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)

Selectivity to aldosterone (Aldo) in mineralocorticoid target tissues has been suggested to be due to the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). This enzyme inactivates the endogenous glucocorticoid cortisol, thus permitting the unhindered access of Aldo to the mineralocorticoid receptor. The 11 beta-HSD activity was measured by the conversion of cortisol to cortisone and vice versa. Concomitant treatment of the cells with either cortisone or cortisol in the presence of the glycyrrhetinic acid derivative carbenoxolone (CBX) blocked both activities of 11 beta-HSD. Dexamethasone and Aldo activated the transcription of transiently transfected mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene in LU-19 cells. The transcription activation by cortisol was synergized by concomitant treatment of the transfectants with CBX. Transactivation with Aldo was inhibited by spironolactone. The enzyme 11 beta-HSD in LU-19 cells is similar to the cloned liver isoform and catalyzes both reduction and dehydrogenation.
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PMID:11 beta-hydroxysteroid dehydrogenase activity in human lung cells and transcription regulation by glucocorticoids. 794 49

The human 11 beta-hydroxysteroid dehydrogenase (h11 beta-HSD) inactivates the active corticosteroid cortisol to its inactive metabolite cortisone. We have developed transactivation analyses of the reporter chimeric gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) to study the catalytic activity of h11 beta-HSD introduced by cotransfection into receptor and 11 beta-HSD deficient CV-1 cells. Assay of 11 beta-HSD expressed in CV-1 cells by cotransfection showed that the catalyzed dehydrogenation of cortisol to cortisone was 2-fold higher in the presence of NADP. The reductase activity was dependent on the coenzyme NADPH. The addition of increasing concentrations of the inhibitor carbenoxolone (CBX) in the incubates blocked the enzyme activity in a dose dependent fashion. In CV-1 cells cotransfected with expression vectors of either human glucocorticoid (hGR1-777) or mineralocorticoid (hMR1-984) and the reporter plasmid MMTV-CAT, dexamethasone (DEX), aldosterone (ALDO), cortisol, and corticosterone induction of CAT activity was dose dependent. Cotransfection of CV-1 cells transfected with 10 micrograms of 11 beta-HSD expression vector reduced the transactivation of MMTV-CAT by hGR or hMR in the presence of either cortisol or corticosterone to basal values. The concomitant addition of 100 nM cortisone and 1 microM NADPH to these transfectants elevated CAT activity. These data show that transactivation analyses can be used to study the 11 beta-HSD-catalyzed regulation of corticosteroid levels, which triggers physiological processes and in certain cases provides an alternative to animal experimentation.
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PMID:Transcription activation of mouse mammary tumor virus-chloramphenicol acetyltransferase: a model to study the metabolism of cortisol. 794 89

Incubation of whole LNCaP cells in suspension with tritium labeled cortisol revealed two major and one minor radioactive product. Of the major products, one migrated with an Rf value identical to cortisol (Kendall's compound "F"), and the second migrated with an Rf value similar to nonradioactive cortisone (Kendall's compound "E"); the third minor product comigrated with 21-acetylated cortisol. The conversion of cortisol to cortisone was linear with respect to cell number, and conversion reached a plateau after 120 min of incubation at 37 degrees C. One half of the cortisol was converted to cortisone within 2 h of incubation at 37 degrees C. This conversion was nicotine amide dinucleotide (NAD) dependent. Low levels of transcription activation by cortisol were documented in LNCaP cells transfected with glucocorticoid and androgen responsive mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene (MMTV-CAT). Hormone binding assay and transactivation analysis revealed the presence of a functional mineralocorticoid receptor in LNCaP cells. Treatment of transfectants with F in the presence of carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in a two orders of magnitude increase in measurable CAT activity. The addition of the reduced form of nicotine amide dinucleotide (NADH) in the presence of 10(-7) M E stimulated measurable CAT activity in LNCaP cells. In conferring aldosterone specificity in mineralocorticoid target tissues, 11 beta-HSD may have an important role as "gate keeper" in allowing a specific androgen response in hormone responsive LNCaP prostate cancer cells.
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PMID:11 beta-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP. 803 14

