Vinay Singh

ABSTRACT Setaria cervi was screened for antifilarial agents by gene identification, behavior and genome organization. H2B gene was identified using primers generated from closely related species Brugia malayi. A 0.75kb amplified product from cDNA of S. cervi was used as template; gene sequences (JQ622388; AFI23673) were 83.3% of Brugia malayi and Loa loa. Functional domain analysis with Interproscan server revealed an amplified putative protein similar to Histone H2B (IPR000558), Histone core (IPR007125) and Histone fold (IPR009072) patterns. Model structure (PM0078101) and classification lineage (1.10.20.10) suggested that the existing protein was a histone subunit consisting of an alpha fold with orthogonal bundle architecture. H2B-DNA interactions stabilize the nucleosome and H2B could modulate chromatin structure during DNA repair, replication, transcription and chromatin compaction.

ABSTRACT Brugia malayi Glutathione Reductase (BmGR), a redox enzyme that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, plays an important role in filariasis and other diseases. BmGR was modeled using a 3DK9 template with 324 hydrogen Bonds, 18 helices, 32 strands and 47 turns and accepted at PMDB database (PM0077742). The model was used to dock and simulate the ant filarial drugs diethylcarbamazine citrate and albendazole, specific inhibitors of GR; 3,4 Dihydroxybenzyleamine and 1,3-bis(2-chloroethyl)-1-nitrosourea and substituted chalcones (SK series compounds). The active site analysis alignment of HsGR revealed ten stretches as active binding sites in BmGR. The amino acid residues SER19, GLU39, GLU40, THR41, THR46, TYR146, ASP162, ARG267, ARG272, ASP307 and THR315 were involved in hydrogen bonding, hydrophobic, polar, cation-pi and other interactions with NADPH. The binding of GSSG with protein especially with amino acids ALA142 and VAL143 and overlapped with NADPH binding site except SER19, ARG267, ARG272, ASP307 and THR315. In-silico investigation revealed that interaction of DEC with amino acids TYR181, ILE182 & ASP307 and SER19, THR46, ALA142, VAL143 & ALA318 interacting with ALB and specific inhibitors; DHBA showing interaction with amino acids GLU39, GLU40, THR41, THR46 & THR146 and SER19, GLY20 & THR46 with BCNU are involved in hydrogen, hydrophobic and polar level interactions. However, the interaction sites of antifilarials are similar to the binding sites of GSSG but positions are different and the binding sites of specific inhibitors are similar to the substrate i.e. active site domain II of BmGR protein. While SK series compounds are interact with highest coordination of the major portion of the active binding site as well as NADPH binding site of the protein. Based on estimated free energy of binding, lowest inhibition constant (Ki) and lower frequency percentage ALB, BCNU and substituted chalcones were showing better interactions. Specific inhibitors are showing competitive type binding (stretch 1 & 2) with substrate while substituted chalcones non-competitive (stretch 1, 2, 3 & 6). However, Stretch 5 and 7 may be the activator sites of the protein, which are the binding sites of antifilarials. Amino acids SER19, CYS47, CYS52 and PHE165 may be better inhibitory sites for drug designing using BmGR as template. Among substituted chalcone SK-3 and SK-5 are showing lower estimated free energy of binding, lowest inhibition constant (Ki) and lower frequency percentage. These substituted chalcones may be good inhibitors of glutathione metabolism and have better antifilarial activity. The BmGR structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis.

Basal cell carcinoma (BCC) is the most common dermatological malignancy. Sunlight being the most important aetiological factor, it remains an exceptional rarity in non-sun exposed parts, including the perianal area. We report an atypical case of BCC of the perianal area ...

Expression of synuclein-gamma (SNCG) protein is elevated in the advanced stages of many types of cancers, including ovarian, lung, liver, esophagus, colon, prostate and, in particular, breast. In breast carcinoma, SNCG is causatively linked to stimulated proliferation, metastasis and drug resistance. To establish SNCG as a potential therapeutic target and to discuss clinical use of SNCG inhibiting peptide. This review focuses on the plausible mechanisms of SNCG activity, SNCG mediated drug resistance and its inhibition. Evidence based research shows that the aberrant expression of SNCG has a strong correlation with breast cancer progression and poor clinical outcome. A peptide based inhibitor counters activity of SNCG, which may be developed as an adjuvant therapy.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling.

