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)

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

A critical role of tumor necrosis factor (TNF)-alpha in irritant contact dermatitis and in the challenge phase of allergic contact dermatitis has recently been demonstrated in vivo. As in situ hybridization studies have indicated that keratinocytes were the cellular source of TNF-alpha in these reactions, we studied the mechanisms of TNF-alpha mRNA induction in keratinocytes by agents that induce contact dermatitis. Murine la-/CD3- epidermal cells were stimulated with phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO), sodium dodecyl sulfate (SDS) and NiSO4, all of which up-regulated epidermal cell TNF-alpha mRNA production. In contrast, trinitrobenzenesulfonic acid and trinitrochlorobenzene did not significantly up-regulate TNF-alpha mRNA. These results were confirmed with murine keratinocyte cell lines. In keratinocytes transfected with a chloramphenicol acetyltransferase construct containing the -1059 to +138 base pair TNF-alpha promoter, increased promoter activity was observed upon stimulation with PMA and DMSO. In addition, PMA stimulation did not affect the stability of TNF-alpha mRNA. The PMA- but also the DMSO- and SDS- induced up-regulation of TNF-alpha mRNA was abolished by an inhibitor of protein kinase C (PKC). In contrast, NiSO4 up-regulated TNF-alpha mRNA by a PKC-independent mechanism, did not increase TNF-alpha promoter activity, but markedly increased the stability of the TNF-alpha mRNA. Co-stimulation with PMA and NiSO4 induced a marked increase in TNF-alpha mRNA over that obtained with each agent alone. Thus, whereas PKC-dependent irritants act by up-regulating TNF-alpha promoter activity, nickel acts via post-transcriptional regulation. Our results also establish that some irritants and irritant sensitizers directly induce TNF-alpha in keratinocytes without intermediate Langerhans cell-derived signals.
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PMID:Nickel and skin irritants up-regulate tumor necrosis factor-alpha mRNA in keratinocytes by different but potentially synergistic mechanisms. 779 16

Regulatory elements important for transcription of the murine interleukin-1 beta (IL-1 beta) gene lie within a DNase I-hypersensitive region located > 2,000 bp upstream from the transcription start site. We have identified within this region a novel positive regulatory element that is required for activation of an IL-1 beta promoter-chloramphenicol acetyltransferase (CAT) fusion gene in the murine macrophage line RAW264.7. Electrophoretic mobility shift analysis of the 3' portion (-2315 to -2106) of the hypersensitive region revealed at least two nuclear factor binding sites, one of which is located between positions -2285 and -2256. Competitive inhibition studies localized the binding site to a 15-bp sequence between -2285 and -2271. Nuclear factor binding was lost by mutation of the 6-bp sequence from -2280 to -2275. The specific retarded complex formed with RAW264.7 nuclear extract was not detected under similar conditions with nuclear extracts from RLM-11, a murine T-cell line which does not express IL-1 beta RNA. Mutation of the 6-bp sequence (-2280 to -2275) in the chimeric IL-1 beta promoter -4093 +I CAT plasmid virtually eliminated the activation of this reporter gene by lipopolysaccharide (LPS) in transfected RAW264.7 cells. Multimerization of the 15-bp sequence containing the core wild-type 6-bp sequence 5' of minimal homologous or heterologous promoters in CAT reporter plasmids resulted in significant enhancement of CAT expression compared with parallel constructs containing the mutant 6-bp core sequence. This element was LPS independent and position and orientation dependent. The multimerized 15-bp sequence did not enhance expression in RLM-11 cells. Methylation interference revealed contact residues from -2281 to -2271, CCAAAAAGGAA. Because a search of the NIH TFD data bank with the 11-bp binding site sequence found no homology to known nuclear factor binding sites, we have designated this sequence the IL1 beta -upstream nuclear factor 1 (IL1 beta -UNF1) target. UV cross-linking and sodium dodecyl sulfate-polyacrylamide electrophoresis identified an IL1 beta -UNF1-specific binding factor approximately 85 to 90 kDa in size.
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PMID:A novel cis-acting element required for lipopolysaccharide-induced transcription of the murine interleukin-1 beta gene. 779 17

