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 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 encoding PTH-related peptide (PTHrP) is expressed in a wide variety of normal and neoplastic tissues. Increased PTHrP gene expression in and secretion of PTHrP by specific tumors directly contributes to the development of malignancy-associated hypercalcemia in vivo. To define the genetic elements important for the control of PTHrP gene transcription, we used the reverse transcription polymerase chain reaction to delineate the control of promoter utilization and the splicing patterns of the exons encoding 5'-untranslated sequences. The majority of normal and neoplastic human tissues contained PTHrP mRNA transcripts initiating from both the up-stream (P1) and down-stream (P2) human PTHrP promoters. Furthermore, the downstream promoter was preferentially used by a factor of more than 30-fold. P1-initiated transcripts contained RNA species both with and without exon 2 (E2) sequences, except in the pancreas, adrenal, and stomach, where E2-containing sequences predominated. The transcriptional activities of P1, P2, and P1 + P2 were assessed by transfection of the corresponding PTHrP-chloramphenicol acetyltransferase (CAT) fusion genes into heterologous cell lines. Fusion genes containing P2 sequences were more transcriptionally active than fusion genes containing P1 sequences. The transcriptional activities of P1 + P2 in their natural tandem orientation were additive in rat keratinocytes and human JEG choriocarcinoma cells. In contrast, the activity of P1 + P2 was less than that of P2 alone in hamster BHK fibroblasts and InR1-G9 cells, and human HeLa cells. Analysis of the transcriptional properties of 5'-deleted human PTHrP-CAT constructs revealed the presence of multiple positive and negative DNA sequences (within both P1 and P2) functionally important for human PTHrP gene transcription. Distinct positive and negative DNA elements were also identified from analysis of 5'-deleted rat PTHrP-CAT fusion genes. The results of these experiments provide evidence for cell- and tissue-specific utilization of 1) distinct human PTHrP transcription start sites and specific patterns of 5'-exon splicing and 2) multiple positive and negative DNA control elements, important for the regulation of human and rat PTHrP gene transcription.
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PMID:Regulation of parathyroid hormone-related peptide (PTHrP) gene transcription: cell- and tissue-specific promoter utilization mediated by multiple positive and negative cis-acting DNA elements. 128 Mar 27

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.
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PMID:Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements. 128 Mar 29

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.
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PMID:Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells. 128 Apr 22

The vascular cell adhesion molecule 1 (VCAM-1) is a 110-kD member of the immunoglobulin gene superfamily expressed on the surface of interleukin 1 beta- or tumor necrosis factor alpha (TNF)-stimulated endothelial cells. The cell surface protein functions as an inducible adhesion receptor for circulating mononuclear leukocytes and some tumor cells. We have previously characterized the genomic organization of the VCAM1 gene and described its chromosomal localization. In this report, the promoter of the VCAM1 gene is characterized. New transcription of the VCAM1 gene occurred when endothelial cells were treated with TNF. Fusion plasmids containing the 5' flanking sequence of the VCAM1 gene and the chloramphenicol acetyltransferase reporter gene were used to identify cis-acting sequences that direct the cytokine-induced transcription. When transfected into bovine aortic endothelial cells, constructs containing 755 bp of the 5' flanking sequence were induced by TNF. Within the cytokine-responsive region of the core promoter were functional NF-kappa B and GATA elements. Upstream of the core promoter, the VCAM1 5' flanking sequence contained a negative regulatory activity. NF-kappa B-mediated activation of VCAM1 gene expression may lead to endothelial expression of a mononuclear leukocyte adhesion molecule associated with initial events in the development of an atherosclerotic lesion.
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PMID:Functional analysis of the human vascular cell adhesion molecule 1 promoter. 128 Dec 11

