open: No conditions. Results ordered Creators, Title. 2024-03-29T03:59:36ZEPrintshttp://crdd.osdd.net/images/sitelogo.gifhttp://crdd.osdd.net/open/2012-01-05T15:12:31Z2012-01-05T15:12:31Zhttp://crdd.osdd.net/open/id/eprint/265This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2652012-01-05T15:12:31ZPromiscuous binding nature of SH3 domains to their target proteins.SH3 domains are small but important domains in cell-signaling and function through protein-protein interactions. Their promiscuous nature in binding to polyproline peptides makes them much more important because many SH3 domains from different proteins bind to different proteins having polyproline template on their surface. Very subtle changes in the sequence of SH3 domains and the binding peptides determine the specificity of the peptide binding. Recent observation that SH3 domains bind to non- proline peptides makes the scenario of peptide binding involving SH3 domains complicated. If domain swapped dimerization as observed in Eps8-SH3 domain also binds different peptides, it proves the versatility of the SH3 domains in binding to peptides in various ways. An overview of the promiscuity of SH3 domains has been discussed.Vishal AgrawalK V Radha Kishan2012-01-05T15:06:53Z2015-01-09T10:54:25Zhttp://crdd.osdd.net/open/id/eprint/256This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2562012-01-05T15:06:53ZCrystallization and preliminary X-ray diffraction analysis of a thermostable D-hydantoinase from the mesophilic Bacillus sp. AR9.D-hydantoinase catalyzes the conversion of DL-hydantoin derivatives to the corresponding optically pure N-carbamoyl amino acids, the first step in the industrial preparation of optically pure amino acids using biological catalysts. A thermostable D-hydantoinase from the mesophilic bacteria Bacillus sp. AR9 has been crystallized in three different crystal forms. The hexagonal faced crystals were the best looking, but did not diffract. One of the crystal forms is star-shaped and appeared to be twinned, but diffracted as a single crystal to a resolution of 2.3 A. These crystals belong to space group P6(4) and have unit-cell parameters a = b = 129.55, c = 102.86 A, alpha = beta = 90, gamma = 120 degrees.Vishal AgrawalRakesh SharmaR M VohraK V Radha Kishan2012-02-13T15:17:49Z2015-01-09T10:35:20Zhttp://crdd.osdd.net/open/id/eprint/921This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9212012-02-13T15:17:49ZDesign, Expression, and Structural Characterization of Hybrid Proteins of Samia cynthia ricini and Bombyx mori Silk Fibroins.Genetically engineering strategies have been applied to obtain the designed proteins [GGAGSGYGGGYGHGYGSDGG(GAGAGS)3]n of varying chain lengths (n=2,4 or 6); SLP2, SLP4, and SLP6, respectively. The amino acid sequence of the monomeric unit represents the incorporation of the consensus repeat sequences of the glycine-rich region of Samia cynthia ricini followed by the repetitive crystalline region of Bombyx mori silk fibroins. The oligonucleotides were designed to encode the desired monomeric unit of silk-like hybrid proteins, which were then polymerised and expressed in Escherichi coli system. The monomer, SLP1, was synthesized chemically using solid phase method. The recombinant proteins were identified by N-terminal amino-acid sequencing and Western blotting. The structural characterizations of SLP1 and SLP6 samples were performed using 13C CP/MAS NMR in the solid state. The structure of SLP1 in the solid state was distorted .BETA.-turn by judging from the chemical shift and line shape of the C.BETA. carbon of Ala residue, and the structural transition from distorted .BETA.-turn to .BETA.-sheet occurred partially in SLP6. The solution structure was studied in hexafluoro-2-propanol (HFIP) using CD method. Although both samples, SLP1 and SLP6, showed indication of the ordered structures, marked change in the band intensities was observed between two CD spectra. Especially. the CD pattern of SLP6 is in agreement with the pattern with right-handed 310-helical conformation. (author abst.)T. AsakuraH. KatoJuming YaoRaghuvansh KishoreM. Shirai2012-02-06T18:18:53Z2012-02-06T18:18:53Zhttp://crdd.osdd.net/open/id/eprint/890This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8902012-02-06T18:18:53ZComparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR.The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.Tetsuo AsakuraRena SuginoJuming YaoHidehiko TakashimaRaghuvansh Kishore2012-02-06T18:19:26Z2012-02-06T18:19:26Zhttp://crdd.osdd.net/open/id/eprint/886This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8862012-02-06T18:19:26ZPiceatannol inhibits TNF-induced NF-kappaB activation and NF-kappaB-mediated gene expression through suppression of IkappaBalpha kinase and p65 phosphorylation.Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Previously, we have shown that resveratrol suppresses the activation of the nuclear transcription factor NF-kappaB. Piceatannol, previously reported as a selective inhibitor of protein tyrosine kinase Syk, is structurally homologous to resveratrol. Whether piceatannol can also suppress NF-kappaB activation was investigated. The treatment of human myeloid cells with piceatannol suppressed TNF-induced DNA binding activity of NF-kappaB. In contrast, stilbene or rhaponticin (another analog of piceatannol) had no effect, suggesting the critical role of hydroxyl groups. The effect of piceatannol was not restricted to myeloid cells, as TNF-induced NF-kappaB activation was also suppressed in lymphocyte and epithelial cells. Piceatannol also inhibited NF-kappaB activated by H(2)O(2), PMA, LPS, okadaic acid, and ceramide. Piceatannol abrogated the expression of TNF-induced NF-kappaB-dependent reporter gene and of matrix metalloprotease-9, cyclooxygenase-2, and cyclin D1. When examined for the mechanism, we found that piceatannol inhibited TNF-induced IkappaBalpha phosphorylation, p65 phosphorylation, p65 nuclear translocation, and IkappaBalpha kinase activation, but had no significant effect on IkappaBalpha degradation. Piceatannol inhibited NF-kappaB in cells with deleted Syk, indicating the lack of involvement of this kinase. Overall, our results clearly demonstrate that hydroxyl groups of stilbenes are critical and that piceatannol, a tetrahydroxystilbene, suppresses NF-kappaB activation induced by various inflammatory agents through inhibition of IkappaBalpha kinase and p65 phosphorylation.Kazuhiro AshikawaSekhar MajumdarSanjeev BanerjeeAlok C BhartiShishir ShishodiaBharat B Aggarwal2012-01-05T15:11:22Z2015-01-08T09:47:11Zhttp://crdd.osdd.net/open/id/eprint/260This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2602012-01-05T15:11:22ZFolded conformation of an immunostimulating tetrapeptide rigin: high temperature molecular dynamics simulation study.Employing high temperature quenched molecular dynamics (QMD) stimulations the conformational energy space of an immunostimulating tetrapeptide rigin: H-Gly341-Gln-Pro-Arg344-OH, is explored. Using distance dependent dielectric (epsilon =r(ij)) 31 different low energy starting structures with identical sequence were computed for their conformational preferences. According to the hypothesis of O'Connors et al. [J. Med. Chem. 35 (1992), 2870], 83 low-energy conformers resulted from unrestrained molecular dynamics (MD) simulations, could be classified into two energy minimized families: A and B, comprised of 64 (Pro C(gamma)-endo orientation) and 19 (Pro C(gamma)-exo orientation) structures, respectively. An examination of these families revealed the existence of a remarkably similar folded backbone conformation: torsion angles being phi(i+1) approximately -65 degrees, psi(i+1) approximately -65 degrees, phi(i+2) approximately -65 degrees, psi(i+2) approximately -60 degrees, characterizing a distorted type III beta-turn structure across the central Gln-Pro segment. The folded conformation of rigin is devoid of a classical 1 <-- 4 intra-molecular hydrogen bond nevertheless, the conformation is stabilized by an effective 'salt-bridge', i.e., Gly H(3)N(+)...C(alpha)OO(-) Arg interaction. Surprisingly, in both the families the unusual folded side-chain dispositions of the Gln residue favor the formation of a unique intra-residue 'main-chain to side-chain' H-bond, i.e., N(alpha)-H...N(epsilon) interaction, encompassing a seven-membered ring motif. The conformational attributes may be valuable in de novo construction of structure-based drug candidates having sufficient stimulating activity.A AshishRaghuvansh Kishore2012-02-06T18:19:42Z2015-01-09T10:46:30Zhttp://crdd.osdd.net/open/id/eprint/884This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8842012-02-06T18:19:42ZEvidence that glycoprotein 96 (B2), a stress protein, functions as a Th2-specific costimulatory molecule.After the engagement of Ag receptor, most of the Th cells for their optimal activation require a second (costimulatory) signal provided by the APCs. We demonstrate the isolation and characterization of a 99- to 105-kDa protein (B2), from LPS-activated B cell surface, and its function as a Th2-specific costimulatory molecule. Appearance of B2 as a single entity on two-dimensional gel electrophoresis and as a distinct peak in reverse-phase HPLC ascertains the fact that B2 is homogeneous in preparation. Electron microscopy as well as competitive binding studies reveal that (125)I-labeled B2 specifically binds anti-CD3-activated T cell surface