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)

Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators (extinguishers) that prevent the expression of functions foreign to their own differentiation. Here, we report genetic evidence of such control. In microcell hybrids between well-differentiated rat hepatoma cells and microcells of mouse fibroblast L cells, the extinction of albumin synthesis is directly related to the presence of a single specific chromosome of the mouse fibroblast parent. The expression of several other hepatic functions is not affected. Transfection of these hybrids with a recombinant plasmid, containing a tissue-specific control element of the upstream region of the rat albumin gene linked to coding sequences of the chloramphenicol acetyltransferase gene, reveals that extinction acts on or via this cis-control element.
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PMID:Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids. 345 17

ATBF1 is a 306-kDa protein containing four homeodomains, 17 zinc finger motifs, and several segments potentially involved in transcriptional regulation (T. Morinaga, H. Yasuda, T. Hashimoto, K. Higashio, and T. Tamaoki, Mol. Cell. Biol. 11:6041-6049, 1991). At least one of the homeodomains of ATBF1 binds to an AT-rich element in the human alpha-fetoprotein (AFP) enhancer (enhancer AT motif). In the present work, we analyzed the transcriptional regulatory activity of ATBF1 with respect to the enhancer AT motif and similar AT-rich elements in the human AFP promoter and the human albumin promoter and enhancer. Gel retardation assays showed that ATBF1 binds to the AFP enhancer AT motif efficiently; however, it binds weakly or not at all to other AT-rich elements in the AFP and albumin regulatory regions studied. Alterations of the enhancer AT motif by site-specific mutagenesis resulted in the loss of binding of ATBF1. Cotransfection experiments with an ATBF1 expression plasmid and the chloramphenicol acetyltransferase (CAT) gene fused to AFP promoter or enhancer fragments showed that ATBF1 suppressed the activity of AFP enhancer and promoter regions containing AT-rich elements. This suppression was reduced when the mutated AT motifs with low affinity to ATBF1 were linked to the CAT gene. The ATBF1 suppression of AFP promoter and enhancer activities appeared to be due, at least in part, to competition between ATBF1 and HNF1 for the same binding site. In contrast to the AFP promoter and enhancer, the albumin promoter and enhancer were not affected by ATBF1, although they contain homologous AT-rich elements. These results show that ATBF1 is able to distinguish AFP and albumin AT-rich elements, leading to selective suppression of the AFP promoter and enhancer activities.
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PMID:ATBF1, a multiple-homeodomain zinc finger protein, selectively down-regulates AT-rich elements of the human alpha-fetoprotein gene. 750 6

In McA-RH 8994 rat hepatoma cells, all-trans-retinoic acid (t-RA) induces expression of the alpha-fetoprotein (AFP) and albumin genes and results in a phenotype similar to differentiated fetal hepatocytes. The present study elucidated the mechanism involved in AFP gene regulation mediated by retinoic acid. Northern blot analyses demonstrated that 9-cis-retinoic acid (c-RA), a ligand for retinoid x receptors (RXRs), also induced expression of the AFP gene in McA-RH 8994 cells. The induction was time- and dose-dependent. Northern blots and transfection assays using the 7.3 kb full-length regulatory region of the AFP gene demonstrated that c-RA was more effective than t-RA in regulating expression of the AFP gene. At 10(-7) M, c-RA increased AFP mRNA 5-fold and chloramphenicol acetyltransferase (CAT) activity 2.5-fold. In contrast, t-RA at a concentration of 10(-7) M exerted no significant effect; 10(-6) to 10(-5) M t-RA was needed to affect AFP gene expression. These data suggested that activation of RXRs is essential for the regulation of the AFP gene. Co-transfection experiments revealed that over-expression of RXR alpha in McA-RH 8994 cells further enhanced the CAT activity induced by c-RA. In addition, c-RA did not alter the half-life of AFP mRNA. Thus, RXR alpha may play a crucial role in transcriptional regulation of the AFP gene and in controlling hepatocyte phenotype.
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PMID:9-cis-retinoic acid is more effective than all-trans-retinoic acid in upregulating expression of the alpha-fetoprotein gene. 753 13

Insulin-like growth factor binding protein-1 (IGFBP-1) is expressed primarily in the liver, kidney, and uterus. Basal IGFBP-1 promoter activity in human HEP G2 hepatoma cells is dependent upon a proximal promoter element that binds hepatic nuclear factor 1 (HNF1), a protein that is likely to be an important factor regulating the expression of many genes in liver and kidney. To test whether HNF1 activates IGFBP-1 transcription, HEP G2 cells and HeLa cells were cotransfected transiently with HNF1 expression vectors and with IGFBP-1 promoter/chloramphenicol acetyltransferase reporter gene constructs. HNF1 increased IGFBP-1 promoter activity in both HEP G2 and HeLa cells. Gel mobility-shift assays and additional transfections in HeLa cells showed that expressed full-length and carboxy-terminal truncated forms of HNF1 could each bind the HNF1 cis element of the IGFBP-1 promoter; however, significant trans-activation only occurred in the presence of the full-length HNF1 protein, similar to past experience with these two HNF1 forms and the albumin promoter. Further studies showed that IGFBP-1 promoter constructs containing mutations with high or low affinity for HNF1 responded to HNF1 expression with increased or decreased activity, respectively, relative to the native promoter. These studies suggest that HNF1 and/or related proteins play a role in hepatic, and perhaps also renal, expression of IGFBP-1.
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PMID:HNF1 activates transcription of the human gene for insulin-like growth factor binding protein-1. 768 29

Asialoglycoproteins (ASG) are internalized by hepatocytes by ASG receptor (ASGR)-mediated endocytosis. We have shown previously that when a plasmid DNA, pAlb(9-12)CAT (expressing chloramphenicol acetyltransferase driven by an albumin promoter enhancer), was complexed with an ASG-polylysine conjugate and injected intravenously in rats, 80% of the DNA was internalized by the liver. In normal recipient rats, over 95% of the internalized DNA was degraded in 4 h; the plasmid was undetectable after 48 h. In contrast, when 66% hepatectomy was performed 20 min after DNA administration, the internalized DNA persisted for several weeks in cytoplasmic vesicles (Chowdhury, N. R., Wu, C. H., Wu, B. Y., Yerneni, P. C., Bommineni, V. R., and Chowdhury, J. R. (1993) J. Biol. Chem. 268, 11265-11271). Since microtubules are required for the translocation of ligand-containing endosomes to lysosomes, the site of ligand degradation, we hypothesized that persistence of the endocytosed DNA might be related to changes in microtubular structure and function. To test this hypothesis, we examined hepatocellular microtubules by immunofluorescence confocal microscopy. Liver from untreated rats or sham-operated controls showed a network of fibrillar microtubules throughout the cytoplasm. The extent of the microtubular network was substantially reduced 3-6 h after 66% hepatectomy. By 24 h, microtubules had regenerated. Intraportal infusion of cycloheximide (250 mg/kg body weight) 15 min before 66% hepatectomy, prevented microtubular disruption, indicating that protein synthesis is required for this process. Immunotransblot analysis showed that hepatic alpha-tubulin concentration remained unchanged through microtubular disassembly and subsequent reassembly, which is consistent with conservation and reutilization of tubulin released by depolymerization of microtubules.
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PMID:Depolymerization of hepatocellular microtubules after partial hepatectomy. 792 9

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.
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PMID:Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors. 793 27

We have developed a novel, highly efficient DNA delivery system to accomplish gene transfer through the asialoglycoprotein receptor-mediated endocytosis pathway. Natural nuclear DNA-binding proteins, the histones (H1, H2a, H2b, H3, and H4), were modified and used as receptor-targeted DNA carriers. Galactosylated with a coupling agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the histones and albumin were conjugated to DNA and then used to transfect HepG2 cells, which display the asialoglycoprotein receptor. The extent of galactosylation was determined for all histone subgroups and albumin with 14C-labeled galactose. A reporter gene for the bacterial chloramphenicol acetyltransferase (CAT), under the control of the 5' long terminal repeat (LTR) of Rous sarcoma virus, was used for comparisons of transfection efficiency of various carrier proteins. The CAT activity resulting from histone H1-mediated transfection was 1.66 unit per 10(6) cells, the highest among histone subgroups. The galactyosylated histone H1 was also eleven times more effective than the asialo-orosomucoid-polylysine. Ten galactosyl units are attached to histone H1 by the galactosylation reaction. Differences in the extent of galactosylation could not explain different transfection efficiencies among various proteins studied in this report. Treatment with galactose oxidase abolished the transfection ability of both the galactosylated histone H1 and asialo-orosomucoid. The intrinsic DNA-binding domains and nuclear location signal sequences are unique to histones as receptor-targeted DNA carriers, and are advantageous for effective gene delivery.
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PMID:Galactosylated histone-mediated gene transfer and expression. 804 1

