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

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previously, we identified a novel human cytochrome P450 cDNA that is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and represents the first member of a new subfamily designated cytochrome P4501B1 (CYP1B1; Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099). Here, we report on the isolation and initial characterization of the CYP1B1 gene. The CYP1B1 gene maps to human chromosome 2 at 2p21-22 and contains three exons and two introns. The putative open reading frame starts in the second exon and is 1629 base pairs in length. Southern analysis using DNA probes directed to each of the three exons confirmed that CYP1B1 is a single copy gene. Human CYP1B1 differs from its two most closely related members of the cytochrome P450 superfamily, CYP1A1 and CYP1A2, in the number of exons (3 versus 7) and chromosome location (2 versus 15). A single transcription initiation site was identified by primer extension analysis and S1 nuclease mapping. Based on nucleotide sequence analysis, the CYP1B1 gene lacks a consensus TATA box in the promoter region and contains nine TCDD-responsive enhancer core binding motifs (5'-GCGTG-3') located within a 2.5-kilobase pair genomic fragment 5'-ward of the transcription initiation start site. Deletion analysis of chloramphenicol acetyltransferase reporter gene constructs containing 5' CYP1B1 genomic fragments indicates that a region from -1022 to -835 containing three of the nine core binding motifs contributes to the TCDD-inducible expression of CYP1B1.
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PMID:Isolation and characterization of the human cytochrome P450 CYP1B1 gene. 891 Apr 54

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.
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PMID:cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression. 901 60

Steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity of SF-1/Ad4BP are not well defined. In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD II), the major gonadal and adrenal isoform. Regulation of the 3beta-HSD II promoter was examined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells. H295R cells were transfected with a series of 5' deletions of 1251 base pairs (bp) of the 3beta-HSD II 5'-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol ester (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated that the region from -101 to -52 bp of the promoter was required for PMA-induced expression. A putative SF-1/Ad4BP regulatory element, TCAAGGTAA, was identified by sequence homology at -64 to -56 bp of the promoter. Cotransfection of HeLa cells with the -101 3beta-HSD-CAT construct and an expression vector for SF-1/Ad4BP increased CAT activity 49-fold. Subsequent treatment with PMA induced an unexpected synergistic increase in transcriptional activity 540-fold over basal. Mutation of the putative response element (TCAAGGTAA to TCAATTTAA) abolished SF-1-induced CAT activity and the synergistic response to PMA. Gel mobility shift assays confirmed that SF-1/Ad4BP interacts with the putative element and transcripts for SF-1/Ad4BP were detected in H295R cells by Northern analysis. These data are the first to demonstrate 1) regulation of a non-cytochrome P450 steroidogenic enzyme promoter by SF-1/Ad4BP, 2) a powerful synergistic effect of PMA on SF-1/Ad4BP-induced transcription, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3beta-HSD II promoter.
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PMID:Synergistic activation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase promoter by the transcription factor steroidogenic factor-1/adrenal 4-binding protein and phorbol ester. 906 66

The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report. Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression. Substantial induction of chloramphenicol acetyltransferase (CAT) activity by iron and salicylate in B. megaterium cells bearing a P450BM-1 promoter-cat transcriptional fusion vector suggests that the induction of P450BM-1 by iron and salicylate occurs at the transcriptional level. Unexpectedly, in contrast to the bm1P1-dependent induction of P450BM-1 by pentobarbital, disruption of bm1P1 gene did not affect induction of P450BM-1 by iron and salicylate. This result suggests that the induction of P450BM-1 by iron and salicylate occurs by a bm1P1-independent mechanism. To our knowledge, this is the first example of an iron-regulated cytochrome P450 gene in prokaryotes.
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PMID:Iron and salicylate induction of cytochrome P450BM-1 in Bacillus megaterium. 917 56

