Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P-450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. The CYP composition of enterocytes could thus affect the potential toxicity or therapeutic efficacy of xenobiotics by modifying systemic uptake. We have characterized human enterocyte CYP composition to enable assessment of its functional roles. An isolation method for enterocytes from human small intestine was developed using EDTA buffer-mediated elution. Villous enterocytes were isolated in high yield, separated from crypt cells. Reverse transcriptase-polymerase chain reaction of total RNA from enterocytes revealed that CYP1A1, 1B1, 2C, 2D6, 2E1, 3A4, and 3A5 mRNA were expressed, but only CYP2C and 3A4 were detectable by Western immunoblotting in enterocyte microsomes from 10 human small intestines, whereas CYP1A1 was weakly detectable in two of eight intestines tested. Microsomal protein content decreased markedly along the small intestine from the duodenum to the ileum, whereas total CYP content and CYP3A4 erythromycin N-demethylase activity increased slightly in progressing from the duodenum to the jejunum and then decreased markedly toward the ileum. Levels of CYP3A4 and 2C protein did not decrease in concert as a function of length along the intestine distally. Maximal CYP content for the 10 intestines varied from 0.06 to 0.18 nmol/mg microsomal protein and maximal CYP3A4 erythromycin N-demethylase activity varied from 0.30 to 0.76 nmol/min/mg microsomal protein. In conclusion, CYP3A4 is the major form of CYP expressed in human small intestine enterocytes, CYP3A5 expression was not detected, CYP2C and, in some intestines, CYP1A1 were expressed. The highest metabolic activity occurred in the proximal intestine.
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PMID:Characterization of human small intestinal cytochromes P-450. 1038 24

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.
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PMID:Ethynylestradiol-mediated induction of hepatic CYP3A9 in female rats: implication for cyclosporine metabolism. 1057 34

Elevated leukotriene (LT)C(4) synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16(+) neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16(+) neutrophils from all tested patients transformed exogenous LTA(4) to LTC(4). These cells also produced LTC(4 )after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC(4) synthase mRNA in CML CD16(+) neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC(4) synthase at the protein level in CML CD16(+) neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC(4) synthase activity or expression of the protein could not be demonstrated in CD16(+) neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16(+) neutrophils, transformed LTA(4) to LTB(4). The results indicate that aberrant expression of LTC(4) synthase is a regular feature of morphologically mature CML CD16(+) neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC(4) synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC(4 )has been demonstrated to stimulate proliferation of human bone marrow-derived myeloid progenitor cells. (Blood. 2000;95:1456-1464)
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PMID:Aberrant expression of active leukotriene C(4) synthase in CD16(+) neutrophils from patients with chronic myeloid leukemia. 1066 25

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.
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PMID:RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice. 1123 May 74

The present study reports the genomic organization and the characterization of a novel cynomolgus monkey UDP-glucuronosyltransferase (UGT) enzyme, UGT2B30. UGT enzymes are microsomal proteins that catalyse the transfer of the glucuronosyl group from UDP-glucuronic acid (UDPGA) to a wide variety of lipophilic compounds, namely hormonal steroids. The 15 kb UGT2B30 gene amplified by PCR showed a genomic organization similar to those encoding UGT2B human enzymes. The cDNA encoding UGT2B30 was isolated from a cynomolgus monkey prostate cDNA library, and the deduced amino acid sequence showed an identity of 94% with UGT2B19, a monkey isoform previously characterized. Stable expression of UGT2B30 protein in human kidney 293 (HK293) cells was assessed by Western-blot analysis and its conjugating activity was screened using 39 potential substrates. The UGT2B30 enzyme is active on many compounds of different classes, including testosterone, dihydrotestosterone, 5alpha-androstane-3alpha,17beta-diol, androsterone, oestradiol, tetrahydroaldosterone and tetrahydrocortisone, with glucuronidation efficiencies (V(max)/K(m) ratios) ranging from 0.6 to 8.8 microl x min(-1) x mg of protein(-1). Reverse-transcriptase-PCR analysis revealed that the UGT2B30 transcript is expressed in several tissues, including prostate, testis, mammary gland, kidney, adrenals and intestine. The relative activity of UGT2B30 in comparison with other simian UGT2B isoforms, as well as its large variety of substrates, strongly suggest that this enzyme is essential to inactivation of several steroids.
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PMID:Isolation and characterization of the monkey UGT2B30 gene that encodes a uridine diphosphate-glucuronosyltransferase enzyme active on mineralocorticoid, glucocorticoid, androgen and oestrogen hormones. 1207 53

