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 chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.
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PMID:Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis. 1 97

The two 4.6 kb chloramphenicol resistance (CmR) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed Mr values of 70,000 on FPLC and were composed of three identical subunits, each of Mr approximately 23,000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The Km values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5 microM and 58.8 microM, respectively, while those of the CAT from pSCS7 were 2.7 microM and 55.5 microM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.
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PMID:Characterization of the chloramphenicol acetyltransferase variants encoded by the plasmids pSCS6 and pSCS7 from Staphylococcus aureus. 156 39

A small chloramphenicol resistance (Cmr) plasmid of approximately 3.75 kb, designated pSCS5, was isolated from Staphylococcus haemolyticus. This plasmid encoded an inducible chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). The cat gene of pSCS5 was cloned into the Escherichia coli plasmid vector pBluescript SKII+. It differed in its nucleotide sequence and deduced amino acid sequence from the cat genes described previously in staphylococci and other gram-positive bacteria. The CAT enzyme was purified from cell-free lysates by ammonium sulfate precipitation, ion-exchange chromatography, and fast protein liquid chromatography. The native enzyme had an Mr of 70,000 and was composed of three identical subunits, each with an Mr of approximately 23,000. Its isoelectric point was at pH 6.15. CAT from pSCS5 exhibited Km values of 2.81 and 51.8 microM for chloramphenicol and acetyl coenzyme A, respectively. The optimum pH for activity was 7.8. CAT encoded by pSCS5 proved to be relatively heat stable, but sensitive to mercury ions. The observed differences in the nucleotide sequence and the biochemical characteristics of the enzyme allowed the identification of the pSCS5-encoded CAT from S. haemolyticus as a CAT variant different from those described previously in gram-positive bacteria.
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PMID:Molecular cloning, purification, and properties of a plasmid-encoded chloramphenicol acetyltransferase from Staphylococcus haemolyticus. 192 82

Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.
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PMID:Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells. 279 44

From the highly chloramphenicol-resistant cytophaga-like bacterium Flavobacterium CB60, which can both acetylate chloramphenicol and degrade it in co-metabolism, the chloramphenicol acetyltransferase (CAT) was purified to homogeneity and characterized. The purification included fractional precipitation with ammonium sulphate and two affinity chromatography steps, eluting CAT the first time with 5 mM-chloramphenicol and the second time with a linear gradient (0-10 mM) of chloramphenicol. The purification was 3979-fold. Properties of this CAT were investigated and compared with CATs from other bacteria. Although CAT from Flavobacterium CB60 shares some properties with the enzymes from Escherichia coli and other Gram-negative bacteria--especially with CATII and CATIII--it has distinct properties like extreme heat lability and the inability to produce diacetylchloramphenicol, so that it might be regarded as a new variant.
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PMID:Purification and characterization of chloramphenicol acetyltransferase from Flavobacterium CB60. 332 15

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.
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PMID:Cyclic AMP signaling pathways are important in IL-1 beta transcriptional regulation. 759 50

Haemophilus influenzae can utilize iron-loaded human transferrin as an iron source for growth in vitro. H. influenzae tonB mutants, containing a chloramphenicol acetyltransferase gene within their tonB genes, could bind iron-charged human transferrin to their cell surfaces, but they were unable to utilize this serum glycoprotein as the sole source of iron for growth in vitro. In contrast, these tonB mutants were able to utilize an iron chelate (ferric ammonium citrate) for growth. Transformation of a tonB mutant with a plasmid encoding a wild-type H. influenzae tonB gene restored the ability of a tonB mutant to utilize iron-charged human transferrin. These results indicate that the uptake of iron from human transferrin by H. influenzae is a TonB-dependent process.
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PMID:Utilization of transferrin-bound iron by Haemophilus influenzae requires an intact tonB gene. 782 47

A cationic peptide amphiphile comprising an L-alanine residue interposed between a charged head group and a double-chain segment, N,N-dihexadecyl-N alpha-[6-(trimethylammonio)- hexanoyl]-L-alaninamide bromide (NC5Ala2C16), was synthesized and used to prepare sonicated liposomes. We examined the efficiency of this liposome in gene transfer according to the transient expression of chloramphenicol acetyltransferase (CAT). This cationic liposome reagent facilitates efficient DNA transfection in COS-7 cells. We determined the optimum conditions for NC5Ala2C16 liposome-mediated transfection. The optimal amounts of the amphiphile and plasmid DNA were determined to be about 100 micrograms and 10 micrograms per 35-mm dish, respectively. The activity of this liposome was greater than that of commercial reagents, lipofectin, and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and it was less toxic than lipofectin and DOTAP in COS-7 cells.
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PMID:Synthetic cationic amphiphile for liposome-mediated DNA transfection with less cytotoxicity. 879 87

We have demonstrated that tracheal insufflation of recombinant plasmid DNA results in transfection of rat lungs to the same extent as insufflation of plasmid-cationic liposome complex. To understand this observation better, we investigated the in vitro gene transfer of plasmid DNA in the presence and absence of cationic liposome and the effect of surfactant on gene transfer. The chloramphenicol acetyltransferase (CAT) expression plasmids pBL-CAT and pSV-CAT were studied in three cell types: rat fetal lung fibroblast (RFL-6), calf pulmonary artery endothelial cell (CPAE), and rat type II alveolar epithelial cell (type II AE). Three cationic liposomes were tested: DDAB (dimethyl-dioctadecyl ammonium bromide)-liposome, DOTAP (dioleoyltrimethyl ammonium propane)-liposome, and lipofectin. The results revealed that (i) plasmid DNA alone caused a dose-dependent, low-level transfection, most efficiently in RFL-6 followed by CPAE and type II AE, (ii) DDAB-liposome markedly enhanced gene transfer, most efficiently in RFL-6 followed by CPAE and type II AE, (iii) Survanta, a naturally derived surfactant preparation, and Exosurf, a synthetic surfactant, while having no effect on in vitro gene transfer by plasmid DNA alone, markedly inhibited cationic liposome-mediated gene transfer, (iv) dipalmitoyl phosphatidylcholine was responsible for the inhibitory effect of Exosurf, and (v) inhibition of cationic liposome-mediated gene transfer by Exosurf was not due to inhibition of plasmid DNA-cationic liposome complex uptake or interference with the promoter and enhancer. The observed inhibition of cationic liposome-mediated gene transfer by surfactant may in part explain our previous observation that tracheal insufflation of plasmid DNA and plasmid-cationic liposome complex results in equal lung gene transfer.
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PMID:Surfactant inhibits cationic liposome-mediated gene transfer. 914 7

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with chloramphenicol acetyltransferase in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival.
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PMID:Role of urease in megasome formation and Helicobacter pylori survival in macrophages. 1654 3


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