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Ma H Jiang WR Robledo N Leveque V Ali S Lara-Jaime T Masjedizadeh M Smith DB Cammack N Klumpp K Symons J 《The Journal of biological chemistry》2007,282(41):29812-29820
beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes. 相似文献
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Guo Y Weller P Farrell E Cheung P Fitch B Clark D Wu SY Wang J Liao G Zhang Z Allard J Cheng J Nguyen A Jiang S Shafer S Usuka J Masjedizadeh M Peltz G 《Nature biotechnology》2006,24(5):531-536
Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice. 相似文献
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Alvandi Amirhooshang Farajzadeh Ahmad Ghaforian Borojerdnia Mehri Jelodar Abbass Aryan Ehsan Gholipour Abolfazl Masjedizadeh Abdolrahim Makvandi Manoochehr 《World journal of microbiology & biotechnology》2011,27(4):969-974
UreB is one of the urease subunits of Helicobacter pylori and can be used as an excellent antigen candidate for H. pylori vaccination. Easy access to highly purified UreB protein, facilitate advances in therapeutic or preventive strategies. To
achieve a simplified purification procedure, the present report represents a novel method of producing recombinant urease
subunit B extracellularly. ureB gene from 26,695 standard strain was amplified by PCR and cloned into pET-26b(+) expression vector. UreB was expressed as
a soluble, N-terminal pelB and C-terminal hexahistidine-tagged fusion protein (UreB-6His) and secreted into the periplasmic
space of Escherichia coli. Expression of the recombinant UreB in E. coli BL21 (DE3) was induced by isopropylthio-β-d-galactoside (IPTG). Expression of UreB was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
and western blot analysis using anti-His monoclonal antibody. UreB-6His protein was extracted from the periplasm by osmotic
shock treatment and was purified in one step by Nickel affinity chromatography. In conclusion, the present protocol is easier
to perform; more time effective and low cost than earlier methods. 相似文献
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