the efficacy of
tamoxifen in ER-positive patients such as the Two-gene Ratio test and the
Cytochrome P450 CYP2D6 genotyping assay. Gene expression assays that are used in daily clinical practice for treating
early breast cancer patients have been introduced in the clinic only recently.
This review discusses the development of these arrays, summarizes the validation
of those that are commercially available and indicates how the information
provided by these assays can help in the care of patients. The review also
provides an extensive overview of commercially available assays focusing on
MammaPrint, the first and only assay for breast cancer management that has been
cleared by the FDA. |
What is known as Von Hippel–Lindau disease or syndrome? | von Hippel-Lindau (VHL) disease is a rare, autosomal dominantly inherited multisystem disorder characterized by development of a variety of benign and malignant tumors, which are usually accompanied with cysts. The spectrum of clinical manifestations of the disease is broad and includes retinal and central nervous system hemangioblastomas, endolymphatic sac tumors, renal cysts and tumors, pancreatic cysts and tumors, pheochromocytomas, and epididymal cystadenomas. The most common causes of death in VHL disease patients are renal cell carcinoma and neurologic complications from cerebellar hemangioblastomas. von Hippel-Lindau (VHL) syndrome is associated with mutations of the VHL tumor suppressor gene (3p25-26). Its estimated incidence ranges from 1 in 36,000 to 1 in 53,000 with a penetrance of up to 95% by age 60. The VHL tumour suppressor gene, responsible for the disease, encodes for a major regulator of the hypoxic response by targeting the transcription factor hypoxia inducible factor (HIF) for degradation. Loss of pVHL leads to activation of the HIF pathway in normoxia with the concomitant increase in tumour vascularisation due to the up-regulation of pro-angiogenic genes.In addition, many HIFalpha-independent functions of pVHL have recently been identified. These include microtubule-based processes, extracellular matrix assembly and suppression of kidney cyst formation. | von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ
line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors
observed in this disorder include retinal and central nervous system
hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL
gene product, pVHL, is a component of a ubiquitin ligase which targets the
transcription factor known as hypoxia-inducible factor (HIF) for degradation in
the presence of oxygen. pVHL also plays roles in the control of extracellular
matrix formation and cell-cycle exit. Different VHL mutations confer different
site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of
pheochromocytoma without the other stigmata of VHL disease. Here we report that
the products of such type 2C VHL alleles retain the ability to down regulate HIF
but are defective for promotion of fibronectin matrix assembly. Furthermore,
pVHL L188V, a well studied type 2C mutant, retained the ability to suppress
renal carcinoma growth in vivo. These studies strengthen the notion that HIF
deregulation plays a causal role in hemangioblastoma and renal carcinoma, and
raises the possibility that abnormal fibronectin matrix assembly contributes to
pheochromocytoma pathogenesis in the setting of VHL disease. PURPOSE OF REVIEW: von Hippel-Lindau disease is an inherited, multisystemic
cancer syndrome often involving the retina. This review will provide an update
for the clinical characterization and treatment of ocular von Hippel-Lindau
disease.
RECENT FINDINGS: A comprehensive and quantitative clinical characterization of
ocular von Hippel-Lindau disease has been limited by small patient numbers and
nonrepresentative sampling. Recently, a large population of patients with
clinically and genetically defined von Hippel-Lindau disease was systemically
characterized in a single center, enabling a quantitative evaluation of the
ocular involvement of this syndrome. Correlations between the nature of von
Hippel-Lindau gene mutations and the ocular phenotype were also examined,
providing clues as to how disruptions in von Hippel-Lindau protein function may
result in eye disease. This understanding may be relevant to the development of
new therapies targeting the molecular biology of von Hippel-Lindau disease, some
of which are presently being investigated.
SUMMARY: Quantitative studies enable a full characterization of the impact of
von Hippel-Lindau disease on eye health and visual function. Establishing
correlations between the genotype of the von Hippel-Lindau mutation and the
phenotype of eye disease may inform us as to how ocular von Hippel-Lindau
disease arises, and help guide molecular interventions in ocular von
Hippel-Lindau disease. von Hippel-Lindau (VHL) disease is an inherited multisystem familial cancer
syndrome caused by mutations of the VHL gene on chromosome 3p25. A wide variety
of neoplastic processes are known to be associated with VHL disease. The
consequences of the VHL mutations and the pathway for tumor development continue
to be elucidated. This paper will detail the variety of tumors associated with
VHL disease and discuss the genetic mechanisms that lead to the predisposition
for neoplasia. Germ line inactivation of the von-Hippel-Lindau (VHL) tumor suppressor gene
causes von Hippel-Lindau hereditary cancer syndrome, and somatic mutations of
this gene have been linked to the development of sporadic hemangioblastomas and
clear cell renal carcinomas. The protein encoded by VHL, pVHL, has no known
enzymatic activities but interacts with various partner proteins. In this
review, various pVHL functions are highlighted. pVHL acts as a multi-purpose
adaptor protein that controls different gene expression programs. Through its
oxygen-dependent regulation of hypoxia-inducible factor alpha (HIFalpha), pVHL
plays a central role in the oxygen-sensing pathway. In addition, many
HIFalpha-independent functions of pVHL have recently been identified. These
include microtubule-based processes, extracellular matrix assembly and
suppression of kidney cyst formation. These complex pVHL functions can explain
the diverse consequences of pVHL dysregulation in tumor formation and
progression. Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene is
responsible for the development of renal carcinomas, pheochromocytomas and
tumours in other organs. The gene product (pVHL) is a central component in the
oxygen-sensing pathway through its role in the regulation of the
hypoxia-inducible factor (HIF). Loss of pVHL leads to activation of the HIF
pathway in normoxia with the concomitant increase in tumour vascularisation due
to the up-regulation of pro-angiogenic genes. However, although the role of pVHL
in the regulation of HIF has proved to be important for tumour growth, other
pVHL functions independent of HIF have been reported and help to explain why
loss of VHL leads to renal cancer. Studies aimed to characterise other molecular
pathways that shed light on its physiological roles as a gatekeeper gene in
kidney and other organs will be very helpful for the development of novel
anticancer therapies. PURPOSE: von Hippel-Lindau (VHL) disease is a domitly inherited,
multisystemic tumor syndrome caused by mutations in the VHL gene. This study was
conducted to establish genotype-phenotype correlations between the positions of
disease-causing missense mutations and the ocular phenotypes of VHL disease.
METHODS: Participants with clinically defined VHL disease and documented
germline missense mutations in the VHL gene were identified in a cross-sectional
study (n=412). Statistical analysis was used to correlate the position of the
missense mutation in either the alpha- or beta-domain of the VHL protein with
the ocular disease phenotype.
RESULTS: Missense mutations among study participants were located in 47 of the
213 possible codons in the VHL gene. Almost all mutations (98.5%) were located
in one of two structural domains of the VHL protein: the alpha- and
beta-domains. alpha-Domain mutations were significantly associated with a higher
prevalence of retinal capillary hemangioblastomas (RCHs) compared with the
beta-domain mutations (P=0.016). Among patients with RCHs, the prevalence of the
lesions in the juxtapapillary position was also significantly higher in patients
with alpha-domain mutations (P=0.0017). Conversely, beta-domain mutations
correlated with a higher prevalence of peripherally located RCHs (P=0.0104).
CONCLUSIONS: The location of missense mutations in the VHL gene correlates
significantly with the prevalence and phenotype of ocular disease, and as such,
influences the risk of visual loss in affected patients. These
genotype-phenotype correlations can assist in the prognostic counseling and
follow-up of VHL patients and may provide a basis for molecular inferences on
ocular VHL disease pathogenesis. Patients with von Hippel-Lindau disease (VHL) often harbor significant disease
burden within the CNS, specifically craniospinal-axis hemangioblastomas and
endolymphatic sac tumors (ELSTs). The majority (60-80%) of patients with VHL
harbor hemangioblastomas, and 10-15% will develop ELSTs. Advances in the
understanding of the natural history and outcomes associated with the surgical
management of VHL-associated tumors have led to improved management of patients
with VHL. Optimizing indications for surgical intervention and refining of
surgical techniques for these lesions can reduce patient morbidity associated
with the management of this syndrome. In this article, we review the various
aspects of perioperative management of patients with VHL, surgical indications
and general operative principles for the management of hemangioblastomas and
ELSTs, and outcomes associated with the surgical treatment of these tumors. PURPOSE: Von Hippel-Lindau (VHL) disease is an inherited syndrome caused by
germline mutations in the VHL tumor suppressor gene, predisposing to a variety
of neoplasms including pancreatic neuroendocrine tumors (PanNET). In VHL
disease, PanNET probably progress according to a specific pathway of
carcinogenesis. Our aim was to characterize by molecular quantitative analysis a
panel of molecules implicated in the VHL pathway and in tumor progression in the
PanNET of patients with VHL.
EXPERIMENTAL DESIGN: The expression of 52 genes was studied by quantitative
reverse transcriptase PCR in 18 patients with VHL operated on for PanNET and
compared with 16 non-VHL PanNET. The VHL and non-VHL tumors were matched
according to their size and cell proliferation. For some genes, we looked for
differences in the protein expression in VHL PanNET (n = 31), microadenomas (n =
22), and non-VHL PanNET (n = 16), included in tissue microarray blocks.
RESULTS: Nineteen (36%) genes were significantly upregulated and three (6%)
downregulated in VHL PanNET. The upregulated genes were related to (i)
hypoxia-inducible factor (HIF) molecules (CA9, HIF2A, and GLUT1), (ii)
angiogenesis (CDH5, VEGFR1, EDNRA, ANGPT2, CD34, VEGFR2, VEGFA, and ANGPT1),
(iii) the processes of epithelial-mesenchymal transition (VIM) and/or metastasis
(LAMA4 and CXCR4), (iv) growth factors and receptors (PDGFB, IRS1, and ERBB1),
or (v) cell cycle (CCND1 and CDKN2A). The downregulated genes were related to
(i) EMT (OCLN) and (ii) signaling pathways (RPS6KB1 and GADD45B).
CONCLUSION: This study shows that the progression of PanNET in patients with VHL
tumors follows a specific pathway and supports that targeting molecules
specifically involved may be of therapeutic importance. von Hippel-Lindau (VHL) disease is an inheritable multisystem tumor syndrome
characterized by multiple benign and maligt tumors affecting multiple organs.
VHL is the result of a germline mutation in the VHL tumor suppressor gene.
Molecular genomic analysis routinely confirms the clinical diagnosis. However,
the use of molecular diagnostic methods can often be insufficient for the
detection of mosaic germline VHL mutations, making the diagnosis of some cases
of VHL difficult. Here, we report the case of a VHL mosaic patient with
bilateral renal lesions in the absence of other VHL-associated lesions. A VHL
mutation was not originally detected by routine molecular testing. Nonetheless,
the detection of a heterozygous c.194C>G (p.Ser65Trp) VHL mutation in the
patient's daughter prompted further genetic assessment and eventually resulted
in the finding of a mosaic c.194C>G (p.Ser65Trp) VHL mutation in the patient.
The mutation rate was 18.8 ± 3.84% in peripheral leukocytes. As the frequency of
VHL mosaicism remains underdetermined, the possibility of a diagnosis of mosaic
VHL should be considered in patients with both typical and atypical
VHL-associated manifestations. von Hippel-Lindau (VHL) disease is an autosomal-domit familial cancer
syndrome associated with mutations of the VHL tumor suppressor gene (3p25-26).
Its estimated incidence ranges from 1 in 36,000 to 1 in 53,000 with a penetrance
of up to 95% by age 60. Genotype-phenotype correlation divides VHL into two
broad clinical subtypes. Type 1 VHL is predomitly associated with large
deletion or truncation mutations which result in an encoded protein with very
little or no activity. It is associated with retinal and CNS hemangioblastoma
and renal cell carcinoma but not pheochromocytoma. Type 2 is usually associated
with missense mutations encoding a protein with limited activity and includes
pheochromocytoma. It is further classified into three other subtypes (2A, 2B,
2C) based on the presence of hemangioblastoma and renal cell carcinoma. Visceral
cysts in the kidney, pancreas and epididymis, nonfunctioning pancreatic
neuroendocrine tumors which often show distinctive clear cell cytology,
endolymphatic sac tumors and head and neck paragangliomas are well recognized
but less common presenting features. Surveillance of carriers can reduce the
burden of disease and is best performed in specialist referral centers with due
consideration given to both the complex molecular pathogenesis and psychosocial
aspects of the disease. BACKGROUND: Von Hippel-Lindau Disease (VHL) is an autosomal domit inherited
systemic cancer syndrome that gives rise to cystic and highly vascularized
tumors in many organs, including the eye. Recent studies have contributed to the
understanding of VHL pathophysiology, genetics, and the role of the VHL protein.
This article reviews recent studies on VHL clinical findings, genetics and
tumorigenesis.
METHODS: Literature review of articles on VHL genetics with correlation to
clinical findings.
RESULTS: Genotype-phenotype correlation studies show that patients with a
complete deletion mutation of the VHL gene, relative to participants with a
missense or protein-truncating mutation, had better visual acuity and decreased
tumorigenesis incidence of retinal hemangioblastomas. It has also been
documented that higher levels of vascular endothelial growth factor (VEGF),
hypoxia induced factor (HIF), and ubiquitin are found in ocular
hemangioblastomas. The stromal foamy vacuolated cells seem to be the true tumor
cells of the disease acting on the surrounding endothelial cells in ocular
hemangioblastomas. Tumor cells and ocular lesions have shown increased levels of
Erythropoietin (Epo), Epo receptor (EpoR), and CD133. Also, CXCR4, a CXC
chemokine receptor, is expressed in retinal VHL hemangioblastomas. Recent
studies suggest that the VHL mutation alone may not be sufficient to develop
VHL-associated neoplasms. Studies suggest that targeting various proteins along
with anti-angiogenesis molecules may be a better therapeutic approach than
targeting VEGF alone.
CONCLUSION: Understanding of the mechanisms and genetics underlying VHL and its
associated retinal hemangioblastomas has increased substantially in recent
years. This knowledge suggests that future advances may include better
identification of individuals at higher risk of vision loss and the development
of novel individualized therapies. Von Hippel-Lindau syndrome is an autosomal domit inherited phacomatosis with
a predisposition for the central nervous system and retina. There is variable
expression with hemangioblastomas in the brain, medulla oblongata, spinal chord,
renal carcinoma, pheochromocytoma, pancreatic cysts and islet cell tumors as
well as tumors of the endolymphatic sac of the inner ear. Clinical symptoms
occur first after an age of approximately 30 years. Magnetic resoce imaging
(MRI) of the neuroaxis is indicated in all patients with a suspicion of von
Hippel-Lindau syndrome. |
Is HER2 active only when it dimerizes? | Yes, HER2 activation is driven by the formation of various dimer complexes between members of this receptor family. | The human epidermal growth factor receptor (HER) family plays an important role
in cell survival and proliferation, and is implicated in oncogenesis.
Overexpression of HER2 is associated with aggressive disease and poor prognosis.
Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven
survival benefit for women with HER2-positive early and metastatic breast
cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization
inhibitor that binds to a different epitope on HER2 than trastuzumab and
inhibits HER2 dimer formation with other HER family members such as HER3 and
HER1. We investigated the antitumor activity of these agents alone and in
combination in HER2-positive breast and non-small cell lung cancer xenografts.
Our data show that the combination of trastuzumab and pertuzumab has a strongly
enhanced antitumor effect and induces tumor regression in both xenograft models,
something that cannot be achieved by either monotherapy. The enhanced efficacy
of the combination was also observed after tumor progression during trastuzumab
monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab
binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we
show by in vitro assay that both trastuzumab and pertuzumab potently activate
antibody-dependent cellular cytotoxicity. However, our data suggest that the
strongly enhanced antitumor activity is mainly due to the differing but
complementary mechanisms of action of trastuzumab and pertuzumab, namely
inhibition of HER2 dimerization and prevention of p95HER2 formation. INTRODUCTION: Patients with human epidermal growth factor receptor 2
(HER2)-positive breast cancer have a poor prognosis. Despite proven efficacy
with the standard of care, trastuzumab, most patients with HER2-positive
metastatic breast cancer (MBC) will eventually progress, requiring further
intervention to prolong life. Pertuzumab, a humanized monoclonal antibody, is
the first HER2 dimerization inhibitor. It binds to the dimerization site on the
HER2 domain and prevents ligand-driven pairing of HER2 with other HER receptors,
thus inhibiting tumor cell growth and survival. Combining trastuzumab and
pertuzumab therefore provides a more comprehensive blockade of the HER signaling
pathways, resulting in greater antitumor efficacy.
STUDY DESIGN: CLEOPATRA (CLinical Evaluation Of Pertuzumab And TRAstuzumab) is
an international, randomized, double-blind, placebo-controlled phase III trial
that aims to recruit approximately 800 patients from around 250 centers
worldwide. Adults with HER2-positive adenocarcinoma of the breast with locally
recurrent or metastatic disease will be randomized (1:1) to receive docetaxel,
trastuzumab, and pertuzumab or docetaxel, trastuzumab, and placebo. The primary
endpoint of the study is progression-free survival (PFS), based on tumor
assessments conducted by an independent review facility. Secondary endpoints
include PFS evaluated by investigator assessment, overall survival, objective
response rate, duration of response, time to symptom progression, and safety.
Data will also be collected to identify biomarkers that may correlate with
clinical outcomes.
CONCLUSION: The CLEOPATRA study will provide important information about the
efficacy and safety of adding pertuzumab to one current standard of care in
patients with HER2-positive MBC. The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine
kinase receptor overexpressed in 30% of human breast cancers. One of the
mechanisms by which tumor cell proliferation can be inhibited consists in
hampering HER2 dimerization by targeting its extracellular domain with specific
antibodies. In recent clinical practice, a valuable alternative to entire IgGs
resides in the use of smaller molecules, such as single-chain variable fragments
(scFv), developed for selective molecular targeting. In this paper, we report on
the production and purification of a soluble anti-HER2 scFv antibody secreted by
Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at
the 3'-end was inserted into the expression vector pPICZα and transformed in P.
pastoris. The highest expression level was obtained in presence of 0.5% methanol
and 0.8% glycerol in the culture medium after 48 h of induction. The use of P.
pastoris proved very valuable as an expression system, allowing the isolation of
10 mg/L of highly purified antibody, remarkably higher than previously reported
data. The functionality of purified anti-HER2 scFv was assessed by
cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer
cells, showing good affinity and high selectivity for the target membrane
receptor. These findings confirm that P. pastoris is a suitable host for high
level expression of antibody fragments and highlight the potential role of
scFv800E6 in diagnostic and therapeutic application. Unlike early stage breast cancer, metastatic breast cancer (MBC) is generally
considered incurable except for a small number of patients with oligometastatic
disease. The goal of treatment of MBC should be the prolongation of life and
improvement of symptoms and quality of life. The prognoses of patients with MBC,
however, have been improved with the introduction of newer, more effective
agents. Therefore, the clinical question arises whether MBC patients can be
cured with these new therapeutic agents. However, there are a couple of problems
in tackling this question, including the duration of follow-up and the presence
of strong adjuvant therapy. Firstly, most trials in MBC have a relatively short
follow-up; long-term surveillance (>3-5 years) is exceptional, so little is
known about the definitive outcome and the exact proportion of long-term
survivors. Secondly, most of the patients have received pre- or postoperative
adjuvant therapy. The cancer cells at metastatic sites are considered to be
relatively resistant to the agents used in metastatic settings. Promisingly, a
number of novel therapeutic agents including antibody-drug conjugates,
irreversible small molecule HER2-tyrosine inhibitors, and HER2 dimerization
inhibitors show promise in the treatment of HER2-overexpressing MBC, as well as
PARP-1 [poly(ADP-ribose) polymerase-1] inhibitors for triple-negative breast
cancer. The expression level of the HER family is unreliable as a predictive marker for
targeted therapies in cancer. Thus, there is a need to develop other biomarkers,
which can be used to accurately select responsive patients for targeted
therapies. The HER dimerization status may be more important than HER receptor
expression per se in determining sensitivity or resistance to a given
therapeutic agent. The aim of the study is to develop a FRET assay using dye
conjugated secondary antibodies to assess HER receptor dimerization. Using
primary antibodies from different species in conjunction with Alexa488 and
Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2
dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2
positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay
to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results
show promise for the assay to be applied to tumor samples in order to assess the
prognostic significance and predictive value of HER receptor dimerization in
various cancers. Following the development of targeted therapies against EGFR and HER2, two
members of the human epidermal receptor (HER) family of receptor tyrosine
kinases, much interest has been focused on their expression in tumors. However,
knowing the expression levels of individual receptors may not be sufficient to
predict drug response. Here, we describe the development of antibody-based
time-resolved Förster resoce energy transfer (TR-FRET) assays for the
comprehensive analysis not only of EGFR and HER2 expression in tumor
cryosections, but also of their activation through quantification of HER homo-
or heterodimers. First, EGFR and HER2 expression levels were quantified in 18
breast tumors and the results were compared with those obtained by using
reference methods. The EGFR number per cell determined by TR-FRET was
significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our
method detected HER2 overexpression with 100% specificity and sensibility, as
confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2
dimerization was then assessed, using as controls xenograft tumors from cell
lines with known dimer expression profiles. Our results show that quantification
of HER dimerization provides information about receptor activation that cannot
be obtained by quantification of single receptors. Quantifying HER expression
and dimerization by TR-FRET assays might help identifying novel clinical markers
for optimizing patients' treatment in oncology. Dimerization among the EGFR family of tyrosine kinase receptors leads to
allosteric activation of the kinase domains of the partners. Unlike other
members in the family, the kinase domain of HER3 lacks key amino acid residues
for catalytic activity. As a result, HER3 is suggested to serve as an allosteric
activator of other EGFR family members which include EGFR, HER2 and HER4. To
study the role of intracellular domains in HER3 dimerization and activation of
downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by
replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the
C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric
receptors in Chinese hamster ovary (CHO) cells. While over expression of the
intact human HER3 transformed CHO cells with oncogenic properties such as
AKT/ERK activation and increased proliferation and migration, CHO cells
expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2
dimerization and decreased phosphorylation of both AKT and ERK1/2 in the
presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2
chimeric receptor resulted in a total loss of downstream AKT activation in
response to NRG-1, but maintained partial activation of ERK1/2. The results
demonstrate that the intracellular domains play a crucial role in HER3's
function as an allosteric activator and its role in downstream signaling. Aberrant expression and activation of EGFR and ERBB2 (HER2) have been
successfully targeted for cancer therapeutics. Recent evidence from both basic
and clinical studies suggests that ERBB3 (HER3) serves as a key activator of
downstream signaling through dimerization with other ERBB proteins and plays a
critical role in the widespread clinical resistance to EGFR and HER2 targeting
cancer therapies. As a result, HER3 is actively pursued as an antibody
therapeutic target for cancer. Ligand binding is thought to be a prerequisite
for dimerization of HER3 with other ERBB proteins, which results in
phosphorylation of its c-terminal tyrosine residues and activation of downstream
AKT and MAPK signaling pathways. In this study, we report that an anti-HER2
monoclonal antibody (HER2Mab), which blocks HER2 dimerization with HER3, induces
HER3 dimerization with EGFR in both low and high HER2 expressing cancer cells.
Treatment of the low HER2 expressing MCF7 cancer cells with HER2Mab promoted
cell proliferation and migration in the absence of HER3 ligand stimulation.
Follow-up studies revealed that HER2Mab-induced HER3 signaling via EGFR/HER3
dimerization and activation of downstream AKT signaling pathways. These results
suggest that equilibrium of dimerization among the ERBB proteins can be
perturbed by HER2Mab and HER3 plays a key role in sensing the perturbation. The therapeutic potential of anticancer antibodies is limited by the resistance
of tumor cells to complement-mediated attack, primarily through the
over-expression of membrane complement regulatory proteins (mCRPs: CD46, CD55
and CD59). Trastuzumab, an anti- HER2 monoclonal antibody, approved for the
treatment of HER2-positive breast and gastric cancers, exerts only minor
complement-mediated cytotoxicity (CDC). Pertuzumab is a novel anti-HER2
monoclonal antibody, which blocks HER2 dimerization with other ligand-activated
HER family members. Here, we explored the complement-mediated anti-tumor effects
of trastuzumab and pertuzumab on HER2-positive tumor cells of various
histological origins. Delivery of chemically stabilized anti-mCRP siRNAs using
cationic lipoplexes, AtuPLEXes, to HER2-over-expressing BT474, SK-BR-3 (breast),
SKOV3 (ovarian) and Calu-3 (lung) cancer cells reduced mCRPs expression by
85-95%. Knockdown of individual complement regulators variably led to increased
CDC only upon combined treatment with trastuzumab and pertuzumab. The combined
down-regulation of all the three regulators augmented CDC by 48% in BT474, 46%
in SK-BR-3 cells, 78% in SKOV3 cells and by 30% in Calu-3 cells and also
increased complement-induced apoptosis and caspase activity on mCRP neutralized
tumor cells. In addition, antibody-induced C3 opsonization of tumor cells was
significantly enhanced after mCRP silencing and further augmented tumor cell
killing by macrophages. Our findings suggest that siRNA-induced inhibition of
complement regulator expression clearly enhances complement- and
macrophage-mediated anti-tumor activity of trastuzumab and pertuzumab on
HER2-positive tumor cells. Thus - if selectively targeted to the tumor -
siRNA-induced inhibition of complement regulation may serve as an innovative
strategy to potentiate the efficacy of antibody-based immunotherapy. Human epidermal growth factor receptor 2 (HER2)-targeted therapy by trastuzumab
has become increasingly important for treating HER2-positive cancers, and
trastuzumab emtansine (T-DM1) is expected to serve as an effective alternative
to trastuzumab. Pertuzumab, a HER2 dimerization inhibitor, showed prolonged
progression-free survival when used with trastuzumab for HER2-positive breast
cancer. In this study, we investigated the effect of combining T-DM1 and
pertuzumab on xenografted gastric tumors. T-DM1 as a single agent showed
significant antitumor activity in all the three HER2-high expression tumor
models tested (NCI-N87, SCH and 4-1ST) but was ineffective against two HER2-low
expression tumors (SNU-16 and MKN-28). Using the T-DM1-sensitive NCI-N87 model,
the combination efficacy of T-DM1 and pertuzumab was elucidated. The combination
induced significant tumor regression, whereas T-DM1 or pertuzumab alone did not.
In cultured NCI-N87 cells stimulated with epidermal growth factor (EGF) or
heregulin-α, concomitant treatment of T-DM1 and pertuzumab significantly
inhibited proliferation and increased caspase 3/7 activity compared to either
agent alone. Only the combination significantly inhibited the phosphorylation of
EGFR or HER3, and its downstream factor AKT. Suppressed HER3 phosphorylation by
the combination was also seen in the NCI-N87 xenografted tumors. Compared to
single agent treatments, the combination treatment significantly enhanced
antibody-dependent cellular cytotoxicity (ADCC) against NCI-N87 cells. These
findings suggest that T-DM1 in combination with pertuzumab shows significant
antitumor activity by increasing AKT signal inhibition and ADCC in HER2-positive
gastric cancers. PURPOSE: The pharmacology, pharmacokinetics, clinical efficacy, safety, and
administration of pertuzumab in patients with metastatic human epidermal growth
factor receptor type 2 (HER2)-positive breast cancer are reviewed.
SUMMARY: Disease progression in HER2-positive breast cancer is often due to
resistance to or a lack of efficacy of trastuzumab-based anti-HER2 therapy.
Pertuzumab is the first humanized monoclonal antibody in a new class of drugs,
the HER dimerization inhibitors, approved by the Food and Drug Administration
for the first-line treatment of patients with metastatic HER2-positive breast
cancer who have not received prior anti-HER2 therapy or chemotherapy for
metastatic disease. Since pertuzumab binds to a different epitope than
trastuzumab, combination therapy with pertuzumab and trastuzumab results in a
more complete blockade of HER2 signaling than trastuzumab monotherapy. The
efficacy of adding pertuzumab to trastuzumab-docetaxel dual therapy was
demonstrated in a pivotal randomized multicenter Phase III trial, which showed a
significant benefit in terms of progression-free survival, with improved overall
survival, in favor of the triple therapy as an initial regimen in
treatment-naive patients with metastatic HER2-positive breast cancer. The
combination of pertuzumab and trastuzumab has been found to have a tolerable
toxicity profile. As clinical trials of pertuzumab for adjuvant, neoadjuvant,
and metastatic-disease treatment continue, its role in the treatment of
HER2-positive breast cancer will continue to evolve.
CONCLUSION: Pertuzumab, a novel HER2 dimerization inhibitor, has been shown to
be effective in the treatment of metastatic HER2-positive breast cancer when
used in combination with trastuzumab and docetaxel and is recommended for
first-line therapy. The HER/ErbB family of receptor tyrosine kinases drives critical responses in
normal physiology and cancer, and the expression levels of the various HER
receptors are critical determits of clinical outcomes. HER activation is
driven by the formation of various dimer complexes between members of this
receptor family. The HER dimer types can have differential effects on downstream
signaling and phenotypic outcomes. We constructed an integrated mathematical
model of HER activation, and trafficking to quantitatively link receptor
expression levels to dimerization and activation. We parameterized the model
with a comprehensive set of HER phosphorylation and abundance data collected in
a panel of human mammary epithelial cells expressing varying levels of
EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple
solutions, predictions for dimer phosphorylation were in agreement with each
other. We validated the model using experiments where pertuzumab was used to
block HER2 dimerization. We used the model to predict HER dimerization and
activation patterns in a panel of human mammary epithelial cells lines with
known HER expression levels in response to stimulations with ligands EGF and
HRG. Simulations over the range of expression levels seen in various cell lines
indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2
dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both
contribute significantly to HER2 activation with the EGFR expression level
determining the relative importance of these species, and iii) the HER2-HER3
dimer is largely responsible for HER3 activation. The model can be used to
predict phosphorylated dimer levels for any given HER expression profile. This
information in turn can be used to quantify the potencies of the various HER
dimers, and can potentially inform personalized therapeutic approaches. |
Which pharmacogenetic test is available for abacavir? | The pharmacogenetic test recommended prior to abacavir administration is the HLA B*5701 genotyping. | OBJECTIVE: Abacavir, a human immunodeficiency virus-1 (HIV-1)
nucleoside-analogue reverse transcriptase inhibitor, causes severe
hypersensitivity in 4-8% of patients. HLA B*5701 is a known genetic risk factor
for abacavir hypersensitivity in Caucasians. Our aim was to confirm the presence
of this genetic factor in our patients, and to determine whether genotyping for
HLA B*5701 would be a cost-effective use of healthcare resources.
METHODS: Patients with and without abacavir hypersensitivity were identified
from a UK HIV clinic. Patients were genotyped for HLA B*5701, and pooled data
used for calculation of test characteristics. The cost-effectiveness analysis
incorporated the cost of testing, cost of treating abacavir hypersensitivity,
and the cost and selection of alternative antiretroviral regimens. A
probabilistic decision analytic model (comparing testing versus no testing) was
formulated and Monte Carlo simulations performed.
RESULTS: Of the abacavir hypersensitive patients, six (46%) were HLA B*5701
positive, compared to five (10%) of the non-hypersensitive patients (odds ratio
7.9 [95% confidence intervals 1.5-41.4], P = 0.006). Pooling of our data on HLA
B*5701 with published data resulted in a pooled odds ratio of 29 (95% CI
6.4-132.3; P < 0.0001). The cost-effectiveness model demonstrated that depending
on the choice of comparator, routine testing for HLA B*5701 ranged from being a
domit strategy (less expensive and more beneficial than not testing) to an
incremental cost-effectiveness ratio (versus no testing) of Euro 22,811 per
hypersensitivity reaction avoided.
CONCLUSIONS: Abacavir hypersensitivity is associated with HLA B*5701, and
pre-prescription pharmacogenetic testing for this appears to be a cost-effective
use of healthcare resources. BACKGROUND: Hypersensitivity reaction to abacavir is strongly associated with
the presence of the HLA-B*5701 allele. This study was designed to establish the
effectiveness of prospective HLA-B*5701 screening to prevent the
hypersensitivity reaction to abacavir.
METHODS: This double-blind, prospective, randomized study involved 1956 patients
from 19 countries, who were infected with human immunodeficiency virus type 1
and who had not previously received abacavir. We randomly assigned patients to
undergo prospective HLA-B*5701 screening, with exclusion of HLA-B*5701-positive
patients from abacavir treatment (prospective-screening group), or to undergo a
standard-of-care approach of abacavir use without prospective HLA-B*5701
screening (control group). All patients who started abacavir were observed for 6
weeks. To immunologically confirm, and enhance the specificity of, the clinical
diagnosis of hypersensitivity reaction to abacavir, we performed epicutaneous
patch testing with the use of abacavir.
RESULTS: The prevalence of HLA-B*5701 was 5.6% (109 of 1956 patients). Of the
patients receiving abacavir, 72% were men, 84% were white, and 18% had not
previously received antiretroviral therapy. Screening eliminated immunologically
confirmed hypersensitivity reaction (0% in the prospective-screening group vs.
2.7% in the control group, P<0.001), with a negative predictive value of 100%
and a positive predictive value of 47.9%. Hypersensitivity reaction was
clinically diagnosed in 93 patients, with a significantly lower incidence in the
prospective-screening group (3.4%) than in the control group (7.8%) (P<0.001).
CONCLUSIONS: HLA-B*5701 screening reduced the risk of hypersensitivity reaction
to abacavir. In predomitly white populations, similar to the one in this
study, 94% of patients do not carry the HLA-B*5701 allele and are at low risk
for hypersensitivity reaction to abacavir. Our results show that a
pharmacogenetic test can be used to prevent a specific toxic effect of a drug.
(ClinicalTrials.gov number, NCT00340080.) HIV infection is a serious but treatable disease, yet current treatment is
limited by development of resistance and high rates of adverse drug reactions.
Antiretroviral therapy is especially suitable for pharmacogenomic investigation
as both drug exposure and treatment response can be reliably measured.
Increasing knowledge about genes implicated in pharmacokinetics, mode of action,
efficacy, and toxicity of drugs has already provided relevant results for
clinical practice, for example: The strong association of the abacavir
hypersensitivity reaction with HLA-B*5701 permits testing patients for the
allele, and if present avoiding the drug and therefore preventing the reaction.
Persons with the allele CYP2B6*6 present higher efavirenz "area under the curve"
and have increased risk of neuropsychological toxicity. Additional gene variants
are being discovered that influence the action of antiretroviral drugs. And,
moreover, it is expected that larger-scale comprehensive genome approaches will
profoundly improve the landscape of knowledge of HIV therapy in the future. The
present article shows some recent patents related to the treatment of viral
infections. The aim of the PREDICT-1 study was to determine the clinical utility of the
pharmacogenetic test identifying HLA-B*5701 to reduce the incidence of
hypersensitivity reaction to abacavir, diagnosed clinically and with
immunological confirmation, as well as to reduce unwarranted withdrawal of this
drug. In the PREDICT-1 study, 1,956 patients were randomized to be screened for
HLA-B*5701 before starting abacavir treatment (excluding participants who were
HLA-B*5701-positive) or to receive abacavir without knowing their HLA-B*5701
status under conventional clinical monitoring. The prevalence of
HLA-B*5701-positivity was 5.7%. In the group that underwent prospective
screening, no hypersensitivity tests with immunological confirmation (by
positive epicutaneous patch testing) were observed compared with an incidence of
2.7% in the group undergoing standard follow-up. The sensitivity of prospective
screening in predicting immunologically confirmed hypersensitivity reaction to
abacavir was 100% and its negative predictive value was 100%. The number of
clinically suspected hypersensitivity reactions to abacavir was also lower in
the screened group (3.4% versus 7.8% in the group undergoing conventional
follow-up). The sensitivity of epicutaneous patch testing for immunological
confirmation was 100%. In the last few years, research in pharmacogenetics and pharmacogenomics has
identified distinct variants or markers that can help to define the benefits and
risk of patients requiring antiretroviral treatment. The beneficial effect of
the deletion 32 allele of the CCR5 coreceptor on the natural history of HIV
infection and, to a certain extent, on treatment response is well known. The
bases of immune reconstitution after initiation of antiretroviral therapy,
although the subject of intense study, are probably multifactorial and
polygenetic and consequently conclusions with clear applicability to clinical
practice are currently lacking. Among the risks, no significant progress has
been made in lipodystrophy. The origin of dyslipidemia associated with
antiretroviral treatment and the excess cardiovascular risk conferred by some
antiretroviral drugs is probably polygenetic and, at present, poorly defined.
The genetic bases of efavirenz-induced neurological toxicity and of
hyperbilirubinemia secondary to atazanavir are fairly well known, although their
application in daily clinical practice has not been adequately assessed. Some
aspects that help to understand the molecular bases of hypersensitivity reaction
to nevirapine and of nevirapine-induced hepatotoxicity have been described but
are not applicable in most cases and consequently further studies are required.
Some data correlate tenofovir-induced renal toxicity with genetic variations in
some transport proteins. The most significant advance for clinical practice is
the correlation between the presence of the HLA-B*5701 allele and
hypersensitivity reaction to abacavir. In particular, one clinical trial with a
large number of patients from distinct ethnic groups found that the probability
of not developing hypersensitivity reaction (immunologically confirmed) was 100%
if the patient was HLA-B*5701-negative. These data suggest the need to implement
this test in daily clinical practice. OBJECTIVE: To evaluate the clinical impact and cost-effectiveness of HLA-B*5701
testing to guide selection of first-line HIV regimens in the United States.
DESIGN: Cost-effectiveness analysis using a simulation model of HIV disease. The
prevalence of HLA-B*5701 and the probabilities of confirmed and unconfirmed
severe systemic hypersensitivity reaction among patients taking abacavir testing
HLA-B*5701 positive and negative were from the Prospective Randomized Evaluation
of DNA Screening in a Clinical Trial study. The monthly costs of abacavir-based
and tenofovir-based regimens were $1135 and $1139, respectively; similar
virologic efficacy was assumed and this assumption was varied in sensitivity
analysis.
PATIENTS: Simulated cohort of patients initiating HIV therapy.
INTERVENTIONS: The interventions are first-line abacavir, lamivudine, and
efavirenz without pretreatment HLA-B*5701 testing; the same regimen with
HLA-B*5701 testing; and first-line tenofovir, emtricitabine, and efavirenz.
MAIN OUTCOME MEASURES: Quality-adjusted life years and lifetime medical costs
discounted at 3% per annum, cost-effectiveness ratios ($/QALY).
RESULTS: Abacavir-based treatment without HLA-B*5701 testing resulted in a
projected 30.93 years life expectancy, 16.23 discounted quality-adjusted life
years, and $472,200 discounted lifetime cost per person. HLA-B*5701 testing
added 0.04 quality-adjusted months at an incremental cost of $110, resulting in
a cost-effectiveness ratio of $36,700/QALY compared with no testing. Initiating
treatment with a tenofovir-based regimen increased costs without improving
quality-adjusted life expectancy. HLA-B*5701 testing remained the preferred
strategy only if abacavir-based treatment had equal efficacy and cost less per
month than tenofovir-based treatment. Results were also sensitive to the cost of
HLA-B*5701 testing and the prevalence of HLA-B*5701.
CONCLUSION: Pharmacogenetic testing for HLA-B*5701 is cost-effective only if
abacavir-based treatment is as effective and costs less than tenofovir-based
treatment. Pharmacogenetics (PGX) is the study of drug response as a function of an
individual's DNA. PGX is often viewed as an extension of disease association
genetics, and although this information may be related, it is not the study of
drug response. Although medicines are used to treat diseases, the value of
strategies that identify and incorporate DNA biomarkers associated with clinical
efficacy, or DNA biomarkers for untoward clinical responses, can be applied
directly to pharmaceutical pipelines. The growth of adverse event PGX studies
involving marketed medicines generally uses relatively large numbers of affected
patients, but has been productive. However, the two critical strategies for
pipeline genetics must make use of fewer patients: (1) the early identification
of efficacy signals so that they can be applied early in development for
targeted therapies and (2) identification of safety signals that can
subsequently be validated prospectively during development using the least
number of patients with adverse responses. Assumptions are often made that large
numbers of patients are necessary to recognize PGX hypotheses and to validate
DNA biomarkers. In some ways, pipeline pharmacogenetics may be viewed as the
opposite of current genome-wide scanning designs. The goal is to obtain PGX
signals in as few patients as possible, and then validate PGX hypotheses for
specificity and sensitivity as development trials go forward--not using hundreds
of thousand of markers to detect strong linkage disequilibrium signals in
thousands of patients and their controls. Drug development takes 5-7 years for a
drug candidate to traverse to registration--and this is similar to the timeframe
for validating genetic biomarkers using sequential clinical trials. Two
important examples are discussed, the association of APOE genotypes to the
demonstration of actionable efficacy signals for the use of rosiglitazone for
Alzheimer's disease; and the identification of HLA-B(*)5701 as a highly
sensitive and specific predictive marker for abacavir treated patients who will
develop hypersensitivity syndrome (HSS). The rosiglitazone study prevented
pipeline attrition by changing the interpretation of a critical Phase IIB proof
of concept study (2005) from a failed study, to a positive efficacy response in
a genetically predictable proportion of patients. Now, three years later, a
Phase III program of clinical trials using pharmacogenetic designs is months
away from completion (late08). If successfully registered (early09), millions of
patients could benefit, and efficacy PGX would have achieved its first
prospective block-buster. The use of safety candidate gene association genetics
in patients who received abacavir therapy and developed HSS starting in 1998
culminated in a double blind clinical trial that determined sensitivity > 97%
and specificity >99% in 2007. Clinical consensus panels rapidly recommended
abacavir as the preferred therapy along with HLA-B(*)5701 pre-testing,
immediately increasing the market share of abacavir with respect to other
reverse transcriptases that are associated with there own adverse events.
