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)

1. Variants of chloramphenicol acetyltransferase from a variety of bacterial species have been isolated and purified to homogeneity. They constitute a heterogeneous group of proteins as judged by analytical affinity and hydrophobic ('detergent') chromatography, native and sodium dodecyl sulfate electrophoresis, sensitivity to sulfhydryl specific reagents, steady state kinetic analysis, and reaction with antisera. 2. The most striking observation is that three variants of chloramphenicol acetyltransferase (R factor type III, Streptomyces acrimycini, and Agrobacterium tumefaciens) possess an apparent subunit molecular weight (24,500) which is significantly greater than that of all other variants examined (22,500). The three atypical variants are not identical since they show marked differences in a number of important parameters. 3. Although the fundamental mechanism of catalysis may prove to be identical for all chloramphenicol acetyltransferase variants, there is a wide range of sensitivity to thiol-directed inhibitors among the enzymes studied. 4. Amino acid sequence analysis of the N-termini of selected variants suggests that the qualitative differences among chloramphenicol acetyltransferase variants is a reflection of structural heterogeneity which is most marked in comparisons between variants from Gram-positive and Gram-negative species.
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PMID:Characterization and comparison of chloramphenicol acetyltransferase variants. 11 49

A strain of Pseudomonas aeruginosa, which was resistant to 400 mug/ml of chloramphenicol (CM), was isolated. The generation time of the resistant strain was the same in the presence or absence of CM and similar to that of the parent strain growing in the absence of chloramphenicol. Resistance is eliminated by treatment with acridine dyes, mitomycin C, and sodium dodecyl sulfate, suggesting that resistance may be expressed by a plasmid. The resistant strain does not produce the pigment pyocyanine and the addition of pyocyanine to this strain eliminates the resistance factor. A strain sensitive to CM was isolated. This strain does not produce the enzyme acetyl CoA : chloramphenicol transacetylase whereas the resistant strain does. The sensitive strain accumulates 14C-CM at a greater rate and to a greater extent than the resistant strain grown in the presence of CM. The results suggest that the resistant strain inactivates CM by acetylation and, in.addition, develops a "permeability" barrier towards chloramphenicol.
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PMID:The resistance of Pseudomonas aeruginosa to chloramphenicol. 80 23

The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibiotic susceptibility and uptake, as well as outer membrane protein patterns, were compared between strain 249-2 grown with and without salicylates. The MICs of chloramphenicol, trimethoprim, ciprofloxacin, and ceftazidime were compared between organisms grown in standard and salicylate-containing medium and are as follows: chloramphenicol, 12.5 versus 100 micrograms/ml; trimethoprim, 0.78 versus 3.125 micrograms/ml; ciprofloxacin, 0.4 versus 1.56 micrograms/ml; ceftazidime, 3.125 versus 3.125 micrograms/ml. The permeability of beta-lactam antibiotics was calculated from the rate of hydrolysis of the chromogenic cephalosporin, PADAC. There was no significant difference between strains grown in the presence and absence of salicylate. By using high-pressure liquid chromatography quantitation of loss from culture medium, the effect of 10 mM salicylate on the cellular permeability of chloramphenicol was measured in strain 249-2 by introduction of a plasmid which encodes production of chloramphenicol acetyltransferase. After 1 h of incubation, 18.5% +/- 1.54% versus 70.1% +/- 3.52%, and after 2 h, 4.20% +/- 1.65% versus 41.90% +/- 2.16% remained in supernatants from organisms grown in the absence and presence of 10 mM salicylate, respectively. Outer membrane protein pattern analysis demonstrated the absence of a protein of apparent molecular weight of 40,000 when strain 249-2 was grown in the presence of 10 mM salicylate. To determine whether this protein functioned as a porin, reconstituted membrane vesicles were constructed to assess antibiotic permeability. Vesicles constructed with this salicylate-suppressible outer membrane protein (OpcS) were permeable to chloramphenicol but not to penicillin G. These findings suggest that OpcS is a selective, antibiotic-permeable porin which can be suppressed by growth in the presence of salicylate. Further investigation will be required to determine the biochemical effects of salicylate on porin synthesis.
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PMID:Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein. 128 56