and also competes with its unlabeled form. Internal amino acid sequences of B2 are found to be identical with stress protein gp96. The identity of B2 as gp96 is also revealed by immunological characterization and by confocal microscopic colocalization studies of B2 and gp96 on LPS-activated B cells. Confocal imaging studies also demonstrate that gp96 can be induced on B cell surface without association of MHC molecules. Furthermore, the novel role of gp96 in Th cell proliferation skewing its differentiation toward Th2 phenotype has also been established. Ab-mediated blocking of gp96-induced signaling not only abrogates in vitro proliferation of CD4(+) T cells, but also diminishes the secretion of Th2-specific cytokines. Notably, the expression of CD91 (receptor of gp96/B2) is up-regulated on anti-CD3-activated Th cells and also found to be present on Th1 and Th2 subsets.Pinaki P BanerjeeD S VinayAjith MathewManoj RajeVrajesh ParekhDurbaka V R PrasadAnil KumarDebashis MitraG C Mishra2012-02-13T15:20:27Z2012-03-28T09:42:50Zhttp://crdd.osdd.net/open/id/eprint/909This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9092012-02-13T15:20:27ZProduction and characterization of a thermostable beta-galactosidase from Bacillus coagulans RCS3.A strain of Bacillus coagulans RCS3 isolated from a hot-water spring produced significant beta-galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 degrees C, the highest activity was at 65 degrees C, where the half-life was 2 h. A 2 degrees C decrease in temperature increased the half-life to 15 h without significantly changing the activity, suggesting that 63 degrees C is the temperature of preference compared with 65 degrees C for a combination of good activity and stability. The beta-galactosidase was also stable over pH 5-8, with peak activity at pH 6-7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu(2+), Ni(2+) and Hg(2+)) in the concentration range of 0.5-2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 degrees C. The thermostability and pH-stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry. Navneet BatraJagtar SinghU C BanerjeeP R PatnaikRanbir C Sobti2012-02-06T18:17:08Z2012-02-06T18:17:08Zhttp://crdd.osdd.net/open/id/eprint/901This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9012012-02-06T18:17:08ZRecombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogenA truncated version of the denguevirustype2envelopeprotein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitisBsurfaceantigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichiapastoris. Both the recombinantproteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybridprotein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybridprotein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.Himani BishtDipti A ChughManoj RajeS SwaminathanNavin Khanna2012-02-13T15:18:22Z2012-03-28T10:09:56Zhttp://crdd.osdd.net/open/id/eprint/919This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9192012-02-13T15:18:22ZMicrobial diversity: Significance, conservation and application.Microorganisms occur nearly everywhere in nature and occupy an important place in human view of life. They exhibit an impressive diversity in their metabolic activities and interactions with other microbes, plants and animals. However, relatively little is known about the diversity of the microorganisms that constitute natural as well as artificial ecosystems and are potentially useful for biotechnology applications. Our knowledge of the microbial diversity has been severely limited by relying on organisms that have been cultured. Fewer than 10 percent of the extant microorganisms have been discovered and culture methods are inadequate for studying microbial community composition. Majority of naturally occurring microbes cannot be cultured using standard techniques. Speculation that more than 90 percent of the microbes remain undiscovered raise the question of how well we know the Earth's biota and its biochemical potential. Conceptual and methodological approaches for dealing with such diversity are now being developed. The application, of molecular phylogenetic methods to study natural microbial ecosystems without the traditional requirements for cultivation has resulted in the discovery of many evolutionary lineages and are opening vast areas of microbial world to exploration.R BudhirajaA BasuR K Jain2012-01-05T15:15:19Z2012-01-05T15:15:19Zhttp://crdd.osdd.net/open/id/eprint/276This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2762012-01-05T15:15:19ZEvidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division.A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.Rachna ChabaManoj RajePradip K Chakraborti2012-01-05T15:13:14Z2012-01-05T15:13:14Zhttp://crdd.osdd.net/open/id/eprint/267This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2672012-01-05T15:13:14ZSite-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C.Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.Radha ChauhanShekhar C Mande2012-02-06T18:19:19Z2012-02-06T18:19:19Zhttp://crdd.osdd.net/open/id/eprint/887This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8872012-02-06T18:19:19ZInvolvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains.The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.Jayeeta DharAbhay H PandeVasudha SundramJagpreet S NandaShekhar C MandeGirish Sahni2013-10-18T06:14:45Z2013-10-18T06:14:45Zhttp://crdd.osdd.net/open/id/eprint/1395This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13952013-10-18T06:14:45ZStudies on Glutathione Mediated Detoxification Pathways in YeastKailash Gulshan2012-01-05T15:11:53Z2012-01-05T15:11:53Zhttp://crdd.osdd.net/open/id/eprint/262This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2622012-01-05T15:11:53ZGWFASTA: server for FASTA search in eukaryotic and microbial genomes.Similarity searches are a powerful method for solving important biological problems such as database scanning, evolutionary studies, gene prediction, and protein structure prediction. FASTA is a widely used sequence comparison tool for rapid database scanning. Here we describe the GWFASTA server that was developed to assist the FASTA user in similarity searches against partially and/or completely sequenced genomes. GWFASTA consists of more than 60 microbial genomes, eight eukaryote genomes, and proteomes of annotatedgenomes. Infact, it provides the maximum number of databases for similarity searching from a single platform. GWFASTA allows the submission of more than one sequence as a single query for a FASTA search. It also provides integrated post-processing of FASTA output, including compositional analysis of proteins, multiple sequences alignment, and phylogenetic analysis. Furthermore, it summarizes the search results organism-wise for prokaryotes and chromosome-wise for eukaryotes. Thus, the integration of different tools for sequence analyses makes GWFASTA a powerful toolfor biologists.Biju IssacG.P.S. Raghava2012-01-09T09:45:55Z2012-01-09T09:45:55Zhttp://crdd.osdd.net/open/id/eprint/104This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/1042012-01-09T09:45:55ZLocating probable genes using Fourier transform approach.FTG is a web server for analyzing nucleotide sequences to predict the genes using Fourier transform techniques. This server implements the existing Fourier transform algorithms for gene prediction and allows the rapid visualization of analysis by output in GIF format.Biju IssacHarpreet SinghHarpreet KaurG.P.S. Raghava2012-02-06T18:18:41Z2012-04-02T09:45:13Zhttp://crdd.osdd.net/open/id/eprint/892This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8922012-02-06T18:18:41ZOrigins of the 2,4-dinitrotoluene pathway.The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds. Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction. The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA). The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from Burkholderia cepacia R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source. Genes for the lower pathway in 2,4-DNT degradation were found downstream from dntD, the gene encoding the extradiol ring fission enzyme of the pathway. The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (dntE), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (dntG). Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that dntD dntE ORF13 dntG composes an operon that encodes the lower pathway. Additional genes that were uncovered encode the 2,4-DNT dioxygenase (dntAaAbAcAd), methylnitrocatechol monooxygenase (dntB), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements. Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes. The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region.G R JohnsonR K JainJim C Spain2012-01-05T15:14:43Z2012-01-05T15:14:43Zhttp://crdd.osdd.net/open/id/eprint/273This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2732012-01-05T15:14:43ZBetaTPred: prediction of beta-TURNS in a protein using statistical algorithms.MOTIVATION: beta-turns play an important role from a structural and functional point of view. beta-turns are the most common type of non-repetitive structures in proteins and comprise on average, 25% of the residues. In the past numerous methods have been developed to predict beta-turns in a protein. Most of these prediction methods are based on statistical approaches. In order to utilize the full potential of these methods, there is a need to develop a web server.