Vitamin D analogs are valuable drugs with established and potential uses in hyperproliferative disorders. Lexacalcitol (KH1060) is over 100 times more active than 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3], as judged by in vitro antiproliferative and cell differentiating assays. The underlying biochemical reasons for the increased biological activity of KH1060 are unknown, but are thought to include 1) metabolic considerations in addition to explanations based upon 2) enhanced stability of KH1060-liganded transcriptional complexes. In this study we explored the in vivo and in vitro metabolism of KH1060. We established by physicochemical techniques the existence of multiple side-chain hydroxylated metabolites of KH1060, including 24-, 24a-, 26-, and 26a-hydroxylated derivatives as well as side-chain truncated forms. KH1060 metabolism could be blocked by the cytochrome P450 inhibitor, ketoconazole. KH1060 was not an effective competitor of C24 oxidation of 1alpha,25-(OH)2D3. Certain hydroxylated metabolites of KH1060 retained significant biological activity in vitamin D-dependent reporter gene systems (chloramphenicol acetyltransferase). Likewise, those metabolites accumulating in the target cell culture models in metabolism studies, particularly 24a-hydroxy-KH1060 and 26-hydroxy-KH1060, retained biological activities superior to those of 1alpha,25-(OH)2D3 in native gene expression systems in vitamin D target cells (osteopontin and P450cc24). We conclude that KH1060 is rapidly metabolized by a variety of cytochrome P450-mediated enzyme systems to products, many of which retain significant biological activity in vitamin D-dependent assay systems. These results provide an explanation for the considerable biological activity advantage displayed by KH1060 compared with 1alpha,25-(OH)2D3 in various in vitro assay systems.
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PMID:The vitamin D analog, KH1060, is rapidly degraded both in vivo and in vitro via several pathways: principal metabolites generated retain significant biological activity. 938 35

Inflammatory cytokines cause the down-regulation of multiple cytochrome P450 mRNAs, but the transcriptional mechanisms involved are not known. We investigated the role of a putative negative NF-kappaB-responsive element, nkappaB-RE1, in the down-regulation of the CYP2C11 gene in rat hepatocytes. This sequence spans the transcription start site of CYP2C11, from positions -2 to +8. Electrophoretic mobility shift assays showed that nuclear extracts from livers of rats treated with bacterial lipopolysaccharide, or from hepatocytes treated with interleukin-1beta, formed a protein complex with an oligonucleotide probe containing the nkappaB-RE1, and that this complex contained predominantly the p50 subunit of NF-kappaB. Binding of NF-kappaB to the nkappaB-RE1 probe was of lower affinity than to a probe containing the prototypic NF-kappaB enhancer of the immunoglobulin kappa chain gene. Mutations in the 5'-end of the nkappaB-RE1, and to a lesser extent the 3'-end, reduced the affinity of NF-kappaB for this element. Introduction of the 5'-mutation into nkappaB-RE1 abolished the response of the -200-CYP2C11-chloramphenicol acetyltransferase reporter construct to interleukin-1 or lipopolysaccharide. We conclude that nkappaB-RE1 is a functional negative regulatory element that participates in the inflammatory suppression of CYP2C11.
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PMID:Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappaB binding at the transcription start site. 1077 59

Progesterone is essential to the sustenance of pregnancy in humans and other mammals. From the second trimester on, the human placenta is the sole origin of de novo synthesized steroid hormones. In mice, placentation at midgestation is accompanied by a temporal rise of steroid hormone synthesis commencing in the giant cells of the mouse trophoblast. In doing so, the giant trophoblasts, as any other steroidogenic cell, express high levels of the key steroidogenic enzyme, cholesterol side-chain cleavage cytochrome P450 (P450scc). Because steroidogenic factor 1 (SF-1), the transcription factor required for expression of P450scc in the adrenals and the gonads, is not expressed in the placenta, we hypothesized that placenta-specific nuclear factor(s) (PNF) assumes the role of SF-1 by binding to the same promoter region that harbors the SF-1 recognition site in the P450scc gene. To address this possibility, we used SCC1, a well conserved proximal region in the P450scc genes (-60/-32 in the rat gene) to purify PNF from human term placenta. Sequencing of the purified PNF revealed that it is the alpha isoform of the human activating protein-2 (AP-2alpha). Specific antibodies tested in EMSA confirmed that AP-2alpha is the predominant isoform that binds SCC1 in the human placenta, whereas AP-2gamma is the only mouse placental protein that binds this oligonucleotide. Functional studies showed that coexpression of the rat P450scc promoter (-378/+8 CAT) and AP-2 isoforms (alpha or gamma) in human embryonic kidney 293 cells results in a marked activation of chloramphenicol acetyltransferase (CAT) transcription that is dependent on an intact AP-2 motif, GCCTTGAGC. This motif conforms with consensus sequences previously determined for binding of the AP-2 alpha and gamma isoforms. Mutations of the AP-2 element ablated binding of AP-2 to SCC1, as well as severely diminished the promoter activity in primary mouse giant trophoblasts and human choriocarcinoma JAR cells. Collectively, these studies suggest that expression of placental P450scc is governed by AP-2 factors that bind to a cis-element that largely overlaps the sequence required for recognition of SF-1 in other steroidogenic tissues.
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PMID:Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element. 1214 40


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