Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1β -511CC genotype, TGFβ1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.     

Association analysis of common variants in FOXO3 with type 2 diabetes in a South Indian Dravidian population

Recent studies have identified common variants in forkhead box O3 gene (FOXO3) to be strongly associated with longevity in different populations. But studies have not been carried out to analyse the role of common variants in FOXO3 with type 2 diabetes. Since type 2 diabetes is an age related disorder and FOXO proteins play an important role in the regulation of metabolism, we studied the role of common variants in FOXO3 for association with type 2 diabetes. The study was carried out in 994 type 2 diabetic samples and 984 normoglycemic control samples from a South Indian Dravidian population. In our analysis, we found that there was no association between any of the selected SNPs in FOXO3 with type 2 diabetes. Analysis of these SNPs with diabetes related biochemical and clinical parameters also did not reveal any significant association. Haplotype association of SNPs in FOXO3 with type 2 diabetes was observed, but the frequency of the haplotypes was considerably lower and they do not remain significant after correction for multiple testing. In conclusion, we did not observe any association of SNPs in FOXO3 with type 2 diabetes and related parameters suggesting an entirely different mechanism by which these SNPs influence longevity. However additional studies in other populations are required to completely rule out the association of common variants in FOXO3 with type 2 diabetes.     

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

A novel cellobiase gene, designated cba3, was cloned from Cellulomonas biazotea. Although cellobiase genes of C. biazotea were previously cloned, published and/or patented, they encoded β-glucosidases all belonging to glycoside hydrolase family 3 (GH3); the new Cba3 cellobiase was identified to be a glycoside hydrolase family 1 (GH1) member, which represents the first discovered GH1 β-glucosidase of C. biazotea. Escherichia coli transformants expressing recombinant Cba3 were shown to grow readily in minimal media using cellobiose as the sole carbon source, supporting the conclusion that Cba3 is a genuine cellobiase. The full-length cba3 gene was revealed by sequencing to be 1344 bp long. Cba3 deletants lacking either the N-terminal 10 amino acids or the C-terminal 10 residues were found to be biologically inactive, supporting the importance of both ends in catalysis. Like other GH1 β-glucosidases, Cba3 was shown to contain the highly conserved NEP and ENG motifs, which are crucial for enzymatic activity. Despite lacking a classical N-terminal signal peptide, Cba3 was demonstrated to be a secretory protein. The findings that Cba3 is a cellobiase, and that it was expressed well as an extracellular protein in E. coli, support the potential of Cba3 for use with other cellulases in the hydrolysis of cellulosic biomass.     

Protein phosphatase-1M and Rho-kinase affect exocytosis from cortical synaptosomes and influence neurotransmission at a glutamatergic giant synapse of the rat auditory system

Protein phosphatase-1M (PP1M, myosin phosphatase) consists of a PP1 catalytic subunit (PP1c) and the myosin phosphatase target subunit-1 (MYPT1). RhoA-activated kinase (ROK) regulates PP1M via inhibitory phosphorylation of MYPT1. Using multidisciplinary approaches, we have studied the roles of PP1M and ROK in neurotransmission. Electron microscopy demonstrated the presence of MYPT1 and ROK in both pre- and post-synaptic terminals. Tautomycetin (TMC), a PP1-specific inhibitor, decreased the depolarization-induced exocytosis from cortical synaptosomes. trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride, a ROK-specific inhibitor, had the opposite effect. Mass spectrometry analysis identified several MYPT1-bound synaptosomal proteins, of which interactions of synapsin-I, syntaxin-1, calcineurin-A subunit, and Ca(2+) /calmodulin-dependent kinase II with MYPT1 were confirmed. In intact synaptosomes, TMC increased, whereas Y27632 decreased the phosphorylation levels of MYPT1(Thr696) , myosin-II light chain(Ser19) , synapsin-I(Ser9) , and syntaxin-1(Ser14) , indicating that PP1M and ROK influence their phosphorylation status. Confocal microscopy indicated that MYPT1 and ROK are present in the rat ventral cochlear nucleus both pre- and post-synaptically. Analysis of the neurotransmission in an auditory glutamatergic giant synapse demonstrated that PP1M and ROK affect neurotransmission via both pre- and post-synaptic mechanisms. Our data suggest that both PP1M and ROK influence synaptic transmission, but further studies are needed to give a full account of their mechanism of action.     

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0 U with a specific activity of 201.0 U/mg in a process control bioreactor. A 21.0 kDa alkaline α-amylase purified from this strain showed optimum activity at 55 °C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The K_m and V_max values for this enzyme towards starch were determined as 1.9 mg/ml and 198.21 μmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.     

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.     

Long-term daily access to alcohol alters dopamine-related synthesis and signaling proteins in the rat striatum

Chronic alcohol exposure can adversely affect neuronal morphology, synaptic architecture and associated neuroplasticity. However, the effects of moderate levels of long-term alcohol intake on the brain are a matter of debate. The current study used 2-DE (two-dimensional gel electrophoresis) proteomics to examine proteomic changes in the striatum of male Wistar rats after 8 months of continuous access to a standard off-the-shelf beer in their home cages. Alcohol intake under group-housed conditions during this time was around 3–4 g/kg/day, a level below that known to induce physical dependence in rats. After 8 months of access rats were euthanased and 2-DE proteomic analysis of the striatum was conducted. A total of 28 striatal proteins were significantly altered in the beer drinking rats relative to controls. Strikingly, many of these were dopamine (DA)-related proteins, including tyrosine hydroxylase (an enzyme of DA biosynthesis), pyridoxal phosphate phosphatase (a co-enzyme in DA biosynthesis), DA and cAMP regulating phosphoprotein (a regulator of DA receptors and transporters), protein phosphatase 1 (a signaling protein) and nitric oxide synthase (which modulates DA uptake). Selected protein expression changes were verified using Western blotting. We conclude that long-term moderate alcohol consumption is associated with substantial alterations in the rat striatal proteome, particularly with regard to dopaminergic signaling pathways. This provides potentially important evidence of major neuroadaptations in dopamine systems with daily alcohol consumption at relatively modest levels.