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Sample records for human cyp isoforms

  1. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  2. Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

    DEFF Research Database (Denmark)

    Redlich, Gorden; Zanger, Ulrich M; Riedmaier, Stephan;

    2008-01-01

    In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based...

  3. Effects of two novel sugar drug candidates on CYP450 isoforms in different sexed Chinese human liver microsome in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Jie; ZHANG Xin-hui; SU Jia-ru

    2008-01-01

    The sex-based differences between the effects of two novel sugar-based drug candidates, a sulfated polymannuroguluronate (SPMG-911) and an acidic oligosaccharide sugar chain compound (AOSC-971), on the enzymes CYP 1A2, CYP2E1 and CYP3A4 of Chinese human liver microsome were investigated. The results showed that neither SPMG-911 nor AOSC-971 have any effect on CYP3A4, AOSC-971 induced the CYP 2E1 in men but have no effect on CYP1A2, SPMG-911 inhibit the CYP1A2 also in men but have no effect on CYP2E1. The results are useful for their safety evaluation, as well as for the prediction of interdrug interactions associated with the two drugs.

  4. Enzyme kinetic study of a new cardioprotective agent, KR-32570 using human liver microsomes and recombinant CYP isoforms.

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    Kim, Hyojin; Seo, Kyung-Ah; Kim, Hyunmi; Lee, Hye Suk; Lee, Choong-Hwan; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2007-04-01

    KR-32570 (5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. Human liver microsomal incubation of KR-32570 in the presence of NADPH resulted in the formation of two metabolites, hydroxy-KR-32570 and O-desmethyl-KR-32570. In this study, a kinetic analysis of the metabolism of two metabolites from KR-32570 was performed in human liver microsomes, and recombinant CYP1A2, and CYP3A4. The metabolism for hydroxy- and O-desmethyl-KR-32570 formation from KR-32570 by human liver microsomes was best described by a Michaelis-Menten equation and a Hill equation, respectively. The Cl(int) values of hydroxy- and O-desmethyl-KR-32570 formation were similar to each other (0.03 vs 0.04 microL/min/pmol CYP, respectively). CYP3A4 mediated the formation of hydroxy-KR-32570 from KR-32570 with Cl(int) = 0.24 microL/min/pmol CYP3A4. The intrinsic clearance for O-desmethyl-KR-32570 formation by CYP1A2 was 0.83 AL/min/pmol CYP1A2. These findings suggest that CYP3A4 and CYP1A2 enzymes are major enzymes contributing to the metabolism of KR-32570.

  5. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

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    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

  6. Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

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    Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike (Vanderbilt)

    2010-11-16

    14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} for fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.

  7. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    Science.gov (United States)

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 micro

  8. Hormonal regulation of Cyp4a isoforms in mouse liver and kidney.

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    Zhang, Youcai; Klaassen, Curtis D

    2013-12-01

    Mouse Cyp4a subfamily, including Cyp4a10, Cyp4a12a, Cyp4a12b and Cyp4a14, demonstrate a gender- and strain-specific expression in liver and kidney. In C57BL/6 mouse liver and kidney, Cyp4a12a and 4a12b are male-predominant, whereas Cyp4a14 is female-predominant. Cyp4a10 is female-predominant in liver, but shows no gender difference in kidney. The present study was aimed to determine whether sex hormones and/or growth hormone (GH) secretion patterns are responsible for the gender-specific Cyp4a expression in C57BL/6 mice. Gonadectomized mice, GH-releasing hormone receptor-deficient little (lit/lit) mice and hypophysectomized mice were used with replacement of sex hormones or GH in male or female secretion patterns. Both androgens and male-pattern GH regulated the gender-divergent Cyp4a10, 4a12a and 4a12b in liver, whereas androgens played an exclusive role in regulating Cyp4a10 and 4a12a in kidney. In contrast, Cyp4a12b was increased by male-pattern GH but not androgens in kidney. The female-predominant Cyp4a14 in liver and kidney was due to a combined effect of male-pattern GH and androgens. In addition, estrogens played a minor role in regulation of Cyp4a isoforms through an indirect pathway. In conclusion, gender-divergent Cyp4a mRNA expression in liver is caused by male-pattern GH secretion pattern and androgens, whereas in kidney, Cyp4a mRNA expression is primarily regulated by androgens.

  9. CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

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    Davies, Benjamin J; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

    2007-01-01

    The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.

  10. Evolution of a TRIM5-CypA splice isoform in old world monkeys.

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    Ruchi M Newman

    2008-02-01

    Full Text Available The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA. We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101 of rhesus macaques (M. mulatta. The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a

  11. Perturbation of murine liver cyp-superfamily of isoforms by different combinations of pesticide mixtures.

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    Canistro, D; Pozzetti, L; Sapone, A; Broccoli, M; Affatato, A A; Stradiotti, A; Longo, V; Menichini, P; Barale, R; Paolini, M

    2008-01-01

    It was previously found that fenarimol, vinclozolin or acephate, three of the most used pesticides worldwide, provoked a marked perturbation of murine cytochrome P450 (CYP)-linked monooxygenases. Here, to more closely mimic human exposure, it was investigated whether different pesticide combinations administered i.p. in male Swiss Albino CD1 mice in single or repeated fashion (daily, for three consecutive days), affect CYP-dependent oxidations. The four simulated mixtures showed a complex pattern of CYP induction and suppression, especially after repeated injection. For example, while fenarimol alone was the most inducing agent--reaching a 79-fold increase over control in testosterone 2alpha-hydroxylase--followed by vinclozolin and acephate, coadministration with the former markedly reduced induction. Coadministration with vinclozolin, determined various positive and negative modulations. An increase of CYP2B1/2 and CYP3A1/2-associated oxidases and a decrease of ethoxycoumarin metabolism was observed in the acephate and vinclozolin mixture. An equivalent or reduced CYP expression, if compared to double combinations, was seen using the complete mixture. Taken as a whole, the unpredictability of the recorded effects with simple mixtures, shrinks the misleading extrapolation performed on a single pesticide. If reproduced in human, such changes, altering either endogenous metabolism or biotransformation of ubiquitous toxins, might have public health implications.

  12. Avian cytochrome P450 (CYP 1-3 family genes: isoforms, evolutionary relationships, and mRNA expression in chicken liver.

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    Kensuke P Watanabe

    Full Text Available Cytochrome P450 (CYP of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.

  13. Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos.

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    Joo, Hyun; Choi, Kyoungju; Rose, Randy L; Hodgson, Ernest

    2007-01-01

    Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.

  14. TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity

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    Na, Lei [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Tang, Yan-Dong [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Biotechnology Institute of Southern Medical University, Guangzhou 510515 (China); Liu, Jian-Dong; Yu, Chang-Qing; Sun, Liu-Ke; Lin, Yue-Zhi; Wang, Xue-Feng [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Wang, Xiaojun, E-mail: xjw@hvri.ac.cn [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Zhou, Jian-Hua, E-mail: jianhua_uc@126.com [Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Harbin Pharmaceutical Group Biovaccine Company, Harbin 150069 (China)

    2014-04-04

    Highlights: • TRIMe7-CypA expresses in rhesus and pig-tailed, but not long-tailed macaques. • TRIMe7-CypA does not show the restriction to a HIV-GFP report virus in vitro. • It acts as a negative modulator to TRIM5α likely by competitive inhibition. - Abstract: The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition.

  15. The OsCYP19-4 Gene Is Expressed as Multiple Alternatively Spliced Transcripts Encoding Isoforms with Distinct Cellular Localizations and PPIase Activities under Cold Stress

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    Areum Lee

    2016-07-01

    Full Text Available Alternative splicing (AS is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1 transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H and bimolecular fluoresence complementation (BiFC assays, although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.

  16. Identification of cytochrome P450 isoforms involved in the metabolism of paroxetine and estimation of their importance for human paroxetine metabolism using a population-based simulator

    DEFF Research Database (Denmark)

    Jornil, Jakob; Jensen, Klaus Gjervig; Larsen, Frank

    2010-01-01

    the importance of the identified paroxetine-metabolizing P450 isoforms for human metabolism, taking mechanism-based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism-based inhibition) was also estimated by Simcyp. For extensive and poor metabolizers of CYP2D6......We identify here for the first time the low-affinity cytochrome P450 (P450) isoforms that metabolize paroxetine, using cDNA-expressed human P450s measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography-tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP......3A4, and CYP3A5 were identified as paroxetine-catechol-forming P450 isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing P450 isoforms by substrate depletion. Michaelis-Menten constants K(m) and V(max) were determined by product formation and substrate depletion. Using selective...

  17. Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

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    Glisic, Branka; Hrubik, Jelena; Fa, Svetlana; Dopudj, Nela; Kovacevic, Radmila; Andric, Nebojsa

    2016-02-01

    Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes.

  18. Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes

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    Sun Joo Kim

    2016-01-01

    Full Text Available Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a Ki value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.

  19. High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS.

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    Lee, Miran; Park, Jeonghyeon; Lim, Mi-sun; Seong, Sook Jin; Lee, Joomi; Seo, Jeong Ju; Park, Yong-Ki; Lee, Hae Won; Yoon, Young-Ran

    2012-01-01

    Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

  20. CYP1A2-mediated biotransformation of cardioactive 2-thienylidene-3,4-methylenedioxybenzoylhydrazine (LASSBio-294) by rat liver microsomes and human recombinant CYP enzymes.

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    Fraga, Aline Guerra M; da Silva, Leandro Louback; Fraga, Carlos Alberto Manssour; Barreiro, Eliezer J

    2011-01-01

    We describe herein the metabolic fate of cardioactive 1,3-benzodioxolyl N-acylhydrazone prototype LASSBio-294 (4) and the structural identification of its major phase I metabolite from rat liver microsomal assays. Our results confirmed the hard-metabolic character of N-acylhydrazone (NAH) framework of LASSBio-294 (4). The development of a reproducible analytical methodology for the major metabolite by using HPLC-MS and the comparison with an authentic synthetic sample, allowed us to identify 2-thienylidene 3,4-dihydroxybenzoylhydrazine derivative (7), formed by oxidative scission of methylenedioxy bridge of LASSBio-294, as the main metabolite formed by action of CYP1A2 isoform. The identification of this isoform in the LASSBio-294 in the clearance of LASSBio-294 (4) oxidation was performed by the use of selective CYP inhibitors or human recombinant CYP enzymes.

  1. Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

    Science.gov (United States)

    Wu, Jing-Jing; Ge, Guang-Bo; He, Yu-Qi; Wang, Ping; Dai, Zi-Ru; Ning, Jing; Hu, Liang-Hai; Yang, Ling

    2016-01-01

    Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

  2. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  3. CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Xiao-ping ZHAO; Jiao ZHONG; Xiao-quan LIU; Guang-ji WANG

    2007-01-01

    Aim: To study the metabolism of vinflunine and the effects of selective cyto-chrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. Methods: Individual selective CYP450 inhibitors were used to inves-tigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M1 and M2 in human liver microsomes.Results: Vinflunine was rapidly metabolized to 2 metabolites: M1 and M2 in human liver microsomes. M1 and M2 were tentatively presumed to be the N-oxide metabo-lite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M1, and competitively inhibited the formation of M2, while α-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M1 and M2. Conclusion: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.

  4. CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

    Science.gov (United States)

    Price, R J; Surry, D; Renwick, A B; Meneses-Lorente, G; Lake, B G; Evans, D C

    2000-08-01

    1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8

  5. CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes.

    Science.gov (United States)

    Cao, Y-F; Zhang, Y-Y; Li, J; Ge, G-B; Hu, D; Liu, H-X; Huang, T; Wang, Y-C; Fang, Z-Z; Sun, D-X; Huo, H; Yin, J; Yang, L

    2010-01-01

    Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), (1)H-nuclear magnetic resonance (NMR), and (13)C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin's metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

  6. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6

    Directory of Open Access Journals (Sweden)

    Li-Na Gao

    2015-01-01

    Full Text Available Peony (Paeonia lactiflora Pall- is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony on cytochrome P450 (CYP 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10−7 M to 10−5 M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application.

  7. Metabolism of sanguinarine in human and in rat: characterization of oxidative metabolites produced by human CYP1A1 and CYP1A2 and rat liver microsomes using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Deroussent, Alain; Ré, Micheline; Hoellinger, Henri; Cresteil, Thierry

    2010-07-08

    The quaternary benzo[c]phenanthridine alkaloid, sanguinarine (SA), has been detected in the mustard oil contaminated with Argemone mexicana, which produced severe human intoxications during epidemic dropsy in India. Today, SA metabolism in human and in rat has not yet been fully elucidated. The goal of this study is to investigate the oxidative metabolites of SA formed during incubations with rat liver microsomes (RLM) and recombinant human cytochrome P450 (CYP) and to tentatively identify the CYP isoforms involved in SA detoxification. Metabolites were analyzed by liquid chromatography combined with electrospray ionization-tandem mass spectrometry. Up to six metabolites were formed by RLM and their modified structure has been proposed using their mass spectra and mass shifts from SA (m/z 332). The main metabolite M2 (m/z 320) resulted from ring-cleavage of SA followed by demethylation, whereas M4 (m/z 348) is oxidized by CYP in the presence of NADPH. The diol-sanguinarine metabolite M6 (m/z 366) formed by RLM might derive from a putative epoxy-sanguinarine metabolite M5 (m/z 348). M4 and M6 could be detected in rat urine as their respective glucuronides. 5,6-Dihydrosanguinarine is the prominent derivative formed from SA in cells expressing no CYP. Oxidative biotransformation of SA was investigated using eight human CYPs: only CYP1A1 and CYP1A2 displayed activity.

  8. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  9. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  10. Determination of the inhibitory potential of 6 fluoroquinolones on CYPIA2 and CYP2C9 in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Min-ji WEI; Cai-yun ZHAO; Hui-min QI

    2008-01-01

    Aim: To determine the inhibitory potential of 2 new fluoroquinolones, caderofloxacin and antofloxacin, together with 4 marketed fluoroquinolones, moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin, on the activity of cytochrome P450 isoforms 1A2 (CYP1A2) and 2C9 (CYP2C9). Methods: Probe substrates, phenacetin (CYP1A2), and tolbutamide (CYP2C9) were incubated with human liver microsomes and the metabolites were analyzed by liquid chromatography/mass spectrometry using electrospray ionization in positive or negative mode. Glipizide was used as the internal standard in both modes. The inhibitory potential of fluoroquinolones on CYP1A2 and CYP2C9 was investigated. Results: The IC50 values (μmol/L) determined with the cocktail were in agreement with individual probe substrates (α-naphthoflavone: 0.27 vs 0.26; sulfaphenazole: 0.49 vs 0.37). Ciprofloxacin showed weak inhibition on both the activity of CYPIA2 (IC50 135 μmol/L) and CYP2C9 (IC50 180 μmol/L), whereas levofloxacin inhibited only CYP2C9 (IC50 210 μmol/L). Caderofloxacin, antofloxacin, moxifloxacin, and gatifloxacin showed little or no inhibition on the activity of CYPIA2 or CYP2C9 when tested at comparable concentrations (0-200 mg/L). Conclusion: Caderofloxacin, antofloxacin, moxifloxacin, and gatifloxacin are negligible inhibitors to CYP1A2 and CYP2C9. The in vitro system can be used as a high-throughput model to screen similar compounds for the early identification of drug-drug interaction potential.

  11. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    Science.gov (United States)

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  12. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    Science.gov (United States)

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

  13. Effects of 4-nonylphenol on hepatic gene expression of peroxisome proliferator-activated receptors and cytochrome P450 isoforms (CYP1A1 and CYP3A4) in juvenile sole (Solea solea).

    Science.gov (United States)

    Cocci, Paolo; Mosconi, Gilberto; Palermo, Francesco Alessandro

    2013-10-01

    The objective of the present study was to investigate the modulatory effects of the xenoestrogen 4-nonylphenol (4-NP) on hepatic peroxisome proliferator-activated receptor (PPAR) α and β gene expression patterns in relation to the detoxification pathways mediated by cytochrome P450 isoforms (CYP1A1 and CYP3A4). Waterborne 4-NP-induced effects were compared with those of 10(-8)M 17β-estradiol (E2) by using in vivo dose-response experiments carried out with juvenile sole (Solea solea). Compared to the controls, significantly higher levels of PPARα mRNAs were found in fish treated with E2 or 4-NP (10(-6)M) 3 d after exposure; the highest dose of 4-NP also caused up-regulation of retinoid X receptor α (RXRα) transcript levels. On the contrary, PPARβ gene expression was not modulated by E2 or 4-NP. Our data show that 4-NP-induced PPARα mRNA levels coincide with suppression of CYP1A1 and CYP3A4 expression similarly to E2. The results from these in vivo studies suggest the presence of cross-talk between nuclear receptor-mediated signaling pathways and PPARα that may result in modulation of CYP450 isoforms expression following 4-NP treatment in sole liver. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

    Energy Technology Data Exchange (ETDEWEB)

    Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won (Toronto); (IBC-Belarus)

    2010-09-22

    The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

  15. Identification of Potent and Selective CYP1A1 Inhibitors via Combined Ligand and Structure-Based Virtual Screening and Their in Vitro Validation in Sacchrosomes and Live Human Cells.

    Science.gov (United States)

    Joshi, Prashant; McCann, Glen J P; Sonawane, Vinay R; Vishwakarma, Ram A; Chaudhuri, Bhabatosh; Bharate, Sandip B

    2017-06-26

    Target structure-guided virtual screening (VS) is a versatile, powerful, and inexpensive alternative to experimental high-throughput screening (HTS). To discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercial library of 50 000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structure-based VS. These 300 compounds were screened, at 10 μM concentration, for in vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-de-ethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 μM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ∼95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity toward inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e., there was no detectable inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 at 10 μM). It was further investigated in live CYP-expressing human cell system, which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC50 values of 269, 30, and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 μM. The compound 119 rescued CYP1A1 overexpressing HEK293 cells from CYP1A1 mediated benzo[a]pyrene (B[a]P) toxicity and also overcame cisplatin resistance in CYP1B1 overexpressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully

  16. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

    Directory of Open Access Journals (Sweden)

    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  17. Oxidation of 1-chloropyrene by human CYP1 family and CYP2A subfamily cytochrome P450 enzymes: catalytic roles of two CYP1B1 and five CYP2A13 allelic variants.

    Science.gov (United States)

    Shimada, Tsutomu; Murayama, Norie; Kakimoto, Kensaku; Takenaka, Shigeo; Lim, Young-Ran; Yeom, Sora; Kim, Donghak; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2017-07-21

    1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.

  18. Herbicide resistance of transgenic rice plants expressing human CYP1A1.

    Science.gov (United States)

    Kawahigashi, Hiroyuki; Hirose, Sakiko; Ohkawa, Hideo; Ohkawa, Yasunobu

    2007-01-01

    Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.

  19. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross...

  20. Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

    Science.gov (United States)

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

    2016-01-01

    Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC50>1 mM) on the activities of five major isoforms of human CYP in vitro.

  1. Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse heart

    Science.gov (United States)

    Edin, Matthew L.; Wang, ZhongJing; Bradbury, J. Alyce; Graves, Joan P.; Lih, Fred B.; DeGraff, Laura M.; Foley, Julie F.; Torphy, Robert; Ronnekleiv, Oline K.; Tomer, Kenneth B.; Lee, Craig R.; Zeldin, Darryl C.

    2011-01-01

    Cytochrome P450 (CYP) epoxygenases CYP2C8 and CYP2J2 generate epoxyeicosatrienoic acids (EETs) from arachidonic acid. Mice with expression of CYP2J2 in cardiomyocytes (αMHC-CYP2J2 Tr) or treated with synthetic EETs have increased functional recovery after ischemia/reperfusion (I/R); however, no studies have examined the role of cardiomyocyte- vs. endothelial-derived EETs or compared the effects of different CYP epoxygenase isoforms in the ischemic heart. We generated transgenic mice with increased endothelial EET biosynthesis (Tie2-CYP2C8 Tr and Tie2-CYP2J2 Tr) or EET hydrolysis (Tie2-sEH Tr). Compared to wild-type (WT), αMHC-CYP2J2 Tr hearts showed increased recovery of left ventricular developed pressure (LVDP) and decreased infarct size after I/R. In contrast, LVDP recovery and infarct size were unchanged in Tie2-CYP2J2 Tr and Tie2-sEH Tr hearts. Surprisingly, compared to WT, Tie2-CYP2C8 Tr hearts had significantly reduced LVDP recovery (from 21 to 14%) and increased infarct size after I/R (from 51 to 61%). Tie2-CYP2C8 Tr hearts also exhibited increased reactive oxygen species (ROS) generation, dihydroxyoctadecenoic acid (DiHOME) formation, and coronary resistance after I/R. ROS scavengers and CYP2C8 inhibition reversed the detrimental effects of CYP2C8 expression in Tie2-CYP2C8 Tr hearts. Treatment of WT hearts with 250 nM 9,10-DiHOME decreased LVDP recovery compared to vehicle (16 vs. 31%, respectively) and increased coronary resistance after I/R. These data demonstrate that increased ROS generation and enhanced DiHOME synthesis by endothelial CYP2C8 impair functional recovery and mask the beneficial effects of increased EET production following I/R.—Edin, M. L., Wang, Z. J., Bradbury, J. A., Graves, J. P., Lih, F. B., DeGraff, L. M., Foley, J. F., Torphy, R., Ronnekleiv, O. K., Tomer, K. B., Lee, C. R., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse

  2. Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

    Science.gov (United States)

    Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

    2015-03-01

    Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

  3. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    Science.gov (United States)

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  4. Inhibition of human aromatase complex (CYP19) by antiepileptic drugs

    DEFF Research Database (Denmark)

    Jacobsen, Naja Wessel; Halling-Sørensen, Bent; Birkved, Franziska Maria A Kramer

    2008-01-01

    transfected insect cells using dibenzylfluorescein as substrate. The drugs inhibiting CYP19 were: lamotrigine, oxcarbazepine, tiagabine, phenobarbital, phenytoin, ethosuximide, and valproate. The inhibitory effects (50% reduction in activity compared to enzymes without inhibitor present) were in the range...... with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered....

  5. Coexpression of CPR from various origins enhances biotransformation activity of human CYPs in S. pombe.

    Science.gov (United States)

    Neunzig, Ina; Widjaja, Maria; Peters, Frank T; Maurer, Hans H; Hehn, Alain; Bourgaud, Frédéric; Bureik, Matthias

    2013-08-01

    Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop's weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.

  6. Characterization of the Human LPIN1-encoded Phosphatidate Phosphatase Isoforms*

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M.

    2010-01-01

    The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218). In this work, we characterized human lipin 1 α, β, and γ isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the α, β, and γ isoforms were dependent on Mg2+ or Mn2+ ions at pH 7.5 at 37 °C. The activities were inhibited by concentrations of Mg2+ and Mn2+ above their optimums and by Ca2+, Zn2+, N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 °C. The α, β, and γ activities followed saturation kinetics with respect to the molar concentration of PA (Km values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number ∼2) kinetics with respect to the surface concentration of PA (Km values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (kcat) for the α, β, and γ isoforms were 68.8 ± 3.5, 42.8 ± 2.5, and 5.7 ± 0.2 s−1, respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity. PMID:20231281

  7. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  8. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  9. Cloning and characterization of the neural isoforms of human dystonin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, A.; Dalpe, G.; Mathieu, M.; Kothary, R. [Univ. of Montreal, Quebec (Canada)

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.

  10. Inhibitory Effect of Selaginellins from Selaginella tamariscina (Beauv. Spring against Cytochrome P450 and Uridine 5′-Diphosphoglucuronosyltransferase Isoforms on Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Jae-Kyung Heo

    2017-09-01

    Full Text Available Selaginella tamariscina (Beauv. has been used for traditional herbal medicine for treatment of cancer, hepatitis, and diabetes in the Orient. Numerous bioactive compounds including alkaloids, flavonoids, lignans, and selaginellins have been identified in this medicinal plant. Among them, selaginellins having a quinone methide unit and an alkylphenol moiety have been known to possess anticancer, antidiabetic, and neuroprotective activity. Although there have been studies on the biological activities of selaginellins, their modulatory potential of cytochrome P450 (P450 and uridine 5′-diphosphoglucuronosyltransferase (UGT activities have not been previously evaluated. In this study, we investigated the drug interaction potential of two selaginellins on ten P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and 3A and six UGT isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7 using human liver microsomes and liquid chromatography-tandem mass spectrometry. Selaginellin and selaginellin M had high inhibitory potential for CYP2C8-mediated amodiaquine O-demethylation with IC50 values of 0.5 and 0.9 μM, respectively. Selaginellin and selaginellin M also showed medium inhibitory potential against CYP2C9, CYP2J2, UGT1A1, and UGT1A3 (1 μM < IC50 < 5 μM. These two selaginellins had low inhibitory potential against CYP1A2, CYP2A6, CYP2E1, and UGT1A6 (IC50 > 25 μM. This information might be helpful to predict possible drug interaction potential of between selaginellins and co-administered drugs.

  11. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Directory of Open Access Journals (Sweden)

    Marina Ilicic

    2017-01-01

    Full Text Available Background. Regulation of myometrial progesterone receptor (PR expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture.

  12. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Science.gov (United States)

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  13. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  14. Expression of aquaporin isoforms during human and mouse tooth development.

    Science.gov (United States)

    Felszeghy, S; Módis, L; Németh, P; Nagy, G; Zelles, T; Agre, P; Laurikkala, J; Fejerskov, O; Thesleff, I; Nielsen, S

    2004-04-01

    Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

  15. The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

    Science.gov (United States)

    Donzelli, Massimiliano; Derungs, Adrian; Serratore, Maria-Giovanna; Noppen, Christoph; Nezic, Lana; Krähenbühl, Stephan; Haschke, Manuel

    2014-03-01

    Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the

  16. Critical Amino Acid Residues for Nicotine 5' -Hydroxylation in Human CYP2A Enzymes

    Institute of Scientific and Technical Information of China (English)

    Xiaoyang He Xiaoyang He; Xu Xu; Jian Shen; Li Sun; Anthony Y. H. Lu; Clifford Weisel; Junyan Hong

    2008-01-01

    Objective: We have continued previous work in which we demonstrated that #117 and #372 amino acids contrib-uted to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-(3-pyridyl)-1-hutanone(NNK) and aflatoxin B1(AFB1) carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods: A series of reciprocally substituted mutants of CYP2A6IIe'300→Phe, CYP2A6Gly'301Ala, CYP2A6Ser'369→Gly, CYP2A13Phe'300→Ile, CYP2A13AIa'301→Gly and CYP2A13Gly'369→Ser were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5'hydroxylatin by wild type and mutant CYP2A proteins was performed. Results:All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile'300→Phe and CYP2A6Gly'301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe'300→Ile and CYP2A13Ala'301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by V,maxvariations rather than K,m changes. Substi-tution of #369 residue significantly affected both K,m and V,max values. CYP2A6Ser'369→Gly increase the catalytic efficiency via a significant Km decrease versus V,max enhancement, while the opposite effects were seen with CYP2A13Gly'369→Ser. Conclusion:#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5' -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Seta69 to Gly substitution indirectly affected

  17. Cocktail 探针药物法评价辣椒素对大鼠CYP450亚型的影响%Effects of Capsaicin on Rat Cytochrome P450 Isoforms by Cocktail Probe Drug Method

    Institute of Scientific and Technical Information of China (English)

    舒舟; 翟学佳; 刘金梅; 吕永宁

    2014-01-01

    目的::采用cocktail 探针药物法研究辣椒素在体外对大鼠肝微粒体CYP 450四种亚型的影响。方法:分为试验组和对照组,试验组采用大鼠肝微粒体孵育体系、探针药物和系列浓度的辣椒素(对照组仅加入缓冲液)共同孵育20 min,反应终止后用HPLC方法检测代谢产物的生成量以代表酶的活性。采用Graphpad prism 5.0计算辣椒素对各亚型的IC50值。将辣椒素与大鼠肝微粒体分别预孵育0,5,10,15,20,30 min,计算不同预孵育时间下各亚型的相对活性百分比。结果:辣椒素对大鼠肝微粒体CYP1A2、CYP2C11、CYP2E1和CYP3A2的IC50值分别为36.21,17.19,51.64,18.86μmol·L-1。预孵育没有增强辣椒素对CYP450的抑制作用。结论:辣椒素在体外对大鼠肝微粒CYP1A2、CYP2C11、CYP2E1和CYP3A2具有抑制作用,且未呈现出预孵育时间依赖性。%Objective:To investigate the in vitro effect of capsaicin on four major rat cytochrome P450 ( CYP) enzymes using a cocktail probe drug method. Methods:The in vitro incubation was divided into capsaicin group and the control group. Rat liver micro-somes, probe drugs, capsaicin at various concentration ( buffer solution in the control group) and cofactors were cultured altogether for 20 min. After the culture, the concentration of metabolites was determined by HPLC to assess the activities of enzymes. IC50 value of capsaicin on every isoform was calculated using Graphpad prism 5. 0. Capsaicin and hepatic microsomess were pre-incubated respec-tively for 0, 5, 10, 15, 20 and 30 min, and then the relative activity of the four isoforms at different time was calculated. Results:The activity of the rat liver microsomes enzyme CYP450 isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2) was all inhibited by capsaicin in vitro with IC50 value of 36. 21, 17. 19, 51. 64 and 18. 86 μmol·L-1 , respectively. Pre-incubation could not increase cap-saicin inhibitory activity against the four CYP enzymes

  18. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    Science.gov (United States)

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

  19. Dehydroepiandrosterone induces human CYP2B6 through the constitutive androstane receptor.

    Science.gov (United States)

    Kohalmy, Krisztina; Tamási, Viola; Kóbori, László; Sárváry, Eniko; Pascussi, Jean-Marc; Porrogi, Pálma; Rozman, Damjana; Prough, Russell A; Meyer, Urs A; Monostory, Katalin

    2007-09-01

    Dehydroepiandrosterone (DHEA), the major precursor of androgens and estrogens, has several beneficial effects on the immune system, on memory function, and in modulating the effects of diabetes, obesity, and chemical carcinogenesis. Treatment of rats with DHEA influences expression of cytochrome P450 (P450) genes, including peroxisome proliferator-activated receptor alpha (PPAR alpha)- and pregnane X receptor (PXR)-mediated induction of CYP4As and CYP3A23, and suppression of CYP2C11. DHEA treatment elevated the expression and activities of CYP3A4, CYP2C9, CYP2C19, and CYP2B6 in primary cultures of human hepatocytes. Induction of CYP3A4 in human hepatocytes was consistent with studies in rats, but induction of CYP2Cs was unexpected. The role of PXR in this response was studied in transient transfection assays. DHEA activated hPXR in a concentration-dependent manner. Because CYP2B6 induction by DHEA in human hepatocytes might involve either PXR or constitutive androstane receptor (CAR) activation, we performed experiments in primary hepatocytes from CAR knockout mice and observed that CAR was required for maximal induction of Cyp2b10 by DHEA. Furthermore, CAR-mediated Cyp2b10 induction by DHEA was inhibited by the inverse agonist of CAR, androstanol (5 alpha-androstan-3 alpha-ol). Further evidence for CAR activation was provided by cytoplasmic/nuclear transfer of CAR upon DHEA treatment. Elucidation of CAR activation and subsequent induction of CYP2B6 by DHEA presented an additional mechanism by which the sterol can modify the expression of P450s. The effect of DHEA on the activation of the xenosensors PPAR alpha, PXR, and CAR, and the consequent potential for adverse drug/toxicant interactions should be considered in humans treated with this nutriceutical agent.

  20. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  1. Interactions between CYP2C9 and CYP2C19 in reconstituted binary systems influence their catalytic activity: possible rationale for the inability of CYP2C19 to catalyze methoxychlor demethylation in human liver microsomes.

    Science.gov (United States)

    Hazai, Eszter; Kupfer, David

    2005-01-01

    Previous studies in our laboratory showed that among cDNA-expressed human cytochrome P450 (P450) supersomes, CYP2C19 was the most active in methoxychlor-O-demethylation. However, based on the lack of inhibition of methoxychlor-O-demethylation by monoclonal anti-CYP2C19 antibodies in human liver microsomes (HLM), CYP2C19 did not seem to catalyze that reaction in HLM. By contrast, CYP2C9, much less active than CYP2C19 in supersomes, was the most active in HLM. The current study examines whether the lack of methoxychlor-O-demethylation by CYP2C19 in HLM was due to CYP2C19 exhibiting inferior competition for the NADPH-cytochrome P450 reductase (CPR) versus CYP2C9 and explores the interactions between CYP2C9 and CYP2C19 in a singular and binary complex of a reconstituted system. When reconstituted with CPR, cytochrome b(5), and lipid, purified CYP2C19 and CYP2C9 catalyzed methoxychlor-O-demethylation. However, whereas equimolar CPR to CYP2C9 supported maximal rates of methoxychlor demethylation and diclofenac hydroxylation, the rate of methoxychlor demethylation by CYP2C19 was not fully saturated, even with a 9-fold molar excess of CPR over CYP2C19. This behavior of CYP2C19 was also observed with S-mephenytoin as the substrate. When a binary reconstitution system was prepared by mixing CYP2C9 and CYP2C19 enzymes, methoxychlor-O-demethylation and S-mephenytoin hydroxylation by CYP2C19 were dramatically inhibited. Inhibition depended on the amount of CPR and substrate used. By contrast, in the incubation containing CYP2C9, diclofenac hydroxylation was activated by the presence of CYP2C19. These results show that interactions among P450 enzymes can modulate their catalytic rates, which depend on the substrate undergoing metabolism.

  2. Identification of CYP3A7 for Glyburide Metabolism in Human Fetal Livers

    Science.gov (United States)

    Shuster, Diana L.; Risler, Linda J.; Prasad, Bhagwat; Calamia, Justina C.; Voellinger, Jenna L.; Kelly, Edward J.; Unadkat, Jashvant D.; Hebert, Mary F.; Shen, Danny D.; Thummel, Kenneth E.; Mao, Qingcheng

    2014-01-01

    Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers. PMID:25450675

  3. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wei [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Guifang [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Cai, Xiaojun [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Yang, Jing, E-mail: yangjingliu@yahoo.com.cn [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  4. Expression of the cytochrome P450 epoxygenase CYP2J2 in human monocytic leukocytes.

    Science.gov (United States)

    Nakayama, Kaeko; Nitto, Takeaki; Inoue, Teruo; Node, Koichi

    2008-08-29

    CYP2J2 is one of the cytochrome P450 epoxygenases involved in the metabolism of arachidonic acid. CYP2J2 has been identified in several tissues, especially cardiovascular tissues. CYP2J2 has cardiovascular effects, as epoxyeicosatrienoic acid, one of its metabolites, has anti-inflammatory and vasodilative activities. We investigated the expression of CYP2J2 in human leukocytes using reverse transcription-polymerase chain reaction, immunoblotting and immunostaining. Human monocytic cells, but not human neutrophils, exhibited constitutive expression of CYP2J2. Furthermore, the expression of CYP2J2 mRNA increased when the human monocytic cell line THP-1 cells and human monocytes were stimulated with phorbol 12-myristate 13-acetate and macrophage-colony stimulating factor in combination with granulocyte/macrophage-colony stimulating factor, respectively. These results suggest that expression of CYP2J2 was up-regulated when human monocytes differentiated into macrophages and that human monocytic cells and macrophages have a pathway to metabolize arachidonic acid using CYP epoxygenases.

