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Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup. 相似文献
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Wu R Ma CX Yang MC Chang M Littell RC Santra U Wu SS Yin T Huang M Wang M Casella G 《Genetical research》2003,81(1):51-64
Growth trajectories are a biological process important to plant and animal breeding, and to evolutionary genetic studies. In this article, we report the detection of quantitative trait loci (QTLs) responsible for growth trajectories in poplars that are used as a model system for the study of forest biology. These QTLs were localized on a genetic linkage map of polymorphic markers using a statistical mapping method incorporating growth-curve models. The effects of the QTLs on growth are described as a function of age, so that age-specific changes in QTL effects can be readily projected throughout the entire growth process. The QTLs identified display increased effects on growth when trees age, yet the timing of QTL activation is earlier for stem height than diameter, which is consistent with the ecological viewpoint of canopy competition. The implications of the results for breeding and silviculture are discussed. 相似文献
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Supratim Ghosh Sumana Mallick Upasana Das Ajay Verma Uttam Pal Sabyasachi Chatterjee Abhishek Nandy Krishna D. Saha Nakul Chandra Maiti Bikash Baishya G. Suresh Kumar William H. Gmeiner 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(3):485-494
We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ? H of curcumin heptadiene chain are closely positioned to the A16-H8 and A17-H8, while G12-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy. 相似文献
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Summary Ten antibiotic resistance mutants inBacillus subtilis have been tested forin vitro resistance in cell-free amino acid incorporating systems. Seven of these mutants,str-1, ole-2, spc-2, mic-1, bry-2, nea-1, andlin-2 are shown to have alterations in ribosomal function.neo-2, kan-2 andery-1 did not showin vitro alterations. Extensive cross-resistance and some cross-sensitivity to ribosomal antibiotics are determined by these mutations. 相似文献
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Genetic mapping of aminoglycoside and fusidic acid resistant mutations in Bacillus subtilis 总被引:6,自引:0,他引:6
Summary Mutations governing resistance to neamine, neomycin, kanamycin and fusidic acid have been mapped within the ribosomal region of theBacillus subtilis genome using PBS1-mediated transduction. The probable order of these closely linked markers is:
This work was supported by a predoctoral training grant, GM-1290, from the National Institutes of Health, administered by the Department of Microbiology, N.Y.U. School of Medicine, and by a research grant from the National Science Foundation, GB-16782, awarded to I.S. 相似文献