Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes.
We report here some efficient biotransformations using Escherichia coli strains with disruptions for the AcrAB-TolC efflux pump system. Biotransformations of compactin into pravastatin (6alpha-hydroxy-iso-compactin) were performed using E. coli strains with tolC and/or acrAB mutations expressing a cytochrome P450 (P450) gene. The production levels of pravastatin using strains with acrAB, tolC, and tolC acrAB mutations increased by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the production levels of 25-hydroxy vitamin D3 and 25-hydroxy 4-cholesten 3-one using tolC acrAB mutant strains expressing an individual P450 gene increased by 2.2- and 16-fold, respectively. The enhancement of this biotransformation efficiency could be explained by increases in the intracellular amounts of substrates and the concentrations of active P450s. These results demonstrate that we have achieved versatile methods for efficient biotransformations using E. coli strains with tolC acrAB mutations expressing P450 genes. (+info)
Measurement of serum 7alpha-hydroxy-4-cholesten-3-one (or 7alphaC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography-tandem mass spectrometry.
Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.
Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. (+info)
Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways.
AIM: To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action. METHODS: The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential (Deltapsim) were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases (PI3K). RESULTS: Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 micromol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt. CONCLUSION: Alisol B acetate exhibits an anti-proliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways. (+info)
Regulation of diurnal variation of cholesterol 7alpha-hydroxylase (CYP7A1) activity in healthy subjects.
Cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory enzyme of bile acid synthesis, displays a pronounced diurnal variation. To better understand the regulation of CYP7A1 activity, three day-long examinations were carried out in 12 healthy men. The concentrations of 7alpha-hydroxycholest-4-en-3-one (C4), a surrogate marker of CYP7A1 activity, bile acids (BA), insulin, glucose, nonesterified fatty acids, triglycerides, and cholesterol were measured in serum in 90-min intervals from 7 AM till 10 PM. To lower and to increase BA concentration during the study, the subjects received cholestyramine and chenodeoxycholic acid (CDCA), respectively, in two examinations. No drug was used in the control examination. There was a pronounced diurnal variation of C4 concentration with a peak around 1 PM in most of the subjects. The area under the curve (AUC) of C4 concentration was five times higher and three times lower when subjects were treated with cholestyramine and CDCA, respectively. No relationship was found between AUC of C4 and AUC of BA concentration, but AUC of C4 correlated positively with that of insulin. Moreover, short-term treatment with cholestyramine resulted in about 10 % suppression of glycemia throughout the day. Our results suggest that insulin is involved in the regulation of diurnal variation of CYP7A1 activity in humans. (+info)
Analyses of biologically active steroids: antitumor active OSW-1 and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.
Naturally occurring constituents of biological or pharmaceutical interest often exist in the form of glycosides or conjugates. Mass spectral investigations of these compounds require soft ionization techniques if information on molecular mass, sugar sequence, or conjugate content is desired. In this study, matrix-assisted laser desorption/ionization (MALDI) quadrupole ion trap (QIT) time-of-flight tandem mass spectrometry (TOF-MS(n)) was used to identify both OSW-1, an acetylated cholestane diglycoside showing antitumor activity, and the cardiotonic steroid, bufotoxin. Each molecular-related ion was identified, and subsequent collision-induced dissociation experiments in which a molecular-related ion was selected as a precursor ion produced the characteristic product ions that are essential for structural elucidation. OSW-1 and its analogue with a modified side chain, thienyl OSW-1, were synthesized, and bufotoxins, i.e., marinobufotoxin and its homologue, marinobufagin 3-pimeloylarginine ester, were isolated from toad venom. On MALDI-TOF-MS, sodium-adduct [M+Na](+) ions were observed in the steroid glycosides, although protonated [M+H](+) ions were relatively more abundant than sodium-adduct [M+Na](+) ions in the bufotoxins. On the basis of tandem MS results, we propose key fragmentation pathways. The sugar moiety or side chain from the precursor ion was eliminated in OSW-1. However, characteristic product ions originating from the cleavage of the side chain with an ester formation were observed in the bufotoxins. Post-source decay (PSD) on MALDI-TOF-MS is also described when evaluating alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as a matrix to obtain useful ions required for the identification of compound. (+info)
Olesoxime (cholest-4-en-3-one, oxime): analgesic and neuroprotective effects in a rat model of painful peripheral neuropathy produced by the chemotherapeutic agent, paclitaxel.