  5. CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响%Effects of CYP2C19 genotype and CYP2C9 on fluoxetine N-demethylation in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    刘昭前; 舒焱; 黄松林; 王连生; 何楠; 周宏灏

    2001-01-01

    目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶.方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学.鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶.结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征.当底物浓度为氟西汀25μmol/L和100 μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r1=0.821,P1=0.001;r2=0.668,P2=0.013),当底物浓度为氟西汀100 μmol/L时,N-FLU的生成率与S-美芬妥因4'-羟化酶活性显著相关(r=0.717,P=0.006).PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73%vs 45%,P<0.01).结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主.%AIM: The present study was designed to define the kinetic behavior of fluoxetine N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P-450 (CYP) involved in this metabolic pathway. METHODS: The kinetics of the formation of norfluoxetine was determined in human liver microsomes from six genotyped CYP2C19 extensive metabolizers (EM). The correlation studies between the fluoxetine N-demethylase activity and various CYP enzyme activities were performed. Selective inhibitors or chemical probes of various cytochrome P-450 isoforms were also employed. RESULTS: The kinetics of norfluoxetine formation in all liver microsomes were fitted by a singleenzyme Michaelis-Menten equation (mean Km=32 μmol/L±7

  6. Metabolism of novel anti-HIV agent 3-cyanomethyl-4-methyl-DCK by human liver microsomes and recombinant CYP enzymes

    Institute of Scientific and Technical Information of China (English)

    Xiao-mei ZHUANG; Jing-ting DENG; Hua LI; Wei-li KONG; Jin-xiu RUAN; Lan XIE

    2011-01-01

    Aim:To investigate the metabolism of 3-cyanomethyl-4-methyl-DCK (CMDCK),a novel anti-HIV agent,by human liver microsomes (HLMs) and recombinant cytochrome P450 enzymes (CYPs).Methods:CMDCK was incubated with HLMs or a panel of recombinant cytochrome P450 enzymes including CYP1A2,2B6,2C8,2C9,2C19,2D6,3A4,and 3A5.LC-ion trap mass spectrometry was used to separate and identify CMDCK metabolites.In the experiments with recombinant cytochrome P450 enzymes,specific chemical inhibitors combined with CYP antibodies were used to identify the CYP isoforms involved in CMDCK metabolism.Results:CMDCK was rapidly and extensively metabolized by HLMs.Its intrinsic hepatic clearance estimated from the in vitro data was 19.4 mL.min-1·kg-1,which was comparable to the mean human hepatic blood flow rate (20.7 mL·min-1·kg-1).The major metabolic pathway of CMDCK was oxidation,and a total of 14 metabolites were detected.CYP3A4 and 3A5 were found to be the principal CYP enzymes responsible for CMDCK metabolism.Conclusion:CMDCK was metabolized rapidly and extensively in human hepatic microsomes to form a number of oxidative metabolites.CYP3A4 and 3A5 were the predominant enzymes responsible for the oxidation of CMDCK.

  7. The landscape of isoform switches in human cancers

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

    2017-01-01

    developed methods for identification and visualization of isoform switches with predicted functional consequences. Using these methods, we characterized isoform switching in RNA-seq data from >5,500 cancer patients covering 12 solid cancer types. Isoform switches with potential functional consequences were...... highly predictive of patient survival independent of cancer types. Our data constitute an important resource for cancer researchers, available through interactive web tools. Moreover, our methods, available as an R package, enable systematic analysis of isoform switches from other RNA-seq datasets...

  8. Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1.

    Science.gov (United States)

    Ozawa, S; Ohta, K; Miyajima, A; Kurebayashi, H; Sunouchi, M; Shimizu, M; Murayama, N; Matsumoto, Y; Fukuoka, M; Ohno, Y

    2000-10-01

    1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

  9. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Lie-Gang Liu; Hong Yan; Ping Yao; Wen Zhang; Li-Jun Zou; Fang-Fang Song; Ke Li; Xiu-Fa Sun

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively.Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of ceilular MDA level, LDH, and AST activities in supernatants.Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.CONCLUSION: A positive relationship between ethanol-induced oxidative aamage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

  10. CYP1A1 and CYP1B1 in human lymphocytes as biomarker of exposure: effect of dioxin exposure and polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Duursen, M. van; Sanderson, T.; Berg, M. van den [Inst. for Risk Assessment Sciences, Utrecht (Netherlands)

    2004-09-15

    There are several known genetic polymorphisms of the CYP1A1 and CYP1B1 genes. A polymorphism in the 3'-untranslated region of the CYP1A1 gene (CYP1A1 MspI or CYP1A1 m1) is often studied in relation with breast or lung cancer, but little is known about the functional effect of this polymorphism. An amino acid substitution in codon 432 (Val to Leu) of the CYP1B1 gene is associated with a lower catalytic activity of the enzyme. However, the involvement of these polymorphisms on the inducibility of CYP1A1 and CYP1B1 gene expression is unclear. CYP1A1 and CYP1B1 mRNA expression levels can be determined in peripheral blood lymphocytes. This makes them potential candidates for use as biomarker of exposure to environmental compounds. Interindividual variations in mRNA expression patterns, catalytic activity and polymorphisms are very important factors when CYP1A1 and CYP1B1 expression patterns are used as biomarker of exposure, but little is known about it. Spencer et al. showed a concentration-dependent increase of CYP1B1 mRNA in lymphocytes upon exposure in vitro to 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), the most potent dioxin. Yet, only a few studies describe the in vivo correlation between polymorphisms, mRNA expression level and exposure to environmental factors. In this study, we wanted to obtain a better insight in the CYP1A1 and CYP1B1 mRNA expression and enzyme activity in human lymphocytes. We determined the constitutive CYP1A1 and CYP1B1 mRNA expression in lymphocytes of ten healthy volunteers and the variability in sensitivity toward enzyme induction by TCDD. Further, the CYP1A1 m1 and CYP1B1 Val432Leu polymorphisms were determined.

  11. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    Science.gov (United States)

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  12. Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

    Science.gov (United States)

    Luo, Wei; Karpf, Adam R.; Deeb, Kristin K.; Muindi, Josephia R.; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.

    2010-01-01

    Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. PMID:20587525

  13. Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans.

    Science.gov (United States)

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; Bermudez, Mario; Martínez-Hernández, Roberto; Ortega, Arturo; Salinas, Juan E; Albores, Arnulfo

    2004-01-01

    CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.

  14. Inhibition of CYP3A4 and CYP2C9 by podophyllotoxin: Implication for clinical drug–drug interactions

    Indian Academy of Sciences (India)

    Jin-Hui Song; Dong-Xue Sun; Bin Chen; Dai-Hong Ji; Jie Pu; Jie Xu; Feng-De Tian; Lin Guo

    2011-12-01

    Podophyllotoxin (PPT) and its derivatives exert significant anti-cancer activities, and one derivative etoposide is often utilized to treat various cancers in the clinic. The aim of the present study is to investigate the inhibitory effects of PPT on major cytochrome P450 (CYP) isoforms in human livers. Inhibition of CYP3A4, CYP2C9, CYP2C8, CYP2D6, CYP2E1 and CYP2A6 by PPT was investigated in the human liver microsomal system. Time-dependent inhibition of CYP3A4 by PPT was also evaluated. The results showed that PPT strongly exhibited inhibitory effects on CYP3A4 and CYP2C9 in a concentration-dependent manner. Half inhibition concentration (IC50) was 1.1±0.3 and 4.6±0.3 M for CYP3A4 and CYP2C9, respectively. Inhibition kinetic analysis showed that PPT exhibited competitive inhibition towards CYP3A4 and CYP2C9 with Ki of 1.6 and 2.0 M, respectively. Additionally, PPT exerted time-dependent inhibition towards CYP3A4 and the kinetic parameters were 4.4±2.1 M and 0.06±0.01 min–1 for KI and kinact, respectively. Our experimental data indicate that potential drug–drug interaction (DDI) might exist when PPT is co-administered with the substrates which mainly undergo CYP3A4- or CYP2C9-mediated metabolism.

  15. Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes.

    Science.gov (United States)

    Kobayashi, Kaoru; Kajiwara, Eri; Ishikawa, Masayuki; Oka, Hidenobu; Chiba, Kan

    2012-11-01

    Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6β-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.

  16. p53 Family: Role of Protein Isoforms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  17. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

  18. Molecular population genetics of human CYP3A locus: signatures of positive selection and implications for evolutionary environmental medicine.

    Science.gov (United States)

    Chen, Xiaoping; Wang, Haijian; Zhou, Gangqiao; Zhang, Xiumei; Dong, Xiaojia; Zhi, Lianteng; Jin, Li; He, Fuchu

    2009-10-01

    The human CYP3A gene cluster codes for cytochrome P450 (CYP) subfamily enzymes that catalyze the metabolism of various exogenous and endogenous chemicals and is an obvious candidate for evolutionary and environmental genomic study. Functional variants in the CYP3A locus may have undergone a selective sweep in response to various environmental conditions. The goal of this study was to profile the allelic structure across the human CYP3A locus and investigate natural selection on that locus. From the CYP3A locus spanning 231 kb, we resequenced 54 genomic DNA fragments (a total of 43,675 bases) spanning four genes (CYP3A4, CYP3A5, CYP3A7, and CYP3A43) and two pseudogenes (CYP3AP1 and CYP3AP2), and randomly selected intergenic regions at the CYP3A locus in Africans (24 individuals), Caucasians (24 individuals), and Chinese (29 individuals). We comprehensively investigated the nucleotide diversity and haplotype structure and examined the possible role of natural selection in shaping the sequence variation throughout the gene cluster. Neutrality tests with Tajima's D, Fu and Li's D* and F*, and Fay and Wu's H indicated possible roles of positive selection on the entire CYP3A locus in non-Africans. Sliding-window analyses of nucleotide diversity and frequency spectrum, as well as haplotype diversity and phylogenetically inferred haplotype structure, revealed that CYP3A4 and CYP3A7 had recently undergone or were undergoing a selective sweep in all three populations, whereas CYP3A43 and CYP3A5 were undergoing a selective sweep in non-Africans and Caucasians, respectively. The refined allelic architecture and selection spectrum for the human CYP3A locus highlight that evolutionary dynamics of molecular adaptation may underlie the phenotypic variation of the xenobiotic disposition system and varied predisposition to complex disorders in which xenobiotics play a role.

  19. Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain.

    Science.gov (United States)

    Miksys, Sharon; Lerman, Caryn; Shields, Peter G; Mash, Deborah C; Tyndale, Rachel F

    2003-07-01

    CYP2B6 metabolizes drugs such as nicotine and bupropion, and many toxins and carcinogens. Nicotine induces CYP2B1 in rat brain and in humans polymorphic variation in CYP2B6 affects smoking cessation rates. The aim of this study was to compare CYP2B6 expression in brains of human smokers and non-smokers and alcoholics and non-alcoholics (n=26). CYP2B6 expression was brain region-specific, and was observed in both neurons and astrocytes. CYP2B6 levels were higher in brains of smokers and alcoholics, particularly in cerebellar Purkinje cells and hippocampal pyramidal neurons, cells known to be damaged in alcoholics. Significantly more (penzyme levels, stability and activity. Preliminary genotyping of this small sample (n=24) suggested that individuals with the CC genotype had higher brain CYP2B6 than those with the CT or TT genotype. Higher brain CYP2B6 activity in smokers and alcoholics may cause altered sensitivity to centrally acting drugs, increased susceptibility to neurotoxins and carcinogenic xenobiotics and contribute to central tolerance to nicotine.

  20. Development of CYP11B1 and CYP11B2 assays utilizing homogenates of adrenal glands: Utility of monkey as a surrogate for human.

    Science.gov (United States)

    Cerny, Matthew A; Csengery, Alexander; Schmenk, Jennifer; Frederick, Kosea

    2015-11-01

    Elevated levels of aldosterone are associated with arterial hypertension, congestive heart failure, chronic kidney disease, and obesity. Aldosterone is produced predominantly in the zona glomerulosa of the cortex of the adrenal gland by the enzyme aldosterone synthase (CYP11B2). Treatment of the above indications by decreasing production of aldosterone is thought to be of therapeutic benefit by lessening the deleterious effects of aldosterone mediated through both the mineralocorticoid receptor and also through so called non-genomic pathways. However, inhibition of the highly similar enzyme, CYP11B1, which is responsible for the production of cortisol, must be avoided in the development of clinically useful aldosterone synthase inhibitors due to the resulting impairment of the cortisol-induced stress response. In efforts to assess the interactions of compounds with the CYP11B enzymes, a variety of cell-based inhibitor screening assays for both CYP11B1 and CYP11B2 have been reported. Herein we report details of assays employing both cynomolgus monkey adrenal homogenate (CAH) and human adrenal homogenate (HAH) as sources of CYP11B1 and CYP11B2 enzymes. Utilizing both CAH and HAH, we have characterized the kinetics of the CYP11B1-mediated conversion of 11-deoxycortisol to cortisol and the CYP11B2-mediated oxidation of corticosterone to aldosterone. Inhibition assays for both CYP11B1 and CYP11B2 were subsequently developed. Based on a comparison of human and monkey amino acid sequences, kinetics data, and inhibition values derived from the HAH and CAH assays, evidence is provided in support of using cynomolgus monkey tissue-derived cell homogenates as suitable surrogates for the human enzymes.

  1. Baculovirus-mediated expression and characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal toxicants.

    Science.gov (United States)

    Liu, C; Zhuo, X; Gonzalez, F J; Ding, X

    1996-10-01

    Cytochrome P450 2A3 (CYP2A3) was previously identified in rat lung by cDNA cloning and recently found to be expressed at a high level in the olfactory mucosa. In the current study, CYP2A3 was expressed in insect cells lacking endogenous cytochrome P450 (P450) activity, and the substrate specificity of the recombinant cytochrome was characterized and compared with that of CYP2A6, a human ortholog of rat CYP2A3, which has been detected in human olfactory mucosa as well as in liver. The CYP2A3 and CYP2A6 cDNAs were cloned into baculovirus, and recombinant viruses were used to produce active enzymes in Spodoptera frugiperta (SF9) cells. The metabolic activities of S. frugiperta cell microsomal fractions containing CYP2A3 or CYP2A6 were studied in a reconstituted system with purified rabbit NADPH-P450 reductase. CYP2A3 was found to be active toward testosterone, producing 15 alpha-hydroxytestosterone and several other metabolites, but it had only low activity toward coumarin. On the other hand, CYP2A6 was active toward coumarin but not toward testosterone. However, both enzymes were active in the metabolic activation of hexamethylphosphoramide, a nasal procarcinogen, and 2,6-dichlorobenzonitrile (DCBN), a herbicide known to cause tissue-specific toxicity in the olfactory mucosa of rodents at very low doses. In addition, both enzymes were active toward 4-nitrophenol, a preferred substrate for CYP2E1. Consistent with CYP2A3 being a major catalyst in microsomal metabolism of DCBN, the activities of both CYP2A3 and rat olfactory microsomes in DCBN metabolism were inhibited strongly by metyrapone and methoxsalen (ID50 50 microM). Thus, rat CYP2A3 and human CYP2A6 have differences in substrate specificity as well as tissue distributor. These findings should be taken into account when assessing the risk of exposure to potential nasal toxicants in humans.

  2. Dietary cholesterol fails to stimulate the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in transgenic mice.

    Science.gov (United States)

    Agellon, Luis B; Drover, Victor A B; Cheema, Sukhinder K; Gbaguidi, G Franck; Walsh, Annemarie

    2002-06-07

    Dietary cholesterol has been shown to have a stimulatory effect on the murine cholesterol 7alpha-hydroxylase gene (Cyp7a1), but its effect on human cholesterol 7alpha-hydroxylase gene (CYP7A1) expression in vivo is not known. A transgenic mouse strain harboring the human CYP7A1 gene and homozygous for the disrupted murine Cyp7a1 gene was created. Cholesterol feeding increased the expression of the endogenous modified Cyp7a1 allele but failed to stimulate the human CYP7A1 transgene. In transfected hepatoma cells, 25-hydroxycholesterol increased murine Cyp7a1 gene promoter activity, whereas the human CYP7A1 gene promoter was unresponsive. Electrophoretic mobility shift assays demonstrated the interaction of the liver X receptor alpha (LXRalpha): retinoid X receptor (RXR) heterodimer, a transcription factor complex that is activated by oxysterols, with the murine Cyp7a1 gene promoter, whereas no binding to the human CYP7A1 gene promoter was detected. The results demonstrate that the human CYP7A1 gene is not stimulated by dietary cholesterol in the intact animal, and this is attributable to the inability of the CYP7A1 gene promoter to interact with LXRalpha:RXR.

  3. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  4. In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes

    Directory of Open Access Journals (Sweden)

    Alice Varghese

    2015-01-01

    Full Text Available Terminalia arjuna is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Alcoholic and aqueous bark extracts of T. arjuna, arjunic acid, arjunetin and arjungenin were evaluated for their potential to inhibit CYP3A4, CYP2D6 and CYP2C9 enzymes in human liver microsomes. We have demonstrated that alcoholic and aqueous bark extract of T. arjuna showed potent inhibition of all three enzymes in human liver microsomes with IC50 values less than 50 μg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP enzymes in human liver microsomes. Enzyme kinetics studies suggested that the extracts of arjuna showed reversible non-competitive inhibition of all the three enzymes in human liver microsomes. Our findings suggest strongly that arjuna extracts significantly inhibit the activity of CYP3A4, CYP2D6 and CYP2C9 enzymes, which is likely to cause clinically significant drug–drug interactions mediated via inhibition of the major CYP isozymes.

  5. Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes.

    Science.gov (United States)

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-05-01

    Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki-64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki-36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC50 values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co-administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb-drug interactions in humans.

  6. Human CYP2E1 mediates the formation of glycidamide from acrylamide.

    Science.gov (United States)

    Settels, Eva; Bernauer, Ulrike; Palavinskas, Richard; Klaffke, Horst S; Gundert-Remy, Ursula; Appel, Klaus E

    2008-10-01

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species.

  7. Human CYP2E1 mediates the formation of glycidamide from acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Settels, Eva; Appel, Klaus E. [Federal Institute for Risk Assessment, Center for Experimental Toxicology, Berlin (Germany); Bernauer, Ulrike; Gundert-Remy, Ursula [Federal Institute for Risk Assessment, Department of Safety of Substances and Preparations, Berlin (Germany); Palavinskas, Richard; Klaffke, Horst S. [Federal Institute for Risk Assessment, Center for Analytical Chemistry, Berlin (Germany)

    2008-10-15

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes trademark, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes trademark, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes trademark; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species. (orig.)

  8. CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol.

    Science.gov (United States)

    Rodríguez-Morató, Jose; Robledo, Patricia; Tanner, Julie-Anne; Boronat, Anna; Pérez-Mañá, Clara; Oliver Chen, C-Y; Tyndale, Rachel F; de la Torre, Rafael

    2017-02-15

    The dietary phenol tyrosol has been reported to be endogenously transformed into hydroxytyrosol, a potent antioxidant with multiple health benefits. In this work, we evaluated whether tyrosine hydroxylase (TH) and cytochrome P450s (CYPs) catalyzed this process. To assess TH involvement, Wistar rats were treated with α-methyl-L-tyrosine and tyrosol. Tyrosol was converted into hydroxytyrosol whilst α-methyl-L-tyrosine did not inhibit the biotransformation. The role of CYP was assessed in human liver microsomes (HLM) and tyrosol-to-hydroxytyrosol conversion was observed. Screening with selective enzymatic CYP inhibitors identified CYP2A6 as the major isoform involved in this process. Studies with baculosomes further demonstrated that CYP2D6 and CYP3A4 could transform tyrosol into hydroxytyrosol. Experiments using human genotyped livers showed an interindividual variability in hydroxytyrosol formation and supported findings that CYP2D6 and CYP2A6 mediated this reaction. The dietary health benefits of tyrosol-containing foods remain to be evaluated in light of CYP pharmacogenetics.

  9. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

    Science.gov (United States)

    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

  10. Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer

    Science.gov (United States)

    2009-02-01

    TITLE: Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer PRINCIPAL INVESTIGATOR: Jianghua Wang, M.D...6 JAN 2009 / / /4. TITLE AND SUBTITLE Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer 5a. CONTRACT NUMBER W81XWH...quantitative RT-PCR arrays we have identified candidate mediators of these phenotypic effects . We propose to extend these studies to primary prostate epithelial

  11. Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

    Science.gov (United States)

    Sams, C; Cocker, J; Lennard, M S

    2004-10-01

    The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

  12. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes

    OpenAIRE

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-01-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine’s metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 ...

  13. Differential expression of cytochrome P450 enzymes from the CYP2C subfamily in the human brain.

    Science.gov (United States)

    Booth Depaz, Iris M; Toselli, Francesca; Wilce, Peter A; Gillam, Elizabeth M J

    2015-03-01

    Cytochrome P450 enzymes from the CYP2C subfamily play a prominent role in the metabolic clearance of many drugs. CYP2C enzymes have also been implicated in the metabolism of arachidonic acid to vasoactive epoxyeicosatrienoic acids. CYP2C8, CYP2C9, and CYP2C19 are expressed in the adult liver at significant levels; however, the expression of CYP2C enzymes in extrahepatic tissues such as the brain is less well characterized. Form-specific antibodies to CYP2C9 and CYP2C19 were prepared by affinity purification of antibodies raised to unique peptides. CYP2C9 and CYP2C19 were located in microsomal fractions of all five human brain regions examined, namely the frontal cortex, hippocampus, basal ganglia, amygdala, and cerebellum. Both CYP2C9 and CYP2C19 were detected predominantly within the neuronal soma but with expression extending down axons and dendrites in certain regions. Finally, a comparison of cortex samples from alcoholics and age-matched controls suggested that CYP2C9 expression was increased in alcoholics.

  14. Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49.

    Directory of Open Access Journals (Sweden)

    Hua Cai

    Full Text Available The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49 was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

  15. CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes.

    Science.gov (United States)

    Buratti, Franca M; Volpe, Maria Teresa; Meneguz, Annarita; Vittozzi, Luciano; Testai, Emanuela

    2003-02-01

    The bioactivation of azinphos-methyl (AZIN), chlorpyrifos (CPF), diazinon (DIA), and parathion (PAR), four widely used organophosphorothioate (OPT) pesticides has been investigated in human liver microsomes (HLM). In addition, the role of human cytochrome P450 (CYPs) in OPT desulfuration at pesticide levels representative of human exposure have been defined by means of correlation and immunoinhibition studies. CYP-mediated oxon formation from the four OPTs is efficiently catalyzed by HLM, although showing a high variability (>40-fold) among samples. Two distinct phases were involved in the desulfuration of AZIN, DIA, and PAR, characterized by different affinity constants (K(mapp1) = 0.13-9 microM and K(mapp2) = 5- 269 microM). Within the range of CPF concentrations tested, only the high-affinity component was evidenced (K(mapp1) = 0.27-0.94 microM). Oxon formation in phenotyped individual HLM showed a significant correlation with CYP1A2-, 3A4-, and 2B6-related activities, at different levels depending on the OPT concentration. Anti-human CYP1A2, 2B6, and 3A4 antibodies significantly inhibited oxon formation, showing the same OPT concentration dependence. Our data indicated that CYP1A2 is mainly involved in OPT desulfuration at low pesticide concentrations, while the role of CYP3A4 is more significant to the low-affinity component of OPT bioactivation. The contribution of CYP2B6 to total hepatic oxon formation was relevant in a wide range of pesticide concentrations, being a very efficient catalyst of both the high- and low-affinity phase. These results suggest CYP1A2 and 2B6 as possible metabolic biomarkers of susceptibility to OPT toxic effect at the actual human exposure levels.

  16. Detection of VEGF-A(xxx)b isoforms in human tissues.

    Science.gov (United States)

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  17. Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line

    Institute of Scientific and Technical Information of China (English)

    Shu-Jie XIA; Xiao-Da TANG; Qing-Zheng MA

    2001-01-01

    Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5,6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3H-DHT) to these three isoforms was inhibited by the addition ofl00-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isofonns is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.

  18. An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Fasullo, Michael; Freedland, Julian; St John, Nicholas; Cera, Cinzia; Egner, Patricia; Hartog, Matthew; Ding, Xinxin

    2017-05-01

    P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside.

    Science.gov (United States)

    Sun, Dong-Xue; Lu, Jin-Cai; Fang, Zhong-Ze; Zhang, Yan-Yan; Cao, Yun-Feng; Mao, Yu-Xi; Zhu, Liang-Liang; Yin, Jun; Yang, Ling

    2010-11-01

    Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8.

  20. Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy

    Science.gov (United States)

    Bae, Sun Sik; Lee, Min Young; Rhee, Harin; Kim, Il Young; Seong, Eun Young; Lee, Dong Won; Lee, Soo Bong; Kwak, Ihm Soo; Lovett, David H.

    2017-01-01

    Background We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2) in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2) and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2) generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms. Methods We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE) and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). The streptozotocin (STZ) murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study. Results Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold). Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively). Conclusions The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for

  1. Two novel human NUMB isoforms provide a potential link between development and cancer.

    Science.gov (United States)

    Karaczyn, Aldona; Bani-Yaghoub, Mahmud; Tremblay, Roger; Kubu, Chris; Cowling, Rebecca; Adams, Tamara L; Prudovsky, Igor; Spicer, Douglas; Friesel, Robert; Vary, Calvin; Verdi, Joseph M

    2010-12-01

    We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  2. Two novel human NUMB isoforms provide a potential link between development and cancer

    Directory of Open Access Journals (Sweden)

    Prudovsky Igor

    2010-12-01

    Full Text Available Abstract We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif GTPase activating protein 1. Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5 and filopodia (NUMB6 in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  3. Differential Regulation of Human Thymosin Beta 15 Isoforms by Transforming Growth Factor Beta 1

    Science.gov (United States)

    Banyard, Jacqueline; Barrows, Courtney; Zetter, Bruce R.

    2009-01-01

    We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080 and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322 and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility. PMID:19296525

  4. Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5

    Science.gov (United States)

    Ye, Ling; Yang, Xiao-Shan; Lu, Lin-lin; Chen, Wei-Ying; Zeng, Shan; Yan, Tong-Meng; Dong, Ling-Na; Peng, Xiao-Juan; Shi, Jian; Liu, Zhong-Qiu

    2013-01-01

    Aconitum, widely used to treat rheumatoid arthritis for thousands of years, is a toxic herb that can frequently cause fatal cardiac poisoning. Aconitum toxicity could be decreased by properly hydrolyzing diester-diterpene alkaloids into monoester-diterpene alkaloids. Monoester-diterpene alkaloids, including benzoylaconine (BAC), benzoylmesaconine (BMA), and benzoylhypaconine (BHA), are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP) enzymes protect the human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzyme. High-resolution mass spectrometry was used to characterize the metabolites. A total of 7, 8, and 9 metabolites were detected for BAC, BMA, and BHA, respectively. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, hydroxylation and didemethylation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound. Taken together, the results of the chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzymes experiments demonstrated that CYP3A4 and CYP3A5 have essential functions in the metabolism of BAC, BMA, and BHA. PMID:23864901

  5. Monoester-Diterpene Aconitum Alkaloid Metabolism in Human Liver Microsomes: Predominant Role of CYP3A4 and CYP3A5

    Directory of Open Access Journals (Sweden)

    Ling Ye

    2013-01-01

    Full Text Available Aconitum, widely used to treat rheumatoid arthritis for thousands of years, is a toxic herb that can frequently cause fatal cardiac poisoning. Aconitum toxicity could be decreased by properly hydrolyzing diester-diterpene alkaloids into monoester-diterpene alkaloids. Monoester-diterpene alkaloids, including benzoylaconine (BAC, benzoylmesaconine (BMA, and benzoylhypaconine (BHA, are the primary active and toxic constituents of processed Aconitum. Cytochrome P450 (CYP enzymes protect the human body by functioning as the defense line that limits the invasion of toxicants. Our purposes were to identify the CYP metabolites of BAC, BMA, and BHA in human liver microsomes and to distinguish which isozymes are responsible for their metabolism through the use of chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzyme. High-resolution mass spectrometry was used to characterize the metabolites. A total of 7, 8, and 9 metabolites were detected for BAC, BMA, and BHA, respectively. The main metabolic pathways were demethylation, dehydrogenation, demethylation-dehydrogenation, hydroxylation and didemethylation, which produced less toxic metabolites by decomposing the group responsible for the toxicity of the parent compound. Taken together, the results of the chemical inhibitors, monoclonal antibodies, and cDNA-expressed CYP enzymes experiments demonstrated that CYP3A4 and CYP3A5 have essential functions in the metabolism of BAC, BMA, and BHA.

  6. Involvement of CYP2B6 in the biotransformation of propofol by human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    TANG Bing; WANG Jun-ke; FENG Wan-yu

    2008-01-01

    Objective To determine whether the cytochrome P4502B6 (CYP2B6) is involved in the oxidation of propofol by human liver microsomes. Methods The change of propofol concentration in an incubation mixture with human liver microsomes was monitored by the high performance liquid chromatography (HPLC), in order to calculate the rate constants of metabolism of propofol. The correlation between the rate constants and the rate of metabolism of CYP2B6 selective substrate bupropion, and the effect of two different CYP2B6 specific inhibitors on the propofol metabolism were examined. Results The mean rate constant of propofol metabolism by liver microsomes obtained from twelve individuals was 3.9 (95 % confidence intervals 3.3, 4.5) nmol·min-1·mg-1 protein. The rate constants of propofol metabolism by liver microsomes were significantly correlated with bupropion hydroxylation (r=0.888, P<0.001). Both selective chemical inhibitors of CYP2B6, orphenadrine and N, N′, N″-triethylenethiophosphoramide (thioTEPA), reduced the rate constants of propofol metabolism by 37.596 (P<0.001) and 42.796 (P<0.001)in liver microsomes, respectively. Conclusions CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.

  7. Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro.

    Science.gov (United States)

    Lammel Lindemann, Jan A; Angajala, Anusha; Engler, David A; Webb, Paul; Ayers, Stephen D

    2014-05-05

    Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients.

  8. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Directory of Open Access Journals (Sweden)

    Niels Jacob Aachmann-Andersen

    Full Text Available The membrane-assisted isoform immunoassay (MAIIA quantitates erythropoietin (EPO isoforms as percentages of migrated isoforms (PMI. We evaluated the effect of recombinant human EPO (rhEPO on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13; high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13; or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3 % (mean (SD. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2% (p<0.00001 and 45.2 (7.3% (p<0.00001. Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8% (p<0.00001 and 46.1 (10.4% (p<0.00001. In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4% (p=0.029; low-dose Epoetin beta: 73.1 (17.8% (p=0.039. In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  9. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Science.gov (United States)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p=0.029); low-dose Epoetin beta: 73.1 (17.8)% (p=0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  10. A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

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    Evangelos D Karousis

    Full Text Available Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02 which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

  11. Endocannabinoids anandamide and 2-arachidonoylglycerol are substrates for human CYP2J2 epoxygenase.

    Science.gov (United States)

    McDougle, Daniel R; Kambalyal, Amogh; Meling, Daryl D; Das, Aditi

    2014-12-01

    The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.

  12. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

    Science.gov (United States)

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D

    2013-02-01

    CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

  13. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

    OpenAIRE

    Erickson, J.D.; Schafer, M K; Bonner, T I; Eiden, L. E.; Weihe, E.

    1996-01-01

    A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpr...

  14. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  15. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

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    Dipak K. Dube

    2016-01-01

    Full Text Available In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4 each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

  16. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Science.gov (United States)

    Abbott, Lynn; Alshiekh-Nasany, Ruham; Mitschow, Charles

    2016-01-01

    In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart. PMID:27703814

  17. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes...... a non-functional FR beta on the surface, and, in addition, nanomolar concentrations of a secretory functional FBP (29 kDa) can be present in the secondary granules. A statistically significant correlation between the concentrations of functional FBP, probably a gamma isoform, in granulocytes and serum......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...

  18. Ultrafine carbon particles down-regulate CYP1B1 expression in human monocytes

    Directory of Open Access Journals (Sweden)

    Ziegler-Heitbrock Loems

    2009-10-01

    Full Text Available Abstract Background Cytochrome P450 monoxygenases play an important role in the defence against inhaled toxic compounds and in metabolizing a wide range of xenobiotics and environmental contaminants. In ambient aerosol the ultrafine particle fraction which penetrates deeply into the lungs is considered to be a major factor for adverse health effects. The cells mainly affected by inhaled particles are lung epithelial cells and cells of the monocyte/macrophage lineage. Results In this study we have analyzed the effect of a mixture of fine TiO2 and ultrafine carbon black Printex 90 particles (P90 on the expression of cytochrome P450 1B1 (CYP1B1 in human monocytes, macrophages, bronchial epithelial cells and epithelial cell lines. CYP1B1 expression is strongly down-regulated by P90 in monocytes with a maximum after P90 treatment for 3 h while fine and ultrafine TiO2 had no effect. CYP1B1 was down-regulated up to 130-fold and in addition CYP1A1 mRNA was decreased 13-fold. In vitro generated monocyte-derived macrophages (MDM, epithelial cell lines, and primary bronchial epithelial cells also showed reduced CYP1B1 mRNA levels. Benzo[a]pyrene (BaP is inducing CYB1B1 but ultrafine P90 can still down-regulate gene expression at 0.1 μM of BaP. The P90-induced reduction of CYP1B1 was also demonstrated at the protein level using Western blot analysis. Conclusion These data suggest that the P90-induced reduction of CYP gene expression may interfere with the activation and/or detoxification capabilities of inhaled toxic compounds.

  19. Aryl hydrocarbon hydroxylase represents CYP1B1, and not CYP1A1, in human freshly isolated white cells: trimodal distribution of Japanese population according to induction of CYP1B1 mRNA by environmental dioxins.

    Science.gov (United States)

    Toide, Kenji; Yamazaki, Hiroshi; Nagashima, Rikako; Itoh, Keisuke; Iwano, Shunsuke; Takahashi, Yoshiki; Watanabe, Shaw; Kamataki, Tetsuya

    2003-03-01

    The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10(7) molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes.

  20. Drug-drug Interaction between Losartan and Paclitaxel in Human Liver Microsomes with Different CYP2C8 Genotypes.

    Science.gov (United States)

    Mukai, Yuji; Senda, Asuna; Toda, Takaki; Hayakawa, Toru; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2015-06-01

    The cytochrome P450 (CYP) 2C8*3 allele is associated with reduced metabolic activity of paclitaxel. This study was aimed to investigate the inhibitory effect of losartan on paclitaxel metabolism in human liver microsomes (HLMs) and to determine the impact of the CYP2C8*3 polymorphism. HLMs that contained the CYP2C8*1 homozygote (HL60) or CYP2C8*3 heterozygote (HL54) genotype were used for the inhibition study. Losartan, at a concentration of 50 μmol/L, significantly inhibited paclitaxel metabolism by 29% and 57% in the HL60 (p losartan and the CYP3A4-selective inhibitors, erythromycin and ketoconazole, caused a greater inhibition of the paclitaxel metabolism than quercetin, a CYP2C8-selective inhibitor. This demonstrated that the paclitaxel metabolism was mainly catalysed by CYP3A4 in HL60. There were no significant differences found for the inhibitory effects caused by the four inhibitors of the paclitaxel metabolism in HL54, indicating that both CYP2C8 and CYP3A4 play important roles in paclitaxel metabolism in HL54. These findings suggest that 50 μmol/L of losartan inhibits both CYP2C8 and CYP3A4 in HLMs. In summary, losartan inhibited paclitaxel metabolism, with concentrations over 50 μmol/L in HLMs. The CYP2C8*3 allele carriers are likely susceptible to the interactions of losartan and CYP3A4 inhibitors to paclitaxel metabolism.