Targeting of medicines during drug development is now possible, practical, and
profitable. Abacavir hypersensitivity (ABC HSR) is a treatment-limiting adverse event
associated with the use of the antiretroviral medicine, abacavir. The objective
of the ABC HSR pharmacogenetics program was to identify clinically useful
genetic risk factors to predict an individual patient's risk for ABC HSR. The
major histocompatibility complex allele, HLA-B*5701, was identified
retrospectively and confirmed with independent sample sets. The clinical utility
of prospective HLA-B*5701 screening was demonstrated in a blinded randomized
clinical trial and in open-label cohorts. Screening has been incorporated into
clinical practice and the ABC HSR pharmacogenetics program has been highlighted
as a success by pharmacogenetics researchers. Important lessons from this
pharmacogenetics program will be discussed in this paper. Abacavir hypersensitivity syndrome (AHS) is a potentially life-threatening
illness occurring in 4-8% of those initiating the drug. Early studies identified
a strong association between the MHC class I allele HLA-B*5701 and AHS. These
studies suggested that HLA-B*5701 holds promise as a screening test to prevent
AHS, but concern arose from HLA-B*5701-negative cases with a clinical diagnosis
of AHS, and particularly from early reports of apparently low sensitivities of
HLA-B*5701 for AHS in patients of non-White race. However, open screening
studies suggested that HLA-B*5701 screening can largely eliminate AHS.
Furthermore, skin-patch testing was used in later-generation studies to separate
those patients with true immunologically mediated AHS from those with
false-positive clinical diagnoses. Currently, high-level evidence suggests that
HLA-B*5701 has a negative predictive value of 100% for patch-test-confirmed AHS,
which is generalizable across White and Black populations. Current HIV treatment
guidelines have been revised to reflect the recommendation that HLA-B*5701
screening be incorporated into routine care for patients who may require
abacavir. New laboratory techniques such as PCR and flow cytometric methods, as
well as an international quality assurance program, have evolved to ensure the
availability of cost-effective screening methods whose consistency and standard
can be maintained over time. An elegant body of basic science has evolved, which
supports and complements the clinical research in suggesting that AHS is
specifically and exquisitely restricted by HLA-B*5701 and mediated by CD8+
lymphocytes. Abrogating factors explaining why 45% of those carrying HLA-B*5701
can tolerate abacavir remain to be defined. The research approach applied to AHS
has led to a genetic screening test being successfully implemented globally in
primary HIV clinical practice. The abacavir 'example' can be applied to other
drugs to facilitate the development and operationalization of genetic tests that
may be useful to predict and prevent otherwise unpredictable drug reactions. OBJECTIVE: Prospective pharmacogenetic screening for the human leucocyte antigen
(HLA) B*5701 allele can significantly reduce the number of cases of
abacavir-related hypersensitivity among HIV-infected patients treated with this
drug. The aim of this study was to establish the frequency of the HLA B*5701
variant in HIV-infected Poles.
METHODS: The sequence-specific primer (SSP) test was used to assess the
feasibility of the introduction of such testing in clinical practice. For this
purpose, 234 randomly selected HIV-positive patients were screened using a
low-resolution SSP assay, with HLA B*5701-positive results confirmed using a
high-resolution test.
RESULTS AND CONCLUSIONS: The HLA B*5701 variant was found in 11 of 234 subjects
(4.7%). Testing with the selected method proved quick and reliable. Pharmacogenomics strives to explain the interindividual variability in response
to drugs due to genetic variation. Although technological advances have provided
us with relatively easy and cheap methods for genotyping, promises about
personalised medicine have not yet met our high expectations. Successful results
that have been achieved within the field of pharmacogenomics so far are, to name
a few, HLA-B*5701 screening to avoid hypersensitivity to the antiretroviral
abacavir, thiopurine S-methyltransferase (TPMT) genotyping to avoid thiopurine
toxicity, and CYP2C9 and VKORC1 genotyping for better dosing of the
anticoagulant warfarin. However, few pharmacogenetic examples have made it into
clinical practice in the treatment of complex diseases. Unfortunately, lack of
reproducibility of results from observational studies involving many genes and
diseases seems to be a common pattern in pharmacogenomic studies. In this
article we address some of the methodological and statistical issues within
study design, gene and single nucleotide polymorphism (SNP) selection and data
analysis that should be considered in future pharmacogenomic research. First, we
discuss some of the issues related to the design of epidemiological studies,
specific to pharmacogenomic research. Second, we describe some of the pros and
cons of a candidate gene approach (including gene and SNP selection) and a
genome-wide scan approach. Finally, conventional as well as several innovative
approaches to the analysis of large pharmacogenomic datasets are proposed that
deal with the issues of multiple testing and systems biology in different ways. Drug hypersensitivity reactions and severe cutaneous adverse drug reactions,
such as Stevens-Johnson syndrome and toxic epidermal necrolysis, are examples of
serious adverse drug reactions mediated through a combination of metabolic and
immunological mechanisms that could traditionally not have been predicted based
on the pharmacological characteristics of the drug alone. The discovery of new
associations between these syndromes and specific HLA has created the promise
that risk for these reactions could be predicted through pharmacogenetic
screening, thereby avoiding serious morbidity and mortality associated with
these types of drug reactions. Despite this, several hurdles exist in the
translation of these associations into pharmacogenetic tests that could be
routinely used in the clinical setting. HLA-B*5701 screening to prevent abacavir
hypersensitivity syndrome is an example of a test now in widespread routine
clinical use in the developed world. The 2010 US FDA-Drug Industry Association (DIA) Pharmacogenomics Workshop, the
fifth in a series of meetings that begun in 2002, brought together
multidisciplinary experts from regulatory authorities, medical research,
healthcare and drug development. This article summarizes the 'Designing
Pharmacogenomic Studies to be Fit for Purpose' track in which considerations
regarding the use of retrospective and prospective studies were examined in
relation to their ability to influence treatment decisions and labeling for
drugs. The aim of the session, using real-world examples (KRAS/panitumumab and
HLA-B*5701/abacavir), was to identify good scientific principles that would
guide the design of studies to identify subgroups of responders during
development programs (including marketed drugs), which could subsequently be
used to guide treatment decisions. AIM: International HIV treatment guidelines recommend HLA-B*57:01 typing before
abacavir administration, in order to reduce the incidence of abacavir
hypersensitivity reactions, the major cause of early therapy discontinuation. A
fast, sensitive and specific test for HLA-B*57:01 detection has been developed
in the present study.
MATERIALS & METHODS: Two sets of sequence-specific primers were designed, and
amplification rapidly detected by real-time PCR.
RESULTS: A total of 108 samples were analyzed in a single-blind fashion, and 41
samples were identified as positive. Complete agreement, with κ = 1 (standard
error = 0.0962, p < 0.0001), was found, with a validated methodology used in the
EPI109367 clinical trial funded by GlaxoSmithKline, and consisting of
low-resolution sequence-specific oligonucleotide PCR, followed by
high-resolution sequence-specific oligonucleotide PCR carried out on the
HLA-B*57-positive samples.
CONCLUSION: We provided a detailed characterization of a novel HLA-B*57:01
screening test, which can be easily implemented by those laboratories already
involved in the detection of viral load and virus genotyping. Original submitted
26 October 2010; Revision submitted 13 December 2010. Pharmacogenomic tests offer a promising strategy to improve the safety and
efficacy of drug treatment. Compelling examples, such as HLA-B*5701 testing to
identify patients at risk for abacavir-associated hypersensitivity, are already
changing clinical care. However, the level of evidence required to establish
clinical utility is often the subject of debate. Determining the most efficient
and effective pathway to benefit for a given test is therefore both a practical
and an ethical concern. Many pharmacogenomic biomarkers (PGBM) were identified and translated into
clinical practice, affecting the usage of drugs via label updates. In this
context, abacavir is one of the most brilliant examples of pharmacogenetic
studies translated into clinical practice. Pharmacogenetic studies have revealed
that abacavir HSRs are highly associated with the major histocompatibility
complex class I. Large studies established the effectiveness of prospective
HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results
the abacavir label has been modified: the European Medicines Agency (EMA) and
the FDA recommend/suggested that the administration of abacavir must be preceded
by a specific genotyping test. The HLA locus is extremely polymorphic,
exhibiting many closely related alleles, making it difficult to discriminate
HLA-B*57:01 from other related alleles, and a number of different molecular
techniques have been developed recently to detect the presence of HLA-B*57:01.
In this review, we provide a summary of the available techniques used by
laboratories to genotype HLA-B*57:01, outlining the scientific and
pharmacoeconomics pros and cons. |
Are microRNA (miR) regulated through DNA methylation of their promoters? | Dysregulation of miRNA expression involved in cancer and Alzheimer's disease can be triggered by multiple mechanisms including aberrant DNA methylation of the miRNA gene promoter. Epigenetic dysregulation of tumor-suppressor miRNA genes by promoter DNA methylation has been implicated in human cancers, including multiple myeloma (MM). | The cellular response to Nutlin-3, a small-molecule inhibitor of the p53
repressor MDM2, varies widely among human cancer-derived cell types. Whereas
HCT116 colorectal carcinoma cells display sustained cell cycle arrest, BV173
leukemia cells undergo rapid apoptosis and other cell lines show an intermediate
response. We found that the expression of the p53 target genes p21, 14-3-3sigma
and the microRNA miR-34a correlates tightly with the cell fate choice adopted.
All three genes were strongly induced in arresting cells, but silenced in cells
undergoing Nutlin-3-induced apoptosis. In contrast, key apoptotic p53 target
genes were equally expressed in arresting and apoptotic cells. Interestingly, we
establish that miR-34a cooperates with p21 and 14-3-3sigma to override the
apoptotic signals generated by p53 activation. Strikingly, p53 binding to
chromatin and p53-mediated recruitment of certain coactivators to all three
target loci does not vary among cell types. Instead, the cell type-specific
silencing of these genes is due to enhanced p21 mRNA degradation, 14-3-3sigma
promoter DNA methylation and reduced processing of the miR-34a primary
transcript. Thus, p53-independent events regulating expression of protein-coding
genes and microRNAs within the network can define the cellular outcome of p53
activation. Aberrant epigenetic regulation has recently been implicated in the
downregulation of tumour suppressor microRNAs (miRNAs). Histone modification and
DNA methylation can have different roles in gene silencing in cancer. To
investigate whether histone modifications would contribute to the dysregulation
of miRNAs in acute lymphoblastic leukaemia (ALL), the effect of a histone
deacetylase inhibitor, trichostatin A (TSA), on miRNA expression profile was
analysed by microarray assay in a precursor B-cell ALL cell line NALM-6. A total
of 10 miRNAs were downregulated and 31 were upregulated significantly following
TSA treatment. Among TSA-upregulated miRNAs, MIR22 is an extronic miRNA and
resides in the second exon of the non-coding transcript MGC14376. Upregulation
of MIR22 transcription was found in both NALM-6 cells and primary human ALL
maligt cells treated with TSA. Whereas a CpG island was identified within the
promoter element of MIR22, no promoter DNA methylation was detected in these
cells. In contrast, accumulation of the repressive histone marker H3K27
trimethylation (H3K27triM) was identified around the transcriptional start point
of the gene, which was reduced by TSA treatment. Thus, accumulation of H3K27triM
independent of promoter DNA methylation may be a novel epigenetic mechanism for
MIR22 silencing in ALL. Dysregulated microRNA (miRNA) expression contributes to the pathogenesis of
hematopoietic maligcies, including chronic lymphocytic leukemia (CLL).
However, an understanding of the mechanisms that cause aberrant miRNA
transcriptional control is lacking. In this study, we comprehensively
investigated the role and extent of miRNA epigenetic regulation in CLL.
Genome-wide profiling conducted on 24 CLL and 10 healthy B cell samples revealed
global DNA methylation patterns upstream of miRNA sequences that distinguished
maligt from healthy cells and identified putative miRNA promoters.
Integration of DNA methylation and miRNA promoter data led to the identification
of 128 recurrent miRNA targets for aberrant promoter DNA methylation. DNA
hypomethylation accounted for more than 60% of all aberrant promoter-associated
DNA methylation in CLL, and promoter DNA hypomethylation was restricted to
well-defined regions. Individual hyper- and hypomethylated promoters allowed
discrimination of CLL samples from healthy controls. Promoter DNA methylation
patterns were confirmed in an independent patient cohort, with 11 miRNAs
consistently showing an inverse correlation between DNA methylation status and
expression level. Together, our findings characterize the role of epigenetic
changes in the regulation of miRNA transcription and create a repository of
disease-specific promoter regions that may provide additional insights into the
pathogenesis of CLL. DNA methylation is one of the heritable epigenetic modifications, leading to
repressed gene expressions and consequent phenotypic alterations without
changing the DNA sequence. MicroRNA (miRNA) is a novel class of short non-coding
RNA molecules regulating a wide range of cellular functions through
translational repression of their target genes. Recently, epigenetic
dysregulation of tumor-suppressor miRNA genes by promoter DNA methylation has
been implicated in human cancers, including multiple myeloma (MM). This article
presents a brief overview of the pathogenesis of MM, the role of DNA methylation
in cancer biology, methods of DNA methylation analysis, miRNA biology and
dysregulation of miRNAs in MM and summaries the current data on the role of DNA
methylation of tumor-suppressive miRNAs in MM. miRNAs are a group of small noncoding RNAs measuring 19-25 nucleotides.
Sequence-specific binding of miRNAs to the 3´ untranslated regions of target
genes leads to translational repressions. Dysregulation of miRNA expression
involved in cancer can be triggered by multiple mechanisms including aberrant
DNA methylation of the miRNA gene promoter. Of note, DNA methylation of tumor
suppressor miRNAs has been implicated in various human cancers. Moreover, miRNA
silencing mediated by aberrant promoter DNA methylation can potentially be
reversed by hypomethylating agents, and hence may pose a new therapeutic target
in cancer. In this review, the authors will focus on the aberrant methylation of
miRNAs in the pathogenesis of lymphoid maligcies including chronic
lymphocytic leukemia, multiple myeloma and acute lymphoblastic leukemia. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous
silencers of target genes. Some tumor-suppressive miRNAs are known to be
epigenetically silenced by promoter DNA methylation in cancer. In the present
study, we aimed to identify miRNA genes that are silenced by DNA
hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes
with promoter DNA hypermethylation using a genome-wide methylation microarray
analysis in HCC cells. It was found that miR-335, which is harbored within an
intron of its protein-coding host gene, MEST, was downregulated by aberrant
promoter hypermethylation via further methylation assays, including
methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite
sequencing analysis and 5-aza-2'-deoxycytidine treatment. The expression levels
of miR-335 significantly correlated with those of MEST, supporting the notion
that the intronic miR-335 is co-expressed with its host gene. The levels of
miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary
HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The
expression levels of miR-335 were significantly lower in 25 (78%) out of 32
primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001).
Furthermore, the expression levels of miR-335 were significantly lower in HCC
tumors with distant metastasis compared to those without distant metastasis
(P=0.02). In conclusion, our results indicate that expression of miR-335 is
reduced by aberrant DNA methylation in HCC. Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal
and differentiation in mouse embryonic stem cells (ESCs). We have previously
shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor
gene constitutively activates Wnt signaling in ESCs and inhibits their capacity
to differentiate towards ecto-, meso-, and endodermal lineages. However, the
underlying molecular and cellular mechanisms through which Wnt regulates lineage
differentiation in mouse ESCs remain to date largely unknown. To this aim, we
have derived and studied the gene expression profiles of several Apc-mutant ESC
lines encoding for different levels of Wnt signaling activation. We found that
down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the
control of self-renewal and pluripotency, represents a specific and primary
response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression
partially restored the neural defects observed in Apc-mutant ESCs, suggesting
that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression
of neural differentiation. We found that Tcf3 down-regulation in the context of
constitutively active Wnt signaling does not result from promoter DNA
methylation but is likely to be caused by a plethora of mechanisms at both the
RNA and protein level as shown by the observed decrease in activating histone
marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel
Wnt-regulated microRNA that targets Tcf3 and attenuates early neural
differentiation in mouse ESCs. Our data show for the first time that Wnt
signaling down-regulates Tcf3 expression, possibly at both the transcriptional
and post-transcriptional levels, and thus highlight a novel mechanism through
which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse
embryonic stem cells. |
Are nucleosomes positioned at DNA replication origins? | No, origins of replication occur in nucleosome-free regions in both budding yeast and Drosophila. Open chromatin domains, characterized by nucleosome depletion, are preferentially permissive for replication. | Circular duplex DNA containing the SV40 replication origin was assembled into
chromosomes in vitro with core histones and nucleosome assembly factor from HeLa
cells. Their ability to serve as a template for replication was examined by
incubating them with SV40 T antigen and HeLa cell extract. Nucleosome assembly
of the template prevented DNA replication. Replication of chromosomes was
severely inhibited at more than two-thirds of physiological nucleosome density.
When the DNA was preincubated with T antigen and then assembled into
chromosomes, however, inhibition of DNA replication was greatly reduced. These
results suggest that nucleosome assembly of the template inhibits initiation of
SV40 DNA replication and that the inhibition can be overcome by formation of an
initiation complex before nucleosome assembly. The distribution of DNA replication origins (ORIs) on eukaryotic chromosomes is
nonrandom, but the reasons behind this are not well understood. Previous studies
have suggested a prominent role of transcriptional activity in determining the
ORI organization. Here, we identify nucleosome occupancy as a likely candidate
to set up ORI distribution. Combining genome-wide data on nucleosome positioning
and ORI organization in yeast and humans, we demonstrate that open chromatin
domains, characterized by nucleosome depletion, are preferentially permissive
for replication. However, contrary to priori claims, the impact of
transcriptional activity is considerably weaker than previously proposed and
could partly be explained by our nucleosome exclusion model. We propose that the
ORI organization imposed by nucleosome positioning is phylogenetically
widespread in eukaryotes. The origin recognition complex (ORC) specifies replication origin location. The
Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating
sequence) consensus sequence (ACS), but only a subset of potential genomic sites
are bound, suggesting other chromosomal features influence ORC binding. Using
high-throughput sequencing to map ORC binding and nucleosome positioning, we
show that yeast origins are characterized by an asymmetric pattern of positioned
nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a
nucleosome-free origin; however, ORC is required for the precise positioning of
nucleosomes flanking the origin. These findings identify local nucleosomes as an
important determit for origin selection and function. Genome-scale mapping of pre-replication complex proteins has not been reported
in mammalian cells. Poor enrichment of these proteins at specific sites may be
due to dispersed binding, poor epitope availability or cell cycle stage-specific
binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding
in G1-synchronized populations of Chinese hamster cells harboring amplified
copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity
purification of biotinylated chromatin followed by high-density microarray
analysis across the DHFR locus. We have identified several sites of significant
enrichment for both complexes distributed throughout the previously identified
initiation zone. Analysis of the frequency of initiations across stretched DNA
fibers from the DHFR locus confirmed a broad zone of de-localized initiation
activity surrounding the sites of ORC and MCM enrichment. Mapping positions of
mononucleosomal DNA empirically and computing nucleosome-positioning information
in silico revealed that ORC and MCM map to regions of low measured and predicted
nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM
enrichment can be detected within a mammalian initiation zone, and suggest that
initiation zones may be regions of generally low nucleosome occupancy where
flexible nucleosome positioning permits flexible pre-RC assembly sites. |
Which are the most under-represented oligonucleotides in higher eukaryote genomes? | The oligonucleotides containing the CG and TA dinucleotide are generally under-represented in higher eukaryote genomes | Strand-symmetric relative abundance functionals for di-, tri-, and
tetranucleotides are introduced and applied to sequences encompassing a broad
phylogenetic range to discern tendencies and anomalies in the occurrences of
these short oligonucleotides within and between genomic sequences. For
dinucleotides, TA is almost universally under-represented, with the exception of
vertebrate mitochondrial genomes, and CG is strongly under-represented in
vertebrates and in mitochondrial genomes. The traditional
methylation/deamination/mutation hypothesis for the rarity of CG does not
adequately account for the observed deficiencies in certain sequences, notably
the mitochondrial genomes, yeast, and Neurospora crassa, which lack the standard
CpG methylase. Homodinucleotides (AA.TT, CC.GG) and larger homooligonucleotides
are over-represented in many organisms, perhaps due to polymerase slippage
events. For trinucleotides, GCA.TGC tends to be under-represented in phage,
human viral, and eukaryotic sequences, and CTA.TAG is strongly under-represented
in many prokaryotic, eukaryotic, and viral sequences. The CCA.TGG triplet is
ubiquitously over-represented in human viral and eukaryotic sequences. Among the
tetranucleotides, several four-base-pair palindromes tend to be
under-represented in phage sequences, probably as a means of restriction
avoidance. The tetranucleotide CTAG is observed to be rare in virtually all
bacterial genomes and some phage genomes. Explanations for these over- and
under-representations in terms of DNA/RNA structures and regulatory mechanisms
are considered. Prokaryotic, eukaryotic and mitochondrial DNA sequences of total Length 300 000
nucleotides have been analyzed to find out whether stretches of alternating
purines and pyrimidines are unusual in terms of occurrence, composition and base
sequence. Alternating runs longer than 5 nucleotides are significantly
under-represented in the natural sequences as compared to random ones.
Octanucleotides are the most deficient, occurring at only 60% of the frequency
expected in random sequences. An unexpectedly high proportion of these octamers
consists of alternating tetramers with the repeat structure (PuPyPuPy)2 or
(PyPuPyPu)2. DNA stretches containing such sequences can potentially form a S1
nuclease sensitive slippage (staggered loop) structure, which might serve as a
locally unstacked intermediate in the B- to Z-DNA conformational transition. Results are described from application of a computer program that compares the
expected and actual incidence of CpG dinucleotides in relation to the codon
reading frame of genes, assuming a conserved amino acid sequence and normalizing
for the third-position incidences of C and G in the remainder of the sequence.
Sequences encoding certain proteins showed a pronounced bias in favour of CpG in
the (3,1) compared with the (2,3) codon position; whereas sequences encoding
related proteins expressed to a similar extent or in the same tissue did not. We
propose that the cases exhibiting this bias reflect a difference between the two
strands of the DNA duplex in their susceptibility to loss of CpG dinucleotides
by mutation. Although in vertebrates this loss of CpG dinucleotides from the
sense strand might reflect strand-asymmetry in deamination of 5-methylcytosine
residues, the fact that a similar CpG codon bias is found in some invertebrates
indicates that other factor(s) must also be involved. Despite being generally under-represented in the genome, CpG sequences represent
a disproportionately high fraction of sites involved in mutational events
leading to human genetic disease. Cytosine within CpG dinucleotides is often
modified to 5-methylcytosine. Deamination of 5-methylcytosine in situ yields a
thymine, which being mispaired with guanine, is potentially mutagenic. Previous
reports have indicated that most mutations recovered at these sites appear to
originate on the non-transcribed strand as C-->T transitions. This trend may
however, reflect the lack of detectable mutant phenotypes resulting from this
transition at the complementary positions on the transcribed strand. To date,
there has not been a good model system in which mutations can be recovered on
both strands at the same CpG site. The human hprt gene has MeCpG sites contained
within arginine codons for which mutations have been recovered on both strands.
From an analysis of a database of hprt mutations, a statistically significant
strand bias is observed in mutations recovered at CpG sites. We describe some
models for the bias of mutation distribution observed at MeCpG sites in light of
this and previous work are described. Lymphocyte recognition of characteristic structural features in microbial DNA
may contribute to immune defense by promoting protective immune responses. The
dinucleotide CpG is significantly under-represented in vertebrate DNA and is
usually methylated. In contrast, CpG dinucleotides are generally present at the
expected frequency in bacterial DNA and are unmethylated. These unmethylated CpG
motifs induce B cells to secrete IL-6 and IgM, and can induce NK and CD4+ T
cells to produce the immunoregulatory Th1 cytokine, IFN-gamma. IFN-gamma
inhibits IgM secretion that is triggered by a different bacterial product, LPS.
The present study demonstrates that in contrast to its antagonistic interaction
with LPS, IFN-gamma causes a dose-dependent increase in the level of IgM
secretion induced by CpG DNA. Like IgM secretion, B cell secretion of IL-6 more
than doubles after the addition of exogenous IFN-gamma. Mice with disrupted
IFN-gamma genes produced less than half as much IL-6 and IgM in response to CpG
DNA, supporting the hypothesis that CpG-induced IFN-gamma production contributes
to the B cell response. In contrast to its promotion of IL-6 and IgM secretion,
IFN-gamma did not significantly affect the spleen cell proliferation activated
by CpG motifs. These results indicate that IFN-gamma produced by T and NK cells
after CpG DNA stimulation contributes to the B cell production of IL-6 and the
subsequent Ig production. These studies provide further evidence that the immune
system responds to CpG motifs in bacterial DNA by activating a coordinated set
of humoral and cellular responses. Vertebrates have evolved innate immune defense mechanisms that recognize and
respond to structural patterns that are specific to microbial molecules. One
such pattern recognition system is based on unmethylated CpG dinucleotides in
particular sequence contexts (CpG motifs); these motifs are common in bacterial
DNA but are under-represented ("CpG suppression") and methylated in vertebrate
DNA. Mice that are injected with bacterial DNA or synthetic
oligodeoxynucleotides (ODNs) containing CpG motifs respond with a rapid
production of IL-12 and IFN-gamma. The serum levels of IL-12 were increased for
at least 8 days after a single injection of CpG ODNs, but IFN-gamma levels
returned to baseline within 24 h. This Th1-like cytokine response to CpG motifs
induces a state of resistance to infection by Listeria monocytogenes in
susceptible specific pathogen-free BALB/c mice. Resistance developed within 48 h
of pretreatment with CpG ODNs, persisted for at least 2 wk, and was dependent
upon IFN-gamma secretion. These data support the hypothesis that CpG DNA motifs
are a "danger signal" that activates protective innate immune defenses and may
have therapeutic potential. CpG and TpA dinucleotides are underrepresented in the human genome. The CpG
deficiency is due to the high mutation rate from C to T in methylated CpG's. The
TpA suppression was thought to reflect a counterselection against TpA's
destabilizing effect in RNA. Unexpectedly, the TpA and CpG deficiencies vary
according to the G+C contents of sequences. It has been proposed that the
variation in CpG suppression was correlated with a particular chromatin
organization in G+C-rich isochores. Here, we present an improved model of
dinucleotide evolution accounting for the overlap between successive
dinucleotides. We show that an increased mutation rate from CpG to TpG or CpA
induces both an apparent TpA deficiency and a correlation between CpG and TpA
deficiencies and G+C content. Moreover, this model shows that the ratio of
observed over expected CpG frequency underestimates the real CpG deficiency in
G+C-rich sequences. The predictions of our model fit well with observed
frequencies in human genomic data. This study suggests that previously published
selectionist interpretations of patterns of dinucleotide frequencies should be
taken with caution. Moreover, we propose new criteria to identify unmethylated
CpG islands taking into account this bias in the measure of CpG depletion. We have systematically varied microsatellite sequence composition to determine
the effects of repeat unit size, G+C content, and DNA secondary structure on
microsatellite stability in human cells. The microsatellites were inserted in
frame within the 5' region of the herpes simplex virus thymidine kinase (HSV-tk)
gene. The polypyrimidine/polypurine microsatellites displayed enhanced S1
nuclease sensitivity in vitro, consistent with the formation of non-B-form DNA
structures. Microsatellite mutagenesis studies were performed with a shuttle
vector system in which inactivating HSV-tk mutations are measured after
replication in a nontumorigenic cell line. A significant increase in the HSV-tk
mutation frequency per cell generation was observed after insertion of
[TTCC/AAGG]9, [TTTC/AAAG]9, or [TCTA/AGAT]9 sequences (P <or= 0.0002), relative
to the HSV-tk gene control. We observed that the G + C content of the
microsatellite may affect mutagenesis, as the mean microsatellite mutation rates
of the [TTTC/AAAG]9 and [TCTA/AGAT]9 alleles were sevenfold and 11-fold higher,
respectively, than the [TTCC/AAGG]9 allele. A bias toward expansion mutations
was noted for the majority of clones bearing the [TTCC/AAGG]9 allele as well as
a [TC/AG]17 microsatellite of similar allele length. The mean microsatellite
mutation rate of the [TTCC/AAGG]9 allele did not differ significantly from that
for a [TC/AG]11 allele, demonstrating that these tetranucleotide and
dinucleotide alleles are of equivalent stability. It is known that
microsatellite mutagenesis is affected by the number of repeat units within an
allele. Our data suggest that additional biochemical factors may regulate both
the rate and specificity of somatic cell microsatellite mutagenesis. The domestic apple (Malus domestica; also known as Malus pumila Mill.) has
become a model fruit crop in which to study commercial traits such as disease
and pest resistance, grafting, and flavor and health compound biosynthesis. To
speed the discovery of genes involved in these traits, develop markers to map
genes, and breed new cultivars, we have produced a substantial expressed
sequence tag collection from various tissues of apple, focusing on fruit tissues
of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been
collected from 43 different cDNA libraries representing 34 different tissues and
treatments. Clustering of these sequences results in a set of 42,938
nonredundant sequences comprising 17,460 tentative contigs and 25,478
singletons, together representing what we predict are approximately one-half the
expressed genes from apple. Many potential molecular markers are abundant in the
apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant
sequences, mainly in the 5'-untranslated region of the gene, with a bias toward
one repeat type (containing AG, 88%) and against another (repeats containing CG,
0.1%). Trinucleotide repeats are most common in the predicted coding regions and
do not show a similar degree of sequence bias in their representation.
Bi-allelic single-nucleotide polymorphisms are highly abundant with one found,
on average, every 706 bp of transcribed DNA. Predictions of the numbers of
representatives from protein families indicate the presence of many genes
involved in disease resistance and the biosynthesis of flavor and
health-associated compounds. Comparisons of some of these gene families with
Arabidopsis (Arabidopsis thaliana) suggest instances where there have been
duplications in the lineages leading to apple of biosynthetic and regulatory
genes that are expressed in fruit. This resource paves the way for a concerted
functional genomics effort in this important temperate fruit crop. The innate immune response provides a first line of defense against pathogens by
targeting generic differential features that are present in foreign organisms
but not in the host. These innate responses generate selection forces acting
both in pathogens and hosts that further determine their co-evolution. Here we
analyze the nucleic acid sequence fingerprints of these selection forces acting
in parallel on both host innate immune genes and ssRNA viral genomes. We do this
by identifying dinucleotide biases in the coding regions of innate immune
response genes in plasmacytoid dendritic cells, and then use this signal to
identify other significant host innate immune genes. The persistence of these
biases in the orthologous groups of genes in humans and chickens is also
examined. We then compare the significant motifs in highly expressed genes of
the innate immune system to those in ssRNA viruses and study the evolution of
these motifs in the H1N1 influenza genome. We argue that the significant
under-represented motif pattern of CpG in an AU context--which is found in both
the ssRNA viruses and innate genes, and has decreased throughout the history of
H1N1 influenza replication in humans--is immunostimulatory and has been selected
against during the co-evolution of viruses and host innate immune genes. This
shows how differences in host immune biology can drive the evolution of viruses
that jump into species with different immune priorities than the original host. |
Does nimotuzumab improve survival of glioblastoma patients? | Yes. Nimotuzumab improves survival of adult and pediatric patients diagnosed with glioblastoma and with other high-grade gliomas. | Radioimmunotherapy (RIT) may improve the management of maligt gliomas. A
Phase I clinical trial was performed to evaluate, for the first time, the
toxicity and clinical effect of an intracavitary administration of a single dose
of Nimotuzumab (h-R3) labeled wit (188)Re. Nimotuzumab is a humanized monoclonal
antibody directed against epidermal growth factor receptors. Three patients with
anaplastic astrocytoma (AA) and 8 with glioblastoma multiforme (GBM) were
intended to be treated with 3 mg of mAb labelled with 10 or 15 mCi of (188)Re.
In patients treated with 10 mCi (n=6) transitory worsening of pre-existing
neurological symptoms were observed. Two patients treated with 15 mCi (n=4)
developed early severe neurological symptoms and one also developed late severe
toxicity (radionecrosis). In the group treated with 10 mCi, 1 GBM patient died
in progression 6 months after the treatment, 2 patients (1 GBM and 1 AA)
developed stable disease during 3 months. One GBM patient had partial response
for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in
complete response after 3 years of treatment. Maximal tolerated dose of the
radioimmunoconjugate (188)Re-Nimotuzumab was 3 mg of the h-R3 labelled with 10
mCi of (188)Re. The radioimmunoconjugate showed a high retention in the surgical
created resection cavity and the brain adjacent tissues with a mean value of
85.5 % of the injected dose one hour post-administration. This
radioimmunoconjugate may be relatively safe and a promising therapeutic approach
for treating high grade gliomas. As epidermal growth factor receptor (EGFR) has been reported to be a radiation
response modulator, HER inhibitors are regarded to act as potential
radiosensitisers. Our study examined the role of nimotuzumab and cetuximab both,
the two monoclonal antibodies (mAbs) to EGFR, as radiosensitisers in a murine
glioma model in vivo. Co-administration of both the antibodies with radiation
increased the radiosensitivity of U87MG, resulting in a significant delay of
subcutaneous (s.c.) tumour growth. Furthermore, the addition of antibodies to
the radiation decreased brain tumour sizes and is inhibited by 40-80% the
increased tumour cell invasion provoked by radiotherapy, although promoted
tumour cell apoptosis. Whereas nimotuzumab led to a reduction in the size of
tumour blood vessels and proliferating cells in s.c. tumours, cetuximab had no
significant antiangiogenic nor antiproliferative activity. In contrast,
cetuximab induced a more marked inhibition of EGFR downstream signalling
compared with nimotuzumab. Moreover, both antibodies reduced the total number of
radioresistant CD133+ cancer stem cells (CSCs). These results were encouraging,
and showed the superiority of combined treatment of mAbs to EGFR and radiation
over each single therapy against glioblastoma multiforme (GBM), confirming the
role of these drugs as radiosensitisers in human GBM. In addition, we first
showed the ability of mAb specifics against EGFR to target radioresistant glioma
CSC, supporting the potential use in patients. INTRODUCTION: Diffuse intrinsic pontine gliomas (DIPG) have a poor prognosis:
the median survival rate is less than one year. Radiotherapy is the only
effective treatment affording an overall survival of 6 - 9 months. So far, no
improvement has been achieved with the addition of single/poly-chemotherapy
regimens. An urgent need is to advance in this field, from both the biological
and the clinical points of view.
AREAS COVERED: Among the few studies providing biological information on DIPG,
Gilbertson's group demonstrated a significant increase in EGFR expression. The
activity of nimotuzumab, a humanized anti-EGFR monoclonal antibody, was
therefore studied within a Phase II trial in 47 relapsing pediatric patients
with DIPG and high-grade gliomas, showing an interesting, persistent response,
especially in the first group treated. A multicenter exploratory study combining
nimotuzumab and radiotherapy showed disease control and an overall patient
survival similar to previous experiences along with an improvement in the
quality of patient survival and no severe side effects.
EXPERT OPINION: We recommend considering this combination in the armamentarium
against DIPG. It might be improved by adding other target drugs/low-toxicity
chemotherapy regimens with a synergistic effect with the anti-EGFR component. OBJECTIVE: Nimotuzumab is a humanized monoclonal antibody targeted against
epidermal growth factor receptor (EGFR). Recent clinical studies show that
patients with maligt gliomas could benefit from nimotuzumab treatment. The
aim of the present study was to evaluate the efficacy and side effects of
nimotuzumab in combination with chemotherapy for patients with maligt
gliomas.
METHODS: The patients received 200 mg of nimotuzumab infusion intravenously over
60 minutes once weekly for the first eight weeks and then once every two weeks
until unacceptable toxicity or tumor progression occurred. Individualized
chemotherapy was administered based on O(6)-methylguanine-DNA methyltransferase
(MGMT) expression and previous chemotherapy responses in combined with
nimotuzumab.
RESULTS: Fourteen patients received a total of 122 times of nimotuzumab ranging
from 2 to 20 (median 7.5 times). Combined chemotherapy regimens included:
continuous 21-day temozolomide (10 cases), standard 5-day temozolomide (2
cases), teniposide plus cisplatin (1 case), and teniposide plus nimustine (1
case). Partial response (PR) and stable disease (SD) were found in 3 patients
(21.4%)and 6 patients (42.9%), respectively. Disease control rate (PR + SD) was
64.3%. The median progression-free survival (PFS) was 4 months (95%CI: 0.7 -
7.3) and PFS at 6 months was 30.6%. The most common toxicities include grade
I-II neutropenia (2 cases), thrombocytopenia (2 cases), lymphopenia (1 case),
nausea and vomitting (3 case) and asymptomatic transaminase increase (1 case).
One patient developed grade IV neutropenia and thrombocytopenia. One patient
developed nimotuzumab-related acneiform rash.
CONCLUSIONS: Nimotuzumab in combination with chemotherapy has moderate activity
in patients with maligt gliomas and the toxicities are well tolerable,
therefore, worth further investigation. Primary metastatic diffuse intrinsic pontine glioma (DIPG) is relatively rare
and associated with a dismal prognosis. Combining craniospinal irradiation (CSI)
with concurrent temozolomide and nimotuzumab therapy may slightly improve tumor
control and overall survival. However, little is known about the feasibility and
toxicity of this treatment approach. Here, we describe the case of an 8-year-old
girl with primary metastatic DIPG who received craniospinal radiotherapy, a
local boost, and concurrent temozolomide and nimotuzumab treatment based on an
individual therapy recommendation. Radiotherapy could be completed without any
interruption. However, concurrent temozolomide had to be disrupted several times
due to considerable acute myelotoxicity (grade III-IV).Maintece
immunochemotherapy could be started with a delay of 5 days and was performed
according to treatment schedule. The disease could be stabilized for a few
months. A routine MRI scan finally depicted disease progression 5.7 months after
the start of irradiation. The patient died 1.9 months later. BACKGROUND: The prognosis of patients bearing high grade glioma remains dismal.
Epidermal Growth Factor Receptor (EGFR) is well validated as a primary
contributor of glioma initiation and progression. Nimotuzumab is a humanized
monoclonal antibody that recognizes the EGFR extracellular domain and reaches
Central Nervous System tumors, in nonclinical and clinical setting. While it has
similar activity when compared to other anti-EGFR antibodies, it does not induce
skin toxicity or hypomagnesemia.
METHODS: A randomized, double blind, multicentric clinical trial was conducted
in high grade glioma patients (41 anaplastic astrocytoma and 29 glioblastoma
multiforme) that received radiotherapy plus nimotuzumab or placebo. Treatment
and placebo groups were well-balanced for the most important prognostic
variables. Patients received 6 weekly doses of 200 mg nimotuzumab or placebo
together with irradiation as induction therapy. Maintece treatment was given
for 1 year with subsequent doses administered every 3 weeks. The objectives of
this study were to assess the comparative overall survival, progression free
survival, response rate, immunogenicity and safety.
RESULTS: The median cumulative dose was 3200 mg of nimotuzumab given over a
median number of 16 doses. The combination of nimotuzumab and RT was
well-tolerated. The most prevalent related adverse reactions included nausea,
fever, tremors, anorexia and hepatic test alteration. No anti-idiotypic response
was detected, confirming the antibody low immunogenicity. The mean and median
survival time for subjects treated with nimotuzumab was 31.06 and 17.76 vs.
21.07 and 12.63 months for the control group.
CONCLUSIONS: In this randomized trial, nimotuzumab showed an excellent safety
profile and significant survival benefit in combination with irradiation.
TRIAL REGISTRATION: Cuban National Register for clinical trials (No. 1745)
(http://registroclinico.sld.cu/ensayos). Nimotuzumab, a humanized antibody targeting epidermal growth factor receptor,
has potent anti-proliferative, anti-angiogenic, and pro-apoptotic effects in
vitro and in vivo. It also reduces the number of radio-resistant CD133(+) glioma
stem cells. The antibody has been extensively evaluated in patients with
advanced head and neck, glioma, lung, esophageal, pancreatic, and gastric
cancer. In this single institution experience, 35 patients with anaplastic
astrocytoma (AA) or glioblastoma multiforme (GBM) were treated with irradiation
and 200 mg doses of nimotuzumab. The first 6 doses were administered weekly,
together with radiotherapy, and then treatment continued every 21 days until 1
year. The median number of doses was 12, and the median cumulative dose was thus
2400 mg of nimotuzumab. The most frequent treatment-related toxicities were
increase in liver function tests, fever, nausea, anorexia, asthenia, dizziness,
and tremors. These adverse reactions were classified as mild and moderate. The
median survival time was 12.4 mo or 27.0 mo for patients with GBM or AA
patients, respectively, who received curative-intent radiotherapy in combination
with the antibody. The survival time of a matched population treated at the same
hospital with irradiation alone was decreased (median 8.0 and 12.2 mo for GBM
and AA patients, respectively) compared with that of the patients who received
nimotuzumab and curative-intent radiotherapy. We have thus confirmed that
nimotuzumab is a very well-tolerated drug, lacking cumulative toxicity after
maintece doses. This study, in a poor prognosis population, validates the
previous data of survival gain after combining nimotuzumab and radiotherapy, in
newly diagnosed high-grade glioma patients. AIM: To evaluate the safety and efficacy of nimotuzumab, a humanized monoclonal
antibody specific for the epidermal growth factor receptor (EGFR), in
combination with temozolomide (TMZ) and radiation therapy (RT) in the treatment
of newly diagnosed glioblastoma (GBM) in Chinese patients.
