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)

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

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.
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PMID:Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity. 972 39

Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 microm NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at approximately 300 microm. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.
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PMID:Nitric oxide donors inhibit luciferase expression in a promoter-independent fashion. 1252 97