Tipranavir (TPV) is a non-peptidic protease inhibitor belonging to the class of 4-hydroxy-5,6-dihydro-2-pyrones, which exhibits potent and specific activity against HIV type I (HIV-1) and 2 (HIV-2). Clinically effective plasma levels of TPV are achieved by concomitant administration of ritonavir (RTV). Therefore, TPV has been coadministered with RTV in clinical trials. TPV has demonstrated antiviral activity against HIV-1 isolates that are resistant to reverse-transcriptase and selected peptidic protease inhibitors. Therefore, TPV is emerging as one of the newer drugs in the armamentarium against HIV-1 in patients demonstrating multi-drug resistance. TPV administered orally to humans exhibits linear pharmacokinetics at doses of 100 - 2000 mg. Steady-state plasma levels are attained within 7 days of initiating multiple dosing. The half-life of the drug is approximately 6 h at steady-state. The plasma concentration is lower with repeated dosing than predicted from single-dose studies due to induction of the cytochrome p450 3A4 isoform of the liver microsomal enzyme system. Phase II clinical trials have shown that the administration of TPV and RTV in combination is safe and generally well-tolerated in HIV-1-infected adults. Phase III trials are underway to compare the efficacy of this drug versus other antiretroviral regimens. Gastrointestinal toxicity has been described with TPV, the most frequently reported side effects being diarrhoea, nausea, vomiting and abdominal pain. There is no known evidence of teratogenicity or effect on fertility. TPV dosed twice-daily, in the range of 500 - 1250 mg and combined with 100 - 200 mg of RTV has been shown to substantially and durably reduce viral load in HIV-1-infected drug-naive and experienced patients.
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PMID:Tipranavir: a novel non-peptidic protease inhibitor for the treatment of HIV infection. 1458 57

The effect of the administration of Thonningia sanguinea (T. S.) on the abundance of individual components of the cytochrome P450 monooxygenase enzyme was examined using Western blotting and competitive reverse-transcriptase-polymerase chain reaction (RT-PCR). We also investigated the time-course of inhibition of T. S. on drug metabolizing enzymes. A single intraperitoneal dose of T. S. extract (5 ml/kg) suppressed CYP, cytochrome b5 and NADPH-CYP reductase activity by 45%, 34% and 22% respectively 24 h after T. S. administration. While T. S. did not have any significant effect on microsomal glutathione S-transferase activity, it inhibited p-nitrophenol hydroxylase (PNPH, CYP2E1) and 7-methoxyresorufin O-demethylase (MROD, CYP 1A2) activities by 37% and 32% respectively at 12 h post-T. S. administration. PNPH, erythromycin N-demethylase (ERDM, CYP 3A1/2) and MROD activities were inhibited by 28-36% 24 h after T. S. injection. Consistent with these observations, the levels of CYP2E1, CYP1A2 and CYP3A2 proteins were also suppressed 24 h post-T. S. administration. While CYP2E1 mRNA was unaffected by T. S. administration, CYP1A2 and CYP3A2 mRNAs were decreased by T. S. Cytosolic glutathione S-transferase activity was increased by 30%, 6 h after T. S injection. These data demonstrate that administration of T. S. differentially affect CYP isoforms in the liver of rats and that T. S. selectively suppresses CYP3A2 and CYP1A2 gene expression.
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PMID:Selective suppression of cytochrome P450 gene expression by the medicinal herb, Thonningia sanguinea in rat liver. 1474 31

This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100 microl. A nominal 30 mM source compound concentration provided high microM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for (125)I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced 'hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle.
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PMID:Origin and evolution of high throughput screening. 1760 42

This study aimed to address the relative contributions of the proinflammatory cytokine interleukin-6 (IL-6) and the cytokine-like hormone leptin to the genomic activation of brain cells during lipopolysaccharide (LPS)-induced systemic inflammation. Wildtype and IL-6KO mice were injected with LPS (50 microg/kg, intraperitoneally) and the brains analyzed by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). LPS induced a pronounced nuclear translocation of the signal transducer and activator of transcription (STAT3) throughout the brains of wildtype mice, an effect that was significantly diminished, but not abolished, in the IL-6KOs. The remnant STAT3-activation, although still observed within some of the same areas activated by IL-6, was most intense in ependymal and meningial cells and along distinct blood vessels throughout the brain. This expression was almost totally abolished in the presence of an anti-leptin antiserum. Interestingly, the induction of cyclooxygenase 2 and microsomal prostaglandin E synthase (mPGES), the rate-limiting enzymes for synthesis of PGE2 by LPS, was diminished to a degree that correlated with the absence of IL-6 but not entirely with leptin. These results demonstrate that the induction of the inflammatory pathway in the brain is mediated by both IL-6 and leptin, which appear to work in tandem. Unlike IL-6, however, the contribution of leptin to this response was limited to distinct cell types/brain areas and STAT3-responsive target genes implicated in the brain-controlled sickness-type response. The physiological significance of leptin's action on meningeal and endothelial cells remains to be clarified but might reflect a role in LPS-induced immune cell infiltration into the brain.
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PMID:Selective contribution of interleukin-6 and leptin to brain inflammatory signals induced by systemic LPS injection in mice. 1880 40

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.
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PMID:Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins. 1914 38


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