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)

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.
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PMID:In vitro inhibition of carnitine acyltransferase activity in mitochondria from rat and mouse liver by a diethylhexylphthalate metabolite. 845 57

The promoter region driving the gene for the 170-kDa heavy subunit of the Entamoeba histolytica galactose-inhibitable lectin was analysed by transient transfection using the chloramphenicol acetyltransferase gene as reporter. S1 mapping confirmed our previous notion that the promoter is located within a 1.35-kb intergenic sequence preceding the structural lectin gene. Transcripts derived from the chloramphenicol acetyltransferase gene of transfected trophozoites were found to be polyadenylated and the transcriptional start mapped to a position similar to that of the wild-type lectin gene. By deletion analysis the entire promoter was restricted to a fragment covering about 550 bp upstream from the start of transcription. On the other hand, residual promoter activity required a sequence of about 140 bp only, encompassing a newly identified CCAAT-box like element around position -100, as well as the amebic specific TATA-box. This 140-bp fragment as well as a stretch of 15 bp, which is located some 100 nt further upstream, were found to be conserved within the 5' noncoding region of a second E. histolytica lectin gene. Point-mutation analyses indicated that the 15-bp fragment, the likely CCAAT-box, as well as the TATA-box are required for full promoter activity.
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PMID:Analysis of the 170-kDa lectin gene promoter of Entamoeba histolytica. 853 79

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme. No effect on glycolate metabolism was detected by insertional mutation in glcG. Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript.
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PMID:glc locus of Escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein. 860 83

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.
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PMID:In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae. 931 63

An Escherichia coli DNA fragment was identified that contained part of the beta-glucoside (bgl) operon. This fragment was identified because it contained a promoter that was responsible for the expression of a reporter gene, the chloramphenicol acetyltransferase gene, in a mouse liver during bacterial infection but not when a bacterial clone was grown in vitro. This fragment contained a promoter and a rho-independent transcription terminator which were flanked by the 3' end of bglG and the 5' end of bglF. Reverse transcription-PCR confirmed that cat-specific mRNA was produced in infected mouse liver but not in vitro. mRNA encoding the positive regulator of the bgl operon, bglG, also was detected in mouse liver infected with an E. coli strain. These results demonstrated that expression of the bgl operon occurs in infected mouse liver and suggests a unique role for this operon in vivo.
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PMID:In vivo expression of the beta-glucoside (bgl) operon of Escherichia coli occurs in mouse liver. 972 21

Galactose was introduced to poly(L-lysine) (PLL) with an average molecular weight of 13,000 to develop a hepatocyte-specific carrier for gene drugs. The pharmacokinetic characteristics of a model plasmid, pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene), complexed with galactosylated PLL (Gal-PLL) was studied in mice in relation to its physicochemical properties. pCAT/Gal-PLL complex at a ratio of 1:0.6 (w/w) has a zeta potential of -20 mV and a mean particle size of about 180 nm. After intravenous injection, [32P]pCAT/Gal-PLL was rapidly eliminated from the circulation and preferentially taken up by the liver's parenchymal cells. The hepatic uptake of [32P]pCAT/Gal-PLL was significantly inhibited by prior administration of Gal-bovine serum albumin, suggesting that the uptake was mediated by the asialoglycoprotein receptors on hepatocytes. In vitro transfection experiments using a hepatoma cell line expressing the asialoglycoprotein receptor revealed that pCAT/Gal-PLL gave a high CAT gene expression whereas pCAT complexed with unmodified PLL failed to transfect the cells.
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PMID:Targeted delivery of plasmid DNA complexed with galactosylated poly(L-lysine). 974 38