METHODS: Twenty-six patients with newly diagnosed GBM were enrolled. All
patients received standard external beam RT after surgery, with a total dose of
60 Gy in 30 fractions. During RT, concurrent TMZ was given daily at 75 mg/m(2)
for 40-42 days, combined with six weekly infusions of nimotuzumab at a 200 mg
dose. After a 4-week interval upon completion of RT, six cycles of adjuvant TMZ
(150 to 200 mg/m(2) for 5 days in each 28-day cycle) were given. The primary end
point was 6-month progression-free survival (PFS) rate. EGFR expression in tumor
tissues was analyzed by immunohistochemistry.
RESULTS: Treatment was well tolerated and no grade III or higher grade toxicity
was observed. Median PFS and overall survival (OS) were 10.0 and 15.9 months,
respectively, while the 6-month PFS and OS rates were 69.2% and 88.5%,
respectively. No correlation between efficacy and EGFR expression was found.
CONCLUSIONS: Combination of Nimotuzumab with RT plus concomitant and adjuvant
TMZ showed favorable safety and tolerability profiles in newly diagnosed GBM in
Chinese patients. The survival times were similar to those seen in historical
data of standard therapy. |
List co-morbidities that may occur together with "Stiff man Syndrome" | SMS (Stiff man Syndrome) is is a rare disorder of the central nervous system of probable autoimmune origin and as such is associated with other autoimmune diseases, such as Insulin Dependent Diabetes Mellitus . GAD-65 is a dominant auto-antigen that is found both in in stiff-man syndrome and insulin-dependent diabetes mellitus. TRAB -positive Graves' disease has been reported to occur together with SMS.
In a subgroup of patients with the stiff-man syndrome, the condition is likely to have an autoimmune paraneoplastic origin. The detection of autoantibodies against the 128-kd antigen in patients with this syndrome should be considered an indication to search for an occult breast cancer.
HCV may be the etiologic virus of progressive encephalomyelitis with rigidity; a rare disorder similar to stiff-man syndrome although different because it is progressive and fatal.
It is possible that the reported case of association of progressive dementia with concomitant development of stiff-man syndrome in an elderly man represents an exaggerated form of such motor disturbances in dementia, and that clinical and electromyographic features of stiff-man syndrome may be present with increased incidence in patients with dementia. | Stiff-man syndrome is a rare disorder of the central nervous system of unknown
pathogenesis. We have previously reported the presence of autoantibodies against
glutamic acid decarboxylase (GAD) in a patient with stiff-man syndrome,
epilepsy, and insulin-dependent diabetes mellitus. GAD is an enzyme selectively
concentrated in neurons secreting the neurotransmitter gamma-aminobutyric acid
(GABA) and in pancreatic beta cells. We subsequently observed autoantibodies to
GABA-ergic neurons in 20 of 33 patients with stiff-man syndrome. GAD was the
principal autoantigen. In the group of patients positive for autoantibodies
against GABA-ergic neurons, there was a striking association with organ-specific
autoimmune diseases, primarily insulin-dependent diabetes mellitus. These
findings support the hypothesis that stiff-man syndrome is an autoimmune disease
and suggest that GAD is the primary autoantigen involved in stiff-man syndrome
and the associated insulin-dependent diabetes mellitus. Our findings also
indicate that autoantibodies directed against GABA-ergic neurons are a useful
marker in the diagnosis of the disease. Stiff-man syndrome is characterized clinically by fluctuating muscular stiffness
and spasm, and electromyographically by continuous motor unit activity at rest,
which is abolished by sleep, general anesthesia, nerve block, curare, and
several centrally-acting medications. A spinal or supraspinal origin has been
proposed for this disorder. Some clinical and electrophysiologic features, along
with an occasional association with encephalopathy, may support a proposed
supraspinal cause. An elderly man with progressive dementia and concomitant
development of stiff-man syndrome is described. He had not had stiff-man
syndrome one year earlier, when he had only mild dementia. An association
between stiff-man syndrome and dementia has not been previously described.
Increased muscle tone and muscular rigidity is frequently encountered in
patients with dementia, however, and pathologic reflexes involving neck and
proximal musculature have been described in dementia. It is possible that this
patient represents an exaggerated form of such motor disturbances in dementia,
and that clinical and electromyographic features of stiff-man syndrome may be
present with increased incidence in patients with dementia. Stiff-man syndrome (SMS) is a rare disorder of the central nervous system of
probable autoimmune origin. Patients with SMS often have other autoimmune
diseases, in particular type I (insulin-dependent) diabetes mellitus (IDDM).
Approximately 60% of patients with SMS have high titers of autoantibodies
against the enzyme glutamic acid decarboxylase. Similar to SMS, the majority of
patients with IDDM have autoantibodies against glutamic acid decarboxylase at or
before diabetes onset, although usually at a lower titer and with a different
reaction pattern than patients with SMS. To investigate the immunogenetic basis
of SMS, we HLA-typed 18 patients with the disease. Seventy-two percent carried
the DQB1*0201 allele (13 of 18, P = 0.02 vs. 18 of 48 controls), indicating that
SMS is associated with this allele. DQB1*0201 is also a susceptibility allele
for IDDM and other autoimmune diseases. Patients with SMS carried the
IDDM-protective DQB1*0602 allele and other sequence-related DQB1*06 alleles with
the same frequency observed in controls. In contrast, these alleles are rarely
found in IDDM. Five of 8 (62.5%) SMS patients lacking a DQB1*06 allele were
diabetic in contrast to only 2 of 10 (20%) with a DQB1*06 allele (P = 0.08),
suggesting that the presence of DQB1*0602 or other DQB1*06 alleles may be
associated with a reduced prevalence of diabetes among patients with SMS. Glutamic acid decarboxylase (GAD) is the enzyme responsible for synthesis of the
neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta cells.
It is represented by two isoforms, GAD-65 and GAD-67, which are the products of
two different genes and differ substantially only at their N-terminal regions.
GAD-65 is a domit autoantigen in stiff-man syndrome and insulin-dependent
diabetes mellitus. In neurons and beta cells, GAD is concentrated around
synaptic vesicles and synaptic-like microvesicles, respectively, as well as in
the area of the Golgi complex. The mechanisms responsible for specific targeting
of GAD to these organelles are not yet understood. The elucidation of the
mechanism of subcellular targeting of GAD may be relevant to understanding its
role as an autoantigen. In this study, the cloned genes for GAD-65 and GAD-67
were expressed separately in Chinese hamster ovary (CHO) cells and COS cells.
While GAD-67 had a diffuse cytoplasmic localization, GAD-65 had a punctate
distribution, with most of the immunoreactivity being concentrated in the area
of the Golgi complex. A chimeric protein in which the 88 N-terminal amino acids
of GAD-67 were replaced by the 83 N-terminal amino acids of GAD-65 was targeted
to the Golgi complex, indicating that the N-terminal region of GAD-65 contains a
targeting signal sufficient for directing the remaining portion of the molecule,
highly similar in GAD-65 and GAD-67, to the Golgi complex-associated structures. Retrospective psychological evaluation of nine patients with stiff-man syndrome
(SMS), seven of whom evidenced autoimmune disease, revealed a characteristic set
of psychological symptoms or features: Major stressful life events preceded the
development of permanent symptoms by 6 months or less (seven patients);
transient motor symptoms occurred in emotionally distressing situations months
or even years before the onset of a permanent motor deficit (five patients);
after onset, similar situations specifically precipitated or augmented stiffness
and spasms (five patients). We also found task-specific fear resembling
agoraphobia (six patients) and loss or invalidation of one or both parents, or
loss of home, in childhood (seven patients). Eight patients were initially
misdiagnosed as having psychogenic movement disorder. We conclude that the
common misdiagnosis of SMS as a psychogenic movement disorder is due to the
compelling association of a set of salient psychological features, bizarre and
fluctuating motor symptoms, and lack of approved neurologic signs. We report a patient who developed stiff-man syndrome, including disabling
shoulder subluxation and wrist ankylosis, in association with breast cancer.
Immunologic investigations disclosed autoimmunity directed against not only
glutamic acid decarboxylase but also amphiphysin, a 128-kd protein located in
the presynaptic compartment of neurons. The patient improved after surgery and
corticosteroid treatment and has been stable for nearly 4 years on only
anti-estrogenics. The triad of stiff-man syndrome, breast cancer, and
autoantibodies against amphiphysin identifies a new autoimmune paraneoplastic
syndrome of the CNS. Autoantibodies to glutamic acid decarboxylase (GAD) are an important marker of
the autoimmune-mediated beta-cell destruction in insulin-dependent (Type I)
diabetes. However, these autoantibodies are also found in patients with
Stiff-man syndrome (SMS) without onset of diabetes and some diabetic patients
who initially present as non-insulin dependent (Type II) diabetes later becoming
insulin-dependent, called as latent autoimmune diabetes in adults (LADA). To
study the immune response to GAD in these LADA patients a competitive
radiobinding assay based on murine monoclonal antibodies recognizing three
different GAD regions was performed. The monoclonal antibodies against GAD
recognize two different linear epitopes localized at the N- (amino acids 4-17)
and C-terminus (amino acids 572-585) and one conformation-dependent epitope
region (amino acids 221-442 IDDM-E1) known to be immunodomit for
diabetes-associated autoantibodies. All LADA sera (20/20) reduced substantially
the 125I-GAD binding of the monoclonal antibodies reactive with the
conformation-dependent epitope region IDDM-E1 and only 20% of these sera
additionally diminished the 125I-GAD65 binding by those monoclonals reactive
with the both linear epitopes. The SMS sera completely abolished the GAD binding
of all three monoclonals, reflecting a broader repertoire including an immune
response against the IDDM-E1, a conformation-dependent GAD65 epitope region,
also revealed if the SMS sera are diluted to equivalent antibody concentrations.
In summary, our results show that diabetes-associated GAD autoantibodies even in
adult patients with a late autoimmune process preferentially recognize a
conformation-dependent middle GAD65 region. An immune response to all three GAD
epitope regions is seldom in these LADA patients and only detectable in
association with high antibody titres. BACKGROUND: Hepatitis C viral (HCV) infection is common in the general
population and can cause disease in the nervous system. This article reviews the
neurologic complications associated with this virus.
REVIEW SUMMARY: A vasculitic neuropathy is the most firmly linked neurologic
illness associated with HCV infection. This type of neuropathy occurs frequently
in the presence of cryoglobulinemia. HCV is considered the most common cause of
cryoglobulinemia. Other types of neuropathy have been rarely reported with HCV
infection and this association is less firm. In the central nervous system,
vasculitis causing stroke appears to complicate HCV infection, usually in the
setting of cryoglobulinemia. Several reports of myelitis, encephalitis,lymphoma
are reviewed. HCV may be the etiologic virus of progressive encephalomyelitis
with rigidity; a rare disorder similar to stiff-man syndrome although different
because it is progressive and fatal. Treatment of the neurologic complications
associated with HCV infection is summarized.
CONCLUSIONS: HCV infection is being increasingly recognized as a probable cause
of a variety of neurologic disorders. Systematic study of the various
therapeutic options remains unexplored. OBJECTIVE: To report an association between two autoimmune conditions, Graves'
disease and stiff-person (stiff-man) syndrome, and discuss the relevant
literature.
METHODS: We present a case of a 52-year-old white woman with stiff-person
syndrome who also had Graves' disease, discuss her management, and review the
related literature. Pertinent published reports from 1950 through 2004 were
researched with use of MEDLINE and PubMed, and cross-references to other
articles were reviewed.
RESULTS: A 52-year-old white woman presented with symptoms of hyperthyroidism
due to Graves' disease. Laboratory data were as follows: thyrotropin <0.005 m
IU/mL, thyroxine 11.1 microg/dL, free thyroxine index (FTI) 10.7, and
triiodothyronine 170 ng/dL. Thyroid-stimulating immunoglobulins (TSI) and
thyrotropin-binding inhibitory immunoglobulins (TBII) were positive at 1,986%
and 82.5 U/L, respectively. The hyperthyroidism was treated with propranolol.
She had a long-standing history of musculoskeletal complaints and was ultimately
diagnosed with stiff-person syndrome. During her thyroid evaluation, she had
severe neurologic deterioration that necessitated hospitalization and treatment
with clonazepam, baclofen, intravenous immunoglobulin, and subsequently
prednisone and azathioprine for appreciable symptomatic relief. The aggressive
immunosuppression had a profound effect on her symptoms of hyperthyroidism,
results of thyroid function tests, and thyrotropin receptor antibodies (TRABs).
Thyrotropin was 0.52 microIU/mL, thyroxine was 6.9 microg/dL, and FTI was 5.7.
The TSI decreased from 1,986% to 248%, and her TBII normalized from 82.5 U/L to
<5 U/L. She was clinically and biochemically euthyroid at last follow-up in May
2004.
CONCLUSION: This case illustrates the association between TRAB-positive Graves'
disease and stiff-person syndrome and the improvement of Graves' disease with
immunosuppressive therapy. |
Which mechanisms underlie adaptive mutagenesis (stationary-phase mutagenesis) in Bacillus subtilis? | Increased transcription levels potentiate adaptive mutagenesis. Central to stationary-phase mutagenesis in B. subtilis is the requirement for Mfd protein (transcription repair coupling factor). The B. subtilis' ability to accumulate chromosomal mutations under conditions of starvation is influenced by cell differentiation and transcriptional derepression, as well as by proteins homologous to transcription and repair factors. Under conditions of nutritional stress, the processing of deaminated bases in B. subtilis may normally occur in an error-prone manner to promote adaptive mutagenesis. A functional RecA protein is not required for adaptive mutagenesis, which seems to be independent of recombination-dependent repair and, in some cases, of the Y DNA polymerases. Oxidative stress-induced DNA damage has been associated with adaptive mutagenesis. The occurrence of such mutations is exacerbated by reactive oxygen species. Starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. The MMR (encoded by the mutSL operon) protects B. subtilis from stationary-phase mutations. The MMR modulation of the mutagenic/antimutagenic properties of MutY regulates stationary-phase mutagenesis. Two of the genes that are involved in the regulation of post-exponential phase prokaryotic differentiation, comA and comK, are involved in adaptive mutagenesis. Also, YqjH, a homolog of DinB protein, plays a role in stationary phase mutagenesis. | Postsynthesis mismatch repair is an important contributor to mutation avoidance
and genomic stability in bacteria, yeast, and humans. Regulation of its activity
would allow organisms to regulate their ability to evolve. That mismatch repair
might be down-regulated in stationary-phase Escherichia coli was suggested by
the sequence spectrum of some stationary-phase ("adaptive") mutations and by the
observations that MutS and MutH levels decline during stationary phase. We
report that overproduction of MutL inhibits mutation in stationary phase but not
during growth. MutS overproduction has no such effect, and MutL overproduction
does not prevent stationary-phase decline of either MutS or MutH. These results
imply that MutS and MutH decline to levels appropriate for the decreased DNA
synthesis in stationary phase, whereas functional MutL is limiting for mismatch
repair specifically during stationary phase. Modulation of mutation rate and
genetic stability in response to environmental or developmental cues, such as
stationary phase and stress, could be important in evolution, development,
microbial pathogenicity, and the origins of cancer. Adaptive (stationary-phase) mutagenesis occurs in the gram-positive bacterium
Bacillus subtilis. Furthermore, taking advantage of B. subtilis as a paradigm
for the study of prokaryotic differentiation and development, we have shown that
this type of mutagenesis is subject to regulation involving at least two of the
genes that are involved in the regulation of post-exponential phase prokaryotic
differentiation, i.e., comA and comK. On the other hand, a functional RecA
protein was not required for this type of mutagenesis. The results seem to
suggest that a small subpopulation(s) of the culture is involved in adaptive
mutagenesis and that this subpopulation(s) is hypermutable. The existence of
such a hypermutable subpopulation(s) raises important considerations with
respect to evolution, the development of specific mutations, the nature of
bacterial populations, and the level of communication among bacteria in an
ecological niche. YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily
of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia
coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the
comparisons of protein structures demonstrate that YqjH has 36% identity to E.
coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA
polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and
the products of the yqjW operon are involved in UV-induced mutagenesis in this
bacterium. Furthermore, resistance to UV-induced damage is significantly reduced
in cells lacking a functional YqjH protein. Analysis of stationary-phase
mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the
ability of B. subtilis to generate revertants at the hisC952 allele via this
system. These data suggest a role for YqjH in the generation of at least some
types of stationary-phase-induced mutagenesis. A reversion assay system previously implemented to demonstrate the existence of
adaptive or stationary-phase-induced mutagenesis in Bacillus subtilis was
utilized in this report to study the influence of the mismatch DNA repair (MMR)
system on this type of mutagenesis. Results revealed that a strain deficient in
MutSL showed a significant propensity to generate increased numbers of
stationary-phase-induced revertants. These results suggest that absence or
depression of MMR is an important factor in the mutagenesis of nongrowing B.
subtilis cells because of the role of MMR in repairing DNA damage. In agreement
with this suggestion, a significant decrease in the number of adaptive revertant
colonies, for the three markers tested, occurred in B. subtilis cells which
overexpressed a component of the MMR system. Interestingly, the single
overexpression of mutS, but not of mutL, was sufficient to decrease the level of
adaptive mutants in the reversion assay system of B. subtilis. The results
presented in this work, as well as in our previous studies, appear to suggest
that an MMR deficiency, putatively attributable to inactivation or saturation
with DNA damage of MutS, may occur in a subset of B. subtilis cells that
differentiate into the hypermutable state. Previously, using a chromosomal reversion assay system, we established that an
adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under
stress, and we demonstrated that multiple mechanisms are involved in generating
these mutations (41, 43). In an attempt to delineate how these mutations are
generated, we began an investigation into whether or not transcription and
transcription-associated proteins influence adaptive mutagenesis. In B.
subtilis, the Mfd protein (transcription repair coupling factor) facilitates
removal of RNA polymerase stalled at transcriptional blockages and recruitment
of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate
that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An
association between Mfd mutagenesis and aspects of transcription is discussed. Previous studies showed that a Bacillus subtilis strain deficient in mismatch
repair (MMR; encoded by the mutSL operon) promoted the production of
stationary-phase-induced mutations. However, overexpression of the mutSL operon
did not completely suppress this process, suggesting that additional DNA repair
mechanisms are involved in the generation of stationary-phase-associated mutants
in this bacterium. In agreement with this hypothesis, the results presented in
this work revealed that starved B. subtilis cells lacking a functional error
prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic
propensity to increase the number of stationary-phase-induced revertants. These
results strongly suggest that the occurrence of mutations is exacerbated by
reactive oxygen species in nondividing cells of B. subtilis having an inactive
GO system. Interestingly, overexpression of the MMR system significantly
diminished the accumulation of mutations in cells deficient in the GO repair
system during stationary phase. These results suggest that the MMR system plays
a general role in correcting base mispairing induced by oxidative stress during
stationary phase. Thus, the absence or depression of both the MMR and GO systems
contributes to the production of stationary-phase mutants in B. subtilis. In
conclusion, our results support the idea that oxidative stress is a mechanism
that generates genetic diversity in starved cells of B. subtilis, promoting
stationary-phase-induced mutagenesis in this soil microorganism. Stress-promoted mutations that occur in nondividing cells (adaptive mutations)
have been implicated strongly in causing genetic variability as well as in
species survival and evolutionary processes. Oxidative stress-induced DNA damage
has been associated with generation of adaptive His(+) and Met(+) but not Leu(+)
revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we
report that an interplay between MutY and MutSL (mismatch repair system [MMR])
plays a pivotal role in the production of adaptive Leu(+) revertants.
Essentially, the genetic disruption of MutY dramatically reduced the reversion
frequency to the leu allele in this model system. Moreover, the increased rate
of adaptive Leu(+) revertants produced by a MutSL knockout strain was
significantly diminished following mutY disruption. Interestingly, although the
expression of mutY took place during growth and stationary phase and was not
under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon
increased the expression of mutY several times. Thus, in starved cells,
saturation of the MMR system may induce the expression of mutY, disturbing the
balance between MutY and MMR proteins and aiding in the production of types of
mutations detected by reversion to leucine prototrophy. In conclusion, our
results support the idea that MMR regulation of the mutagenic/antimutagenic
properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells. The disruption of ung, the unique uracil-DNA-glycosylase-encoding gene in
Bacillus subtilis, slightly increased the spontaneous mutation frequency to
rifampin resistance (Rif(r)), suggesting that additional repair pathways
counteract the mutagenic effects of uracil in this microorganism. An alternative
excision repair pathway is involved in this process, as the loss of YwqL, a
putative endonuclease V homolog, significantly increased the mutation frequency
of the ung null mutant, suggesting that Ung and YwqL both reduce the mutagenic
effects of base deamination. Consistent with this notion, sodium bisulfite (SB)
increased the Rif(r) mutation frequency of the single ung and double ung ywqL
strains, and the absence of Ung and/or YwqL decreased the ability of B. subtilis
to eliminate uracil from DNA. Interestingly, the Rif(r) mutation frequency of
single ung and mutSL (mismatch repair [MMR] system) mutants was dramatically
increased in a ung knockout strain that was also deficient in MutSL, suggesting
that the MMR pathway also counteracts the mutagenic effects of uracil. Since the
mutation frequency of the ung mutSL strain was significantly increased by SB, in
addition to Ung, the mutagenic effects promoted by base deamination in growing
B. subtilis cells are prevented not only by YwqL but also by MMR. Importantly,
in nondividing cells of B. subtilis, the accumulations of mutations in three
chromosomal alleles were significantly diminished following the disruption of
ung and ywqL. Thus, under conditions of nutritional stress, the processing of
deaminated bases in B. subtilis may normally occur in an error-prone manner to
promote adaptive mutagenesis. Scientists have been aware for many years of genetic programs that get activated
under stress and produce genetic variants in cells that escape non-proliferating
conditions. These programs are well conserved in all organisms and expand our
view of evolution. They mediate genome instability, create diversity in antibody
formation, expand metabolism and increase fitness of pathogens within host
environments. Error-prone DNA replication and repair are genetic
variability-causing agents that get stimulated by the onset of cellular
stresses. Embedded in these programs is the ability to limit mutagenesis to
defined genomic regions and times, ensuring integrity of most of the genome.
Recent evidence suggests that factors involved in RNA polymerase (RNAP)
processivity or transcriptional derepression contribute to the generation of
stress-induced mutations. In Bacillus subtilis, transcription-associated
mutagenesis has been shown to be independent of recombination-dependent repair
and, in some cases, of the Y DNA polymerases. Central to stationary-phase
mutagenesis in B. subtilis is the requirement for Mfd, transcription coupling
repair factor, which suggests a novel mechanism from those described in other
model systems. In growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or
resulting from the enzymatic elimination of oxidized bases must be processed by
AP endonucleases before they compromise cell integrity. Here, we investigated
how AP sites and the processing of these noncoding lesions by the AP
endonucleases Nfo, ExoA, and Nth contribute to the production of mutations
(hisC952, metB5, and leuC427) in starved cells of the Bacillus subtilis YB955
strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA,
and Nth accumulated a greater amount of AP sites in the stationary phase than
during exponential growth. Moreover, under growth-limiting conditions, the
triple nfo exoA nth knockout strain significantly increased the amounts of
adaptive his, met, and leu revertants produced by the B. subtilis YB955 parental
strain. Of note, the number of stationary-phase-associated reversions in the
his, met, and leu alleles produced by the nfo exoA nth strain was significantly
decreased following disruption of polX. In contrast, during growth, the
reversion rates in the three alleles tested were significantly increased in
cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX).
Therefore, we postulate that adaptive mutations in B. subtilis can be generated
through a novel mechanism mediated by error-prone processing of AP sites
accumulated in the stationary phase by the PolX DNA polymerase. |
What personality traits can be evaluated with the Ten Item Personality Inventory. | The Ten Item Personality Inventory measures each of the five major facets of personality: openness, extroversion, conscientiousness, agreeableness and neuroticism. | In their definition of modern nursing, the Royal College of Nursing emphasizes
the importance of caring. However, there is little other than anecdotal evidence
that female qualified staff nurses are more caring and compassionate than
average individuals. A study was carried out to test, under scientific
conditions with a case control study, the hypothesis that staff nurses are no
more caring than average female individuals. Using the ten-item personality
inventory (TIPI) questionnaire, a statistical comparison was made between 174
volunteer female staff nurses and data for 760 adult female controls extracted
from the TIPI instrument's original validation study. The questionnaire measures
each of the five major facets of personality: openness, extroversion,
conscientiousness, agreeableness and neuroticism. Agreeableness, which is a
tendency to be compassionate, considerate and cooperative, was used as a proxy
measure for 'caring'. Data were analysed using unpaired Student's t-tests.
Female staff nurses recorded significantly higher scores than female controls
concerning the personality traits extroversion, agreeableness, conscientiousness
and emotional stability (P<0.05). The analysis demonstrates that in direct
comparison to normal adult females, staff nurses are significantly more caring,
conscientious and resilient individuals. The personality traits found in female
staff nurses complement their profession and to some extent justify the caring,
compassionate nurse stereotype. Whether career nursing self-selects these
qualities or to what extent nursing staff develop aspects of their personality
as a product of experience is a subject for debate. BACKGROUND: There are approximately 2 million adoptive parents in the United
States and some struggle with depressive symptoms postplacement. We know little
about personality traits that may be associated with depression in adoptive
parents.
OBJECTIVES: This study describes the relationships between personality traits,
unmet expectations, and maternal postadoption depression.
DESIGN: Adoptive mothers (N = 136) were surveyed for depressive symptoms using
the Center for Epidemiologic Studies-Depression Scale (CES-D) and the Edinburgh
Postnatal Depression Scale (EPDS). Analyses included correlations and a
regression analysis between depressive symptoms and unmet expectations with the
Five-Factor Model personality traits (extraversion, agreeableness,
conscientiousness, emotional stability, openness to experience) as measured by
the Ten-Item Personality Inventory.
RESULTS: Both the CES-D and EPDS were significantly, negatively correlated with
all five personality dimensions. Mothers whose expectations of themselves as
parents matched pre- and postplacement were more likely to be emotionally stable
and extraverted. Approximately 36% of the variance in depressive symptoms was
explained by personality traits (emotional stability: p < .0001).
CONCLUSIONS: The postadoption period is a crucial time of transition for mothers
and their children. Maternal emotional stability, depressive symptoms, and unmet
expectations may affect this period. Mothers who are lower in emotional
stability may be at risk for postadoptive depressive symptoms. In planning and
providing innovative care that promotes positive mother-child relationships,
nurses should assess adoptive mothers for depressive symptoms, emotional
stability, and unmet expectations. |
Which are the enzymes involved in the control of tubulin acetylation? | Acetyltransferase MEC-17, and deacetylases SIRT2 (Sirtuin 2), HDAC6 (histone deacetylase 6) and dTip60 are known to control the levels of tubulin acetylation. | It has been shown that Wallerian degeneration, an anterograde degeneration of
transected axons, is markedly delayed in a mutant mouse called slow Wallerian
degeneration (Wld(S)). These mice also show resistance to axonal degeneration
caused by microtubule depolymerizing drugs, suggesting that axonal microtubules
are stabilized. Here, we have focused on tubulin acetylation, a
post-translational modification associated with microtubule stability. We found
that the basal level of microtubule acetylation was increased in cultured
cerebellar granule cells from Wld(S) mice. Nicotinamide but not
3-aminobenzamide, an inhibitor for poly(ADP)ribose polymerase, enhanced tubulin
acetylation and resistance to axonal degeneration in cultured cerebellar granule
cells from wild-type (WT) mice, suggesting that mammalian Sir2-related protein
(SIRT) 2, a nicotinamide adenine dinucleotide (NAD)--dependent tubulin
deacetylase, could modulate resistance to axonal degeneration. Indeed, the
levels of NAD and SIRT2 were decreased in the cytoplasm from Wld(S) granule
cells. Moreover, SIRT2 overexpression abrogated microtubule hyperacetylation and
resistance to axonal degeneration in these cells. Conversely, SIRT2 knockdown by
using a lentiviral vector expressing small interfering RNA, enhanced microtubule
acetylation and resistance to axonal degeneration in WT granule cells. Taken
together, these results suggest that SIRT2-mediated tubulin deacetylation is
involved in both microtubule hyperacetylation and resistance to axonal
degeneration in Wld(S) granule cells. The human HDAC (histone deacetylase) family, a well-validated anticancer target,
plays a key role in the control of gene expression through regulation of
transcription. While HDACs can be subdivided into three main classes, the class
I, class II and class III HDACs (sirtuins), it is presently unclear whether
inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific
isoforms that show aberrant levels in tumours, will prove more effective as an
anticancer strategy in the clinic. To address the above issues, we have tested a
number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC
(recombit human HDAC) isoforms. Eight rhHDACs were expressed using a
baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC
assay was optimized for each purified isoform. The potency and selectivity of
ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class
II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275
was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was
HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I
HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824,
panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC
inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that
both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results
also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted
in increased acetylation of histones, but only pan-HDAC inhibitor treatment
resulted in increased tubulin acetylation, which is in agreement with their
activity towards the HDAC6 isoform. We have been using polarized, hepatic WIF-B cells to examine ethanol-induced
liver injury. These cells polarize in culture and maintain numerous
liver-specific activities including the ability to metabolize alcohol.
Previously, we found that microtubules were more highly acetylated and more
stable in ethanol-treated WIF-B cells and that increased microtubule acetylation
required ethanol metabolism and was likely mediated by acetaldehyde. This study
was aimed at identifying the mechanism responsible for increased microtubule
acetylation. We examined the expression of two known microtubule deacetylases,
histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF-B cells.
Immunoblotting, immunofluorescence microscopy, and assays using the SirT2
inhibitor nicotinamide revealed that WIF-B cells do not express SirT2. In
contrast, HDAC6 was highly expressed in WIF-B cells. Addition of trichostatin A
(TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as
in ethanol-treated cells (approximately threefold). Although immunofluorescence
labeling revealed that HDAC6 distribution did not change in ethanol-treated
cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6
solubility was increased in nocodazole-treated cells, suggesting impaired
microtubule binding. Direct microtubule binding assays confirmed this
hypothesis. The decreased microtubule binding was partially prevented by
4-methyl pyrazole, indicating the effect was in part mediated by acetaldehyde.
Interestingly, HDAC6 from ethanol-treated cells was able to bind and deacetylate
exogenous tubulin to the same extent as control, suggesting that ethanol-induced
tubulin modifications prevented HDAC6 binding to endogenous microtubules.
CONCLUSION: We propose that lower HDAC6 levels combined with decreased
microtubule binding lead to increased tubulin acetylation in ethanol-treated
cells. BACKGROUND: Tubulin is a major substrate of the cytoplasmic class II histone
deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated
tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which
promotes transport of cargoes along microtubules. Microtubule-dependent
intracellular trafficking may therefore be regulated by modulating the activity
of HDAC6. We have shown previously that the neuromodulator serotonin increases
mitochondrial movement in hippocampal neurons via the Akt-GSK3beta signaling
pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway.
METHODOLOGY/PRINCIPAL FINDINGS: We found that the presence of tubacin, a
specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in
hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no
effect. Compared to control cultures, higher levels of acetylated tubulin were
found in neurons treated with tubacin, and more kinesin-1 was associated with
mitochondria isolated from these neurons. Inhibition of GSK3beta decreased
cytoplasmic deacetylase activity and increased tubulin acetylation, whereas
blockade of Akt, which phosphorylates and down-regulates GSK3beta, increased
cytoplasmic deacetylase activity and decreased tubulin acetylation.
Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor
agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin
acetylation. GSK3beta was found to co-localize with HDAC6 in hippocampal
neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to
phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6. GSK3beta
may therefore regulate HDAC6 activity by phosphorylation.
CONCLUSIONS/SIGNIFICANCE: This study demonstrates that HDAC6 plays an important
role in the modulation of mitochondrial transport. The link between HDAC6 and
GSK3beta, established here, has important implications for our understanding of
neurodegenerative disorders. In particular, abnormal mitochondrial transport,
which has been observed in such disorders as Alzheimer's disease and Parkinson's
disease, could result from the misregulation of HDAC6 by GSK3beta. HDAC6 may
therefore constitute an attractive target in the treatment of these disorders. Angiotensin II has been implicated in vascular remodeling. Microtubule composed
of tubulins regulates cell shape, migration and survival. Tubulin acetylation
has an important role in the control of microtubule structure and
microtubule-based cellular functions. In this study, angiotensin II induced
disassembly and deacetylation of α-tubulin, which were blocked by pretreatment
with an angiotensin II type 1 receptor blocker losartan and a sirtuin class
deacetylase inhibitor sirtinol, and by depletion of a deacetylase SIRT2 using
RNA interference. We investigated the involvement of SIRT2 in angiotensin
II-induced endothelial cell migration using the Boyden chamber method.
Angiotensin II caused a significant increase in cell migration, which was
blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported
that angiotensin II is involved in cytoskeletal reorganization stimulated by
mechanical stretch in endothelial cells. We also demonstrated that endothelial
cells subjected to a 10% uniaxial stretch showed vertical alignment to the
direction of tension and tubulin deacetylation in the peripheral side of cells,
in comparison with control static cells. The mechanical stretch-induced changes
of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion.
Immunofluorescence microscopy showed that acetylated tubulin was decreased in
platelet-endothelial cell adhesion molecule-1-positive cells in the intima of
the aortic walls in mice loaded with angiotensin II, in comparison with mice
loaded with control vehicle. These data show that angiotensin II and mechanical
stretch stimulate microtubule redistribution and deacetylation via SIRT2 in
endothelial cells, suggesting the emerging role of SIRT2 in hypertension-induced
vascular remodeling. BACKGROUND: Histone acetylation of chromatin plays a key role in promoting the
dynamic transcriptional responses in neurons that influence the neuroplasticity
linked to cognitive ability, yet the specific histone acetyltransferases (HATs)
that create such epigenetic marks remain to be elucidated.
METHODS AND FINDINGS: Here we use the Drosophila neuromuscular junction (NMJ) as
a well-characterized synapse model to identify HATs that control synaptic
remodeling and structure. We show that the HAT dTip60 is concentrated both pre
and post-synaptically within the NMJ. Presynaptic targeted reduction of dTip60
HAT activity causes a significant increase in synaptic bouton number that
specifically affects type Is boutons. The excess boutons show a suppression of
the active zone synaptic function marker bruchpilot, suggesting defects in
neurotransmission function. Analysis of microtubule organization within these
excess boutons using immunohistochemical staining to the microtubule associated
protein futsch reveals a significant increase in the rearrangement of
microtubule loop architecture that is required for bouton division. Moreover,
α-tubulin acetylation levels of microtubules specifically extending into the
terminal synaptic boutons are reduced in response to dTip60 HAT reduction.
CONCLUSIONS: Our results are the first to demonstrate a causative role for the
HAT dTip60 in the control of synaptic plasticity that is achieved, at least in
part, via regulation of the synaptic microtubule cytoskeleton. These findings
have implications for dTip60 HAT dependant epigenetic mechanisms underlying
cognitive function. Neurotensin (NT), an intestinal peptide secreted from N cells in the small
bowel, regulates a variety of physiological functions of the gastrointestinal
tract, including secretion, gut motility, and intestinal growth. The class IA
phosphatidylinositol 3-kinase (PI3K) family, which comprised of p110 catalytic
(α, β and δ) and p85 regulatory subunits, has been implicated in the regulation
of hormone secretion from endocrine cells. However, the underlying mechanisms
remain poorly understood. In particular, the role of PI3K in intestinal peptide
secretion is not known. Here, we show that PI3K catalytic subunit, p110α,
negatively regulates NT secretion in vitro and in vivo. We demonstrate that
inhibition of p110α, but not p110β, induces NT release in BON, a human endocrine
cell line, which expresses NT mRNA and produces NT peptide in a manner analogous
to N cells, and QGP-1, a pancreatic endocrine cell line that produces NT
peptide. In contrast, overexpression of p110α decreases NT secretion.
Consistently, p110α-inhibition increases plasma NT levels in mice. To further
delineate the mechanisms contributing to this effect, we demonstrate that
inhibition of p110α increases NT granule trafficking by up-regulating α-tubulin
acetylation; NT secretion is prevented by overexpression of HDAC6, an α-tubulin
deacetylase. Moreover, ras-related protein Rab27A (a small G protein) and kinase
D-interacting substrate of 220 kDa (Kidins220), which are associated with NT
granules, play a negative and positive role, respectively, in
p110α-inhibition-induced NT secretion. Our findings identify the critical role
and novel mechanisms for the PI3K signaling pathway in the control of intestinal
hormone granule transport and release. Neuronal migration is a fundamental process during the development of the
cerebral cortex and is regulated by cytoskeletal components. Microtubule
dynamics can be modulated by posttranslational modifications to tubulin
subunits. Acetylation of α-tubulin at lysine 40 is important in regulating
microtubule properties, and this process is controlled by acetyltransferase and
deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has
been found to play a major role in the acetylation of α-tubulin in different
species in vivo. However, the physiological function of MEC-17 during neural
development is largely unknown. Here, we report that MEC-17 is critical for the
migration of cortical neurons in the rat. MEC-17 was strongly expressed in the
cerebral cortex during development. MEC-17 deficiency caused migratory defects
in the cortical projection neurons and interneurons, and perturbed the
transition of projection neurons from the multipolar stage to the
unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore,
knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to
mimic acetylated α-tubulin could reduce the migratory and morphological defects
caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which
regulates the acetylation of α-tubulin, appears to control the migration and
morphological transition of cortical neurons. This finding reveals the
importance of MEC-17 and α-tubulin acetylation in cortical development. Cytoskeleton remodelling is a prerequisite step for the morphological transition
from preadipocytes to mature adipocytes. Although microtubules play a pivotal
role in organizing cellular structure, regulation of microtubule dynamics during
adipogenesis remains unclear. In the present paper we show that acetylation of
α-tubulin is up-regulated during adipogenesis, and adipocyte development is
dependent on α-tubulin acetylation, as expression of an acetylation-resistant
α-tubulin mutant significantly inhibits adipogenesis. Moreover, acetylation of
α-tubulin is under the control of the acetyltransferase MEC-17 and deacetylases
SIRT2 (Sirtuin 2) and HDAC6 (histone deacetylase 6). Adipocyte development is
inhibited in MEC-17-knockdown cells, but enhanced in MEC-17-overexpressing
cells. Finally, we show that katanin, a microtubule-severing protein with
enhanced activity on acetylated α-tubulin, is actively involved in adipogenesis.
We propose that co-ordinated up-regulation of α-tubulin acetylation initiates
cytoskeleton remodelling by promoting α-tubulin severing by katanin which, in
turn, allows expansion of lipid droplets and accelerates the morphological
transition toward mature adipocytes. The post-translational modification of tubulin appears to be a highly controlled
mechanism that regulates microtubule functioning. Acetylation of the ε-amino
group of Lys-40 of α-tubulin marks stable microtubules, although the causal
relationship between tubulin acetylation and microtubule stability has remained
poorly understood. HDAC6, the tubulin deacetylase, plays a key role in
maintaining typical distribution of acetylated microtubules in cells. Here, by
using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of
HDAC6, we have explored whether tubulin acetylation has a role in regulating
microtubule stability. We found that whereas both pharmacological inhibition of
HDAC6 as well as its depletion enhance microtubule acetylation, only
pharmacological inhibition of HDAC6 activity leads to an increase in microtubule
stability against cold and nocodazole-induced depolymerizing conditions.
Tubastatin A treatment suppressed the dynamics of individual microtubules in
MCF-7 cells and delayed the reassembly of depolymerized microtubules.
Interestingly, both the localization of HDAC6 on microtubules and the amount of
HDAC6 associated with polymeric fraction of tubulin were found to increase in
the tubastatin A-treated cells compared with the control cells, suggesting that
the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to
microtubules. The evidence presented in this study indicated that the increased
binding of HDAC6, rather than the acetylation per se, causes microtubule
stability. The results are in support of a hypothesis that in addition to its
deacetylase function, HDAC6 might function as a MAP that regulates microtubule
dynamics under certain conditions. |
Is there a relation between ANP and transcapillary albumin escape? | A possible role of ANP gene in conferring protection from nephropathy and microvascular damage in type 1 diabetes is present.