RESULTS: This paper describes a web server called BetaTPred, developed for predicting beta-TURNS in a protein from its amino acid sequence. BetaTPred allows the user to predict turns in a protein using existing statistical algorithms. It also allows to predict different types of beta-TURNS e.g. type I, I', II, II', VI, VIII and non-specific. This server assists the users in predicting the consensus beta-TURNS in a protein
Harpreet KaurG.P.S. Raghava2012-01-05T15:07:18Z2012-01-05T15:07:18Zhttp://crdd.osdd.net/open/id/eprint/258This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2582012-01-05T15:07:18ZAn evaluation of beta-turn prediction methods.MOTIVATION: beta-turn is an important element of protein structure. In the past three decades, numerous beta-turn prediction methods have been developed based on various strategies. For a detailed discussion about the importance of beta-turns and a systematic introduction of the existing prediction algorithms for beta-turns and their types, please see a recent review (Chou, Analytical Biochemistry, 286, 1-16, 2000). However at present, it is still difficult to say which method is better than the other. This is because of the fact that these methods were developed on different sets of data. Thus, it is important to evaluate the performance of beta-turn prediction methods. RESULTS: We have evaluated the performance of six methods of beta-turn prediction. All the methods have been tested on a set of 426 non-homologous protein chains. It has been observed that the performance of the neural network based method, BTPRED, is significantly better than the statistical methods. One of the reasons for its better performance is that it utilizes the predicted secondary structure information. We have also trained, tested and evaluated the performance of all methods except BTPRED and GORBTURN, on new data set using a 7-fold cross-validation technique. There is a significant improvement in performance of all the methods when secondary structure information is incorporated. Moreover, after incorporating secondary structure information, the Sequence Coupled Model has yielded better results in predicting beta-turns as compared with other methods. In this study, both threshold dependent and independent (ROC) measures have been used for evaluation.Harpreet KaurG.P.S. Raghava2012-01-05T15:11:11Z2012-03-29T09:48:02Zhttp://crdd.osdd.net/open/id/eprint/259This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2592012-01-05T15:11:11ZPre-screening for antigen detectability in cells: a TEM-based solid phase digital immunogold detection method utilizing ultra low volumes of reagents.Rapid and sensitive pre-screening for the presence of antigens in cell samples and confirmation of reactivity of antibodies, before proceeding with electron microscopy, is highly desirable. Most of the methods developed for this purpose are generally not very efficient and suitable for dealing with very small volumes of sample and reagents. In this work we present a simple, sensitive and rapid solid phase transmission electron microscope (TEM) based method for the detection of picogram (pg) levels of soluble antigens using as little as 10 micro L of reagents. Protein was adsorbed onto grids coated with polystyrene films to form the solid phase. The presence of antigen was detected using immunogold labelling. Gold particles adhering to the film were visualized and counted in a TEM providing a digital signal. This method was 100-fold more sensitive than dot blot in detection of rabbit IgG. We have demonstrated the utility of this technique by screening for Vitreoscilla haemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold labelling transmission electron microscopy of cell sections.R KaurKanak L DikshitManoj Raje2012-01-05T15:13:52Z2012-01-05T15:13:52Zhttp://crdd.osdd.net/open/id/eprint/269This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2692012-01-05T15:13:52ZOptimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen.We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.Ramandeep KaurKanak L DikshitManoj Raje2012-01-05T15:16:04Z2012-01-05T15:16:04Zhttp://crdd.osdd.net/open/id/eprint/280This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2802012-01-05T15:16:04ZChimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.Ramandeep KaurRanjana PathaniaVishwamitra SharmaShekhar C MandeKanak L Dikshit2012-02-06T18:18:32Z2012-02-06T18:18:32Zhttp://crdd.osdd.net/open/id/eprint/893This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8932012-02-06T18:18:32ZChimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.Ramandeep KaurRanjana PathaniaVishwamitra SharmaShekhar C MandeKanak L Dikshit2012-02-06T18:17:43Z2015-07-22T10:30:38Zhttp://crdd.osdd.net/open/id/eprint/898This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8982012-02-06T18:17:43ZPurification and Characterization of a Human Pancreatic Adenocarcinoma MucinPancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 andMUC4mRNA, and moderate levels ofMUC5ACandMUC5BmRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.Ali M. KhorramiAnirban Roy ChoudhuryMahefatiana AndrianifahananaGrish C VarshneySambhu N. BhattacharyyaMichael A. HollingsworthBernard KaufmanS K Batra2013-10-18T06:22:05Z2013-10-18T06:22:05Zhttp://crdd.osdd.net/open/id/eprint/1396This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13962013-10-18T06:22:05ZBiochemical and Genetic Analysis of Gaseous Alkane Utilizing BacteriaJitendra Kumar2012-01-05T15:13:30Z2012-01-05T15:13:30Zhttp://crdd.osdd.net/open/id/eprint/268This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2682012-01-05T15:13:30ZUse of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase.Understanding protein aggregation may hold important clues to understanding what goes wrong with protein folding in neurodegenerative disorders and in bioreactors in which proteins are overexpressed. Unfortunately, aggregates tend to be intractable to most standard methods of biochemical investigation. Thus, relatively little is even now known about the micro- and macro-structural features of aggregates. To gain insights into the thermal aggregation of a model globular protein [bovine carbonic anhydrase (BCA)], we have used spectrofluorimetry to examine the binding of a hydrophobic dye, 8-anilinonaphthalene sulfonate (ANS), to hydrophobic clusters on the protein's surface both before and after heat-induced aggregation and upon cooling. Whereas native BCA shows no surface hydrophobicity, thermally aggregated BCA displays significant hydrophobicity both in the heated state and upon cooling. The timing of the addition of ANS in the course of aggregation makes no net difference to the ANS bound; we argue that this suggests that aggregates are essentially porous. Cooling of aggregates results in a dramatic, fully reversible increase in ANS binding that cannot be explained by the temperature dependence of fluorescence quantum yield alone; we argue that the enhancement of fluorescence upon cooling indicates possible structural consolidation of unfolded regions within aggregates (akin to refolding), with the required structural reorganization being facilitated by porosity. Finally, implications of porosity in aggregates are discussed, in particular, for the possible immobilization of enzymes through fusion with aggregation-prone protein domains.Bishwajit KunduPurnananda Guptasarma2012-01-05T15:15:10Z2012-01-05T15:15:10Zhttp://crdd.osdd.net/open/id/eprint/275This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2752012-01-05T15:15:10ZManipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening.A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates.Bishwajit KunduAnshuman ShuklaPurnananda Guptasarma2012-01-05T15:06:34Z2012-01-05T15:06:34Zhttp://crdd.osdd.net/open/id/eprint/254This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2542012-01-05T15:06:34ZPhenomenological perspectives on the folding of beta/alpha-barrel domains through the modular formation and assembly of smaller structural elements.The beta/alpha-barrel motif was once considered to be a single protein domain. In recent years, however, it has been shown to consist of smaller substructures displaying the ability to fold autonomously. Here we review the current status of experimental findings concerning the motif's folding behavior in the light of what is currently known about (a) the relative rates of formation of helices and sheets in proteins, in general, and (b) the peculiarities of topology and architecture of the motif, in particular, to develop a detailed phenomenological understanding of how beta/alpha-barrels might form through the modular folding and assembly of substructures.Sankar MaitiManni Luthra-GuptasarmaPurnananda Guptasarma2012-02-06T18:19:33Z2012-02-06T18:19:33Zhttp://crdd.osdd.net/open/id/eprint/885This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8852012-02-06T18:19:33ZThalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester.Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.Sekhar MajumdarBetty LamotheBharat B Aggarwal2012-02-13T15:18:31Z2015-01-08T10:30:57Zhttp://crdd.osdd.net/open/id/eprint/918This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9182012-02-13T15:18:31ZEffects of various nutritional supplements on biosurfactant production by a strain of Bacillus subtilis at 45°CEffects of various factors on growth and biosurfactant production by Bacillus subtilis MTCC 2423 were studied. Sucrose (2%) and potassium nitrate (0.3%) were the best carbon and nitrogen sources. The addition of various metal supplements (magnesium, calcium, iron, and trace elements) greatly affected growth and biosurfactant production. The effect of the metal cations, used together, is greater than when they are used individually. The biosurfactant production increased considerably (almost double) by addition of metal supplements. Very high concentrations of metal supplements, however, inhibited biosurfactant production. Amino acids such as aspartic acid, asparagine, glutamic acid, valine, and lysine increased the final yield of biosurfactant by about 60%. The organism could produce biosurfactant at 45°C and within the pH range of 4.5–10.5. The biosurfactant was thermostable and pH stable (from 4.0 to 12.0). The capability of the organism to produce biosurfactant under thermophilic, alkaliphilic, and halophilic conditions makes it a suitable candidate for field applications. Infrared, nuclear magnetic resonance, and mass spectroscopy studies showed the surfactant to be identical to surfactin. R S MakkarSwaranjit Singh Cameotra2012-01-05T15:14:33Z2012-03-29T06:26:05Zhttp://crdd.osdd.net/open/id/eprint/272This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2722012-01-05T15:14:33ZAn update on the use of unconventional substrates for biosurfactant production and their new applications.Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes. Microbes synthesize them, especially during growth on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. Because of their structural diversity (i.e., glycolipids, lipopeptides, fatty acids, etc.), low toxicity, and biodegradability, these molecules could be widely used in cosmetic, pharmaceutical, and food processes as emulsifiers, humectants, preservatives, and detergents. Moreover, they are ecologically safe and can be applied in bioremediation and waste treatments. They can be produced from various substrates, mainly renewable resources such as vegetable oils, distillery and dairy wastes, which are economical but have not been reported in detail. In this review, we report advances made in using renewable substrates for biosurfactant production and their newer applications.R S MakkarSwaranjit Singh Cameotra2013-10-18T04:27:47Z2015-01-09T08:46:09Zhttp://crdd.osdd.net/open/id/eprint/1384This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13842013-10-18T04:27:47ZAnti-inflammatory Agents regulated Nitric Oxide Production in Murine MacrophagesJ Mittal2012-01-05T15:05:24Z2012-02-15T12:07:56Zhttp://crdd.osdd.net/open/id/eprint/251This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2512012-01-05T15:05:24ZIn vitro effects of cAMP-elevating agents and glucocorticoid either alone or in combination on the production of nitric oxide, interleukin-12 and interleukin-10 in IFN-gamma- and LPS-activated mouse peritoneal macrophages.The effects of cAMP-elevating agents, N6-2'-O-dibutyryl cAMP (Bu2cAMP), and glucocorticoid (dexamethasone) on the production of inflammatory mediators--nitric oxide and interleukin-12 (IL-12) and anti-inflammatory mediator interleukin-10 (IL-10) were demonstrated in murine peritoneal macrophages. Inducible nitric oxide synthase (iNOS) and iNOS mRNA were detected by northern blot and western blot, respectively. The cAMP elevating agents Bu2cAMP and prostaglandin E2 each alone did not show any effect on NO production but along with IFN-gamma and lipolysaccharide (LPS) they slightly enhanced NO production. Dexamethasone inhibited NO production in IFN-gamma- and LPS-treated cells; cAMP elevating agents interfered with the NO production inhibited by dexamethasone. Inhibition was revealed at the mRNA level as well as at protein level. Bu2cAMP or dexamethasone either alone or synergistically inhibited IL-12 production; Bu2cAMP interfered with dexamethasone-mediated inhibition of IL-10 production in IFN-gamma- and LPS-treated macrophages. The use of glucocorticoids along with cAMP elevating agents was beneficial in lowering the level of inflammatory mediator IL-12 and producing high levels of the anti-inflammatory mediator IL-10 active in cell protection. On the other hand, interference of Bu2cAMP with dexamethasone-mediated NO inhibition may have adverse effect. Therefore, adverse effects due to cAMP-mediated interference (inhibition) with NO synthesis may occur in many inflammatory diseases during combined drug therapy by glucocorticoids and cAMP elevating agents.J MittalN DograR DassS Majumdar2012-02-13T15:21:07Z2015-01-09T08:58:45Zhttp://crdd.osdd.net/open/id/eprint/905This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9052012-02-13T15:21:07ZCloning, characterization, and expression studies in Escherichia coli of growth hormone cDNAs from Indian zebu cattle, reverine buffalo, and beetal goat.The growth hormone cDNAs from three different economically important animal species of indian origin viz., indian zebu cattle (Bos indicus), indian reverine buffalo (Bubalus bubalis), and beetal goat (Capra hircus) were isolated by the RT-PCR technique. The amplified product was then cloned into phagemid pBluescriptIIKS- and the nucleotide sequence of the entire 573 base coding region for each product was determined. The genetic sequences as well as the translated protein sequence of these ruminant species were compared to that of closely related species like taurine cattle (Bos taurus) and sheep (Ovis aries). A very high degree of nucleotide sequence homology, ranging between 97-98%, was observed. Subsequently, the buffalo and goat cDNAs were used for expression studies in Escherichia coli. Very low levels of expression resulted when the growth hormone cDNAs were directly placed under the strong E. coli (trc) or phage (T7) promoters with the approximate level being less than 0.1% and 1% of the intracellular E. coli proteins, respectively. The nearly 10-fold enhancement of the level of expression as observed was attributable to the nature of the untranslated leader sequence donated by the individual expression element. High level (about 20% of soluble E. coli protein) expression of buffalo/goat growth hormone was achieved as a fusion protein with glutathione-s-transferase (GST) in pGEX-KT. Further, although attempts at converting the GST-GH fusion protein system to a two-cistronic gene expression system were unsuccessful, the utilization of a short synthetic first cistron in the two-cistronic mode of expression resulted in high levels (approximately 30% of soluble protein cell fraction) of GH polypeptide with a native N-terminus in E. coli for all three cDNAs.Utpal Kumar MukhopadhyayGirish Sahni2012-02-06T18:17:28Z2012-02-06T18:17:28Zhttp://crdd.osdd.net/open/id/eprint/900This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9002012-02-06T18:17:28ZProduction of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E. coli strainsThe growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (⩾30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield (≈40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.Utpal Kumar MukhopadhyayGirish Sahni2012-01-05T15:12:54Z2012-01-05T15:12:54Zhttp://crdd.osdd.net/open/id/eprint/266This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2662012-01-05T15:12:54ZProduction of recombinant buffalo (Bubalus bubalis) and goat (Capra hircus) growth hormones from genetically modified E. coli strains.The growth hormone cDNAs of Indian reverine buffalo (Bubalus bubalis) and beetal goat (Capra hircus) were cloned in Escherichia coli through RT-PCR technique. Nucleotide sequencing revealed several silent mutations in both cDNAs and only one amino acid change in the case of goat when compared to reported bovine (Bos taurus) sequence. The high level expression of both the polypeptide hormones was achieved in E. coli (> or =30% of soluble intracellular proteins) through the construction of two-cistronic gene expression system. The solubilisation of recombinant growth hormones from inclusion bodies and subsequent oxidation to correctly folded monomeric form was also carried out. A combination of reverse-phase HPLC and non-reducing SDS-PAGE was successfully applied to distinguish between reduced and oxidised forms of growth hormones. A moderate yield ( approximately 40% of starting material, with potential for upscaling), two-step purification process comprising of hydrophobic interaction and ion-exchange chromatographies was developed. The process eliminates the need for costly, laborious and time-consuming steps of ultrafiltration and dialysis, as reported