02308nas a2200241   4500008004100000022001400041245015900055210006900214260007900283300001000362490000700372520137500379653002001754653001901774653001401793653001401807653001901821100002501840700003401865700002301899700001401922856013001936       2010                        eng d  a0196-978100aMolecular cloning, over expression, and activity studies of a peptidic HIV-1 protease inhibitor: designed synthetic gene to functional recombinant peptide0 aMolecular cloning over expression and activity studies of a pept  a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcJAN  a16-210 v313 a<p>The aspartic protease inhibitor (ATBI) purified from a Bacillus sp. is a potent inhibitor of several proteases including recombinant HIV-1 protease, pepsin, and fungal aspartic protease. In this study, we report the cloning, and over expression of a synthetic gene coding for ATBI in Escherichia coli and establish a purification Protocol. The ATBI molecule consists of eleven amino acids and is peptidic in nature. We used the peptide sequence data of ATBI to synthesize complementary oligonucleotides, which were annealed and subsequently cloned in-frame with the gene for glutathione-S-transferase (GST). The expression of the resulting fusion protein was induced in E. coli BL21-A1 cells using arabinose. The recombinant peptide was Purified using a reduced glutathione column, and cleaved with Factor Xa to remove the GST tag. The resultant product was further purified to homogeneity using RP-HPLC. Mass spectroscopy analysis revealed that the purified peptide had a molecular weight of 1186 Da which matches the theoretical molecular weight of the amino acids present in the synthetic gene. The recombinant peptide was found to be active in vitro against HIV-1 protease, pepsin, and fungal aspartic protease. The protocol described in this study may be used to clone pharmaceutically important peptide molecules. (C) 2009 Elsevier Inc. All rights reserved.</p>10aGateway cloning10aHIV-1 protease10ainhibitor10aProteases10aSynthetic gene1 aVathipadiekal, Vinod1 aUmasankar, Perunthottathu, K.1 aPatole, Milind, S.1 aRao, Mala  uhttps://library.ncl.res.in/content/molecular-cloning-over-expression-and-activity-studies-peptidic-hiv-1-protease-inhibitor-002223nas a2200217   4500008004100000022001400041245016600055210006900221260007600290300001200366490000700378520134900385653003001734653001401764653003101778653002201809653000901831100001801840700001901858856012801877       2012                        eng d  a1087-057100aPotential use of nitrate reductase as a biomarker for the identification of active and dormant inhibitors of mycobacterium tuberculosis in a THP1 infection model0 aPotential use of nitrate reductase as a biomarker for the identi  a2455 TELLER RD, THOUSAND OAKS, CA 91320 USAbSAGE PUBLICATIONS INCcAUG  a966-9730 v173 a<p>The development of a macrophage-based, antitubercular high-throughput screening system could expedite discovery programs for identifying novel inhibitors. In this study, the kinetics of nitrate reduction (NR) by Mycobacterium tuberculosis during growth in Thp1 macrophages was found to be almost parallel to viable bacilli count. NR in the culture medium containing 50 mM of nitrate was found to be optimum on the fifth day after infection with M. tuberculosis. The signal-to-noise (S/N) ratio and Z-factor obtained from this macrophage-based assay were 5.4 and 0.965, respectively, which confirms the robustness of the assay protocol. The protocol was further validated by using standard antitubercular inhibitors such as rifampicin, isoniazid, streptomycin, ethambutol, and pyrazinamide, added at their IC90 value, on the day of infection. These inhibitors were not able to kill the bacilli when added to the culture on the fifth day after infection. Interestingly, pentachlorophenol and rifampicin killed the bacilli immediately after addition on the fifth day of infection. Altogether, this assay protocol using M. tuberculosis-infected Thp-1 macrophages provides a novel, cost-efficient, robust, and easy-to-perform screening platform for the identification of both active and hypoxic stage-specific inhibitors against tuberculosis.</p>10ahigh-throughput screening10ainhibitor10aMycobacterium tuberculosis10anitrate reductase10aTHP11 aSarkar, Sampa1 aSarkar, Dhiman  uhttps://library.ncl.res.in/content/potential-use-nitrate-reductase-biomarker-identification-active-and-dormant-inhibitors-0