ANP infusion in healthy subjects caused a shift of plasma water and electrolytes from the circulation, with albumin escape as a secondary phenomenon | Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and
diuretic effects. To better define the ANP hormonal system in the nephrotic
syndrome, a condition associated with renal sodium retention, we undertook a
study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced
nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly
decreased serum albumin and total protein and significantly increased serum
cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level
of atrial natriuretic peptide was similar in both groups of rats. Competition
binding inhibition studies in isolated glomeruli demonstrated one binding site
in both groups of rats. The density of ANP binding sites in isolated glomeruli
was similar in nephrotic and pair-fed rats while the binding affinity was
increased significantly in the nephrotic rats. This is the first study to
demonstrate alterations in renal ANP receptors in the nephrotic syndrome.
Further studies will be necessary to determine whether alterations in glomerular
ANP receptors contribute to renal sodium retention in the nephrotic syndrome. The authors investigated the role of atrial natriuretic peptide (alpha-hANP
99-126) in essential hypertension by evaluating some hemodynamic and renal
effects of acute peptide infusion (1 micrograms/kg for 1 min + 50 ng/kg for the
following 20 min) in fourteen subjects: eight mild to moderate, untreated,
essential hypertensives (EH) and six normotensive (N) controls, during 2
hour-clearance periods, the 1st after ANP infusion, the 2nd during placebo (PL)
administration. The double-blind study was carried out after the patients had
rested and fasted overnight. It showed no significant changes in heart rate
(HR); instead, compared with placebo, mean blood pressure (MBP) decreased
significantly in both groups, beginning from the 3rd min after ANP infusion was
begun (N: PL = 87.04 +/- 1.7 mmHg, ANP = 80.9 +/- 3.7 mmHg, p less than 0.0001;
EH: PL = 102.6 +/- 3.2 mmHg, ANP = 97.7 +/- 5.9 mmHg, p less than 0.01). Among
the urinary parameters we considered, cyclic GMP (cGMP) increased after ANP
infusion in all subjects (N: PL = 129.1 +/- 56.3 pmol/mL, ANP = 199.2 +/- 85.4
pmol/mL; EH: PL = 106.55 +/- 56.2 pmol/mL, ANP = 220.03 +/- 92.7 pmol/mL, p less
than 0.05); diuresis showed a prompt and significant increase in EH (N: PL = 837
+/- 368 mL, ANP = 1066 +/- 340 mL; EH: PL = 713 +/- 286 mL, ANP = 1043 +/- 280
mL, p less than 0.005), and so did natriuresis (N: PL = 23 +/- 14.3 mEq/L, ANP =
33 +/- 14.6 mEq/L; EH: PL = 25.6 +/- 8.9 mEq/L, ANP = 41.9 +/- 13.8 mEq/L, p
less than 0.01); urinary potassium excretion was significantly reduced in EH (N:
PL = 18.7 +/- 12.9 mEq/L, ANP = 14.2 +/- 6.9 mEq/L; EH: PL = 16.5 +/- 7.9 mEq/L,
ANP = 10.7 +/- 4.8 mEq/L, p less than 0.005), while no changes were noted in
glomerular filtration rate (GFR), estimated as creatinine clearance, urinary
magnesium, albumin and aldosterone excretion. To investigate other potential
mechanisms involved in renal effects of ANP, the urinary excretion of both
prostaglandins 6-cheto PGF1-alpha and thromboxane B2 (TXB2), and dopamine were
studied. The results showed only a significant decrease of dopamine urinary
excretion in EH after ANP administration (N: PL = 50.4 +/- 28.7 micrograms/L,
ANP = 45.0 +/- 29.7 micrograms/L; EH: PL = 47.3 +/- 21.5 micrograms/L, ANP =
27.1 +/- 12.7 micrograms/mL, p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS) Low-dose infusions of atrial natriuretic peptide (ANP) into humans reduce
cardiac filling pressures without enhancing renal excretion or producing
vasodilation. The present human study was undertaken to seek an effect of ANP on
capillary filtration in humans and to determine its relationship to reductions
in cardiac filling pressures. Heart rate (electrocardiogram), blood pressure
(cuff method), and renal excretion of salt and water were determined, and
central venous pressure (jugular vein cannulation, strain-gauge transducer) and
forearm venous compliance and capillary filtration coefficient (strain-gauge
plethysmography) were derived by computer. Forearm girth and venous pressure
(peripheral vein catheter) measurements were obtained while the arm was elevated
above heart level, and an upper arm blood pressure cuff was intermittently
inflated to venous occluding pressures of 20, 30, and 40 mmHg. Forearm
measurements, hematocrit, plasma proteins, albumin and plasma levels of ANP were
determined from euvolemic volunteers before and during 60 min intravenous
infusions of ANP (5 ng.kg-1.min-1, n = 9) or placebo (isotonic saline, n = 7).
ANP infusions produced physiological increases (4- to 5-fold) in plasma ANP
(from a base line 35 +/- 6 pg/ml) (P less than 0.05). Hemodynamic responses to
ANP consisted of a reduction in central venous pressure (P less than 0.05) and
no change in heart rate, mean arterial pressure, or renal excretory parameters.
ANP increased forearm capillary filtration between 37 and 63% (P less than 0.05)
from base line but did not significantly alter forearm compliance
measurements.(ABSTRACT TRUNCATED AT 250 WORDS) Changes in plasma levels of atrial natriuretic peptide (ANP) and albumin, and
blood hematocrit (Hct) during treadmill exercise were studied in 6 healthy men
before and after beta-adrenergic blockade. Plasma ANP levels increased during
exercise and then gradually decreased. There was a concomitant increase in both
Hct and plasma albumin concentrations. Prior administration of a long-acting
propranolol, 160 mg daily for 3 consecutive days, markedly elevated plasma ANP
levels before, during and after exercise. In addition, the mean basal Hct
increased significantly and further rose during exercise after propranolol
administration. When increments in plasma ANP concentrations during exercise in
individual subjects were compared with those in Hct, there was a significant
positive correlation between the two variables before and after propranolol
administration. The results indicate a close relationship between the changes in
plasma ANP and those in Hct during exercise at different ANP levels, and suggest
that ANP may be at least one of the factors involved in the hemoconcentration
associated with exercise. Plasma atrial natriuretic peptide (ANP) levels were examined in 66 patients with
non-insulin-dependent diabetes mellitus (NIDDM), and in 9 age-matched normal
controls and 18 hypertensive controls. The diabetic patients were classified
into three groups according to random urine albumin/creatinine ratio (ACR, mg/g
or mg/88.4 nM); group 1 (normo-albuminuria, ACR less than 20, n = 34), group 2
(borderline microcalbuminuria, 20 less than or equal to ACR less than 100, n =
17) and group 3 (manifest microalbuminuria and macroalbuminuria, 100 less than
or equal to ACR less than 2000, n = 15). Plasma ANP levels (pg per ml) were
significantly elevated in group 2 (46.0 +/- 19.0 SD) when compared with either
normal controls (23.8 +/- 14.2), group 1 (28.9 +/- 15.6) or group 3 (26.0 +/-
12.9). This increase in plasma ANP levels was not related to hypertension.
Furthermore, plasma ANP levels correlated positively with log(ACR) among the
patients with ACR under 100 (groups 1 and 2 combined, r = 0.4701, p less than
0.01). These results suggest that an elevated plasma ANP level in the early
phase of microalbuminuria possibly plays a pathophysiological role in the
development of nephropathy in NIDDM patients. Previous studies in our laboratory showed that ANP inhibits increases in
endothelial monolayer permeability to macromolecules induced by thrombin. In
this present study, we investigated the second messenger system involved in the
influence of ANP on monolayer permeability. In bovine aortic endothelial cells
(BAEC), ANP (100 nM) caused increased cGMP levels which were measurable at 30
sec and maximal at 3 min. Addition of 8-bromo cGMP (1 mM) to BAEC monolayers
mimicked the actions of ANP by inhibiting thrombin- mediated increases in
permeability to [125I]-labeled bovine serum albumin. Inhibition of increases in
permeability by lower concentrations of ANP was enhanced by the cGMP-selective
phosphodiesterase inhibitor, M&B 22948 (100 microM). The use of ANP structural
analogs which stimulate cGMP production (AP III or BNP) prevented
thrombin-induced increases in monolayer permeability, whereas AP-I, which does
not increase cGMP levels, was ineffective. The role of atrial natriuretic peptide (ANP) on jejunal net fluid transport was
studied in intact rats as well as in rats subjected to a perivascular
denervation of the intestinal segment. In rats with intact nerves, an acute
volume expansion with 5% albumin (10% of estimated blood volume) decreased
jejunal net fluid absorption by approximately 70% compared to control animals
not subjected to volume expansion. After a perivascular denervation of the
intestinal segment, the acute volume expansion reversed net fluid absorption
into a net fluid secretion. In order to reduce the volume expansion-induced
endogenous release of ANP, one group of rats was subjected to a right atrial
appendectomy 7 days prior to the experiments. In these animals, the intestinal
response to the same 10% volume load was blunted compared to controls.
Administration of rat alpha-ANP (99-126; 5 micrograms kg-1 i.v.) induced effects
similar to those of volume expansion both in rats with intact perivascular
nerves as well as in denervated animals. Volume expansion increased mean
arterial pressure (MAP) as well as central venous pressure and decreased heart
rate (HR) in all groups. When exogenous ANP was administered, a fall in MAP was
seen, while HR remained unchanged. In conclusion, these data strongly indicate a
physiological role for ANP in jejunal fluid transfer in response to acute volume
expansion. Endogenous alpha-atrial natriuretic peptide (ANP) in plasma is elevated in
various hypervolaemic conditions. Possible relationships between circulating
immunoreactive ANP and cardiovascular and splanchnic haemodynamics were
therefore studied in patients with cirrhosis (n = 16) and controls (n = 12).
Arterial plasma concentration of ANP in supine patients was (mean +/- SEM) 33
+/- 4 vs 41 +/- 10 pg/ml (9.9 +/- 1.2 vs 12.3 +/- 3.0 fmol/l) in controls
(n.s.), and there was a weak direct correlation with right atrial pressure (r =
0.36, P = 0.05). There was no relationship with the presence of ascites or
diuretic treatment. Central blood volume (CBV, i.e. the blood volume in the
heart cavities, lungs, and aorta), determined from the mean transit time of
125I-labelled of 125I-labelled albumin and cardiac output, was significantly
reduced in cirrhotics compared to controls (1.45 +/- 0.12 vs. 1.83 +/- 0.10 l, P
less than 0.02) and inversely correlated with portal pressure (r = 0.42, P less
than 0.05), whereas total plasma volume was somewhat increased (3.51 +/- 0.2 vs.
3.19 +/- 0.2, 0.05 less than P less than 0.1). A high arterio-venous extraction
of ANP was found in the splanchnic system (extraction ratio 0.44 vs 0.28),
kidney (0.45 vs 0.54), lower limb (0.53 vs 0.40), and forearm (0.27 vs 0.18) in
patients and controls, respectively (n.s.). Our results suggest that the lack of
elevation of circulating ANP in cirrhosis, even in the presence of actual fluid
retention, may be explained by central hypovolaemia in these patients. Turnover
and degradation of ANP is rapid and normal, as evaluated from the tissue
extraction ratios. Plasma levels of atrial natriuretic peptide (ANP), arginine vasopressin (AVP),
renin activity (PRA), aldosterone (PA), catecholamines and urinary
prostaglandins (PG), as well as renal function were measured in children in the
edematous state of the nephrotic syndrome before and after infusion of human
serum albumin. Before albumin infusion, plasma levels of AVP, PRA, PA and
noradrenaline (NA) and urinary excretion of PGE2, PGE-Met, PGF2 alpha were
elevated. The mean value of plasma ANP was in the normal range. Albumin infusion
produced a 36% increase in the calculated plasma volume. It was associated with
a fivefold rise in the plasma level of ANP (31.6 +/- 22.6 vs. 151.4 +/- 52
fmol/ml mean, SD), and a significant fall in the levels of PRA, AVP, PA, and NA.
Similarly, urinary concentration of PGE2, PGE-Met and PGF2 alpha fell. Urine
flow, GFR, UNaV, FENa, and COsm increased significantly, while CH2O remained
unchanged. The diuresis, natriuresis and GFR correlated with the level of plasma
ANP, while urinary sodium excretion did not correlate with PA or NA levels.
These findings suggest that ANP plays an important role in albumin induced
natriuresis in children with nephrotic syndrome. 1. Plasma immunoreactive atrial natriuretic peptide (ANP) and urinary sodium
excretion were measured in six normal male subjects before, during and for 195
min after a 60 min infusion of 2 litres of saline (0.9% NaCl, 308 mmol of Na+).
2. During the saline infusion, there was a significant increase in plasma ANP
and urinary sodium excretion and a significant decrease in plasma renin
activity, aldosterone, albumin, creatinine and packed cell volume. 3. The
maximal rise in mean plasma ANP occurred 15 min after stopping the infusion and
the maximal rise in mean urinary sodium excretion in the collection period 30
min later. 4. Plasma ANP then decreased so that by the end of the study the
level was the same as before the saline infusion. However, at this time, 195 min
after the saline infusion was stopped, there was still a net positive sodium
balance of 220 mmol and urinary sodium excretion remained significantly
elevated. 5. Our results are compatible with the concept that increased ANP
secretion may play a role in the immediate increase in sodium excretion after a
saline load. However, they also suggest that other mechanisms may be more
important for the longer term increase in sodium excretion. To study the effect of atrial natriuretic peptide (ANP) on vascular permeation
of albumin in the fetus, ANP (167-600 ng/min) was infused into eight ovine
fetuses and saline vehicle was infused into eight twin controls (gestational age
127 +/- 3 d) over a 50-min period. Using two different radiolabeled albumin
markers, we determined the tissue to blood isotope ratio (TBIR), an index of
albumin permeation, and the albumin clearance. Although ANP had no hemodynamic
effect, a marked increase in the hematocrit was observed in ANP-infused fetuses
compared with initial values (0.37 +/- 0.04 vs 0.42 +/- 0.04, p < 0.005) but was
unchanged in the twin fetuses receiving saline vehicle (0.35 +/- 0.03 versus
0.35 +/- 0.02). TBIR and albumin permeation were increased in combined tissues
of ANP-infused fetuses compared with saline controls (TBIR: 1.49 +/- 0.58 versus
1.29 +/- 0.3, p < 0.001; albumin clearance: 1091 +/- 1279 versus 827 +/- 1464
nL/g/min, p < 0.01). In individual tissues, TBIR was significantly increased in
skin (2.88 +/- 0.67 versus 1.55 +/- 0.35, p < 0.02), muscle (1.6 +/- 0.27 versus
1.24 +/- 0.26, p < 0.02), adrenal (1.33 +/- 0.10 versus 1.13 +/- 0.15, p <
0.02), bone (1.67 +/- 0.45 versus 1.20 +/- 0.40, p < 0.02), kidney (1.52 +/-
0.25 versus 1.24 +/- 0.26, p < 0.03), and gut (1.69 +/- 0.20 versus 1.39 +/-
0.34, p < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS) Low dose infusion of alpha human atrial natriuretic peptide (ANP) has been shown
to cause a shift of intravascular fluid to the interstitial space. No studies
have been reported on the effect of ANP on capillary permeability to plasma
proteins. We studied the effect of low dose ANP (2.5 pmol/kg.min) or equal
volume of saline control when infused over 90 min, on plasma volume,
transcapillary escape rate of intravascular albumin, and the rate of reentry of
albumin to the vascular space in eight normal subjects. Intravenous injection of
125I-human serum albumin was used for measurement of plasma volume and
intravascular albumin escape rate. A significant ANP-induced fall in plasma
volume (P < 0.01) was observed. Transcapillary escape rate of intravascular
albumin when corrected for concurrent plasma volume changes showed a
significantly greater escape during ANP infusion than during saline control (P <
0.05). The mean +/- SEM changes in plasma albumin concentration were +0.36 +/-
0.22 g/L.h during ANP infusion and -0.46 +/- 0.50 g/L.h during placebo. Plasma
sodium, red cell volume, and urinary albumin and radioactivity remained
unchanged. The mass of albumin reentering the circulation per hour showed no
significant difference between the 2 days. In summary, low dose ANP infusion in
healthy subjects caused a shift of plasma water and electrolytes from the
circulation, with albumin escape as a secondary phenomenon. The effects of endopeptidase inhibition and right atrial appendectomy (AA) on
plasma immunoreactive atrial natriuretic peptide (irANP) concentrations and
extravascular accumulation of 131I-labeled bovine serum albumin (CBSA) during
volume expansion (VE) were examined in pentobarbital sodium-anesthetized rats (n
= 5 per group). Compared with controls, infusion of isoncotic albumin (30 ml/kg)
increased central venous pressure (CVP) by 5.5 mmHg, plasma irANP by 7-fold, and
CBSA in multiple tissues by 1.6- to 4-fold (P < or = 0.05). Inhibition of ANP
metabolism with thiorphan (40 mg/kg) had no effect in control animals but
increased plasma irANP (+118%) and CBSA (+30-100%) compared with VE alone (P <
or = 0.05). Likewise, interference with ANP release by AA reduced plasma irANP
(-80%) and CBSA (-30 to -80%) response to VE in parallel (P < or = 0.05). The
peak increase in CVP during VE was not affected by either of these
manipulations. It is concluded that endogenous ANP increases plasma protein
extravasation independent of volume and pressure disturbances during acute
intravascular expansion. Renal function was measured by clearance technique before and after acute
myocardial infarction (MI) induced by left coronary artery ligation in male
Sprague-Dawley rats. The animals were anaesthetized with halothane-nitrous
oxide, paralysed with pancuronium and artificially ventilated. All parameters
were stable throughout the experiment in sham-operated time control animals (n =
8). After MI, rats developed left ventricular dysfunction with increased left
ventricular end-diastolic pressure and decreased mean arterial pressure. MI
produced antidiuresis and antinatriuresis without changes in glomerular
filtration rate (GFR), lithium clearance or renal albumin excretion (n = 8). The
antidiuretic and antinatriuretic responses to MI were similar in rats with
chronic bilateral renal denervation (n = 5). Three additional rats with chronic
bilateral renal denervation had cardiac arrest and were resuscitated with
cardiac massage, i.v. lidocaine and intracardiac adrenaline administration.
These animals showed a transient increase in urine flow rate, sodium and albumin
excretion with maximum 30-60 min after resuscitation, while GFR and lithium
clearance were normal. Since cardiac ischaemia and sympathetic stimulation are
strong stimuli for the release of atrial natriuretic peptide (ANP), we examined
if ANP (0.25, 0.50, and 1.00 microg kg(-1) min(-1), n = 8 per dose) affects
urinary albumin excretion. ANP increased dose-dependently the urine/plasma
concentration ratio of albumin relative to inulin, which suggests that ANP
increases the glomerular permeability for albumin. We conclude that MI causes
stimulation of renal tubular sodium and water reabsorption by a mechanism which
is independent of intact renal innervation. MI does not produce any change in
renal albumin excretion in rats, but transient albuminuria may be observed in
rats following cardiac arrest and/or manoeuvres used in cardiac resuscitation.
Since ANP produces albuminuria, we speculate that ANP may be an important
mediator of albuminuria in states with elevated plasma concentrations of ANP. Raised plasma concentrations of atrial natriuretic peptide (ANP) have been
reported in patients with Type 1 (insulin dependent) diabetes mellitus (DM) who
have poor glycaemic control and are associated with the presence of
microalbuminuria. To test the hypothesis that elevations in plasma ANP
concentration increase urinary albumin excretion in Type 1 DM, we have studied
the effects of intravenous infusions of ANP in eight such subjects with
established microalbuminuria. Blood glucose was maintained between 4 and 7 mmol
l-1 in all subjects for the duration of studies; after euglycaemia had been
established, a standard oral water load (20 ml kg-1 plus replacement of urinary
losses) was given. Once steady state diuresis was attained, subjects received
intravenous infusion of either placebo (0.9% saline), low dose (2.5 pmol kg-1
min-1) or high dose (5.0 pmol kg-1 kg min-1) ANP solution in a randomized,
double-blind protocol. Infusion of ANP caused a dose-dependent increase in
urinary albumin excretion rate (placebo, 11.3 (SD 8.9) to 8.7 (SD 6.8)
micrograms min-1; low dose ANP, 12.4 (SD 9.9) to 26.5 (SD 27.5) micrograms
min-1, p < 0.01; high dose ANP 10.3 (SD 7.3) to 36.6 (SD 28.5) micrograms min-1,
p < 0.001, ANOVA). Only high dose ANP caused an increase in urine flow. Blood
glucose remained unchanged in all studies. We conclude that intravenous
infusions of ANP cause a dose-dependent increase in urinary albumin excretion
rate in Type 1 DM subjects with microalbuminuria. These data support the
hypothesis that ANP has albuminuric actions which may contribute to
microalbuminuria in Type 1 DM. BACKGROUND: It has been suggested that atrial natriuretic peptide (ANP)
contributes to the glomerular hyperfiltration of diabetes mellitus. Infusion of
ANP increases the urinary excretion of albumin in patients with type I diabetes
mellitus (IDDM). Although the increased albuminuria is attributed to a rise in
glomerular pressure, alterations in tubular protein handling might be involved.
PATIENTS AND METHODS: We have studied the effects of ANP in nine
microalbuminuric IDDM patients. After obtaining baseline parameters, ANP was
infused over a 1-h period (bolus 0.05 microgram kg-1, infusion rate 0.01
microgram kg-1 min-1). Renal haemodynamics, sodium and water clearance and
tubular protein handling were studied.
RESULTS: The glomerular filtration rate (GFR) increased from 116.4 +/- 8.9 to
128.3 +/- 8.8 mL min-1 1.73 m-2, whereas the effective renal plasma flow (ERPF)
decreased from 534.3 +/- 44.3 to 484.9 +/- 33.3 mL min-1 1.73 m-2 (P < 0.05). As
a result, the filtration fraction was significantly higher during infusion of
ANP. ANP attenuated proximal tubular sodium reabsorption. Urinary albumin
excretion rose from 87.57 +/- 21.03 to 291.40 +/- 67.86 micrograms min-1 (P <
0.01). Changes in the urinary excretion of beta 2-microglobulin and free kappa
light chains were more marked, the excretion of beta 2-microglobulin increasing
from 0.28 +/- 0.21 to 51.87 +/- 10.51 micrograms min-1 (P < 0.01), and of free
kappa-light chains from 4.73 +/- 1.74 to 46.14 +/- 6.19 micrograms min-1 (P <
0.01).
CONCLUSIONS: The observed rise in albuminuria during infusion of ANP does not
simply reflect a change in glomerular pressure, but might at least partly result
from an attenuation of tubular protein reabsorption. Approximately 30% of diabetic patients develop nephropathy, the appearance of
which is partially under genetic control. Atrial natriuretic peptide (ANP) has
associated physiologic effects on the kidney. This study was conducted to
examine the relationship between a newly identified and known polymorphism at
the pronatriodilatin (PND) gene locus and renal involvement in type 1 diabetic
subjects. Of 454 type 1 diabetic patients (219 men, 235 women), 323 showed no
sign of nephropathy, 79 had incipient renal involvement, and 52 established
nephropathy; 58 healthy control subjects were examined for comparison. Allele
frequencies (C708 versus T708) were: 0.95 and 0.05 in normoalbuminuric patients,
respectively; 0.88 and 0.12 in microalbuminuric patients; 0.96 and 0.04 both in
those with overt nephropathy and in healthy control subjects (P = 0.011).
Patients with incipient nephropathy were in disequilibrium compared with the
total diabetic cohort (P = 0.02). In the same populations, an additional
genotype for ScaI polymorphism of the PND gene was tested. The A1 and A2 allele
frequencies were: 0.21 and 0.79 in normoalbuminuric patients; 0. 13 and 0.87 in
microalbuminuric patients; 0.06 and 0.94 in type 1 diabetic subjects with overt
nephropathy; and 0.20 and 0.80 in healthy control subjects, respectively (P <
0.0001). A subset of 55 normotensive patients with type 1 diabetes, well matched
for clinical features, plasma ANP levels, and microvascular permeability to
macromolecules, was investigated on the basis of the C708/T and A2/A1
polymorphisms. Both transcapillary escape rate of albumin (TERalb) and plasma
ANP levels were significantly lower in patients with the T708 than with C708
allele, as well as in the A1 than in A2 allele (TERalb: T708 versus C708:
5.5+/-1.7 versus 7.8+/-2.0%/h, P = 0.0001; plasma ANP levels: 8.3+/-3.9 versus
15.3+/-7.7 pg/ml, P = 0.0003; A1 versus A2: 6.05+/-2.2 versus 7.3+/-2.1%/h, P =
0.044; 8.53+/-4.6 versus 14.5+/-7.4 pg/ml, P = 0.0024, respectively). Thus, in a
large ethnically homogeneous cohort of diabetic subjects, our data show: (1) a
significant association of C708/T polymorphism with microalbuminuria in
long-term diabetes and with both lower plasma ANP levels and widespread albumin
leakage; and (2) a strong association between ScaI polymorphism and both
diabetic nephropathy and plasma ANP concentrations. These results suggest a
possible role of PND gene in conferring protection from nephropathy and
microvascular damage in type 1 diabetes. BACKGROUND: Microalbuminuria reflects widespread vascular dysfunction in type 1
diabetes mellitus and results from increased glomerular sieving caused by
changes in transglomerular pressure and/or permselectivity characteristics of
the glomerular basement membrane. Increased tubular reabsorption or degradation
of albumin will offset an early increase in albuminuria. We hypothesized that
the infusion of atrial natriuretic peptide (ANP) as a tool to increase
glomerular permeability might uncover changes in permselectivity in patients
with uncomplicated type 1 diabetes.
METHODS: We investigated whether these patients were characterized by
endothelial and/or vascular dysfunction. We therefore studied 46
normoalbuminuric patients (urinary albumin excretion [UAE] < 10 microg/min) with
type 1 diabetes and 44 healthy controls. Measurements of renal hemodynamics and
albuminuria were performed before (baseline) and during the infusion of ANP
(0.01 microg/kg/min). On a separate occasion, endothelial function was assessed
by the intra-arterial infusion of acetylcholine (ACh), an endothelial-dependent
vasodilator.
RESULTS: At baseline, glomerular filtration rate (GFR) and effective renal
plasma flow (ERPF) were greater in patients with diabetes (GFR, 121 +/- 3 versus
106 +/- 2 mL/min/1.73 m(2); ERPF, 558 +/- 16 versus 527 +/- 13 mL/min/1.73 m(2);
P < 0.001). The infusion of ANP increased filtration fraction. There were no
differences in these responses between groups. UAE was significantly greater in
patients with diabetes after the ANP infusion (15.8 +/- 1.4 [+183%] versus 9.5
+/- 1.3 microg/min [+96%]; P < 0.01). A subgroup of patients with diabetes with
an enhanced albuminuric response (change in UAE > 2 SD of controls) to ANP
infusion (mean UAE, 30.3 +/- 1.0 microg/min; 425% +/- 61%) was characterized by
a diminished vasodilatory response to ACh (maximal forearm blood flow, 17.2 +/-
2.9 [+563%] versus 26.3 +/- 2.3 mL/min/dL [+800%] in patients with diabetes with
a normal albuminuric response; P < 0.05).
CONCLUSIONS: In a subgroup of patients with uncomplicated type 1 diabetes, an
increase in glomerular permselectivity can be unmasked by the infusion of ANP.
These patients are characterized by a diminished vascular response to ACh. OBJECTIVE: To study the regulation of antidiuretic hormone (ADH) and atrial
natriuretic peptide (ANP) in obese and lean women with a swelling syndrome.
PATIENTS: Thirty-four obese women and 12 lean women with a swelling syndrome and
an abnormal isotopic test of capillary permeability to albumin were
investigated.
MEASUREMENTS: After 10 nocturnal hours of fluid restriction, subjects were asked
at 8am to ingest a tap water load of 20 ml/kg within 10 min and to remain
strictly recumbent until twelve noon on the first day, and to remain standing
and to walk around until twelve noon on the second day. Free water clearance and
the cGMP/creatinine and albumin/creatinine ratios were determined hourly in the
morning.
RESULTS: The total 4 h-urinary volume/ingested water volume ratio was
significantly lower on the second day both in the lean and the obese patients,
the differences being slightly larger in the obese patients. The increase in
free water clearance was significantly less on the second day in the obese
patients. The increase in cGMP/creatinine ratio was also significantly lower on
the second day in the obese patients. The maximum level of the urinary
albumin/creatinine ratio was significantly higher on the second day in the obese
patients.
CONCLUSION: In obese women with a swelling syndrome: (1) The higher increase in
the urinary albumin excretion rate after water loading followed by a sustained
upright position suggests a widespread alteration in capillary function, which
is also indicated by the isotopic test of capillary permeability to albumin. (2)
The water load-induced inhibition of ADH secretion and stimulation of ANP
secretion or ANP activity, more defective in the upright position than in the
recumbent one, is probably another major contributing factor to orthostatic
oedema. BACKGROUND: Microalbuminuria, a powerful predictor of cardiovascular events, is
thought to reflect widespread subclinical vascular abnormalities. To explore the
pathogenesis of increased urinary albumin excretion in primary hypertension we
evaluated systemic capillary permeability and ambulatory blood pressure (BP)
measurement in two groups of matched untreated patients with (n = 11) and
without (n = 29) microalbuminuria.
METHODS: Albuminuria was measured as the mean of albumin-to-creatinine ratio
(ACR) in three nonconsecutive first morning urine samples. Systemic capillary
permeability was evaluated by transcapillary escape rate of albumin (TERalb)
(ie, the 1-h decline rate of intravenous (125)I-albumin). Twenty-four-hour
ambulatory BP, renal hemodynamics, and hormones of the
renin-angiotensin-aldosterone system (RAAS) were also assessed.
RESULTS: Patients with microalbuminuria showed greater body mass index (BMI) (P
< .04), higher 24-h systolic and diastolic BP levels (P = .02), and higher
capillary permeability to albumin (P < .02) as compared to normoalbuminurics.
Renal hemodynamics and RAAS hormones were similar in the two groups. Univariate
analysis showed that urinary ACR was related to ambulatory pressure components
(P < .02), TERalb (r = 0.31, P < .05), smoking habits (r = 0.36, P = .02), and
left ventricular mass index (LVMI) (r = 0.57, P < .001) among the whole study
group. Logistic regression analysis showed that each 1% increment in TERalb or
10 mm Hg increase in systolic BP entailed an almost three times higher risk of
having microalbuminuria.
CONCLUSIONS: Microalbuminuria is associated with greater systemic BP load and
increased vascular permeability in patients with primary hypertension. Increased arterial endothelial cell permeability (ECP) is considered an initial
step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly
degraded by neprilysin (NEP) may reduce injury-induced endothelial cell
leakiness. Omapatrilat represents a first in class of pharmacological agents
which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized
that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs)
and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance
ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined
by I(125) albumin flux was assessed in HAECs with and without ANP pretreatment.
Next we examined the effects of chronic oral administration of omapatrilat (12
mg/kg/day, n=13) or placebo (n=13) for 8 weeks on aortic leakiness,
atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit
model of atherosclerosis produced by high cholesterol diet. In HAECs,
thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the
area of increased aortic leakiness determined by Evans-blue dye and area of
atheroma formation assessed by Oil-Red staining compared to placebo. In isolated
arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo
with and without the endothelium. ANP prevents thrombin-induced increases in ECP
in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness
and atheroma formation with enhanced endothelial independent vasorelaxation to
ANP. These studies support the therapeutic potential of dual inhibition of NEP
and ACE in the prevention of increased arterial ECP and atherogenesis which may
be linked to the ANP/cGMP system. |
List all approved indications for Glivec | CML - blast crisis, in accelerated phase, and in chronic phase after interferon failure or intolerance. Glivec received orphan drug status from the U.S. Food and Drug Administration (FDA) Office of Orphan Products Development on January 31, 2001, and accelerated approval from the FDA for the above three indications on May 10, 2001.
Gastrointestinal stromal tumor (GIST
Treatment with adjuvant imatinib following surgical resection of localized Kit-positive GIST
In locally advanced inoperable patients and metastatic patients, Imatinib is the standard treatment. | BACKGROUND: Imatinib (STI571 or Glivec, Novartis) is a new type of tyrosine
kinase inhibitor that selectively inhibits various tyrosine kinases including
ABL, BCR-ABL, KIT and PDGF receptors.
IMATINIB IN CML: Earlier studies have shown that Imatinib is highly effective in
the treatment of chronic myeloid leukemia (CML), which is characterized by
translocation of chromosome material from chromosome 9 to chromosome 22 with
formation of the so-called Philadelphia chromosome. During this process, an
abnormal fusion protein, tyrosine kinase BCR-ABL, is formed. In a phase I study
it was shown that a daily dose of 300 mg Imatinib resulted in a complete
hematological response in almost 98% of the patients.
IMATINIB IN GIST: Gastrointestinal stromal tumors (GIST) are also suitable
indications for treatment with Imatinib, the prerequisite being overexpression
by the tumor of c-KIT (CD117). This tumor entity responds extremely poorly to
polychemotherapy. Initial case reports and various study approaches appear to
indicate that treatment of GIST with Imatinib is a highly promising therapeutic
option in this entity. Follow-up studies with FDG-PET have shown an appreciable
decrease in uptake by tumor tissue, in some cases within only a few days, which
may well indicate an inhibition of intratumoral metabolism and growth. Most
patients with metastatic GIST achieve durable responses with imatinib, and the
disappearance of cancer-related symptoms is often rapid.
CONCLUSION: Imatinib is the first effective systemic therapy for advanced GIST. Gleevec (imatinib mesylate), a highly promising new drug for the treatment of
chronic myelogenous leukemia in blast crisis, in accelerated phase, and in
chronic phase after interferon failure or intolerance, received orphan drug
status from the U.S. Food and Drug Administration (FDA) Office of Orphan
Products Development on January 31, 2001, and accelerated approval from the FDA
for the above three indications on May 10, 2001. The purpose of this report is
to summarize FDA regulatory mechanisms, i.e., accelerated approval and orphan
drug regulations, that have permitted patients to receive this drug as rapidly
as possible. Imatinib mesylate (Gleevec, Novartis Pharmaceuticals East Manruer, NJ) received
accelerated approval on May 10, 2001 for the treatment of patients with chronic
myeloid leukemia (CML) in (a) chronic phase after failure of IFN-alpha therapy,
(b) accelerated phase, and (c) blast crisis. The accelerated approval was
accompanied by a postmarketing commitment by Novartis Pharmaceuticals to
continue patient follow-up to determine duration of treatment response and
survival. The present review, based on a safety and efficacy report submitted on
December 20, 2002, summarizes data applicable to the conversion of these three
CML indications to full approval status.
RESULTS: Chronic phase CML: Five hundred thirty-two chronic phase CML patients
who had not benefited from prior IFN therapy were treated at a starting imatinib
mesylate dose of 400 mg p.o. qd; dose escalation to 800 mg p.o. qd was allowed.
Patients had received a median of 14 months of IFN therapy at doses > or =25
million IU/wk and were all in late chronic phase, with a median time from
diagnosis of 32 months. Median duration of imatinib mesylate treatment was 29
months, with 81% of patients treated for > or =24 months (maximum 31.5 months).
Initial favorable treatment responses were sustained. An estimated 87.8% of
patients who had a major cytogenetic response maintained their response 2 years
after their initial response. After 2 years of treatment, an estimated 85.4% of
patients were free of progression to accelerated phase or blast crisis, and the
estimated overall survival was 90.8% (95% confidence interval, 88.3-93.2).
Accelerated phase CML: Patients enrolled totaled 293: 235 with CML accelerated
phase, 48 with relapsed/refractory acute lymphocytic leukemia, 2 with
relapsed/refractory acute myelocytic leukemia, and 8 with relapsed/refractory
CML in lymphoid blast crisis. Patients received imatinib mesylate 400 or 600 mg
p.o. qd. Dose escalation was permitted, to a maximum of 800 mg/d, taken as 400
mg bid. Efficacy results were improved in patients receiving imatinib mesylate
600 mg qd versus patients receiving 400 mg qd. The median duration of
hematologic response was 29 versus 17 months and the estimated 24-month
maintained hematologic response rate was 61% versus 42%. The median survival of
patients treated with imatinib mesylate 600 mg qd was not reached versus 20.9
months for patients receiving 400 mg qd. Estimated 24-month survival rate was
66% versus 46%. The median survival in the advanced leukemia population (acute
lymphocytic leukemia, acute myelocytic leukemia, and lymphoid blast crisis) was
only 5 months, and only two patients are still on treatment. Blast crisis CML: A
total of 260 patients were recruited. The imatinib mesylate dose was initially
400 mg qd (37 patients) but was subsequently increased to 600 mg qd (223
patients). Patients receiving imatinib mesylate 600 mg qd had a higher
hematologic response rate than did patients receiving 400 mg (33% versus 16%).
Major cytogenetic responses occurred in 15% of the 260 study patients. The
overall median survival was 6.9 months: 7.1 months for patients treated with
imatinib mesylate 600 mg and 4.7 months for patients receiving imatinib mesylate
400 mg. Estimated 12-month survival rate for all study patients was 32.1% and
estimated 24-month survival rate was 18.3%.
SAFETY: Imatinib mesylate was generally well tolerated, but relatively frequent
reports of common toxicity criteria grade 3/4 neutropenia and thrombocytopenia
were encountered. The most frequently reported adverse events included
gastrointestinal disturbances, edema, rash, and musculoskeletal complaints.
These rarely led to discontinuation of therapy.
CONCLUSIONS: The results confirm those of the interim analysis and suggest that
imatinib mesylate represents an effective therapeutic agent for the treatment of
patients with CML in chronic phase after failure of IFN-alpha therapy, in blast
crisis, and in accelerated phase. The Accreditation Subcommittee of the EBMT regularly publishes special reports
on current practice of haemopoietic stem cell transplantation for haematological
diseases, solid tumours and immune disorders in Europe. Major changes have
occurred since the first report was published in 1996. Haemopoietic stem cell
transplantation today includes grafting with allogeneic and autologous stem
cells derived from bone marrow, peripheral blood and cord blood. With reduced
intensity conditioning regimens in allogeneic transplantation, the age limit has
increased, permitting the inclusion of older patients. New indications have
emerged such as autoimmune disorders and AL amyloidosis for autologous, and
solid tumours for allogeneic transplants. The introduction of alternative
therapies has challenged well-established indications such as imatinib for
chronic myeloid leukaemia. An updated report with revised tables and operating
definitions is presented here. The European Group for Blood and Marrow Transplantation regularly publishes
special reports on the current practice of haematopoietic SCT for haematological
diseases, solid tumours and immune disorders in Europe. Major changes have
occurred since the first report was published. HSCT today includes grafting with
allogeneic and autologous stem cells derived from BM, peripheral blood and cord
blood. With reduced-intensity conditioning regimens in allogeneic
transplantation, the age limit has increased, permitting the inclusion of older
patients. New indications have emerged, such as autoimmune disorders and AL
amyloidosis for autologous HSCT and solid tumours, myeloproliferative syndromes
and specific subgroups of lymphomas for allogeneic transplants. The introduction
of alternative therapies, such as imatinib for CML, has challenged
well-established indications. An updated report with revised tables and
operating definitions is presented. Drug plasma monitoring has emerged as an important tool to obtain optimal levels
of a particular drug among individual patients. Plasma monitoring of imatinib
levels would appear to be practical in cases where there is lack of response,
heightened toxicity, or evidence of poor adherence to therapy. However, the
potential role of monitoring plasma drug concentrations in guiding treatment
decisions and optimizing patient therapy has yet to be established. Currently,
there are no clinical recommendations regarding how to incorporate imatinib drug
plasma monitoring in patients with either chronic myeloid leukemia or
gastrointestinal stromal tumors, indications for which imatinib is approved.
Here, the latest research and evidence regarding imatinib drug plasma monitoring
is discussed. Three cases are presented to illustrate the most common examples
where monitoring imatinib plasma concentrations may help to guide treatment
decisions. These cases include a suboptimal response to imatinib treatment, lack
of patient adherence to imatinib, and imatinib-related toxicity. By
understanding the potential role of monitoring plasma imatinib concentrations in
patients with chronic myeloid leukemia or gastrointestinal stromal tumors,
physicians can identify patients who may benefit from drug plasma monitoring and
consider incorporating the data in order to improve patient outcomes. BACKGROUND: Imatinib mesylate, an orally administered kinase inhibitor that
targets the Kit (CD117) protein, currently has 10 approved indications including
treatment of chronic myelogenous leukemia and metastatic gastrointestinal
stromal tumors (GIST). Treatment with adjuvant imatinib following surgical
resection of localized Kit-positive GIST, the most recent FDA-approved
indication (December 2008), has been shown to significantly improve
recurrence-free survival (RFS) compared with surgical resection alone. Although
adjuvant imatinib has proven effective in clinical trials, it is important to
consider the economic impact to health plans of introducing imatinib in
accordance with its new labeled indication.
OBJECTIVE: To evaluate the budgetary impact over a 3-year time horizon of
treating patients with localized Kit-positive GIST with 1 year of adjuvant
imatinib following surgical resection.