Butachlor an extensively used rice field herbicide negatively affects the cyanobacterial proliferation, yet the molecular mechanism underlying its toxicity in diazotrophic cyanobacteria is largely unknown. The present study focuses on the comparative proteomics to decode the molecular basis of butachlor toxicity/tolerance in three Anabaena species e.g. Anabaena sp. PCC 7120, Anabaena doliolum and Anabaena L31. 75 differentially expressed proteins from each Anabaena sp. included those involved in photosynthesis, C, N and protein metabolism, redox homeostasis, and signal transduction. While early accumulated proteins related to photosynthesis (atpA, atpB), carbon metabolism (glpx, fba and prk), protein folding (groEL, PPIase), regulation (orrA) and other function (OR, akr) appeared crucial for tolerance of Anabaena L31, the late accumulated proteins in Anabaena 7120 presumably offer acclimation during prolonged exposure to butachlor. Contrary to the above, a multitude of down-accumulated proteins vis-a-vis metabolisms augment sensitivity of A. doliolum to butachlor. A cluster of high abundant proteins (atpA, groEL, OR, AGTase, Alr0803, Alr0806, Alr3090, Alr3199, All4050 and All4051) common across the three species may be taken as markers for butachlor tolerance and deserve exploitation for stress management and transgenic development. Cyanobacteria offer an eco-friendly alternative to chemical fertilizers for increasing productivity, especially in rice cultivation. This study is the first to compare the proteome of three diazotrophic cyanobacteria subjected to butachlor, a pre-emergent herbicide extensively used in rice paddy. Changes in protein dynamics over time along with physiological and biochemical attributes clearly provide a comprehensive overview on differential tolerance of Anabaena species to butachlor. Molecular docking further added a new dimension in identification of potential protein candidates for butachlor stress management in cyanobacteria. This study strongly recommends combined application of Anabaena spp. A. L31 and A. PCC7120 as biofertilizer in paddy fields undergoing butachlor treatment.

The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.

We describe the development of a user friendly tool that would assist in the retrieval of information relating to Cry genes in transgenic crops. The tool also helps in detection of transformed Cry genes from Bacillus thuringiensis present in transgenic plants by providing suitable designed primers for PCR identification of these genes. The tool designed based on relational database model enables easy retrieval of information from the database with simple user queries. The tool also enables users to access related information about Cry genes present in various databases by interacting with different sources (nucleotide sequences, protein sequence, sequence comparison tools, published literature, conserved domains, evolutionary and structural data). http://insilicogenomics.in/Cry-btIdentifier/welcome.html.

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.

ABSTRACT The ability to rapidly adapt to changes in their environment is essential for the survival of microorganisms. DnaK is the prokaryotic analogue of eukaryotic Hsp70. These proteins protect the cell against heat injury or other forms of stress. In the present research, 78 rhizobial isolates were collected from different geographical regions of India and tested for their sensitivity. Forty-two out of 78 were shown to be susceptible to eight isolated phages. Only six Rhizobium strains out of 42 (MPSR033, MPSR041, MPSR052, MPSR084, MPSR220 and UPSR095) showed susceptibility to a single phage strain separately. All of these six rhizobial strains were slow growing and had the capability of Ex-planta nitrogenase activities. The PCR amplification of the dnaK region of the selected strain showed a single band~ 600 bp in length. The restriction endonuclease analysis of dnaK regions produced six restriction patterns. Sequence BLASTn analysis of DnaK showed linkage with Actinobacterium sp. and Bradyrhizobium sp. Multiple sequence alignment of all six Rhizobium dnaK regions exhibited a 99 % similarity between MPSR041 and MPSR220, and formed a close cluster with UPSR095 and MPSR033. The Prosite database showed that the functional domain of the DnaK protein presented three signatures, PS00297 IDLGTTNS (HSP70_1), PS00329 VYDLGGGTFDISIL (HSP70_2) and PS01036 VVLVGGMSRMPKVQE (HSP70_3), which belong to the HSP70 protein family. An in silico interaction study revealed that DnaK protein is involved in interactions with blr, gap, grpE, pgk, hrcA and recA heat-shock proteins.

The Dof (DNA binding with One Finger) family represents a classic zinc-finger transcription factors involved with multifarious roles exclusively in plants. There exists great diversity in terms of number of Dof genes observed in different crops. In current study, a total of 28 putative Dof genes have been predicted in silico from the recently available whole genome shotgun sequence of Sorghum bicolor (L.) Moench (with assigned accession numbers TPA:BK006983-BK007006 and TPA:BK007079-BK007082). The predicted SbDof genes are distributed on nine out of ten chromosomes of sorghum and most of these genes lack introns based on canonical intron/exon structure. Phylogenetic analysis of 28 SbDof proteins resulted in four subgroups constituting six clusters. The comparative phylogenetic analysis of these Dof proteins along with 30 rice and 36 Arabidopsis Dof proteins revealed six major groups similar to what has been observed earlier for rice and Arabidopsis. Motif analysis revealed the presence of conserved 50-52 amino acids Dof domain uniformly distributed across all the 28 Dof proteins of sorghum. The in silico cis-regulatory elements analysis of these SbDof genes suggested its diverse functions associated with light responsiveness, endosperm specific gene expression, hormone responsiveness, meristem specific expression and stress responsiveness.

Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. G. oxydans is an obligate aerobe, having a respiratory type of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, baker's soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5-diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed.