Browsing by Author "Indra Mani"

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Analysis of the multicopper oxidase gene regulatory network of Aeromonas hydrophila
(Kluwer Academic Publishers, 2012) Vijai Singh; Indra Mani; Dharmendra Kumar Chaudhary
Multicopper oxidase (MCO) is an enzyme which involves in reducing the oxygen in a four electron reduction to water with concomitant one electron oxidation of reducing the substrate. We have generated the 3-D structure of MCO by homology modeling and validated on the basis of free energy while 90. 4 % amino acid residues present in allowed regions of Ramachandran plot. The screening of potential hazardous aromatic compounds for MCO was performed using molecular docking. We obtained Sulfonaphthal, Thymolphthalein, Bromocresol green and Phloretin derivatives of phenol and aromatic hydrocarbon were efficient substrates for MCO. The phylogeny of MCO reveals that other bacteria restrain the homologous gene of MCO may play an important role in biodegradation of aromatic compounds. We have demonstrated the gene regulatory network of MCO with other cellular proteins which play a key role in gene regulation. These findings provide a new insight for oxidization of phenolic and aromatic compounds using biodegradation process for controlling environmental pollution. © 2012 Springer Science+Business Media B.V.
ATP4A gene regulatory network for fine-tuning of proton pump and ion channels
(2013) Vijai Singh; Indra Mani; Dharmendra Kumar Chaudhary
The ATP4A encodes α subunit of H+, K+-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H+, K+-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H+, K+ and Cl-) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H+, K+-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H+, K+-ATPase on the basis of their affinity between drug-protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers. © 2013 Springer Science+Business Media Dordrecht.
Chromosomal distribution of constitutive heterochromatin in eight species of mahseers (Family: Cyprinidae) from India
(2013) Indra Mani; Ravindra Kumar; Mamta Singh; B. Kushwaha; N.S. Nagpure; P.K. Srivastava; W.S. Lakra
The chromosomal distributions of constitutive heterochromatin were analyzed in 8 species of mahseer, namely, Tor chelynoides (McClelland), T. khudree (Sykes), T. mosal mahanadicus (David), T. mussullah (Sykes), T. putitora (Hamilton), T. progeneius (McClelland), T. tor (Hamilton) and Neolissochilus hexagonolepis (McClelland) using C-banding technique. The constitutive heterochromatin bands were observed on several pairs of chromosomes with maximum numbers in T. progeneius (11 pairs), followed by T. putitora (7 pairs), T. mosal mahanadicus (4), T. khudree (3), T. mussullah (3), N. hexagonolepis (3), T. chelynoides (3) and T. tor (2). In all species, the C-bands were observed mainly on p arm with few on centromeric region of chromosomes. This seems to be the characteristic feature for characterization of these species. The presence of similar chromosome numbers (2n=100) with diverse karyotypes in the mahseer species suggests evolution among the species through pericentric inversions and/or heterochromatin processes. The number and location of the heterochromatic bands were found species-specific and thus useful for the cytotaxonomy of these species. The findings of the present study can also help to understand the evolutionary relationships among the mahseer species.
Codon optimization of the major antigen encoding genes of diverse strains of influenza a virus
(International Association of Scientists in the International Association of Scientists in the, 2011) Indra Mani; Vijai Singh; Dharmendra Kumar Chaudhary; Pallavi Somvanshi; M.P.S. Negi
A large number of influenza A virus outbreaks and mortality occurred in the world recently, an urgent attention to develop effective and sufficient quantity of vaccines are needed. Vaccines are generally protein with immunogenic properties and are not expressed in sufficient quantity because of the codon bias, so it is necessary to optimize its codon in the expression host. Codon optimization was used to improve the protein expression in living organisms by increasing the translational efficiency of gene of interest. Two surface antigenic glycoproteins, hemagglutinin (HA) and neuraminidase (NA) are present in influenza A viruses. We have used HA and NA genes from 19 strains of influenza A viruses for codon optimization in E. coli. Both genes of the influenza virus show that the codon adaptation index (CAI) and GC content of the genes in optimized DNA were enhanced significantly (p <0.01) as compared to wild type. CAI and GC of HA in optimized DNA was enhanced by 3.2 (68.5%) and 1.2 (16.2%) fold respectively, while in NA it was increased by 3.3 (69.7%) and 1.2 (15.8%) fold respectively. Our finding demonstrates that the optimized genes could be useful for better expression in host without any truncated proteins and also helpful for protein folding and function. This work provides new insight in the synthetic biology research. © 2011 International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg.