Serum colloid osmotic pressure is believed to control the hepatic output of plasma proteins, including albumin. The present study was aimed at identifying the molecular basis for feedback control of albumin gene expression in highly differentiated hepatoma cells. The steady-state level of albumin mRNA and the activity of a 282-bp albumin promoter-chloramphenicol acetyltransferase reporter gene in cells incubated in the presence of increasing amounts of serum albumin or dextran were significantly and selectively decreased. When nuclear extracts from cells exposed to 5% (wt/vol) serum albumin were tested in a gel-retardation assay with six oligonucleotide probes containing DNA elements of the albumin promoter, only the element B-retarded band, which contains the nucleotide recognition sequence for hepatocyte nuclear transcription factor 1 alpha (HNF-1 alpha), was consistently decreased as compared to nuclear extract from cells not exposed to serum albumin. Moreover, the activity of a reporter gene with a basal TATA-promoter driven by multiple HNF-1 alpha recognition elements was selectively inhibited in cells incubated in the presence of 5% serum albumin. A reduction of HNF-1 alpha mRNA appears to be responsible for this response to a change in the level of macromolecules in the incubation medium. These results indicate that activity of a dominant liver transcription factor, HNF-1 alpha, controlling the transcription of several liver-specific genes, is modulated by a fluctuation in the level of oncotically active macromolecules.
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PMID:Homeostatic regulation of hepatocyte nuclear transcription factor 1 expression in cultured hepatoma cells. 827 61

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a critical role in adipose tissue glyceroneogenesis. We have previously shown that transcription of the PEPCK gene was stimulated by isoprenaline and retinoic acid in 3T3-F442A adipocytes. We also showed that oleate increased PEPCK mRNA. Here, we analysed the effect that fatty acids of various chain lengths and unsaturation degrees exerted on PEPCK gene expression in 3T3-F442A adipocytes. When maintained in serum-free, glucose-free medium, differentiated cells responded to unsaturated long-chain fatty acids by a large increase in PEPCK mRNA whereas saturated fatty acids were inefficient. A maximum fivefold stimulation by oleate was attained at 4 h of treatment with 1 mM fatty acid bound to albumin in a 6:1 ratio. The poly-unsaturated very long-chain fatty acid all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) was even more potent and produced a tenfold increase. The expression of the genes encoding glycerol-3-phosphate dehydrogenase, hormone-sensitive lipase or actin remained unaffected by oleate exposure. A 4-h treatment by the hypolipidemic drug clofibrate, 0.5-2 mM, also produced a large (3-9-fold) increase in PEPCK mRNA. When used at non-saturating concentrations, oleate and clofibrate acted in an additive manner. At maximally effective concentrations, additivity was lost, suggesting that fatty acids and fibrates might act through similar mechanisms. Nuclear transcription experiments showed that oleate and clofibrate stimulated the transcription rate of the gene. 3T3-F442A cells were stably transfected with a plasmid containing the base pairs -2100 to +69 of the PEPCK gene promoter fused to the chloramphenicol acetyltransferase gene. These differentiated stable transfectants responded to oleate and clofibrate by a specific increase in chloramphenicol acetyltransferase activity. Adipocytes express various isoforms of peroxisome-proliferator-activated receptors that can be activated by fibrates and fatty acids. Potential recognition sequences for peroxisome-proliferator-activated receptors are present in the -2100 to +69 fragment of the PEPCK gene promoter. Thus, this gene represents an ideal molecular target for understanding the complex transcriptional control exerted by fatty acids and peroxisome proliferators.
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PMID:Fatty acids and fibrates are potent inducers of transcription of the phosphenolpyruvate carboxykinase gene in adipocytes. 853 80

Peroxisome proliferators induce stearoyl-CoA desaturase activity (EC 1.14.99.5) in liver [Kawashima, Y., Hanioka, N., Matsumura, M. & Kozuka, H. (1983) Biochim. Biophys. Acta 752, 259-264]. We analyzed the changes in stearoyl-CoA desaturase 1 (SCD1) mRNA to further define the molecular mechanism for the induction of stearoyl-CoA desaturase by peroxisome proliferators. SCD1 mRNA was analyzed from the livers of BALB/c mice that had been fed diets supplemented with clofibrate or gemfibrozil. Clofibrate was found to induce liver SCD1 mRNA levels 3-fold within 6 hr to a maximum of 22-fold in 30 hr. Gemfibrozil administration resulted in a similar induction pattern. This induction is primarily due to an increase in transcription of the SCD1 gene, as shown by nuclear run-on transcription assays and DNA deletion analysis of transfected SCD1-chloramphenicol acetyltransferase fusion genes. The cis-linked response element for peroxisome proliferator-activated receptor (PPAR) was localized to an AGGTCA consensus sequence between base pairs -664 to -642 of the SCD1 promoter. Clofibrate-mediated induction of SCD1 mRNA was shown to be independent of polyunsaturated fatty acids, with peroxisome proliferators and arachidonic acid having opposite effects on SCD1 mRNA levels. Additionally, the activation of SCD1 mRNA by clofibrate was inhibited 77% by cycloheximide administration. Levels of liver beta-actin and albumin mRNAs were unchanged by these dietary manipulations. Our data show that hepatic SCD1 gene expression is regulated by PPARs and suggest that peroxisome proliferators and poly-unsaturated fatty acids act through distinct mechanisms.
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PMID:Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. 879 Mar 49


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