earlier for the purification of many recombinant animal growth hormones. The biophysical, biochemical and functional analyses of purified refolded polypeptides showed that the hormones produced in this study were identical to natural pituitary bovine growth hormone.Utpal Kumar MukhopadhyayGirish Sahni2012-02-13T15:19:27Z2012-02-13T15:19:27Zhttp://crdd.osdd.net/open/id/eprint/913This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9132012-02-13T15:19:27ZAn insight into the possible mechanism of working of two-cistronic gene expression systems and rational designing of newer systems.The initial attempts at hyper-expressing buffalo/goat growth hormone (GH)-ORFs in Escherichia coli directly under various strong promoters were not successful despite the presence of a functional gene. High level expression of GH was achieved as a fusion protein with glutathione-S-transferase (GST). To produce native GH in an unfused state, we adapted an established strategy of two-cistronic approach in our system. In this strategy, utilizing one of the highly efficient reported sequences as the first cistron led to a nearly 1000-fold enhancement in the level of expression under an E. coli promoter (trc). In search of a newer first-cistron sequence as well as to see the generality of the two-cistronic approach, we explored the ability of different lengths of a highly expressing natural gene to act as an efficient first cistron. Surprisingly, GST, which is naturally highly expressible in E. coli, could not be fitted into a successful two-cistronic construct. In addition, placement of the entire two-cistronic expression cassette (which had earlier given high-level GH expression under trc promoter) under the T7 promoter in E. coli failed to hyper-express GH. These results suggest that the successful exploitation of the two-cistron arrangement for hyper-expression of eukaryotic ORFs in bacteria is not as straightforward as was previously thought. It appears probable that factors such as the sequence context, together with the length and codons used in the first cistron are important as well.Utpal Kumar MukhopadhyayGirish Sahni2012-02-13T15:16:29Z2012-02-13T15:16:29Zhttp://crdd.osdd.net/open/id/eprint/924This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9242012-02-13T15:16:29ZNon-Deposit of Voucher CulturesD PADMANABAN2012-01-05T15:07:08Z2015-01-09T11:21:41Zhttp://crdd.osdd.net/open/id/eprint/257This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2572012-01-05T15:07:08ZChemotaxis of a Ralstonia sp. SJ98 toward co-metabolizable nitroaromatic compounds.We have earlier reported chemotaxis of a Gram-negative, motile Ralstonia sp. SJ98 towards p-nitrophenol (PNP), 4-nitrocatechol (NC), o-nitrobenzoate (ONB), p-nitrobenzoate (PNB), and 3-methyl-4-nitrophenol (MNP) that also served as sole source of carbon and energy to the strain [S.K. Samanta, B. Bhushan, A. Chauhan, R.K. Jain, Biochem. Biophy. Res. Commun. 269 (2000) 117; B. Bhushan, S.K. Samanta, A. Chauhan, A.K. Chakraborti, R.K. Jain, Biochem. Biophy. Res. Commun. 275 (2000) 129]. In this paper, we report chemotaxis of a Ralstonia sp. SJ98 toward seven different nitroaromatic compounds (NACs) by drop assay, swarm plate assay, and capillary assay. These NACs do not serve as sole carbon and energy source to strain SJ98 but are partially transformed in the presence of an alternate carbon source such as succinate. This is the first report showing chemotaxis of a bacterial strain toward co-metabolizable NACs.Gunjan PandeyAshvini ChauhanS K SamantaR K Jain2012-02-06T18:18:15Z2012-04-03T07:27:10Zhttp://crdd.osdd.net/open/id/eprint/895This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8952012-02-06T18:18:15ZBacterial chemotaxis toward environmental pollutants: role in bioremediation.Gunjan PandeyR K Jain2012-01-05T15:11:40Z2015-01-09T08:39:18Zhttp://crdd.osdd.net/open/id/eprint/261This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2612012-01-05T15:11:40ZPseudomonas indica sp. nov., a novel butane-utilizing species.The taxonomic position of two butane-utilizing bacteria was studied using a polyphasic approach. Biochemical and physiological characteristics indicated these to be members of the genus Pseudomonas, showing more similarity to Pseudomonas mendocina than to any other species. The major fatty acids found in these two strains also pointed to their similarity to P. mendocina. On the other hand, DNA-DNA hybridization studies with seven related Pseudomonas species belonging to the gamma-Proteobacteria and the deltaTm values of reassociated molecules clearly showed that these two strains do not belong to any of the seven species tested. The 16S rRNA gene was sequenced and compared with the sequences available in the GenBank database. Phylogenetic analysis using the region covering positions 31-1488 (Escherichia coli numbering) confirmed these observations and placed these two strains as members of the authentic Pseudomonas, but not in any existing species of the genus. On the basis of biochemical characteristics, fatty acid profiles, DNA-DNA reassociation and deltaTm values, as well as 16S rRNA gene sequence analyses, these two isolates were shown to belong to one species but to have characteristics distinct from those of validly described species of Pseudomonas (sensu stricto). These strains, therefore, should be recognized as a novel species, for which the name Pseudomonas indica sp. nov. is proposed. The type strain is strain IMT37T (= MTCC 3713T = DSM 14015T).K K PandeyShanmugam MayilrajT Chakrabarti2013-10-18T08:49:32Z2013-10-18T08:49:32Zhttp://crdd.osdd.net/open/id/eprint/1401This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/14012013-10-18T08:49:32ZMolecualr Biochemical and Genetic Studies on Truncated HamoglobinsRanjana Pathania2012-01-05T15:12:10Z2012-01-05T15:12:10Zhttp://crdd.osdd.net/open/id/eprint/263This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2632012-01-05T15:12:10ZNitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli.Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.Ranjana PathaniaNaveen K NavaniAnne M GardnerPaul R GardnerKanak L Dikshit2012-01-05T15:15:39Z2012-01-05T15:15:39Zhttp://crdd.osdd.net/open/id/eprint/278This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2782012-01-05T15:15:39ZMycobacterium tuberculosis hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli.The truncated hemoglobins HbN and HbO of Mycobacterium tuberculosis H37Rv share little sequence similarity and display structural differences in their EF-loop regions, suggesting distinct function(s) for these hemoglobins. HbO of M. tuberculosis was expressed in Escherichia coli and Mycobacterium smegmatis as a 14.5-kDa homodimeric heme protein exhibiting nearly 50-fold (P(50) approximately 0.51) lower oxygen affinity than HbN. 40-50% of HbO remained associated with the cell membranes and significantly enhanced its respiration in comparison with the membrane fractions of control cells or cells overproducing HbN. Oxygen uptake of HbO-associated membranes was decreased by washing and restored by adding HbO. Additionally, membrane vesicles prepared from terminal oxidase-deficient (cyo(-), cyd(-)) mutants of E. coli did not exhibit significant enhancement in oxygen uptake in the presence of HbO, suggesting its interaction(s) with the electron transport chain. Expression of HbO in Mycobacterium bovis bacillus Calmette-Guérin, an experimental model of M. tuberculosis, was observed (0.2-0.5% of total cellular proteins) throughout its aerobic growth. These results provided evidence for the involvement of HbO with the component of aerobic electron transport chain, suggesting that its function may be related to the facilitation of oxygen transfer during aerobic metabolism of M. tuberculosis. Membrane association properties of HbO may thus play a crucial role in sequestering oxygen and facilitating its availability to internalized M. tuberculosis (an obligate aerobe) under the hypoxic conditions of its intracellular habitat.Ranjana PathaniaNaveen K NavaniGovindan RajamohanKanak L Dikshit2012-02-13T15:20:56Z2015-01-09T09:58:41Zhttp://crdd.osdd.net/open/id/eprint/906This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9062012-02-13T15:20:56ZComparative evaluation of batch and fed-batch bioreactors for GAPDH production by recombinant Escherichia coli with distributed plasmid copy numberIn large bioreactors, there is normally a distribution of plasmid copy numbers among the recombinant cells. This has been modelled here by a Gaussian distribution. For the production of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Escherichia coli containing the plasmid pBR Eco gap, the performances of batch and fed-batch fermentation have been compared for mean copy numbers between 8 and 12 per cell. In both modes of operation, GAPDH activity increased with copy number but biomass concentration decreased, suggesting that optimum performance may be possible by having a distribution of copy numbers in the starting culture.