METHODS: A Markov model was developed to predict patients' transitions across
health states defined by initial treatment (surgical resection followed by
adjuvant imatinib 400 milligrams [mg] daily versus surgical resection alone),
recurrence, and progression. Treatments for a first recurrence were (a) imatinib
400 mg daily for recurrences following resection only or after completion of 1
year of treatment with imatinib 400 mg daily and (b) imatinib 800 mg daily for
recurrence during active treatment with imatinib 400 mg daily. Treatments for
further progression were imatinib 800 mg daily, sunitinib, or best supportive
care (BSC) following imatinib 400 mg per day, and sunitinib or BSC following
imatinib 800 mg daily. Recurrence rates were derived from the American College
of Surgeons Oncology Group (ACOSOG) Z9001 clinical trial, which compared 1 year
of adjuvant imatinib following surgical resection with surgical resection only.
The total number of patients with localized and surgically resected GIST
(incidence rate of 0.36 per 100,000) was estimated from epidemiologic studies of
GIST. Uptake of treatment with imatinib was estimated from unpublished data from
qualitative market research funded by the study sponsor. The uptake rate
assumptions reflected both (a) the percentage of patients with Kitpositive
disease and (b) the percentage of clinically eligible patients who would use
imatinib. Costs were estimated by combining unit costs from published sources
with expected resource utilization based on the clinical trial publication and
National Comprehensive Cancer Network guidelines on the treatment of patients
with GIST. To obtain estimates of the budgetary impact, we compared estimated
health care costs with versus without adjuvant imatinib, where health care costs
with imatinib reflected the costs of treatment minus cost offsets associated
with delayed or avoided recurrence or progression. All "with" scenarios assumed
no additional uses other than surgically resected localized Kit-positive GIST
(i.e., no change in off-label use of imatinib). The budgetary impact was
estimated for the first 3 years after the introduction of adjuvant imatinib in
accordance with its new labeled indication in a hypothetical plan population of
10 million persons. Results were calculated both as total budgetary impact and
as per member per month (PMPM) cost in 2009 dollars. Sensitivity analyses were
performed to test the robustness of model results to changes in parameter
estimates.
RESULTS: The model predicted 36 incident resected GIST cases per year in a
health plan of 10 million members. The estimated counts of cases treated with
adjuvant imatinib were 10.8, 16.2, and 21.6 in the first, second, and third
years after introduction, respectively, with the annual increases attributable
to changes in the proportion of patients with resected GIST assumed to use
imatinib (30% in year 1, rising to 45% in year 2 and 60% in year 3). The model
predicted that treatment of these cases with imatinib will increase pharmacy
costs by an additional $505,144 in the first year, $757,717 in the second year,
and $1,010,289 in the third year. Increased resource use associated with
monitoring patients during and after treatment with adjuvant imatinib would cost
an additional $21,564, $38,145, and $56,605 in the first, second, and third
years, respectively. Recurrence would be avoided or delayed in 7 patients over
the 3-year period. Avoided or delayed recurrences would result in cost offsets
of $61,583 in the first year, $156,702 in the second year, and $233,849 in the
third year. The net budgetary impact was estimated to be $465,126 in the first
year (less than $0.01 PMPM), $639,159 in the second year ($0.01 PMPM), and
$833,044 in the third year ($0.01 PMPM). Results of sensitivity analyses
indicated that the budgetary impact in the third year is most sensitive to
changes in the price of adjuvant imatinib and recurrence rates.
CONCLUSIONS: The model predicted that the introduction of adjuvant imatinib for
treatment of surgically resected, localized, Kit-positive GIST will lead to a
net budgetary impact of $0.01 PMPM in the third year after introduction assuming
change in use only in accordance with the new labeled indication. Approximately
11.7%-21.9% of the cost of adjuvant imatinib is offset by the reduction in costs
associated with GIST recurrence. INTRODUCTION: The Orphan Drug Act encourages drug development for rare
conditions. However, some orphan drugs become top sellers for unclear reasons.
We sought to evaluate the extent and cost of approved and unapproved uses of
orphan drugs with the highest unit sales.
METHODS: We assessed prescription patterns for four top-selling orphan drugs:
lidocaine patch (Lidoderm) approved for post-herpetic neuralgia, modafinil
(Provigil) approved for narcolepsy, cinacalcet (Sensipar) approved for
hypercalcemia of parathyroid carcinoma, and imatinib (Gleevec) approved for
chronic myelogenous leukemia and gastrointestinal stromal tumor. We pooled
patient-specific diagnosis and prescription data from two large US state
pharmaceutical benefit programs for the elderly. We analyzed the number of new
and total patients using each drug and patterns of reimbursement for approved
and unapproved uses. For lidocaine patch, we subcategorized approved
prescriptions into two subtypes of unapproved uses: neuropathic pain, for which
some evidence of efficacy exists, and non-neuropathic pain.
RESULTS: We found that prescriptions for lidocaine patch, modafinil, and
cinacalcet associated with non-orphan diagnoses rose at substantially higher
rates (average monthly increases in number of patients of 14.6, 1.45, and 1.58)
than prescriptions associated with their orphan diagnoses (3.12, 0.24, and 0.03,
respectively (p<0.001 for all)). By contrast, for imatinib, approved uses
increased significantly over off-label (0.97 vs. 0.47 patients, p<0.001).
Spending on off-label uses was highest for lidocaine patch and modafinil (>75%).
Increases in lidocaine patch use for non-neuropathic pain far exceeded
neuropathic pain (10.2 vs. 3.6 patients, p<0.001).
DISCUSSION: In our sample, three of four top-selling orphan drugs were used more
commonly for non-orphan indications. These orphan drugs treated common clinical
symptoms (pain and fatigue) or laboratory abnormalities. We should continue to
monitor orphan drug use after approval to identify products that come to be
widely used for non-FDA approved indications, particularly those without
adequate evidence of efficacy. |
Which proteins induce inhibition of LINE-1 and Alu retrotransposition? | It was demonstrated that antiretroviral restriction factors, human APOBEC3 proteins A to H, differentially inhibit LINE-1 and Alu retrotransposition. The same effect was shown to be induced by the Aicardi-Goutières syndrome gene product SAMHD1. | The non-LTR retrotransposon LINE-1 (L1) comprises approximately 17% of the human
genome, and the L1-encoded proteins can function in trans to mediate the
retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably
SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we
have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that
inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other
L1-mediated retrotransposition processes. We demonstrate that APOBEC3G
selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An
active cytidine deaminase site is not required for the inhibition of Alu
retrotransposition and the resultant integration events lack G to A or C to T
hypermutation. These data demonstrate a differential restriction of L1 and Alu
retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein
complex may be targeted by APOBEC3G. Members of the apolipoprotein B mRNA editing complex polypeptide 1-like (APOBEC)
family of enzymes exhibit inhibitory activity against a variety of exogenous and
endogenous retroviruses including retrotransposons, such as long interspersed
element 1 (LINE-1). Indeed, human APOBEC3A, APOBEC3B, and APOBEC3F inhibit
retrotransposition of human LINE-1, mouse IAP and MusD retrotransposons. In our
study, we examined whether the inhibitory effect of APOBEC3 proteins correlates
with APOBEC3 ability to bind the LINE-1 ORF1 protein. We examined the
interactions between the LINE-1 ORF1 protein and the most potent LINE-1
retrotransposon inhibitors, human APOBEC3A and APOBEC3B, by immunofluorescence
and immunoprecipitation. Although human APOBEC3A shows the highest inhibitory
potency against LINE-1 retrotransposon, no direct interactions were identified
either by immunofluorescence or by co-immunoprecipitation. APOBEC3B binds to
LINE-1 ORF1 protein, yet no co-localization was detected. We concluded that
APOBEC3 proteins interfere indirectly with the LINE-1 retrotransposition
pathway, probably through interference with RNA targeting. Human APOBEC3B (A3B) has been described as a potent inhibitor of retroviral
infection and retrotransposition. However, we found that the predomitly
nuclear A3B only weakly restricted infection by HIV-1, HIV-1Δvif, and human
T-cell leukemia virus type 1 (HTLV-1), while significantly inhibiting LINE-1
retrotransposition. The chimeric construct A3G/B, in which the first 60 amino
acids of A3B were replaced with those of A3G, restricted HIV-1, HIV-1Δvif, and
HTLV-1 infection, as well as LINE-1 retrotransposition. In contrast to the
exclusively cytoplasmic A3G, which is inactive against LINE-1
retrotransposition, the A3G/B protein, while localized mainly to the cytoplasm,
was also present in the nucleus. Further mutational analysis revealed that
residues 18, 19, 22, and 24 in A3B were the major determits for nuclear
versus cytoplasmic localization and antiretroviral activity. HIV-1Δvif packages
A3G, A3B, and A3G/B into particles with close-to-equal efficiencies. Mutation
E68Q or E255Q in the active centers of A3G/B resulted in loss of the inhibitory
activity against HIV-1Δvif, while not affecting activity against LINE-1
retrotransposition. The low inhibition of HIV-1Δvif by A3B correlated with a low
rate of G-to-A hypermutation. In contrast, viruses that had been exposed to
A3G/B showed a high number of G-to-A transitions. The mutation pattern was
similar to that previously reported for A3B, with a preference for the GA
context. In summary, these observations suggest that changing 4 residues in the
amino terminus of A3B not only retargets the protein from the nucleus to the
cytoplasm but also enhances its ability to restrict HIV while retaining
inhibition of retrotransposition. Long interspersed elements 1 (LINE-1) occupy at least 17% of the human genome
and are its only active autonomous retrotransposons. However, the host factors
that regulate LINE-1 retrotransposition are not fully understood. Here, we
demonstrate that the Aicardi-Goutières syndrome gene product SAMHD1, recently
revealed to be an inhibitor of HIV/simian immunodeficiency virus (SIV)
infectivity and neutralized by the viral Vpx protein, is also a potent regulator
of LINE-1 and LINE-1-mediated Alu/SVA retrotransposition. We also found that
mutant SAMHD1s of Aicardi-Goutières syndrome patients are defective in LINE-1
inhibition. Several domains of SAMHD1 are critical for LINE-1 regulation. SAMHD1
inhibits LINE-1 retrotransposition in dividing cells. An enzymatic active site
mutant SAMHD1 maintained substantial anti-LINE-1 activity. SAMHD1 inhibits
ORF2p-mediated LINE-1 reverse transcription in isolated LINE-1
ribonucleoproteins by reducing ORF2p level. Thus, SAMHD1 may be a cellular
regulator of LINE-1 activity that is conserved in mammals. |
Can chronological age be predicted by measuring telomere length? | No, telomere length measurement by real-time quantitative PCR cannot be used to predict age of a person, due to the presence of large inter-individual variations in telomere lengths. | Currently DNA profiling methods only compare a suspect's DNA with DNA left at
the crime scene. When there is no suspect, it would be useful for the police to
be able to predict what the person of interest looks like by analysing the DNA
left behind in a crime scene. Determination of the age of the suspect is an
important factor in creating an identikit. Human somatic cells gradually lose
telomeric repeats with age. This study investigated if one could use a
correlation between telomere length and age, to predict the age of an individual
from their DNA. Telomere length, in buccal cells, of 167 individuals aged
between 1 and 96 years old was measured using real-time quantitative PCR.
Telomere length decreased with age (r=-0.185, P<0.05) and the age of an
individual could be roughly determined by the following formula: (age=relative
telomere length -1.5/-0.005). The regression (R(2)) value between telomere
length and age was approximately 0.04, which is too low to be use for forensics.
The causes for the presence of large variation in telomere lengths in the
population were further investigated. The age prediction accuracies were low
even after dividing samples into non-related Caucasians, males and females (5%,
9% and 1%, respectively). Mean telomere lengths of eight age groups representing
each decade of life showed non-linear decrease in telomere length with age.
There were variations in telomere lengths even among similarly aged individuals
aged 26 years old (n=10) and age 54 years old (n=9). Therefore, telomere length
measurement by real-time quantitative PCR cannot be used to predict age of a
person, due to the presence of large inter-individual variations in telomere
lengths. |
What is a benefit of being g6PD-deficient? | Increased resistance to malaria, reduces the risk of coronary diseases, beneficial effect in terms of longevity | Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity
were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls.
It is confirmed that, in contrast to the high stimulation of GR by FAD commonly
found in in thalassaemia indicating red-cell deficiency of FAD, and shown here
to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD
deficiency, leading to high in vitro activity. Unexpectedly, on the other hand,
low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN,
confirmed by response to oral riboflavin, was found in the majority of subjects
with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia.
Whereas this is explained in thalassaemia by an inherited slow red-cell
metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an
increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and
high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is
usually saturated with FAD as in G6PD deficiency alone, unless there is an
inherited, very slow red-cell metabolism of riboflavin to FMN. The part played
by GR in haemolytic crises in G6PD deficiency is discussed. In the present study we examined gene expression and glucose-6-phosphate
dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and
deficient subjects. The results have shown that G6PD activity strongly increases
in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when
compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD
gene expression were observed in leukemic cells from G6PD deficient patients
compared to G6PD normal. A similar pattern of gene expression was also observed
for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results
support the hypothesis that G6PD deficient cell, in order to sustain their
growth, must respond to the low activity of their mutant enzyme with an increase
in quantity through an induction of gene expression. To establish the neonatal screening method of glucose-6 phosphate dehydrogenase
(G6PD) deficiency, G6PD activity was measured using the fluorescence spot test
(FST) using dried blood samples on filter paper. The G6PD/6PGD rate test of
venous blood samples was further performed for confirmation. The positive G6PD
deficiency rate was 4.2% and its detection rates were 3.7% for all neonates and
5.2% only for male newborns when FST was used for neonatal screening.
Conformation rates by use of G6PD/ 6PGD ratio test for G6PD deficiency were
86.8% and 100% particularly in the severely deficient groups. Both sensitivity
and specificity were very high in the severely deficient groups. FST can be used
in neonatal screening of G6PD deficiency because of its high accuracy,
applicability, and simplicity. Moreover, a high volume of dried blood samples on
filter paper can be tested quickly. It is very favorable to diagnose and treat
G6PD deficiency early in high incidence districts. BACKGROUND AND OBJECTIVES: The cause of hyperbilirubinemia cannot be found in
about 45% of cases of neonatal jaundice. Gilbert syndrome (GS) is the most
common congenital disease associated with bilirubin metabolism in the liver.
Since the screening value of genetic tests cannot be fully determined until
accurate data on the prevalence and penetrance of the GS genotype are known, we
sought to estimate whether the prevalence of GS is higher in the parents of
neonates with severe unexplained indirect hyperbilirubinemia.
DESIGN AND SETTING: Case-control study of parents of neonates with severe
unexplained indirect hyperbilirubinemia admitted to a neonatal ward.
METHODS: We used the rifampin test (checked bilirubin before and 4 hours after
administration of 600 mg rifampin) for diagnosis of GS in parents of 115
neonates with severe unexplained indirect hyperbilirubinemia. We compared the
prevalence of GS in these parents with that of a control group of 115 couples
referred for premarital counseling.
RESULTS: The 115 neonates were aged 5.2 (1.6) days (mean, standard deviation),
all were breast-fed, and males constituted 56.5%. Mean total serum bilirubin
(TSB) level was 20.96 (5.48) mg/dL. 14.8% were glucose 6 phosphate dehydrogenase
(G6PD) deficiency was present in 14.8%, and 10.4% had A, B or O blood group
(ABO) incompatibilities with their mothers. There was no difference in the
prevalence of GS between parents of the group with hyperbilirubinemia (22.2%)
and the control group (19.13%) (P=.42). Mean TSB in neonates with parents who
had GS was more (about 3 mg/dL) than in neonates with normal parents (P=.004).
Fathers had GS twice as often as the mothers among the parents of neonates with
hyperbilirubinemia (P=.003), among the control group (P=.009) and among neonates
(P=.014).
CONCLUSION: This study showed that GS cannot cause severe indirect
hyperbilirubinemia by itself, but it may have a summative effect on rising
bilirubin when combined with other factors, for example, G6PD. Our results
showed that in GS, males are affected about twice as much as the females. Reliable and accurate epidemiological data is a prerequisite for a cost
effective screening program for inherited disorders, which however, is lacking
in a number of developing countries. Here we report the first detailed
population study in the Republic of Guinea, a sub-Saharan West African country,
designed to assess the frequency of glucose-6-phosphate dehydrogenase (G6PD)
deficiency and hemoglobinopathies, including screening for thalassemia.
Peripheral blood samples from 187 Guinean adults were screened for hemoglobin
(Hb) variants by standard hematological methods. One hundred and ten samples
from males were screened for G6PD deficiency by the fluorescent spot test.
Molecular analysis was performed for the most common α-thalassemia (α-thal)
deletions, β-globin gene mutations, G6PD variants B (376A), A (376G), A-
(376G/202A) and Betica (376G/968C), using polymerase chain reaction (PCR),
restriction fragment length polymorphism (RFLP) or sequencing. Of the 187
subjects screened, 36 were heterozygous for Hb S [β6(A3)Glu→Val, GAG>GTG]
(allele frequency 9.62%). Sixty-four subjects were heterozygous and seven were
homozygous for the -α(3.7) kb deletion (allele frequency 20.85%). β-Thalassemia
alleles were detected in five subjects, four with the -29 (A>G) mutation (allele
frequency 1.07%) and one with codon 15 (TGG>TAG) (allele frequency 0.96%). The
G6PD A- and G6PD Betica deficient variants were highly prevalent with a
frequency of 5.7 and 3.3%, respectively. While we did not test for ferritin
levels or α(0)-thal, four females (5.2%) had red cell indices strongly
suggestive of iron deficient anemia: Hb <9.7 g/dL; MCH <19.3 pg; MCV <68.2; MCHC
<31.6 g/dl; RDW >19.8%. Our results are consistent with high frequency of
alleles such as Hb S, α-thal and G6PD deficient alleles associated with malaria
resistance. Finding a 9.6% Hb S allele frequency supports the notion for a
proficient neonatal screening to identify the sickle cell patients, who might
benefit from early prophylactic treatment for infections. The incidence of
significant iron deficient anemia in women is lower than expected in an under
developed country. BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd) has been shown
to protect against malaria infection and severe manifestations in African and
Asia, but there is a scarcity of studies in the Americas. This study aimed to
study the prevalence of G6PDd and its association with malaria occurrence in the
Brazilian Amazon.
METHODS: A cross-sectional study was conducted in the male population to
estimate the prevalence of G6PDd and malaria infection. G6PD deficient samples
were genotyped to identify the deficient variant. Number of previous malaria
episodes and need for blood transfusion during malaria episodes were recorded by
applying a standardized questionary.
RESULTS: From a sample of 1478 male individuals, 66 were detected as G6PD
deficient, resulting in a prevalence of of 4.5% (95% CI = 3.44-5.56%). Fifty six
G6PD deficient individuals (3.8%; 95% CI = 2.82-4.77) presented the G6PD
A-variant mutation, while 10 individuals (0.7%; 95% CI = 0.42-0.97) severely
deficient were genotyped as carriers of the G6PD Mediterranean variant. After
adjusting for age, G6PD deficient individuals were less likely to report the
occurrence of malaria episodes, and the protective effect was related to the
enzyme activity, with carriers of the GG6PD A-variant presenting a 88% reduction
(AOR: 0.119; 95% CI = 0.057-0.252; p < 0.001) and carriers of the Meditarrenean
variant presenting 99% lower risk (AOR: 0.010; 95% CI = 0.002-0.252; p < 0.001)
when compared to non-deficient individuals. On the other hand, G6PD deficient
subjects reported higher need of transfusion during malaria episodes (p <
0.001).
CONCLUSION: G6PD enzyme activity was directly related to susceptibility to
malaria in the Brazilian Amazon, where P. vivax predominates. Severe G6PDd was
associated with considerable higher risk of malaria-related transfusions. We review here some recent data about Glucose-6-phosphate dehydrogenase (G6PD),
the housekeeping X-linked gene encoding the first enzyme of the pentose
phosphate pathway (PPP), a NADPH-producing dehydrogenase. This enzyme has been
popular among clinicians, biochemists, geneticists and molecular biologists
because it is the most common form of red blood cell enzymopathy. G6PD deficient
erythrocytes do not generate NADPH in any other way than through the PPP and for
this reason they are more susceptible than any other cells to oxidative damage.
Moreover, this enzyme has also been of crucial importance in many significant
discoveries; indeed, G6PD polymorphisms have been instrumental in studying
X-inactivation in the human species, as well as in establishing the clonal
nature of certain tumors. G6PD deficiency, generally considered as a mild and
benign condition, is significantly disadvantageous in certain environmental
conditions like in presence of certain drugs. Nevertheless, G6PD deficiency has
been positively selected by malaria, and recent knowledge seems to show that it
also confers an advantage against the development of cancer, reduces the risk of
coronary diseases and has a beneficial effect in terms of longevity. BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is rare among
Japanese ethnicity although it is known as one of the most common hereditary
disorders of erythrocytes, causing intravascular hemolysis. It is well-known
that G6PD deficiency may cause hemolysis even in the neonatal period. However,
most cases are asymptomatic, and the frequency of severe anemia is low.
FINDINGS: We describe a Japanese male neonatal case of G6PD deficiency
presenting as severe, persistent indirect hyperbilirubinemia on day 2 and
hemolytic anemia. He was born to non-consanguineous Japanese parents without any
family history. We could not find any triggers that could have induced hemolysis
during pregcy.
CONCLUSIONS: This case encouraged us to investigate G6PD deficiency as a
differential diagnosis of severe neonatal jaundice and hemolytic anemia despite
the low prevalence in Japan. Five bromophenols isolated from three Rhodomelaceae algae (Laurencia nipponica,
Polysiphonia morrowii, Odonthalia corymbifera) showed inhibitory effects against
glucose 6-phosphate dehydrogenase (G6PD). Among them, the symmetric bromophenol
dimer (5) showed the highest inhibitory activity against G6PD. Malaria elimination will be possible only with serious attempts to address
asymptomatic infection and chronic infection by both Plasmodium falciparum and
Plasmodium vivax. Currently available drugs that can completely clear a human of
P. vivax (known as "radical cure"), and that can reduce transmission of malaria
parasites, are those in the 8-aminoquinoline drug family, such as primaquine.
Unfortunately, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency
risk having severe adverse reactions if exposed to these drugs at certain doses.
G6PD deficiency is the most common human enzyme defect, affecting approximately
400 million people worldwide.Scaling up radical cure regimens will require
testing for G6PD deficiency, at two levels: 1) the individual level to ensure
safe case management, and 2) the population level to understand the risk in the
local population to guide Plasmodium vivax treatment policy. Several technical
and operational knowledge gaps must be addressed to expand access to G6PD
deficiency testing and to ensure that a patient's G6PD status is known before
deciding to administer an 8-aminoquinoline-based drug.In this report from a
stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in
support of radical cure is discussed in detail. The focus is on challenges to
the development and evaluation of G6PD diagnostic tests, and on challenges
related to the operational aspects of implementing G6PD testing in support of
radical cure. The report also describes recommendations for evaluation of
diagnostic tests for G6PD deficiency in support of radical cure. Red blood cells (RBCs) contain large amounts of iron and operate in highly
oxygenated tissues. As a result, these cells encounter a continuous oxidative
stress. Protective mechanisms against oxidation include prevention of formation
of reactive oxygen species (ROS), scavenging of various forms of ROS, and repair
of oxidized cellular contents. In general, a partial defect in any of these
systems can harm RBCs and promote senescence, but is without chronic hemolytic
complaints. In this review we summarize the often rare inborn defects that
interfere with the various protective mechanisms present in RBCs. NADPH is the
main source of reduction equivalents in RBCs, used by most of the protective
systems. When NADPH becomes limiting, red cells are prone to being damaged. In
many of the severe RBC enzyme deficiencies, a lack of protective enzyme activity
is frustrating erythropoiesis or is not restricted to RBCs. Common hereditary
RBC disorders, such as thalassemia, sickle-cell trait, and unstable hemoglobins,
give rise to increased oxidative stress caused by free heme and iron generated
from hemoglobin. The beneficial effect of thalassemia minor, sickle-cell trait,
and glucose-6-phosphate dehydrogenase deficiency on survival of malaria
infection may well be due to the shared feature of enhanced oxidative stress.
This may inhibit parasite growth, enhance uptake of infected RBCs by spleen
macrophages, and/or cause less cytoadherence of the infected cells to capillary
endothelium. |
Describe the usefulness of Macrostomum lignano in ion channel and stem cell research | Bioelectrical signals generated by ion channels play crucial roles in many cellular processes in both excitable and nonexcitable cells. Some ion channels are directly implemented in chemical signaling pathways, the others are involved in regulation of cytoplasmic or vesicular ion concentrations, pH, cell volume, and membrane potentials. Together with ion transporters and gap junction complexes, ion channels form steady-state voltage gradients across the cell membranes in nonexcitable cells. These membrane potentials are involved in regulation of such processes as migration guidance, cell proliferation, and body axis patterning during development and regeneration. While the importance of membrane potential in stem cell maintenance, proliferation, and differentiation is evident, the mechanisms of this bioelectric control of stem cell activity are still not well understood, and the role of specific ion channels in these processes remains unclear. The flatworm Macrostomum lignano is a versatile model organism for addressing these topics. Experimental tools have been developed which demonstrate how manipulation of membrane potential influences regeneration in M. lignano. | Bioelectrical signals generated by ion channels play crucial roles in many
cellular processes in both excitable and nonexcitable cells. Some ion channels
are directly implemented in chemical signaling pathways, the others are involved
in regulation of cytoplasmic or vesicular ion concentrations, pH, cell volume,
and membrane potentials. Together with ion transporters and gap junction
complexes, ion channels form steady-state voltage gradients across the cell
membranes in nonexcitable cells. These membrane potentials are involved in
regulation of such processes as migration guidance, cell proliferation, and body
axis patterning during development and regeneration. While the importance of
membrane potential in stem cell maintece, proliferation, and differentiation
is evident, the mechanisms of this bioelectric control of stem cell activity are
still not well understood, and the role of specific ion channels in these
processes remains unclear. Here we introduce the flatworm Macrostomum ligo as
a versatile model organism for addressing these topics. We discuss biological
and experimental properties of M. ligo, provide an overview of the recently
developed experimental tools for this animal model, and demonstrate how
manipulation of membrane potential influences regeneration in M. ligo. |
Is there an association between c-reactive protein concentrations and outcomes of subarachnoid hemorrhage patients? | Yes. Higher concentrations of C-reactive protein are associated with worse outcomes of subarachnoid hemorrhage patients. | Subarachnoid hemorrhage (SAH) elicits an inflammatory response in the
subarachnoid space, which is mediated by the release of various cytokines. To
assess their involvement in post-hemorrhagic complications, we determined the
source and time-course of the release of inflammatory cytokines and acute-phase
proteins in cerebrospinal fluid (CSF) following SAH. Concentrations of
interleukin (IL)- 1beta, IL-6, transforming growth factor-beta1 (TGF-beta1) and
C-reactive protein (CRP) in CSF of 36 patients with SAH were measured by
enzyme-linked immunoabsorbent assay (ELISA). Floating cells collected from the
CSF were centrifuged four to six days after SAH, and examined
immunohistochemically. Intracellular IL-1beta and IL-6 were examined by flow
cytometric analysis. The molecular weight of TGF-beta1 in CSF of 30 patients was
examined by Western blot analysis. The TGF-beta1 levels of patients who had
undergone ventriculoperitoneal (VP) shunt (n = 19) was significantly higher than
nonshunt group (n = 16). The CRP levels of VP shunt group was significantly
higher than nonshunt group. IL-6 concentration was maximal within day 0-1 and it
was secreted by neutrophils and monocytes. ELISA showed consistently low levels
of IL-1beta, whereas a proportion of monocytes and lymphcytes were IL-
1beta-positive by flow cytometric analysis. TGF-beta1 levels were also maximal
on day 0-1 according to ELISA, although it tended to be in the inactive form
derived from platelets. A 25 kDa band of TGF-1 was detectable for at least 13
days after SAH, which may have been secreted in part by neutrophils and
monocytes. CRP levels in CSF peaked on day 2-3. The present results suggest that
leukocytes induced by SAH play an important role in post-hemorrhagic
inflammation in the subarachnoid space by releasing IL-6 and TGF-beta1. The CRP
and TGF-beta1 levels in CSF are strongly concerned with communicating
hydrocephalus after SAH. BACKGROUND: The purpose of the present study was to investigate the relationship
between serum concentrations of the immunoglobulin-like superfamily, selectins,
hsCRP, and the development of DIND in patients with aneurysmal SAH.
METHODS: Serum ICAM-1, VCAM-1, E-selectin, P-selectin, L-selectin, and hsCRP
were measured in 33 patients with SAH who underwent aneurysmal clipping within
48 hours of the onset of symptoms. Serum samples were obtained during the early
period (day 0) and the late period (day 7).
RESULTS: The serum concentrations of ICAM-1 (P = .009), VCAM-1 (P = .0383) and
hsCRP (P = .0014) during the early period were significantly higher in patients
with SAH than in control patients. Further, serum hsCRP concentration during the
late period was significantly higher in patients with SAH than in control
patients (P = 0033). Finally, serum concentrations of ICAM-1, VCAM-1, and hsCRP
during the early (P = .0055, P = .0266, and P = .0266) and late (P = .0423, P =
.0041, and P = .0004) period were significantly higher in patients with DIND
than in patients without DIND.
CONCLUSIONS: Serum levels of ICAM-1, VCAM-1 and hsCRP during the early and late
period following SAH correlate with DIND. OBJECT: Cerebral vasospasm is a common and potentially devastating complication
of aneurysmal subarachnoid hemorrhage (aSAH). Inflammatory processes seem to
play a major role in the pathogenesis of vasospasm. The C-reactive protein (CRP)
constitutes a highly sensitive inflammatory marker. The association of elevated
systemic CRP and coronary vasospasm has been well established. Additionally,
elevation of the serum CRP levels has been demonstrated in patients with aSAH.
The purpose of the current study was to evaluate the possible relationship
between elevated CRP levels in the serum and CSF and the development of
vasospasm in patients with aSAH.
METHODS: A total of 41 adult patients in whom aSAH was diagnosed were included
in the study. Their demographics, the admitting Glasgow Coma Scale (GCS) score,
Hunt and Hess grade, Fisher grade, CT scans, digital subtraction angiography
studies, and daily neurological examinations were recorded. Serial serum and CSF
CRP measurements were obtained on Days 0, 1, 2, 3, 5, 7, and 9. All patients
underwent either surgical or endovascular treatment within 48 hours of their
admission. The outcome was evaluated using the Glasgow Outcome Scale and the
modified Rankin Scale.
RESULTS: The CRP levels in serum and CSF peaked on the 3rd postadmission day,
and the CRP levels in CSF were always higher than the serum levels. Patients
with lower admission GCS scores and higher Hunt and Hess and Fisher grades had
statistically significantly higher levels of CRP in serum and CSF. Patients with
angiographic vasospasm had higher CRP measurements in serum and CSF, in a
statistically significant fashion (p < 0.0001). Additionally, patients with
higher CRP levels in serum and CSF had less favorable outcome in this cohort.
CONCLUSIONS: Patients with aSAH who had high Hunt and Hess and Fisher grades and
low GCS scores showed elevated CRP levels in their CSF and serum. Furthermore,
patients developing angiographically proven vasospasm demonstrated significantly
elevated CRP levels in serum and CSF, and increased CRP measurements were
strongly associated with poor clinical outcome in this cohort. OBJECTIVE: There is a rising debate about the role of inflammation in the
pathogenesis of complications after aneurysmal subarachnoid hemorrhage (SAH)
such as intracranial hypertension (intracranial pressure, ICP >20 mmHg). This
study aimed to analyse the origin of interleukin-6 (IL-6) in respect to ICP and
cerebral metabolism in SAH patients.
METHODS: Prospectively, IL-6 was measured in three compartments, the
extracellular fluid (ECF) monitored by cerebral microdialysis (MD),
cerebrospinal fluid (CSF) and plasma for 10 days after SAH (days 0-4, three
times daily; days 5-10, two times daily). Patients were classified having
intracranial hypertension (n=7) or normal ICP (n=17) during 10 days after
bleeding. Glasgow outcome scale (GOS) was assessed after 3 and 6 months.
RESULTS: Patient groups were comparable for age, WFNS and Fisher grade.
Intracranial hypertension was associated with an inflammatory response,
indicating activation of the inflammatory cascade in the brain (ECF) and
systemic circulation with high IL-6 and C-reactive protein (CRP) plasma levels
after SAH, the latter associated with unfavourable outcome. The data suggest the
ECF but not the CSF as main origin of IL-6 in the systemic circulation in the
presence of intracranial hypertension in SAH.
DISCUSSION: Intracranial hypertension is associated with a strong activation of
the inflammatory cascade in the brain and systemic circulation, and might be
underestimated as proinflammmatory trigger in the pathogenesis of complications
after SAH. Future therapies targeting anti-inflammatory response in plasma may
help to reduce the inflammatory cascade responsible for development of
intracranial hypertension. BACKGROUND AND PURPOSE: A proinflammatory prothrombotic state may increase the
risk of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage
(SAH). We studied the relationship of levels of leukocytes, platelets,
C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) with the
development of DCI and with clinical outcome in patients with aneurysmal SAH.
METHODS: In 125 patients admitted within 72 h after aneurysmal SAH, we
dichotomized initial blood levels at their median values and investigated the
prediction of DCI with Cox proportional hazard analysis and of poor clinical
outcome with logistic regression analysis. We also analyzed concentrations
before and after onset of DCI with the paired-samples t test and compared
changes with those in patients without DCI.
RESULTS: During the development of DCI (unrelated to treatment), patients had a
larger increase in counts of platelets (difference 49 x 10(9)/l; 95% CI: 2-98)
and leukocytes (difference 2.6 x 10(9)/l; 95% CI: 0.4-5.0) than patients without
DCI during the same period. CRP increased during DCI and decreased in patients
without DCI (difference 14 mg/l; 95% CI: -29 to 58). ESR increased slightly in
both groups (difference 3 mm/h; 95% CI: -15 to 20). None of the determits at
baseline predicted the development of DCI. An increased risk of poor outcome
predicted by a high initial leukocyte count (OR 2.5; 95% CI: 1.1-5.7) decreased
after adjustment for clinical variables (OR 2.1; 95% CI: 0.8-5.5).
CONCLUSION: Counts of platelets and leukocytes disproportionally increase during
the occurrence of DCI after aneurysmal SAH. Drugs with anti-thrombotic or
anti-inflammatory properties should be studied for prevention and treatment of
DCI. BACKGROUND: This study examines the inflammatory response via interleukin-6
(IL-6) in aneurysmal subarachnoid hemorrhage (aSAH) patients and its association
with their clinical course (occurrence of acute focal neurological deficits,
AFND; and delayed cerebral ischemia, DCI).
METHODS: A total of 38 consecutive aSAH patients were studied prospectively
within 14 days after admission and classified as asymptomatic (n = 9; WFNS grade
1 (1-2), median and quartiles) and symptomatic (n = 29; WFNS grade 4 (2-5)); the
latter presenting with AFND (n = 13), DCI (n = 10) or both (n = 6). Levels of
pro-inflammatory cytokine IL-6 were determined in cerebral extracellular fluid
(ECF, using cerebral microdialysis), cerebrospinal fluid (CSF) and plasma for 10
days after aSAH. Additionally, C-reactive protein (CRP) levels were measured in
plasma.
RESULTS: High IL-6 levels in CSF, ECF and plasma were found in all patients,
reflecting a pronounced local inflammatory response after aSAH, followed only in
symptomatic patients by a delayed systemic inflammation (CRP P < 0.025, days 7-9
after aSAH). In all compartments, IL-6 levels appeared to be higher in
symptomatic patients, accompanied also by a higher ECF lactate-pyruvate ratio (P
= 0.04). Cerebral, but not plasma IL-6, levels were indicative of the
development of DCI in symptomatic patients (ECF P = 0.003; CSF P = 0.001).
CONCLUSIONS: A pronounced initial cerebral inflammatory state was observed in
patients of all WFNS grades, suggesting that IL-6 elevations are not necessarily
detrimental. Cerebral, but not plasma IL-6, levels were predictive of the
development of delayed ischemic deficits in symptomatic patients, suggesting
that CSF or ECF are the best sampling media for future studies. BACKGROUND: Ninety-three patients undergoing surgical or endovascular operation
secondary to aneurysmal subarachnoid hemorrhage (SAH) were retrospectively
analyzed to determine the influence of the different time points of C-reactive
protein (CRP) measurement on the prediction of vasospasm and clinical outcome.
METHODS: Laboratory data such as the CRP level and the white blood cell count,
preoperative demographic and clinical data, intraoperative and postoperative
data, and complications such as intracerebral hemorrhage, hydrocephalus,
vasospasm, and surgical decompression were collected at hospital discharge or
symptomatic vasospasm and used as predictable factors for poor outcome (Modified
Rankin Scale score 4 to 6).
RESULTS: Twenty-three and 28 patients showed poor outcome and symptomatic
vasospasm after SAH, respectively. Both preoperative and postoperative CRP
levels were significantly higher in patients with a poor outcome compared with
patients with a good outcome (P<0.05). The area under the receiver operating
characteristic curve of CRP measured on postoperative day 1 or 2 (CRP POD1-2)
for predicting a poor clinical outcome was 0.870, and its cutoff point of 4
mg/dL had a sensitivity of 0.826 and a specificity of 0.843. A high CRP level
after aneurysm treatment was associated with severe neurological deterioration
on admission, cerebral infarction, intracerebral hemorrhage, and surgical
decompression (P<0.05). CRP POD1-2, and not the preoperative CRP, was an
independent factor in predicting symptomatic vasospasm (P<0.05). In patients
with symptomatic vasospasm, an increase in the postoperative CRP was associated
with the time profile of developing symptomatic vasospasm.
CONCLUSION: Postoperative CRP, especially CRP POD1-2, can be a useful prognostic
factor for both poor outcome and symptomatic vasospasm in patients with
aneurysmal SAH. BACKGROUND: The mechanism of development of delayed cerebral ischemia (DCI)
after aneurysmal subarachnoid hemorrhage (SAH) is poorly understood.
Inflammatory processes are implicated in the development of ischemic stroke and
may also predispose to the development of DCI following SAH. The objective of
this study was to test whether concentrations of circulating inflammatory
markers (C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin 1
receptor antagonist (IL-1Ra)) were predictive for DCI following SAH. Secondary
analyses considered white cell count (WCC) and erythrocyte sedimentation rate
(ESR).
METHODS: This was a single-center case-control study nested within a prospective
cohort. Plasma inflammatory markers were measured in patients up to 15 days
after SAH (initial, peak, average, final and rate of change to final). Cases
were defined as those developing DCI. Inflammatory markers were compared between
cases and randomly selected matched controls.
RESULTS: Among the 179 participants there were 46 cases of DCI (26%). In primary
analyses the rate of change of IL-6 was associated with DCI (OR 2.3 (95% CI 1.1
to 5.0); p=0.03). The final value and rate of change of WCC were associated with
DCI (OR 1.2 (95% CI 1.0 to 1.3) and OR 1.3 (95% CI 1.0 to 1.6), respectively).
High values of ESR were associated with DCI (OR 2.4 (95% CI 1.3 to 4.6) initial;
OR 2.3 (95% CI 1.3 to 4.2) average; OR 2.1 (95% CI 1.1 to 3.9) peak; and OR 2.0
(95% CI 1.2 to 3.3) final value).
CONCLUSIONS: Leucocytosis and change in IL-6 prior to DCI reflect impending
cerebral ischemia. The time-independent association of ESR with DCI after SAH
may identify this as a risk factor. These data suggest that systemic
inflammatory mechanisms may increase the susceptibility to the development of
DCI after SAH. BACKGROUND: The role and impact of systemic inflammatory response after
aneurysmal subarachnoid hemorrhage remain to be elucidated.
OBJECTIVE: To assess the time course and correlation of systemic inflammatory
parameters with outcome and the occurrence of delayed ischemic neurological
deficits (DINDs) after subarachnoid hemorrhage.
METHODS: Besides the baseline characteristics, daily interleukin-6 (IL-6),
procalcitonin, C-reactive protein levels, and leukocyte counts were
prospectively measured until day 14 after subarachnoid hemorrhage. Occurrence of
infectious complications and application of therapeutic hypothermia were
assessed as confounding factors. The primary end point was outcome after 3
months, assessed by Glasgow outcome scale; the secondary end point was the
occurrence of DINDs.
RESULTS: During a 3-year period, a total of 138 patients were included. All
inflammatory parameters measured were higher in patients with unfavorable
outcome (Glasgow outcome scale score, 1-3). After adjustment for confounding
factors, elevated IL-6 and leukocyte counts remained significant risk factors
for unfavorable outcome. The odds ratio for log IL-6 was 4.07 (95% confidence
interval, 1.18 to 14.03; P = .03) and for leukocyte counts was 1.24 (95%
confidence interval, 1.06-1.46, P = .008). The analysis of the time course
established that IL-6 was the only significantly elevated parameter in the early
phase in patients with unfavorable outcome. Higher IL-6 levels in the early
phase (days 3-7) were associated with the occurrence of DINDs. The adjusted odds
ratio for log IL-6 was 4.03 (95% confidence interval, 1.21-13.40; P = .02).
