Role of TNF-α -308G/A Polymorphism in Bipolar Disorder and its Relationship with Clinical and Demographic Variables.

Objective: Gene-environment interactions might play a significant role in the development of bipolar disorder (BD). The objective of the current study was to investigate the association between tumor necrosis factor (TNF)-α -308 G/A polymorphism and BD and conduct a bioinformatics analysis of the protein-protein network of TNF-α. Gene-environment interactions and the relationship between stressful life events (SLEs) and substance abuse with TNF genotypes and other characteristics were analyzed. Methods: The genomic deoxyribonucleic acid (DNA) of 400 patients with BD and 200 control subjects were extracted and genotyped for TNF-α -308 G/A polymorphism. SLEs and substance abuse were evaluated using the Life Event and Difficulty Schedule (LEDS) and a self-designed substance abuse questionnaire for the events six months prior to the onset of the disease, respectively. Gene-environment interactions were assessed by multiple statistical tools. Bioinformatics analysis of the TNF-α network and its interacting proteins was carried out using STRING and Cytoscape softwares. Results: Genotyping analysis revealed a significant association between TNF-α -308 G/A polymorphism and BD (p<0.009). Furthermore, analysis of gene-environment interaction revealed a significant association between TNF-α -308 G/A and SLEs (p=0.001) and TNF-α -308 G/A and substance abuse (p=0.001). Three distinct proteins, RELA, RIPK1, and BIRC3, were identified through hub analysis of the protein network. Conclusion: TNF-α -308 G/A polymorphism is positively associated with BD. SLEs and substance abuse might trigger the early onset of BD. Proteins identified through bioinformatics analysis might contribute to the TNF-α mediated pathophysiology of BD and can be the potential therapeutic targets.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Pcal_0768, a hyperactive 4-α-glucanotransferase from Pyrobacculum calidifontis.

Genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame, Pcal_0768, corresponding to a putative 4-α-glucanotranferase belonging to glycoside hydrolases (GH) family 77. We have produced, in Escherichia coli, and purified recombinant Pcal_0768 which exhibited high disproportionation (690 U mg(-1)) activity. To the best of our knowledge, this is the highest ever reported activity for any member of family GH77. Maltooligosaccharides, when used as sole substrates, were disproportionated into linear maltooligohomologues. The analysis of the reaction end products revealed no evidence for the production of cycloamyloses. Catalytic activity of the enzyme remained unchanged in the presence or the absence of ionic and nonionic detergents. γ-cyclodextrin, an inhibitor of 4-α-glucanotransferases, did not show any inhibitory effect on Pcal_0768 activity. These properties make Pcal_0768 a potential candidate for starch processing industry.