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Crude Cell Extracts (crude + cell_extract)

Distribution by Scientific Domains
Life Sciences100%

Selected Abstracts

High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection

ELECTROPHORESIS, Issue 1-2 2003
Yan He
Abstract Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC50 value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases. [source]

Mutagenicity of nitroaromatic degradation compounds

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2003
Ranjit S. Padda
Abstract The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100. A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system. A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds. The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100. This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test. Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic. In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic. These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate. [source]

Mechanism of H-8 inhibition of Cyclin-dependent kinase 9: study using inhibitor-immobilized matrices

GENES TO CELLS, Issue 3 2003
Daisuke Shima
Background: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-,-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. Results: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. Conclusions: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data. [source]

,-Galactosidase from Lactobacillus pentosus: Purification, characterization and formation of galacto-oligosaccharides

BIOTECHNOLOGY JOURNAL, Issue 8 2010
Thomas Maischberger
Abstract A novel heterodimeric ,-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o -nitrophenyl-,-D-galactopyranoside (oNPG) were 38 mM, 20 s -1, 530 M -1·s -1 and 1.67 mM, 540 s -1, 325 000 M -1·s -1, respectively. The temperature optimum of ,-galactosidase activity was 60,65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal ,-galactosidases. Mg2+ ions enhanced both activity and stability significantly. L. pentosus ,-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of ,-(1,3) and ,-(1,6) linkages, and the main transgalactosylation products identified were the disaccharides ,-D-Galp -(1,6)- D -Glc, ,-D-Galp -(1,3)- D -Glc, ,- D -Galp -(1,6)- D -Gal, ,- D -Galp -(1,3)- D -Gal, and the trisaccharides ,- D -Galp -(1,3)- D -Lac, ,- D -Galp -(1,6)- D -Lac. [source]