Expanded CAG repeats activate the DNA damage checkpoint pathway

Trinucleotide repeats (TNRs) are sequences whose expansion causes several genetic diseases and chromosome breakage. We report a novel finding that expanded CAG repeats activate the DNA damage response. Mutations in yeast MEC1, RAD9, or RAD53 genes result in increased rates of fragility of a CAG repeat tract while single or double deletions of RAD17 or RAD24 have only a modest effect on TNR fragility, indicating that signaling down the Rad9 pathway and not the Rad17-Rad24 pathway plays a major role in sensing and repairing CAG-tract breaks. Deletion of CHK1 had no effect on CAG fragility, suggesting that a Chk1-mediated G2 arrest is not required for TNR repair. Absence of Mec1, Ddc2, Rad17, Rad24, or Rad53 also gives rise to increased frequency of CAG repeat contractions, indicating that components of the checkpoint machinery play an active role in the maintenance of both chromosomal integrity and repeat stability at expanded CAG sequences.     

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.     

Membrane biochips

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.     

Changes in free polyamines and related enzymes during stipule and pod wall development in Pisum sativum

Level of free polyamines, their key metabolic enzymes, and other features related to ageing were examined during stipule and pod wall development in pea (Pisum sativum). Free polyamine titre (per unit fresh mass) in both the organs, the specific activities of arginine decarboxylase and ornithine decarboxylase in the pod wall, gradually decreased with maturation. In stipule, these enzymes attained peak activity at 15 days after pod emergence and declined thereafter. Ornithine decarboxylase activity was greater in pod wall than in stipule; while, arginine decarboxylase activity was higher in stipule. Activity of degradative enzyme diamine oxidase increased with the onset of senescence in both the organs. Chlorophyll and electrical conductance had a inverse relationship throughout the experimental period, whereas, the chlorophyll content was directly related with polyamine levels in both stipule and pod wall during aging. On the other hand, protein and RNA contents were positively correlated with free polyamines throughout the test period in stipule, but in the pod wall this was true only for the later stages of development.     

Atrial natriuretic peptide stimulates cat carotid body chemoreceptors in vivo

It is known that atrial natriuretic peptide (ANP) is released from cardiac myocyte and other stores during hypoxia and is involved in pulmonary-cardiovascular reflexes and in natriuresis and diuresis. Since the carotid body initiates hypoxic chemoreflexes, we hypothesized that ANP could potentiate the hypoxic stimulation of the carotid body chemoreceptor in vivo. We studied the effect of close intra-arterial injection of ANP on carotid chemoreceptor activity in anesthetized male cats which were paralyzed and artificially ventilated. Graded doses of ANP (0-10 nmoles) were administered by intra-arterial injections and they produced an excitatory response. Single dose of ANP (6.5 nmoles) at four steady-state levels of arterial PO(2), at constant PCO(2), produced increases of chemoreceptor activity. This increase of chemoreceptor activity with ANP in the presence of CO(2)-HCO(3)(-) in vitro could make a difference from those without CO(2)-HCO(3)(-) in vivo.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

Isolation and purification of a lethal scorpion toxin with neuromuscular blocking activity

Toxin-L a lethal neuromuscular blocking agent was isolated from the venom of the scorpion, Lychas laevifrons (Pocock), by the CM-cellulose ion-exchange chromatography. It was a homogenous, thermolabile and low molecular weight protein. The toxin produced irreversible blockade of indirect stimulation induced twitch responses on innervated rat phrenic nerve-diaphragm and chick biventer cervicis preparation. The toxin did not produce any contractile response on toad rectus abdominis muscle preparation. On chronically denervated rat diaphragm, the toxin failed to alter the responses induced by direct stimulation, exogenous acetylcholine, potassium chloride and caffeine. Acetylcholine and carbachol induced contractions on isolated chick biventer cervicis remained unaltered by the toxin. Neostigmine failed to alter toxin induced neuromuscular blockade on innervated rat diaphragm. The toxin released a significant amount of acetylcholine from innervated rat diaphragm. It may be concluded that the toxin acts presynaptically through the release of acetylcholine, thereby producing neuromuscular blockade.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Impaired Glucose Tolerance or Newly Diagnosed Diabetes Mellitus Diagnosed during Admission Adversely Affects Prognosis after Myocardial Infarction: An Observational Study

Objective

To investigate the prognostic effect of newly diagnosed diabetes mellitus (NDM) and impaired glucose tolerance (IGT) post myocardial infarction (MI).

Research Design and Methods

Retrospective cohort study of 768 patients without preexisting diabetes mellitus post-MI at one centre in Yorkshire between November 2005 and October 2008. Patients were categorised as normal glucose tolerance (NGT n = 337), IGT (n = 279) and NDM (n = 152) on pre- discharge oral glucose tolerance test (OGTT). Primary end-point was the first occurrence of major adverse cardiovascular events (MACE) including cardiovascular death, non-fatal MI, severe heart failure (HF) or non-haemorrhagic stroke. Secondary end-points were all cause mortality and individual components of MACE.

Results

Prevalence of NGT, impaired fasting glucose (IFG), IGT and NDM changed from 90%, 6%, 0% and 4% on fasting plasma glucose (FPG) to 43%, 1%, 36% and 20% respectively after OGTT. 102 deaths from all causes (79 as first events of which 46 were cardiovascular), 95 non fatal MI, 18 HF and 9 non haemorrhagic strokes occurred during 47.2 ± 9.4 months follow up. Event free survival was lower in IGT and NDM groups. IGT (HR 1.54, 95% CI: 1.06–2.24, p = 0.024) and NDM (HR 2.15, 95% CI: 1.42–3.24, p = 0.003) independently predicted MACE free survival. IGT and NDM also independently predicted incidence of MACE. NDM but not IGT increased the risk of secondary end-points.

Conclusion

Presence of IGT and NDM in patients presenting post-MI, identified using OGTT, is associated with increased incidence of MACE and is associated with adverse outcomes despite adequate secondary prevention.     

Evidence for <Emphasis Type="Italic">Wolbachia</Emphasis> symbiosis in microfilariae of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> from West Bengal,India

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.