In vivo receptor-mediated targeting of plasmid DNA to hepatocytes was achieved through optimizing the physicochemical and pharmacokinetic properties of a plasmid DNA/carrier complex. Galactosylated poly(L-lysine) (Gal-PLL) was synthesized using PLL with a molecular weight of 1,800, 13,000 or 29,000 without loss of the cationic charge. Plasmid DNA encoding chloramphenicol acetyltransferase was complexed with each Gal-PLL. A larger amount of PLL1800 is required for the complex formation than with PLL13000 and PLL29000, and increasing the number of galactose units on Gal-PLL resulted in reduced binding to plasmid DNA. The particle size and zeta-potential of the complexes varied depending on the mixing ratio and Gal-PLL used. Then, plasmid DNA/Gal-PLL complexes having diameters of 200 nm or less and a weak negative charge were prepared. After i.v. injection of [32P]plasmid DNA/Gal13-PLL13000 and [32P]plasmid DNA/Gal26-PLL29000, almost 80% of the radioactivity rapidly accumulated in the liver, preferentially in the parenchymal cells. The hepatic uptake clearances (CLliver) were much greater than any of the other tissue uptake clearances. Compared with these complexes, [32P]plasmid DNA/Gal5-PLL1800 and [32P]plasmid DNA/Gal5-PLL13000 had a smaller CLliver, suggesting that both the molecular weight of PLL and the degree of galactose modification determine the hepatic targeting of plasmid DNA. In vitro and in vivo gene expression studies revealed that plasmid DNA/Gal13-PLL13000 and plasmid DNA/Gal26-PLL29000 complexes are superior to plasmid DNA/Gal5-PLL1800 complex for introducing DNA into cells. These results demonstrated that an optimal design of a DNA/carrier complex based on physicochemical properties and a pharmacokinetic analysis of the distribution properties leads to successful in vivo gene delivery.
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PMID:Targeted delivery of plasmid DNA to hepatocytes in vivo: optimization of the pharmacokinetics of plasmid DNA/galactosylated poly(L-lysine) complexes by controlling their physicochemical properties. 976 63

The versatile plant acyltransferase (VPAT) family is a recently identified protein family consisting of acyltransferases involved in secondary metabolism in plants along with numerous homologues with as yet unidentified biochemical functions. Malonyl-CoA:anthocyanin 5-O-glucoside-6' "-O-malonyltransferase of Salvia splendens flowers (Ss5MaT1) is a member of this family that catalyzes the regiospecific transfer of the malonyl group from malonyl-CoA to the 6' "-hydroxyl group of the 5-glycosyl moiety of anthocyanins. To elucidate the mechanism and functional amino acid residues of VPAT family enzymes, steady-state kinetic analyses and site-directed mutagenesis of Ss5MaT1 guided by sequence comparison studies were carried out. On the basis of the results of product and dead-end inhibition studies as well as sequence comparison studies, the kinetic mechanism of Ss5MaT1 could be most consistently described in terms of a ternary complex mechanism in which both substrates and the enzyme form a complex before catalysis can occur, as in the case of chloramphenicol O-acetyltransferase (CAT) and histone acetyltransferase (HAT). Eight polar or ionizable amino acid residues that are invariant among 12 VPAT family enzymes were replaced by alanine, and the mutant enzymes were kinetically characterized. A significant diminution of the k(cat) value was observed with the substitution of His167 (relative k(cat), 0.02%) and Asp390 (<0.01%), strongly suggesting that His167 and Asp390 are very important for catalytic activity. The log k(cat) versus pH plots of the Ss5MaT1-catalyzed malonyl transfer suggested that a deprotonated active site group of pK(a) = 7.0 +/- 0.1 may be involved in the catalytic steps of the "substrate to product" conversion in the ternary enzyme-substrate complex. Taking these lines of evidence together with the suggested similarity of the kinetic and catalytic mechanisms of Ss5MaT1 to those of CAT and HAT, the following Ss5MaT1 mechanism based on general acid/base catalysis was proposed: in the ternary complex, a general base deprotonates the 6' "-hydroxyl group of the anthocyanin substrate, thereby promoting a nucleophilic attack on the carbonyl of the thioester of malonyl-CoA; His167 and Asp390 appear to be involved in the general acid/base mechanism of Ss5MaT1.
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PMID:Proposed mechanism and functional amino acid residues of malonyl-CoA:anthocyanin 5-O-glucoside-6'''-O-malonyltransferase from flowers of Salvia splendens, a member of the versatile plant acyltransferase family. 1257 91

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.
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PMID:Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system. 1290 69

When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. Although the Escherichia coli lacZ gene, encoding beta-galactosidase (beta-gal), can be used as a standard reporter for monitoring the strength of a promoter or enhancer in a transient or stable transfection assay, it is predominantly used as an internal control during transient transfection experiments. When used in this manner, cells are usually transfected with the control plasmid (containing a ubiquitously active viral promoter fused to the E. coli lacZ gene) and an experimental plasmid containing another reporter gene (e.g., luciferase or chloramphenicol acetyltransferase [CAT]) under the control of the promoter or enhancer of interest. The basic colorimetric assay described here is the simplest and least expensive assay for quantifying beta-gal activity. The cells are lysed and, after determining the total protein concentration in the extracts, an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-beta-D-galactopyranoside (ONPG), in a buffer containing sodium phosphate and magnesium chloride. When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.
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PMID:Beta-galactosidase assay. 2043 10


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