CONCLUSION: Higher IL-6 levels are associated with worse clinical outcome and
the occurrence of DINDs. Because IL-6 levels were significantly elevated in the
early phase, they might be a useful parameter to monitor. |
Which drug is benserazide usually co-administered with? | Co-administration of L-Dopa with carbidopa or benserazide is the most effective symptomatic treatment for Parkinson Disease (PD). | The effect of levodopa (L-dopa), alone or in combination with a peripheral
decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid
decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD),
dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been
studied. In healthy subjects and in patients with parkinsonism plasma ALAAD
level fell after administration of L-dopa + benserazide, but returned to
previous levels within 90 min. In a cross-sectional study blood was obtained, 2
h after dosing, from 104 patients with idiopathic parkinsonism, divided into
four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa +
benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4).
Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups
3 and 4, indicating that there was induction of ALAAD by the co-administration
of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily
L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times
higher, while plasma DA levels were not different. The DA/L-dopa ratio was
decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group
1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence
of on-off fluctuations. In a longitudinal study of three patients started on
Madopar treatment the induction of plasma ALAAD was found to occur gradually
over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and
cerebrospinal fluid are required in order to elucidate whether the ALAAD
induction by PDI may be related to the loss of clinical efficacy of combination
therapy in some patients and how it is related to end-of-dose deterioration and
on-off phenomena. In vivo brain microdialysis was used to assess the effects of tolcapone, a novel
central and peripheral inhibitor of catechol-O-methyltransferase on striatal
3,4-dihydroxyphenyl-L-alanine (L-dopa) and dopamine metabolism. The oral
administration of 30 mg/kg of tolcapone failed to change dopamine output but
elicited a marked and long-lasting decrease of the extracellular levels of
homovanillic acid (HVA) and 3-methoxytyramine with a concomitant increase of
3,4-dihydroxyphenylacetic acid (DOPAC). The administration of L-dopa (20 and 60
mg/kg p.o.) + benserazide (15 mg/kg p.o.) resulted in dose-dependent increase of
dialysate levels of L-dopa and 3-O-methyl-DOPA. Tolcapone (30 mg/kg p.o.), given
as adjunct to both doses of L-dopa, markedly enhanced the elevation or
extracellular L-dopa, while it completely prevented the formation of
3-O-methyl-DOPA. In another experiment, the administration of L-dopa +
benserazide (30 + 15 mg/kg p.o.) resulted in increased extracellular levels of
dopamine, DOPAC, HVA and 3-methoxytyramine. The co-administration of tolcapone
(30 mg/kg p.o.) further increased dopamine and DOPAC levels, whereas HVA and
3-methoxytyramine effluxes were reduced. These findings support the notion that
tolcapone has the ability to enhance striatal dopamine neurotransmission by
increasing L-dopa bioavailability through peripheral and central inhibition of
L-dopa O-methylation, as well as by blocking the central conversion of dopamine
into 3-methoxytyramine. Clozapine reduces L-3,4-dihydroxyphenylalanine (L-Dopa)-induced dyskinesias in
parkinsonian patients. To test if the antidyskinetic effect of clozapine is
related to antagonism at the dopamine D(4) receptor, we investigated the effect
of 8-methyl-6-(4-methyl-1-piperazinyl)-11H-pyrido[2,3-b][1, 4]benzodiazepine
(JL-18), a structural analog of clozapine which is more selective for this
receptor. Four 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-treated
cynomolgus monkeys with a stable parkinsonian syndrome and reproducible
dyskinesias to L-Dopa were used in this study. They were injected subcutaneously
(s.c.) with L-Dopa methyl ester (125 mg per animal) plus benserazide (50 mg per
animal; L-Dopa/benserazide) alone or in combination with JL-18 (at the doses of
0.1, 0.3, or 0.9 mg/kg, s.c.). Subcutaneous injection of sterile saline was used
as control. L-Dopa/benserazide increased locomotion and improved parkinsonism
but also induced dyskinesias. Co-administration of JL-18, at low doses (0.1, 0.3
mg/kg) with L-Dopa/benserazide, produced a dose-dependent reduction in
L-Dopa-induced dyskinesias without a parallel return to parkinsonism. The
present results suggest that novel selective dopamine D(4) receptor antagonists
may represent a useful tool to reduce L-Dopa-induced dyskinesias. (S)-(-)-3-(3-(methylsulfonyl)phenyl)-1-propylpiperidine ((-)-OSU6162) is a
phenylpiperidine derivative which exhibits low affinity to the dopamine D2
receptor in vitro. However, in vivo, positron emission tomography scanning
studies show that the compound displaces the selective dopamine D2 receptor
antagonist, raclopride. We have evaluated, in this study, the effect of
(-)-OSU6162, on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias in a
primate model of Parkinson's disease. Five
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus monkeys
with a stable parkinsonian syndrome and reproducible dyskinesias to L-DOPA were
used in this study. The monkeys were housed in observation cages equipped with
an electronic motility monitoring system. They were injected subcutaneously
(s.c.) with L-DOPA methyl ester (125 mg per animal) plus benserazide (50 mg per
animal; L-DOPA/benserazide) alone or in combination with (-)-OSU6162 (1.0, 3.0,
6.0 or 10 mg/kg, s.c.). Subcutaneous injection of sterile saline was used as
control. L-DOPA/benserazide increased locomotion and improved parkinsonism but
also induced dyskinesias. Co-administration of (-)-OSU6162 with
L-DOPA/benserazide produced a significant reduction in L-DOPA-induced
dyskinesias. This improvement in L-DOPA-induced dyskinesias occurred mainly at
the onset of the L-DOPA/benserazide effect as reflected by an increase in the
duration of the "ON" state without dyskinesias up to 3.4 fold after (-)-OSU6162
co-administration as compared to L-DOPA/benserazide alone. The anti-dyskinetic
effect of (-)-OSU6162 was maintained during 14 days and no tolerance to this
effect was observed. Our data suggests that (-)-OSU6162 could be of significant
clinical value to reduce L-DOPA-induced dyskinesias in fluctuating advanced
Parkinson's disease patients. The mechanism of L-DOPA for antinociception was investigated. Nociceptive
behaviors in mice after an intrathecal (i.t.) administration of substance P were
evaluated. L-DOPA (i.t.) dose-dependently attenuated the substance P-induced
nociceptive behaviors. Co-administration of benserazide (i.t.), a DOPA
decarboxylase inhibitor, abolished the antinociceptive effect of L-DOPA. The
L-DOPA-induced antinociception was antagonized by sulpiride, a D2 blocker, but
not by SCH 23390, a D1 blocker. These results suggest that L-DOPA relieves pain
after conversion to dopamine, with the dopamine sedating pain transmission by
way of the dopamine D2 receptor. Using a rat model of L-DOPA-induced dyskinesia (LID), the contributions of
dopamine D1 and D2 receptors to axial, limb, and orolingual (ALO) abnormal
involuntary movements (AIMs) elicited by L-DOPA were examined. Chronic
L-DOPA-treated rats received the D1 receptor antagonist SCH23390 (0.01, 0.1, and
1.0 mg/kg; i.p.), the D2 receptor antagonist Eticlopride (0.01, 0.1, and 1.0
mg/kg; i.p.), a mixture of both antagonists (0.01, 0.1, 1.0 mg/kg each; i.p.),
or vehicle 30 min prior to L-DOPA (6 mg/kg; i.p.)+Benserazide (15 mg/kg; i.p.).
SCH23390 (0.1 and 1.0 mg/kg) significantly reduced axial and limb AIMs, while
the same doses of Eticlopride significantly decreased axial, limb, and
orolingual AIMs. Co-administration of SCH23390+Eticlopride significantly reduced
axial (0.01, 0.1 and 1.0 mg/kg), limb (0.1 and 1.0 mg/kg), and orolingual (0.1
and 1.0 mg/kg) AIMs. These results indicate the importance of D1 and D2
receptors to LID and further validate the rat AIMs model. 5-HT1A autoreceptor stimulation can act to attenuate supraphysiological swings
in extracellular dopamine levels following long-term levodopa treatment and may
be useful in the treatment and prevention of the motor complications. The
purpose of this study was to investigate cellular and behavioral effects of
5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor
complications. Two sets of experiments were performed. First, animals were
treated with levodopa (50 mg/kg with benserazide 12.5 mg/kg, twice daily),
intraperitoneally (i.p.) for 22 days. On day 23, animals received either
8-OH-DPAT (1 mg/kg, i.p.) or 8-OH-DPAT plus WAY-100635 (0.1 mg/kg, i.p) or
vehicle with each levodopa dose. In the second set, animals were treated either
with levodopa (50 mg/kg, i.p.) plus 8-OH-DPAT (1 mg/kg, i.p.) or levodopa (50
mg/kg, i.p.) plus vehicle, administered twice daily for 22 consecutive days. Our
study showed that 8-OH-DPAT plus levodopa both prolonged the duration of the
motor response and reduced peak turning. 8-OH-DPAT plus levodopa also decreased
the frequency of failures to levodopa. Co-administration of WAY-100635, a 5-HT1A
receptor antagonist, with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor
complications indicating that the observed 8-OH-DPAT responses were probably
mediated at the 5-HT1A autoreceptor. Moreover, 8-OH-DPAT plus levodopa
significantly reduced hyperphosphorylation of GluR1 at serine 845, which was
closely associated with levodopa-induced motor complications. L-DOPA-induced dyskinesia is a common side effect developed after chronic
treatment with 3,4-dihydroxyphenyl-l-alanine (l-DOPA) in Parkinson's disease.
The biological mechanisms behind this side effect are not fully comprehended
although involvement of dopaminergic, serotonergic, and glutamatergic systems
has been suggested. The present study utilizes in vivo amperometry to
investigate the impact from unilateral 6-hydroxydopamine lesions and l-DOPA (4
mg/kg, including benserazide 15 mg/kg) -induced dyskinetic behavior on striatal
basal extracellular glutamate concentration and potassium-evoked glutamate
release in urethane-anesthetized rats. Recordings were performed before and
after local L-DOPA application in the striatum. In addition, effects from the
5-HT(1A) receptor agonist (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin
hydrobromide (8-OHDPAT; 1 mg/kg) was assessed on glutamate release and on
dyskinetic behavior. The results revealed a bilateral ≈ 30% reduction of basal
extracellular glutamate concentration and attenuated potassium-evoked glutamate
release after a unilateral dopamine-depletion in L-DOPA naïve animals. In
dyskinetic subjects, basal glutamate concentration was comparable to normal
controls, although potassium-evoked glutamate release was reduced to similar
levels as in drug naïve dopamine-lesioned animals. Furthermore, acute striatal
L-DOPA administration attenuated glutamate release in all groups, except in the
dopamine-lesioned striatum of dyskinetic animals. Co-administration of 8-OHDPAT
and L-DOPA decreased dyskinesia in dopamine-lesioned animals, but did not affect
potassium-evoked glutamate release, which was seen in normal animals. These
findings indicate altered glutamate transmission upon dopamine-depletion and
dyskinesia. |
What is the inheritance pattern of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) caused by RYR2 mutations? | Autosomal dominant catecholaminergic polymorphic ventricular tachycardia (CPVT) was mapped to chromosome 1q42-43 with identification of pathogenic mutations in RYR2. | Ryanodine receptor (RyR), a homotetrameric Ca2+ release channel, is one of the
main actors in the generation of Ca2+ signals that trigger muscle contraction.
Three genes encode three isoforms of RyRs, which have tissue-restricted
distribution. RyR1 and RyR2 are typical of muscle cells, with RyR1 originally
considered the skeletal muscle type and RyR2 the cardiac type. However, RyR1 and
RyR2 have recently been found in numerous other cell types, including, for
instance, peripheral B and T lymphocytes. In contrast, RyR3 is widely
distributed among cells. RyR1 and RyR2 are localized in a specialized portion of
the sarcoplasmic reticulum (SR), the terminal cisternae, which is the portion of
the SR Ca2+ store that releases Ca2+ to control the process of muscle
contraction. A specific role for RyR3 has not yet been established: probably,
its co-expression with the other RyR isoforms contributes to qualitatively
modulate Ca2+-dependent processes in muscle cells and in neurons. Several
mutations in the genes encoding RyR1 and RyR2 have been identified in autosomal
domit diseases of skeletal and cardiac muscle, such as maligt hyperthermia
(MH), central core disease (CCD), catecholaminergic polymorphic ventricular
tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia type 2
(ARVD2). More recently, CCD cases with recessive inheritance have also been
described. MH is a pharmacogenetic disease, but the others manifest as
congenital myopathies. Even if their clinical phenotypes are well established,
particularly in skeletal muscle, the molecular mechanisms that generate the
conditions are not clear. A number of studies on cellular models have attempted
to elucidate the molecular defects associated with the different mutations, but
the problem of understanding how mutations in the same gene generate such an
array of diverse pathological traits and diseases of widely different degrees of
severity is still open. This review will consider the molecular and cellular
effects of RyR mutations, summarizing recent data in the literature on Ca2+
dysregulation, which may lead to a better understanding of the functioning of
RyRs. |
What is the INSURE procedure in premature babies. | The INSURE procedure includes intubation, surfactant administration, and extubation (InSurE). It is used to treat respiratory distress syndrome in newborns. | Forty-five patients with aneurysms of the anterior cerebral artery distal to the
anterior communicating artery were operated on by a direct approach method in
the years 1960-1973. The incidence of aneurysms in this location was 4.8% of the
total 1,000 aneurysms. It is of upmost importance in the treatment of aneurysms
to insure the parent artery for the purpose of temporary occlusion. This makes
it easier and safer to approach the aneurysmal neck and to handle possible
premature aneurysmal rupture. From this technical standpoint, the aneurysms in
this location were classified into two types, ascending and horizontal.
Aneurysms of the pericallosal artery between the origin of the anterior
communicating artery and the knee of the corpus callosum were designated as the
aneurysms of the ascending portion, whereas the aneurysms of the pericallosal
artery from the knee of the corpus callosum and beyond were designated as the
aneurysms of the horizontal portion. Depending on the location of the aneurysm,
craniotomy was performed at one of two different sites. For aneurysms of the
ascending portion, bifrontal craniotomy was determined and applied as the safest
approach. A small parasagittal craniotomy was determined to be sufficient for
aneurysms of the horizontal portion. Although the total operative mortality was
4 of 45 cases (9.0%), no mortalities nor morbidities occurred in the last 12
cases since 1972, when the sites of craniotomies were differenciated. INTRODUCTION: The purpose of this study was to examine the efficiency of a
single dose of fast-acting exogenous surfactant (Curosurf) given to premature
babies with progressive respiratory insufficiency. Curosurf was given as early
rescue therapy during a brief intubation (INSURE method:
INtubation-SURfactant-Extubation) during treatment with nasal Continuous
Positive Airway Pressure (CPAP).
MATERIALS AND METHODS: Retrospective study of 115 premature infants with
respiratory distress syndrome born and treated at Odense University Hospital
during the years 1999 to 2004. The criterion for surfactant treatment was a
decrease in the arterial/alveolar oxygen tension ratio (or corresponding values
for the fraction of inspired oxygen) below 0.36 in infants with gestational age
(GA) < 30 weeks and 0.22 in more mature infants.
RESULTS: The primary end point was survival during the first week of life
without mechanical ventilation. This end point was reached by 51% of the infants
with GA < 30 weeks. However, the effect proved to be GA-dependent, increasing
from 22% in infants with a GA of 24-25 weeks to 86% in week 29. Seventy percent
survived to be discharged. In infants with GA > 29 weeks the effect was 87%, and
all survived.
CONCLUSION: The effect of surfactant therapy administered per the INSURE method
is GA-dependent, and the method works best after GA week 25. The question is
whether earlier therapy will increase its efficiency or the method has reached
its limit. OBJECTIVE: To study the effects of implementing a method for surfactant
administration by transient intubation, INSURE (i.e. INtubation SURfactant
Extubation) during nasal continuous positive airway pressure (nCPAP) for
moderately preterm infants with respiratory distress syndrome (RDS).
STUDY DESIGN: A descriptive, retrospective, bi-center study in Stockholm,
Sweden, comparing mechanical ventilation (MV) rates, surfactant use, treatment
response and outcome of all inborn infants with gestational age 27 to 34 weeks
and RDS, (n=420), during the 5-year periods before and after the introduction of
the INSURE-strategy at one of the centers (Karolinska Huddinge) in 1998. The
other center (Karolinska Solna) continued conventional surfactant therapy in
conjunction with MV throughout the study.
RESULTS: Implementation of INSURE at Karolinska Huddinge reduced the number of
infants requiring MV by 50% (P<0.01), resulted in earlier surfactant
administration and increased overall surfactant use. INSURE-treatment improved
oxygenation and the treatment response was sustained over time with only 17% of
the infants requiring >1 dose of surfactant. At Karolinska Solna, the MV rates
were unaltered between the first and second 5-year period.
CONCLUSION: Implementing a strategy of surfactant administration by transient
intubation during nCPAP reduces the need for MV without adverse effects on
outcome and may be an option to more effectively treat RDS, particularly in a
care setting where transfer is necessary to provide MV. BACKGROUND: In preterm infants with respiratory distress syndrome (RDS) nasal
continuous positive airway pressure (nCPAP) with the "InSurE" procedure
(intubation, surfactant, extubation) is increasingly used. However, its effect
on cerebral oxygenation and brain function is not known.
OBJECTIVE: To evaluate the effects of the "InSurE" procedure in infants with RDS
on regional cerebral oxygen saturation (rScO(2)) and relative cerebral
fractional tissue oxygen extraction (cFTOE) using near infrared spectroscopy and
on electrical brain activity using amplitude-integrated electroencephalography
(aEEG).
METHODS: Sixteen infants with RDS, treated with the "InSurE" procedure, and 16
matched controls with nCPAP, were monitored for mean arterial blood pressure
(MABP), arterial oxygen saturation (SaO(2)), rScO(2), cFTOE and aEEG. Ten-minute
periods were selected and averaged at 120 and 20 minutes before, during the
procedure and at 30 minutes, 1, 2, 6, 12 and 24 h after the start of the
"InSurE" procedure. aEEG was analysed by quantitative and qualitative (Burdjalov
score) methods.
RESULTS: MABP was not different between groups on all time points. rScO(2) and
cFTOE were comparable between groups, but there was a trend towards lower
rScO(2) and higher cFTOE 30 minutes after opioid administration in the "InSurE"
infants compared with controls (62% (SD 11) vs 68% (SD 10) and 0.30 (SD 0.10 )
vs 0.28 (SD 0.11), respectively). aEEG amplitudes and Burdjalov scores were
significantly lower in "InSurE" infants from 30 minutes after opioid
administration up to 24 h after the start of the procedure (p<0.05).
CONCLUSION: In the present study, the "InSurE" procedure did not induce
perturbation of cerebral oxygen delivery and extraction, whereas electrical
brain activity decreased for a prolonged period of time. BACKGROUND: Sustained lung inflation (SLI) applied at birth has been
demonstrated to lead to clearance of lung fluid and achievement of a precocious
functional residual capacity in animal studies.
OBJECTIVES: To verify if the application of SLI in preterm infants at birth may
reduce the need for mechanical ventilation and improve their respiratory
outcome.
METHODS: We prospectively studied 89 infants with respiratory distress
(gestational age (GA) 28.1 ± 2.2 weeks) treated at birth with a SLI (25 cm
H(2)O, sustained for 15 s) in addition to AAP recommendations versus a
historical control group (n = 119; GA 28.1 ± 2.0 weeks) treated without SLI with
the same device (controlled positive end-expiratory pressure of 5 cm H(2)O).
RESULTS: The SLI group had less need for (51 vs. 76%, p < 0.0001) and shorter
duration of mechanical ventilation (5 ± 11 vs. 11 ± 19 days, p = 0.008), a more
frequent occurrence of exclusive nasal continuous airway pressure support (49
vs. 24%, p < 0.0001) and INtubation-SURfactant-Extubation (INSURE) treatment (16
vs. 3%, p = 0.01), less need for surfactant (45 vs. 61%, p = 0.027) and
postnatal steroids (10 vs. 25%, p = 0.01), a shorter duration of oxygen therapy
(21 ± 27 vs. 31 ± 31 days, p = 0.016), and, finally, a lower occurrence of
bronchopulmonary dysplasia in survivors (7 vs. 25%, p = 0.004). Multiple
regression analysis showed that 23-27 weeks of GA and birth weight <750 g
increased the risk of mechanical ventilation, while a clinical risk index for
babies (CRIB) score <3 as well as INSURE strategy and SLI treatment in the
delivery room decreased it.
CONCLUSIONS: The application of a SLI at birth in preterm infants with
respiratory distress may decrease the need for mechanical ventilation without
inducing evident adverse effects. BACKGROUND: Propofol is gaining increasing popularity as induction agent for
pediatric endotracheal intubation. Recently, propofol has been described for the
first time as induction agent for endotracheal intubation in preterm neonates.
Propofol seemed to be efficient, safe and ideally suited for the INSURE
(Intubation SURfactant Extubation) procedure in preterm neonates. The purpose of
this study was to document intubating conditions, vital signs, extubation times
and outcome in preterm neonates receiving propofol as induction agent for the
INSURE procedure.
PATIENTS AND METHODS: Preterm neonates with a gestational age of 29-32 weeks and
respiratory distress were eligible for INSURE with propofol if their postnatal
age was <8 h. Exclusion criteria were any kind of disease not allowing early
extubation.
RESULTS: There were 13 inborn neonates enrolled for INSURE, mean gestational age
was 30 weeks + 3 days, and mean birth weight was 1428 g (range 1170-1780 g). We
stopped our observational study ahead of time as a result of significant
cardiovascular side effects. Propofol generally offered good intubating
conditions, but we encountered severe problems with arterial hypotension. A low
propofol bolus of 1 mg kg(-1) caused a distinctive decline in mean arterial
blood pressure from 38 mmHg (range 29-42 mmHg) prior premedication to 24 mmHg
(22-40 mmHg) 10 min after propofol application.
CONCLUSIONS: Our experience with propofol as induction agent for endotracheal
intubation in preterm neonates reveals distinctive cardiovascular effects, which
represent an important risk factor for serious complications of prematurity like
intraventricular hemorrhage or periventricular leucomalacia. Propofol should be
used with caution in very preterm neonates with respiratory distress during the
first hours of life. OBJECTIVES: Our aim was to evaluate whether single and multiple
intubation-surfactant-extubation (INSURE) procedures have similar effects on the
need of mechanical ventilation (MV) and occurrence of bronchopulmonary dysplasia
(BPD) in extremely preterm infants.
METHODS: We studied infants of <30 weeks of gestation with respiratory distress
syndrome (RDS) who were treated with single (FiO(2)>0.30 without need of MV) or
multiple (FiO(2)>0.40 without need of MV) INSURE procedures.
RESULTS: Seventy-five infants were studied: 53 (71%) received single INSURE and
22 (29%) received multiple INSURE procedures. Infants in the single and multiple
groups had similar rates of need of MV (15 vs. 23%) and occurrence of BPD (9 vs.
9%), although the latter were more immature and affected by more severe RDS
(higher FiO(2), lower a/ApO(2), and pO(2)/FiO(2)) than the former.
CONCLUSIONS: Single and multiple INSURE procedures were followed by similar
respiratory outcome in a cohort of extremely preterm infants. Further studies
are warranted to evaluate whether the multiple INSURE strategy enhances the
success rate of INSURE in preventing the need of MV and the occurrence of BPD. BACKGROUND AND OBJECTIVE: Nasal continuous positive airway pressure (NCPAP) plus
intubation, surfactant, and extubation (InSurE) with the option of back-up
ventilation for those infants for whom noninvasive ventilatory support failed
resulted in a significant increase in survival in extremely low birth weight
(ELBW) infants. The authors sought to determine the outcome of ELBW infants
treated with NCPAP and InSurE in a neonatal high care ward with limited back-up
ventilation.
METHODS: Three hundred eighteen inborn infants with birth weight 500-1000 g and
gestational age ≥25 weeks who were admitted to the neonatal high care ward were
included in this observational study. InSurE was administered to infants with
respiratory distress syndrome on NCPAP who had severe in-drawing and recession,
apneic spells, or an Fio(2) >0.4 within 1 hour of birth.
RESULTS: Two hundred twelve (68.6%) infants could be treated with NCPAP only and
97 (31.4%) required InSurE. Seventeen infants were admitted to the NICU; 90%,
87%, and 74.8% of the infants survived until day 3, 7, and discharge,
respectively. Only 2 infants developed a pneumothorax and 2 had chronic lung
disease. Seventy-nine percent of the infants of ≥750 g or >26 weeks' gestation
survived to discharge compared with 56% and 60% of the infants of <750 g or ≤26
weeks' gestation, respectively. Maternal antenatal steroid administration
contributed significantly to the survival of the infants (P = 0.0017, odds ratio
2.7, 95% confidence interval 1.44-5.07).
CONCLUSIONS: The use of NCPAP and InSurE in a neonatal high care ward with
limited resources can improve the survival of ELBW infants. Maternal antenatal
steroid administration contributed significantly to survival. OBJECTIVE: To evaluate the clinical effectiveness and safety of
intubation-surfactant-extubation (INSURE) method in the treatment of neonatal
respiratory distress syndrome (NRDS), and to investigate its possible
mechanisms.
METHODS: Sixty-four premature infants, who were admitted for NRDS and treated
with pulmonary surfactant from March 2010 to March 2012, were enrolled in the
study. They were randomly divided into INSURE (n=32) and conventional mechanical
ventilation (CMV) groups (n=32). The two groups were compared in terms of
respiratory function, ventilation time, duration of oxygen therapy,
complications, and prognosis, as well as expression of interleukin-10 (IL-10),
tumor necrosis factor-α (TNF-α) and serum ferritin (SF).
RESULTS: Oxygenation index in the INSURE group was significantly higher than in
the CMV group at 48 hours after treatment (P<0.05). Compared with the CMV group,
the INSURE group showed significantly lower incidence of ventilator-associated
pneumonia (VAP) and significantly shorter duration of oxygen therapy (P<0.05 for
all comparisons). There were no significant differences in ventilation time and
the incidence of pneumothorax, intracranial hemorrhage, necrotizing
enteroolitis, bronchopulmonary dysplasia, and pneumorrhagia between the two
groups (P>0.05). The levels of TNF-α and SF were significantly lower in the
INSURE group than in the CMV group at 6, 24, 48, and 72 hours after treatment
(P<0.05), while the level of IL-10 was significantly higher in the INSURE group
than in the CMV group (P<0.05).
CONCLUSIONS: INSURE method can improve the oxygenation function of the lung,
decrease the incidence of VAP and shorten the duration of oxygen therapy in
neonates with NRDS, which is probably due to the fact that this method can
reduce the production of TNF-α and SF and inhibit the decrease of IL-10. BACKGROUND: The primary aim of this randomized study was to describe the
feasibility of early administration of surfactant via a thin catheter during
spontaneous breathing (Take Care) and compare early mechanical ventilation (MV)
requirement with the InSurE (Intubate, Surfactant, Extubate) procedure.
METHODS: Preterm infants, who were <32 weeks and stabilized with nasal
continuous positive airway pressure (nCPAP) in the delivery room, were
randomized to receive early surfactant treatment either by the Take Care or
InSurE technique. Tracheal instillation of 100 mg/kg poractant α via 5-F
catheter during spontaneous breathing under nCPAP was performed in the
intervention group. In the InSurE procedure, infants were intubated, received
positive pressure ventilation for 30 seconds after surfactant instillation, and
placed on nCPAP immediately.
RESULTS: One hundred infants in each group were analyzed. The MV requirement in
the first 72 hours of life was significantly lower in the Take Care group when
compared with the InSurE group (30% vs 45%, P = .02, odds ratio -0.52, 95%
confidence interval -0.94 to -0.29). Mean duration of both nCPAP and MV were
significantly shorter in the Take Care group (P values .006 and .002,
respectively). Bronchopulmonary dysplasia rate was significantly lower among the
infants treated with the Take Care technique (relative risk -0.27, 95%
confidence interval -0.1 to -0.72)
CONCLUSIONS: The Take Care technique is feasible for the treatment of
respiratory distress syndrome in infants with very low birth weight. It
significantly reduces both the need and duration of MV, and thus the
bronchopulmonary dysplasia rate in preterm infants. |
Which are the available biomedical text mining tools for the detection of protein-protein interactions? | Protein-protein interactions (PPI) can be extracted from biomedical literature using text mining approaches. These approaches have been classified into two categories, the statistical calculation of the co-occurrence of proteins and the computational linguistic method. Moreover, bioinformatics methods based on sequence, structural, or evolutionary information have been developed to predict protein-protein interactions. The available state-of-the-art biomedical text mining tools are: eFIP (Extracting Functional Impact of Phosphorylation), a system for text mining of protein interaction networks of phosphorylated proteins; GeneView, a suite of state-of-the-art text-mining tools designed for the automated identification of protein–protein interactions; PPI finder, a text mining tool for human protein-protein interactions based on computational linguistic methods. PPI Finder system consists of the Information Retrieval module and Information Extraction module; PreBIND and Textomy are two components of a biomedical literature-mining system optimized to discover protein-protein interactions using a support vector machine; BioMap system is optimized for the identification of protein-protein interactions from large biomedical literature datasets; Protopia searches for and integrates protein-protein interactions and the information about them contained in five different Protein Interaction Web Databases; STRING uses Natural Language Processing to extract a subset of semantically specified known and predicted protein-protein interactions. | BACKGROUND: The rapid publication of important research in the biomedical
literature makes it increasingly difficult for researchers to keep current with
significant work in their area of interest.
RESULTS: This paper reports a scalable method for the discovery of
protein-protein interactions in Medline abstracts, using a combination of text
analytics, statistical and graphical analysis, and a set of easily implemented
rules. Applying these techniques to 12,300 abstracts, a precision of 0.61 and a
recall of 0.97 were obtained, (f = 0.74) and when allowing for two-hop and
three-hop relations discovered by graphical analysis, the precision was 0.74 (f
= 0.83).
CONCLUSION: This combination of linguistic and statistical approaches appears to
provide the highest precision and recall thus far reported in detecting
protein-protein relations using text analytic approaches. Text-mining in molecular biology -- defined as the automatic extraction of
information about genes, proteins and their functional relationships from text
documents -- has emerged as a hybrid discipline on the edges of the fields of
information science, bioinformatics and computational linguistics. A range of
text-mining applications have been developed recently that will improve access
to knowledge for biologists and database annotators. BACKGROUND: Information extraction (IE) efforts are widely acknowledged to be
important in harnessing the rapid advance of biomedical knowledge, particularly
in areas where important factual information is published in a diverse
literature. Here we report on the design, implementation and several evaluations
of OpenDMAP, an ontology-driven, integrated concept analysis system. It
significantly advances the state of the art in information extraction by
leveraging knowledge in ontological resources, integrating diverse text
processing applications, and using an expanded pattern language that allows the
mixing of syntactic and semantic elements and variable ordering.
RESULTS: OpenDMAP information extraction systems were produced for extracting
protein transport assertions (transport), protein-protein interaction assertions
(interaction) and assertions that a gene is expressed in a cell type
(expression). Evaluations were performed on each system, resulting in F-scores
ranging from .26-.72 (precision .39-.85, recall .16-.85). Additionally, each of
these systems was run over all abstracts in MEDLINE, producing a total of 72,460
transport instances, 265,795 interaction instances and 176,153 expression
instances.
CONCLUSION: OpenDMAP advances the performance standards for extracting
protein-protein interaction predications from the full texts of biomedical
research articles. Furthermore, this level of performance appears to generalize
to other information extraction tasks, including extracting information about
predicates of more than two arguments. The output of the information extraction
system is always constructed from elements of an ontology, ensuring that the
knowledge representation is grounded with respect to a carefully constructed
model of reality. The results of these efforts can be used to increase the
efficiency of manual curation efforts and to provide additional features in
systems that integrate multiple sources for information extraction. The open
source OpenDMAP code library is freely available at
http://bionlp.sourceforge.net/ A major challenge in text mining for biomedicine is automatically extracting
protein-protein interactions from the vast amount of biomedical literature. We
have constructed an information extraction system based on the Hidden Vector
State (HVS) model for protein-protein interactions. The HVS model can be trained
using only lightly annotated data whilst simultaneously retaining sufficient
ability to capture the hierarchical structure. When applied in extracting
protein-protein interactions, we found that it performed better than other
established statistical methods and achieved 61.5% in F-score with balanced
recall and precision values. Moreover, the statistical nature of the pure
data-driven HVS model makes it intrinsically robust and it can be easily adapted
to other domains. A particular challenge in biomedical text mining is to find ways of handling
'comprehensive' or 'associative' queries such as 'Find all genes associated with
breast cancer'. Given that many queries in genomics, proteomics or metabolomics
involve these kind of comprehensive searches we believe that a web-based tool
that could support these searches would be quite useful. In response to this
need, we have developed the PolySearch web server. PolySearch supports >50
different classes of queries against nearly a dozen different types of text,
scientific abstract or bioinformatic databases. The typical query supported by
PolySearch is 'Given X, find all Y's' where X or Y can be diseases, tissues,
cell compartments, gene/protein names, SNPs, mutations, drugs and metabolites.
PolySearch also exploits a variety of techniques in text mining and information
retrieval to identify, highlight and rank informative abstracts, paragraphs or
sentences. PolySearch's performance has been assessed in tasks such as gene
synonym identification, protein-protein interaction identification and disease
gene identification using a variety of manually assembled 'gold standard' text
corpuses. Its f-measure on these tasks is 88, 81 and 79%, respectively. These
values are between 5 and 50% better than other published tools. The server is
freely available at http://wishart.biology.ualberta.ca/polysearch. Protein-protein interaction (PPI) extraction has been an important research
topic in bio-text mining area, since the PPI information is critical for
understanding biological processes. However, there are very few open systems
available on the Web and most of the systems focus on keyword searching based on
predefined PPIs. PIE (Protein Interaction information Extraction system) is a
configurable Web service to extract PPIs from literature, including
user-provided papers as well as PubMed articles. After providing abstracts or
papers, the prediction results are displayed in an easily readable form with
essential, yet compact features. The PIE interface supports more features such
as PDF file extraction, PubMed search tool and network communication, which are
useful for biologists and bio-system developers. The PIE system utilizes natural
language processing techniques and machine learning methodologies to predict PPI
sentences, which results in high precision performance for Web users. PIE is
freely available at http://bi.snu.ac.kr/pie/. BACKGROUND: The goal of text mining is to make the information conveyed in
scientific publications accessible to structured search and automatic analysis.
Two important subtasks of text mining are entity mention normalization - to
identify biomedical objects in text - and extraction of qualified relationships
between those objects. We describe a method for identifying genes and
relationships between proteins.
RESULTS: We present solutions to gene mention normalization and extraction of
protein-protein interactions. For the first task, we identify genes by using
background knowledge on each gene, namely annotations related to function,
location, disease, and so on. Our approach currently achieves an f-measure of
86.4% on the BioCreative II gene normalization data. For the extraction of
protein-protein interactions, we pursue an approach that builds on classical
sequence analysis: motifs derived from multiple sequence alignments. The method
achieves an f-measure of 24.4% (micro-average) in the BioCreative II interaction
pair subtask.
CONCLUSION: For gene mention normalization, our approach outperforms strategies
that utilize only the matching of genes names against dictionaries, without
invoking further knowledge on each gene. Motifs derived from alignments of
sentences are successful at identifying protein interactions in text; the
approach we present in this report is fully automated and performs similarly to
systems that require human intervention at one or more stages.
AVAILABILITY: Our methods for gene, protein, and species identification, and
extraction of protein-protein are available as part of the BioCreative Meta
Services (BCMS), see http://bcms.bioinfo.cnio.es/. BACKGROUND: The biomedical literature is the primary information source for
manual protein-protein interaction annotations. Text-mining systems have been
implemented to extract binary protein interactions from articles, but a
comprehensive comparison between the different techniques as well as with manual
curation was missing.
RESULTS: We designed a community challenge, the BioCreative II protein-protein
interaction (PPI) task, based on the main steps of a manual protein interaction
annotation workflow. It was structured into four distinct subtasks related to:
(a) detection of protein interaction-relevant articles; (b) extraction and
normalization of protein interaction pairs; (c) retrieval of the interaction
detection methods used; and (d) retrieval of actual text passages that provide
evidence for protein interactions. A total of 26 teams submitted runs for at
least one of the proposed subtasks. In the interaction article detection
subtask, the top scoring team reached an F-score of 0.78. In the interaction
pair extraction and mapping to SwissProt, a precision of 0.37 (with recall of
0.33) was obtained. For associating articles with an experimental interaction
detection method, an F-score of 0.65 was achieved. As for the retrieval of the
PPI passages best summarizing a given protein interaction in full-text articles,
19% of the submissions returned by one of the runs corresponded to
curator-selected sentences. Curators extracted only the passages that best
summarized a given interaction, implying that many of the automatically
extracted ones could contain interaction information but did not correspond to
the most informative sentences.
CONCLUSION: The BioCreative II PPI task is the first attempt to compare the
performance of text-mining tools specific for each of the basic steps of the PPI
extraction pipeline. The challenges identified range from problems in full-text
format conversion of articles to difficulties in detecting interactor protein
pairs and then linking them to their database records. Some limitations were
also encountered when using a single (and possibly incomplete) reference
database for protein normalization or when limiting search for interactor
proteins to co-occurrence within a single sentence, when a mention might span
neighboring sentences. Finally, distinguishing between novel, experimentally
verified interactions (annotation relevant) and previously known interactions
adds additional complexity to these tasks. BACKGROUND: In the absence of consolidated pipelines to archive biological data
electronically, information dispersed in the literature must be captured by
manual annotation. Unfortunately, manual annotation is time consuming and the
coverage of published interaction data is therefore far from complete. The use
of text-mining tools to identify relevant publications and to assist in the
initial information extraction could help to improve the efficiency of the
curation process and, as a consequence, the database coverage of data available
in the literature. The 2006 BioCreative competition was aimed at evaluating
text-mining procedures in comparison with manual annotation of protein-protein
interactions.
RESULTS: To aid the BioCreative protein-protein interaction task, IntAct and
MINT (Molecular INTeraction) provided both the training and the test datasets.
Data from both databases are comparable because they were curated according to
the same standards. During the manual curation process, the major cause of data
loss in mining the articles for information was ambiguity in the mapping of the
gene names to stable UniProtKB database identifiers. It was also observed that
most of the information about interactions was contained only within the
full-text of the publication; hence, text mining of protein-protein interaction
data will require the analysis of the full-text of the articles and cannot be
restricted to the abstract.
CONCLUSION: The development of text-mining tools to extract protein-protein
interaction information may increase the literature coverage achieved by manual
curation. To support the text-mining community, databases will highlight those
sentences within the articles that describe the interactions. These will supply
data-miners with a high quality dataset for algorithm development. Furthermore,
the dictionary of terms created by the BioCreative competitors could enrich the
synonym list of the PSI-MI (Proteomics Standards Initiative-Molecular
Interactions) controlled vocabulary, which is used by both databases to annotate
their data content. This article collects opinions from leading scientists about how text mining can
provide better access to the biological literature, how the scientific community
can help with this process, what the next steps are, and what role future
BioCreative evaluations can play. The responses identify several broad themes,
including the possibility of fusing literature and biological databases through
text mining; the need for user interfaces tailored to different classes of users
and supporting community-based annotation; the importance of scaling text mining
technology and inserting it into larger workflows; and suggestions for
additional challenge evaluations, new applications, and additional resources
needed to make progress. BACKGROUND: Reliable information extraction applications have been a long sought
goal of the biomedical text mining community, a goal that if reached would
provide valuable tools to benchside biologists in their increasingly difficult
task of assimilating the knowledge contained in the biomedical literature. We
present an integrated approach to concept recognition in biomedical text.
Concept recognition provides key information that has been largely missing from
previous biomedical information extraction efforts, namely direct links to well
defined knowledge resources that explicitly cement the concept's semantics. The
BioCreative II tasks discussed in this special issue have provided a unique
opportunity to demonstrate the effectiveness of concept recognition in the field
of biomedical language processing.
RESULTS: Through the modular construction of a protein interaction relation
extraction system, we present several use cases of concept recognition in
biomedical text, and relate these use cases to potential uses by the benchside
biologist.
CONCLUSION: Current information extraction technologies are approaching
performance standards at which concept recognition can begin to deliver high
quality data to the benchside biologist. Our system is available as part of the
BioCreative Meta-Server project and on the internet
http://bionlp.sourceforge.net. BACKGROUND: Human protein-protein interaction (PPIs) data are the foundation for
understanding molecular signalling networks and the functional roles of
biomolecules. Several human PPI databases have become available; however,
comparisons of these datasets have suggested limited data coverage and poor data
quality. Ongoing collection and integration of human PPIs from different
sources, both experimentally and computationally, can enable disease-specific
network biology modelling in translational bioinformatics studies.
RESULTS: We developed a new web-based resource, the Human Annotated and
Predicted Protein Interaction (HAPPI) database, located at
http://bio.informatics.iupui.edu/HAPPI/. The HAPPI database was created by
extracting and integrating publicly available protein interaction databases,
including HPRD, BIND, MINT, STRING, and OPHID, using database integration
techniques. We designed a unified entity-relationship data model to resolve
semantic level differences of diverse concepts involved in PPI data integration.