Development of diagnostic and vaccine markers through cloning, expression, and regulation of putative virulence-protein-encoding genes of Aeromonas hydrophila
(2013) Vijai Singh; Dharmendra Kumar Chaudhary; Indra Mani; Rohan Jain; B.N. Mishra
Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of diseases in fish, amphibians, reptiles, and humans. In fish it causes several disease symptoms including tail and skin rot, and haemorrhagic septicemia; in human it causes soft-tissue wound infection and diarrhoea. The pathogenesis of A. hydrophila is multifactorial, but the mechanism is unknown so far. It is considered to be mediated by expression and secretion of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. A number of the putative virulence-protein-encoding genes that are present in the genome of A. hydrophila have been targeted by PCR for molecular diagnosis. These significant genes are also targeted for over-production of proteins by cloning and expression methods. In this review, we emphasize recent progress in the cloning, expression, and regulation of putative virulence-protein-encoding genes of A. hydrophila for a better understanding of the pathogenesis and also help to provide effective strategies for control of diseases. © 2013 The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
Gene cloning and homology modeling of the 3-oxoacyl-ACP synthase from Aeromonas hydrophila for drug discovery
(2011) Vijai Singh; Indra Mani; Dharmendra Kumar Chaudhary
Fatty acid biosynthesis in the Aeromonas hydrophila plays an important role in the formation of cell membrane and cell viability. The 3-oxoacyl- acyl carrier protein (ACP) synthase fadH and fadB from A. hydrophila have contributed in the initiation and elongation of fatty acid biosynthesis. In this study, we have designed new set of primers for amplification of the open reading frame of fadH and fadB. Cloning and sequencing of these amplified genes, analyzed that the G+C content of fadH and fadB were 63.3% and 68.79% respectively. The homology modeling was performed to generate the 3- D structure of fadH and fadB; and showed that more than 90% amino acid residues in allowed region of Ramachandranan plot. These findings provide a new avenue for better understanding, the role of genes encoding proteins involved in the fatty acid biosynthesis and thereby using as a potential and novel target for structure based drug designing to control the infections of A. hydrophila. © 2011 Bentham Science Publishers Ltd.
Gene network analysis of Aeromonas hydrophila for novel drug target discovery
(2012) Vijai Singh; Dharmendra Kumar Chaudhary; Indra Mani
Increasing the multi-drug resistance Aeromonas hydrophila creates a health problem regularly thus, an urgent needs to develop and screen potent antibiotics for controlling of the infections. There are many studies have focused on interactions between specific drugs, little is known about the system properties of a full drug interaction in gene network. Thus, an attractive approach for developing novel antibiotics against DNA gyrase, an enzyme essential for DNA replication, transcription, repair and recombination mechanisms which is important for bacterial growth and cell division. Homology modeling method was used to generate the 3-D structure of B subunit of DNA gyrase (gyrB) using known crystal structure. The active amino acids in 3-D structure of gyrB were targeted for structure based virtual screening of potent drugs by molecular docking. Number of drugs and analogs were selected and used for docking against gryB. The drugs Cinodine I, Cyclothialidine and Novobiocin were found to be more binding affinity with gyrB-drug interaction. The homology of gyrB protein sequence of A. hydrophila resembles with other species of Aeromonas closely showed relationship in phylogenetic tree. We have also demonstrated the gene network interactions of gyrB with other cellular proteins which are playing the key role in gene regulation. These findings provide new insight to understand the 3-D structure of gyrB which can be used in structure-based drug discovery; and development of novel, potent and specific drug against B subunit of DNA gyrase. © 2012 Springer Science+Business Media B.V.