  1. Biotransformation of OH-PBDEs by pig liver microsomes: Investigating kinetics, identifying metabolites, and examining the role of different CYP isoforms.

    Science.gov (United States)

    Li, Jianhua; Zhang, Ya; Du, Zhongkun; Peng, Jianbiao; Mao, Liang; Gao, Shixiang

    2016-04-01

    Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are of great concern due to their potential risk to animal and human health. The biotransformation potential of OH-PBDEs in organisms is important for the understanding of their health risk. In the present study, the biotransformation of 3'-OH-2,4-di-BDE (3'-OH-BDE-7), 4'-OH-2,2',4-tri-BDE (4'-OH-BDE-17) and 3-OH-2,2',4,4'-tetra-BDE (3-OH-BDE-47) by pig liver microsomes was studied. Compared with their precursor PBDEs, the three OH-PBDEs were more readily biotransformed by pig liver microsomes, and the biotransformation rate followed the order: 3'-OH-BDE-7 > 4'-OH-BDE-17 > 3-OH-BDE-47. These results revealed that the biotransformation rate of OH-PBDEs was decreased with an increase in the number of bromine substituents. Cleavage of the diphenyl ether bond was the dominant pathway for biotransformation of the three OH-PBDEs by pig liver microsomes, while debromination and hydroxylation were found to be of less importance. CYP3A4 was suggested to be the specific enzyme responsible for the biotransformation of OH-PBDEs via associated inhibition assay. These findings may enrich our understanding of health risk associated with OH-PBDEs in mammals and human beings.

  2. CYP1A induction and human risk assessment: an evolving tale of in vitro and in vivo studies.

    Science.gov (United States)

    Ma, Qiang; Lu, Anthony Y H

    2007-07-01

    CYP1A1 and 1A2 play critical roles in the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines/amides (HAAs), respectively, to electrophilic reactive intermediates, leading to toxicity and cancer. CYP1As are highly inducible by PAHs and halogenated aromatic hydrocarbons via aryl hydrocarbon receptor-mediated gene transcription. The impact of CYP1A induction on the carcinogenic and toxic potentials of environmental, occupational, dietary, and therapeutic chemicals has been a central focus of human risk evaluation and has broadly influenced the fields of cancer research, toxicology, pharmacology, and risk assessment over the past half-century. From the early discovery of CYP1A induction and its role in protection against chemical carcinogenesis in intact animals, to the establishment of CYP1A enzymes as the principal cytochromes P450 for bioactivation of PAHs and HAAs in in vitro assays, to the recent realization of an essential protective role of CYP1A in benzo[a]pyrene-induced lethality and carcinogenesis with CYP1A knockout mice, the understanding of the interrelation between CYP1A induction and chemical safety has followed a full circle. This unique path of CYP1A research underscores the importance of whole animal and human studies in chemical safety evaluation.

  3. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  4. Diazinon is activated by CYP2C19 in human liver.

    Science.gov (United States)

    Kappers, W A; Edwards, R J; Murray, S; Boobis, A R

    2001-11-15

    Phosphorothioate compounds are used throughout the world as agricultural and domestic pesticides. Here, the activation of the phosphorothioate diazinon to diazoxon in human liver is described. In an initial study using three human liver microsomal samples, K(m) for diazoxon formation varied markedly (31, 208, and 660 microM; V(max) 1125, 685, and 1028 pmol/min/mg protein, respectively), suggesting the involvement of more than one P450 enzyme. A wide variation in activity was found using 50 microM diazinon as substrate, (11-648 pmol/min/mg protein, n = 15), whereas, with 500 microM, variation was less (164-978 pmol/min/mg protein). Among eight P450-catalyzed reactions, the putative high-affinity component (50 microM diazinon) correlated with S-mephenytoin 4'-hydroxylase activity (r = 0.686, p diazinon) correlated with both S-mephenytoin 4'-hydroxylase (r = 0.714; p diazinon (500 microM) at the fastest rate, followed by CYP3A4, CYP1A2, and CYP2C9. Both hepatic microsomal S-mephenytoin 4'-hydroxylase and high-affinity phenacetin O-deethylase activities were strongly inhibited by diazinon (IC50 diazinon activation in human liver, while other enzymes including CYP1A2 may play a more minor role.

  5. [Heterogeneous expression of human CYP and its application in early stage of new drug research and development].

    Science.gov (United States)

    Xie, Zhangming; Chen, Shuqing

    2013-01-01

    Human cytochrome P450 (CYP) has a pivotal role on metabolism of xenobiotics and endogenous substances in clinical practice. Since the CYP from human tissue is very complex, and the human tissue itself is not easy to obtain, investigators begin to use all kinds of expression system to heterogeneously express the CYP. The single CYP expressed was then used for drug metabolism and drug-drug interaction research, to improve the efficiency of high-throughput drug screening greatly. Besides, since the polymorphism of drug-metabolizing enzymes makes efficacy variance for some drugs in different population, the heterogeneous expression and drug metabolizing research of certain CYP mutants will be helpful to guide the optimization of therapeutic regimen and conduct the personalized medication.

  6. Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

    Science.gov (United States)

    Hu, Yiding; Kupfer, David

    2002-12-01

    Methoxychlor, a currently used pesticide that in mammals elicits proestrogenic/estrogenic activity and reproductive toxicity, has been classified as a prototype endocrine disruptor. Methoxychlor is prochiral, and its metabolites 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M); 1,1,1-trichloro- 2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M); and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M) are chiral; whereas 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M) is achiral. These metabolites are formed during methoxychlor incubation with liver microsomes or recombinant cytochrome p450s (rp450s). Since methoxychlor-metabolite enantiomers may have different estrogenic/antiestrogenic/antiandrogenic activities than corresponding racemates, the possibility that p450s preferentially generate or use R or S enantiomers, was examined. Indeed, rCYP1A2 and r2A6 mono-demethylated methoxychlor primarily into (R)-mono-OH-M at 91 and 75%, respectively, whereas rCYP1A1, 2B6, 2C8, 2C9, 2C19, and 2D6 formed the (S)-enantiomer at 69, 66, 75, 95, 96, and 80%, respectively. However, rCYP3A4, 3A5, and 2B1(rat) weakly demethylated methoxychlor without enantioselectivity. Human liver microsomes generated (S)-mono-OH-M (77-87%), suggesting that CYP1A2 and 2A6 display only minor catalytic contribution. P450 inhibitors demonstrated that CYP2C9 and possibly 2C19 are major hepatic catalysts forming (S)-mono-OH-M, and CYP1A2 is primarily involved in forming the (R)-mono-OH-M. Demethylation rate of (S)-mono-OH-M versus (R)-mono-OH-M forming achiral bis-OH-M by rCYP1A2 was 97/3, compared with 15/85 and 17/83 for rCYP2C9 and 2C19, respectively, indicating opposite substrate enantioselectivity of rCYP1A2 versus 2C9 and 2C19. Also, rCYP1A2 preferentially O-demethylated (R)-catechol-M into (R)-tris-OH-M (at 80%), contrasting r2C9 and r2C19 that yielded (S)-tris-OH-M at 80 and 77%, respectively. Ortho-hydroxylation of

  7. Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11.

    Science.gov (United States)

    Choi, Seunghye; Han, Songhee; Lee, Hwayoun; Chun, Young-Jin; Kim, Donghak

    2013-11-01

    Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

  8. Investigation of the CYP2C9 induction profile in human hepatocytes by combining experimental and modelling approaches.

    Science.gov (United States)

    Belic, Ales; Temesvári, Manna; Kohalmy, Krisztina; Vrzal, Radim; Dvorak, Zdenek; Rozman, Damjana; Monostory, Katalin

    2009-12-01

    The goal of the present review is to characterise the induction profile of CYP2C9, a member of the cytochrome P450 superfamily. Since the mechanism of CYP2C9 induction is fairly complex, with parallel processes triggered by various inducers, an evaluation of the experimental results is often a great challenge. At least three nuclear receptors, the glucocrticoid receptor (GR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to mediate the CYP2C9 gene induction in man. However, mathematical modelling and simulation can provide an appropriate tool for the interpretation of CYP2C9 regulatory mechanisms. As an example, we present modelling and simulation approaches of the CYP2C9 gene expression in human hepatocytes treated with well-known CYP2C9 inducers: the steroid hormone precursor dehydroepiandrosterone (DHEA) and the synthetic glucocorticoid dexamethasone (DXM). The results of the analysis suggest that in addition to the potent function of GR and the further involvement of PXR and CAR activated by DXM or DHEA, an additional factor might play a role in CYP2C9 regulation by DHEA. The novel potential candidate for DHEA action in CYP2C9 induction is likely to be the estrogen receptor. Additionally, the balance of DHEA sulphation-desulphation processes should also be considered in any description of DHEA-induced CYP2C9 profiles.

  9. Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors.

    Science.gov (United States)

    Valve, E; Martikainen, P; Seppänen, J; Oksjoki, S; Hinkka, S; Anttila, L; Grenman, S; Klemi, P; Härkönen, P

    2000-12-01

    FGF-8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF-8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF-8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF-8 is over-expressed in human breast and prostate cancers. Expression has also been found in RT-PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF-8 isoforms are expressed in ovarian cancer and whether FGF-8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT-PCR and immunohistochemistry. FGF-8 isoform b was expressed in all ovarian tumors and in all 7 ovarian-cancer cell lines studied. Isoform a was co-expressed in 9 malignant ovarian tumors. FGF-8 mRNA was not detected by RT-PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF-8 protein to cancer cells. In general, the increased intensity of FGF-8 staining was associated with loss of differentiation within the tumors (Bowker's test, p = 0.37). FGF-8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT-PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF-8 receptors expressed in normal ovaries and in ovarian tumors. FGF-8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF-8 have acquired the capacity of expressing FGF-8. This suggests that FGF-8 has an important role in ovarian tumorigenesis.

  10. Skatole (3-Methylindole Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Martin Krøyer Rasmussen

    Full Text Available Skatole (3-methylindole is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR regulated genes, such as cytochrome P450 1A1 (CYP1A1, in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.

  11. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Science.gov (United States)

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  12. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    Science.gov (United States)

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  13. In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

    Directory of Open Access Journals (Sweden)

    Kristin R Wildsmith

    Full Text Available Apolipoprotein E (ApoE is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4 each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD. Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS, we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

  14. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    Science.gov (United States)

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. The human-specific invariant chain isoform Iip35 modulates Iip33 trafficking and function.

    Science.gov (United States)

    Sand, Kine Marita Knudsen; Landsverk, Ole J B; Berg-Larsen, Axel; Bakke, Oddmund; Gregers, Tone F

    2014-10-01

    The invariant chain (Ii) is a multifunctional protein, which has an essential role in the assembly and transport of major histocompatibility complex class II (MHC II) molecules. From a single gene, Ii is synthesized as four different isoforms: Iip33, Iip35, Iip41 and Iip43. Iip35 and Iip43 are specific to humans, and are formed due to an upstream alternative translation site, resulting in an N-terminal extension of 16 amino acids. This extension harbors a strong endoplasmic reticulum (ER) retention motif. Consequently, Iip35 or Iip43 expressed alone are retained in the ER, whereas Iip33 and Iip41 rapidly traffic to the endosomal pathway. Endogenously expressed, the four isoforms form mixed heterotrimers in the ER; however, mainly due to the absence of the Iip35/p43 isoforms in mice, little is known about how they influence general Ii function. In this study, we have co-expressed Iip33 and Iip35 in human cells with and without MHC II to gain a better understanding of how Iip35 isoform influences the cellular properties of Iip33. We find that Iip35 significantly affects the properties of Iip33. In the presence of Iip35, the transport of Iip33 out of the ER is delayed, its half-life is dramatically prolonged and its ability to induce enlarged endosomes and delayed endosomal maturation is abrogated.

  16. Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

    Science.gov (United States)

    U, M; Miyashita, T; Shikama, Y; Tadokoro, K; Yamada, M

    2001-11-30

    Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

  17. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...

  18. Investigation of Orlistat effects on PXR activation and CYP3A4 expression in primary human hepatocytes and human intestinal LS174T cells.

    Science.gov (United States)

    Novotna, Aneta; Doricakova, Aneta; Vrzal, Radim; Maurel, Patrick; Pavek, Petr; Dvorak, Zdenek

    2010-10-09

    Drugs for weight loss have been in use for nearly hundred years. Orlistat (Xenical) is a non-centrally acting anti-obesity drug that inactivates gastric and intestinal lipases, thus, preventing absorption of dietary triglycerides. There are reports indicating that Orlistat reduces bioavailability of Cyclosporin to a clinically relevant degree. Since Cyclosporin is metabolized by cytochrome P450 CYP3A4, we examined whether interaction between Orlistat and Cyclosporin involves induction of CYP3A4. Human Caucasian colon adenocarcinoma cells LS174T and primary cultures of human hepatocytes were used, as in vitro models of intestinal and hepatic cells, respectively. Treatment of LS174T cells for 24h with Orlistat (1-100mg/L) did not cause induction of CYP3A4 mRNA levels as compared to control cells while Orlistat (100mg/L) slightly induced CYP3A4 mRNA in human hepatocytes. Rifampicin, a model CYP3A4 inducer, significantly induced CYP3A4 mRNA in both types of cells. The level of CYP3A4 protein in human hepatocytes was increased by Orlistat after 48h, while rifampicin strongly induced CYP3A4 protein level. In addition, Orlistat moderately dose-independently activated pregnane X receptor (PXR) in LS174T cells transiently transfected with p3A4-luc reporter construct containing the basal promoter (-362/+53) with proximal PXR response element and the distal xenobiotic responsive enhancer module (-7836/-7208) of the CYP3A4 gene 5'-flanking region. In conclusion, we report here that Orlistat is weak PXR activator and CYP3A4 inducer in human hepatocytes, but it has no effect on CYP3A4 in intestinal cells, implying no role of CYP3A4 induction in the interaction between Orlistat and Cyclosporin in absorption process.

  19. CYP2C subfamily, primarily CYP2C9, catalyses the enantioselective demethylation of the endocrine disruptor pesticide methoxychlor in human liver microsomes: use of inhibitory monoclonal antibodies in P450 identification.

    Science.gov (United States)

    Hu, Y; Krausz, K; Gelboin, H V; Kupfer, D

    2004-02-01

    1. The endocrine disruptor pesticide methoxychlor undergoes O-demethylation by mammalian liver microsomes forming chiral mono-phenolic (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane, i.e. mono-OH-M) and achiral bis-phenolic oestrogenic metabolites. Human liver microsomes (HLM) generated primarily the S-mono-OH-M. 2. Inhibitory monoclonal antibodies (MAb) identified those P450s catalysing the enantioselective O-demethylation of methoxychlor. In HLM, O-demethylation was inhibited by MAb anti-2C9 (30-40%), diminishing the per cent of S-mono-OH-M from about 80 to 55-60%. MAb anti-CYP1A2, 2A6, 2B6, 2C8, 2C19, 2D6 and 3A4 did not affect the demethylation rate in HLM. Nevertheless, MAb anti-CYP1A2 decreased the formation of R-mono-OH-M from 21-23 to 10-17%, indicating that CYP1A2 exhibits a role in generating the R-enantiomer. 3. Among cDNA-expressed human P450s (supersomes), CYP2C19 was the most active in demethylation, but in HLM, CYP2C19 appeared inactive (no inhibition by MAb anti-CYP2C19). There was a substantial difference in the per cent inhibition of demethylation by MAb anti-CYP2C9 and anti-rat CYP2C (MAb inhibiting all human CYP2C forms) and in altering the enantioselectivity, suggesting that demethylation by combined CYP2C8, 2C18 and 2C19 was significant (20-30%). 4. Polymorphism of methoxychlor demethylation was examined with supersomes and HLM-expressing CYP2C9 allelic variants. CYP2C9*1 and 2C9*2 were highly active; however, CYP2C9*3 appeared inactive.

  20. Ospemifene metabolism in humans in vitro and in vivo: metabolite identification, quantitation, and CYP assignment of major hydroxylations.

    Science.gov (United States)

    Tolonen, Ari; Koskimies, Pasi; Turpeinen, Miia; Uusitalo, Jouko; Lammintausta, Risto; Pelkonen, Olavi

    2013-01-01

    The metabolism of ospemifene, a novel nonsteroidal selective estrogen receptor modulator, was investigated as part of its development. Metabolite identification, tentative quantitation, and CYP assignment of ospemifene were performed in human liver microsomes or homogenate incubations and in plasma samples from volunteer humans. The potential contributions of CYP enzymes were determined by recombinant human CYPs. Metabolite identification and tentative quantification were performed by liquid chromatography-mass spectrometry. The relative abundances of metabolites produced were dependent on ospemifene concentration and liver preparation, but the largest quantities of 4- and 4'-hydroxy-ospemifene (and their glucuronides in smaller quantities) were produced in human liver microsomes at low ospemifene concentrations. Other metabolites were detected in in vitro incubation with human liver including a direct glucuronide of ospemifene and some metabolites with only minor abundance. In human plasma samples, 4-hydroxy-ospemifene was the most abundant metabolite, representing about 25% of the abundance of the parent compound. All the other metabolites detected in plasma, including 4'-hydroxy-ospemifene, represented <7% of the abundance of ospemifene. Several CYP enzymes participated in 4-hydroxylation, including CYP2C9, CYP2C19, CYP2B6, and CYP3A4, whereas CYP3A enzymes were the only ones to catalyze 4'-hydroxylation. In vitro incubations with liver preparations provided a rather reliable starting point in the search for potential metabolites in clinical settings. The in vitro metabolite profile is informative for the in vivo metabolite profile, especially regarding the major hydroxylated metabolites. However, it is anticipated that extended in vivo exposures may result in an increased production of more distal metabolites from major metabolites.

  1. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

    Directory of Open Access Journals (Sweden)

    Maléne E Lindholm

    2016-09-01

    Full Text Available Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  2. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zorka Milićević

    2014-01-01

    Full Text Available In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa and Δ133p53β (35 kDa, known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.

  3. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...... major and several minor subpopulations of SAP. IEF of all SAP isolates showed a previously unreported degree of heterogeneity with six isoelectric forms (pKi range 5.5-6.1) and with minor interindividual differences in respect of isoelectric points. Total enzymatic deglycosylation of SAP reduced...

  4. Human CYP4Z1 catalyzes the in-chain hydroxylation of lauric acid and myristic acid.

    Science.gov (United States)

    Zöllner, Andy; Dragan, Calin-Aurel; Pistorius, Dominik; Müller, Rolf; Bode, Helge B; Peters, Frank T; Maurer, Hans H; Bureik, Matthias

    2009-04-01

    Overexpression of human CYP4Z1, a cytochrome P450 enzyme, has been correlated with poor prognosis in human cancer. However, its catalytic properties are not yet known. We expressed this P450 in Schizosaccharomyces pombe and demonstrate by whole-cell biotransformation assays CYP4Z1-dependent in-chain hydroxylation of lauric and myristic acid, which in both cases leads to the formation of four different monohydroxylated products at positions omega-2, omega-3, omega-4, and omega-5, respectively. The CYP4Z1-expressing fission yeast should be a new valuable tool for testing cancer drugs or for the development of new prodrug strategies.

  5. Secreted Isoform of Human Lynx1 (SLURP-2)

    DEFF Research Database (Denmark)

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we......-evoked currents at concentrations via...... interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs....

  6. Effects of norfloxacin on hepatic genes expression of P450 isoforms (CYP1A and CYP3A), GST and P-glycoprotein (P-gp) in Swordtail fish (Xiphophorus Helleri).

    Science.gov (United States)

    Liang, Ximei; Wang, Lan; Ou, Ruikang; Nie, Xiangping; Yang, YuFeng; Wang, Fang; Li, Kaibin

    2015-10-01

    The presence of antibiotics including norfloxacin in the aquatic environment may cause adverse effects in non-target organisms. But the toxic mechanisms of fluoroquinolone to fish species are still not completely elucidated. Thus, it is essential to investigate the response of fish to the exposure of fluoroquinolone at molecular or cellular level for better and earlier prediction of these environmental pollutants toxicity. The sub-chronic toxic effects of norfloxacin (NOR) on swordtail fish (Xiphophoru s helleri) were investigated by measuring mRNA expression of cytochrome P450 1A (CYP1A), cytochrome P450 3A (CYP3A), glutathione S-transferase (GST) and P-glycoprotein (P-gp) and their corresponding enzyme activities (including ethoxyresorufin O-deethylase, erythromycin N-demethylase and GST. Results showed that NOR significantly affected the expression of CYP1A, CYP3A, GST and P-gp genes in swordtails. The gene expressions were more responsive to NOR exposure than their corresponding enzyme activities. Moreover, sexual differences were found in gene expression and enzyme activities of swordtails exposed to NOR. Females displayed more dramatic changes than males. The study further demonstrated that the combined biochemical and molecular parameters were considered as useful biomarkers to improve our understanding of potential ecotoxicological risks of NOR exposure to aquatic organisms.

  7. Docking studies of piperine - iron conjugate with human CYP450 3A4

    OpenAIRE

    2013-01-01

    Piperine, a major constituent of Piper nigrum (Black pepper), is one of the well known components in many Ayurvedic formulations. Piperine is most studied bioenhancer because it inhibits drug metabolizing enzymes in rodents and increases plasma concentrations of several drugs, including P-glycoprotein substrates. However, there areno evidences on piperine-iron conjugate to inhibit human CYP450 3A4. We therefore investigated the influence of piperine-Fe conjugate to study the metabolism of iro...

  8. Human septin isoforms and the GDP-GTP cycle.

    Science.gov (United States)

    Zent, Eldar; Wittinghofer, Alfred

    2014-02-01

    Septins form oligomeric complexes consisting of septins from different subgroups, which form filaments that are involved in a number of biological processes. They are GTP-binding proteins that contain all the necessary elements to perform the general GDP-to-GTP conformational switch. It is however unclear whether or not such a switch is important for the dynamics of septin filaments. Here we investigate the complex GTPase reaction of members of each of the four human septin groups, which is dominated by the stability of dimer formation via the nucleotide binding or so-called G-interface. The results also show that the actual hydrolysis reaction is very similar for three septin groups in the monomeric state while the Sept6 has no GTPase activity. Sept7, the only member of the Sept7 subgroup, forms a very tight G-interface dimer in the GDP-bound state. Here we show that the stability of the interface is dramatically decreased by exchanging GDP with a nucleoside triphosphate, which is believed to influence filament formation and dynamics via Sept7.

  9. Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.

    Science.gov (United States)

    Fukuda, Mitsunori; Ogata, Yukie; Saegusa, Chika; Kanno, Eiko; Mikoshiba, Katsuhiko

    2002-07-01

    Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

  10. Multiple, but Concerted Cellular Activities of the Human Protein Hap46/BAG-1M and Isoforms

    Directory of Open Access Journals (Sweden)

    Ulrich Gehring

    2009-03-01

    Full Text Available The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types.

  11. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    National Research Council Canada - National Science Library

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    .... There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms...

  12. Inhibitory potency of 8-methoxypsoralen on cytochrome P450 2A6 (CYP2A6 allelic variants CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22: differential susceptibility due to different sequence locations of the mutations.

    Directory of Open Access Journals (Sweden)

    Kai Hung Tiong

    Full Text Available Human cytochrome P450 2A6 (CYP2A6 is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22 have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP, in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.

  13. Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

    Science.gov (United States)

    Pu, Qiang-Hong; Shi, Liang; Yu, Chao

    2015-03-01

    1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

  14. Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors.

    Science.gov (United States)

    Desbans, C; Hilgendorf, C; Lutz, M; Bachellier, P; Zacharias, T; Weber, J C; Dolgos, H; Richert, L; Ungell, A-L

    2014-01-01

    1.  It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2.  The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3.  We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4.  It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

  15. WhichCyp

    DEFF Research Database (Denmark)

    Rostkowski, Michal; Spjuth, Ola; Rydberg, Patrik

    2013-01-01

    SUMMARY: In this work we present WhichCyp, a tool for prediction of which cytochromes P450 isoforms (among 1A2, 2C9, 2C19, 2D6 and 3A4) a given molecule is likely to inhibit. The models are built from experimental high-throughput data using support vector machines and molecular signatures...

  16. Differential expression of human cytochrome P450 enzymes from the CYP3A subfamily in the brains of alcoholic subjects and drug-free controls.

    Science.gov (United States)

    Booth Depaz, Iris M; Toselli, Francesca; Wilce, Peter A; Gillam, Elizabeth M J

    2013-06-01

    Cytochrome P450 enzymes are responsible for the metabolism of most commonly used drugs. Among these enzymes, CYP3A forms mediate the clearance of around 40-50% of drugs and may also play roles in the biotransformation of endogenous compounds. CYP3A forms are expressed both in the liver and extrahepatically. However, little is known about the expression of CYP3A proteins in specific regions of the human brain. In this study, form-selective antibodies raised to CYP3A4 and CYP3A5 were used to characterize the expression of these forms in the human brain. Both CYP3A4 and CYP3A5 immunoreactivity were found to varying extents in the microsomal fractions of cortex, hippocampus, basal ganglia, amygdala, and cerebellum. However, only CYP3A4 expression was observed in the mitochondrial fractions of these brain regions. N-terminal sequencing confirmed the principal antigen detected by the anti-CYP3A4 antibody in cortical microsomes to be CYP3A4. Immunohistochemical analysis revealed that CYP3A4 and CYP3A5 expression was primarily localized in the soma and axonal hillock of neurons and varied according to cell type and cell layer within brain regions. Finally, analysis of the frontal cortex of chronic alcohol abusers revealed elevated expression of CYP3A4 in microsomal but not mitochondrial fractions; CYP3A5 expression was unchanged. The site-specific expression of CYP3A4 and CYP3A5 in the human brain may have implications for the role of these enzymes in both normal brain physiology and the response to drugs.

  17. Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Soraya Scuderi

    2014-01-01

    Full Text Available Parkinson protein 2, E3 ubiquitin protein ligase (PARK2 gene mutations are the most frequent causes of autosomal recessive early onset Parkinson’s disease and juvenile Parkinson disease. Parkin deficiency has also been linked to other human pathologies, for example, sporadic Parkinson disease, Alzheimer disease, autism, and cancer. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification. To date several PARK2 splice variants have been identified; however, the expression and distribution of parkin isoforms have not been deeply investigated yet. Here, the currently known PARK2 gene transcripts and relative predicted encoded proteins in human, rat, and mouse are reviewed. By analyzing the literature, we highlight the existing data showing the presence of multiple parkin isoforms in the brain. Their expression emerges from conflicting results regarding the electrophoretic mobility of the protein, but it is also assumed from discrepant observations on the cellular and tissue distribution of parkin. Although the characterization of each predicted isoforms is complex, since they often diverge only for few amino acids, analysis of their expression patterns in the brain might account for the different pathogenetic effects linked to PARK2 gene mutations.

  18. Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2*

    Science.gov (United States)

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. PMID:20139082

  19. Resveratrol inhibits androgen production of human adrenocortical H295R cells by lowering CYP17 and CYP21 expression and activities

    Science.gov (United States)

    Marti, Nesa; Bouchoucha, Nadia; Sauter, Kay-Sara

    2017-01-01

    Resveratrol, a natural compound found in grapes, became very popular for its suggested protective effects against aging. It was reported to have similar positive effects on the human metabolism as caloric restriction. Recently, positive effects of resveratrol on steroid biosynthesis in cell systems and in humans suffering from polycystic ovary syndrome have also been reported, but the exact mechanism of this action remains unknown. Sirtuins seem targeted by resveratrol to mediate its action on energy homeostasis. In this study, we investigated the mechanisms of action of resveratrol on steroidogenesis in human adrenal H295R cells. Resveratrol was found to inhibit protein expression and enzyme activities of CYP17 and CYP21. It did not alter CYP17 and CYP21 mRNA expression, nor protein degradation. Only SIRT3 mRNA expression was found to be altered by resveratrol, but SIRT1, 3 and 5 overexpression did not result in a change in the steroid profile of H295R cells, indicating that resveratrol may not engage sirtuins to modulate steroid production. Previous studies showed that starvation leads to a hyperandrogenic steroid profile in H295R cells through inhibition of PKB/Akt signaling, and that resveratrol inhibits steroidogenesis of rat ovarian theca cells via the PKB/Akt pathway. Therefore, the effect of resveratrol on PKB/Akt signaling was tested in H295R cells and was found to be decreased under starvation growth conditions, but not under normal growth conditions. Overall, these properties of action together with recent clinical findings make resveratrol a candidate for the treatment of hyperandrogenic disorders such as PCOS. PMID:28323907

  20. Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

    Directory of Open Access Journals (Sweden)

    Katherine J D A Excoffon

    Full Text Available Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7 and CAR(Ex8. We found low-level expression of the CAR(Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7, CAR(Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7, CAR(Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8 providing a potential mechanism for the apical localization of CAR(Ex8 in airway epithelial. In summary, apical localization of CAR(Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.

  1. Isoform-specific targeting and interaction domains in human nicotinamide mononucleotide adenylyltransferases.

    Science.gov (United States)

    Lau, Corinna; Dölle, Christian; Gossmann, Toni I; Agledal, Line; Niere, Marc; Ziegler, Mathias

    2010-06-11

    Several important signaling pathways require NAD as substrate, thereby leading to significant consumption of the molecule. Because NAD is also an essential redox carrier, its continuous resynthesis is vital. In higher eukaryotes, maintenance of compartmentalized NAD pools is critical, but so far rather little is known about the regulation and subcellular distribution of NAD biosynthetic enzymes. The key step in NAD biosynthesis is the formation of the dinucleotide by nicotinamide/nicotinic acid mononucleotide adenylyltransferases (NMNATs). The three human isoforms were localized to the nucleus, the Golgi complex, and mitochondria. Here, we show that their genes contain unique exons that encode isoform-specific domains to mediate subcellular targeting and post-translational modifications. These domains are dispensable for catalytic activity, consistent with their absence from NMNATs of lower organisms. We further demonstrate that the Golgi-associated NMNAT is palmitoylated at two adjacent cysteine residues of its isoform-specific domain and thereby anchored at the cytoplasmic surface, a potential mechanism to regulate the cytosolic NAD pool. Insertion of unique domains thus provides a yet unrecognized enzyme targeting mode, which has also been adapted to modulate subcellular NAD supply.

  2. PPARG in Human Adipogenesis: Differential Contribution of Canonical Transcripts and Dominant Negative Isoforms

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    M. Aprile

    2014-01-01

    Full Text Available The nuclear receptor PPARγ is a key regulator of adipogenesis, and alterations of its function are associated with different pathological processes related to metabolic syndrome. We recently identified two PPARG transcripts encoding dominant negative PPARγ isoforms. The existence of different PPARG variants suggests that alternative splicing is crucial to modulate PPARγ function, underlying some underestimated aspects of its regulation. Here we investigate PPARG expression in different tissues and cells affected in metabolic syndrome and, in particular, during adipocyte differentiation of human mesenchymal stem cells. We defined the transcript-specific expression pattern of PPARG variants encoding both canonical and dominant negative isoforms and identified a novel PPARG transcript, γ1ORF4. Our analysis indicated that, during adipogenesis, the transcription of alternative PPARG variants is regulated in a time-specific manner through differential usage of distinct promoters. In addition, our analysis describes—for the first time—the differential contribution of three ORF4 variants to this process, suggesting a still unexplored role for these dominant negative isoforms during adipogenesis. Therefore, our results highlight crucial aspects of PPARG regulation, suggesting the need of further investigation to rule out the differential impact of all PPARG transcripts in both physiologic and pathologic conditions, such as metabolism-related disorders.

  3. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

    Science.gov (United States)

    Shafqat, Naeem; Turnbull, Andrew; Zschocke, Johannes; Oppermann, Udo; Yue, Wyatt W

    2010-05-14

    3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.

  4. Functional role of human NK cell receptor 2B4 (CD244) isoforms.

    Science.gov (United States)

    Mathew, Stephen O; Rao, Krithi K; Kim, Jong R; Bambard, Nowland D; Mathew, Porunelloor A

    2009-06-01

    2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

  5. In vitro inhibition and induction of human liver cytochrome P450 enzymes by gentiopicroside: potent effect on CYP2A6.

    Science.gov (United States)

    Deng, Yating; Wang, Lu; Yang, Yong; Sun, Wenji; Xie, Renming; Liu, Xueying; Wang, Qingwei

    2013-01-01

    Gentiopicroside (GE), a naturally occurring iridoid glycoside, has been developed into a Novel Traditional Chinese Drug named gentiopicroside injection, and it was approved for the treatment of acute jaundice and chronic active hepatitis by SFDA. However, the inhibitory and inducible effects of GE on the activity of cytochrome P450 (CYP450) are unclear. The purpose of this study was to evaluate the ability of GE to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, GE inhibited CYP2A6 and CYP2E1 in a concentration-dependent manner, with IC₅₀ values of 21.8 µg/ml and 594 µg/ml, respectively, and the IC₅₀ of CYP2A6 was close to the C(max) value observed clinically. GE was a non-competitive inhibitor of CYP2A6 at lower concentrations and a competitive inhibitor at higher concentrations. GE did not produce inhibition of CYP2C9, CYP2D6, CYP1A2 or CYP3A4 activities. However, a significant increase of CYP1A2 and CYP3A4 activity was observed at high concentrations. In cultured human hepatocytes no significant induction of CYP1A2, CYP3A4 or CYP2B6 was observed. Given these results, the in vivo potential inhibition of GE on CYP2A6 deserves further investigation, and it seems that the hepatoprotective effect of GE is irrelevant to its effect on P450s.

  6. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  7. Mice expressing the human CYP7A1 gene in the mouse CYP7A1 knock-out background lack induction of CYP7A1 expression by cholesterol feeding and have increased hypercholesterolemia when fed a high fat diet.

    Science.gov (United States)

    Chen, Jean Y; Levy-Wilson, Beatriz; Goodart, Sheryl; Cooper, Allen D

    2002-11-08

    Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the pathway responsible for the formation of the majority of bile acids. Transcription of the gene is regulated by the size of the bile acid pool and dietary and hormonal factors. The farnesoid X receptor and the liver X receptor (LXR) are responsible for regulation by bile acids and cholesterol, respectively. To study the effects of dietary cholesterol and fat upon expression of the human CYP7A1 gene, mice were generated by crossing transgenic mice carrying the human CYP7A1 gene with mice that were homozygous knock-outs (CYP7A1(-/-)). The mice (mCYP7A1(-/-)/hCYP7A1) expressed the human gene at much higher levels than did the transgenics bred in the wild-type background. A diet containing 1% cholic acid reduced the expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice to undetectable levels. Cholestyramine (5%) increased the level of expression of the human gene and the mouse gene. Thus, farnesoid X receptor-mediated regulation was preserved. A diet containing 2% cholesterol increased expression of the mouse gene in wild-type mice, but it did not affect expression of the human gene in mCYP7A1(-/-)/hCYP7A1 mice. None of the diets altered the serum cholesterol or triglyceride levels in these mice; 1% cholic acid caused a redistribution of cholesterol from the high density lipoprotein to the low density lipoprotein density in the humanized mice but not in wild-type mice. A diet containing 30% saturated fat and 2% cholesterol caused a decrease in CYP7A1 levels in mCYP7A1(-/-)/hCYP7A1 mice. The serum cholesterol levels rose in all mice fed this diet. The increase was greater in the mCYP7A1(-/-)/hCYP7A1 mice. Together, these data suggest that the lack of an LXR element in the region from -56 to -49 of the human CYP7A1 promoter may account for some of the differences in response to diets between humans and rodents.