We applied a unified scoring model to give each PPI a measure of its reliability
that can place each PPI at one of the five star rank levels from 1 to 5. We
assessed the quality of PPIs contained in the new HAPPI database, using
evolutionary conserved co-expression pairs called "MetaGene" pairs to measure
the extent of MetaGene pair and PPI pair overlaps. While the overall quality of
the HAPPI database across all star ranks is comparable to the overall qualities
of HPRD or IntNetDB, the subset of the HAPPI database with star ranks between 3
and 5 has a much higher average quality than all other human PPI databases. As
of summer 2008, the database contains 142,956 non-redundant, medium to
high-confidence level human protein interaction pairs among 10,592 human
proteins. The HAPPI database web application also provides ..." should be "The
HAPPI database web application also provides hyperlinked information of genes,
pathways, protein domains, protein structure displays, and sequence feature maps
for interactive exploration of PPI data in the database.
CONCLUSION: HAPPI is by far the most comprehensive public compilation of human
protein interaction information. It enables its users to fully explore PPI data
with quality measures and annotated information necessary for emerging network
biology studies. BACKGROUND: Protein-protein interactions can be considered the basic skeleton
for living organism self-organization and homeostasis. Impressive quantities of
experimental data are being obtained and computational tools are essential to
integrate and to organize this information. This paper presents Protopia, a
biological tool that offers a way of searching for proteins and their
interactions in different Protein Interaction Web Databases, as a part of a
multidisciplinary initiative of our institution for the integration of
biological data http://asp.uma.es.
RESULTS: The tool accesses the different Databases (at present, the free version
of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with
biological protein names or databases codes and can be depicted as a vector or a
matrix. They can be represented and handled interactively as an organic graph.
Comparison among databases is carried out using the Uniprot codes annotated for
each protein.
CONCLUSION: The tool locates and integrates the current information stored in
the aforementioned databases, and redundancies among them are detected. Results
are compatible with the most important network analysers, so that they can be
compared and analysed by other world-wide known tools and platforms. The
visualization possibilities help to attain this goal and they are especially
interesting for handling multiple-step or complex networks. MOTIVATION: Identification and characterization of protein-protein interactions
(PPIs) is one of the key aims in biological research. While previous research in
text mining has made substantial progress in automatic PPI detection from
literature, the need to improve the precision and recall of the process remains.
More accurate PPI detection will also improve the ability to extract
experimental data related to PPIs and provide multiple evidence for each
interaction.
RESULTS: We developed an interaction detection method and explored the
usefulness of various features in automatically identifying PPIs in text. The
results show that our approach outperforms other systems using the AImed
dataset. In the tests where our system achieves better precision with reduced
recall, we discuss possible approaches for improvement. In addition to test
datasets, we evaluated the performance on interactions from five human-curated
databases-BIND, DIP, HPRD, IntAct and MINT-where our system consistently
identified evidence for approximately 60% of interactions when both proteins
appear in at least one sentence in the PubMed abstract. We then applied the
system to extract articles from PubMed to annotate known, high-throughput and
interologous interactions in I(2)D.
AVAILABILITY: The data and software are available at: http://www.cs.utoronto.ca/
approximately juris/data/BI09/. BACKGROUND: Biological knowledge is represented in scientific literature that
often describes the function of genes/proteins (bioentities) in terms of their
interactions (biointeractions). Such bioentities are often related to biological
concepts of interest that are specific of a determined research field.
Therefore, the study of the current literature about a selected topic deposited
in public databases, facilitates the generation of novel hypotheses associating
a set of bioentities to a common context.
RESULTS: We created a text mining system (LAITOR: Literature Assistant for
Identification of Terms co-Occurrences and Relationships) that analyses
co-occurrences of bioentities, biointeractions, and other biological terms in
MEDLINE abstracts. The method accounts for the position of the co-occurring
terms within sentences or abstracts. The system detected abstracts mentioning
protein-protein interactions in a standard test (BioCreative II IAS test data)
with a precision of 0.82-0.89 and a recall of 0.48-0.70. We illustrate the
application of LAITOR to the detection of plant response genes in a dataset of
1000 abstracts relevant to the topic.
CONCLUSIONS: Text mining tools combining the extraction of interacting
bioentities and biological concepts with network displays can be helpful in
developing reasonable hypotheses in different scientific backgrounds. The most important way of conveying new findings in biomedical research is
scientific publication. Extraction of protein-protein interactions (PPIs)
reported in scientific publications is one of the core topics of text mining in
the life sciences. Recently, a new class of such methods has been proposed -
convolution kernels that identify PPIs using deep parses of sentences. However,
comparing published results of different PPI extraction methods is impossible
due to the use of different evaluation corpora, different evaluation metrics,
different tuning procedures, etc. In this paper, we study whether the reported
performance metrics are robust across different corpora and learning settings
and whether the use of deep parsing actually leads to an increase in extraction
quality. Our ultimate goal is to identify the one method that performs best in
real-life scenarios, where information extraction is performed on unseen text
and not on specifically prepared evaluation data. We performed a comprehensive
benchmarking of nine different methods for PPI extraction that use convolution
kernels on rich linguistic information. Methods were evaluated on five different
public corpora using cross-validation, cross-learning, and cross-corpus
evaluation. Our study confirms that kernels using dependency trees generally
outperform kernels based on syntax trees. However, our study also shows that
only the best kernel methods can compete with a simple rule-based approach when
the evaluation prevents information leakage between training and test corpora.
Our results further reveal that the F-score of many approaches drops
significantly if no corpus-specific parameter optimization is applied and that
methods reaching a good AUC score often perform much worse in terms of F-score.
We conclude that for most kernels no sensible estimation of PPI extraction
performance on new text is possible, given the current heterogeneity in
evaluation data. Nevertheless, our study shows that three kernels are clearly
superior to the other methods. MOTIVATION: Protein-protein interactions (PPIs) play an important role in
understanding biological processes. Although recent research in text mining has
achieved a significant progress in automatic PPI extraction from literature,
performance of existing systems still needs to be improved.
RESULTS: In this study, we propose a novel algorithm for extracting PPIs from
literature which consists of two phases. First, we automatically categorize the
data into subsets based on its semantic properties and extract candidate PPI
pairs from these subsets. Second, we apply support vector machines (SVMs) to
classify candidate PPI pairs using features specific for each subset. We obtain
promising results on five benchmark datasets: AIMed, BioInfer, HPRD50, IEPA and
LLL with F-scores ranging from 60% to 84%, which are comparable with the
state-of-the-art PPI extraction systems. Furthermore, our system achieves the
best performance on cross-corpora evaluation and comparative performance in
terms of computational efficiency.
AVAILABILITY: The source code and scripts used in this article are available for
academic use at http://staff.science.uva.nl/~bui/PPIs.zip
CONTACT: [email protected]. Keeping up with the rapidly growing literature has become virtually impossible
for most scientists. This can have dire consequences. First, we may waste
research time and resources on reinventing the wheel simply because we can no
longer maintain a reliable grasp on the published literature. Second, and
perhaps more detrimental, judicious (or serendipitous) combination of knowledge
from different scientific disciplines, which would require following disparate
and distinct research literatures, is rapidly becoming impossible for even the
most ardent readers of research publications. Text mining - the automated
extraction of information from (electronically) published sources - could
potentially fulfil an important role - but only if we know how to harness its
strengths and overcome its weaknesses. As we do not expect that the rate at
which scientific results are published will decrease, text mining tools are now
becoming essential in order to cope with, and derive maximum benefit from, this
information explosion. In genomics, this is particularly pressing as more and
more rare disease-causing variants are found and need to be understood. Not
being conversant with this technology may put scientists and biomedical
regulators at a severe disadvantage. In this review, we introduce the basic
concepts underlying modern text mining and its applications in genomics and
systems biology. We hope that this review will serve three purposes: (i) to
provide a timely and useful overview of the current status of this field,
including a survey of present challenges; (ii) to enable researchers to decide
how and when to apply text mining tools in their own research; and (iii) to
highlight how the research communities in genomics and systems biology can help
to make text mining from biomedical abstracts and texts more straightforward. There is an increasing interest in developing ontologies and controlled
vocabularies to improve the efficiency and consistency of manual literature
curation, to enable more formal biocuration workflow results and ultimately to
improve analysis of biological data. Two ontologies that have been successfully
used for this purpose are the Gene Ontology (GO) for annotating aspects of gene
products and the Molecular Interaction ontology (PSI-MI) used by databases that
archive protein-protein interactions. The examination of protein interactions
has proven to be extremely promising for the understanding of cellular
processes. Manual mapping of information from the biomedical literature to
bio-ontology terms is one of the most challenging components in the curation
pipeline. It requires that expert curators interpret the natural language
descriptions contained in articles and infer their semantic equivalents in the
ontology (controlled vocabulary). Since manual curation is a time-consuming
process, there is strong motivation to implement text-mining techniques to
automatically extract annotations from free text. A range of text mining
strategies has been devised to assist in the automated extraction of biological
data. These strategies either recognize technical terms used recurrently in the
literature and propose them as candidates for inclusion in ontologies, or
retrieve passages that serve as evidential support for annotating an ontology
term, e.g. from the PSI-MI or GO controlled vocabularies. Here, we provide a
general overview of current text-mining methods to automatically extract
annotations of GO and PSI-MI ontology terms in the context of the BioCreative
(Critical Assessment of Information Extraction Systems in Biology) challenge.
Special emphasis is given to protein-protein interaction data and PSI-MI terms
referring to interaction detection methods. Extracting protein-protein interaction (PPI) from biomedical literature is an
important task in biomedical text mining (BioTM). In this paper, we propose a
hash subgraph pairwise (HSP) kernel-based approach for this task. The key to the
novel kernel is to use the hierarchical hash labels to express the structural
information of subgraphs in a linear time. We apply the graph kernel to compute
dependency graphs representing the sentence structure for protein-protein
interaction extraction task, which can efficiently make use of full graph
structural information, and particularly capture the contiguous topological and
label information ignored before. We evaluate the proposed approach on five
publicly available PPI corpora. The experimental results show that our approach
significantly outperforms all-path kernel approach on all five corpora and
achieves state-of-the-art performance. Research results are primarily published in scientific literature and curation
efforts cannot keep up with the rapid growth of published literature. The
plethora of knowledge remains hidden in large text repositories like MEDLINE.
Consequently, life scientists have to spend a great amount of time searching for
specific information. The enormous ambiguity among most names of biomedical
objects such as genes, chemicals and diseases often produces too large and
unspecific search results. We present GeneView, a semantic search engine for
biomedical knowledge. GeneView is built upon a comprehensively annotated version
of PubMed abstracts and openly available PubMed Central full texts. This
semi-structured representation of biomedical texts enables a number of features
extending classical search engines. For instance, users may search for entities
using unique database identifiers or they may rank documents by the number of
specific mentions they contain. Annotation is performed by a multitude of
state-of-the-art text-mining tools for recognizing mentions from 10 entity
classes and for identifying protein-protein interactions. GeneView currently
contains annotations for >194 million entities from 10 classes for ∼21 million
citations with 271,000 full text bodies. GeneView can be searched at
http://bc3.informatik.hu-berlin.de/. MOTIVATION: Although the amount of data in biology is rapidly increasing,
critical information for understanding biological events like phosphorylation or
gene expression remains locked in the biomedical literature. Most current text
mining (TM) approaches to extract information about biological events are
focused on either limited-scale studies and/or abstracts, with data extracted
lacking context and rarely available to support further research.
RESULTS: Here we present BioContext, an integrated TM system which extracts,
extends and integrates results from a number of tools performing entity
recognition, biomolecular event extraction and contextualization. Application of
our system to 10.9 million MEDLINE abstracts and 234 000 open-access full-text
articles from PubMed Central yielded over 36 million mentions representing 11.4
million distinct events. Event participants included over 290 000 distinct
genes/proteins that are mentioned more than 80 million times and linked where
possible to Entrez Gene identifiers. Over a third of events contain contextual
information such as the anatomical location of the event occurrence or whether
the event is reported as negated or speculative.
AVAILABILITY: The BioContext pipeline is available for download (under the BSD
license) at http://www.biocontext.org, along with the extracted data which is
also available for online browsing. Protein phosphorylation is a central regulatory mechanism in signal transduction
involved in most biological processes. Phosphorylation of a protein may lead to
activation or repression of its activity, alternative subcellular location and
interaction with different binding partners. Extracting this type of information
from scientific literature is critical for connecting phosphorylated proteins
with kinases and interaction partners, along with their functional outcomes, for
knowledge discovery from phosphorylation protein networks. We have developed the
Extracting Functional Impact of Phosphorylation (eFIP) text mining system, which
combines several natural language processing techniques to find relevant
abstracts mentioning phosphorylation of a given protein together with
indications of protein-protein interactions (PPIs) and potential evidences for
impact of phosphorylation on the PPIs. eFIP integrates our previously developed
tools, Extracting Gene Related ABstracts (eGRAB) for document retrieval and name
disambiguation, Rule-based LIterature Mining System (RLIMS-P) for Protein
Phosphorylation for extraction of phosphorylation information, a PPI module to
detect PPIs involving phosphorylated proteins and an impact module for relation
extraction. The text mining system has been integrated into the curation
workflow of the Protein Ontology (PRO) to capture knowledge about phosphorylated
proteins. The eFIP web interface accepts gene/protein names or identifiers, or
PubMed identifiers as input, and displays results as a ranked list of abstracts
with sentence evidence and summary table, which can be exported in a spreadsheet
upon result validation. As a participant in the BioCreative-2012 Interactive
Text Mining track, the performance of eFIP was evaluated on document retrieval
(F-measures of 78-100%), sentence-level information extraction (F-measures of
70-80%) and document ranking (normalized discounted cumulative gain measures of
93-100% and mean average precision of 0.86). The utility and usability of the
eFIP web interface were also evaluated during the BioCreative Workshop. The use
of the eFIP interface provided a significant speed-up (∼2.5-fold) for time to
completion of the curation task. Additionally, eFIP significantly simplifies the
task of finding relevant articles on PPI involving phosphorylated forms of a
given protein. |
How thyrocyte destruction is induced in autoimmune thyroiditis? | Thyrocytes from Hashimoto's thyroiditis (HT) glands, but not from nonautoimmune thyroids, expressed Fas (CD95), therefore autonomous interaction between thyrocyte Fas (CD95) and FasL (CD95L) has been proposed as a major mechanism of thyrocyte depletion in HT. Moreover, experimental evidence has showed that Infiltrating T Lymphocytes (ITLs) do not express significant amounts of FasL, suggesting that ITLs are not directly involved in thyrocyte destruction. | Hashimoto's thyroiditis (HT) is a chronic autoimmune disease resulting from
Fas-mediated thyrocyte destruction. Although autocrine/paracrine Fas-Fas ligand
(FasL) interaction is responsible for thyrocyte cell death during the active
phases of HT, the role of infiltrating T lymphocytes (ITL) in this process is
still unknown. Therefore, we investigated the expression and function of Fas and
FasL in ITL. All ITL expressed high levels of Fas and CD69, an early marker of T
cell activation associated with functional Fas expression in T cells in vivo. In
contrast to thyrocytes that were found to produce high levels of FasL, ITL did
not express significant amounts of FasL, suggesting that ITL are not directly
involved in thyrocyte destruction. The analysis of ITL purified from HT thyroids
showed that ITL were massively killed by Fas crosslinking and that a
considerable number (24-36%) underwent spontaneous apoptosis within 36 h of
culture. Accordingly, in situ TUNEL (terminal deoxynucleotidyl transferase dUTP
nick end labeling) staining revealed that a significant number (10-15%) of ITL
in proximity to FasL-producing thyroid follicles were apoptotic. Moreover,
virtually all ITL in proximity to thyroid follicles were preapoptotic, as they
expressed high levels of GD3 ganglioside, a killer glycolipid responsible for
the generation of irreversible apoptotic signals that accumulate in
hematopoietic cells shortly after Fas crosslinking. These data demonstrate that
ITL are not directly involved in thyrocyte cell death during HT, suggesting that
autocrine/paracrine Fas-FasL interaction is a major mechanism in autoimmune
thyrocyte destruction. Hashimoto's thyroiditis is a common chronic autoimmune disease characterized by
the loss of thyroid follicular cells (thyrocytes) that are gradually replaced by
lymphocytic infiltration and diffuse fibrosis. These morphological findings
suggested that autoreactive T-cell clones were responsible for thyrocyte
destruction and hypothyroidism through effector-target cytotoxic recognition.
Later, autonomous interaction between thyrocyte Fas and FasL has been proposed
as a major mechanism of thyrocyte depletion in Hashimoto's thyroiditis. Here, we
analyze the possible role of Fas and FasL in the pathogenesis of Hashimoto's
thyroiditis. We suggest that the Fas-FasL system dictates the outcome of the
autoimmune response by acting on both immune and target cells. |
Do A-type lamins bind euchromatin or heterochromatin? | These data reveal that the domain encoded by exon 9 is important to maintain telomere homeostasis and heterochromatin structure but does not play a role in DNA repair, thus pointing to other exons in the lamin A tail as responsible for the genomic instability phenotype in Lmna(Δ8-11/Δ8-11) mice | Certain DNA binding proteins are thought to organize the mammalian genome into
distinct 3 dimensional structures, each characteristic of a given differentiated
state. Autoantibodies to 2 types of DNA binding protein complexes, the nuclear
lamina and p70/p80 (Ku), were identified in sera of patients with collagen
vascular diseases. The intranuclear distribution, DNA binding, and behavior
during mitosis of these antigens were examined using autoimmune sera and murine
monoclonal antibodies. In vivo, the antigens have different intranuclear
distributions and solubility characteristics. However, both antigens appear to
reversibly bind to DNA during interphase and to rapidly dissociate from DNA
during mitosis. Although the binding affinity of p70/p80 to DNA is
heterogeneous, the interaction between p70/p80 and DNA in vitro is stable over 2
h or more. The rapid dissociation of p70/p80 from DNA during mitosis may
therefore be mediated by a modification in either chromatin structure or in the
p70/p80 antigen itself. Other proteins that reversibly interact with DNA, such
as the lamins and nuclear pores, may have a role in the organization of DNA into
transcribable euchromatin and nontranscribable heterochromatin. Autoantibodies
to these proteins, and possibly those reactive with p70/p80, or other DNA
binding proteins may be useful probes for studying both chromatin organization
and the causes of autoimmune diseases such as systemic lupus erythematosus. To study the role of caspase-6 during nuclear disassembly, we generated a
chicken DT40 cell line in which both alleles of the caspase-6 gene were
disrupted. No obvious morphological differences were observed in the apoptotic
process in caspase-6- deficient cells compared with the wild type. However,
examination of apoptosis in a cell-free system revealed a block in chromatin
condensation and apoptotic body formation when nuclei from HeLa cells expressing
lamin A or lamin A-transfected Jurkat cells were incubated in
caspase-6-deficient apoptotic extracts. Transfection of exogenous caspase-6 into
the clone reversed this phenotype. Lamins A and C, which are caspase-6-only
substrates, were cleaved by the wild-type and heterozygous apoptotic extracts
but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor
z-VEID-fmk mimicked the effects of caspase-6 deficiency and prevented the
cleavage of lamin A. Taken together, these observations indicate that caspase-6
activity is essential for lamin A cleavage and that when lamin A is present it
must be cleaved in order for the chromosomal DNA to undergo complete
condensation during apoptotic execution. Posttranslational modification of histones is known to regulate chromatin
structure and transcriptional activity, and the nuclear lamina is thought to
serve as a site for heterochromatin maintece and transcriptional silencing.
In this report, we show that the nuclear lamina can also play a role in the
downregulation of heterochromatin and in gene activation. Herpes simplex virus
DNA initiates replication in replication compartments near the inner edge of the
nucleus, and histones are excluded from these structures. To define the role of
nuclear lamins in HSV replication, we examined HSV infection in wild-type and
A-type lamin-deficient (Lmna-/-) murine embryonic fibroblasts (MEFs). In Lmna-/-
cells, viral replication compartments are reduced in size and fail to target to
the nuclear periphery, as observed in WT cells. Chromatin immunoprecipitation
and immunofluorescence studies demonstrate that HSV DNA is associated with
increased heterochromatin in Lmna-/- MEFs. These results argue for a functional
role for A-type lamins as viral gene expression, DNA replication, and growth are
reduced in Lmna-/- MEFs, with the greatest effect on viral replication at low
multiplicity of infection. Thus, lamin A/C is required for targeting of the
viral genome and the reduction of heterochromatin on viral promoters during
lytic infection. The nuclear lamina can serve as a molecular scaffold for DNA
genomes and the protein complexes that regulate both euchromatin and
heterochromatin histone modifications. The nuclear lamins function in the regulation of replication, transcription, and
epigenetic modifications of chromatin. However, the mechanisms responsible for
these lamin functions are poorly understood. We demonstrate that A- and B-type
lamins form separate, but interacting, stable meshworks in the lamina and have
different mobilities in the nucleoplasm as determined by fluorescence
correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically
increases the lamina meshwork size and the mobility of nucleoplasmic lamin A
(LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich
nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH)
analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and
immunofluorescence localization of modified histones demonstrate that gene-rich
euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated
RNA polymerase II (Pol II) and histone marks for active transcription suggest
that blebs are transcriptionally active. However, in vivo labeling of RNA
indicates that transcription is decreased, suggesting that the LA/C-rich
microenvironment induces promoter proximal stalling of Pol II. We propose that
different lamins are organized into separate, but interacting, microdomains and
that LB1 is essential for their organization. Our evidence suggests that the
organization and regulation of chromatin are influenced by interconnections
between these lamin microdomains. After in vitro maturation (IVM), isolation of the healthiest oocytes is
essential for successful in vitro fertilization. As germinal vesicle (GV)
oocytes resume meiosis through healthy or apoptotic pathways without discernable
morphological criteria, we checked for an apoptotic element acting at the
nucleus level. We hypothesized that caspase-6 with its corresponding substrate,
lamin A/C, could be a potential target candidate, because caspase-6 is the only
functional caspase for lamin A/C. We used immunohistochemistry methods, Western
blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C
and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated
that these proteins were always present and that their distributions were
related to oocyte maturity, determined by chromatin configuration and oocyte
diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the
involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and
caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny. Embryonic stem cells are characterized by unique epigenetic features including
decondensed chromatin and hyperdynamic association of chromatin proteins with
chromatin. Here we investigate the potential mechanisms that regulate chromatin
plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic
stem cells lacking various chromatin proteins, we find that histone acetylation,
G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all
affect chromatin protein dynamics. Histone acetylation controls, almost
exclusively, euchromatin protein dynamics; lamin A expression regulates
heterochromatin protein dynamics, and G9a regulates both euchromatin and
heterochromatin protein dynamics. In contrast, we find that DNA methylation and
nucleosome repeat length have little or no effect on chromatin-binding protein
dynamics in embryonic stem cells. Altered chromatin dynamics associates with
perturbed embryonic stem cell differentiation. Together, these data provide
mechanistic insights into the epigenetic pathways that are responsible for
chromatin plasticity in embryonic stem cells, and indicate that the genome's
epigenetic state modulates chromatin plasticity and differentiation potential of
embryonic stem cells. Over 300 mutations in the LMNA gene, encoding A-type lamins, are associated with
15 human degenerative disorders and premature aging syndromes. Although genomic
instability seems to contribute to the pathophysiology of some laminopathies,
there is limited information about what mutations cause genomic instability and
by which molecular mechanisms. Mouse embryonic fibroblasts depleted of A-type
lamins or expressing mutants lacking exons 8-11 (Lmna(Δ8-11/Δ8-11)) exhibit
alterations in telomere biology and DNA repair caused by cathepsin L-mediated
degradation of 53BP1 and reduced expression of BRCA1 and RAD51. Thus, a region
encompassing exons 8-11 seems essential for genome integrity. Given that
deletion of lamin A exon 9 in the mouse (Lmna(Δ9/Δ9)) results in a progeria
phenotype, we tested if this domain is important for genome integrity.
Lmna(Δ9/Δ9) MEFs exhibit telomere shortening and heterochromatin alterations but
do not activate cathepsin L-mediated degradation of 53BP1 and maintain
expression of BRCA1 and RAD51. Accordingly, Lmna(Δ9/Δ9) MEFs do not present
genomic instability, and expression of mutant lamin A Δexon9 in lamin-depleted
cells restores DNA repair factors levels and partially rescues nuclear
abnormalities. These data reveal that the domain encoded by exon 9 is important
to maintain telomere homeostasis and heterochromatin structure but does not play
a role in DNA repair, thus pointing to other exons in the lamin A tail as
responsible for the genomic instability phenotype in Lmna(Δ8-11/Δ8-11) mice. Our
study also suggests that the levels of DNA repair factors 53BP1, BRCA1 and RAD51
could potentially serve as biomarkers to identify laminopathies that present
with genomic instability. |
Which is the most abundant membrane protein on Earth? | LHCII, the largest plant photosynthetic pigment-protein complex of photosystem II, is a most abundant membrane protein in living organisms and comprises approximately half of the pool of chlorophyll molecules in the biosphere. | Many factors trigger the degradation of proteins, including changes in
environmental conditions, genetic mutations, and limitations in the availability
of cofactors. Despite the importance for viability, still very little is known
about protein degradation and its regulation. The degradation of the most
abundant membrane protein on Earth, the light-harvesting complex of Photosystem
II (LHC II), is highly regulated under different environmental conditions, e.g.
light stress, to prevent photochemical damage of the reaction center. However,
despite major effort to identify the protease/proteases involved in the
degradation of the apoproteins of LHC II the molecular details of this important
process remain obscure. LHC II belongs to the family of chlorophyll a/b binding
proteins (CAB proteins) and is located in the thylakoid membrane of the plant
chloroplast. The results of biochemical experiments to isolate and characterize
the protease degrading LHC II are summarized here and compared to our own recent
finding indicating that a metalloprotease of the FtsH family is involved in this
process. The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII)
is the most abundant membrane protein in green plants, and its degradation is a
crucial process for the acclimation to high light conditions and for the
recovery of nitrogen (N) and carbon (C) during senescence. However, the
molecular mechanism of LHCII degradation is largely unknown. Here, we report
that chlorophyll b reductase, which catalyzes the first step of chlorophyll b
degradation, plays a central role in LHCII degradation. When the genes for
chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana,
chlorophyll b and LHCII were not degraded during senescence, whereas other
pigment complexes completely disappeared. When purified trimeric LHCII was
incubated with recombit chlorophyll b reductase (NOL), expressed in
Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl
chlorophyll a. Accompanying this conversion, chlorophylls were released from
LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from
the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into
monomeric forms but remained in the trimeric form. Based on these results, we
propose the novel hypothesis that chlorophyll b reductase catalyzes the initial
step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII
degradation. LHCII is the most abundant membrane protein on earth. It participates in the
first steps of photosynthesis by harvesting sunlight and transferring excitation
energy to the core complex. Here we have analyzed the LHCII complex of the green
alga Chlamydomonas reinhardtii and its association with the core of Photosystem
II (PSII) to form multiprotein complexes. Several PSII supercomplexes with
different antenna sizes have been purified, the largest of which contains three
LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13Å
resolution was obtained allowing the reconstruction of the 3D structure of the
supercomplex. The position and orientation of the S trimer are the same as in
plants; trimer M is rotated by 45° and the additional trimer (named here as
LHCII-N), which is taking the position occupied in plants by CP24, is directly
associated with the core. The analysis of supercomplexes with different antenna
sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the
trimers in the PSII supercomplex, while LhcbM5 is part of the "extra" LHCII pool
not directly associated with the supercomplex. It is also shown that
Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex
from plants and a blue shifted absorption spectrum. Finally the data indicate
that there are at least six LHCII trimers per dimeric core in the thylakoid
membranes, meaning that the antenna size of PSII of C. reinhardtii is larger
than that of plants. LHCII, the largest plant photosynthetic pigment-protein complex of photosystem
II, is a most abundant membrane protein in living organisms and comprises
approximately half of the pool of chlorophyll molecules in the biosphere. The
principal role of this pigment-protein complex is to collect sunlight quanta and
transfer electronic excitations toward the reaction centers, where the primary
photosynthetic electric charge separation reactions take place. The LHCII
protein, as a major protein component of the photosynthetic membranes, modulates
also the structural and dynamic properties of the lipid phase of the membranes.
According to the recent concepts, one of the physiological roles of LHCII is
also a protection of the photosynthetic apparatus against oxidative damage
caused by illumination with high intensity light. Detailed examination of all
those physiological functions of LHCII, in relation to the complex structure,
was possible owing to the application of several molecular spectroscopy
techniques. Some examples of such studies are presented in this chapter. The
examples of application of steady-state and time-resolved fluorescence
spectroscopy, Fourier-transform infrared absorption spectroscopy, and resoce
Raman scattering spectroscopy are presented and discussed. |
Can RNASeq be used for the analysis of nascent transcripts? | Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues. Here we show that RNA-seq can also be used for studying nascent RNAs undergoing transcription. We utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. | During the late stage of adenovirus 2 infection, RNA chains are initiated at a
site near coordinate 16 (Evans et al., 1977) and transcribed approximately
30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs
processed from these transcripts contain a common spliced tripartite leader
(Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at approximately 16,
20 and 27, and protein coding sequences which map downstream. This report maps
the late promoter and the capped 5' end of nuclear and cytoplasmic RNAs from
this transciption unit, and analyzes their structures. We show that nascent RNA
chains pulse-labeled in vivo are initiated at coordinate 16.5 +/- 0.5 and
contain the sequences intervening between the leader segments. We map the capped
5' terminus of late nuclear transcripts at a site between 16.4 and 16.6 by
aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of
the promoter region. The structure of the first eleven residues of the capped 5'
terminus of late mRNA was determined by direct RNA sequencing. This structure
corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions
the mRNA cap template within the promoter region. These results suggest that the
promoter and the cap template sites are coincident, and that the initiating
residues of the primary transcript are precursors of the capped 5' end of mRNA.
Residues removed from transcripts by splicing were identified. These plus caps
were detected in large polyadenylated nuclear RNA, indicating that capping and
polyadenylation can occur on unspliced molecules. Residues retained in the mRNA
first leader contain a nine residue sequence adjacent to the cap which is
complementary to the 3' end of 18S rRNA, suggesting that the first leader
functions in ribosome binding. Nucleotide sequences from the promoter region are
compared with cellular counterparts. Strong homologies at cap sites and splice
points suggest that for the noted cases, the virus and cell share closely
related mechanisms for mRNA 5' end synthesis and splicing. Here we propose a new general method for directly determining RNA sequence based
on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the
chain terminators (RdRP sequencing). The following properties of the polymerase
render it appropriate for this application: (1) the phi6 polymerase can
replicate a number of single-stranded RNA templates in vitro. (2) In contrast to
the primer-dependent DNA polymerases utilized in the sequencing procedure by
Sanger et al. (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent
strand synthesis without a primer, starting the polymerization on the very
3'-terminus of the template. (3) The polymerase can incorporate
chain-terminating nucleotide analogs into the nascent RNA chain to produce a set
of base-specific termination products. Consequently, 3' proximal or even
complete sequence of many target RNA molecules can be rapidly deduced without
prior sequence information. The new technique proved useful for sequencing
several synthetic ssRNA templates. Furthermore, using genomic segments of the
bluetongue virus we show that RdRP sequencing can also be applied to naturally
occurring dsRNA templates. This suggests possible uses of the method in the RNA
virus research and diagnostics. RNA polymerases are highly regulated molecular machines. We present a method
(global run-on sequencing, GRO-seq) that maps the position, amount, and
orientation of transcriptionally engaged RNA polymerases genome-wide. In this
method, nuclear run-on RNA molecules are subjected to large-scale parallel
sequencing and mapped to the genome. We show that peaks of promoter-proximal
polymerase reside on approximately 30% of human genes, transcription extends
beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent.
Additionally, most promoters have an engaged polymerase upstream and in an
orientation opposite to the annotated gene. This divergent polymerase is
associated with active genes but does not elongate effectively beyond the
promoter. These results imply that the interplay between polymerases and
regulators over broad promoter regions dictates the orientation and efficiency
of productive transcription. 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. We have previously demonstrated that the KH-domain protein αCP binds to a 3'
untranslated region (3'UTR) C-rich motif of the nascent human alpha-globin
(hα-globin) transcript and enhances the efficiency of 3' processing. Here we
assess the genome-wide impact of αCP RNA-protein (RNP) complexes on 3'
processing with a specific focus on its role in alternative polyadenylation
(APA) site utilization. The major isoforms of αCP were acutely depleted from a
human hematopoietic cell line, and the impact on mRNA representation and poly(A)
site utilization was determined by direct RNA sequencing (DRS). Bioinformatic
analysis revealed 357 significant alterations in poly(A) site utilization that
could be specifically linked to the αCP depletion. These APA events correlated
strongly with the presence of C-rich sequences in close proximity to the
impacted poly(A) addition sites. The most significant linkage was the presence
of a C-rich motif within a window 30 to 40 bases 5' to poly(A) signals (AAUAAA)
that were repressed upon αCP depletion. This linkage is consistent with a
general role for αCPs as enhancers of 3' processing. These findings predict a
role for αCPs in posttranscriptional control pathways that can alter the coding
potential and/or levels of expression of subsets of mRNAs in the mammalian
transcriptome. Monitoring protein synthesis is essential to our understanding of gene
expression regulation, as protein abundance is thought to be predomitly
controlled at the level of translation. Mass-spectrometric and RNA sequencing
methods have been recently developed for investigating mRNA translation at a
global level, but these still involve technical limitations and are not widely
applicable. In this study, we describe a novel system-wide proteomic approach
for direct monitoring of translation, termed puromycin-associated nascent chain
proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin
into newly synthesized proteins under cell-free conditions followed by
streptavidin affinity purification and liquid chromatography-tandem mass
spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific
fluctuations in synthesis for >5000 proteins in mammalian cells, identified
proteins not previously implicated in cell cycle processes, and generated the
first translational profile of a whole mouse brain. This simple and economical
technique is broadly applicable to any cell type and tissue, enabling the
identification and quantification of rapid proteome responses under various
biological conditions. A technique is described to directly measure ongoing transcription from
individual genes in permeabilized cells of either the budding yeast
Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe.
Transcription run-on (TRO) analysis is used to compare the relative rates of
synthesis for specific transcripts in cells grown under different environmental
conditions or harvested at different stages of development. As the amount of an
individual RNA species present at any given time is determined by its net rate
of synthesis and degradation, an accurate picture of transcription per se can be
obtained only by directly measuring de novo synthesis of RNA (if you are
interested in RNA degradation, see Method for measuring mRNA decay rate in
Saccharomyces cerevisiae). Most techniques employed to measure changes in the
relative levels of individual transcripts present under different conditions,
including Northern analysis (see Northern blotting), RT-PCR (see
Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory
Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray
analysis and high throughput RNA sequencing, measure changes in the steady-state
level of a transcript, which may or may not reflect the actual changes in
transcription of the gene. Recent studies carried out in fission yeast have
demonstrated that increases in the steady-state level (accumulation) of many
individual mRNAs occur without any significant changes in transcription rates
(McPheeters et al., 2009), highlighting the important role of regulated RNA
stability in determining gene expression programs (Harigaya et al., 2006). Dosage compensation in Drosophila is mediated by the MSL complex, which
increases male X-linked gene expression approximately 2-fold. The MSL complex
preferentially binds the bodies of active genes on the male X, depositing
H4K16ac with a 3' bias. Two models have been proposed for the influence of the
MSL complex on transcription: one based on promoter recruitment of RNA
polymerase II (Pol II), and a second featuring enhanced transcriptional
elongation. Here, we utilize nascent RNA sequencing to document dosage
compensation during transcriptional elongation. We also compare X and autosomes
from published data on paused and elongating polymerase in order to assess the
role of Pol II recruitment. Our results support a model for differentially
regulated elongation, starting with release from 5' pausing and increasing
through X-linked gene bodies. Our results highlight facilitated transcriptional
elongation as a key mechanism for the coordinated regulation of a diverse set of
genes. |
Do RNA:DNA hybrids preferentially form in high or low GC regions? | Transcription through a GC-rich region favors R-loop formation, with the nascent (G-rich) RNA forming a stable RNA:DNA hybrid with the template DNA strand. | Cloned human apo-C-II cDNA was used as a hybridization probe to identify the
human apo-C-II gene in a genomic library constructed in our laboratory. The
isolated apo-C-II DNA was studied both by electron microscopy and by direct
sequence analysis. Ultrastructural morphological analysis of RNA-DNA hybrids
revealed that the apo-C-II gene had complex structures because of regions of
inverted complementary sequences in and around the gene forming stem-and-loop
structures which interfere with the formation of stable RNA:DNA hybrids.
Extensive morphological analysis revealed a minimum of 3 intervening sequences
(IVS), and their lengths were measured. Direct sequence analysis of the cloned
gene confirmed the presence of 3 IVS. There are 4 Alu type sequences in IVS-I.
We sequenced 4340 nucleotides which include 545 nucleotides in the 5' flanking
region, the entire gene which spans 3320 nucleotides, and 475 nucleotides in the
3' flanking region which also encompasses an additional Alu sequence. The 5' end
of the gene was identified by primer extension and sequencing of the primer
extended cDNA. Apo-C-II mRNA structure was deduced from the cDNA sequence, the
primer extension experiments, and the genomic sequence. It is 494 nucleotides in
length. Its sequence differs from previously published sequences in that there
are 7 additional nucleotides before the polyadenylate tail. In the 5' flanking
region, nucleotides -234 to -213 encompass a GC-rich region which exhibits high
homology (greater than 70%) to the 5' flanking regions of the genes of all the
apolipoproteins published to date, namely, apo-A-II (-497 to -471), apo-A-I
(approximately -196 to -179), apo-E (-409 to -391), and apo-C-III (approximately
-116 to -103). This highly conserved region might represent some evolutionarily
conserved sequences from these related genes and/or might represent a region
with regulatory function. Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences
coding for a GC-rich RNA hairpin followed by a U-rich tract. We used
single-molecule techniques to investigate the mechanism by which three
representative terminators (his, t500, and tR2) destabilize the elongation
complex (EC). For his and tR2 terminators, loads exerted to bias translocation
did not affect termination efficiency (TE). However, the force-dependent
kinetics of release and the force-dependent TE of a mutant imply a forward
translocation mechanism for the t500 terminator. Tension on isolated U-tracts
induced transcript release in a manner consistent with RNA:DNA hybrid shearing.
We deduce that different mechanisms, involving hypertranslocation or shearing,
operate at terminators with different U-tracts. Tension applied to RNA at
terminators suggests that closure of the final 2-3 hairpin bases destabilizes
the hybrid and that competing RNA structures modulate TE. We propose a
quantitative, energetic model that predicts the behavior for these terminators
and mutant variants. Expansion of tandem repeat sequences is responsible for more than 20 human
diseases. Several cis elements and trans factors involved in repeat instability
(expansion and contraction) have been identified. However no comprehensive model
explaining large intergenerational or somatic changes of the length of the
repeating sequences exists. Several lines of evidence, accumulated from
different model studies, indicate that transcription through repeat sequences is
an important factor promoting their instability. The persistent interaction
between transcription template DNA and nascent RNA (RNA•DNA hybrids, R loops)
was shown to stimulate genomic instability. Recently, we demonstrated that
cotranscriptional RNA•DNA hybrids are preferentially formed at GC-rich
trinucleotide and tetranucleotide repeat sequences in vitro as well as in human
genomic DNA. Additionally, we showed that cotranscriptional formation of RNA•DNA
hybrids at CTG•CAG and GAA•TTC repeats stimulate instability of these sequences
in both E. coli and human cells. Our results suggest that persistent RNA•DNA
hybrids may also be responsible for other downstream effects of expanded
trinucleotide repeats, including gene silencing. Considering the extent of
transcription through the human genome as well as the abundance of GC-rich
and/or non-canonical DNA structure forming tandem repeats, RNA•DNA hybrids may
represent a common mutagenic conformation. Hence, R loops are potentially
attractive therapeutic target in diseases associated with genomic instability. Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs)
encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract,
and it has been proposed that steric clash of the stem-loop with the exit pore
of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA
hybrid, resulting in misalignment of the active site. The structurally unrelated
T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common
mechanism for termination. In the absence of a hairpin (passive conditions), T7
RNAP slips efficiently in both homopolymeric A and U tracts, and we have found
that replacement of the U tract in TΦ with a slippage-prone A tract still allows
efficient termination. Under passive conditions, incorporation of a single G
residue following a poly(U) tract (which is the situation during termination at
TΦ) results in a "locked" complex that is unable to extend the transcript. Our
results support a model in which transmission of the shearing force generated by
steric clash of the hairpin with the exit pore is promoted by the presence of a
slippery tracts downstream, resulting in alterations in the active site and the
formation of a locked complex that represents an early step in the termination
pathway. |
Which are the most commonly reported pathological states associated with the formation of DNA G-quadruplexes? | There is a growing recognition for the profound role of G-quadruplexes in a wide spectrum of diseases, such as cancer, diabetes and cardiovascular disease. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) were recently shown to be caused by expansion of a (GGGGCC)n/(GGCCCC)n repeat in the C9ORF72 gene. Treatment with a G-quadruplex interactive ligand was shown to achieve antifibrotic action. G-quadruplex forming sequences have also been linked with ADAM10 a primary candidate for anti-amyloidogenic activity in Alzheimer's. A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia. | Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic
chromosomes, is up-regulated in the vast majority of human neoplasias but not in
normal somatic tissues. Therefore, the telomerase complex represents a promising
universal therapeutic target in cancer. Telomeric G-rich single-stranded DNA can
adopt in vitro an intramolecular quadruplex structure, which has been shown to
inhibit telomerase activity. We examined G-quadruplex interactive agent,
telomestatin (SOT-095), for its ability to inhibit the proliferation of human
leukemia cells, including freshly obtained leukemia cells. Telomere length was
determined by either the terminal restriction fragment method or flow-FISH, and
apoptosis was assessed by flow cytometry. Moreover, chemosensitivity was
examined in telomestatin-treated U937 cells before ultimate telomere shortening.