Genotyping of Aeromonas hydrophila by Box elements
(2010) Vijai Singh; Dharmendra Kumar Chaudhary; Indra Mani; Pallavi Somvanshi; Gaurav Rathore; Neeraj Sood
PCR-based DNA fingerprinting techniques were evaluated to genotype eight diseased, particularly normal and environmental isolates of Aeromonas hydrophila. PCR-based fingerprinting method has an advantage of having repetitive sequence also called Box elements that are interspersed throughout the genome in diverse bacterial species. The BOX-PCR fingerprinting technique was evaluated for the discrimination of different isolates of A. hydrophila. All the studied isolates have shown major banding patterns ranged from 500-3000 bp. These finding could be advantageous to investigate the strain level specific fingerprints of A. hydrophila as potential genotypic markers. © 2010 Pleiades Publishing, Ltd.
Molecular detection and cloning of thermostable hemolysin gene from Aeromonas hydrophila
(2011) Vijai Singh; Indra Mani; Dharmendra Kumar Chaudhary; Pallavi Somvanshi
Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines. © 2011 Pleiades Publishing, Ltd.
Nucleotide variation and physical mapping of ribosomal genes using FISH in genus Tor (Pisces, Cyprinidae)
(2011) Indra Mani; Ravindra Kumar; Mamta Singh; N.S. Nagpure; B. Kushwaha; P.K. Srivastava; D.S. Krishna Rao; W.S. Lakra
Molecular cytogenetic studies were carried out for localization of 18S and 5S ribosomal DNAs on chromosomes of three cyprinid fish species viz., T. khudree, T. mussullah and T. mosal mahanadicus using two color fluorescence in situ hybridization (FISH). All the species typically possessed 100 diploid chromosomes with minor variation in karyo-morphology. The 18S rDNA signals were observed on two pair of chromosomes in T. khudree and T. mussullah, and three pairs in T. mosal mahanadicus. The location of 18S signals also showed affinity to silver nitrate and chromomycin A3 staining. Similarly, variation in localization of 5S rDNA among the three species has been detected with the presence of FISH signals on one pair of chromosome in T. khudree and T. mussullah, and on two pairs in T. mosal mahanadicus. These molecular markers could be used as species specific markers for taxonomic identification and can further add in understanding the dynamics of genome organization and karyotypic evolution of these species. The 18S rDNA region was sequenced that generated 1811, 1810 and 1776 bp long 18S sequence in T. khudree, T. mussullah and T. mosal mahanadicus, respectively. The 18S rDNA sequence showed 95-98% identity among the subject species. Similarly, 5S sequencing generated 203 bp long fragments in these species with 100% identity in coding and 9.63% variability in non-transcribed spacer regions. The nucleotide sequence variations could be used for understanding the genetic diversity and will add new informative characters in comparative genomics. These results, in general, would enhance the value and interpretation of ecological assessment data for conservation of Tor species. © Springer Science+Business Media B.V. 2010.
Role of metabolites and significance of SH groups in the action of NADP+-linked isocitrate dehydrogenase of urdbean seeds (Phaseolus mungo L.)
(2011) Pramod Kumar Srivastava; Govind Kant Srivastava; Indra Mani; Sharawan Yadav; Asha Anand
NADP+-linked-isocitrate dehydrogenase (EC 1.1.1.42) is a key enzyme of the Tricarboxylic Acid Cycle (TCA) and has been purified from urdbean seeds and it is inhibited by ATP in a competitive manner having inhibitor constant (Ki 1.32 mM. Phosphoenol-pyruvate, an energy rich compound plays an important role in the regulation of this enzyme and this metabolite inhibited the enzyme activity of NADP+-linked-isocitrate dehydrogenase of urdbean with inhibitor constant (Ki 2.66 mM in a competitive manner. The mode of inhibition by various metabolites of Krebs cycle has been carried out and found that oxaloacetate and succinate inhibit ICDH urdbean enzyme in a competitive manner with respect to isocitrate and their Ki values are found to be 7.27 and 10.67 mM, respectively. Citrate inhibits the urdbean ICDH enzyme non competitively with Ki revalue equal to 3.33 mM. The SH groups play a important role in the activity of NADP+-linked-isocitrate dehydrogenase and blocking of this group with SH-reagents, leads to inactivation of urdbean ICDH enzyme. With excess iodoacetamide (1.00 mM) and N-ethylmaleimide (4.0 mM) inhibition of this enzyme follows first order kinetics, suggesting that there are four reactive SH groups per mole of enzyme which are equally reactive and there is no site- site interaction among the tetrameric isoicitrate dehydrogenase of urdbean. © 2011 Academic Journals Inc.