  8. 大鼠体外肝星状细胞活化过程中代谢活化相关 CYP450亚型的变化%MuItipIe xenobiotic metaboIism-associated cytochrome P450 isoforms might be invoIved in earIy activation of rat hepatic steIIate ceIIs in vitro

    Institute of Scientific and Technical Information of China (English)

    廖长秀; 吴勇; 平洁; 敖英; 印宪; 汪晖

    2014-01-01

    OBJECTIVE To investigate the role of several xenobiotic metabolism-associated CYP450 isoforms in the auto-activation of hepatic stellate cells(HSCs) invitro. METHODS HSCs were isolated from adult Wistar rats and cultured on plastic as an in vitroauto-activation model. Positive expression of α-smooth muscle actin(α-SMA)was used as an activated marker of HSCs and detected by immunocytochemical staining in HSCs cultured for 1,2,5 or 11 d. The expressions of CYP450 isoforms were analyzed by real-time RT-PCR in the HSCs. RESULTS The immunocytochemical stai-ning showed no α-SMA expression in HSCs cultured for 1 d,while its expression gradually increased during culture since day 2. ln terms of α-SMA expression,HSCs cultured for 1,2,5,and 11 d were classified as the quiescent,early,middle and later stages of activation,respectively. The RT-PCR results revealed that CYP1B1,CYP2B1/ 2,and CYP2E1 mRNA were expressed at high levels in the early stage of HSCs activation(at day 2),which were 2.1-,1.6-,and 23.9-fold those in the quiescent HSCs(day 1),respectively. Further study revealed that mRNA expressions of these up-regulated CYPs in the early stage of activation were diminished at the subsequent two stages. The levels of CYP1A1 and CYP1A2 mRNA decreased constantly throughtout the activation process. CONCLUSION Multiple xenobi-otic metabolism-associated CYP450 isoforms might be involved in the auto-activation of rat HSCs invitro.%目的:观察大鼠肝星状细胞(HSC)活化不同时期外源活化相关 CYP450亚型CYP1A1, CYP1B1,CYP1A2,CYP2B1/2和 CYP2E1 mRNA 表达水平的变化规律。方法分离大鼠 HSC,接种在无包被塑料培养瓶中作为 HSC 自氧化活化模型,免疫细胞化学法检测不同培养时间 HSC 活化标志物α-平滑肌肌动蛋白(α-SMA)表达。实时定量 RT-PCR 法测定 HSC 自氧化激活的不同时期多种外源物活化相关 CYP450亚型 mRNA 的表达变化。结果免疫细胞化学结果表明

  9. The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

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    Junting Hu

    2014-12-01

    Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

  10. Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

    Science.gov (United States)

    Shimada, Tsutomu; Kakimoto, Kensaku; Takenaka, Shigeo; Koga, Nobuyuki; Uehara, Shotaro; Murayama, Norie; Yamazaki, Hiroshi; Kim, Donghak; Guengerich, F Peter; Komori, Masayuki

    2016-12-01

    2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with Ks values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute(-1)) than CYP2A13 (∼0.02 minute(-1)) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute(-1)) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute(-1)) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute(-1)). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  11. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  12. Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

    Institute of Scientific and Technical Information of China (English)

    Pei-pei DONG; Chang-xiao LIU; Ling YANG; Zhong-ze FANG; Yan-yan ZHANG; Guang-bo GE; Yu-xi MAO; Liang-liang ZHU; Yan-qing QU; Wei Li; Li-ming WANG

    2011-01-01

    Aim: To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo.Methods:The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A.Results: The activity of CYP2C8 was inhibited with an IC50 value of 6.17±2.0μmol/L. Erlotinib stimulated the midazolam 1'-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substratedependent: the IC50 values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of K1 and k1nack were 6.3μmol/L and 0.035 min-1 for midazolam; 9.0μmol/L and 0.045 min-1 for testosterone; and 10.1μmol/L and 0.058 min-1 for nifedipine.Conclusion: The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrateindependent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib's safety, especially in the context of combination therapy.

  13. Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo.

    Science.gov (United States)

    Jiang, Weiwu; Couroucli, Xanthi I; Wang, Lihua; Barrios, Roberto; Moorthy, Bhagavatula

    2011-04-01

    Supplemental oxygen administration is frequently administered to pre-term and term infants having pulmonary insufficiency. However, hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Cytochrome P450 (CYP)A enzymes have been implicated in hyperoxic lung injury. In this study, we tested the hypothesis that hyperoxia induces CYP1A1 and 1A2 enzymes by transcriptional activation of the corresponding promoters in vivo, and transgenic mice expressing the human CYP1A1 or the mouse 1A2 promoter would be more susceptible to hyperoxic lung injury than wild type (WT) mice. Adult WT (CD-1) (12week-old) mice, transgenic mice carrying a 10kb human CYP1A1 promoter and the luciferase (luc) reporter gene (CYP1A1-luc), or mice expressing the mouse CYP1A2 promoter (CYP1A2-luc) were maintained in room air or exposed to hyperoxia for 24-72h. Hyperoxia exposure of CYP1A1-luc mice for 24 and 48h resulted in 2.5- and 1.25-fold increases, respectively, in signal intensities, compared to room air controls. By 72h, the induction had declined to control levels. CYP1A2-luc mice also showed enhanced luc expression after 24-48h, albeit to a lesser extent than those expressing the CYP1A1 promoter. Also, these mice showed decreased levels of endogenous CYP1A1 and 1A2 expression after prolonged hyperoxia, and were also more susceptible to lung injury than similarly exposed WT mice, with CYP1A2-luc mice showing the greatest injury. Our results support the hypothesis that hyperoxia induces CYP1A enzymes by transcriptional activation of its corresponding promoters, and that decreased endogenous expression of these enzymes contribute to the increased susceptibilities to hyperoxic lung injury in the transgenic animals. In summary, this is the first report providing direct evidence of hyperoxia-mediated induction of CYP1A1 and CYP1A2 expression in vivo by mechanisms entailing transcriptional activation of the corresponding promoters, a phenomenon that has

  14. Inhibition of Human Cytochrome P450 Enzymes by Allergen Removed Rhus verniciflua Stoke Standardized Extract and Constituents

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    Hyunsik Jung

    2014-01-01

    Full Text Available Objective. Potential interactions between herbal extracts and the cytochrome P450 (CYP system lead to serious adverse events or decreased drug efficacy. Rhus verniciflua stoke (RVS and its constituents have been reported to have various pharmacological properties. We evaluated the inhibitory potential of RVS and its constituents on the major CYP isoforms. Methods. The effects of allergen removed RVS (aRVS standardized extract and major components, fustin and fisetin isolated from aRVS, were evaluated on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzyme activity by a luminescent CYP recombinant human enzyme assay. Results. The aRVS extract showed relative potent inhibitory effects on the CYP2C9 (IC50, <0.001 μg/mL, CYP2C19 (IC50, 9.68 μg/mL, and CYP1A2 (IC50, 10.0 μg/mL. However, it showed weak inhibition on CYP3A4 and CYP2D6. Fustin showed moderate inhibitory effects on the CYP2C19 (IC50, 64.3 μg/mL and weak inhibition of the other CYP isoforms similar to aRVS. Fisetin showed potent inhibitory effects on CYP2C9, CYP2C19, and CYP1A2. Fisetin showed moderate inhibition of CYP2D6 and weak inhibition of CYP3A4. Conclusions. These results indicate that aRVS, a clinically available herbal medicine, could contribute to herb-drug interactions when orally coadministered with drugs metabolized by CYP2C9, CYP2C19, and CYP1A2.

  15. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    Energy Technology Data Exchange (ETDEWEB)

    Levova, Katerina; Moserova, Michaela [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic); Nebert, Daniel W. [Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati (United States); Phillips, David H. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Frei, Eva [Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg (Germany); Schmeiser, Heinz H. [Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg (Germany); Arlt, Volker M. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Stiborova, Marie, E-mail: stiborov@natur.cuni.cz [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  16. Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution.

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    Eva M Schmidt

    Full Text Available CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5-exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207. We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76% in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.

  17. Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity.

    Science.gov (United States)

    Cheng, Jie; Ma, Xiaochao; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2009-08-01

    Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

  18. Dual roles of nuclear receptor liver X receptor α (LXRα) in the CYP3A4 expression in human hepatocytes as a positive and negative regulator.

    Science.gov (United States)

    Watanabe, Keisuke; Sakurai, Kaori; Tsuchiya, Yuri; Yamazoe, Yasushi; Yoshinari, Kouichi

    2013-08-01

    CYP3A4 is a major drug-metabolizing enzyme in humans, whose expression levels show large inter-individual variations and are associated with several factors such as genetic polymorphism, physiological and disease status, diet and xenobiotic exposure. Nuclear receptor pregnane X receptor (PXR) is a key transcription factor for the xenobiotic-mediated transcription of CYP3A4. In this study, we have investigated a possible involvement of liver X receptor α (LXRα), a critical regulator of cholesterol homeostasis, in the hepatic CYP3A4 expression since several recent reports suggest the involvement of CYP3A enzymes in the cholesterol metabolism in humans and mice. Reporter assays using wild-type and mutated CYP3A4 luciferase reporter plasmids and electrophoretic mobility shift assays revealed that LXRα up-regulated CYP3A4 through the known DNA elements critical for the PXR-dependent CYP3A4 transcription, suggesting LXRα as a positive regulator for the CYP3A4 expression and a crosstalk between PXR and LXRα in the expression. In fact, reporter assays showed that LXRα activation attenuated the PXR-dependent CYP3A4 transcription. Moreover, a PXR agonist treatment-dependent increase in CYP3A4 mRNA levels was suppressed by co-treatment with an LXRα agonist in human primary hepatocytes and HepaRG cells. The suppression was not observed when LXRα expression was knocked-down in HepaRG cells. In conclusion, the present results suggest that sterol-sensitive LXRα positively regulates the basal expression of CYP3A4 but suppresses the xenobiotic/PXR-dependent CYP3A4 expression in human hepatocytes. Therefore, nutritional, physiological and disease conditions affecting LXRα might be one of the determinants for the basal and xenobiotic-responsive expression of CYP3A4 in human livers.

  19. Isoform switch of pyruvate kinase M1 indeed occurs but not to pyruvate kinase M2 in human tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Cheng Zhan

    Full Text Available Muscle type of pyruvate kinase (PKM is one of the key mediators of the Warburg effect and tumor metabolism. Due to alternative splicing, there are at least 12 known isoforms of the PKM gene, of which PKM1 and PKM2 are two major isoforms with only a 23 amino acid sequenced difference but quite different characteristics and functions. It was previously thought the isoform switch from PKM1 to PKM2 resulted in high PKM2 expression in tumors, providing a great advantage to tumor cells. However, this traditional view was challenged by two recent studies; one study claimed that this isoform switch does not occur during the Warburg effect; the other study asserted that the isoform switch is tissue-specific. Here, we re-analyzed the RNA sequencing data of 25 types of human tumors from The Cancer Genome Atlas Data Portal, and confirmed that PKM2 was the major isoform in the tumors and was highly elevated in addition to the entire PKM gene. We further demonstrated that the expression level of PKM1 significantly declined even though there was substantially increased expression of the entire PKM gene. The proportion of PKM1 in total transcript variants also significantly declined in tumors but the proportion of PKM2 did not change accordingly. Therefore, we conclude that the isoform switch of PKM1 does indeed occur, but it switches to other isoforms rather than PKM2. Considering the change in the expression levels of PKM1, PKM2 and the entire PKM gene, we propose that the upregulation of PKM2 is primarily due to elevated transcriptional levels of the entire PKM gene, instead of the isoform switch.

  20. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  1. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  2. Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability*

    Science.gov (United States)

    Wegmann, Susanne; Jung, Yu Jin; Chinnathambi, Subashchandrabose; Mandelkow, Eva-Maria; Mandelkow, Eckhard; Muller, Daniel J.

    2010-01-01

    Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility. PMID:20566652

  3. Human Tau isoforms assemble into ribbon-like fibrils that display polymorphic structure and stability.

    Science.gov (United States)

    Wegmann, Susanne; Jung, Yu Jin; Chinnathambi, Subashchandrabose; Mandelkow, Eva-Maria; Mandelkow, Eckhard; Muller, Daniel J

    2010-08-27

    Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the "thin twisted" and "thin smooth" fibrils resemble flat ribbons (cross-section approximately 10 x 15 nm) with diverse twist periodicities. "Thick fibrils" show periodicities of approximately 65-70 nm and thicknesses of approximately 9-18 nm such as routinely reported for "paired helical filaments" but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of approximately 17-19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures ( approximately 2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the "fuzzy coat" of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.

  4. Design and synthesis of benzodiazepine analogs as isoform-selective human lysine deacetylase inhibitors.

    Science.gov (United States)

    Reddy, D Rajasekhar; Ballante, Flavio; Zhou, Nancy J; Marshall, Garland R

    2017-02-15

    A comprehensive investigation was performed to identify new benzodiazepine (BZD) derivatives as potent and selective human lysine deacetylase inhibitors (hKDACis). A total of 108 BZD compounds were designed, synthesized and from that 104 compounds were biologically evaluated against human lysine deacetylases (hKDACs) 1, 3 and 8 (class I) and 6 (class IIb). The most active compounds showed mid-nanomolar potencies against hKDACs 1, 3 and 6 and micromolar activity against hKDAC8, while a promising compound (6q) showed selectivity towards hKDAC3 among the different enzyme isoforms. An hKDAC6 homology model, refined by molecular dynamics simulation was generated, and molecular docking studies performed to rationalize the dominant ligand-residue interactions as well as to define structure-activity-relationships. Experimental results confirmed the usefulness of the benzodiazepine moiety as capping group when pursuing hKDAC isoform-selectivity inhibition, suggesting its continued use when designing new hKDACis.

  5. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu-Ge; Ying-Nian Yu; Yu-Li Qian; Xin Li

    2002-01-01

    AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

  6. Effects of green tea catechins on cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A activities in human liver and intestinal microsomes.

    Science.gov (United States)

    Misaka, Shingen; Kawabe, Keisuke; Onoue, Satomi; Werba, José Pablo; Giroli, Monica; Tamaki, Sekihiro; Kan, Toshiyuki; Kimura, Junko; Watanabe, Hiroshi; Yamada, Shizuo

    2013-01-01

    The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

  7. Contribution of NADH increases to ethanol’s inhibition of retinol oxidation by human ADH isoforms

    Science.gov (United States)

    Chase, Jennifer R.; Poolman, Mark G.; Fell, David A.

    2010-01-01

    Background A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free NADH in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. Method To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of four reactions: (1) ADH oxidation of ethanol and NAD+ (2) ADH oxidation of retinol and NAD+ (3) oxidation of ethanol by a generalized Ethanoloxidase that uses NAD+ (4) NADHoxidase which carries out NADH turnover. Results Using the metabolic modeling package SCRUMPY, we have shown that the ethanol-induced increase in NADH contributes from 0–90% of the inhibition by ethanol, depending on [ethanol] and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanoloxidase and the NADHoxidase contribute as well. Discussion Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases. PMID:19183134

  8. Comparison of chlorzoxazone one-sample methods to estimate CYP2E1 activity in humans

    DEFF Research Database (Denmark)

    Kramer, Iza; Dalhoff, Kim; Clemmesen, Jens O

    2003-01-01

    OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one...... estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.......-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl...

  9. In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

    Science.gov (United States)

    Yamane, Mizuki; Kawashima, Kosuke; Yamaguchi, Koji; Nagao, Shunsuke; Sato, Mika; Suzuki, Masayuki; Honda, Kiyofumi; Hagita, Hitoshi; Kuhlmann, Olaf; Poirier, Agnes; Fowler, Stephen; Funk, Christoph; Simon, Sandrine; Aso, Yoshinori; Ikeda, Sachiya; Ishigai, Masaki

    2015-03-01

    Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

  10. Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

    Directory of Open Access Journals (Sweden)

    Eric K Hoobler

    Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

  11. Myosin heavy chain isoform expression in human extraocular muscles: longitudinal variation and patterns of expression in global and orbital layers.

    Science.gov (United States)

    Park, Kyung-Ah; Lim, Jeonghee; Sohn, Seongsoo; Oh, Sei Yeul

    2012-05-01

    We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs. Copyright © 2012 Wiley Periodicals, Inc.

  12. CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin.

    Science.gov (United States)

    John, Kaarthik; Divi, Rao L; Keshava, Channa; Orozco, Christine C; Schockley, Marie E; Richardson, Diana L; Poirier, Miriam C; Nath, Joginder; Weston, Ainsley

    2010-06-28

    Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4muM) in the absence or presence of chlorophyllin (5muM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (pchlorophyllin followed by administration of the carcinogen with chlorophyllin (pchlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.

  13. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    Science.gov (United States)

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.

  14. V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

    Science.gov (United States)

    Miquel-Serra, Laia; Serra, Montserrat; Hernández, Daniel; Domenzain, Clelia; Docampo, María José; Rabanal, Rosa M; de Torres, Inés; Wight, Thomas N; Fabra, Angels; Bassols, Anna

    2006-09-01

    Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.

  15. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    Science.gov (United States)

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions.

  16. [Isolation of isoforms of apolipoprotein CIII from human serum by chromatofocusing].

    Science.gov (United States)

    Fang, D; Gong, R; O, K

    1999-03-01

    This study aimed to isolate isoforms of apolipoprotein (apo) C III from human serum. 24-hour fasting serum from normal and hyperlipidemic subjects was pooled and subjected to ultracentrifugation at plasma density for 20 hours. Very low density lipoprotein (VLDL) was collected at density of d < 1.006 g/ml, and it was delipidated by ethanol and ether. The delipidated apo-VLDL was dissolved in a solution containing 7.2 mol/L urea and 20 mmol/L dithiothreitol. The insoluble apo B was removed by centrifugation. The soluble apo-VLDL was applied to PBE94 column, and eluted with elution buffer containing polybuffer 74 and 8 mol/L urea (1:8, pH4.0). After pooled, the eluted peaks of apolipoproteins were applied to column chromatography of hydroxylapatite to remove the polybuffer. The purified isoforms of apoC III and the purified apo C I, C II and E, were characterized by isoelectrofocusing and west blot. The results showed that the purified apoC III1, C III2, and C II were pure.

  17. Different enzyme kinetics of midazolam in recombinant CYP3A4 microsomes from human and insect sources.

    Science.gov (United States)

    Christensen, Hege; Mathiesen, Liv; Postvoll, Lillian W; Winther, Bjørn; Molden, Espen

    2009-01-01

    In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1'- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b(5)) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean K(m) for the formation of 1'-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (pmicrosomes was reflected by significantly lower Cl(int) compared to insect microsomes (pmicrosomes displayed Michaelis-Menten kinetics, while insect microsomes showed substrate inhibition kinetics. The different enzyme kinetics of midazolam observed in recombinant CYP3A4 microsomes from human and insect sources, especially the substantially higher K(m) obtained in human microsomes compared to insect microsomes, should be further evaluated since it may have implications for correlations to in vivo situation.

  18. Temporal, Diagnostic, and Tissue-Specific Regulation of NRG3 Isoform Expression in Human Brain Development and Affective Disorders.

    Science.gov (United States)

    Paterson, Clare; Wang, Yanhong; Hyde, Thomas M; Weinberger, Daniel R; Kleinman, Joel E; Law, Amanda J

    2017-03-01

    Genes implicated in schizophrenia are enriched in networks differentially regulated during human CNS development. Neuregulin 3 (NRG3), a brain-enriched neurotrophin, undergoes alternative splicing and is implicated in several neurological disorders with developmental origins. Isoform-specific increases in NRG3 are observed in schizophrenia and associated with rs10748842, a NRG3 risk polymorphism, suggesting NRG3 transcriptional dysregulation as a molecular mechanism of risk. The authors quantitatively mapped the temporal trajectories of NRG3 isoforms (classes I-IV) in the neocortex throughout the human lifespan, examined whether tissue-specific regulation of NRG3 occurs in humans, and determined if abnormalities in NRG3 transcriptomics occur in mood disorders and are genetically determined. NRG3 isoform classes I-IV were quantified using quantitative real-time polymerase chain reaction in human postmortem dorsolateral prefrontal cortex from 286 nonpsychiatric control individuals, from gestational week 14 to 85 years old, and individuals diagnosed with either bipolar disorder (N=34) or major depressive disorder (N=69). Tissue-specific mapping was investigated in several human tissues. rs10748842 was genotyped in individuals with mood disorders, and association with NRG3 isoform expression examined. NRG3 classes displayed individually specific expression trajectories across human neocortical development and aging; classes I, II, and IV were significantly associated with developmental stage. NRG3 class I was increased in bipolar and major depressive disorder, consistent with observations in schizophrenia. NRG3 class II was increased in bipolar disorder, and class III was increased in major depression. The rs10748842 risk genotype predicted elevated class II and III expression, consistent with previous reports in the brain, with tissue-specific analyses suggesting that classes II and III are brain-specific isoforms of NRG3. Mapping the temporal expression of genes

  19. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    Science.gov (United States)

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  20. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    Science.gov (United States)

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR.

  1. Application of Mice Humanized for CYP2D6 to the Study of Tamoxifen Metabolism and Drug–Drug Interaction with Antidepressants

    Science.gov (United States)

    MacLeod, A. Kenneth; McLaughlin, Lesley A.; Henderson, Colin J.

    2017-01-01

    Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. It is a prodrug that is converted by several cytochrome P450 enzymes to a primary metabolite, N-desmethyltamoxifen (NDT), which is then further modified by CYP2D6 to a pharmacologically potent secondary metabolite, 4-hydroxy-N-desmethyltamoxifen (endoxifen). Antidepressants (ADs), which are often coprescribed to patients receiving tamoxifen, are also metabolized by CYP2D6 and evidence suggests that a drug–drug interaction between these agents adversely affects the outcome of tamoxifen therapy by inhibiting endoxifen formation. We evaluated this potentially important drug–drug interaction in vivo in mice humanized for CYP2D6 (hCYP2D6). The rate of conversion of NDT to endoxifen by hCYP2D6 mouse liver microsomes (MLMs) in vitro was similar to that of the most active members of a panel of 13 individual human liver microsomes. Coincubation with quinidine, a CYP2D6 inhibitor, ablated endoxifen generation by hCYP2D6 MLMs. The NDT-hydroxylation activity of wild-type MLMs was 7.4 times higher than that of hCYP2D6, whereas MLMs from Cyp2d knockout animals were inactive. Hydroxylation of NDT correlated with that of bufuralol, a CYP2D6 probe substrate, in the human liver microsome panel. In vitro, ADs of the selective serotonin reuptake inhibitor class were, by an order of magnitude, more potent inhibitors of NDT hydroxylation by hCYP2D6 MLMs than were compounds of the tricyclic class. At a clinically relevant dose, paroxetine pretreatment inhibited the generation of endoxifen from NDT in hCYP2D6 mice in vivo. These data demonstrate the potential of ADs to affect endoxifen generation and, thereby, the outcome of tamoxifen therapy. PMID:27756789

  2. Differential ligand binding affinities of human estrogen receptor-α isoforms.

    Directory of Open Access Journals (Sweden)

    Amanda H Y Lin

    Full Text Available Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66 and the truncated isoforms, estrogen receptor-α46 (ER46 and estrogen receptor-α36 (ER36. However, the binding affinities of the membrane estrogen receptors (mERs remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

  3. Functional refolding and characterization of two Tom40 isoforms from human mitochondria.

    Science.gov (United States)

    Mager, Frauke; Gessmann, Dennis; Nussberger, Stephan; Zeth, Kornelius

    2011-07-01

    Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

  4. Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

    Science.gov (United States)

    Vemu, Annapurna; Atherton, Joseph; Spector, Jeffrey O; Szyk, Agnieszka; Moores, Carolyn A; Roll-Mecak, Antonina

    2016-06-17

    Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

  5. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction

    NARCIS (Netherlands)

    Martignoni, Marcella; Groothuis, Geny M. M.; de Kanter, Ruben

    2006-01-01

    Animal models are commonly used in the preclinical development of new drugs to predict the metabolic behaviour of new compounds in humans. It is, however, important to realise that humans differ from animals with regards to isoform composition, expression and catalytic activities of drug-metabolisin

  6. Isoforms of Hsp70-binding human LDL in adult Schistosoma mansoni worms.

    Science.gov (United States)

    Pereira, Adriana S A; Cavalcanti, Marília G S; Zingali, Russolina B; Lima-Filho, José L; Chaves, Maria E C

    2015-03-01

    Schistosoma mansoni is one of the most common parasites infecting humans. They are well adapted to the host, and this parasite's longevity is a consequence of effective escape from the host immune system. In the blood circulation, lipoproteins not only help to conceal the worm from attack by host antibodies but also act as a source of lipids for S. mansoni. Previous SEM studies showed that the low-density lipoprotein (LDL) particles present on the surface of adult S. mansoni worms decreased in size when the incubation time increased. In this study, immunocytochemical and proteomic analyses were used to locate and identify S. mansoni binding proteins to human plasma LDL. Ultrathin sections of adult worms were cut transversely from the anterior, medial and posterior regions of the parasite. Immunocytochemical experiments revealed particles of gold in the tegument, muscle region and spine in male worms and around vitelline cells in females. Immunoblotting and 2D-electrophoresis using incubations with human serum, anti-LDL antibodies and anti-chicken IgG peroxidase conjugate were performed to identify LDL-binding proteins in S. mansoni. Analysis of the binding proteins using LC-MS identified two isoforms of the Hsp70 chaperone in S. mansoni. Hsp70 is involved in the interaction with apoB in the cytoplasm and its transport to the endoplasmic reticulum. However, further studies are needed to clarify the functional role of Hsp70 in S. mansoni, mainly related to the interaction with human LDL.

  7. Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.

    Science.gov (United States)

    Bandiera, Silvio; Weidlich, Simone; Harth, Volker; Broede, Peter; Ko, Yun; Friedberg, Thomas

    2005-02-01

    Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues.

  8. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  9. Endosulfan-alpha Induces CYP26 and CYP3A4 by Activating the Pregnane X Receptor But Not the Constitutive Androstane Receptor

    Science.gov (United States)

    2006-01-01

    of cadmium, endosulfan, and atrazine in adrenal steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana. Environ Toxicol...1519. Dai D, Cao Y, Falls G, Levi PE, Hodgson E and Rose RL (2001) Modulation of mouse P450 isoforms CYP 1A2, CYP2B 10, CYP2E1, and CYP3A by the

  10. Cocktail探针药物法评价生、醋莪术对CYP450酶亚型的影响%Application of probe drugs for detecting influences of Rhizoma Curcumae and processed Rhizoma Curcumae on cytochrome P450 isoforms

    Institute of Scientific and Technical Information of China (English)

    王菊; 陆兔林; 毛春芹; 肖永庆; 顾娟娟

    2012-01-01

    Aim To research and compare the influences of Rhizoma Curcumae and processed Rhizoma Curcumae on the cytochrome P450 isoforms, and to explore the tropism processed of Rhizoma Curcumae to enhance or change the role. Methods Rats were randomly divided into three groups. The treatment groups were received Rhizoma Curcumae and processed Rhizoma Curcumae respectively. The other group were received saline as blank control group. Blood samples were collected aL different time points and the plasma concentration of cocktail probe drugs was determined by IIPLC. Results There was insignificant difference of the ophylline, dapsone, chlorzoxazone between the Rhizoma Curcumae group and the blank group. Compared with the blank group, the half-life time,Tmax and the area under curve increased significantly and the clearance lowered significantly of ophylline and chlorzoxazone in the processed Rhizoma Curcumae group; There was insignificant difference in the half-life time of dapsone , the area under curve lowered significantly and the clearance increased significantly in the processed Rhizoma Curcumae group. Conclusion Rhizoma Curcumae has no significant effect on the cytochrome P450 isoforms of CYP1A2, CYP3A4 and CYP2E1. But the processed Rhizoma Curcumae can in-hibit CYP1A2 and CYP2E1 and has an indue tive effect on the CYP3A4.%目的 用Cocktail探针药物法,研究生、醋莪术对大鼠CYP1A2、CYP3A4和CYP2E1活性的影响并进行比较,探讨其通过炮制增强或改变作用的趋向性.方法 将SD大鼠随机分组,生、醋莪术组分别给予生、醋莪术提取液(9 g·kg-1),以生理盐水为空白对照,在一定时间点采集血样,用HPLC检测探针药物在大鼠体内的代谢情况,以评价生、醋莪术对CYP450酶的影响.结果 生莪术的茶碱、氨苯酚和氯唑沙宗的代谢情况与空白组相比差异无显著性;但醋莪术与空白组相比,茶碱和氯唑沙宗的T12、Tmax 和AUC明显增加,CL/F明显降低;氨苯酚的T12 变

  11. Differential subcellular localizations of two human Sgo1 isoforms: implications in regulation of sister chromatid cohesion and microtubule dynamics.

    Science.gov (United States)

    Wang, Xiaoxing; Yang, Yali; Dai, Wei

    2006-03-01

    Sgo1 is an evolutionarily conserved protein that functions as a protector of centromeric cohesin during mitosis. Recent studies show that Sgo1 is kinetochorelocalized and required for accurate segregation of mitotic chromosomes because depletion of Sgo1 in mammalian cells results in precocious initiation of anaphase and mis-segregation of chromosomes. Through analysis of GFP fusion proteins, we observe that two major isoforms of human Sgo1 exhibit entirely different subcellular localization patterns. The short isoform of Sgo1 (sSgo1) that lacks exon 6 does not localize to kinetochores during any stages of the cell cycle. Instead, it is enriched at mitotic spindles. On the other hand, the longer isoform of Sgo1 primarily localizes to kinetochores during G(2) phase and mitotic prophase, metaphase, and anaphase. During late mitosis, Sgo1 does not appear to be associated with kinetochores. Intriguingly, the longer isoform of Sgo1 forms discrete foci during S phase, some of which are apparently in the nucleoli. However, a majority of these foci colocalize with CREST, a kinetochore antigen, indicating that Sgo1 is loaded onto kinetochores during or immediately after DNA replication. Together, our studies suggest that different isoforms of Sgo1 may play distinct roles during the cell cycle and that Sgo1 may have an interphase function as well.

  12. Significance of redox-active cysteines in human FAD synthase isoform 2.

    Science.gov (United States)

    Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

    2014-12-01

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

  13. Inhibitory and inductive effects of Polygonum capitatum water extract on CYP450%头花蓼水提取物对CYP450酶诱导和抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    陆苑; 潘洁; 谢玉敏; 郑林; 黄勇; 王永林; 李勇军

    2015-01-01

    Aim To evaluate the inhibitive and induc-tive effects of Polygonum capitatum water extract on main cytochrome P450 isoforms in human and liver mi-crosomes of mouse in vitro for predicting the herb-drug interactions in clinical application. Methods The in vitro inhibitory effect was evaluated by incubating Po-lygonum capitatum water extract with the probe sub-strates of main phase I metabolic enzymes in human liver microsomes, including CYP1A2, CYP2E1, CYP2C9,CYP2C19 and CYP3A4. Mice were adminis-tered with Polygonum capitatum water extract at dosage of 0 . 58 g · kg-1 and 1 . 16 g · kg-1 by gastric lavage for successive 7 days and 14 days, then the cocktail-LC-MS/MS method was applied to assess the inductive effect of main CYP450 isoforms in mouse liver micro-somes. Results The IC50 values of Polygonum capita-tum water extract on main CYP450 isoforms in human liver microsomes were from 849 . 6 mg · L-1 to 2 287 mg·L-1 . Compared with the blank control group, the activites of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 7 d group were about 49 . 9 % and 21. 1 % higher ( P <0. 01, P < 0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 0. 58 g·kg-1 7 d group were 27. 6 % and 15. 5 % higher ( P <0. 01 , P <0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 14 d group were 67. 5 % and 32. 1 %higher (P<0. 01) respectively, while the activities of CYP1 A2 , CYP2 E1 and CYP2 C19 were not increased significantly in Polygonum capitatum treatment group. Conclusions Polygonum capitatum water extract do not show the inhibitory effect on main CYP450 in hu-man liver microsomes. There is induction on CYP2C9 and CYP3 A4 in mouse liver microsomes by Polygonum capitatum water extract.%目的:考察头花蓼水提取物对人肝微粒体CYP4505种亚型酶的体外抑制作用和对小鼠的体内诱导作用,从而预测发生药物相互作用的可能性,为临床合理用药提供科学依据。方法以探针底物代谢物生成法,考察头花

  14. Statistical evaluation of the isoform patterns of N-acetyl-beta-hexosaminidase from human renal cancer tissue separated by isoelectrofocusing.

    Science.gov (United States)

    Borzym-Kluczyk, Malgorzata; Radziejewska, Iwona; Olszewska, Ewa; Szajda, Sławomir; Knaś, Małgorzata; Zwierz, Krzysztof

    2007-03-01

    Isoenzymes of HEX from human renal carcinoma and neighbouring macroscopically normal renal tissue can show different patterns on isoelectrofocusing gels. The aim of our work was to elaborate the method for statistical evaluation of differences. Isoenzymes of N-acetyl-beta-hexosaminidase were separated from renal (control and cancerous) tissues of 15 patients. Isoenzymes were electrofocused in Multiphor II, with ampholine pH 3.5-9.0 (2%) and then evaluated densitometrically and analysed statistically. A similar pattern in activity of isoforms of isoenzymes A and B in normal and cancerous renal tissue was observed. The proposed method of statistical evaluation of differences in isoforms of N-acetyl-beta-glucosaminidase can also be adapted to estimate the isoforms of other enzymes in different tissues.

  15. Influence of various polymorphic variants of cytochrome P450 oxidoreductase (POR on drug metabolic activity of CYP3A4 and CYP2B6.

    Directory of Open Access Journals (Sweden)

    Xuan Chen

    Full Text Available Cytochrome P450 oxidoreductase (POR is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.