Treatment with telomestatin reproducibly inhibited telomerase activity in U937
and NB4 cells followed by telomere shortening. Enhanced chemosensitivity toward
daunorubicin and cytosine-arabinoside was observed in telomestatin-treated U937
cells, before ultimate telomere shortening. Telomere shortening associated with
apoptosis by telomestatin was evident in some freshly obtained leukemia cells
from acute myeloid leukemia patients, regardless of sub-types of AML and
post-myelodysplasia AML. These results suggest that disruption of telomere
maintece by telomestatin limits the cellular lifespan of AML cells, as well.
However, in a minority of AML patients apoptosis was not evident, thus
indicating that resistant mechanism might exist in some freshly obtained AML
cells. Therefore, further investigation of telomestatin as a therapeutic agent
is warranted. The telomerase complex is responsible for telomere maintece and represents a
promising neoplasia therapeutic target. Recently, we have demonstrated that
treatment with a G-quadruplex-interactive agent, telomestatin reproducibly
inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines. In
the present study, we investigated the mechanisms of apoptosis induced by
telomerase inhibition in acute leukemia. We have found the activation of
caspase-3 and poly-(ADP-ribose) polymerase in telomestatin-treated U937 cells
(PD20) and domit-negative DN-hTERT-expressing U937 cells (PD25). Activation
of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in
telomestatin-treated U937 cells (PD20) and domit-negative DN-hTERT-expressing
U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these
cells. To examine the effect of p38 MAP kinase inhibition on growth properties
and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing
U937 cells with or without SB203580. Domit-negative-hTERT-expressing U937
cells stopped proliferation on PD25; however, a significant increase in growth
rate was observed in the presence of SB203580. Treatment of SB203580 also
reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25).
These results suggest that p38 MAP kinase has a critical role for the induction
of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the
effect of telomestatin on the growth of U937 cells in xenograft mouse model.
Systemic intraperitoneal administration of telomestatin in U937 xenografts
decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from
telomestatin-treated animals exhibited marked apoptosis. None of the mice
treated with telomestatin displayed any signs of toxicity. Taken together, these
results lay the foundations for a program of drug development to achieve the
dual aims of efficacy and selectivity in vivo. FANCJ mutations are associated with breast cancer and genetically linked to the
bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant
cells suggests that FANCJ helicase functions in the replicational stress
response. A putative helicase with sequence similarity to FANCJ in
Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract
maintece, suggesting its involvement in the resolution of alternate DNA
structures that impede replication. Under physiological conditions, guanine-rich
sequences spontaneously assemble into four-stranded structures (G quadruplexes
[G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an
ATPase-dependent manner. FANCJ G4 unwinding is specific since another
superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under
conditions in which the helicase unwound duplex DNA. Replication protein A
stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6
inhibited this activity. FANCJ-depleted cells treated with the G4-interactive
compound telomestatin displayed impaired proliferation and elevated levels of
apoptosis and DNA damage compared to small interfering RNA control cells,
suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA
pathway has been classically described in terms of interstrand cross-link (ICL)
repair, the cellular defects associated with FANCJ mutation extend beyond the
reduced ability to repair ICLs and involve other types of DNA structural
roadblocks to replication. Anti-amyloidogenic processing of the amyloid precursor protein APP by
α-secretase prevents formation of the amyloid-β peptide, which accumulates in
senile plaques of Alzheimer disease patients. α-Secretase belongs to the family
of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary
candidate for this anti-amyloidogenic activity. We recently demonstrated that
ADAM10 translation is repressed by its 5'-UTR and that in particular the first
half of ADAM10 5'-UTR is responsible for translational repression. Here, we
asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able
to form complex secondary structures and thus potentially inhibit ADAM10
translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich
region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable,
intramolecular, parallel G-quadruplex secondary structure under physiological
conditions. Mutation of guanines in this sequence abrogates the formation of the
G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits
translation of a luciferase reporter in in vitro translation assays and in
living cells, inhibition of G-quadruplex formation fails to do so. Moreover,
expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of
the G-quadruplex motif results in a significant increase of ADAM10 levels and
consequently APPsα secretion. Thus, we identified a critical RNA secondary
structure within the 5'-UTR, which contributes to the translational repression
of ADAM10. β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the
transmembrane aspartyl protease that catalyzes the first cleavage step in the
proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the
pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative
splicing, the regulation of which is not well understood. We identified a G-rich
sequence within exon 3 of BACE1 involved in controlling splice site selection.
Mutation of the G-rich sequence decreased use of the normal 5' splice site of
exon 3, which leads to full-length and proteolytically active BACE1, and
increased use of an alternative splice site, which leads to a shorter,
essentially inactive isoform. Nuclease protection assays, nuclear magnetic
resoce, and circular dichroism spectroscopy revealed that this sequence folds
into a G-quadruplex structure. Several proteins were identified as capable of
binding to the G-rich sequence, and one of these, heterogeneous nuclear
ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and
in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear
ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as
well as a decrease in Aβ production from APP, suggesting new possibilities for
therapeutic approaches to Alzheimer's disease. Large expansions of a non-coding GGGGCC-repeat in the first intron of the
C9orf72 gene are a common cause of both amyotrophic lateral sclerosis (ALS) and
frontotemporal dementia (FTD). G-rich sequences have a propensity for forming
highly stable quadruplex structures in both RNA and DNA termed G-quadruplexes.
G-quadruplexes have been shown to be involved in a range of processes including
telomere stability and RNA transcription, splicing, translation and transport.
Here we show using NMR and CD spectroscopy that the C9orf72 hexanucleotide
expansion can form a stable G-quadruplex, which has profound implications for
disease mechanism in ALS and FTD. |
Are shadow enhancers associated with development? | Yes. Critical developmental control genes sometimes contain shadow enhancers that can be located in remote positions, including the introns of neighboring genes | Genes include cis-regulatory regions that contain transcriptional enhancers.
Recent reports have shown that developmental genes often possess multiple
discrete enhancer modules that drive transcription in similar spatio-temporal
patterns: primary enhancers located near the basal promoter and secondary, or
'shadow', enhancers located at more remote positions. It has been proposed that
the seemingly redundant activity of primary and secondary enhancers contributes
to phenotypic robustness. We tested this hypothesis by generating a deficiency
that removes two newly discovered enhancers of shavenbaby (svb, a transcript of
the ovo locus), a gene encoding a transcription factor that directs development
of Drosophila larval trichomes. At optimal temperatures for embryonic
development, this deficiency causes minor defects in trichome patterning. In
embryos that develop at both low and high extreme temperatures, however, absence
of these secondary enhancers leads to extensive loss of trichomes. These
temperature-dependent defects can be rescued by a transgene carrying a secondary
enhancer driving transcription of the svb cDNA. Finally, removal of one copy of
wingless, a gene required for normal trichome patterning, causes a similar loss
of trichomes only in flies lacking the secondary enhancers. These results
support the hypothesis that secondary enhancers contribute to phenotypic
robustness in the face of environmental and genetic variability. Critical developmental control genes sometimes contain "shadow" enhancers that
can be located in remote positions, including the introns of neighboring genes
[1]. They nonetheless produce patterns of gene expression that are the same as
or similar to those produced by more proximal primary enhancers. It was
suggested that shadow enhancers help foster robustness in gene expression in
response to environmental or genetic perturbations [2, 3]. We critically tested
this hypothesis by employing a combination of bacterial artificial chromosome
(BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence
is presented that the snail gene is regulated by a distal shadow enhancer
located within a neighboring locus. Removal of the proximal primary enhancer
does not significantly perturb snail function, including the repression of
neurogenic genes and formation of the ventral furrow during gastrulation at
normal temperatures. However, at elevated temperatures, there is sporadic loss
of snail expression and coincident disruptions in gastrulation. Similar defects
are observed at normal temperatures upon reductions in the levels of Dorsal, a
key activator of snail expression (reviewed in [5]). These results suggest that
shadow enhancers represent a novel mechanism of canalization whereby complex
developmental processes "bring about one definite end-result regardless of minor
variations in conditions" [6]. The products of Hox genes function in assigning positional identity along the
anterior-posterior body axis during animal development. In mouse embryos, Hox
genes located at the 3' end of HoxA and HoxB complexes are expressed in nested
patterns in the progenitors of the secondary heart field during early
cardiogenesis and the combined activities of both of these clusters are required
for proper looping of the heart. Using Hox bacterial artificial chromosomes
(BACs), transposon reporters, and transgenic analyses in mice, we present the
identification of several novel enhancers flanking the HoxB complex which can
work over a long range to mediate dynamic reporter expression in the endoderm
and embryonic heart during development. These enhancers respond to exogenously
added retinoic acid and we have identified two retinoic acid response elements
(RAREs) within these control modules that play a role in potentiating their
regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these
control modules, spread throughout the flanking intergenic region, have
regulatory activities that overlap with other local enhancers. This suggests
that they function as shadow enhancers to modulate the expression of genes from
the HoxB complex during cardiac development. Regulatory analysis of the HoxA
complex reveals that it also has enhancers in the 3' flanking region which
contain RAREs and have the potential to modulate expression in endoderm and
heart tissues. Together, the similarities in their location, enhancer output,
and dependence on retinoid signaling suggest that a conserved cis-regulatory
cassette located in the 3' proximal regions adjacent to the HoxA and HoxB
complexes evolved to modulate Hox gene expression during mammalian cardiac and
endoderm development. This suggests a common regulatory mechanism, whereby the
conserved control modules act over a long range on multiple Hox genes to
generate nested patterns of HoxA and HoxB expression during cardiogenesis. |
Does prudent diet reduce cardiovascular risk? | a high adherence to prudent diet is associated with reduced risk of CVD. The adherence to prudent diet was associated to a 28% lower risk of cardiovascular mortality and a 17% lower risk of all-cause mortality in a large cohort of healthy women | BACKGROUND: Although substantial information on individual nutrients or foods
and risk of coronary heart disease (CHD) is available, little is known about the
role of overall eating pattern.
METHODS: Using dietary information from a food frequency questionnaire in 1984
from the Nurses' Health Study, we conducted factor analysis and identified 2
major dietary patterns-"prudent" and "Western"-and calculated factor scores of
each pattern for individuals in the cohort. We used logistic regression to
examine prospectively the associations between dietary patterns and CHD risk
among 69 017 women aged 38 to 63 years in 1984 without history of major chronic
diseases.
RESULTS: The prudent pattern was characterized by higher intakes of fruits,
vegetables, legumes, fish, poultry, and whole grains, while the Western pattern
was characterized by higher intakes of red and processed meats, sweets and
desserts, french fries, and refined grains. Between 1984 and 1996, we documented
821 CHD cases. After adjusting for coronary risk factors, the prudent diet score
was associated with a relative risk (RR) of 0.76 (95% confidence interval (CI),
0.60-0.98; P for trend test,.03) comparing the highest with lowest quintile.
Extreme quintile comparison yielded an RR of 1.46 (95% CI, 1.07-1.99; P for
trend test,.02) for the Western pattern. Those who were jointly in the highest
prudent diet quintile and lowest Western diet quintile had an RR of 0.64 (95%
CI, 0.44-0.92) compared with those with the opposite pattern profile.
CONCLUSION: A diet high in fruits, vegetables, whole grains, legumes, poultry,
and fish and low in refined grains, potatoes, and red and processed meats may
lower risk of CHD. Cardiovascular diseases (CVD) are growing contributors to global disease
burdens, with epidemics of CVD advancing across many regions of the world which
are experiencing a rapid health transition. Diet and nutrition have been
extensively investigated as risk factors for major cardiovascular diseases like
coronary heart disease (CHD) and stroke and are also linked to other
cardiovascular risk factors like diabetes, high blood pressure and obesity. The
interpretation of evidence needs to involve a critical appraisal of
methodological issues related to measurement of exposures, nature of outcome
variables, types of research design and careful separation of cause, consequence
and confounding as the basis for observed associations. Adequate evidence is
available, from studies conducted within and across populations, to link several
nutrients, minerals, food groups and dietary patterns with an increased or
decreased risk of CVD. Dietary fats associated with an increased risk of CHD
include trans-fats and saturated fats, while polyunsaturated fats are known to
be protective. Dietary sodium is associated with elevation of blood pressure,
while dietary potassium lowers the risk of hypertension and stroke. Regular
frequent intake of fruits and vegetables is protective against hypertension, CHD
and stroke. Composite diets (such as DASH diets, Mediterranean diet, 'prudent'
diet) have been demonstrated to reduce the risk of hypertension and CHD.
Sufficient knowledge exists to recommend nutritional interventions, at both
population and individual levels, to reduce cardiovascular risk. That knowledge
should now be translated into policies which promote healthy diets and
discourage unhealthy diets. This requires coordinated action at the level of
governments, international organizations, civil society and responsible sections
of the food industry. Cardiovascular disease (CVD) accounts for almost 50% of all deaths in
industrialized nations. As much as 70% of CVD can be prevented or delayed with
dietary choices and lifestyle modifications. Western-style diets, sedentary
lifestyles, and cigarette smoking are key modifiable CVD risk factors. Although
CVD mortality was trending downward for almost 50 years, a resurgence, both
nationally and globally, has occurred. A growing epidemic of obesity
("globesity"), decreasing physical activity, and persistent cigarette smoking
are major behavioral factors underlying this change. Diet and lifestyle increase
CVD risk both directly and indirectly. Direct effects include biological,
molecular, and physiologic alterations, including inflammatory stimuli and
oxidative stresses. Indirect effects include diabetes, dyslipidemias, and
hypertension. However, trials studying links between diet and CVD remain
notoriously difficult to execute and interpret. Diet interventions are typically
confounded by other aspects of an overall diet as well as by lifestyle.
Furthermore, benefits derived from a specific dietary or lifestyle intervention
may not be proportional to the degree of risk posed by the unhealthy diet or
lifestyle. Nonetheless, therapeutic rationale for diet and lifestyle are
supported by basic and clinical research. Key components of a healthy aggregate
diet include 1) reduced caloric intake; 2) reduced total fat, saturated fat,
trans fat, and cholesterol with proportional increases in monosaturated, n-3
(omega-3), and n-6 fatty acids; 3) increased dietary fiber, fruit, and
vegetables; 4) increased micronutrients (eg, folate, B6, B12); 5) increased
plant protein in lieu of animal protein; 6) reduced portions of highly processed
foods; and 7) adopting a more Mediterranean or "prudent" dietary pattern over
the prevailing "western" dietary pattern. Key lifestyle interventions include
increased physical activity and smoking cessation. Translation of the benefits
of healthy diet and lifestyle to the wider population requires both individual
and public health strategies targeting at-risk groups. BACKGROUND: Diet is a major modifiable risk factor for cardiovascular disease,
but it varies markedly in different regions of the world. The objectives of the
present study were to assess the association between dietary patterns and acute
myocardial infarction (AMI) globally.
METHODS AND RESULTS: INTERHEART is a standardized case-control study involving
participants from 52 countries. The present analysis included 5761 cases and 10
646 control subjects. We identified 3 major dietary patterns using factor
analysis: Oriental (high intake of tofu and soy and other sauces), Western (high
in fried foods, salty snacks, eggs, and meat), and prudent (high in fruit and
vegetables). We observed an inverse association between the prudent pattern and
AMI, with higher levels being protective. Compared with the first quartile, the
adjusted ORs were 0.78 (95% CI 0.69 to 0.88) for the second quartile, 0.66 (95%
CI 0.59 to 0.75) for the third, and 0.70 (95% CI 0.61 to 0.80) for the fourth (P
for trend <0.001). The Western pattern showed a U-shaped association with AMI
(compared with the first quartile, the adjusted OR for the second quartile was
0.87 [95% CI 0.78 to 0.98], whereas it was 1.12 [95% CI 1.00 to 1.25] for the
third quartile and 1.35 [95% CI 1.21 to 1.51] for the fourth quartile; P for
trend <0.001), but the Oriental pattern demonstrated no relationship with AMI.
Compared with the first quartile, the OR of a dietary risk score derived from
meat, salty snacks, fried foods, fruits, green leafy vegetables, cooked
vegetables, and other raw vegetables (higher score indicating a poorer diet)
increased with each quartile: second quartile 1.29 (95% CI 1.17 to 1.42), third
quartile 1.67 (95% CI 1.51 to 1.83), and fourth quartile 1.92 (95% CI 1.74 to
2.11; P for trend <0.001). The adjusted population-attributable risk of AMI for
the top 3 quartiles compared with the bottom quartile of the dietary risk score
was 30%.
CONCLUSIONS: An unhealthy dietary intake, assessed by a simple dietary risk
score, increases the risk of AMI globally and accounts for approximately 30% of
the population-attributable risk. BACKGROUND AND AIMS: Dietary habits have been associated with cardiovascular
disease (CVD) risk factors. This study aimed at evaluating the association of
non-predefined dietary patterns with CVD risk profile and C-reactive protein
(CRP).
METHODS AND RESULTS: We analyzed 7646 healthy subjects from the Moli-sani
project, an on-going cross-sectional cohort study of men and women aged >or=35,
randomly recruited from a general Italian population. The Italian EPIC food
frequency questionnaire was used. Food patterns were generated using principal
factor analysis (PFA) and reduced rank regression (RRR). Three dietary patterns
were identified by PFA. The "Olive Oil and Vegetables" pattern, characterized by
high intake of olive oil, vegetables, legumes, soups, fruits and fish, was
associated with relatively lower values of glucose, lipids, CRP, blood pressure
and individual global CVD risk score. The "Pasta and Meat" pattern,
characterized by high intake of pasta, tomato sauce, red meat, animal fats and
alcohol, was positively associated with glucose, lipids, CRP and CVD risk score.
The "Eggs and Sweets" pattern, characterized by positive loadings of eggs,
processed meat, margarines, butter, sugar and sweets, was associated with high
values of CRP. The first RRR pattern was similar to the "Pasta and Meat" pattern
both in composition and association with CVD risk profile.
CONCLUSIONS: In a large healthy Italian population, non-predefined dietary
patterns including foods considered to be rather unhealthy, were associated with
higher levels of cardiovascular risk factors, CRP and individual global CVD
risk, whereas a "prudent-healthy" pattern was associated with lower levels. Prudent dietary patterns are associated with reduced risk of arterial
cardiovascular diseases (CVD). Limited data exist on the relation between diet
and venous thromboembolism (VTE). The aim of our prospective, population based
study was to investigate the association of a heart healthy diet on risk of
myocardial infarction (MI) and VTE. Information on dietary habits was available
in 18,062 subjects, aged 25-69, who participated in the fourth Tromsø study,
1994-1995. Dietary patterns were assessed by a slightly modified version of the
validated SmartDiet score; a 13-item questionnaire producing a diet score based
on the intakes of fat, fibre, fruit and vegetables. Incident events of MI
(n=518) and VTE (n=172) were recorded to the end of follow-up December 31, 2005
(median follow-up 10.8 years). Cox-regression models were used to calculate
hazard ratios (HR). A healthy diet score of >27 points (upper tertile) was
associated with 17% reduced risk of MI (HR: 0.83, 95% confidence interval [CI]:
0.66-1.06), and no association with VTE (HR: 1.01; 95%CI: 0.66-1.56), compared
to <24 points (lower tertile) in multivariable analysis. High intake of fish,
fruit, vegetables and polyunsatured fat had a 23% reduced risk of MI (HR 0.77;
95%CI: 0.60-0.98), but no association with VTE (HR 0.95; 95%CI: 0.64-1.40). A
heart healthy diet showed an even more favourable association with MI in obese
subjects (HR: 0.62; 95%CI: 0.41-0.95), but not with VTE. Our findings suggest
that a heart healthy dietary pattern is associated with moderately reduced risk
of MI, but not related to risk of VTE. |
Is Crohn's disease (CD) linked to the consumption of refrigerated food? | All findings point to refrigeration as a potential risk factor for Crohn's disease. Environmental risk factors playing a causative role in Crohn's Disease (CD) remain largely unknown. Recently, it has been suggested that refrigerated food could be involved in disease development. Patients were exposed earlier than controls to the refrigerator (X2 = 9.9, df = 3, P = 0.04) and refrigerator exposure at birth was found to be a risk factor for CD (OR = 2.08 (95% CI: 1.01-4.29), P = 0.05). Comparable results were obtained looking for the exposure to freezer at home. | Mice were inoculated with human sarcoid tissue homogenates or with a first or a
second passage homogenate of mouse tissue (including 0.2 mum membrane filtrates)
originating from the inoculation of human sarcoid, Crohn's disease, or control
tissue homogenates. Epithelioid and giant cell granulomas were present in the
footpads and/or viscera of some of the mice given homogenates originating from
each sarcoid or Crohn's disease tissue 15 months after inoculation but were not
present in mice given control homogenates. Among mice given homogenates
originating from human sarcoidosis, granulomas were present in many organs and
tissues; in contrast, a pattern of selective dissemination of visceral
granulomas was found among mice given homogenates originating from Crohn's
disease. This differential distribution of visceral granulomas also followed the
inoculation of 0.2 mum membrane filtrates. Granulomatous responses at Kveim test
sites in the ear 9-17 months after inoculation of homogenatesoriginating from
human sarcoidosis or Crohn's disease were confined to mice showing granulomas in
footpads of viscera. The ability of the transmissible agents to induce
granulomas in mice was destroyed when sarcoid or Crohn's tissues were autoclaved
or when sarcoid homogenates were stored at -20degreesC for 1 week or exposed to
60Co irradiation (2.5 MR). Crohn's disease is the result of an abnormal immune response of the gut mucosa
triggered by one or more environmental risk factors in people with predisposing
gene variations, including CARD15 mutations. Epidemiological data allow
assessment of familial environmental risk factors related to western lifestyle,
diet, bacteria, and domestic hygiene. All findings point to refrigeration as a
potential risk factor for Crohn's disease. Furthermore, cold-chain development
paralleled the outbreak of Crohn's disease during the 20th century. The cold
chain hypothesis suggests that psychrotrophic bacteria such as Yersinia spp and
Listeria spp contribute to the disease. These bacteria have been identified in
Crohn's disease lesions and we discuss their pathogenic properties with respect
to our knowledge of the disease. From a molecular perspective, we postulate that
the disease is a result of a defect in host recognition by pathogenic bacterial
components that usually escape the immune response (eg, Yop molecules), which
results in an excessive host response to these bacteria. A recent published hypothesis proposed that Crohn's disease was provoked by
infantile exposure to micro-organisms that can survive refrigerator temperature.
A case-control study was accordingly devised. The mean age at first fridge was
5.6 years amongst 88 patients with Crohn's disease, 5.5 years in 88 patients
with ulcerative colitis (UC) and 7.6 years in 88 controls, but a majority of
individuals had always been exposed to refrigerated food. Differences were more
striking in subjects aged above the median (10.3, 10.9 and 15.0 years for
Crohn's disease, UC and controls, respectively). This support for the hypothesis
reached statistical significance for those with Crohn's disease compared to the
controls (p=0.045). BACKGROUND: Environmental risk factors playing a causative role in Crohn's
Disease (CD) remain largely unknown. Recently, it has been suggested that
refrigerated food could be involved in disease development. We thus conducted a
pilot case control study to explore the association of CD with the exposure to
domestic refrigeration in childhood.
METHODOLOGY/PRINCIPAL FINDINGS: Using a standard questionnaire we interviewed
199 CD cases and 207 age-matched patients with irritable bowel syndrome (IBS) as
controls. Cases and controls were followed by the same gastroenterologists of
tertiary referral clinics in Tehran, Iran. The questionnaire focused on the date
of the first acquisition of home refrigerator and freezer. Data were analysed by
a multivariate logistic model. The current age was in average 34 years in CD
cases and the percentage of females in the case and control groups were
respectively 48.3% and 63.7%. Patients were exposed earlier than controls to the
refrigerator (X2 = 9.9, df = 3, P = 0.04) and refrigerator exposure at birth was
found to be a risk factor for CD (OR = 2.08 (95% CI: 1.01-4.29), P = 0.05).
Comparable results were obtained looking for the exposure to freezer at home.
Finally, among the other recorded items reflecting the hygiene and comfort at
home, we also found personal television, car and washing machine associated with
CD.
CONCLUSION: This study supports the opinion that CD is associated with exposure
to domestic refrigeration, among other household factors, during childhood. |
Which drugs are included in TAS-102? | TAS-102 is a novel oral nucleoside antitumor agent consisting of trifluridine and tipiracil hydrochloride at a molar ratio of 1:0.5. | TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD)
and tipiracil hydrochloride (TPI). The compound improves overall survival of
colorectal cancer (CRC) patients who are insensitive to standard chemotherapies.
FTD possesses direct antitumor activity since it inhibits thymidylate synthase
(TS) and is itself incorporated into DNA. However, the precise mechanisms
underlying the incorporation into DNA and the inhibition of TS remain unclear.
We found that FTD-dependent inhibition of TS was similar to that elicited by
fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However,
washout experiments revealed that FTD-dependent inhibition of TS declined
rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was
significantly higher than that of other antitumor nucleosides. Additionally,
orally administered FTD had increased antitumor activity and was incorporated
into DNA more effectively than continuously infused FTD. When TAS-102 was
administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent
manner, and significantly delayed tumor growth and prolonged survival, compared
to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell
fraction, and swollen nuclei were observed in treated tumor tissue. The amount
of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft
models were positively and significantly correlated. These results suggest that
TAS-102 exerts its antitumor activity predomitly due to its DNA
incorporation, rather than as a result of TS inhibition. The persistence of FTD
in the DNA of tumor cells treated with TAS-102 may underlie its ability to
prolong survival in cancer patients. INTRODUCTION: Intensive laboratory, preclinical and clinical studies have
identified and validated molecular targets in cancers, leading to a shift toward
the development of novel, rationally designed and specific therapeutic agents.
However, gastrointestinal cancers continue to have a poor prognosis, largely due
to drug resistance.
AREAS COVERED: Here, we discuss the current understanding of DNA synthesis
inhibitors and their mechanisms of action for the treatment of gastrointestinal
maligcies.
EXPERT OPINION: Conventional agents, including DNA synthesis inhibitors such as
fluoropyrimidines and platinum analogs, remain the most effective therapeutics
and are the standards against which new drugs are compared. Novel DNA synthesis
inhibitors for the treatment of gastrointestinal maligcies include a
combination of the antimetabolite TAS-102, which consists of trifluorothymidine
with a thymidine phosphorylase inhibitor, and a novel micellar formulation of
cisplatin NC-6004 that uses a otechnology-based drug delivery system. The
challenges of translational cancer research using DNA synthesis inhibitors
include the identification of drugs that are specific to tumor cells to reduce
toxicity and increase antitumor efficacy, biomarkers to predict pharmacological
responses to chemotherapeutic drugs, identification of ways to overcome drug
resistance and development of novel combination therapies with DNA synthesis
inhibitors and other cancer therapies, such as targeted molecular therapeutics.
Here, we discuss the current understanding of DNA synthesis inhibitors and their
mechanisms of action for the treatment of gastrointestinal maligcies. TAS-102 is a novel oral nucleoside antitumor agent consisting of trifluridine
and tipiracil hydrochloride at a molar ratio of 1:0.5. TAS-102 was approved in
Japan in March 2014 for the treatment of patients with unresectable, advanced or
recurrent colorectal cancer that is refractory to standard therapies. In the
present study, enhancement of the therapeutic efficacy using a combination
therapy of TAS-102 and irinotecan hydrochloride (CPT-11) was evaluated in a
colorectal and gastric cancer xenograft-bearing nude mouse model. TAS-102 was
orally administered twice a day from day 1 to 14, and CPT-11 was administered
intravenously on days 1 and 8. The growth-inhibitory activity was evaluated
based on the tumor volume and the growth-delay period, which was estimated based
on the period required to reach a tumor volume that was five-times greater than
the initial volume (RTV5). The tumor growth-inhibitory activity and the RTV5 of
the group receiving TAS-102 with CPT-11 were significantly superior to those of
both agents as monotherapy for mice with KM12C, KM12C/5-FU, DLD-1/5-FU, and SC-2
xenografts (p<0.01). No significant decrease in body weight was observed. The
present pre-clinical findings indicated that the combination of TAS-102 and
CPT-11 is a promising treatment option for colorectal or gastric cancer, not
only for chemo-naïve tumors, but also for recurrent tumors after 5-fluorouracil
(5-FU)-based chemotherapy. PURPOSE: TAS-102 is an orally administered anticancer agent composed of
α,α,α-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). This
study assessed 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake after
TAS-102 administration.
METHODS: The human colorectal carcinoma cell lines HCT116, HT29, HCT8 and SW620
were exposed to FTD for 2 h, further incubated for 0, 2 and 24 h, and assayed
for [(3)H]FLT uptake, nucleoside transport, thymidine kinase 1 (TK1) expression
and TK1 activity. Static and 2-h dynamic [(18)F]FLT positron emission tomography
(PET) was performed in mice bearing HT29 or SW620 tumours orally administered
with vehicle or TAS-102.
RESULTS: FTD decreased the viability of all cell lines, whereas increased
[(3)H]FLT uptake (P < 0.05). Increased nucleoside transport and/or TK1
expression were observed 24 h after FTD, but not in 0-2 h. Static [(18)F]FLT PET
in mice bearing HT29 tumours showed accumulation of [(18)F]FLT in tumours 1 h
(day 1) after TAS-102. Two-hour dynamic PET in mice bearing SW620 tumours showed
increased influx constant and volume of distribution of phosphorylated
[(18)F]FLT on days 1 and 8 (P < 0.05) after TAS-102 with decreased
dephosphorylation on day 1 (P < 0.001). Ex vivo studies showed that SW620
tumours after TAS-102 had higher TK1 expression than those with vehicle on days
8 and 15.
CONCLUSION: TAS-102 administration induces an increase in [(18)F]FLT uptake.
Mechanisms may involve decreased dephosphorylation of [(18)F]FLT phosphate early
after TAS-102 administration. Increased TK1 expression and/or nucleoside
transporter may be related to increased [(18)F]FLT uptake at a later time.
[(18)F]FLT PET has a potential to assess the pharmacodynamics of TAS-102 in
cancer patients. PURPOSE: Trifluridine (TFT) is an antitumor component of a novel nucleoside
antitumor agent, TAS-102, which consists of TFT and tipiracil hydrochloride
(thymidine phosphorylase inhibitor). Incorporation of TFT into DNA is a probable
mechanism of antitumor activity and hematological toxicity. The objective of
this study was to examine the TFT incorporation into tumor- and white blood
cell-DNA, and to elucidate the mechanism of TFT-related effect and toxicity. TFT
effect on the colony formation of mouse bone marrow cells was also investigated.
METHODS: Pharmacokinetics of TFT was determined in nude mice after single oral
administration of TAS-102, while the antitumor activity and body weight change
were evaluated in the tumor-bearing nude mice after multiple oral
administrations for 2 weeks. TFT concentrations in the blood- and tumor-DNA were
determined by LC/MS/MS. The colony formation was evaluated by CFU-GM assay.
RESULTS: TFT systemic exposure in plasma increased dose-dependently. The tumor
growth rate and body weight gain decreased dose-dependently, but TFT
concentrations in the DNA of tumor tissues and white blood cells increased
dose-dependently. TFT inhibited colony formation of bone marrow cells in a
concentration-dependent manner.
CONCLUSIONS: A significant relationship between systemic exposure of TFT and
pharmacological effects including the antitumor activity and body weight change
was well explained by the TFT incorporation into DNA. TFT inhibited
proliferations of mouse bone marrow cells and human colorectal carcinoma cells
implanted to nude mice dose-dependently. The highest tolerable TFT exposure
provides the highest antitumor activity, and the hematological toxicity may
serve as a potential surrogate indicator of TAS-102 efficacy. Fluoropyrimidines form the mainstay in treatment of gastrointestinal
maligcies. For decades 5-fluorouracil (5FU), was the major fluoropyrimidine.
Currently it is usually given in a combination with leucovorin and oxaliplatin,
i.e. FOLFOX, or irinotecan, i.e. FOLFIRI, or all three, i.e. FOLFIRINOX, but
gradually it has been replaced by oral fluoropyrimidine prodrug formulations,
such as tegafur-uracil and S-1 (both contain ftorafur), and capecitabine
(Xeloda®). Novel drugs such as the antivascular endothelial growth factor
antibody, bevacizumab, and the anti-epidermal growth factor receptor antibody,
cetuximab, are often combined with one of these treatment options. However, when
resistance emerged, no alternatives were available. TAS-102, a combination of
trifluorothymidine and the thymidine phosphorylase inhibitor TPI in a 1:0.5
ratio, is a novel oral formulation, which is active in 5FU-resistant models,
both in vitro and in xenograft models. In addition to inhibition of thymidylate
synthase, the major mechanism of action of classical fluoropyrimidines,
TAS-102's major mechanism of action is incorporation into DNA, thereby causing
DNA damage. TAS-102 also follows an alternative activation pathway via thymidine
kinase, and is not a substrate for dihydropyrimidine dehydrogenase. All together
this explains the efficacy in 5FU-resistant models. In early clinical studies,
the twice-daily schedule (5 days on, 2 days rest) for 2 weeks every 4 weeks, led
to a significant disease control rate in various maligcies. This schedule
showed consistent activity in two randomized trials on fluoropyrimidine
refractory colorectal cancer patients, reflected by an increase of 2-3 months in
overall survival in the TAS-102 group compared with placebo. Considering the
impressive preclinical potential of various combinations TAS-102 has the promise
to become an alternative for 5FU-resistant cancer. |
What is the most probable defect underlying triple negative breast cancer? | The most probable defect underlying triple negative breast cancer is BRCA1 dysfunction. | BACKGROUND: We have previously reported an array comparative genomic
hybridization profile that identifies triple-negative breast cancers (TNBC),
with BRCA1 dysfunction and a high sensitivity to intensified dose bifunctional
alkylating agents. To determine the effect of conventional-dose chemotherapy in
patients with this so-called BRCA1-like profile, clinical characteristics and
survival were studied in a large group of TNBC patients.
PATIENTS AND METHODS: DNA was isolated and BRCA1-like status was assessed in 101
patients with early-stage TNBC receiving adjuvant cyclophosphamide-based
chemotherapy. Clinical characteristics and survival were compared between
BRCA1-like and non-BRCA1-like groups. Results Sixty-six tumors (65%) had a
BRCA1-like profile. Patients with BRCA1-like tumors tended to be younger and had
more often node-negative disease (P = 0.06 and P = 0.03, respectively).
Five-year recurrence-free survival was 80% for the BRCA1-like group and 75% for
the non-BRCA1-like group (P = 0.35). T stage was the only variable significantly
associated with survival.
CONCLUSIONS: BRCA1-like tumors share clinical features, like young age at
diagnosis and similar nodal status, with breast cancers in BRCA1 mutation
carriers. Their prognosis is similar to that of non-BRCA1-like tumors when
conventional-dose chemotherapy is administered. TNBCs that are classified as
BRCA1-like may contain a defect in homologous recombination and could, in
theory, benefit from the addition of poly ADP ribose polymerase inhibitors. |
What causes erucism? | Erucism is defined as urtication by Lepidoptera larvae. It is a skin reaction to envenomation from certain poisonous caterpillar bristles. The hair on the dorsum of the last instar larvae of the moth may cause urticarial reactions (erucism) as well as eye problems and temporary blindness. | A scanning electron microscope study has enabled an explanation as to why the
brown-tail moth provokes Lepidopterism. The brown-tail moth only provokes
Lepidopterism via a transmission of the urticating hairs of its caterpillar.
Urticating moths (genus Hylesia and Anaphae) protect their eggs and young
caterpillars with urticating hairs, thus it is very ambiguous to label erucism
as the contact dermatitis produced by caterpillar production or Lepidopterism as
the contact dermatitis caused by moth urticating hairs. Larvae of the caterpillar Thaumetopoea wilkinsoni are widely distributed in pine
groves throughout Israel. Erucism is defined as urtication by Lepidoptera
larvae. Both irritating effects on contact with skin and eyes and toxic effects
on ingestion have been described after exposure to several species of
Lepidoptera. We report the case of a 4-year-old child who vomited repeatedly and
developed symmetrical swelling of both hands after touching a larva of
Thaumetopoea wilkinsoni. Human poisoning by Lepidoptera (butterflies, caterpillars and moths) has been
known to physicians since old times. In the last few years, several factors have
been causative of a great number of contacts between different stages of these
insects and humans, occasionally with epidemic features. Species of medical
interest in Argentina are those related to Megalopygidae, Saturniidae,
Hemileucidae, Arctiidae, Notodentidae and Eucliedae families. Among all the
species involved, it is important to describe the pathology produced by contact
with Lonomia obliqua caterpillar (Saturniidae). The caterpillar Lonomia has
several enzymes with procoagulant and fibrinolytic activities, acting on various
components of the hemostatic system inducing hemorrhage that can lead to death.
In the South of Brazil during the last years caterpillars of this butterfly
caused a great number of cases of erucism including some deaths. In the past two
years, there have been some cases of poisoning by Lonomia obliqua in Misiones,
in the North East of Argentina on the border with Southern Brazil. This presents
a potential risk, taking into account the poor knowledge regarding this insect
and its venom in Argentina. This brief actualization gives some biochemical,
clinical and epidemiologic tools towards understanding human Lepidopterism, an
uncommon medical problem in this country. The pine processionary caterpillar Thaumetopoea pityocampa (Lepidoptera:
Notodontidae) is considered to be a serious pest of medical importance. The hair
on the dorsum of the last instar larvae of the moth may cause urticarial
reactions (erucism) as well as eye problems and temporary blindness. In Israel,
the pest occurs in all pine plantations as well as on ornamental pine trees in
urban areas. The biology, ecology and management of the moth population are
discussed as well as the mechanism of action of the urticarial hairs and their
medical significance. Awareness of the life cycle and ecology of the pest may
reduce the contact of the population with the urticarial hairs and prevent the
morbidity caused by it. We present a case of erucism (caterpillar dermatitis) in a British serviceman
deployed in Croatia on Operation Resolute. The aetiology, clinical features and
diagnosis of erucism are discussed. Erucism should be highlighted as an
environmental hazard in future troop deployments to the Mediterranean area. This is a case report of a 44-year-old male living in Teresópolis, RJ, Brazil,
probably poisoned by contact with a Lonomia caterpillar, who presented hemolytic
anemia, decreased platelet count and acute renal insufficiency. Lonomia erucism
diagnosis was established by anamnesis and clinical and laboratory
manifestations. Therapeutic measures consisting of hemotransfusion and
hemodialysis were successful. Physiopathologic and clinical features of erucism
by Lonomia are discussed. Lepidoptera (moths, butterflies, and caterpillars) are an uncommon cause of
adverse reactions in humans. Most reactions to Lepidoptera are mild and
self-limited; however, reactions in sensitive individuals and reactions to
particular species can be severe and life threatening. Specific syndromes caused
by Lepidoptera include erucism (cutaneous reactions from contact with
caterpillars, moths, or cocoons), lepidopterism (systemic involvement),
ophthalmia nodosa (ocular involvement), dendrolimiasis and pararamose (each with
joint symptoms relating to a specific species of caterpillar), lonomism (a
severe hemorrhagic disease related to Lonomia species), and seasonal ataxia
(related to ingestion of Anaphe venata). In most cases, reactions to Lepidoptera
can be treated symptomatically with prompt removal of offending hairs.
Antipruritic or anesthetic topical preparations, topical steroids, and oral
antihistamines are often used. In the case of potentially fatal Lonomia
envenomation, an effective antivenin has been manufactured. Although many tropical insects carry infectious diseases, cutaneous injury can
occur by other mechanisms, for example erucism (envenomation by caterpillars) or
lepidopterism (dermatitis from moths). Pararama is a unique form of erucism seen
in workers in contact with rubber trees in the Amazon, and it is caused by
Premolis larvae, resulting in progressive periarticular fibrosis, ankylosis, and
the loss of articulation. Ants and aquatic insects of the Belostomatidae family
can cause painful bites and stings. Anaphylactic shock and death can result from
the venom of bees and wasps. Beetles can cause vesicular dermatitis via
cantharidin or paederin. Myiasis results from fly larvae (maggots) feeding on
live or necrotic tissue of humans or other hosts, while New World screwworm fly
larvae feed only on living tissue and burrow (ie, screw) more deeply when
attempts are made to remove them. Tungiasis is characterized by very pruritic
and painful papules and ulcers resulting from a Tunga flea penetrating the
host's skin. Dermatologists should be able to diagnose and treat the cutaneous
manifestations of these tropical insects and educate their patients on
prevention. |
Subsets and Splits