Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.     

Reexamination of the difference in susceptibility to adjuvant-induced arthritis among LEW/Crj, Slc/Wistar/ST and Slc/SD rats.

The present investigations were performed to assess the differences among rat colonies commonly used for neurophysiological research regarding the development of complete Freund's adjuvant (CFA)-induced arthritis. Inflammatory signs including edema in the paw fluctuated remarkably among individual Wistar (Slc/Wistar/ST) and Sprague-Dawley (Slc/SD) rats, while the inflammatory signs of Lewis (LEW/Crj) rats appeared earlier and was severer and more consistent than Slc/Wistar/ST and Slc/SD rats. Edema in the hind paw developed in 100% of LEW/Crj rats with the lowest dose of CFA (0.6 mg/rat) used as compared with 64% of Slc/Wistar/ST (CFA 1 mg/rat) and 38% of Slc/SD rats (CFA 1.2 mg/rat). Retardation of weight gain was observed in Slc/Wistar/ST and Slc/SD rats in contrast to a severe weight decrease in inflamed LEW/Crj rats after the development of arthritis.     

Alterations in Digestive Enzymes of Three Freshwater Teleostean Fishes by Almix Herbicide: A Comparative Study

Three freshwater teleostean fishes viz., Anabas testudineus (Bloch), Heteropneustes fossilis (Bloch) and Oreochromis niloticus (Linnaeus) were exposed to almix (66.67 mg/l) herbicide for 30 days to investigate the activity of digestive enzymes (amylase, lipase and protease) in stomach, intestine and liver. Amylase activity showed significantly high (p < 0.05) in all the fishes compared to control value and highest activity was observed in liver of A. testudineus (721.99 %) and minimum in intestine of H. fossilis (195.37 %). Lipase activity was also significantly increased (p < 0.05) in all the tissues; but highest in intestine of O. niloticus (235.51 %) and minimum in intestine of A. testudineus (130.51 %). Protease activity also showed similar trends of enhancement; it was maximum in stomach of O. niloticus (362.69 %), whereas in liver of H. fossilis it was rather less (173.72 %). Increased activity of digestive enzymes resulting from tissue damage ultimately affected the fish health due to impairment of digestive physiology confirming the herbicidal contamination on fish species. The sensitivity to the almix herbicide was pronounced in the order of O. niloticus > A. testudineus > H. fossilis.     

Nitrogen Fixation in <Emphasis Type="Italic">Asaia</Emphasis> sp. (Family Acetobacteraceae)

The genus Asaia (family Acetobacteraceae) was first introduced with a single species—Asaia bogorensis and later six more species were described namely A. siamensis, A. krungthepensis, A. lannaensis, A. platycodi, A. prunellae, and A. astilbes. Acetobacteraceae family has been divided into ten genera but, only three of them include nitrogen fixing species: Gluconacetobacter, Acetobacter, and Swaminathania. This article originated from our study primarily aimed to isolate new endosymbiotic nitrogen fixer among Acetobacteraceae during which we have isolated, for the first time in India, four different strains of Asaia sp. from three different sources: Michalia champaca flower, Anopheles mosquito, and ant Tetraponera rufonigra. All the endosymbiotic strains isolated possess the ability to fix nitrogen. Evidence for both nitrogenase activity and the presence of nifH gene in isolated Asaia sp. is presented. Asaia bogorensis (MTCC 4041^T) and A. siamensis (MTCC 4042^T), two of the validated type strains available from the repository, were tested positive for the presence of functional nitrogenase. The nifH gene sequences from these type strains were also confirmed and compared with other nitrogen fixing members of the family Acetobacteraceae. Our result corroborate with the previous reports that Asaia sp. are indeed widely distributed in nature but this is the first time demonstration of their functional nitrogenase activity. This study shows Asaia sp. as fourth genera of nitrogen fixing bacteria in the family Acetobacteraceae.     

Association of Common Genetic Variants with Lipid Traits in the Indian Population

Genome-wide association studies (GWAS) have been instrumental in identifying novel genetic variants associated with altered plasma lipid levels. However, these quantitative trait loci have not been tested in the Indian population, where there is a poorly understood and growing burden of cardiometabolic disorders. We present the association of six single nucleotide polymorphisms in 1671 sib pairs (3342 subjects) with four lipid traits: total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). We also investigated the interaction effects of gender, location, fat intake and physical activity. Each copy of the risk allele of rs964184 at APOA1 was associated with 1.06 mmol/l increase in triglycerides (SE = 0.049; p = 0.006), rs3764261 at CETP with 1.02 mmol/l increase in both total cholesterol (SE = 0.042; p = 0.017) and HDL-C (SE = 0.041; p = 0.008), rs646776 at CELSR2-PSRC1-SORT1 with 0.96 mmol/l decrease in cholesterol (SE = 0.043; p = 0.0003) and 0.15 mmol/l decrease in LDL-C levels (SE = 0.043; p = 0.0003) and rs2954029 at TRIB1 with 1.02 mmol/l increase in HDL-C (SE = 0.039; p = 0.047). A combined risk score of APOA1 and CETP loci predicted an increase of 1.25 mmol/l in HDL-C level (SE = 0.312; p = 0.0007). Urban location and sex had strong interaction effects on the genetic association of most of the studied loci with lipid traits. To conclude, we validated four genetic variants (identified by GWAS in western populations) associated with lipid traits in the Indian population. The interaction effects found here may explain the sex-specific differences in lipid levels and their heritability. Urbanization appears to influence the nature of the association with GWAS lipid loci in this population. However, these findings will require replication in other Indian populations.     

Comparison of Sequencing Platforms for Single Nucleotide Variant Calls in a Human Sample

Next-generation sequencings platforms coupled with advanced bioinformatic tools enable re-sequencing of the human genome at high-speed and large cost savings. We compare sequencing platforms from Roche/454(GS FLX), Illumina/HiSeq (HiSeq 2000), and Life Technologies/SOLiD (SOLiD 3 ECC) for their ability to identify single nucleotide substitutions in whole genome sequences from the same human sample. We report on significant GC-related bias observed in the data sequenced on Illumina and SOLiD platforms. The differences in the variant calls were investigated with regards to coverage, and sequencing error. Some of the variants called by only one or two of the platforms were experimentally tested using mass spectrometry; a method that is independent of DNA sequencing. We establish several causes why variants remained unreported, specific to each platform. We report the indel called using the three sequencing technologies and from the obtained results we conclude that sequencing human genomes with more than a single platform and multiple libraries is beneficial when high level of accuracy is required.