  16. Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

    Directory of Open Access Journals (Sweden)

    Saori Tsuji

    Full Text Available Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

  17. Discoidin domain receptor 1: isoform expression and potential functions in cirrhotic human liver.

    Science.gov (United States)

    Song, Sunmi; Shackel, Nicholas A; Wang, Xin M; Ajami, Katerina; McCaughan, Geoffrey W; Gorrell, Mark D

    2011-03-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

  18. Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

    The differential availability of thyroxine (T/sub 4/) and 3,5,3'-triiodothyronine (T/sub 3/) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of (/sup 125/I)T/sub 4/ and (/sup 125/I)T/sub 3/ to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, (/sup 125/I)T/sub 4/ was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. (/sup 125/I)T/sub 3/ was localized to only two isoforms of TBG, TBG-III, and -IV, with pI's that were identical with those for (/sup 125/I)T/sub 4/. In normal female serum, (/sup 125/I)T/sub 4/ was localized to the same four isoforms of TBG as those of normal male serum, while (/sup 125/I)T/sub 3/ was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, (/sup 125/I)T/sub 4/ was localized to five isoforms, whereas (/sup 125/I)T/sub 3/ was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T/sub 3/. The K/sub i/ values of T/sub 3/ binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and the chromatofocusing (pH range of 3.5-5.0). T/sub 4/ preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T/sub 4/ and T/sub 3/. In conclusion, this study shows that T/sub 4/ and T/sub 3/ do not bind to a single competitive binding site on TBG. Instead, T/sub 4/ is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T/sub 3/ for these proteins.

  19. Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

    Science.gov (United States)

    Rodriguez, R J; Miranda, C L

    2000-09-01

    N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (DAK to N-hydroxy-DAK (15.9%) and metabolite 1 (6.6%). Last, DAK did not appear to be a substrate for human or rabbit FMO5. Heat inactivation of cDNA-expressed FMOs abolished DAK metabolite formation. These results suggest that DAK is a substrate for human and rabbit FMO1 and FMO3, rabbit FMO2, but not human or rabbit FMO5.

  20. Human invariant chain isoform p35 restores thymic selection and antigen presentation in CD74-deficient mice.

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    Genève, Laetitia; Chemali, Magali; Desjardins, Michel; Labrecque, Nathalie; Thibodeau, Jacques

    2012-10-01

    The invariant chain (Ii) has pleiotropic functions and is a key factor in antigen presentation. Ii associates with major histocompatibility complex class II molecules in the endoplasmic reticulum (ER) and targets the complex in the endocytic pathway to allow antigenic peptide loading. The human Iip35 isoform includes a cytoplasmic extension containing a di-arginine motif causing ER retention. This minor isoform does not exist in mice and its function in humans has not been thoroughly investigated. We have recently generated transgenic mice expressing Iip35 and these were crossed with Ii-deficient mice to generate animals (Tgp35/mIiKO) expressing exclusively the human isoform. In these mice, we show that Iip35 is expressed in antigen presenting cells and is inducible by interferon gamma (IFN-γ). Despite the low constitutive expression of the protein and some minor differences in the Vβ repertoire of Tgp35/mIiKO mice, Iip35 restored thymic selection of CD4(+) T cells and of invariant natural killer T cells. In vitro functional assays using purified primary macrophages treated with IFN-γ showed that Iip35 allows presentation of an Ii-dependent ovalbumin T-cell epitope. Altogether, our results suggest that Iip35 is functional and does not require co-expression of other isoforms for antigen presentation.

  1. [Isoforms of the human histamine H3 receptor: Generation, expression in the central nervous system and functional implications].

    Science.gov (United States)

    García-Gálvez, Ana Maricela; Arias-Montaño, José Antonio

    2016-01-01

    Histamine plays a significant role as a neuromodulator in the human central nervous system. Histamine-releasing neurons are exclusively located in the tuberomammillary nucleus of the hypothalamus, project to all major areas of the brain, and participate in functions such as the regulation of sleep/wakefulness, locomotor activity, feeding and drinking, analgesia, learning, and memory. The functional effects of histamine are exerted through the activation of four G protein-coupled receptors (H1, H2, H3 and H4), and in the central nervous system the first three receptors are widely expressed. The H3 receptor (H3R) is found exclusively in neuronal cells, where it functions as auto- and hetero-receptor. One remarkable characteristic of the H3R is the existence of isoforms, generated by alternative splicing of the messenger RNA. For the human H3R, 20 isoforms have been reported; although a significant number lack those regions required for agonist binding or receptor signaling, at least five isoforms appear functional upon heterologous expression. In this work we review the evidence for the generation of human H3R isoforms, their expression, and the available information regarding the functionality of such receptors.

  2. Inhibitory effect of salvianolate on human cytochrome P450 3A4 in vitro involving a noncompetitive manner.

    Science.gov (United States)

    Qin, Chong-Zhen; Ren, Xian; Zhou, Hong-Hao; Mao, Xiao-Yuan; Liu, Zhao-Qian

    2015-01-01

    Salvianolic acid B (Sal B), which is purified from Danshen, is a popular herb extract. Sal B has anti-oxidative, anti-inflammatory, anti-hypoxic, anti-arteriosclerotic and anti-apoptotic properties. This substance can also ameliorate brain injury or neurodegenerative diseases. The listed drug Salvianolate, which contains a substantial amount of Sal B, has been used for the treatment of coronary heart disease. Our present work aimed to evaluate the inhibitory effect of salvianolate on seven cytochrome P450 isoforms (CYP450), namely, CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, in human liver microsomes (HLMs) and recombinant enzymes through high-performance liquid chromatography (HPLC) assay. Salvianolate have a potent inhibitory effect on CYP3A4 activity with IC50 values of 1.438 (HLMs) and 3.582 (recombinant cDNA-expressed CYP3A4) mg/L, respectively. Salvianolate strongly dose, but not time-dependently decreased CYP3A4 activity in HLMs. The typical Lineweaver-Burk plots showed that Salvianolate inhibited CYP3A4 activity noncompetitively, with a Ki value of 2.27 mg/L in HLMs. Other CYP450 isoforms are not markedly affected by Salvianolate. These findings indicate that salvianolate may be involved in potential drug interactions when co-administrated with CYP3A4 substrates.

  3. Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase.

    Science.gov (United States)

    Laurent, Laetitia; Deroy, Kathy; St-Pierre, Joey; Côté, Francine; Sanderson, J Thomas; Vaillancourt, Cathy

    2017-09-01

    The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. Co-expression of SERCA isoforms, phospholamban and sarcolipin in human skeletal muscle fibers.

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    Val A Fajardo

    Full Text Available Sarcolipin (SLN and phospholamban (PLN inhibit the activity of sarco(endoplasmic reticulum Ca(2+-ATPases (SERCAs by reducing their apparent affinity for Ca(2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg to vastus lateralis homogenates increased the apparent Ca(2+ affinity of SERCA (K Ca, pCa units (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02 and maximal SERCA activity (μmol/g protein/min (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11 demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.

  5. Increased CYP1A1 expression in human exfoliated urothelial cells of cigarette smokers compared to non-smokers

    Energy Technology Data Exchange (ETDEWEB)

    Doerrenhaus, Angelika; Roos, Peter H. [Institute for Occupational Physiology at the University Dortmund, Dortmund (Germany); Mueller, Tina [Institute for Occupational Physiology at the University Dortmund, Dortmund (Germany); University Dortmund, Department of Statistics, Mathematical Statistics with Applications in Biometrics, Dortmund (Germany)

    2007-01-15

    Polycyclic aromatic hydrocarbons, arylamines and nitrosamines, constituents of cigarette smoke, are known inducers of bladder cancer. The biochemical response of the target tissue, the bladder urothelium, following inhalation of cigarette smoke has not been studied so far. We used exfoliated transitional urothelial cells from human urine samples to analyze effects of smoking on induction of the cytochrome P450 enzyme CYP1A1. Samples of 40 subjects, including male and female smokers and non-smokers, were examined. A prerequisite for the immunofluorescence microscopic analysis of the cells was the enrichment of the urothelial cell population. This was achieved by a new method which is based on magnetic cell sorting exploiting specific binding of immobilized Griffonia simplicifolia lectin to the surface of urothelial cells. Immunostaining of the final cell preparation with a monoclonal antibody to CYP1A1 showed that about 6% of the urothelial cells of non-smokers stained positive for CYP1A1. However, this fraction of positive cells was more than 44% of the urothelial cells in samples from cigarette smokers. In spite of the individual variation, the difference was statistically significant. There were no gender-related differences in the portion of CYP1A1 expressing urothelial cells of smokers and non-smokers. In essence, we show for the first time that human urothelial cells respond to cigarette smoking by induction of CYP1A1. The approach opens new fields of mechanistic and biomarker research with respect to the pathogenetic processes of cancer development in the human bladder. (orig.)

  6. Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To study the gene expression of metallothionein 1 (MT-1) isoforms in human peripheral blood lymphocytes (HPBLs). Methods The expression of mRNA representing the seven active MT-1 genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-1X, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT-1H, 1F, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference (P<0.05). in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, 1F, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1G, MT-1H, MT-1F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

  7. Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

    Science.gov (United States)

    Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

    2017-01-01

    Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features.

  8. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane, tumor-associated isoforms IX and XII with boronic acids.

    Science.gov (United States)

    Winum, Jean-Yves; Innocenti, Alessio; Scozzafava, Andrea; Montero, Jean-Louis; Supuran, Claudiu T

    2009-05-15

    A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K(I) of 3.7-4.5 microM, whereas the remaining derivatives showed inhibition constants in the range of 6.0-1560 microM for hCA I and of 6.0-1050 microM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K(I)s of 7.6-12.3 microM) whereas the arylalkenyl and aryl-alkyl derivatives generally showed weaker inhibitory properties (K(I)s of 34-531 microM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)(2) moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.

  9. Altered CYP19A1 and CYP3A4 Activities Due to Mutations A115V, T142A, Q153R and P284L in the Human P450 Oxidoreductase

    Directory of Open Access Journals (Sweden)

    Sameer S. Udhane

    2017-08-01

    Full Text Available All cytochromes P450s in the endoplasmic reticulum rely on P450 oxidoreductase (POR for their catalytic activities. Mutations in POR cause metabolic disorders of steroid hormone biosynthesis and affect certain drug metabolizing P450 activities. We studied mutations A115V, T142A, Q153R identified in the flavin mononucleotide (FMN binding domain of POR that interacts with partner proteins and P284L located in the hinge region that is required for flexibility and domain movements in POR. Human wild-type (WT and mutant POR as well as CYP3A4 and CYP19A1 proteins in recombinant form were expressed in bacteria, and purified proteins were reconstituted in liposomes for enzyme kinetic assays. Quality of POR protein was checked by cytochrome c reduction assay as well as flavin content measurements. We found that proteins carrying mutations A115V, T142A located close to the FMN binding site had reduced flavin content compared to WT POR and lost almost all activity to metabolize androstenedione via CYP19A1 and showed reduced CYP3A4 activity. The variant P284L identified from apparently normal subjects also had severe loss of both CYP19A1 and CYP3A4 activities, indicating this to be a potentially disease causing mutation. The mutation Q153R initially identified in a patient with disordered steroidogenesis showed remarkably increased activities of both CYP19A1 and CYP3A4 without any significant change in flavin content, indicating improved protein–protein interactions between POR Q153R and some P450 proteins. These results indicate that effects of mutations on activities of individual cytochromes P450 can be variable and a detailed analysis of each variant with different partner proteins is necessary to accurately determine the genotype-phenotype correlations of POR variants.

  10. Effects of Neonicotinoids on Promoter-Specific Expression and Activity of Aromatase (CYP19) in Human Adrenocortical Carcinoma (H295R) and Primary Umbilical Vein Endothelial (HUVEC) Cells.

    Science.gov (United States)

    Caron-Beaudoin, Élyse; Denison, Michael S; Sanderson, J Thomas

    2016-01-01

    The enzyme aromatase (CYP19; cytochrome P450 19) in humans undergoes highly tissue- and promoter-specific regulation. In hormone-dependent breast cancer, aromatase is over-expressed via several normally inactive promoters (PII, I.3, I.7). Aromatase biosynthesizes estrogens, which stimulate breast cancer cell proliferation. The placenta produces estrogens required for healthy pregnancy and the major placental CYP19 promoter is I.1. Exposure to certain pesticides, such as atrazine, is associated with increased CYP19 expression, but little is known about the effects of neonicotinoid insecticides on CYP19. We developed sensitive and robust RT-qPCR methods to detect the promoter-specific expression of CYP19 in human adrenocortical carcinoma (H295R) and primary umbilical vein endothelial (HUVEC) cells, and determined the potential promoter-specific disruption of CYP19 expression by atrazine and the commonly used neonicotinoids imidacloprid, thiacloprid, and thiamethoxam. In H295R cells, atrazine concentration-dependently increased PII- and I.3-mediated CYP19 expression and aromatase catalytic activity. Thiacloprid and thiamethoxam induced PII- and I.3-mediated CYP19 expression and aromatase activity at relatively low concentrations (0.1-1.0 µM), exhibiting non-monotonic concentration-response curves with a decline in gene induction and catalytic activity at higher concentrations. In HUVEC cells, atrazine slightly induced overall (promoter-indistinct) CYP19 expression (30 µM) and aromatase activity (≥ 3 µM), without increasing I.1 promoter activity. None of the neonicotinoids increased CYP19 expression or aromatase activity in HUVEC cells. Considering the importance of promoter-specific (over)expression of CYP19 in disease (breast cancer) or during sensitive developmental periods (pregnancy), our newly developed RT-qPCR methods will be helpful tools in assessing the risk that neonicotinoids and other chemicals may pose to exposed women.

  11. Seminal vesicles and urinary bladder as sites of aromatization of androgens in men, evidenced by a CYP19A1-driven luciferase reporter mouse and human tissue specimens.

    Science.gov (United States)

    Strauss, Leena; Rantakari, Pia; Sjögren, Klara; Salminen, Anu; Lauren, Eve; Kallio, Jenny; Damdimopoulou, Pauliina; Boström, Minna; Boström, Peter J; Pakarinen, Pirjo; Zhang, FuPing; Kujala, Paula; Ohlsson, Claes; Mäkelä, Sari; Poutanen, Matti

    2013-04-01

    The human CYP19A1 gene is expressed in various tissues by the use of tissue-specific promoters, whereas the rodent cyp19a1 gene is expressed mainly in the gonads and brain. We generated a transgenic mouse model containing a >100-kb 5' region of human CYP19A1 gene connected to a luciferase reporter gene. The luciferase activity in mouse tissues mimicked the CYP19A1 gene expression pattern in humans. Interestingly, the reporter gene activity was 16 and 160 times higher in the urinary bladder and seminal vesicles, respectively, as compared with the activity in the testis. Accordingly, CYP19A1 gene and P450arom protein expression was detected in those human tissues. Moreover, the data revealed that the expression of CYP19A1 gene is driven by promoters PII, I.4, and I.3 in the seminal vesicles, and by promoters PII and I.4 in the urinary bladder. Furthermore, the reporter gene expression in the seminal vesicles was androgen dependent: Castration decreased the expression ∼20 times, and testosterone treatment restored it to the level of an intact mouse. This reporter mouse model facilitates studies of tissue-specific regulation of the human CYP19A1 gene, and our data provide evidence for seminal vesicles as important sites for estrogen production in males.

  12. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    Directory of Open Access Journals (Sweden)

    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  13. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  14. The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

    Science.gov (United States)

    Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

    2017-04-01

    Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites.

  15. Selective inhibition of cytochrome P450 2D6 by Sarpogrelate and its active metabolite, M-1, in human liver microsomes.

    Science.gov (United States)

    Cho, Doo-Yeoun; Bae, Soo Hyeon; Lee, Joeng Kee; Kim, Yang Weon; Kim, Bom-Taeck; Bae, Soo Kyung

    2014-01-01

    The present study was performed to evaluate the in vitro inhibitory potential of sarpogrelate and its active metabolite, M-1, on the activities of nine human cytochrome (CYP) isoforms. Using a cocktail assay, the effects of sarpogrelate on nine CYP isoforms and M-1 were measured by specific marker reactions in human liver microsomes. Sarpogrelate potently and selectively inhibited CYP2D6-mediated dextromethorphan O-demethylation with an IC50 (Ki) value of 3.05 μM (1.24 μM), in a competitive manner. M-1 also markedly inhibited CYP2D6 activity; its inhibitory effect with an IC50 (Ki) value of 0.201 μM (0.120 μM) was more potent than that of sarpogrelate, and was similarly potent as quinidine (Ki, 0.129 μM), a well-known typical CYP2D6 inhibitor. In addition, sarpogrelate and M-1 strongly inhibited both CYP2D6-catalyzed bufuralol 1'-hydroxylation and metoprolol α-hydroxylation activities. However, sarpogrelate and M-1 showed no apparent inhibition of the other following eight CYPs: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5. Upon 30-minute preincubation of human liver microsomes with sarpogrelate or M-1 in the presence of NADPH, no obvious shift in IC50 was observed in terms of inhibition of the nine CYP activities, suggesting that sarpogrelate and M-1 are not time-dependent inactivators. Sarpogrelate strongly inhibited the activity of CYP2D6 at clinically relevant concentrations in human liver microsomes. These observations suggest that sarpogrelate could have an effect on the metabolic clearance of drugs possessing CYP2D6-catalyzed metabolism as a major clearance pathway, thereby eliciting pharmacokinetic drug-drug interactions.

  16. Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology.

    Science.gov (United States)

    Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A

    2008-06-01

    The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.

  17. A reporter gene assay to assess the molecular mechanisms of xenobiotic-dependent induction of the human CYP3A4 gene in vitro.

    Science.gov (United States)

    Ogg, M S; Williams, J M; Tarbit, M; Goldfarb, P S; Gray, T J; Gibson, G G

    1999-03-01

    1. A plasmid containing 1 kb of the CYP3A4 regulatory (promoter) region coupled to a reporter gene for secretary placental alkaline phosphatase (SPAP) was transfected into HepG2 cells. Transfected cells were dosed with several known inducers of CYP3A4 and the levels of SPAP were measured. The effect of co-transfecting a plasmid encoding the human glucocorticoid receptor on reporter gene activity was also examined. 2. Dexamethasone induced CYP3A4-dependent reporter gene expression in a concentration-dependent manner and induction was approximately doubled in the presence of the glucocorticoid receptor. Dexamethasone-dependent induction was blocked by RU-486 (a glucocorticoid receptor antagonist), in the presence of the co-transfected glucocorticoid receptor. 3. Induction of CYP3A4-dependent reporter gene expression and enhancement of the induction by the glucocorticoid receptor was also observed with pregnenolone-16alpha-carbonitrile (PCN), rifampicin, phenytoin, carbamazepine, phenylbutazone and phenobarbitone, all known in vivo inducers of CYP3A4 in man. 4. Metyrapone and sulfinpyrazone induced CYP3A4-dependent reporter gene expression, but induction was not enhanced by the glucocorticoid receptor. 5. Clotrimazole, erythromycin and triacetyloleandomycin (TAO) did not induce CYP3A4-dependent reporter gene expression, consistent with the observation that these inducers act through post-transcriptional mechanisms. 6. These results highlight differences in the molecular mechanisms of induction of CYP3A4 by the xenobiotics studied and indicate that the glucocorticoid receptor is involved in the induction of the CYP3A4 gene by some, but not all, CYP3A4 inducers. 7. We propose that the approach described here provides a useful in vitro approach for the identification of transcriptional regulators of the CYP3A4 gene.

  18. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

    Science.gov (United States)

    Farr, Christine J; Antoniou-Kourounioti, Melissa; Mimmack, Michael L; Volkov, Arsen; Porter, Andrew C G

    2014-04-01

    As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

  19. Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

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    Robert A Eagle

    Full Text Available BACKGROUND: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. METHODOLOGY/PRINCIPAL FINDINGS: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have

  20. Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

    OpenAIRE

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Michael H Gelb; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h...

  1. High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal

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    Denis Kole

    2017-05-01

    Full Text Available Basic fibroblast growth factor (FGF2 is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006, and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011. A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18 kDa low molecular weight (LMW isoform and four larger high molecular weight (HMW isoforms (Arese et al., 1999; Arnaud et al., 1999. As they are not generally secreted, high molecular weight (HMW FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18 kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

  2. CYP19A1 single nucleotide polymorphism associations with CYP19A1, NFκB1, and IL6 gene expression in human normal colon and normal liver samples

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    Penney RB

    2014-07-01

    Full Text Available Rosalind B Penney,1 Abbie Lundgreen,2 Aiwei Yao-Borengasser,3 Vineetha K Edavana,3 Suzanne Williams,3 Ishwori Dhakal,4 Roger K Wolff,2 Susan Kadlubar,3 Martha L Slattery2 1Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR, 2Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT, 3Division of Medical Genetics, University of Arkansas for Medical Sciences, Little Rock, AR, 4Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Background: Estrogen is known to decrease the risk of colon cancer in postmenopausal women, and may exert its actions by decreasing interleukin-6 (IL6 production via stabilization of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB. Estrogens are biosynthesized by CYP19A1 (aromatase, so it is possible that genetic variations in CYP19A1 influences the risk of colon cancer by altering expression of CYP19A1. Further, studies on gene-gene interactions suggest that single nucleotide polymorphisms in one gene may affect expression of other genes. The current study aims to explore the role of CYP19A1 single nucleotide polymorphisms on CYP19A1, NFκB1 and IL6 gene expression. Methods: Phenotype–genotype associations, cross-associations between genes, and haplotype analyses were performed in both normal human colon (n=82 and liver (n=238 samples. Results: CYP19A1 rs10459592, rs1961177, and rs6493497 were associated with CYP19A1 expression in colon samples (P=0.042, P=0.041, and P=0.013, respectively. CYP19A1 single nucleotide polymorphisms (rs12908960, rs730154, rs8025191, and rs17523880 were correlated with NFκB1 expression (P=0.047, P=0.04, P=0.05, and P=0.03, respectively, and CYP19A1 rs11856927, rs2470152, and rs2470144 (P=0.049, P=0.025, P=0.047, respectively were associated with IL6 expression in the colon. While rs730154 and rs17523880

  3. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

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    Nathalie Esber

    Full Text Available The progesterone receptor (PR with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4 and ulipristal acetate (UPA, a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.

  4. Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain.

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    Phillip B Jones

    Full Text Available We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aβ-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aβ than ApoE4, but a greater proportion of Aβ molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aβ deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aβ and thereby increase the risk of AD for carriers of the APOEε4 allele.

  5. A negative cooperativity mechanism of human CYP2E1 inferred from molecular dynamics simulations and free energy calculations.

    Science.gov (United States)

    Li, Jue; Wei, Dong-Qing; Wang, Jing-Fang; Li, Yi-Xue

    2011-12-27

    Human cytochrome P450 2E1 (CYP2E1) participates in the metabolism of over 2% of all the oral drugs. A hallmark peculiar feature of this enzyme is that it exhibits a pronounced negative cooperativity in substrate binding. However the mechanism by which the negative cooperativity occurs is unclear. Here, we performed molecular dynamics simulations and free energy calculations on human CYP2E1 to examine the structural differences between the substrate-free and the enzymes with one and two aniline molecules bound. Our results indicate that although the effector substrate does not bind in the active site cavity, it still can directly interact with the active site residues of human CYP2E1. The interaction of the effector substrate with the active site leads to a reorientation of active site residues, which thereby weakens the interactions of the active substrate with this site. We also identify a conserved residue T303 that plays a crucial role in the negative cooperative binding on the short-range effects. This residue is a key factor in the positioning of substrates and in proton delivery to the active site. Additionally, a long-range effect of the effector substrate is identified in which F478 is proposed to play a key role. As located in the interface between the active and effector sites, this residue structurally links the active and effector sites and is found to play a significant role in affecting substrate access and ligand positioning within the active site. In the negative cooperative binding, this residue can decrease the interactions of the active substrate with the active site by π-π stacking which then lowers the hydroxylation activity for the active substrate. These findings are in agreement with previous experimental observations and thus provide detailed atomistic insight into the poorly understood mechanism of the negative cooperativity in human CYP2E1.

  6. Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein.

    Science.gov (United States)

    Ramos, Adrián M; Duschak, Vilma G; Gerez de Burgos, Nelia M; Barboza, Mariana; Remedi, María S; Vides, Miguel A; Chiabrando, Gustavo A

    2002-02-01

    Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.

  7. Metabolic interactions of agrochemicals in humans.

    Science.gov (United States)

    Hodgson, Ernest; Rose, Randy L

    2008-06-01

    Agrochemicals and other xenobiotics are metabolized by xenobiotic-metabolizing enzymes (XMEs) to products that may be more or less toxic than the parent chemical. In this regard, phase-I XMEs such as cytochrome P450s (CYPs) are of primary importance. Interactions at the level of metabolism may take place via either inhibition or induction of XMEs. Such interactions have often been investigated, in vitro, in experimental animals, using subcellular fractions such as liver microsomes, but seldom in humans or at the level of individual XME isoforms. The authors have been investigating the metabolism of a number of agrochemicals by human liver microsomes and recombinant CYP isoforms and have recently embarked on studies of the induction of XMEs in human hepatocytes. The insecticides chlorpyrifos, carbaryl, carbofuran and fipronil, as well as the repellant DEET, are all extensively metabolized by human liver microsomes and, although a number of CYP isoforms may be involved, CYP2B6 and CYP3A4 are usually the most important. Permethrin is hydrolyzed by esterase(s) present in both human liver microsomes and cytosol. A number of metabolic interactions have been observed. Chlorpyrifos and other phosphorothioates are potent inhibitors of the CYP-dependent metabolism of both endogenous substrates, such as testosterone and estradiol, and exogenous substrates, such as carbaryl, presumably as a result of the interaction of highly reactive sulfur, released during the oxidative desulfuration reaction, with the heme iron of CYP. The hydrolysis of permethrin in human liver can be inhibited by chlorpyrifos oxon and by carbaryl. Fipronil can inhibit testosterone metabolism by CYP3A4 and is an effective inducer of CYP isoforms in human hepatocytes.

  8. Identification and analysis of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 in cynomolgus macaques.

    Science.gov (United States)

    Uno, Yasuhiro; Hosaka, Shinya; Yamazaki, Hiroshi

    2014-12-01

    Cytochromes P450 (P450) are important for not only drug metabolism and toxicity, but also biosynthesis and metabolism of cholesterol and bile acids, and steroid synthesis. In cynomolgus macaques, widely used in biomedical research, we have characterized P450 cDNAs, which were isolated as expressed sequence tags of cynomolgus macaque liver. In this study, cynomolgus CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 cDNAs were characterized by sequence analysis, phylogenetic analysis and tissue expression pattern. By sequence analysis, these five cynomolgus P450s had high sequence identities (94-99%) to the human orthologs in amino acids. By phylogenetic analysis, each cynomolgus P450 was more closely related to the human ortholog as compared with the dog or rat ortholog. By quantitative polymerase chain reaction, among the 10 tissue types, CYP7A1 and CYP17A1 mRNAs were preferentially expressed in liver and adrenal gland, respectively. Cynomolgus CYP27A1 and CYP51A1 mRNAs were most abundantly expressed in liver and testis, respectively. Cynomolgus CYP20A1 mRNA was expressed in all the tissues, including brain and liver. Tissue expression patterns of each cynomolgus P450 were generally similar to that of the human ortholog. These results suggest the molecular similarities of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 between cynomolgus macaques and humans.

  9. Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population.

    Science.gov (United States)

    Yousef, Al-Motassem; Bulatova, Nailya R; Newman, William; Hakooz, Nancy; Ismail, Said; Qusa, Hisham; Zahran, Farah; Anwar Ababneh, Nidaa; Hasan, Farah; Zaloom, Imad; Khayat, Ghada; Al-Zmili, Rawan; Naffa, Randa; Al-Diab, Ola

    2012-10-01

    Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (2 and 3), CYP2C19 (2 and 3), CYP3A4 5, CYP3A5 (3 and 6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1 1, 0.165 for CYP1A1 2A and 0.071 for CYP1A1 2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9 1, 0.135 for CYP2C9 2 and 0.068 for CYP2C9 3. For CYP2C19, the frequencies of the wild type (CYP2C19 1) and the nonfunctional (2 and 3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16 %) were homozygous for 2/2. Regarding CYP3A4 1B, only 12 subjects out of 173 subjects (6.9 %) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5 1, 3 and 6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.

  10. In vitro characterization of the cytochrome P450 isoforms involved in the metabolism of 6-methoxy-2-napthylacetic acid, an active metabolite of the prodrug nabumetone.

    Science.gov (United States)

    Matsumoto, Kaori; Nemoto, Eiichi; Hasegawa, Tetsuya; Akimoto, Masayuki; Sugibayashi, Kenji

    2011-01-01

    The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

  11. Changes in CYP1A2 activity in humans after 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) administration using caffeine as a probe drug.

    Science.gov (United States)

    Yubero-Lahoz, Samanta; Pardo, Ricardo; Farre, Magí; Mathuna, Brian Ó; Torrens, Marta; Mustata, Cristina; Perez-Mañá, Clara; Langohr, Klaus; Carbó, Marcel Lí; de la Torre, Rafael

    2012-01-01

    3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is a ring-substituted amphetamine widely used for recreational purposes. MDMA is predominantly O-demethylenated in humans by cytochrome P450 (CYP) 2D6, and is also a potent mechanism-based inhibitor of the enzyme. After assessing the inhibition and recovery of CYP2D6 in a previous study, the aim of this work was to study in humans the activity of CYP1A2 in vivo after CYP2D6 had been inhibited by MDMA, using caffeine as a probe drug. Twelve male and nine female recreational MDMA users were included. In session 1, 100 mg of caffeine was given at 0 h. In session 2, a 1.5 mg/kg MDMA dose (range 75-100 mg) was given at 0 h followed by a 100 mg dose of caffeine 4 h later. Aliquots of plasma were assayed for caffeine (137X) and paraxanthine (17X) and statistically significant differences were assessed with a one-way ANOVA. There were significant gender differences at basal condition, which persisted after MDMA administration. CYP1A2 activity was higher in both genders after drug administration, with an increase in 40% in females and 20% in males. Results show an increase in CYP1A2 activity when CYP2D6 is inhibited by MDMA in both genders, being more pronounced in females.

  12. Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation

    Directory of Open Access Journals (Sweden)

    Hartmann-Fatu Cristina

    2006-03-01

    Full Text Available Abstract Background The peptidyl prolyl cis/trans isomerase (PPIase Parvulin (Par14/PIN4 is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro. Results In this study we confirm by RT-PCR the existence of a longer Parvulin isoform expressed in all tissues examined so far. This isoform contains a 5' extension including a 75 bp extended open reading frame with two coupled SNPs leading to amino acid substitutions Q16R and R18S. About 1% of all Parvulin mRNAs include the novel extension as quantified by real-time PCR. The human Parvulin promoter is TATA-less and situated in a CpG island typical for house keeping genes. Thus, different Parvulin mRNAs seem to arise by alternative transcription initiation. N-terminally extended Parvulin is protected from rapid proteinaseK degradation. In HeLa and HepG2 cell lysates two protein species of about 17 and 28 KDa are detected by an antibody against an epitope within the N-terminal extension. These two bands are also recognized by an antibody towards the PPIase domain of Parvulin. The longer Parvulin protein is encoded by the human genome but absent from rodent, bovine and non-mammalian genomes. Conclusion Due to its molecular weight of 16.6 KDa we denote the novel Parvulin isoform as Par17 following the E. coli Par10 and human Par14 nomenclature. The N-terminal elongation of Par17-QR and Par17-RS suggests these isoforms to perform divergent functions within the eukaryotic cell than the well characterized Par14.

  13. Absence of the human CYP2C8*3 allele in Ugandan children exposed to Plasmodium falciparum malaria.

    Science.gov (United States)

    Paganotti, Giacomo Maria; Cosentino, Valentina; Russo, Gianluca; Tabacchi, Francesca; Gramolelli, Silvia; Coluzzi, Mario; Romano, Rita

    2014-10-01

    Study of host pharmacogenetics can improve our knowledge of mechanisms of drug resistance selection and spread. This issue has recently been addressed with respect to chloroquine and amodiaquine in malaria endemic areas of West and East Africa. Here we report, from surveys performed in two different areas of Uganda, that the human CYP2C8*3 allele, which had been reported to be strongly associated with parasite drug resistance in Zanzibar, is absent, being a marker of genetic admixture of the Zanzibari population with a Caucasoid component. Moreover, a retrospective analysis of CYP2C8*2 and the Plasmodium falciparum drug resistant pfmdr1 86Y allele does not show any association, which may be related to the high level of drug resistance.

  14. Linkage disequilibrium blocks, haplotype structure, and htSNPs of human CYP7A1 gene

    Directory of Open Access Journals (Sweden)

    Wan Yu-Jui

    2006-05-01

    Full Text Available Abstract Background Cholesterol 7-alpha-hydroxylase (CYP7A1 is the rate limiting enzyme for converting cholesterol into bile acids. Genetic variations in the CYP7A1 gene have been associated with metabolic disorders of cholesterol and bile acids, including hypercholesterolemia, hypertriglyceridemia, arteriosclerosis, and gallstone disease. Current genetic studies are focused mainly on analysis of a single nucleotide polymorphism (SNP at A-278C in the promoter region of the CYP7A1 gene. Here we report a genetic approach for an extensive analysis on linkage disequilibrium (LD blocks and haplotype structures of the entire CYP7A1 gene and its surrounding sequences in Africans, Caucasians, Asians, Mexican-Americans, and African-Americans. Result The LD patterns and haplotype blocks of CYP7A1 gene were defined in Africans, Caucasians, and Asians using genotyping data downloaded from the HapMap database to select a set of haplotype-tagging SNPs (htSNP. A low cost, microarray-based platform on thin-film biosensor chips was then developed for high-throughput genotyping to study transferability of the HapMap htSNPs to Mexican-American and African-American populations. Comparative LD patterns and haplotype block structure was defined across all test populations. Conclusion A constant genetic structure in CYP7A1 gene and its surrounding sequences was found that may lead to a better design for association studies of genetic variations in CYP7A1 gene with cholesterol and bile acid metabolism.

  15. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    Science.gov (United States)

    Girolamo, Francesco; Dallatomasina, Alice; Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  16. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    Directory of Open Access Journals (Sweden)

    Francesco Girolamo

    Full Text Available NG2/CSPG4 is a complex surface-associated proteoglycan (PG recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS

  17. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    Science.gov (United States)

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

  18. Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity

    Energy Technology Data Exchange (ETDEWEB)

    Lepont, Pierig; Stickney, John T.; Foster, Lauren A.; Meng, Jin-Jun; Hennigan, Robert F. [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Ip, Wallace [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States)], E-mail: wallace.ip@uc.edu

    2008-01-01

    Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2{sup -/-} mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

  19. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  20. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  1. Contribution of CYP3A4 to catalysis of ketamine in human hepatic microsome%人肝微粒体中CYP3A4对氯胺酮代谢的催化作用

    Institute of Scientific and Technical Information of China (English)

    赵芸慧; 田阿勇; 马虹; 王俊科

    2012-01-01

    目的 研究人肝脏微粒体中细胞色素P4503A4(CYP3A4)对氯胺酮代谢的催化作用.方法 用高效液相色谱法测定氯胺酮在人肝脏微粒体孵育液中的浓度变化,计算其代谢速率;分析该代谢速率与CYP3A4特异性底物硝苯地平代谢速率的相关性;并应用CYP3A4特异性抑制剂孕二烯酮检测CYP3A4对氯胺酮代谢的催化作用.结果 20例人肝脏微粒体中氯胺酮的代谢速率均值为(12.6±3.8)μmol·min-1·g-1 protein.该速率与CYP3A4活性探针硝苯地平代谢速率呈明显正相关(r=0.917,P<0.01).加入特异性抑制剂孕二烯酮组,氯胺酮的平均代谢速率明显低于正常孵育组,为(4.7±1.6)μmol·min-1·g-1 protein(P<0.01),抑制率为62.7%.结论 人肝微粒体中CYP3A4对氯胺酮代谢具有催化作用.%Aim To investigate the contribution of CYP3A4 to catalysis of ketamine in human hepatic mi-crosome. Methods The change of ketamine concentration in an incubation mixture with human hepatic microsomes was determined by high performance liquid chromatography ( HPLC) , and then the metabolic rate of ketamine was calculated. The correlation of the rate with rates of metabolism of CYP3A4 selective substrate nifedipine, and the effect of CYP3A4 specific inhibitor gestodene on ketamine metabolism were examined. Results The metabolic rate of ketamine in the twenty ca -ses of microsomes was ( 12. 6 ± 3. 8 μmol · min-1 ·g-1 protein) on average. The average rate of ketamine metabolism showed obvious positive correlation to that of nifedipine (activity probe of CYP3A4) (r =0. 917, P < 0. 01). After addition of gestodene (the specific inhibitor of CYP3A4) , the average metabolic rate of ketamine (4.7 ±1.6 μmol· min-1 · g-1 protein) was slower than that without gestodene (P < 0. 01). The inhibition degree was 62.7%. Conclusion CYP3A4 is responsible for metabolism of ketamine in human he -patic microsome.