Since GAPDH activity per unit biomass attained maximum values at the same point in time (3.12 h) for all copy numbers, the effect of a Gaussian distribution of copy numbers with different variances on the performance at this time was studied. Fed-batch required smaller optimum variances than batch fermentation, which allows easier preparation of seed cultures and greater segregational stability in the former case. Although fed-batch operation also generated larger peak activities of GAPDH, this improvement decreased as the copy number increased, again indicating an optimum copy number and its variance.
P R Patnaik2012-02-06T18:16:50Z2015-01-09T09:59:19Zhttp://crdd.osdd.net/open/id/eprint/903This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9032012-02-06T18:16:50ZCondition number analysis for a preliminary evaluation of recombinant fermentation performance Fermentations utilizing genetically modified microbes require accurate monitoring and control. This is done through a control algorithm based on a process model; control usually involves continual measurements and manipulations of the substrate feed rate and its concentration. Since variations in the data, due to the limitations on measurements or external influences, affect the performance, it is useful to have a quick evaluation of these effects before sensitivities or control strategies are studied in detail. Condition numbers provide a convenient way to do this. They have been employed here for a continuous fermentation for -galactosidase production by Saccharomyces cerevisiae containing the plasmid pSXR125. When all recombinant cells have the same number of copies of the plasmid, a selective medium permits less precision in measurement and manipulation than a non-selective medium, but this is reversed when there are two sub-populations with different numbers of plasmids. The more stringent requirement for a selective medium in the latter case is, however, offset by its greater productivity.P R Patnaik2012-02-13T15:20:37Z2012-02-13T15:20:37Zhttp://crdd.osdd.net/open/id/eprint/908This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9082012-02-13T15:20:37ZSteady state performance and sensitivity of a continuous recombinant fermentation with plasmid copy number multiplicity. Fermentations utilizing genetically modified cells usually contain two or more fractions of plasmid-bearing cells differing in the plasmid copy number. Competition among these cells and those without the plasmid may lead to a different performance than in the more 'ideal' situation where all recombinant cells have the same number of plasmids. A recent study considered the steady state sensitivities to the feed concentration of substrate and the dilution rate for a continuous fermentation with Saccharomyces cerevisiae XK1-C2 harbouring the plasmid pSXR125, which synthesizes b-galactosidase, with a uniform copy number. A similar analysis has been applied here to the same fermentation with a binomial distribution in the copy number. Although the steady states and their sensitivities are different from those for a uniform distribution, the dilution rate is still the favoured manipulated variable for control. While a selective medium generates a superior performance, its higher sensitivities require rapid on-line monitoring and anticipatory control.
P R Patnaik2012-01-05T15:14:54Z2012-01-05T15:14:54Zhttp://crdd.osdd.net/open/id/eprint/274This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2742012-01-05T15:14:54ZCan imperfections help to improve bioreactor performance?Pilot-scale and larger bioreactors differ from small laboratory-scale reactors in terms of a greater occurrence of noise and incomplete mixing of the broth. Conventional control tries to induce good mixing and to filter out the noise as completely as possible. As such an 'ideal' operation is difficult to achieve, recent work has tried to exploit the non-ideal features to improve the performance. Using artificial neural networks, the degree of mixing, the extent of filtering of noise and the distribution of plasmid copy number (in a recombinant fermentation) can be controlled effectively on-line. This strategy generates better productivities than well-mixed noise-free operations, which suggests that deviations from ideal behaviour should be gainfully harnessed and not suppressed.Pratap R Patnaik2012-02-13T15:20:46Z2012-02-13T15:20:46Zhttp://crdd.osdd.net/open/id/eprint/907This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9072012-02-13T15:20:46ZTemperature optima of enzymes: sifting fact from fictionThe classic approach to the description of the temperature-dependence of enzyme activity lumps both reversible and irreversible inactivations into one step. This generates an erroneous dependence of the enzymatic rate on temperature and the duration of assay. A two-step ‘equilibrium model’ has been proposed recently to correct these weaknesses. However, this model too has some drawbacks. At short assay times, there are two peaks on the rate–temperature–time surface. This difficulty with fast assays is avoided by slow assays; however, the latter carry a greater risk of irreversible denaturation. This problem disappears on increasing the order of the irreversible step from one to two, suggesting that a second-order equilibrium model may be appropriate for some thermal inactivations.Pratap R Patnaik2013-10-18T06:03:26Z2013-10-22T11:08:25Zhttp://crdd.osdd.net/open/id/eprint/1392This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13922013-10-18T06:03:26ZStructural and Functional Studies on Mycobacterium Tuberculosis Alkylhydroperoxidase C (Ahpc).. Radha Devi2012-02-06T18:19:03Z2012-02-06T18:19:03Zhttp://crdd.osdd.net/open/id/eprint/889This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8892012-02-06T18:19:03ZFunction of the 90-loop (Thr90-Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion.Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr(90)-Glu(100)) for the docking of the substrate PG to the SAK-Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr(92)-Glu(100)) from the SAK 90-loop, resulted in approximately 60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKDelta90) and Pm. The preformed activator complex of SAKDelta90 with Pm, however, displayed a 50-60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK-Pm complex displayed approximately 50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKDelta90. Lysine residues (Lys(94), Lys(96), Lys(97) and Lys(98)) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAK(K97A) and SAK(K98A) exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the K(m) values of PG towards the activator complex of these two SAK mutants were 4-6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys(97) and Lys(98) residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK-Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.Govindan RajamohanMonika DahiyaShekhar C MandeKanak L Dikshit2013-10-18T04:30:39Z2013-10-18T04:30:39Zhttp://crdd.osdd.net/open/id/eprint/1385This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13852013-10-18T04:30:39ZVitreoscilla Hemoglobin: Engineering of Oxygen affinity and studies on its Role in Cellular K Ramandeep2012-02-06T18:16:58Z2015-01-07T04:48:02Zhttp://crdd.osdd.net/open/id/eprint/902This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9022012-02-06T18:16:58ZSandwich microgravimetric immunoassay: Sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystalA multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM)
principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is
sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and
the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin
was used in this study, Mr ∼ 6000 Da) was correlated with the shift of resonant frequency of QCM system before
and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity,
and is sensitive to approx. 1 ng insulin ml−1 with a linear response for insulin from 10 μg ml−1 to 10 ng ml−1 in