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) regulate steroid hormone levels. cDNA cloning indicates that the rat and human liver isoforms display high sequence identity and that they belong to the aldo-keto reductase (AKR) superfamily. Of these the most extensively characterized is rat liver 3 alpha-HSD. The recently solved X-ray crystal structure shows that this enzyme adopts an (alpha/beta)8-barrel scaffold (Hoog et al. 1994). NAD(P)H binds in an extended anti-conformation and lies along the inner surface of the barrel. The nicotinamide ring is stabilized by interaction with Y216. The 4-pro(R)-hydrogen transferred in the reaction is in close proximity to Y55. K84, D50 and H117 which are implicated in catalysis. These residues are located at the base of a hydrophobic pocket which is presumed to be involved in binding steroid hormone. This catalytic tetrad is conserved in members of the AKR superfamily. Mutant enzymes support roles for Y55 in steroid binding and for K84 as the general acid involved in catalysis. The gene for rat 3 alpha-HSD has been cloned and is 47 kb in length and contains 9 exon-intron boundaries which are highly conserved in the human gene(s). The 5'-flanking regions of the rat and human genes contain consensus sequences for AP-1, Oct-1 and multiple copies of perfect and imperfect steroid hormone response elements (REs) (estrogen, glucocorticoid (GRE), and progesterone) which may comprise a steroid response unit (SRU) (Lin & Penning 1995). Constitutive and regulated expression of the rat 3 alpha-HSD gene has been studied by transiently transfecting reporter gene (chloramphenicol acetyltransferase, CAT) constructs into human hepatoma (HepG2) cells. With respect to the transcription start-site (+1), a proximal (-498 to -199bp) and distal (-20 to -4.0kb) enhancer, as well as a powerful silencer (-755 to -498 bp) were located in the promoter. Band-shift and supershift assays provide evidence that Oct-1 binds to the silencer. Tandem repeats of the imperfect proximal and distal GREs that reside in the SRU were inserted into tk-CAT vectors and transiently transfected. Stimulation of transfected cells with dexamethasone resulted in robust CAT activity. These data indicate that glucocorticoids may positively regulate transcription of the rat 3 alpha-HSD gene from the SRU.
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PMID:3 alpha-hydroxysteroid dehydrogenase: three dimensional structure and gene regulation. 894 1

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

Apparent mineralocorticoid excess (AME) characterized by early-onset hypertension and hypokalemia is due to congenital deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). Two isoforms of human 11 beta HSD are known, and the type 2 isoform (11 beta HSD2) has been recently shown to be responsible for AME. In this study we have analyzed the 11 beta HSD2 gene of a Japanese patient with AME. PCR amplification and subsequent nucleotide sequencing of the 11 beta HSD2 gene from the patient and his family members revealed that the patient has a compound heterozygous mutation of this gene. In 1 allele, an undescribed single nucleotide transition in codon 208 in exon 3 resulted in a substitution of arginine to histidine (CGC to CAC: R208H). In the other allele, a deletion of 3 nucleotides in codons 337-338 in exon 5 resulted in a substitution of arginine to histidine and a deletion of tyrosine residue (CGCTAT to CAT: R337H, delta Y338), which has been previously shown to abolish 11 beta HSD2 enzyme activity. A chloramphenicol acetyltransferase assay-based expression study involving the mineralocorticoid receptor indicated that the novel R208H mutation eliminates the enzymatic activity of 11 beta HSD2. From the genetic analysis of 50 healthy subjects, the novel R208H mutation was unlikely to be due to polymorphism. Together, these results indicate that this patient is a compound heterozygote for the mutation in the 11 beta HSD2 gene (R208H and R337H, delta Y338) and that these mutations inactivate the 11 beta HSD2 function and give rise to clinically manifest AME.
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PMID:A new compound heterozygous mutation in the 11 beta-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess. 939 12

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.
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PMID:Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin. 1040 60

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