  2. Differential ligand binding affinities of human estrogen receptor-α isoforms

    OpenAIRE

    Amanda H.Y. Lin; Li, Rachel W. S.; Ho, Eva Y. W.; George P H Leung; Susan W S Leung; Paul M Vanhoutte; Man, Ricky Y K

    2013-01-01

    Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cel...

  3. The role of human cytochrome P450 enzymes in the formation of 2-hydroxymetronidazole: CYP2A6 is the high affinity (low Km) catalyst.

    Science.gov (United States)

    Pearce, Robin E; Cohen-Wolkowiez, Michael; Sampson, Mario R; Kearns, Gregory L

    2013-09-01

    Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

  4. Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation*

    Science.gov (United States)

    Su, Shifeng; Blackwelder, Amanda J.; Grossman, Gail; Minges, John T.; Yuan, Lingwen; Young, Steven L.; Wilson, Elizabeth M.

    2012-01-01

    Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH2-terminal 110LLXXVLXXLL119 motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium. PMID:22891251

  5. Highly sensitive LC-MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human.

    Science.gov (United States)

    Grangeon, Alexia; Gravel, Sophie; Gaudette, Fleur; Turgeon, Jacques; Michaud, Veronique

    2017-01-01

    Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3μm PFP(2) 150×3mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small

  6. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

    NARCIS (Netherlands)

    Rennel, E.; Waine, E.; Guan, H.; Schuler, Y.; Leenders, W.P.J.; Woolard, J.; Sugiono, M.; Gillatt, D.; Kleinerman, E.; Bates, D.; Harper, S.

    2008-01-01

    Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the

  7. Inhibition of human cytochrome P450 enzymes by Bacopa monnieri standardized extract and constituents.

    Science.gov (United States)

    Ramasamy, Seetha; Kiew, Lik Voon; Chung, Lip Yong

    2014-02-24

    Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  8. Inhibition of Human Cytochrome P450 Enzymes by Bacopa monnieri Standardized Extract and Constituents

    Directory of Open Access Journals (Sweden)

    Seetha Ramasamy

    2014-02-01

    Full Text Available Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL, CYP2C9 (36.49/12.5 µg/mL, CYP1A2 (52.20/25.1 µg/mL; competitive inhibition of CYP3A4 (83.95/14.5 µg/mL and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL. However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day, B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%. These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  9. Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

    Science.gov (United States)

    Wanga, Shaynah; Ceron, Carla S; Delgado, Cynthia; Joshi, Sunil K; Spaulding, Kimberly; Walker, Joy P; Song, Sangheon; Olson, Jean L; Lovett, David H

    2015-01-01

    Delayed graft function (DGF) is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2), as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2) that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC), in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are associated with

  10. Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

    Directory of Open Access Journals (Sweden)

    Shaynah Wanga

    Full Text Available Delayed graft function (DGF is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2, as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2 that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC, in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are

  11. Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

    Science.gov (United States)

    Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

    2013-12-01

    Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1.

  12. Isolation and Identification of Intestinal CYP3A Inhibitors from Cranberry (Vaccinium macrocarpon) using Human Intestinal Microsomes

    Science.gov (United States)

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N.; Brantley, Scott J.; Paine, Mary F.; Oberlies, Nicholas H.

    2010-01-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, a cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC50) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study. PMID:20717876

  13. Markers of genetic susceptibility in human environmental hygiene and toxicology: the role of selected CYP, NAT and GST genes.

    Science.gov (United States)

    Thier, Ricarda; Brüning, Thomas; Roos, Peter H; Rihs, Hans-Peter; Golka, Klaus; Ko, Yon; Bolt, Hermann M

    2003-06-01

    Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of

  14. DNMT3B7, a truncated DNMT3B isoform expressed in human tumors, disrupts embryonic development and accelerates lymphomagenesis

    Science.gov (United States)

    Shah, Mrinal Y.; Vasanthakumar, Aparna; Barnes, Natalie Y.; Figueroa, Maria E.; Kamp, Anna; Hendrick, Christopher; Ostler, Kelly R.; Davis, Elizabeth M.; Lin, Shang; Anastasi, John; Le Beau, Michelle M.; Moskowitz, Ivan; Melnick, Ari; Pytel, Peter; Godley, Lucy A.

    2010-01-01

    Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the C-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic are bred with Eμ-Myc transgenic mice, which model aggressive B cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared to Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the re-distribution of DNA methylation characterizing virtually every human tumor. PMID:20587527

  15. Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue.

    Science.gov (United States)

    Gregoraszczuk, E L; Ptak, A; Karpeta, A; Fiedor, E; Wróbel, A; Milewicz, T; Falandysz, J

    2014-01-01

    Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2 ng/ml HCBz or PeCBz for 6-72 h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

  16. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

    Directory of Open Access Journals (Sweden)

    Sandy Azzi

    2015-06-01

    Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

  17. Active site proton delivery and the lyase activity of human CYP17A1

    Energy Technology Data Exchange (ETDEWEB)

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    2014-01-03

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  18. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan); Ohta, Miho [Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033 (Japan); Sakaki, Toshiyuki, E-mail: tsakaki@pu-toyama.ac.jp [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)

    2013-05-03

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.

  19. Epidermal Growth Factor Receptor Kinase Inhibitors Synergize with TCDD to Induce CYP1A1/1A2 in Human Breast Epithelial MCF10A Cells.

    Science.gov (United States)

    Joiakim, Aby; Mathieu, Patricia A; Shelp, Catherine; Boerner, Julie; Reiners, John J

    2016-05-01

    CYP1A1 and CYP1A2 are transcriptionally activated in the human normal breast epithelial cell line MCF10A following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Shifting MCF10A cultures to medium deficient in serum and epidermal growth factor (EGF) caused rapid reductions in the activated (i.e., phosphorylated) forms of extracellular regulated kinases (ERKs) and the epidermal growth factor receptor (EGFR). Shifting to serum/EGF-deficient medium also enhanced TCDD-mediated induction of cytochrome P450 (CYP)1A1 Treatment of cells cultured in complete medium with the EGFR inhibitors gefitinib (Iressa), AG1478, and CI-1033 resulted in concentration-dependent reductions of active EGFR and ERKs, and increased CYP1A1 mRNA content ∼3- to 18-fold above basal level. EGFR inhibitors synergized with TCDD and resulted in transient CYP1A1 and CYP1A2 mRNA accumulations ∼8-fold greater (maximum at 5 hours) than that achieved with only TCDD. AG1478, gefitinib, and TCDD individually induced small increases (∼1.2- to 2.5-fold) in CYP1A1 protein content but did not cause additive or synergistic accumulations of CYP1A1 protein when used in combination. The mitogen-activated protein kinase kinase inhibitor PD184352 inhibited ERK and EGFR activation in a concentration-dependent fashion without causing CYP1A1 mRNA accumulation. However, cotreatment with PD184352 potentiated TCDD-mediated CYP1A1 induction. TCDD-mediated induction of CYP1A1 in MCF7-TET on-EGFR cells, a MCF7 variant in which EGFR expression can be controlled, was not affected by the activity status of EGFR or ERKs. Hence, EGFR signaling mutes both basal and ligand-induced expression of two aryl hydrocarbon receptor-responsive P450s in MCF10A cultures. However, these effects are cell context-dependent. Furthermore, CYP1A1 mRNA and protein abundance are not closely coupled in MCF10A cultures.

  20. Binding and repressive activities of apolipoprotein E3 and E4 isoforms on the human ApoD promoter.

    Science.gov (United States)

    Levros, Louis-Charles; Labrie, Marilyne; Charfi, Cyndia; Rassart, Eric

    2013-12-01

    Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer's disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV-VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.

  1. Human heterochromatin protein 1 isoforms regulate androgen receptor signaling in prostate cancer.

    Science.gov (United States)

    Itsumi, Momoe; Shiota, Masaki; Yokomizo, Akira; Kashiwagi, Eiji; Takeuchi, Ario; Tatsugami, Katsunori; Inokuchi, Junichi; Song, Yoohyun; Uchiumi, Takeshi; Naito, Seiji

    2013-06-01

    Androgen receptor (AR) signaling is critical for the tumorigenesis and development of prostate cancer, as well as the progression to castration-resistant prostate cancer. We previously showed that the heterochromatin protein 1 (HP1) β isoform plays a critical role in transactivation of AR signaling as an AR coactivator that promotes prostate cancer cell proliferation. However, the roles of other HP1 isoforms, HP1α and HP1γ, in AR expression and prostate cancer remain unclear. Here, we found that knockdown of HP1γ, but not HP1α, reduced AR expression and cell proliferation by inducing cell cycle arrest at G1 phase in LNCaP cells. Conversely, overexpression of full-length HP1α and its C-terminal deletion mutant increased AR expression and cell growth, whereas overexpression of HP1γ had no effect. Similarly, HP1α overexpression promoted 22Rv1 cell growth, whereas HP1γ knockdown reduced the proliferation of CxR cells, a castration-resistant LNCaP derivative. Taken together, HP1 isoforms distinctly augment AR signaling and cell growth in prostate cancer. Therefore, silencing of HP1β and HP1γ may be a promising therapeutic strategy for treatment of prostate cancer.

  2. STIM and Orai isoform expression in pregnant human myometrium: a potential role in calcium signaling during pregnancy.

    Directory of Open Access Journals (Sweden)

    Evonne eChin-Smith

    2014-05-01

    Full Text Available Store-operated calcium (Ca2+ entry (SOCE can be mediated by two novel proteins, STIM/Orai. We have previously demonstrated that members of the TRPC family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labor associated stimuli IL-1β and mechanical stretch. Although STIM and Orai isoforms (1-3 have been reported in other smooth muscle cell types, there is little known about the expression or gestational regulation of STIM and Orai expression in human myometrium. Total RNA was isolated from lower segment human myometrial biopsies obtained at caesarean section from women at the time of preterm no labor (PTNL, preterm labor (PTL, term non-labor (TNL and term with labor (TL; primary cultured human uterine smooth muscle cells, and a human myometrial cell line (hTERT-HM. STIM1-2, and Orai1-3 mRNA expression was assessed by quantitative real-time PCR. All five genes were expressed in myometrial tissue and cultured cells. Orai2 was the most abundant Orai isoform in human myometrium. Expression of STIM1-2/Orai1-3 did not alter with the onset of labor. Orai1 mRNA expression in cultured cells was enhanced by IL-1β treatment. This novel report of STIM1-2 and Orai1-3 mRNA expression in pregnant human myometrium and Orai1 regulation by IL-1β indicates a potential role for these proteins in calcium signaling in human myometrium during pregnancy.

  3. Khellin and visnagin differentially modulate AHR signaling and downstream CYP1A activity in human liver cells.

    Directory of Open Access Journals (Sweden)

    Radim Vrzal

    Full Text Available Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1, which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR, a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones.

  4. Identification of the human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of the phytochemical ferulic acid.

    Science.gov (United States)

    Li, Xiaojun; Shang, Liang; Wu, Yaohua; Abbas, Suzanne; Li, Dong; Netter, Patrick; Ouzzine, Mohamed; Wang, Hui; Magdalou, Jacques

    2011-01-01

    Ferulic acid (FA), a member of the hydroxycinnamate family, is an abundant dietary antioxidant that may offer beneficial effects against cancer, cardiovascular disease, diabetes, osteoarthritis and Alzheimer's disease. In this study, evidence for sulfation and glucuronidation of FA was investigated upon incubation with human liver microsomes and cytosol. Two main glucuronides, M1 (ether O-glucuronide) and M2 (ester acylglucuronide), were formed with a similar affinity (apparent K(m) 3.53 and 5.15 mM, respectively). A phenol sulfoconjugate was also formed with a higher affinity (K(m) 0.53 mM). Identification of the UDP-glucuronosyltransferase (UGT) isoforms involved in FA glucuronidation was investigated with 12 human recombinant enzymes. FA was mainly glucuronidated by UGT1A isoforms and by UGT2B7. UGT1A4, 2B4, 2B15 and 2B17 failed to glucuronidate the substance. Examination of the kinetic constants revealed that FA was mainly glucuronidated by UGT1A1 at the two nucleophilic groups. UGT1A3 was able to glucuronidate these two positions with the same, but low, efficiency. UGT1A6 and 1A8 were involved in the formation of the ether glucuronide only, whereas UGT1A7, 1A10 and 2B7 preferentially glucuronidated the carboxyl group. Moreover, octyl gallate, a marker substrate of UGT1A1, competitively inhibited FA glucuronidation mediated by this isoform. Altogether, the results suggest that FA glucuronidation is primarily mediated by UGT1A1.

  5. Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51).

    Science.gov (United States)

    Lamb, David C; Warrilow, Andrew G S; Rolley, Nicola J; Parker, Josie E; Nes, W David; Smith, Stephen N; Kelly, Diane E; Kelly, Steven L

    2015-08-01

    In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.

  6. Human metabolic interactions of environmental chemicals.

    Science.gov (United States)

    Hodgson, Ernest; Rose, Randy L

    2007-01-01

    Investigations utilizing recombinant human xenobiotic-metabolizing enzymes as well as human hepatocytes have revealed a number of interactions not only between different environmental chemicals (ECs) but also between ECs and endogenous metabolites. Organophosphorus insecticides (OPs) are potent inhibitors of the human metabolism of carbaryl, carbofuran, DEET and fipronil, as well as the jet fuel components, nonane and naphthalene. OPs are potent irreversible inhibitors of testosterone metabolism by cytochrome P450 (CYP) 3A4 and of estradiol metabolism by CYP3A4 and CYP1A2. All of these CYP inhibitions are believed to be due to the release of reactive sulfur during CYP-catalyzed oxidative desulfuration. It has also been shown that the esterase(s) responsible for the initial step in permethrin metabolism in human liver is inhibited by both chlorpyrifos oxon and carbaryl. A number of pesticides, including chlorpyrifos, fipronil and permethrin, and the repellent, DEET, have been shown to be inducers of CYP isoforms in human hepatocytes, with fipronil being the most potent. Several agrochemicals, including fipronil and the pyrethroids, permethrin and deltamethrin, show toxicity toward human hepatocytes with fipronil being the most potent in this regard. Endosulfan-alpha, which has shown promise as a model substrate for phenotyping CYP3A4 and CYP2B6 in human liver microsomes, is also an inducer of CYP2B6, acting through the PXR receptor.

  7. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    Science.gov (United States)

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

  8. Sex differences in constitutive mRNA levels of CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46 in the pig liver: Comparison between Meishan and Landrace pigs.

    Science.gov (United States)

    Kojima, Misaki; Degawa, Masakuni

    2016-06-01

    Breed and sex differences in hepatic mRNA levels of cytochrome P450 (CYP) isoforms (CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46) were examined in 5-month-old Meishan, Landrace, and their crossbred F1 (LM and ML) pigs. Serum testosterone levels in male Meishan, LM, and ML pigs were 2.5-3.5-fold higher than in Landrace pigs. CYP3A46 mRNA was breed-specifically detected only in Landrace, LM, and ML pigs. In Meishan, LM, and ML pigs only, male-predominant expressions of CYP2B22, CYP2C33, CYP2C49 and CYP3A29 mRNAs were observed; CYP3A22 mRNA expression showed the opposite pattern. Male-dominant mRNA expression was also observed in LM and ML pigs for CYP3A46. The sex differences in CYP mRNA levels in Meishan pigs disappeared when males were castrated and were restored by testosterone propionate (TP) administration to the castrated males. In Landrace pigs, TP administration to castrated males and intact females significantly increased the levels of CYP2B22, CYP2C33, and CYP3A46 mRNAs. Immature (1-month-old) pigs showed no breed or sex differences in CYP mRNA expressions. The results demonstrated that androgen is an important determinant of sex-associated expression of several CYPs and suggested that breed differences in sex-associated expression could be caused by differences in serum androgen level and by other genetic traits.

  9. Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

    DEFF Research Database (Denmark)

    Kononenko, Olga; Bazov, Igor; Watanabe, Hiroyuki

    2017-01-01

    of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of the signal peptide. ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted...... to the cell nuclei in a model cellular system. This may be driven by bipartite nuclear localization signal (NLS) that is cryptic in the full-length PDYN molecule and becomes functional when signal peptide is truncated. Nuclear PDYN isoform was identified by western blot and radioimmunoassay in neuronal nuclei...

  10. Expression of the WT1 gene -KTS domain isoforms suppresses the invasive ability of human lung squamous cell carcinoma cells.

    Science.gov (United States)

    Moriya, Shogo; Takiguchi, Masaki; Seki, Naohiko

    2008-02-01

    Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(Waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that p21(Waf1/Cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of p21(Waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.

  11. CD99 isoforms regulate CD1a expression in human monocyte-derived DCs through ATF-2/CREB-1 phosphorylation.

    Science.gov (United States)

    Mahiddine, Karim; Mallavialle, Aude; Bziouech, Hanen; Larbret, Frédéric; Bernard, Alain; Bernard, Ghislaine

    2016-06-01

    CD1a expression is considered one of the major characteristics qualifying in vitro human dendritic cells (DCs) during their generation process. Here, we report that CD1A transcription is regulated by a mechanism involving the long and short isoforms of CD99. Using a lentiviral construct encoding for a CD99 short hairpin RNA, we were able to inhibit CD99 expression in human primary DCs. In such cells, CD1a membrane expression increased and CD1A transcripts were much higher in abundance compared to cells expressing CD99 long form (CD99LF). We also show that CD1A transcription is accompanied by a switch in expression from CD99LF to expression at comparable levels of both CD99 isoforms during immature DCs generation in vitro. We demonstrate that CD99LF maintains a lower level of CD1A transcription by up-regulating the phosphorylated form of the ATF-2 transcription factor and that CD99 short form (SF) is required to counteract this regulatory mechanism. Elucidation of the molecular mechanisms related to CD99 alternative splicing will be very helpful to better understand the transcriptional regulatory mechanism of CD1a molecules during DCs differentiation and its involvement in the immune response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  13. A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic vivid p450 substrate.

    Science.gov (United States)

    Marks, Bryan D; Smith, Ronald W; Braun, Heidi A; Goossens, Tony A; Christenson, Marie; Ozers, Mary S; Lebakken, Connie S; Trubetskoy, Olga V

    2002-11-01

    Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.

  14. Soluble human leukocyte antigen-G isoforms in maternal plasma in early and late pregnancy

    DEFF Research Database (Denmark)

    Rizzo, Roberta; Andersen, Anita Sylvest; Lassen, Michael Rud

    2009-01-01

    published studies that a high, detectable soluble HLA-G concentration in maternal plasma or serum is not mandatory for a successful pregnancy. However, complications during pregnancy, such as (severe) pre-eclampsia, spontaneous abortion, IUGR, and premature birth, are associated with a low or undetectable...... was to investigate the levels of different soluble HLA-G isoforms in maternal plasma in early and late pregnancy. METHOD OF STUDY: Soluble HLA-G (sHLA-G) can be detected in maternal blood, and in this study, two different isoforms of sHLA-G, namely sHLA-G1 generated by shedding of membrane-bound HLA-G1 and HLA......-G generated by specific HLA-G transcripts, have been investigated early [median of 16.4 weeks of gestation (GW)] and late (median: 38.9 GW) in pregnancy in an original cohort of 580 pregnant Caucasian women. RESULTS: Lower concentrations of sHLA-G1 were found late in pregnancy (>32 GW) in a group of women...

  15. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

    Science.gov (United States)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

    2009-04-05

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

  16. Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis.

    Science.gov (United States)

    Novotna, Aneta; Dvorak, Zdenek

    2014-01-01

    Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

  17. The inhibition of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor alpha and peroxisome proliferator-activated receptor alpha heterodimer.

    Science.gov (United States)

    Gbaguidi, G Franck; Agellon, Luis B

    2004-01-01

    In previous work, we showed that the binding of the liver x receptor alpha:peroxisome proliferator-activated receptor alpha (LXRalpha:PPARalpha) heterodimer to the murine Cyp7a1 gene promoter antagonizes the stimulatory effect of their respective ligands. In this study, we determined if LXRalpha:PPARalpha can also regulate human CYP7A1 gene promoter activity. Co-expression of LXRalpha and PPARalpha in McArdle RH7777 hepatoma cells decreased the activity of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol. In vitro, the human CYP7A1 Site I bound LXRalpha:PPARalpha, although with substantially less affinity compared with the murine Cyp7a1 Site I. The binding of LXRalpha:PPARalpha to human CYP7A1 Site I was increased in the presence of either LXRalpha or PPARalpha ligands. In HepG2 hepatoblastoma cells, fibrates and 25-hydroxycholesterol inhibited the expression of the endogenous CYP7A1 gene as well as the human CYP7A1 gene promoter when co-transfected with plasmids encoding LXRalpha and PPARalpha. However, a derivative of the human CYP7A1 gene promoter that contains a mutant form of Site I that does not bind LXRalpha:PPARalpha was not inhibited by WY 14,643 or 25-hydroxycholesterol in both McArdle RH7777 and HepG2 cells. The ligand-dependent recruitment of LXRalpha:PPARalpha heterodimer onto the human CYP7A1 Site I can explain the inhibition of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol.

  18. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.

    Science.gov (United States)

    Cai, Shi-Ying; He, Hongwei; Nguyen, Trong; Mennone, Albert; Boyer, James L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRalpha by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1alpha plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.

  19. E3B1/ABI-1 Isoforms Are Down-Regulated in Cancers of Human Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    Rafia A. Baba

    2012-01-01

    Full Text Available The expression of E3B1/ABI-1 protein and its role in cancer progression and prognosis are largely unknown in the majority of solid tumors. In this study, we examined the expression pattern of E3B1/ABI-1 protein in histologically confirmed cases of esophageal (squamous cell carcinoma and adenocarcinoma, gastro-esophageal junction, colorectal cancers and corresponding normal tissues freshly resected from a cohort of 135 patients, by Western Blotting and Immunofluorescence Staining. The protein is present in its phosphorylated form in cells and tissues. Depending on the extent of phosphorylation it is either present in hyper-phosphorylated (M. Wt. 72 kDa form or in hypo-phosphorylated form (M. Wt. 68 kDa and 65 kDa. A thorough analysis revealed that expression of E3B1/ABI-1 protein is significantly decreased in esophageal, gastro-esophageal junction and colorectal carcinomas irrespective of age, gender, dietary and smoking habits of the patients. The decrease in expression of E3B1/ABI-1 was consistently observed for all the three isoforms. However, the decrease in the expression of isoforms varied with different forms of cancers. Down-regulation of E3B1/ABI-1 expression in human carcinomas may play a critical role in tumor progression and in determining disease prognosis.

  20. The highly conserved human cytomegalovirus UL136 ORF generates multiple Golgi-localizing protein isoforms through differential translation initiation.

    Science.gov (United States)

    Liao, Huanan; Lee, Jung-Hyun; Kondo, Rikita; Katata, Marei; Imadome, Ken-Ichi; Miyado, Kenji; Inoue, Naoki; Fujiwara, Shigeyoshi; Nakamura, Hiroyuki

    2014-01-22

    The UL133-UL138 locus in the unique long b' (ULb') region of the human cytomegalovirus (HCMV) genome is considered to play certain roles in viral replication, dissemination and latency in a host cell type-dependent manner. Here we characterized the proteins encoded by UL136, one of the open reading frames (ORFs) in the locus. Comparative sequence analysis of UL136 among clinical isolates and laboratory strains indicates that its predicted amino-acid sequence is highly conserved. A polyclonal antibody against UL136 proteins (pUL136s) was raised against its carboxy-terminal region and this antibody specifically recognized at least five UL136-encoded protein isoforms of 29-17 kDa both in HCMV-infected cells and in cells transfected with a construct expressing pUL136. Immunofluorescence analysis with this antibody revealed localization of pUL136 in the Golgi apparatus. Analysis of several pUL136 mutants indicated that the putative transmembrane domain of pUL136 is required for its Golgi localization. Mutational analysis of multiple AUG codons in UL136 demonstrated that translation initiation from these AUG codons contributes in the generation of pUL136 isoforms.

  1. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Negoro, Ryosuke [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Takayama, Kazuo [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), Kyoto University, Kyoto 606-8302 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Nagamoto, Yasuhito [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Sakurai, Fuminori [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871 (Japan); Tachibana, Masashi [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Mizuguchi, Hiroyuki, E-mail: mizuguch@phs.osaka-u.ac.jp [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871 (Japan)

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. - Highlights: • The hiPS-ELCs were matured by Matrigel overlay. • The hiPS-ELCs expressed intestinal nuclear receptors, such as PXR, GR and VDR. • The hiPS-ELC is a useful model for the drug-mediated CYP3A4 induction test.

  2. Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies.

    Science.gov (United States)

    Dávila-Borja, Víctor M; Belmont, Javier A; Espinosa, J Javier; Moreno-Sánchez, Rafael; Albores, Arnulfo; Montero, Regina D

    2007-10-01

    Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresorufin indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a significant NADPH oxidation catalyzed by CYP2E1 supersomes, strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present findings may contribute to understand better the physiological function of CYP450 in relation with inflammation and other physiological processes in which HA may have a relevant role.

  3. Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5'-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the “gt-ag” rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 17b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context.

  4. An examination of IC50 and IC50-shift experiments in assessing time-dependent inhibition of CYP3A4, CYP2D6 and CYP2C9 in human liver microsomes.

    Science.gov (United States)

    Berry, Loren M; Zhao, Zhiyang

    2008-01-01

    The relationship between time-dependent inactivation (TDI) and IC50 is examined using a consolidated method for evaluating CYP450 inhibition during drug discovery. An IC50 fold-shift of >1.5 indicated significant TDI potency. Further, the "shifted IC50" could be used to estimate, the K(I) and TDI potency ratio k(inact)/K(I) to within 2-fold in most cases.

  5. Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro

    Science.gov (United States)

    Imai, Shunji; Yoshioka, Yasuo; Morishita, Yuki; Yoshida, Tokuyuki; Uji, Miyuki; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2014-12-01

    Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70 nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

  6. 淫羊藿总黄酮对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响%Effect of total flavonoids of epimedium on liver microsomal CYP1A2, CYP3A4 and CYP2E1 activities in rats

    Institute of Scientific and Technical Information of China (English)

    胡道德; 姚慧娟; 顾磊; 王松坡; 刘皋林

    2008-01-01

    To assess the potential effect of total flavonoids of epimedium (TFE) on cytochrome P450 and activity of its main isoforms in rat liver microsomes. TFE (300 mg/kg) was administered once daily to male Sprague-Dowley rats by gavage for fifteen days. The total cytochrome P450 content and its main isoforms CYP1A2, CYP3A4 and CYP2E1 activities in rat liver microsomes were detected. The activity of CYP1A2 was measured by fluorometry and the activities of CYP3A4 and CYP2E1 were determined by measuring the amount of methanal and p-aminophenol formed using UV/Vis spectrophotometer, respec- tively. Administration of TFE significantly increased the total CYP450 content and activities of CYP 1A2, CYP3A4 and CYP2E1 in rat liver microsomes, compared with the control group. Partieularly, the activities of CYP1A2 and CYP2E1 were enhanced significantly (P<0.01). TFE induced the increase in total CYP450 content and its main isoforms CYP1A2, CYP3A4 and CYP2EI activities in rat liver microsomes.%评估淫羊藿总黄酮对大鼠肝细胞色素P450及其主要亚型活性的潜在影响.淫羊藿总黄酮以300 mg/kg/d的剂量对SD大鼠进行连续灌胃处理15天,测定肝微粒体中CYP450含量与CYP1A2、CYP3A4和CYP2E1亚型活性,观察淫羊藿总黄酮的效应.CYP1A2的活性用荧光比色法进行测定,CYP3A4和CYP2E1的活性用紫外可见分光光度法测定.淫羊藿总黄酮处理后的大鼠肝脏CYP450含量及CYP1A2、CYP3A4和LICYP2E1亚型活性均明显增高,其中CYP1A2和CYP2E1活性升高显著(P<0.01).淫羊藿总黄酮对大鼠肝脏CYP450及主要亚型CYP1A2、CYP3A4和CYP2E1活性均有诱导效应.

  7. Expression of the vitamin D metabolizing enzyme CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of semen quality

    DEFF Research Database (Denmark)

    Jensen, Martin Blomberg; Jørgensen, A; Nielsen, J E;

    2012-01-01

    Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression...

  8. The Effects of Acute Hydrogen Sulfide Poisoning on Cytochrome P450 Isoforms Activity in Rats

    OpenAIRE

    Xianqin Wang; Mengchun Chen; Xinxin Chen; Jianshe Ma; Congcong Wen; Jianchun Pan; Lufeng Hu; Guanyang Lin

    2014-01-01

    Hydrogen sulfide (H2S) is the second leading cause of toxin related death (after carbon monoxide) in the workplace. H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe d...

  9. Expression profiles of muscle disease-associated genes and their isoforms during differentiation of cultured human skeletal muscle cells

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    Abdul-Hussein Saba

    2012-12-01

    Full Text Available Abstract Background The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins. Methods We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development. Results Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development. Conclusions In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.

  10. Midazolam microdose to determine systemic and pre-systemic metabolic CYP3A activity in humans

    Science.gov (United States)

    Hohmann, Nicolas; Kocheise, Franziska; Carls, Alexandra; Burhenne, Jürgen; Haefeli, Walter E; Mikus, Gerd

    2015-01-01

    Aim We aimed to establish a method to assess systemic and pre-systemic cytochrome P450 (CYP) 3A activity using ineffective microgram doses of midazolam. Methods In an open, one sequence, crossover study, 16 healthy participants received intravenous and oral midazolam at microgram (0.001 mg intravenous and 0.003 mg oral) and regular milligram (1 mg intravenous and 3 mg oral) doses to assess the linearity of plasma and urine pharmacokinetics. Results Dose-normalized AUC and Cmax were 37.1 ng ml−1 h [95% CI 35.5, 40.6] and 39.1 ng ml−1 [95% CI 30.4, 50.2] for the microdose and 39.0 ng ml−1 h [95% CI 36.1, 42.1] and 37.1 ng ml−1 [95% CI 26.9, 51.3] for the milligram dose. CLmet was 253 ml min−1 [95% CI 201, 318] vs. 278 ml min−1 [95% CI 248, 311] for intravenous doses and 1880 ml min−1 [95% CI 1590, 2230] vs. 2050 ml min−1 [95% CI 1720, 2450] for oral doses. Oral bioavailability of a midazolam microdose was 23.4% [95% CI 20.0, 27.3] vs. 20.9% [95% CI 17.1, 25.5] after the regular dose. Hepatic and gut extraction ratios for microgram doses were 0.44 [95% CI 0.39, 0.49] and 0.53 [95% CI 0.45, 0.63] and compared well with those for milligram doses (0.43 [95% CI 0.37, 0.49] and 0.61 [95% CI 0.53, 0.70]). Conclusion The pharmacokinetics of an intravenous midazolam microdose is linear to the applied regular doses and can be used to assess safely systemic CYP3A activity and, in combination with oral microdoses, pre-systemic CYP3A activity. PMID:25588320

  11. Paracetamol-induced spindle disturbances in V79 cells with and without expression of human CYP1A2

    DEFF Research Database (Denmark)

    Jensen, K G; Poulsen, H E; Doehmer, J

    1996-01-01

    Spindle disturbing effects in terms of c-mitosis and cytotoxicity of paracetamol were investigated in two Chinese hamster V79 cell lines, one of which (V79MZh1A2) was transfected with human CYP1A2. This enzyme catalyses the oxidative formation of the reactive paracetamol metabolite, NAPQI, believed...... to initiate hepatoxicity by covalent binding to proteins after overdose. In the native V79 cell line paracetamol increased c-mitosis frequency in a concentration dependent manner from 8.7 + or - 3.5% (control) to 66 + or - 18% at 20 mM. A significant increase to 13.3 + or - 3.5% was first seen at 2.5 m......M in the native cell line (Pparacetamol. At 5 mM paracetamol the c-mitosis frequency was 14.4 + or - 5.0% and 19.0 + or - 3...

  12. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

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    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  13. 14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

    Science.gov (United States)

    Noutsios, Georgios T; Ghattas, Paul; Bennett, Stephanie; Floros, Joanna

    2015-07-15

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

  14. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes

    Science.gov (United States)

    Eagling, Victoria A; Tjia, John F; Back, David J

    1998-01-01

    Aims Chemical inhibitors of cytochrome P450 (CYP) are a useful tool in defining the role of individual CYPs involved in drug metabolism. The aim of the present study was to evaluate the selectivity and rank the order of potency of a range of isoform-selective CYP inhibitors and to compare directly the effects of these inhibitors in human and rat hepatic microsomes. Methods Four chemical inhibitors of human cytochrome P450 isoforms, furafylline (CYP1A2), sulphaphenazole (CYP2C9), diethyldithiocarbamate (CYP2E1), and ketoconazole (CYP3A4) were screened for their inhibitory specificity towards CYP-mediated reactions in both human and rat liver microsomal preparations. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, chlorzoxazone 6-hydroxylation and testosterone 6β-hydroxylation were monitored for enzyme activity. Results Furafylline was a potent, selective inhibitor of phenacetin O-deethylation (CYP1A2-mediated) in human liver microsomes (IC50 = 0.48 μm), but inhibited both phenacetin O-deethylation and tolbutamide 4-hydroxylation (CYP2C9-mediated) at equimolar concentrations in rat liver microsomes (IC50 = 20.8 and 24.0 μm respectively). Sulphaphenazole demonstrated selective inhibition of tolbutamide hydroxylation in human liver microsomes but failed to inhibit this reaction in rat liver microsomes. DDC demonstrated a low level of selectivity as an inhibitory probe for chlorzoxazone 6-hydroxylation (CYP2E1-mediated). DDC also inhibited testosterone 6β-hydroxylation (CYP3A-mediated) in man and rat, and tolbutamide 4-hydroxylase activity in rat. Ketoconazole was a very potent, selective inhibitor of CYP3A4 activity in human liver (IC50 = 0.04 μm). Although inhibiting CYP3A in rat liver it also inhibited all other reactions at concentrations ≤5 μm. Conclusions It is evident that CYP inhibitors do not exhibit the same selectivity in human and rat liver microsomes. This is due to differential selectivity of the inhibitors and/or differences in the CYP

  15. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    Science.gov (United States)

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

  16. Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.