Cytokine modulation of elastin gene expression was examined by assay of elastin mRNA abundance and by transient transfections of cultured human skin fibroblasts and rat aortic smooth muscle cells with elastin promoter/reporter gene (chloramphenicol acetyltransferase, CAT) constructs. Incubation of cells with human recombinant tumor necrosis factor-alpha (TNF-alpha) markedly suppressed the elastin mRNA levels in a time- and dose-dependent manner by up to 91%. TNF-alpha also suppressed the expression of the elastin promoter/CAT construct by up to 70% in transiently transfected cells, indicating regulation at the transcriptional level. This suppression was temporally preceded by rapid and transient up-regulation of c-jun and c-fos genes. The down-regulatory effect of TNF-alpha on elastin promoter activity was abolished by co-transfections with a synthetic double-stranded AP-1 oligomer. Furthermore, co-transfection of the elastin promoter construct with c-jun and c-fos expression plasmids resulted in a marked decrease in the promoter activity. Elucidation of the cis-regulatory elements in the elastin promoter by 5' deletion construct analysis implicated a region -290 to -198 containing one AP-1 binding site. The functional role of this AP-1 site was further tested by gel retardation assays which indicated formation of a DNA-protein complex specific for TNF-alpha treated cells. This complex could be partially dissociated by a competing oligomer containing the consensus AP-1 binding site. These observations suggest that the inhibitory effects of TNF-alpha on elastin gene expression involve the transcription factor AP-1. Interferon-gamma also suppressed the elastin gene expression at the mRNA level by approximately 52%, but it had no effect on the elastin promoter activity, suggesting post-transcriptional mechanisms. These results indicate that mediators released from inflammatory cells can modulate elastin gene expression, and such modulation may play a role in diseases characterized by altered accumulation of elastic fibers in tissues.
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PMID:Tumor necrosis factor-alpha down-regulates human elastin gene expression. Evidence for the role of AP-1 in the suppression of promoter activity. 128 83

Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(L-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 microM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 microM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.
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PMID:Targeted delivery of antisense oligonucleotides by molecular conjugates. 128 54

We previously used mice bearing a myosin light chain-chloramphenicol acetyltransferase (MLC1-CAT) transgene to show that adult muscle cells bear a heritable, cell autonomous memory of their rostrocaudal position. CAT mRNA and protein are expressed in a > 100-fold rostrocaudal gradient in skeletal muscles of developing and adult MLC1-CAT mice (Donoghue, M. J., Merlie, J. P., Rosenthal, N. and Sanes, J. R. (1991). Proc. Natl. Acad. Sci. USA 88, 5847-5851; Donoghue, M. J., Alvarez, J. D., Merlie, J. P. and Sanes, J. R. (1991). J. Cell Biol. 115, 423-434). Moreover, both in primary cultures and in myogenic cell lines prepared from individual muscles of these mice, CAT levels reflect the body position from which the myoblasts were derived (Donoghue, M.J., Morris-Valero, R., Johnson, Y.R., Merlie, J.P. and Sanes, J. R. (1992). Cell 69, 67-77). Here, we show that the methylation state of the MLC1-CAT transgene in skeletal muscles is also graded along the rostrocaudal axis: methylation levels decrease and expression levels increase in the order, jaw-->neck-->chest and forelimb-->hindlimb. Methylation levels are also approx. 10-fold higher in rostrally derived than in caudally derived myogenic cell lines, which express low and high levels of CAT, respectively. Within each cell line, undifferentiated cells (myoblasts), which do not express the transgene, and differentiated cells (myotubes), which do, are indistinguishable in methylation state. Thus, differentiation-related changes in transgene expression do not affect position-related levels of transgene methylation. On the other hand, treatment of rostrally derived lines with the demethylating agent, 5-azacytidine, decreases methylation and increases expression of the transgene. Thus, perturbation of methylation affects expression. Taken together, these results suggest that methylation provides a genomic imprint of rostrocaudal body position that may serve as a component of the positional memory that mammalian cells retain into adulthood.
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PMID:An axial gradient of transgene methylation in murine skeletal muscle: genomic imprint of rostrocaudal position. 129 32

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.
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PMID:Functional analysis of the promoter of a sea urchin metallothionein gene. 129 38

The ability of cis-urocanic acid to mimic the effects of ultraviolet (UV) was tested in two systems that show cellular responses to direct UV irradiation. First, cultured mouse keratinocytes were treated with cis-urocanic acid and the cell culture supernatants were injected into mice to test for the ability to block spleen cell proliferation in the mixed lymphocyte reaction. Second, human fibroblasts carrying the chloramphenicol acetyltransferase gene under the control of the human immunodeficiency virus (HIV) long terminal repeat promoter were tested for the ability of cis-urocanic acid to induce gene expression. In these tests cis-urocanic acid did not induce a response comparable with UV irradiation alone, although at toxic concentrations cis-UCA did induce low expression from the viral promoter. These data suggest that, unlike UVB radiation, cis-urocanic acid does not activate keratinocytes to produce immunosuppressive cytokines nor does it induce expression of the HIV promoter.
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PMID:Urocanic acid isomers, immunosuppressive cytokines and the induction of human immunodeficiency virus. 130 Jan 41


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