standard solutions. The technique may also be applied for the detection of other small biomoleculesSatish SahaManoj RajeC Raman Suri2012-02-06T18:17:34Z2012-02-06T18:17:34Zhttp://crdd.osdd.net/open/id/eprint/899This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8992012-02-06T18:17:34ZEffect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I(2)B.The influence of oxygen on glycerol production by an osmophilic yeast, Candida magnoliae I(2)B, was studied in a bioreactor. Oxygen transfer rates (OTRs) and volumetric oxygen transfer coefficients (k(L)a) were determined at different aeration and agitation rates. Cell growth as well as glycerol production was strongly affected by oxygen supply. Improvement in OTRs resulted in increased cell growth and glycerol yield. However, at high OTRs, there was a reduction in glucose uptake rate, indicating Pasteur Effect, and glycerol accumulation was also reduced at k(L)a of 253 h(-1). The availability of oxygen per unit of cell mass was found to be the most important factor that controlled cell growth, glucose uptake, and glycerol yield. The overall productivity and yield of glycerol could be related with k(L)a. The biosynthesis of glycerol was found to both growth- and non-growth-associated, although glycerol was mainly produced in post-exponential phase.Debendra K SahooGopal P Agarwal2012-01-05T15:14:08Z2015-01-09T11:23:21Zhttp://crdd.osdd.net/open/id/eprint/270This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2702012-01-05T15:14:08ZPolycyclic aromatic hydrocarbons: environmental pollution and bioremediation.Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. Although various physicochemical methods have been used to remove these compounds from our environment, they have many limitations. Xenobiotic-degrading microorganisms have tremendous potential for bioremediation but new modifications are required to make such microorganisms effective and efficient in removing these compounds, which were once thought to be recalcitrant. Metabolic engineering might help to improve the efficiency of degradation of toxic compounds by microorganisms. However, efficiency of naturally occurring microorganisms for field bioremediation could be significantly improved by optimizing certain factors such as bioavailability, adsorption and mass transfer. Chemotaxis could also have an important role in enhancing biodegradation of pollutants. Here, we discuss the problems of PAH pollution and PAH degradation, and relevant bioremediation efforts.S K SamantaOm V SinghR K Jain2013-10-18T04:43:29Z2013-10-18T04:43:29Zhttp://crdd.osdd.net/open/id/eprint/1387This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13872013-10-18T04:43:29ZMolecular and Biochemical Characterization of ATP-Binding Subunit of Mycobacterial Phosphate Specific TransporterJyoti Sarin2012-02-06T18:19:11Z2012-02-06T18:19:11Zhttp://crdd.osdd.net/open/id/eprint/888This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8882012-02-06T18:19:11ZDifferential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.Dibyendu SarkarMarco A AzaroHideki AiharaChristie V PapagiannisRadhakrishna TirumalaiSimone E Nunes-DübyReid C JohnsonTom EllenbergerArthur Landy2012-02-13T15:18:52Z2015-01-19T05:29:44Zhttp://crdd.osdd.net/open/id/eprint/917This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9172012-02-13T15:18:52ZLocalization, Regulation, and Substrate Transport Properties of Bpt1p, a Saccharomyces cerevisiae MRP-Type ABC TransporterSaccharomyces cerevisiae Bpt1p is an ATP-binding cassette (ABC) protein that belongs to the MRP subfamily and is a close homologue of the glutathione conjugate (GS conjugate) transporter Ycf1p. The function of Bpt1p has previously been evaluated only in vitro, by using nonphysiological substrates. In the present study we examined the localization, regulation, and transport properties of Bpt1p in vivo, as well as its capacity to transport a set of prototypical MRP substrates in vitro. Our results show that Bpt1p, like Ycf1p, localizes to the yeast vacuolar membrane, plays a role in cadmium detoxification and ade2 pigmentation in vivo, and can participate in the transport of GS conjugates and glucuronate conjugates, as well as free glutathione, in vitro. However, in all of these cases the contribution of Bpt1p is substantially less than that of Ycf1p. In addition, the expression patterns of YCF1 and BPT1 differ significantly. Whereas YCF1 expression is markedly increased by cadmium, adenine limitation in an ade2 strain, or overexpression of the stress-responsive transcription factor Yap1p, BPT1 expression is only modestly affected under these conditions. Thus, although the functional capabilities of Bpt1p and Ycf1p overlap, their differences in regulation and substrate preference imply that they contribute to cellular detoxification processes in different ways. K G SharmaD. L. MasonG. LiuP. A. ReaAnand K BachhawatS. Michaelis2012-02-13T15:17:31Z2015-01-09T10:51:05Zhttp://crdd.osdd.net/open/id/eprint/923This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9232012-02-13T15:17:31ZPurification and characterisation of a thermostable alkaline lipase from a new thermophilic Bacillus sp. RSJ-1An extracellular alkaline lipase from a new thermophilic Bacillus sp. RSJ-1 was purified to homogeneity by ultrafiltration, followed by ammonium sulfate precipitation, dialysis, Q-Sepharose ion exchange chromatography and Sephacryl S-200 SF gel filtration chromatography. This purification protocol resulted in a 201-fold purification of lipase with 19.7% final yield and the relative molecular weight of the enzyme was determined to be 37 kDa by SDS-PAGE. The kinetic characterisation of the purified enzyme exhibited maximum activity at 50 °C and pH 8.0–9.0. It was stable at 50 °C for 60 min and retained >90% of its original activity for 120 min. The half lives at 55, 60, 65, 70 and 75 °C were 240, 150, 90, 45 and 30 min, respectively. The enzyme was also highly stable in a pH range of 8.0–9.0 for 120 min. The enzyme activity was promoted in the presence of Ca2+, Na+, Mg2+ and Ba2+ and was strongly inhibited by Cs+, K+, Co2+ and Zn2+. EDTA did not affect the enzyme activity, whereas the presence of various oxidizing agents, reducing agents and some surfactants, reduced the enzyme activity. The enzyme was highly stable in the presence of some commercial detergent formulations. The values of Km and Vmax, as calculated from the Lineweaver–Burk plot, were 2.2 mg/ml and 1429 U/ml, respectively.R SharmaS.K SoniR M VohraL.K. GuptaJ.K. Gupta2012-02-13T15:15:41Z2012-02-13T15:15:41Zhttp://crdd.osdd.net/open/id/eprint/926This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9262012-02-13T15:15:41ZProduction of an extracellular alkaline lipase from a new Bacillus sp. RSJ1 and its application in ester hydrolysis.A thermophilic isolate, Bacillus sp. RSJ1 from hot-spring, grew in temperature range of 40 - 65°C and a pH range of 5 -12 with the optima at 50°C and pH 9. The organism grew logarithmically at 50°C from 2 to 13 h in shakecultures before entering into the stationary phase. The pattern of extracellular alkaline lipase production was found to be growth associated and its secretion started as soon as the organism entered the logarithmic phase with the maximum release (9.2 U mL-1) in the late exponential phase (at 12 h). Production of lipase was substantially enhanced when the type, concentration and sources of carbon, nitrogen and surfactant were consecutively optimised. Cotton-seed oil at a concentration of 0.75% in combination with 0.5% Tween 80 and yeast extract in combination with peptone (0.75% each) were found best sources of carbon and nitrogen giving maximum lipase yield. None of the metal ion salts, other than calcium chloride and sodium chloride, incorporated in the production medium improved the lipase yield. The enzyme was found to catalyze reactions of ester hydrolysis with lighter substituents like the chloro and methyl, but was unable to recognize the bulkier substituents like the bromo and the phenyl. Lipase catalyzed the hydrolysis of the esters - butyl phenylacetate, butyl 2-chloropropanoate and butyl 2-methylbutyrate.Rohit SharmaS K SoniR M VohraR S JollyL.K. GuptaJ.K. Gupta2012-02-13T15:18:10Z2012-02-28T16:15:32Zhttp://crdd.osdd.net/open/id/eprint/920This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9202012-02-13T15:18:10ZDetection of Orientation of MHC Class II Binding Peptides Using Bioinformatics ToolsHarpreet SinghG.P.S. Raghava2012-02-13T15:19:12Z2012-02-13T15:19:12Zhttp://crdd.osdd.net/open/id/eprint/914This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9142012-02-13T15:19:12ZTwo-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p.The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1)--containing the first NBD domain--and also the C-terminal region of Pdr5p, interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein.G Subba RaoAnand K BachhawatC M Gupta2012-01-05T15:14:18Z2015-07-22T05:35:46Zhttp://crdd.osdd.net/open/id/eprint/271This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2712012-01-05T15:14:18ZImmunosensors for pesticide analysis: antibody production and sensor development.Immunosensors, a type of affinity biosensor, are based on the binding interactions between an immobilized biomolecule (antibody/antigen) on the electronic transducer surface with the analyte of interest (antigen/antibody), resulting in a detectable signal. The sensor system takes advantage of the high selectivity provided by the molecular recognition characteristic of an antibody, which binds reversibly with a specific antigen. This review article presents the current status of immunosensors, highlighting their potential benefits and limitations for pesticide analysis. The basic criteria for generating specific antibodies against low-molecular-mass pesticides, which are usually nonimmunogenic in nature, are briefly discussed. The article also describes the fundamentals of important transducer technologies and their use in immunosensor development.C Raman SuriManoj RajeGrish C Varshney2012-01-05T15:16:13Z2015-01-09T10:48:29Zhttp://crdd.osdd.net/open/id/eprint/281This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2812012-01-05T15:16:13ZDistinct role of CD80 and CD86 in the regulation of the activation of B cell and B cell lymphoma.To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.Susmit SuvasVinod SinghSudhir SahdevH VohraJ N Agrewala2012-02-13T15:17:41Z2015-01-07T05:27:32Zhttp://crdd.osdd.net/open/id/eprint/922This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9222012-02-13T15:17:41ZRegulation of memory CD4 T cells: generation, localization and persistence.Susan L SwainJ N AgrewalaDeborah M BrownEulogia Román2012-02-06T18:18:05Z2012-04-02T09:39:36Zhttp://crdd.osdd.net/open/id/eprint/896This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8962012-02-06T18:18:05ZMUC4 expression increases progressively in pancreatic intraepithelial neoplasia.Pancreatic adenocarcinoma is believed to develop from histologically identifiable intraductal lesions known as pancreatic intraepithelial neoplasias (PanINs) that undergo a series of architectural, cytologic, and genetic changes, a progression model similar to the adenoma-carcinoma sequence in the colon. The apomucin MUC4 has been implicated in invasive pancreatic adenocarcinoma. MUC4 expression is not detectable at the RNA level in normal pancreas but is detectable at high levels in invasive pancreatic adenocarcinoma. We documented the pattern of expression of MUC4 in PanINs by studying a series of 71 PanIN lesions immunohistochemically using a new monoclonal antibody to MUC4. Five (17%) of 30 PanIN-1 lesions, 10 (36%) of 28 PanIN-2 lesions, 11 (85%) of 13 PanIN-3 lesions, and 25 (89%) of 28 invasive adenocarcinomas labeled with the MUC4 antibody used in the study. In addition, afew nonneoplastic lesions labeled with the MUC4 antibody, including reactive ducts in chronic pancreatitis, atrophic ducts filled with inspissated secretions, and ducts showing squamous metaplasia. Our data help establish the patterns of MUC4 expression in neoplastic precursors in the pancreas and add further support to the progression model for pancreatic adenocarcinoma.Michael J SwartzSurinder K BatraGrish C VarshneyMichael A. HollingsworthCharles J YeoJohn L CameronRobb E WilentzRalph H HrubanPedram Argani2012-01-05T15:15:32Z2012-01-05T15:15:32Zhttp://crdd.osdd.net/open/id/eprint/277This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2772012-01-05T15:15:32ZStructure of Mycobacterium tuberculosis chaperonin-10 at 3.5 A resolution.Chaperonin-60 (cpn60) and chaperonin-10 (cpn10) are essential proteins involved in ATP-dependent folding of several intracellular proteins in the bacterial cell. Folding of the nascent substrate polypeptide takes place in the large central cavity formed by each ring of the tetradecameric cpn60. This large cavity is closed upon capping by the heptameric cpn10. Cpn10s interact with cpn60s primarily through a 17-residue mobile loop and regulate the release and binding of the substrate polypeptide from the cpn60 surface. Here, the structure of M. tuberculosis cpn10 is reported at 3.5 A resolution. The overall structure of the cpn10 monomer is formed of a four-stranded beta-barrel and two long stretches of highly flexible segments: the dome loop and the mobile loop. The seven subunits in the heptamer show very little conformational difference and exhibit nearly perfect sevenfold geometry. The binding sites for metal ions in the dome loop of cpn10 have been identified, suggesting the role of metal ions in the stabilization of the protein. Comparisons with the available cpn10 structures indicate several interesting features.Bhupesh TanejaShekhar C Mande2012-01-05T15:12:18Z2015-01-09T11:45:15Zhttp://crdd.osdd.net/open/id/eprint/264This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/2642012-01-05T15:12:18ZOccurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India.Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.M Thungapathral AmitaKislay Kumar SinhaSaumya Ray ChaudhuriP. GargThandavarayan RamamurthyG B NairAmit Ghosh2013-10-18T04:52:43Z2013-10-18T04:52:43Zhttp://crdd.osdd.net/open/id/eprint/1388This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/13882013-10-18T04:52:43ZStructure -Function Studies of StreptokinaseS Vasudha2012-02-06T18:18:23Z2012-02-06T18:18:23Zhttp://crdd.osdd.net/open/id/eprint/894This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/8942012-02-06T18:18:23ZMatrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge.An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.Joachim VaterBärbel KablitzChristopher WildePeter FrankeNeena MehtaSwaranjit Singh Cameotra2012-02-06T18:16:32Z2012-03-28T09:39:50Zhttp://crdd.osdd.net/open/id/eprint/904This item is in the repository with the URL: http://crdd.osdd.net/open/id/eprint/9042012-02-06T18:16:32ZStudies on the production of phytase by a newly isolated strain of Aspergillus niger var teigham obtained from rotten wood-logsA hyper-producing strain of Aspergillus niger var teigham was used for the production of phytase in shake flask and fermenter. This newly isolated strain maximally produced 184 nkat/ml phytase activity in minimal medium with a specific activity of 21367 nkat/mg protein. The optimum growth temperature of A. niger was 37 °C while maximum enzyme activity was obtained when the organism was grown at 30 °C and at an initial pH of 6.5. Among the various carbon sources used, a combination of glucose and starch (3 and 1%, respectively) was found to be optimum for phytase production. Although biopeptone and ammonium nitrate gave the maximum enzyme yield, the specific activity of phytase was comparatively higher when ammonium nitrate was used as sole source of nitrogen. A sharp decline in phytase production was observed even at 0.05% phosphorous in the medium with no production at 0.1% and above, indicating end product inhibition in phytase synthesis. In shake flasks, the productivity of phytase was 10824 nkat/l.d while fermenter productivity was 20 000 nkat/l.d.Purva VatsU C Banerjee