    Science.gov (United States)

    Boros, Eszter; Szabó, András; Zboray, Katalin; Héja, Dávid; Pál, Gábor; Sahin-Tóth, Miklós

    2017-02-17

    Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

  17. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    Science.gov (United States)

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology.

  18. The effect of organic solvents on enzyme kinetic parameters of human CYP3A4 and CYP1A2 in vitro.

    Science.gov (United States)

    Rokitta, Dennis; Pfeiffer, Kay; Streich, Christina; Gerwin, Henrik; Fuhr, Uwe

    2013-10-01

    Abstract Enzyme kinetic parameters provide essential quantitative information about characterization of individual steps in drug metabolism. Such enzymes are located in a (partially) aqueous environment. For in vitro measurements potential lipophilic substrates regularly require organic solvents to achieve concentrations sufficient for access of the drug to the binding site of the enzyme. However, solvents may interact with the enzymes. In this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethyl sulfoxide (1% to 4%) on the assessment of km, Vmax and Clint for the metabolism of midazolam via CYP3A4 to 1-hydroxymidazolam and the metabolism of caffeine to paraxanthine via CYP1A2 using expressed enzymes in vitro. The presence of acetonitrile proved the highest apparent Vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. The km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to 8-fold lower for solvent free samples compared to solvent containing samples. The present example suggests that effects of solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. These effects illustrate a difference for individual enzyme--substrate pairs, solvents, and solvent concentrations. What remains is the determination to which extent these effects compromise in vitro-in vivo extrapolations, and which solvents are most appropriate.

  19. Differential Expression of IGF Components and Insulin Receptor Isoforms in Human Seminoma Versus Normal Testicular Tissue

    Directory of Open Access Journals (Sweden)

    Tanja Pascale Neuvians

    2005-05-01

    Full Text Available Insulin-like growth factors (IGF have mitogenic and antiapoptotic functions, and may be involved in tumor growth. The purpose of the study was to investigate the role of IGF components in seminoma compared to normal testis. Normal testicular tissues from autopsy cases and seminoma from surgery cases were obtained for microarray and real-time reverse transcription polymerase chain reaction (RT-PCR analysis of IGF-1, IGF-2, IGF receptor type 1 (IGF-R1, IGF-R2, insulin receptor isoforms A (IR-A and B (IR-B, and IGF-binding proteins (IGFBP 1-6. IGF-2 was localized by immunohistochemistry. IGFBP-5 protein expression was evaluated by Western blot analysis. mRNA expression in microarray and real-time RT-PCR showed similar tendencies: IGF-1, IGF-R1, IGF-R2, IR-A, and IGFBP-2 were not different in both groups. IGF-2, IR-B, IGFBP-1, IGFBP-4, and IGFBP-6 mRNA were downregulated in seminoma. IGFBP-3 tended to be upregulated in pT1 seminoma, but downregulated in pT2 stages. IGFBP-5 and IGF-2 protein expression correlated with mRNA expression. In conclusion, downregulation of mainly inhibiting IGFBPs may allow a stimulated tumor growth. The downregulated IGF-2 does not seem to be involved in the growth regulation of seminoma. Constantly expressed genes (e.g., IGF-1, IGF-R1, IR-A, and IGFBP-2 may reflect an involvement in spermatogenesis, but may also play a major role in tumor growth as their expression is not downregulated despite the lack of spermatogenesis in tumor tissue.

  20. Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

    Science.gov (United States)

    de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

    2013-09-30

    We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test.

  1. Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4, and 6 in distinct membrane microdomains.

    Science.gov (United States)

    Bogard, Amy S; Xu, Congfeng; Ostrom, Rennolds S

    2011-04-01

    Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

  2. An evolutionary test of the isoform switching hypothesis of functional progesterone withdrawal for parturition: humans have a weaker repressive effect of PR-A than mice.

    Science.gov (United States)

    Wagner, Günter P; Tong, Yingchun; Emera, Deena; Romero, Roberto

    2012-03-27

    A decrease in maternal serum progesterone (P4) concentrations precedes the onset of labor in most placental mammals. Humans differ by maintaining high levels of P4 throughout birth. Parturition in humans probably includes mechanisms that undercut the pregnancy sustaining function of P4. One attractive hypothesis is the isoform switching hypothesis (ISH). ISH is supported by in vitro evidence that progesterone receptor isoform A (PR-A) inhibits PR-B and that the PR-A/PR-B ratio increases towards term. Here, we test the hypothesis that isoform switching is an adaptation to high levels of P4 at term, predicting that, in humans, PR-A mediated repression of PR-B is stronger than in mouse. We use reporter assays with human and mouse PRs to detect species differences in the repressive effects of PR-A. We found that human PR-B is less sensitive to repression by human PR-A than mouse PR-B, contrary to our prediction. The difference between human and mouse PR-B sensitivity is most pronounced at PR-A/PR-B ratios typical for the preterm myometrium. Our results are inconsistent with the ISH. We speculate that, instead, the lower sensitivity of human PR-B to PR-A may be relevant for the maintenance of pregnancy at high progesterone levels and increasing PR-A concentrations towards term.

  3. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Science.gov (United States)

    Liu, Wenqin; Shi, Jian; Zhu, Lijun; Dong, Lingna; Luo, Feifei; Zhao, Min; Wang, Ying; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2015-01-01

    Oxymatrine (OMT) is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT), and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs) and human intestinal microsomes (HIMs) and the cytochrome P450 (CYP) isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT. PMID:26586934

  4. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili

    2015-04-01

    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  5. Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

    Science.gov (United States)

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Lee, M; Yang, C S; Hu, W Y; Pan, J

    1999-10-01

    To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the

  6. 1α,25-dihydroxyvitamin D3 modulates CYP2R1 gene expression in human oral squamous cell carcinoma tumor cells.

    Science.gov (United States)

    Sundaram, Kumaran; Sambandam, Yuvaraj; Tsuruga, Eichi; Wagner, Carol L; Reddy, Sakamuri V

    2014-04-01

    Oral squamous cell carcinomas (OSCC) are the most common malignant neoplasms associated with mucosal surfaces of the oral cavity and oropharynx. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is implicated as an anticancer agent. Cytochrome P450 2R1 (CYP2R1) is a microsomal vitamin D 25-hydroxylase which plays an important role in converting dietary vitamin D to active metabolite, 25-(OH)D3. We identified high levels of CYP2R1 expression using tissue microarray of human OSCC tumor specimens compared to normal adjacent tissue. Therefore, we hypothesize that 1,25(OH)2D3 regulates CYP2R1 gene expression in OSCC tumor cells. Interestingly, real-time RT-PCR analysis of total RNA isolated from OSCC cells (SCC1, SCC11B, and SCC14a) treated with 1,25(OH)2D3 showed a significant increase in CYP2R1 and vitamin D receptor (VDR) mRNA expression. Also, Western blot analysis demonstrated that 1,25(OH)2D3 treatment time-dependently increased CYP2R1 expression in these cells. 1,25(OH)2D3 stimulation of OSCC cells transiently transfected with the hCYP2R1 promoter (-2 kb)-luciferase reporter plasmid demonstrated a 4.3-fold increase in promoter activity. In addition, 1,25(OH)2D3 significantly increased c-Fos, p-c-Jun expression, and c-Jun N-terminal kinase (JNK) activity in these cells. The JNK inhibitor suppresses 1,25(OH)2D3, inducing CYP2R1 mRNA expression and gene promoter activity in OSCC cells. Furthermore, JNK inhibitor significantly decreased 1,25(OH)2D3 inhibition of OSCC tumor cell proliferation. Taken together, our results suggest that AP-1 is a downstream effector of 1,25(OH)2D3 signaling to modulate CYP2R1 gene expression in OSCC tumor cells, and vitamin D analogs could be potential therapeutic agents to control OSCC tumor progression.

  7. HPLC separation of human serum albumin isoforms based on their isoelectric points

    OpenAIRE

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2013-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the differ...

  8. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M; Kaul, A; Goke, M; Beil, W.; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  9. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M.; Kaul, A.; GOKE, M; Beil, W; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  10. Pharmacokinetic-pharmacodynamic modeling of rifampicin-mediated Cyp3a11 induction in steroid and xenobiotic X receptor humanized mice.

    Science.gov (United States)

    Raybon, Joseph J; Pray, Devin; Morgan, Daniel G; Zoeckler, Mary; Zheng, Ming; Sinz, Michael; Kim, Sean

    2011-04-01

    The purpose of this study was to develop a mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of rifampicin on hepatic Cyp3a11 RNA, enzymatic activity, and triazolam pharmacokinetics. Rifampicin was administered to steroid and xenobiotic X receptor (SXR) humanized mice at 10 mg/kg p.o. (every day for 3 days) followed by triazolam (4 mg/kg p.o.) 24 h after the last dose of rifampicin. Rifampicin and triazolam concentrations and Cyp3a11 RNA expression and activity in the liver were measured over the 4-day period. Elevations in Cyp3a11 RNA expression were observed 24 h after the first dose of rifampicin, reaching a maximum (∼10 times baseline) after the third dose and were sustained until day 4 and began declining 48 h after the last rifampicin dose. Similar changes in enzymatic activity were also observed. The triazolam serum area under the curve (AUC) was 5-fold lower in mice pretreated with rifampicin, consistent with enzyme induction. The final PK-PD model incorporated rifampicin liver concentration as the driving force for the time-delayed Cyp3a11 induction governed by in vitro potency estimates, which in turn regulated the turnover of enzyme activity. The PK-PD model was able to recapitulate the delayed induction of Cyp3a11 mRNA and enzymatic activity by rifampicin. Furthermore, the model was able to accurately anticipate the reduction in the triazolam plasma AUC by integrating a ratio of the predicted induced enzyme activity and basal activity into the equations describing triazolam pharmacokinetics. In conjunction with the SXR humanized mouse model, this mathematical approach may serve as a tool for predicting clinically relevant drug-drug interactions via pregnane X receptor-mediated enzyme induction and possibly extended to other induction pathways (e.g., constitutive androstane receptor).

  11. Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes.

    Science.gov (United States)

    Zhang, Shimin; Pillai, Venkateswaran C; Mada, Sripal Reddy; Strom, Steve; Venkataramanan, Raman

    2012-05-01

    Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6β-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6β-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6β-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole.

  12. Use of glucuronidation fingerprinting to describe and predict mono- and dihydroxyflavone metabolism by recombinant UGT isoforms and human intestinal and liver microsomes.

    Science.gov (United States)

    Tang, Lan; Ye, Ling; Singh, Rashim; Wu, Baojian; Lv, Chang; Zhao, Jie; Liu, Zhongqiu; Hu, Ming

    2010-06-07

    The present study aims to predict the regiospecific glucuronidation of three dihydroxyflavones and seven monohydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven monohydroxyflavones (or HFs), 2'-, 3'-, 4'-, 3-, 5-, 6-, and 7-hydroxyflavone, and three dihydroxyflavones (or diHFs), 3,7-dihydroxyflavone (3,7-diHF), 3,5-dihydroxyflavone (3,5-diHF), and 3,4'-dihydroxyflavone (3,4'-diHF), were chosen, and rates were measured at 2.5, 10, and 35 microM. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 are the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used, 3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intramolecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoform-specific metabolism rates generally correlated well with the rates of glucuronidation in human intestinal and liver microsomes at each of the three tested concentrations. In conclusion, the glucuronidation "fingerprint" of seven selected monohydroxyflavones was affected by UGT isoforms used, positions of the -OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.

  13. Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.

    Science.gov (United States)

    Kotnis, Smita; Bingham, Brendan; Vasilyev, Dmitry V; Miller, Scott W; Bai, Yuchen; Yeola, Sarita; Chanda, Pranab K; Bowlby, Mark R; Kaftan, Edward J; Samad, Tarek A; Whiteside, Garth T

    2010-06-01

    P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

  14. Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

    Directory of Open Access Journals (Sweden)

    McGuire John J

    2005-09-01

    Full Text Available Abstract Background The rTS gene (ENOSF1, first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.

  15. Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

    Science.gov (United States)

    Tan, Bo; Cai, Weimin; Zhang, Jinlian; Zhou, Ning; Ma, Guo; Yang, Ping; Zhu, Qing; Zhu, Yizhun

    2014-09-01

    Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

  16. Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1.

    Science.gov (United States)

    Root, L L; Shipley, G D

    2000-02-01

    Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which alpha FGF and beta FGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.

  17. Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

    OpenAIRE

    Giuliani, A.L.; Colognesi, D.; T. Ricco; Roncato, C.; Capece, M.; Amoroso, F; Wang, Q. G.; Marchi, E.; Gartland, A.; Di Virgilio, F; Adinolfi, E.

    2014-01-01

    The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tu...

  18. OPIOID PRECURSOR PROTEIN ISOFORM IS TARGETED TO THE CELL NUCLEI IN THE HUMAN BRAIN

    DEFF Research Database (Denmark)

    Kononenko, Olga; Bazov, Igor; Watanabe, Hiroyuki;

    2016-01-01

    Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. We here describe two novel splicing variants of human PDYN mRNA. Expression of one...

  19. Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues

    NARCIS (Netherlands)

    Coers, W; Timens, W; Kempinga, C; Klok, PA; Moshage, H

    1998-01-01

    Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several an

  20. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characte

  1. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    Science.gov (United States)

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

  2. ACC2 is expressed at high levels in human white adipose and has an isoform with a novel N-terminus [corrected].

    Directory of Open Access Journals (Sweden)

    John C Castle

    Full Text Available Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2 that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

  3. Ikaros isoforms in human pituitary tumors: distinct localization, histone acetylation, and activation of the 5' fibroblast growth factor receptor-4 promoter.

    Science.gov (United States)

    Ezzat, Shereen; Yu, Shunjiang; Asa, Sylvia L

    2003-09-01

    Targeted expression of a human pituitary tumor derived-fibroblast growth factor receptor-4 (FGFR4) recapitulates pituitary tumorigenesis. We have shown that FGFR4 is a target for Ikaros, a zinc finger-containing transcription factor that localizes to heterochromatin regions and participates in higher order chromatin complexes and control of gene expression. We report here the expression of Ikaros and functional differences between its alternatively spliced variants in human pituitary tumors. Ik1 expression was detected in human pituitary tumors and we also identified a truncated isoform consistent with the non-DNA-binding Ik6 isoform in a subset of adenomas by reverse transcriptase-polymerase chain reaction, sequencing, and Western immunoblotting. Transfection of Ik6 in GH4 pituitary cells resulted in predominantly cytoplasmic expression as compared to Ik1, which resulted in exclusively nuclear expression as determined by immunofluorescence and immunoblotting of fractionated protein. Immunohistochemistry of primary human pituitary adenomas localized Ikaros expression to the nuclear compartment but also in the cytoplasm, the latter consistent with Ik6. Expression of Ikaros and truncated non-DNA-binding isoforms was also suggested by electromobility shift assays using nuclear proteins from primary human pituitary adenomas. Ik6 resulted in reversal of the effects of Ik1 on wild-type 5' FGFR4 promoter activity, histone acetylation, and regulation of the endogenous gene. We conclude that dominant-negative Ik6 isoforms with their distinct localization and effects on Ik1 action may contribute to the altered expression of FGFR4 and possibly other target genes in human pituitary tumors.

  4. The inhibition of the human cholesterol 7α-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor α and peroxisome proliferator-activated receptor α heterodimer

    OpenAIRE

    Gbaguidi, G. Franck; Agellon, Luis B.

    2004-01-01

    In previous work, we showed that the binding of the liver x receptor α:peroxisome proliferator-activated receptor α (LXRα:PPARα) heterodimer to the murine Cyp7a1 gene promoter antagonizes the stimulatory effect of their respective ligands. In this study, we determined if LXRα:PPARα can also regulate human CYP7A1 gene promoter activity. Co-expression of LXRα and PPARα in McArdle RH7777 hepatoma cells decreased the activity of the human CYP7A1 gene promoter in response to fibrates and 25-hydrox...

  5. Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription.

    Science.gov (United States)

    Michelini, Elisa; Donati, Manuela; Aldini, Rita; Cevenini, Luca; Mezzanotte, Laura; Nardini, Paola; Foschi, Claudio; Zvi, Ido Ben; Cevenini, Monica; Montagnani, Marco; Marangoni, Antonella; Roda, Aldo; Cevenini, Roberto

    2012-11-01

    Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7α-hydroxylase (CYP7A1) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C. pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33±10% and 32±4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line.

  6. Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1).

    Science.gov (United States)

    Qiao, Cheng; Jiang, Yajie; Deng, Cuilan; Huang, Zebo; Teng, Kaixuan; Chen, Lan; Liu, Xin

    2015-03-01

    TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA binding and C-terminal PRD and STY domains for transcriptional activation. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-11-66 and TEF3-1197-434 functioned as two independent activation domains, suggesting that N-terminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197-434 showed that only the peptides TEF3-1197-265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-11-66 and four β sheets in TEF3-1197-265. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber, cell proliferation, migration and targeted gene expression. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes.

  7. Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 Infection

    Directory of Open Access Journals (Sweden)

    Yu-Ping Chiang

    2014-12-01

    Full Text Available Individual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1 infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L, a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31 than their uninfected counterparts (n = 27 (p = 0.009. Fifty male homosexual subjects (20 of them are HIV-1 positive were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Bayesian multiple logistic regression revealed that playing a receptive role and increased levels of MRJ-L in macrophages were two risk factors for HIV-1 infection. A 1% rise in MRJ-L expression was associated with a 1.13 fold (95% CrI 1.06–1.29 increase in odds of contracting HIV-1 infection. Ex vivo experiments revealed that MRJ-L facilitated Vpr-dependent nuclear localization of virus. Infection of macrophage-tropic strain is a critical step in HIV-1 transmission. MRJ-L is a critical factor in this process; hence, subjects with higher macrophage MRJ-L levels are more vulnerable to HIV-1 infection.

  8. Hydrogen peroxide modifies both activity and isoforms of acetylcholinesterase in human neuroblastoma SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Alba Garcimartín

    2017-08-01

    Human neuroblastoma SH-SY5Y cells were treated with H2O2 (1–1000 µM for 24 h and AChE activity and AChE and cytochrome c levels were evaluated. AChE activity was strongly increased from 1 µM to 1000 µM of H2O2. The results of the kinetic study showed that H2O2 affected Vmax but not Km; and also that H2O2 changed the sigmoid kinetic observed in control samples to hyperbolic kinetic. Thus, results suggest that H2O2 acts as an allosteric activators. In addition, H2O2, (100–1000 µM reduced the total AChE content and modified its isoform profile (mainly 50-, 70-, and 132-kDa·H2O2 from 100 µM to 1000 µM induced cytochrome c release confirming cell death by apoptosis. All these results together suggest: a the involvement of oxidative stress in the imbalance of AChE; and b treatment with antioxidant agents may be a suitable strategy to protect cholinergic system alterations promoted by oxidative stress.

  9. Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins.

    Science.gov (United States)

    Torchetti, Enza M; Bonomi, Francesco; Galluccio, Michele; Gianazza, Elisabetta; Giancaspero, Teresa A; Iametti, Stefania; Indiveri, Cesare; Barile, Maria

    2011-11-01

    A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

  10. Gender, but not CYP7A1 or SLCO1B1 polymorphism, affects the fasting plasma concentrations of bile acids in human beings.

    Science.gov (United States)

    Xiang, Xiaoqiang; Backman, Janne T; Neuvonen, Pertti J; Niemi, Mikko

    2012-03-01

    Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production in human beings, and organic anion-transporting polypeptide 1B1 (OATP1B1) may influence bile acid hepatic uptake and cholesterol and bile acid synthesis rate. Our purpose was to investigate the effects of gender and CYP7A1 and SLCO1B1 polymorphisms on the fasting plasma concentrations of bile acids, bile acid synthesis marker and total cholesterol in a Finnish population. Fasting plasma concentrations of 16 endogenous bile acids, their synthesis marker (7α-hydroxy-4-cholesten-3-one) and total cholesterol were measured in 243 samples from 143 healthy volunteers. The volunteers were genotyped for 6 haplotype-tagging single-nucleotide polymorphisms (SNPs) of CYP7A1 and two functionally relevant SNPs in SLCO1B1. The mean plasma concentrations of chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid were 61-111% higher in men than in women (P ≤ 0.001). Accordingly, the mean concentration of total bile acids was 51% higher in men than in women (P = 0.001). The CYP7A1 rs8192879 and rs1023652 SNPs were associated with deoxycholic acid and hyodeoxycholic acid concentrations, respectively, but the associations were not significant after correction for multiple testing. None of the six CYP7A1 SNPs was associated with the plasma concentrations of cholesterol or 7α-hydroxy-4-cholesten-3-one. SLCO1B1 genotype was associated with total plasma cholesterol concentration only, but the association was not significant after correction for multiple testing. In general, the gender contributes substantially more to variation in fasting plasma bile acid concentrations than CYP7A1 or SLCO1B1 polymorphism do. Common genetic variability in CYP7A1 is unlikely to play a significant role in cholesterol metabolism and bile acid homeostasis under normal physiological conditions.

  11. Expression profile of CYP1A1 and CYP1B1 enzymes in colon and bladder tumors.

    Directory of Open Access Journals (Sweden)

    Vasilis P Androutsopoulos

    Full Text Available BACKGROUND: The cytochrome P450 CYP1A1 and CYP1B1 enzymes are involved in carcinogenesis via activation of pro-carcinogenic compounds to carcinogenic metabolites. CYP1A1 and CYP1B1 have shown elevated levels in human tumors as determined by qRT-PCR and immunohistochemical studies. However studies that have examined CYP1 expression by enzyme activity assays are limited. RESULTS: In the current study the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of bladder and colorectal origin by qRT-PCR and enzyme activity assays. The results demonstrated that 35% (7/20 of bladder tumors and 35% (7/20 of colon tumors overexpressed active CYP1 enzymes. CYP1B1 mRNA was overexpressed in 65% and 60% of bladder and colon tumors respectively, whereas CYP1A1 was overexpressed in 65% and 80% of bladder and colon tumors. Mean mRNA levels of CYP1B1 and CYP1A1 along with mean CYP1 activity were higher in bladder and colon tumors compared to normal tissues (p<0.05. Statistical analysis revealed CYP1 expression levels to be independent of TNM status. Moreover, incubation of tumor microsomal protein in 4 bladder and 3 colon samples with a CYP1B1 specific antibody revealed a large reduction (72.5 ± 5.5 % for bladder and 71.8 ± 7.2% for colon in catalytic activity, indicating that the activity was mainly attributed to CYP1B1 expression. CONCLUSIONS: The study reveals active CYP1 overexpression in human tumors and uncovers the potential use of CYP1 enzymes and mainly CYP1B1 as targets for cancer therapy.

  12. IL-13 Receptor Alpha 2 Membrane and Soluble Isoforms Differ in Human and Mouse1

    OpenAIRE

    Chen, Weiguo; Sivaprasad, Umasundari; Tabata, Yasuhiro; Gibson, Aaron M.; Stier, Matthew T.; Finkelman, Fred D.; Khurana Hershey, Gurjit K.

    2009-01-01

    Although mice have ng/ml serum levels of soluble (s) IL-13Rα2, humans lack sIL-13Rα2 in serum. Our data provide a mechanism for this biologic divergence. In mice, discrete transcripts encoding s and membrane (mem) forms of IL-13Rα2 are generated by alternative splicing. We utilized siRNA to specifically deplete the transcript encoding memIL-13Rα2 (full-length) or sIL-13Rα2 (ΔEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Rα2, but had no effect on the level of...

  13. Captan impairs CYP-catalyzed drug metabolism in the mouse.

    Science.gov (United States)

    Paolini, M; Barillari, J; Trespidi, S; Valgimigli, L; Pedulli, G F; Cantelli-Forti, G

    1999-11-30

    To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The

  14. The Expression of MTUS1/ATIP and Its Major Isoforms, ATIP1 and ATIP3, in Human Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Louis, Simon N.S., E-mail: simonnsl@unimelb.edu.au; Chow, Laurie T.C.; Varghayee, Naghmeh; Rezmann, Linda A.; Frauman, Albert G.; Louis, William J. [Clinical Pharmacology and Therapeutics Unit, Department of Medicine, University of Melbourne, Austin Health, Heidelberg 3084, Victoria (Australia)

    2011-10-11

    Angiotensin II (Ang II), the main effector of the renin angiotensin system, acts upon two distinct transmembrane receptors, the Ang II type 1 and the type 2 (AT{sub 2}-) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT{sub 2}-receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT{sub 2}-receptor interacting protein), inhibits the mitogenic effects of EGF in prostate cancer cell lines representing both early and late stage disease. This is the first report on the expression of ATIP in normal and malignant human prostatic biopsies. The expression of ATIP and its major isoforms, ATIP1 and ATIP3, in normal prostatic cells and three prostate cancer cell lines was examined using QPCR and immunohistochemistry. Human biopsies containing benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and well, moderately and poorly differentiated prostate cancer were also examined. Overall, ATIP1 and ATIP3 mRNA expression was increased in malignant compared to normal tissues and cell lines. ATIP immunostaining was low or absent in both the basal and columnar epithelial cell layers surrounding BPH acini; however, it was observed in high concentration in neoplastic epithelial cells of HGPIN and was clearly evident in cytoplasms of malignant cells in all prostate cancer grades. ATIP immunostaining was also identified in the cytoplasms of LNCaP and PC3 prostate cancer cells. As the AT{sub 2}-receptor/ATIP inhibitory signaling pathway exists in malignant cells in all grades of prostate cancer, enhancement of this pathway may be a therapeutic target even after the development of androgen-independence.

  15. In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4

    Directory of Open Access Journals (Sweden)

    Qiao-Li Lv

    2015-12-01

    Full Text Available Glycyrrhetinic acid (GA has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450 cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

  16. Expression of differential nitric oxide synthase isoforms in human gastric mucosa infected with Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    屠振兴; 龚燕芳; 丁华; 许国铭; 李兆申; 满晓华

    2003-01-01

    Objective: To study the relationship between nitric oxide synthase (NOS) expression in human gastric mucosa and Helicobacter pylori (H.pylori) infection. Methods: Gastric mucosa samples were obtained from antrum of 33 patients received gastroendoscopy. H.pylori infection was confirmed by Giems staining and bacteria culture under microaerophilic conditions. Expression of iNOS, eNOS and nitrotyrosine were detected by immunohistochemistry. Results: (1) The positive rate of H. pylori infection was 66.7%(22/33). (2) iNOS positive staining in inflammatory cells was detected in 77.3%(17/22) of samples with H.pylori and 27.3%(3/11) without H.pylori infection (P0.05). (5) Moderate and severe infiltrations of inflammatory cells were found in 86.4%(19/22) of gastric biopsies with H. pylori and 9.1%(1/11) of samples without H. pylori infection (P<0.01). Conclusion: H.pylori infection might promote infiltration of mononuclear cells and macrophages in gastric mucosa and induce iNOS expression in these cells. The accumulated nitric oxide in local area may result in gastric mucosa damage.

  17. Crystal structure of the first WW domain of human YAP2 isoform.

    Science.gov (United States)

    Martinez-Rodriguez, Sergio; Bacarizo, Julio; Luque, Irene; Camara-Artigas, Ana

    2015-09-01

    The WW domains are the smallest modular domains known. The study of the structural basis of their stability is important to understand their physiological role. These domains are intrinsically flexible, which makes them difficult to crystallize. The first WW domain of the human Yes tyrosine kinase Associated Protein (YAP) has been crystallized and its structure has been solved by X-ray diffraction at 1.6 Å resolution. Crystals belong to the orthorhombic space group P21212 with unit cell parameters a=42.67, b=43.10 and c=21.30. The addition of proline and other small-molecule additives improves drastically the quality of the crystals. The interactions that stabilize this minimal modular domain have been analysed. This crystal structure reveals that, besides the stabilization of the hydrophobic core of the protein by the aromatic cluster formed by Trp177-Phe189-Pro202, some salt-bridges interactions might affect the stability of the domain. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.

    Science.gov (United States)

    Jones, M; Tussey, L; Athanasou, N; Jackson, D G

    2000-03-17

    The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.

  19. Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Vorrink, Sabine U. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Severson, Paul L. [Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ (United States); Kulak, Mikhail V. [Department of Surgery, The University of Iowa, Iowa City, IA (United States); Futscher, Bernard W. [Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ (United States); Domann, Frederick E., E-mail: frederick-domann@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Department of Surgery, The University of Iowa, Iowa City, IA (United States)

    2014-02-01

    The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). Exposure to 1% O{sub 2} prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity. - Highlights: • Significant crosstalk exists between AhR and HIF-1α signaling. • Hypoxia perturbs PCB 126 induced AhR function and

  20. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Directory of Open Access Journals (Sweden)

    Liu W

    2015-10-01

    Full Text Available Wenqin Liu,1,2,* Jian Shi,1,2,* Lijun Zhu,2 Lingna Dong,1 Feifei Luo,2 Min Zhao,2 Ying Wang,2 Ming Hu,2,3 Linlin Lu,2 Zhongqiu Liu1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 2International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 3Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA *These authors contributed equally to this work Abstract: Oxymatrine (OMT is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT, and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs and human intestinal microsomes (HIMs and the cytochrome P450 (CYP isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT

  1. Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

    Science.gov (United States)

    Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

    2016-11-01

    The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.

  2. The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2 upregulates galectin-7 expression in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Carole G Campion

    Full Text Available Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2, the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

  3. Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines.

    Science.gov (United States)

    Vorrink, Sabine U; Severson, Paul L; Kulak, Mikhail V; Futscher, Bernard W; Domann, Frederick E

    2014-02-01

    The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity.

  4. A chiral HPLC-MS/MS method for simultaneous quantification of warfarin enantiomers and its major hydroxylation metabolites of CYP2C9 and CYP3A4 in human plasma

    OpenAIRE

    2014-01-01

    Warfarin is an oral anticoagulant that requires frequent therapeutic drug monitoring due to a narrow therapeutic window, considerable interindividual variability in drug response, and susceptibility to drug-drug and drug-diet interactions. Enantiomeric separation and quantification of warfarin enantiomers and clinically important major hydroxylation metabolites are essential for drug interaction studies and phenotypic characterization of CYP2C9 and CYP3A4, the major cytochrome P450 (CYP) enzy...

  5. Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    Institute of Scientific and Technical Information of China (English)

    Guang-Jing Hu; Jia Chen; Xiao-Nan Zhao; Jia-Jia Xu; Dong-Qing Guo; Ming Lu; Ming Zhu

    2013-01-01

    Trans-splicing,a process involving the cleavage and joining of two separate transcripts,can expand the transcriptome and proteome in eukaryotes.Chimeric RNAs generated by trans-splicing are increasingly described in literatures.The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health.Certain antibiotic resistance genes,such as ampicillin resistance gene (Amp),are frequently used in recombinant plasmids.Until now,trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported.Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis.The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities.Here,we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA,which is presumably processed through a prior event of interchromosomal trans-splicing.Strikingly,DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells.Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production,reveal an exogenous-endogenous trans-splicing system,in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells,and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

  6. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    Science.gov (United States)

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans.

  7. Aryl hydrocarbon receptor regulates CYP1B1 but not ABCB1 and ABCG2 in hCMEC/D3 human cerebral microvascular endothelial cells after TCDD exposure.

    Science.gov (United States)

    Jacob, Aude; Potin, Sophie; Chapy, Hélène; Crete, Dominique; Glacial, Fabienne; Ganeshamoorthy, Kayathiri; Couraud, Pierre-Olivier; Scherrmann, Jean-Michel; Declèves, Xavier

    2015-07-10

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor activated by a variety of widespread persistent environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It can transactivate the expression of several target genes. Recently AhR transcripts were detected in isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line, an in vitro model of the human cerebral endothelium. To date AhR implication in the co-regulation of ABCB1, ABCG2 and CYP1B1 at human cerebral endothelium has not been addressed. Here we investigated whether AhR could co-regulate ABCB1, ABCG2 and CYP1B1 expressions in the hCMEC/D3 cell line. Exposure to TCDD induced a concentration-dependent increase in CYP1B1 expression. We demonstrated AhR involvement in the TCDD-mediated increase in CYP1B1 expression by using small interfering RNA against AhR. Western blotting analysis also revealed an increase in CYP1B1 protein expression following TCDD exposure in hCMEC/D3. Regarding ABCB1 and ABCG2, exposure to TCDD had no effect on their protein expressions and functional activities. In conclusion our data indicated a differential modulation of CYP1B1 and ABCB1/ABCG2 expressions in hCMEC/D3 cells following TCDD exposure.

  8. Identification of a novel splice variant of human PD-L1 Mrna encoding an isoform-lacking Igv-like domain

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2005-01-01

    Aim: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). Methods: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. Results: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon 2 encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intmcellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. Conclusion: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  9. Heterogeneous filament network formation by myosin light chain isoforms effects on contractile energy output of single cardiomyocytes derived from human induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Takeomi Mizutani

    2016-03-01

    Full Text Available Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs are expected to play an important role in heart therapies, in which hiPSC-CMs should generate sufficient contractile force to pump blood. However, recent studies have shown that the contractility of myocardial mimics composed of hiPSC-CMs is lower than that of adult human myocardium. To examine the mechanism by which contractile force output of hiPSC-CMs is weakened, we measured the contractile force of single hiPSC-CMs and observed the fibrous distribution of myosin II regulatory light chain (MRLC of cardiac (contributes to beating and non-cardiac (does not contribute to beating isoforms. Single hiPSC-CMs were cultured on an extracellular matrix gel, and the contractile force and strain energy exerted on the gel were measured. Strain energy was not uniform between cells and ranged from 0.2 to 5.8 pJ. The combination of contractile force measurement and immunofluorescent microscopy for MRLC isoforms showed that cells with higher strain energy expressed the weakened non-cardiac myosin II fibers compared to those of cells with lower strain energy. Observation of cardiac and non-cardiac MRLC showed that the MRLC isoforms formed heterogeneous filament networks. These results suggest that strain energy output from single hiPSC-CMs depends both cardiac and non-cardiac myosin fibers, which prevent deformation of the cell body.

  10. hMena+11a Isoform Serves as a Marker of Epithelial Phenotype and Sensitivity to EGFR Inhibition in Human Pancreatic Cancer Cell Lines

    Science.gov (United States)

    Pino, Maria S.; Balsamo, Michele; Di Modugno, Francesca; Mottolese, Marcella; Alessio, Massimo; Melucci, Elisa; Milella, Michele; McConkey, David J.; Philippar, Ulrike; Gertler, Frank B.; Natali, Pier Giorgio; Nistico, Paola

    2008-01-01

    Purpose hMena, member of the Ena/VASP protein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. Experimental design Human pancreatic ductal adenocarcinoma (PDAC) cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition (EMT), and expression of hMena and its isoform hMena+11a. The effects of EGF and erlotinib on hMena expression as well as the effect of hMena knock-down on cell proliferation were also evaluated. Results hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena+11a isoform was specifically associated with an epithelial phenotype, EGFR dependency and sensitivity to erlotinib. In epithelial BxPC3 cells EGF upregulated hMena/hMena+11a and erlotinib downregulated expression. hMena knock-down reduced cell proliferation and MAPK and AKT activation in BxPC3 cells and promoted the growth inhibitory effects of erlotinib. Conclusions Collectively, our data indicate that the hMena+11a isoform is associated with an epithelial phenotype and identifies EGFR dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena+11a specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients. PMID:18676769

  11. Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species.