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.
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PMID:Regulation of the Epstein-Barr virus DNA polymerase gene. 131 4

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.
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PMID:Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. 131 49

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.
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PMID:Immortalization of virus-free human placental cells that express tissue-specific functions. 131 3

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail. 159 93

Replication of human cytomegalovirus (HCMV) in a human epithelial thyroid papillary carcinoma cell line (TPC-1) was restricted. However, pretreatment of these cells with 5 mM sodium butyrate (NaB) for 24 hr before infection enhanced both HCMV yield and infectious center titer to a similar level of that seen in human embryonic lung fibroblast cells. Immunofluorescence staining, gel electrophoresis, and Northern blot analysis revealed that TPC-1 cells are nonpermissive for expression of HCMV major immediate early (IE1) functions, but many of the cells become permissive after being treated with NaB. The presence of cycloheximide during NaB pretreatment of the cells efficiently diminished the stimulatory effect of NaB on expression of the IE1 gene. Therefore, it appeared that NaB induces the synthesis of a cellular protein(s) which apparently plays an important role in the conversion of nonpermissive cells to a permissive state for expression of this critical viral gene. Transient chloramphenicol acetyltransferase (CAT) assay experiments indicated that in TPC-1 cells the HCMV-CAT construct which contains the complete IE1 promoter regulatory region was expressed poorly, whereas a high level of CAT activity was detectable in the NaB-treated cells. Therefore, these results suggest that the enhancing effect of NaB on HCMV replication is expressed through some host cellular factor(s), and the HCMV IE1 promoter regulatory region is most likely to be the primary target of NaB action.
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PMID:Sodium butyrate-inducible replication of human cytomegalovirus in a human epithelial cell line. 165 87

Previous studies have suggested that MM creatine kinase is a muscle-specific protein and is not present in adult brain tissue. We have isolated a protein from human brain with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is identical to the muscle M creatine kinase isoenzyme subunit at all 30 sequenced amino acid residues and possesses creatine kinase enzymatic activity following nondenaturing agarose-gel electrophoresis. Immunohistochemistry localizes M creatine kinase to discrete areas of adult human brain. Northern blot analysis of both total and poly(A)-selected RNA isolated from brain did not detect M creatine kinase mRNA. However, polymerase chain reaction amplification of cDNA synthesized from human placenta, heart, and brain mRNA detected M creatine kinase message in both heart and brain but not placenta which contains no detectable M creatine kinase protein. N1E115 and NS20Y, mouse neuroblastoma cell lines which have been used as models of neural cell differentiation, were found also to express MM creatine kinase. Moreover, a transiently transfected reporter gene with 4,800 base pairs of M creatine kinase upstream region fused to chloramphenicol acetyltransferase was expressed during differentiation of these neural cell lines. In summary, MM creatine kinase is present in human brain and we suggest the M creatine kinase upstream region is sufficient to modulate M creatine kinase expression in certain neuronal cells and may be regulated independently from other muscle genes.
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PMID:Muscle creatine kinase isoenzyme expression in adult human brain. 169 Jul 25

Sequence similarity between alpha B-crystallin and small heat shock proteins (HSPs) has prompted us to investigate whether alpha B-crystallin expression is induced by heat shock. Indeed, accumulation of alpha B-crystallin was detected immunologically in NIH 3T3 cells after incubation at elevated temperatures and after addition of Cd2+ or sodium arsenite to these cells. Two-dimensional gel electrophoresis revealed identity between alpha B-crystallin from eye lenses and from heat-treated fibroblasts. The promoter of the alpha B-crystallin gene was fused to the bacterial chloramphenicol acetyltransferase gene and was shown to confer heat inducibility on this reporter gene in transient transfection assays. A perfect heat shock element within the promoter region is likely to mediate this response. Small HSPs and alpha B-crystallin were shown to share the following two physical properties: (i) they form supramolecular structures with sedimentation values around 17 S and (ii) they are associated with the nucleus at high temperatures and are localized in the cytoplasm under normal conditions. We conclude that alpha B-crystallin has to be considered a member of the class of small HSPs.
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PMID:Alpha B-crystallin is a small heat shock protein. 202 14


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