    Science.gov (United States)

    Mascarello, Francesco; Toniolo, Luana; Cancellara, Pasqua; Reggiani, Carlo; Maccatrozzo, Lisa

    2016-09-01

    In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species. To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b).

  12. A predominate role of CYP1A2 for the metabolism of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid, in human liver microsomes.

    Science.gov (United States)

    Turpeinen, Miia; Hofmann, Ute; Klein, Kathrin; Mürdter, Thomas; Schwab, Matthias; Zanger, Ulrich M

    2009-05-01

    Nabumetone, a widely used nonsteroidal anti-inflammatory drug, requires biotransformation into 6-methoxy-2-naphthylacetic acid (6-MNA), a close structural analog to naproxen, to achieve its analgesic and anti-inflammatory effects. Despite its wide use, the enzymes involved in metabolism have not been identified. In the present study, several in vitro approaches were used to identify the cytochrome P450 (P450) enzyme(s) responsible for 6-MNA formation. In human liver microsomes (HLMs) 6-MNA formation displayed monophasic Michaelis-Menten kinetics with apparent K(m) and V(max) values (mean +/- S.D.) of 75.1 +/- 15.3 microM and 1304 +/- 226 pmol/min/mg protein, respectively, and formation rate of 6-MNA varied approximately 5.5-fold (179-983 pmol/min/mg protein). 6-MNA activity correlated strongly with both CYP1A2-mediated phenacetin O-deethylation activity and CYP1A2 protein content (r = 0.85 and 0.74, respectively; p nabumetone, 6-MNA, is predominantly catalyzed by CYP1A2 in HLMs with only minor contribution of other P450s.

  13. Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations†

    Science.gov (United States)

    Pearson, Josh T.; Hill, John J.; Swank, Jennifer; Isoherranen, Nina; Kunze, Kent L.; Atkins, William M.

    2008-01-01

    The heme-containing Cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is ∼ 25 Å from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed and one of the two binding components resolved by curve-fitting exhibits ‘equilibrium overshoot’. Pre-loading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low spin spectra and KD values that are consistent with the high affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly-dissociating inhibitory mode. Most importantly, they provide the first example of an SPR-based method for the kinetic characterization of drug binding to any human CYP, including mechanistic insight not available from other methods. PMID:16700545

  14. Cloning and functional studies of a splice variant of CYP26B1 expressed in vascular cells.

    Directory of Open Access Journals (Sweden)

    Ali Ateia Elmabsout

    Full Text Available BACKGROUND: All-trans retinoic acid (atRA plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs. CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene. METHODOLOGY/PRINCIPAL FINDINGS: The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells. CONCLUSIONS/SIGNIFICANCE: Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1

  15. Analysis of the rs10046 Polymorphism of Aromatase (CYP19 in Premenopausal Onset of Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Karin Zins

    2014-01-01

    Full Text Available The CYP19 gene encodes aromatase, an enzyme catalyzing the conversion of androgens to estrogens. Studies analyzing associations between single nucleotide polymorphisms in CYP19 and breast cancer risk have shown inconsistent results. The rs10046 polymorphism is located in the 3' untranslated region of the CYP19 gene, but the influence of this polymorphism on breast cancer risk is unclear. In this study, we investigated the impact of rs10046 SNP on breast cancer risk, age at onset and association with clinical characteristics in an Austrian population of 274 breast cancer patients and 253 controls. The results show that a significantly increased fraction of patients with the TT genotype of rs10046 develop breast cancer under the age of 50 (41.8% of TT patients, compared to 26.6% of C carriers; p = 0.018, Chi-square test. No rs10046 genotypes were significantly associated with increased breast cancer risk or patient characteristics other than age at onset. These results suggest that the rs10046 polymorphism in the CYP19 gene may have an effect on breast cancer susceptibility at an age under 50 in the investigated population.

  16. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Institute of Scientific and Technical Information of China (English)

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song

    2008-01-01

    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  17. Induction of the expression of genes encoding TGF-beta isoforms and their receptors by inositol hexaphosphate in human colon cancer cells.

    Science.gov (United States)

    Kapral, Małgorzata; Wawszczyk, Joanna; Hollek, Andrzej; Weglarz, Ludmiła

    2013-01-01

    Transforming growth factors-beta (TGF-beta) are multifunctional cytokines involved in the regulation of cell development, differentiation, survival and apoptosis. They are also potent anticancer agents that inhibit uncontrolled proliferation of cells. Incorrect TGF-beta regulation has been implicated in the pathogenesis of many diseases including inflammation and cancer. In humans, the TGF-beta family consists of three members (TGF-beta1, 2, 3) that show high similarity and homology. TGF-betas exert biological activities on various cell types including neoplastic cells via their specific receptors. Inositol hexaphosphate (phytic acid, IP6), a phytochemical has been reported to possess various health benefits. The aim of this study was to examine the effect of IP6 on the expression of genes encoding TGF-beta1, TGF-beta2, TGF-beta3 isoforms and their receptors TbetaRI, TbetaRII, TbetaRIII in human colorectal cancer cell line Caco-2. The cells were treated with 0.5, 1 and 2.5 mM IP6 for 3, 6 and 12 h. The untreated Caco-2 cells were used as the control. Quantification of genes expression was performed by real time QRT-PCR technique with a SYBR Green I chemistry. The experimental data revealed that the TGF-beta1 mRNA was the predominant isoform in Caco-2 cells and that IP6 enhanced transcriptional activity of genes of all three TGF-beta isoforms and their receptors TbetaRI, TbetaRII TbetaRIII in these cells. At concentrations up to 1 mM, IP6 over-expressed the genes in 6 h lasting cultures, and its higher dose (2.5 mM) caused successively increasing transcript level of TGF-beta isoforms and receptors with the duration of experiment up to 12 h. The findings of this study indicate that one of anti-cancer abilities of IP6 can be realized by enhancing the gene expression of TGF-beta isoforms and their receptors at the transcriptional level.

  18. CYP2C8-mediated interaction between repaglinide and steviol acyl glucuronide: In vitro investigations using rat and human matrices and in vivo pharmacokinetic evaluation in rats.

    Science.gov (United States)

    Xu, Yunting; Zhou, Dandan; Wang, Yedong; Li, Jiajun; Wang, Meiyu; Lu, Jia; Zhang, Hongjian

    2016-08-01

    CYP2C8 is involved in the metabolic clearance of several important drugs and recent reports have shown that acyl glucuronides of gemfibrozil and clopidogrel are potent time-dependent inhibitors of CYP2C8 activity. In the present study, the inhibitory effect of steviol acyl glucuronide (SVAG), a circulating metabolite formed after the ingestion of rebaudioside A, was investigated using in vitro and in vivo systems. Results indicated that SVAG was a reversible but not a time-dependent inhibitor of CYP2C8-mediated paclitaxel 6α-hydroxylation. SVAG was also capable of inhibiting CYP2C8-mediated repaglinide 3'-hydroxylation in human liver microsomes and recombinant human CYP2C8, with Ki values of 15.8 μM and 11.6 μM, respectively. In contrast, SVAG did not exhibit inhibitory effect on CYP2C8 activity in rat liver microsomes. In addition, co-administration of rebaudioside A with repaglinide in rats did not lead to AUC and Cmax changes of repaglinide. Although mathematic prediction using a simplified mechanistic model revealed a moderate interaction potential between repaglinide and SVAG, cautions should be given to patients with hypoglycemia if repaglinide and rebaudioside A are used in combination for the blood sugar control.

  19. Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice

    Science.gov (United States)

    Winter, J. C.; Amorosi, D. J.; Rice, Kenner C.; Cheng, Kejun; Yu, Ai-Ming

    2011-01-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387

  20. Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.

    Science.gov (United States)

    Winter, J C; Amorosi, D J; Rice, Kenner C; Cheng, Kejun; Yu, Ai-Ming

    2011-09-01

    In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

    OpenAIRE

    Ostler, Kelly R.; Yang, Qiwei; Looney, Timothy J.; Li ZHANG; Vasanthakumar, Aparna; Tian, Yufeng; Kocherginsky, Masha; Stacey L. Raimondi; DeMaio, Jessica G.; Salwen, Helen R.; Gu, Song; Chlenski, Alexandre; Naranjo, Arlene; Gill, Amy; Peddinti, Radhika

    2012-01-01

    Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7 which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tum...

  2. Tissue- and Condition-Specific Isoforms of Mammalian Cytochrome c Oxidase Subunits: From Function to Human Disease

    Directory of Open Access Journals (Sweden)

    Christopher A. Sinkler

    2017-01-01

    Full Text Available Cytochrome c oxidase (COX is the terminal enzyme of the electron transport chain and catalyzes the transfer of electrons from cytochrome c to oxygen. COX consists of 14 subunits, three and eleven encoded, respectively, by the mitochondrial and nuclear DNA. Tissue- and condition-specific isoforms have only been reported for COX but not for the other oxidative phosphorylation complexes, suggesting a fundamental requirement to fine-tune and regulate the essentially irreversible reaction catalyzed by COX. This article briefly discusses the assembly of COX in mammals and then reviews the functions of the six nuclear-encoded COX subunits that are expressed as isoforms in specialized tissues including those of the liver, heart and skeletal muscle, lung, and testes: COX IV-1, COX IV-2, NDUFA4, NDUFA4L2, COX VIaL, COX VIaH, COX VIb-1, COX VIb-2, COX VIIaH, COX VIIaL, COX VIIaR, COX VIIIH/L, and COX VIII-3. We propose a model in which the isoforms mediate the interconnected regulation of COX by (1 adjusting basal enzyme activity to mitochondrial capacity of a given tissue; (2 allosteric regulation to adjust energy production to need; (3 altering proton pumping efficiency under certain conditions, contributing to thermogenesis; (4 providing a platform for tissue-specific signaling; (5 stabilizing the COX dimer; and (6 modulating supercomplex formation.

  3. Isoforms of human C4b-binding protein. II. Differential modulation of the C4BPA and C4BPB genes by acute phase cytokines

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, O.C.; Sanchez-Corral, P.; Rodriguez de Cordoba, S. [Center for Biological Investigations (CSIC), Velazquez, Madrid (Spain)

    1995-10-15

    Human C4b-binding protein (C4BP) controls activation of the complement system and inactivates the anticoagulant vitamin K-dependent protein S using two distinct polypeptides known as C4BP{alpha} and C4BP{beta}, respectively. C4BP presents three isoforms, {alpha}7{beta}1, {alpha}7{beta}0, and {alpha}6{beta}1, the proportion of which depends on the relative levels of C4BP{alpha} and C4BP{beta}. To better understand the regulation of C4BP during the acute phase response we analyzed the C4BP isoforms in 23 serial samples of acute phase patients and characterized the effect of various acute phase cytokines on the expression of the C4BPA and C4BPB genes using Hep3B cells. We show that the elevation of C4BP during acute phase response leads to changes in the proportion of the C4BP isoforms. However, there are striking differences among acute phase individuals. Some of them present a pattern of induction that primarily affects the {alpha}7{beta}0 isoform, whereas others present the opposite situation, increasing the C4BP{beta}-containing isoforms. In vitro studies demonstrate that IL-6, IL-1{beta}, and INF-{gamma} increase the levels of both C4BP{alpha} and C4BP{beta}-mRNAs, whereas TNF-{alpha} down-regulates these mRNAs. INF-{gamma} shows, in addition, a differential effect on the C4BP{alpha} and C4BP{beta}-mRNAs. Differential modulation of the C4BPA and C4BPB genes has been postulated as an efficient mechanism to maintain steady concentrations of C4BP{beta} when C4BP is induced. A synergistic 10-fold induction of C4BP{alpha}-mRNA, but a marginal increase of C4BP{beta}-mRNA, was observed when INF-{gamma} was used together with TNF-{alpha}, suggesting that association of these cytokines is critical to avoid elevation of C4BP{beta} during the acute phase induction of C4BP. 33 refs., 6 figs.

  4. Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion.

    Science.gov (United States)

    Zheng, Hao; Li, Ying; Wang, Yuzhong; Zhao, Haixia; Zhang, Jing; Chai, Hongyan; Tang, Tian; Yue, Jiang; Guo, Austin M; Yang, Jing

    2014-10-01

    Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr(458)), phospho-PDK (Ser(241)) and phospho-Akt (Thr(308)). Conversely, the exogenous addition of PGE2, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE2, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE2, 20-HETE and phospho-Akt (Thr(308)). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities.

  5. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hua; Shen, Shuijie [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States); Chen, Xiaoyan; Zhong, Dafang [Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203 (China); Zheng, Jiang, E-mail: jiang.zheng@seattlechildrens.org [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States)

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  6. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    Science.gov (United States)

    Doricakova, Aneta; Vrzal, Radim

    2015-11-04

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A.

  7. Itraconazole cis-diastereoisomers activate aryl hydrocarbon receptor AhR and pregnane X receptor PXR and induce CYP1A1 in human cell lines and human hepatocytes.

    Science.gov (United States)

    Stepankova, Martina; Pastorkova, Barbora; Bachleda, Petr; Dvorak, Zdenek

    2017-04-05

    Triazole antimycotic itraconazole contains in its structure three chiral centres; therefore, it forms eight stereoisomers. Commercial preparations of itraconazole are a mixture of four cis-diastereoisomers. There is much evidence that efficacy, adverse effects, and toxicity of chiral drugs may be stereospecific. Therefore, we have prepared 4 pure cis-diastereoisomers of itraconazole and investigated their effects on transcriptional activities of xenoreceptors aryl hydrocarbon receptor AhR and pregnane X receptor PXR. Gene reporter assays showed that itraconazole dose-dependently activated both AhR and PXR, and the activation of AhR but not of PXR was enantiospecific. Itraconazole diastereoisomers transformed AhR and PXR into their DNA-binding forms, as demonstrated by electromobility shift assays. Cytochrome P450 CYP1A1 mRNA and protein were induced by itraconazole diastereoisomers in human hepatoma cells HepG2, human skin cells HaCaT, and in primary human hepatocytes. The expression of CYP3A4 in human intestinal LS180 cells was not influenced by itraconazole, but we observed downregulation of CYP3A4 in human hepatocytes. Collectively, we show that itraconazole is a dual activator of AhR and PXR, with differential effects on the target genes for xenoreceptors. The enantiospecific pattern was observed only in gene reporter assays for AhR. The data presented here might be of toxicological and clinical importance.

  8. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome.

    Science.gov (United States)

    Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J; Ramos, Feliciano J; Ström, Lena; Pié, Juan

    2017-02-23

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

  9. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

    Directory of Open Access Journals (Sweden)

    Beatriz Puisac

    2017-02-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction. Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys, showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

  10. Development and validation of a LC-MS/MS method for the in vitro analysis of 1-hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures.

    Science.gov (United States)

    Mooiman, K D; Maas-Bakker, R F; Rosing, H; Beijnen, J H; Schellens, J H M; Meijerman, I

    2013-09-01

    Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1-hydroxymidazolam in plasma are often determined with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS). However, validated LC-MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1-hydroxymidazolam. Therefore, the aim was to validate and optimize an LC-MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid-liquid extraction with tert-butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed-phase chromatography consisting of a Zorbax Extend-C18 column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC-MS/MS method was validated over linear range of 1.0-500 nm for 1-hydroxymidazolam. The results revealed good inter-assay accuracy (≥85% and ≤115%) and within-day and between-day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β-carotene, green tea, milk thistle and St. John's wort) was determined.

  11. Improved Homology Model of the Human all-trans Retinoic Acid Metabolizing Enzyme CYP26A1

    Directory of Open Access Journals (Sweden)

    Mohamed K. A. Awadalla

    2016-03-01

    Full Text Available A new CYP26A1 homology model was built based on the crystal structure of cyanobacterial CYP120A1. The model quality was examined for stereochemical accuracy, folding reliability, and absolute quality using a variety of different bioinformatics tools. Furthermore, the docking capabilities of the model were assessed by docking of the natural substrate all-trans-retinoic acid (atRA, and a group of known azole- and tetralone-based CYP26A1 inhibitors. The preferred binding pose of atRA suggests the (4S-OH-atRA metabolite production, in agreement with recently available experimental data. The distances between the ligands and the heme group iron of the enzyme are in agreement with corresponding distances obtained for substrates and azole inhibitors for other cytochrome systems. The calculated theoretical binding energies agree with recently reported experimental data and show that the model is capable of discriminating between natural substrate, strong inhibitors (R116010 and R115866, and weak inhibitors (liarozole, fluconazole, tetralone derivatives.

  12. Influence of the Cyp1B1 L432V gene polymorphism and exposure to tobacco smoke on Cyp1B1 mRNA expression in human leukocytes.

    Science.gov (United States)

    Helmig, Simone; Hadzaad, Bahar; Döhrel, Juliane; Schneider, Joachim

    2009-07-01

    Cytochrome P450 1B1 (CYP1B1), a phase I enzyme, is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 L432V polymorphism with diverse types of cancer, as well as an impact on the catalytic activity of the enzyme, has been described. To show the functional impact of the allelic variant Cyp1B1*3, we investigated the quantitative Cyp1B1 mRNA expression in a population of smokers, nonsmokers, and ex-smokers and determined their genotypes. Detection of the L432V polymorphism in exon 3 of the Cyp1B1 gene was performed by rapid capillary polymerase chain reaction (PCR) with melting curve analysis. For quantitative comparison of Cyp1B1 mRNA levels, real-time PCR was performed using SYBR Green fluorescence in a LightCycler system. Calculations of expression were made with the 2(-DeltaDeltaCT) method. In comparing relative Cyp1B1 mRNA expression, highly significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.185 +/- 0.027, n = 118 versus 0.071 +/- 0.013, n = 56; p = 0.000), as well as between the heterozygote genotype *1/*3 and the homozygote genotype *3/*3 (0.178 +/- 0.025, n = 171 versus 0.071 +/- 0.013, n = 56; p = 0.000), were revealed. Significant differences between the genotypes were also detected within the subgroups of smokers, nonsmokers, and ex-smokers. No significant differences were determined in comparing the relative Cyp1B1 mRNA expression with regard to tobacco smoke exposure. Our results suggest that genotypes carrying the C allele (*1/*1 and *1/*3) at Cyp1B1 L432V polymorphism have a significantly higher Cyp1B1 mRNA expression compared with the genotype without the C allele (*3/*3). Gene expression of Cyp1B1 mRNA cannot be used as a biomarker for exposure of tobacco smoke.

  13. Leptin receptor isoform 219.1: an example of protein evolution by LINE-1-mediated human-specific retrotransposition of a coding SVA element.

    Science.gov (United States)

    Damert, Annette; Löwer, Johannes; Löwer, Roswitha

    2004-04-01

    Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

  14. Understanding the Mechanism of Human P450 CYP1A2 Using Coupled Quantum-Classical Simulations in a Dynamical Environment

    Energy Technology Data Exchange (ETDEWEB)

    Draeger, E W; Bennion, B; Gygi, F; Lightstone, F

    2006-02-10

    The reaction mechanism of the human P450 CYP1A2 enzyme plays a fundamental role in understanding the effects of environmental carcinogens and mutagens on humans. Despite extensive experimental research on this enzyme system, key questions regarding its catalytic cycle and oxygen activation mechanism remain unanswered. In order to elucidate the reaction mechanism in human P450, new computational methods are needed to accurately represent this system. To enable us to perform computational simulations of unprecedented accuracy on these systems, we developed a dynamic quantum-classical (QM/MM) hybrid method, in which ab initio molecular dynamics are coupled with classical molecular mechanics. This will provide the accuracy needed to address such a complex, large biological system in a fully dynamic environment. We also present detailed calculations of the P450 active site, including the relative charge transfer between iron porphine and tetraphenyl porphyrin.

  15. Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Hao; Li, Ying [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Wang, Yuzhong [Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Zhao, Haixia [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Tang, Tian [Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Yue, Jiang [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Guo, Austin M., E-mail: Austin_Guo@nymc.edu [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Department of Pharmacology, New York Medical College, Valhalla, NY 10595 (United States); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China)

    2014-10-01

    Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E{sub 2} (PGE{sub 2}) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr{sup 458}), phospho-PDK (Ser{sup 241}) and phospho-Akt (Thr{sup 308}). Conversely, the exogenous addition of PGE{sub 2}, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE{sub 2}, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE{sub 2}, 20-HETE and phospho-Akt (Thr{sup 308}). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities. - Highlights: • Isoliquiritigenin induces anoikis and suppresses

  16. Effect of Flavonoids on Glutathione Level, Lipid Peroxidation and Cytochrome P450 CYP1A1 Expression in Human Laryngeal Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Lidija Vuković

    2007-01-01

    Full Text Available Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical pathways. Using human laryngeal carcinoma HEp2 cells and their drug-resistant CK2 subline, we examined the effect of five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin, one flavonol (luteolin and one glycosilated flavanone (naringin for: (i their ability to inhibit mitochondrial dehydrogenases as an indicator of cytotoxic effect, (ii their influence on glutathione level, (iii antioxidant/prooxidant effects and influence on cell membrane permeability, and (iv effect on expression of cytochrome CYP1A1. Cytotoxic action of the investigated flavonoids after 72 hours of treatment follows this order: luteolin>quercetin>fisetin>naringin>myricetin. Our results show that CK2 were more resistant to toxic concentrations of flavonoids as compared to parental cells. Quercetin increased the total GSH level in both cell lines. CK2 cells are less perceptible to lipid peroxidation and damage caused by free radicals. Quercetin showed prooxidant effect in both cell lines, luteolin only in HEp2 cells, whereas other tested flavonoids did not cause lipid peroxidation in the tested cell lines. These data suggest that the same compound, quercetin, can act as a prooxidant, but also, it may prevent damage in cells caused by free radicals, due to the induction of GSH, by forming less harmful complex. Quercetin treatment damaged cell membranes in both cell lines. Fisetin caused higher cell membrane permeability only in HEp2 cells. However, these two compounds did not enhance the damage caused by hydrogen peroxide. Quercetin, naringin, myricetin and fisetin increased the expression of CYP1A1 in both cell lines, while luteolin decreased basal level of CYP1A1 only in HEp2 cells. In conclusion, small

  17. Single-fiber expression and fiber-specific adaptability to short-term intense exercise training of Na+-K+-ATPase α- and β-isoforms in human skeletal muscle.

    Science.gov (United States)

    Wyckelsma, V L; McKenna, M J; Serpiello, F R; Lamboley, C R; Aughey, R J; Stepto, N K; Bishop, D J; Murphy, R M

    2015-03-15

    The Na(+)-K(+)-ATPase (NKA) plays a key role in muscle excitability, but little is known in human skeletal muscle about fiber-type-specific differences in NKA isoform expression or adaptability. A vastus lateralis muscle biopsy was taken in 17 healthy young adults to contrast NKA isoform protein relative abundance between type I and IIa fibers. We further investigated muscle fiber-type-specific NKA adaptability in eight of these adults following 4-wk repeated-sprint exercise (RSE) training, comprising three sets of 5 × 4-s sprints, 3 days/wk. Single fibers were separated, and myosin heavy chain (I and IIa) and NKA (α1-3 and β1-3) isoform abundance were determined via Western blotting. All six NKA isoforms were expressed in both type I and IIa fibers. No differences between fiber types were found for α1-, α2-, α3-, β1-, or β3-isoform abundances. The NKA β2-isoform was 27% more abundant in type IIa than type I fibers (P training increased β1 in type IIa fibers (pretraining 0.70 ± 0.25, posttraining 0.84 ± 0.24 arbitrary units, 42%, P training effects were found for other NKA isoforms. Thus human skeletal muscle expresses all six NKA isoforms and not in a fiber-type-specific manner; this points to their different functional roles in skeletal muscle cells. Detection of elevated NKA β1 after RSE training demonstrates the sensitivity of the single-fiber Western blotting technique for fiber-type-specific intervention effects.

  18. Human CD72 splicing isoform responsible for resistance to systemic lupus erythematosus regulates serum immunoglobulin level and is localized in endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Hitomi Yuki

    2012-12-01

    Full Text Available Abstract Background CD72 is an inhibitory co-receptor expressed on B cells. We previously demonstrated significant association of the polymorphism of the CD72 gene with susceptibility to human systemic lupus erythematosus (SLE in individuals carrying a SLE-susceptible FCGR2B genotype (FCGR2B-232Thr/Thr. The human CD72 locus generates a splicing isoform that lacks exon 8 (CD72Δex8 as well as full-length CD72 (CD72fl, and the CD72 polymorphism regulates exon 8 skipping. Results Here we demonstrated that individuals carrying the disease-protective CD72 genotype exhibit significantly lower serum immunoglobulin levels than do individuals carrying other CD72 genotypes (P CD72 genotype, the protein level of CD72Δex8 was increased in individuals carrying the disease-protective CD72 genotype, suggesting a crucial role of CD72Δex8 in regulation of antibody production. By expressing these human CD72 isoforms in mouse cell lines, we further demonstrated that CD72Δex8 is accumulated in endoplasmic reticulum (ER and fails to regulate BCR signaling whereas human CD72fl is efficiently transported to the cell surface and inhibits signaling through the B cell antigen receptor (BCR, as is the case for mouse CD72. Conclusion Human CD72 polymorphism appears to regulate antibody production as well as susceptibility to SLE by regulating expression of ER-localizing CD72Δex8.

  19. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram; Finerty, Jr., Patrick J.; MacKenzie, Farrell; Dimov, Svetoslav; Vedadi, Masoud; Park, Hee-Won (Toronto)

    2010-07-06

    Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

  20. High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates.

    Science.gov (United States)

    Marks, Bryan D; Thompson, David V; Goossens, Tony A; Trubetskoy, Olga V

    2004-08-01

    CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

  1. Modeling Chemical Interaction Profiles: I. Spectral Data-Activity Relationship and Structure-Activity Relationship Models for Inhibitors and Non-inhibitors of Cytochrome P450 CYP3A4 and CYP2D6 Isozymes

    Directory of Open Access Journals (Sweden)

    Richard D. Beger

    2012-03-01

    techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.

  2. Modeling chemical interaction profiles: I. Spectral data-activity relationship and structure-activity relationship models for inhibitors and non-inhibitors of cytochrome P450 CYP3A4 and CYP2D6 isozymes.

    Science.gov (United States)

    McPhail, Brooks; Tie, Yunfeng; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Valerio, Luis G; Fuscoe, James C; Tong, Weida; Buzatu, Dan A; Wilkes, Jon G; Fowler, Bruce A; Demchuk, Eugene; Beger, Richard D

    2012-03-15

    techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.

  3. Multiple isoforms of the tumor protein p73 are expressed in the adult human telencephalon and choroid plexus and present in the cerebrospinal fluid.

    Science.gov (United States)

    Cabrera-Socorro, Alfredo; Pueyo Morlans, Mercedes; Suarez Sola, Maria Luisa; Gonzalez Delgado, Francisco J; Castañeyra-Perdomo, Agustin; Marin, Maria C; Meyer, Gundela

    2006-04-01

    p73, a homolog of the p53 tumor suppressor, codes for full-length transactivating (TA) and N-terminally truncated (DeltaN) isoforms, with pro- and anti-apoptotic activities, respectively. We examined the expression of the main p73 isoforms in adult human and mouse telencephalon and choroid plexus by immunohistochemistry on paraffin sections, and immunoblotting (IB) of tissue extracts and cerebrospinal fluid (CSF), using antibodies against different protein domains. Cortical neurons expressed TAp73 predominantly in the cytoplasm and DeltaNp73 mainly in the nucleus, with partial overlap in the cytoplasm. Highest expression was found in the hippocampus. IB showed an array of TAp73 variants in adult human cortex and hippocampus. IB of human choroid plexus and CSF using TAp73-specific antibodies revealed the presence of a approximately 90-kDa protein whose molecular weight was reduced after N-deglycosylation, suggesting that glycosylated TAp73 is exported into the CSF. In the mouse, high expression of TAp73 was also detected in the subcommissural organ (SCO), an ependymal gland absent in adult humans. TAp73 colocalized with anti-fibra-Reissner-antibody (AFRU), which is a marker of Reissner's fiber, the secreted SCO product. p73-deficient mice had generalized cortical hypoplasia and hydrocephalus; in addition, we observed a dramatic size reduction of the choroid plexus. However, the SCOs were apparently unaltered and continued to secrete Reissner's fiber. Our findings point to complex and widespread p73 activities in the maintenance of adult cortical neurons and in brain homeostasis. TAp73 in the CSF may play important roles in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium.

  4. Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping.

    Science.gov (United States)

    Jordan, Nan Yeun; Mimpen, Jolet Y; van den Bogaard, Willie J M; Flesch, Frits M; van de Meent, Michiel H M; Torano, Javier Sastre

    2015-07-15

    Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6min. The method was validated from 0.05 to 5μgmL(-1) CAF and 0.025-2.5μgmL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions concentration ratios from volunteers were 0.26-1.09 with mean ratios of 0.78±0.26 and 0.38±0.10 for regular and light/non-coffee drinkers, respectively.

  5. Carbonic anhydrase inhibitors: synthesis and inhibition of the human carbonic anhydrase isoforms I, II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties.

    Science.gov (United States)

    Sethi, Kalyan K; Verma, Saurabh M; Tanç, Muhammet; Purper, Gaultier; Calafato, Gaetan; Carta, Fabrizio; Supuran, Claudiu T

    2014-03-01

    A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (Kis in the range of 295-10,000 nM), but were more effective hCA II inhibitors (Kis of 1.7-887 nM). The tumor-associated hCA IX was also inhibited, with Kis in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90-3,746 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. 人CYP的异源表达及其在新药研发早期的作用%Heterogeneous expression of human CYP and its application in early stage of new drug research and development

    Institute of Scientific and Technical Information of China (English)

    谢章明; 陈枢青

    2013-01-01

    人细胞色素P450(cytochrome P450,CYP)在临床上对外源及内源性的物质代谢起着至关重要的作用.由于从人组织中获取的CYP成分较为复杂,且人组织本身不易获得,人们开始用各种表达系统来进行CYP的异源表达,并将表达得到的单一CYP用于药物代谢及药物-药物相互作用的研究,从而大大提高了药物高通量筛选的效率.另外,由于药物代谢酶的多态性使不同人群表现出对某种药物药效的差异,因此对某种CYP突变体的异源表达和药物代谢研究,将有助于指导治疗方案的优化,实施个体化药物治疗.%Human cytochrome P450 ( CYP) has a pivotal role on metabolism of xenobiotics and endogenous substances in clinical practice. Since the CYP from human tissue is very complex, and the human tissue itself is not easy to obtain, investigators begin to use all kinds of expression system to heterogeneously express the CYP. The single CYP expressed was then used for drug metabolism and drug-drug interaction research, to improve the efficiency of high-throughput drug screening greatly. Besides, since the polymorphism of drug-metabolizing enzymes makes efficacy variance for some drugs in different population, the heterougeneous expression and drug metabolizing research of certain CYP mutanes will be helpful to guide the optimization of therapeutic regimen and conduct the personalized medication.

  7. Relative Expression of Vitamin D Hydroxylases, CYP27B1 and CYP24A1, and of Cyclooxygenase-2 and Heterogeneity of Human Colorectal Cancer in Relation to Age, Gender, Tumor Location, and Malignancy: Results from Factor and Cluster Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Brozek, Wolfgang, E-mail: wolfgang.brozek@gmx.at; Manhardt, Teresa; Kállay, Enikö; Peterlik, Meinrad; Cross, Heide S. [Department of Pathophysiology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2012-07-26

    Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH){sub 2}D{sub 3} and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1α-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression.

  8. Genetic analysis of colon tumors induced by a dietary carcinogen PhIP in CYP1A humanized mice: Identification of mutation of β-catenin/Ctnnb1 as the driver gene for the carcinogenesis.

    Science.gov (United States)

    Wang, Hong; Zhou, Hong; Liu, Anna; Guo, Xiangyi; Yang, Chung S

    2015-11-01

    Replacing mouse Cyp1a with human CYP1A enables the humanized CYP1A mice to mimic human metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), by N(2) -hydroxylation to a proximate carcinogen. Our previous study demonstrated that PhIP, combined with the dextrin sulfate sodium (DSS)-induced colitis, induces colon carcinogenesis in hCYP1A mice. Here, we employed whole exome sequencing and found multiple gene mutations in PhIP/DSS-induced colon tumors. Mutations in the exon 3 of Ctnnb1/β-catenin, however, were the predominant events. We further sequenced the key fragments of Apc, Ctnnb1, and Kras, because mutations of these genes in the humans are commonly found as the drivers of colorectal cancer. Mutations on either codon 32 or 34 in the exon 3 of Ctnnb1 were found in 39 out of 42 tumors, but no mutation was found in either Apc or Kras. The sequence context of codons 32 and 34 suggests that PhIP targets +3G in a TGGA motif of Ctnnb1. Since mutations that activate Wnt signal is a major driving force for human colorectal cancers, we conclude that the mutated β-catenin is the driver in PhIP/DSS-induced colon carcinogenesis. This result suggests that the colon tumors in hCYP1A mice mimic human colorectal carcinogenesis not only in the dietary etiology involving PhIP, but also in the aberrant activation of the Wnt signaling pathway as the driving force.

  9. Loss of the SxxSS motif in a human T-cell factor-4 isoform confers hypoxia resistance to liver cancer: an oncogenic switch in Wnt signaling.

    Directory of Open Access Journals (Sweden)

    Hironori Koga

    Full Text Available PURPOSE: Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. METHODS: We investigated the TCF-4J and K isoform pair characterized by the presence (K or absence (J of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells and K (K cells to grow as solid tumors in nude mice was explored. RESULTS: TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells. The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. CONCLUSIONS: Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.

  10. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    Science.gov (United States)

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  11. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    Science.gov (United States)

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

  12. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

    Science.gov (United States)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

    2017-03-01

    A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [(3) H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of (13) C and (14) C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.

  13. Metabolism of Oral Turinabol by Human Steroid Hormone-Synthesizing Cytochrome P450 Enzymes.

    Science.gov (United States)

    Schiffer, Lina; Brixius-Anderko, Simone; Hannemann, Frank; Zapp, Josef; Neunzig, Jens; Thevis, Mario; Bernhardt, Rita

    2016-02-01

    The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17β-hydroxy-17α-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min(-1) mM(-1) for CYP11A1, 741 min(-1) mM(-1) for CYP11B1, and 3338 min(-1) mM(-1) for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli-based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography-tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies. Copyright © 2016 by The

  14. 肝药酶CYP3A4与抗癫(癎)药物的代谢研究进展%Progress on the metabolism of antiepileptic drugs by human hepatic CYP3A4 enzymes

    Institute of Scientific and Technical Information of China (English)

    张君梅

    2011-01-01

    癫(癎)是危害人类健康的常见神经系统疾病,临床常用抗癫(癎)药多通过细胞色素P450等肝药酶代谢.CYP3A4是P450酶系中最重要的代谢酶,参与40%~60%的药物代谢,与临床常用抗癫(癎)药的代谢也有非常紧密的关系.该文对CYP3A4的一般特性及其与抗癫(癎)药物的代谢研究进展作一概述.%Epilepsy is a common disease in nervous system.Most antiepileptic drugs are metabolized by liver enzymes.such as cytochrome P450 enzyme.