Estrone sulfate is the predominant form of estrogens found in the circulation in women and could thus serve as precursor for active estrogens in target tissues by removal of the sulfate group through the action of endogenous steroid sulfatase. Recently, we isolated a cDNA encoding human placental estrogen sulfotransferase that differs from brain aryl sulfotransferase only in the 5'-noncoding sequence. To increase our knowledge of the regulation and tissue-specific expression of sulfotransferase gene, we screened a lambda EMBL3 library of human leucocyte genomic DNA using the estrogen sulfotransferase cDNA as probe and isolated a clone containing almost the whole gene sequence. Sequencing of the gene indicates that it is included in approximately 7.7 kilobases and contains nine short exons separated by eight introns. The two first exons, named exon 1a and exon 1b, are noncoding and correspond to the 5'-untranslated sequences of human brain and human placental estrogen sulfotransferase cDNAs, respectively. Transfection of chloramphenicol acetyltransferase reporter gene vectors containing the 5'-flanking sequence upstream from exon 1a and exon 1b in human adrenal adenocarcinoma cells indicates that both sequences possess promoter activity. The present results thus indicate that brain aryl sulfotransferase and placental human placental estrogen sulfotransferase mRNA species are transcribed from a single gene by alternate exon 1a and exon 1b promoters, respectively. Using DNA from panels of human/rodent somatic cell hybrids and amplification of the gene by polymerase chain reaction, the human placental estrogen sulfotransferase gene was assigned to chromosome 16.
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PMID:Structure of human estrogen and aryl sulfotransferase gene. Two mRNA species issued from a single gene. 796 57

We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins. The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.
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PMID:Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat. 798 30

NF-R2 is a DNA-binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF-R2 binds within the promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF-R2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S-300, DEAE-Sepharose, heparin-Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF-R2 runs as a single protein of 75 kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF-R2 binding. These results suggest that NF-R2 may be involved in the positive regulation of the MDR1 gene transcription.
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PMID:Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene. 809 26

Thyroid hormone (TH) receptor number in red blood cells (RBCs) from Rana catesbeiana (RC) tadpoles increases 4-fold during both spontaneous and TH-induced metamorphosis, an effect that we have previously shown to be preceded by an increase in the level of c-erbA-related mRNA. The goals of the present study were to obtain an RC c-erbA alpha cDNA that contains the entire open reading frame for a putative TH receptor protein, to determine if this protein has characteristics typical of a TH receptor, and to assess its contribution to the developmentally related increase in TH receptor number. To accomplish this, the missing 5'-sequence of a previously isolated partial RC c-erbA alpha cDNA (RC12) was synthesized by polymerase chain reaction (PCR) and spliced to RC12 to yield a 1490-basepair cDNA (RC15) that contained the entire coding sequence of the receptor protein. Transcription of RC15 followed by translation of its mRNA in a rabbit reticulolysate system yielded a 50-kilodalton protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds T3 with high affinity (Kd, approximately 0.1 nM), and its affinity for T3 is at least 5 times that for T4. The results of cotransfection studies indicate that RC15 can function as a TH receptor; when COS cells were cotransfected with a construct consisting of RC15 cloned in the expression vector CMV4 and TK28 mult, a construct containing rat GH gene TH response element sequences up-stream of a chloramphenicol acetyltransferase reporter gene, chloramphenicol acetyltransferase activity is expressed in the presence, but not in the absence, of T3. To determine whether RBCs contain any c-erbA beta mRNA transcripts that might contribute to the developmentally related increase in the transcripts detected using RC c-erbA alpha cDNAs, alpha- and beta-specific cDNAs were synthesized by PCR and used as probes in a variety of hybridization assays. In all experiments using conditions in which c-erbA beta transcripts were detectable in other tissues, there was no evidence that tadpole RBCs contained such species. Lack of any beta-specific transcripts was confirmed by PCR, using as template cDNA prepared by reverse transcription of RC RBC RNA. It was also noted that the RBC at metamorphic climax is the tissue with the highest content of alpha-specific c-erbA transcripts. It is concluded that the c-erbA alpha gene encodes a TH receptor, and that only the alpha-gene is expressed in tadpole RBCs and subject to regulation during development and by TH.
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PMID:Rana catesbeiana tadpole red blood cells express an alpha, but not a beta, c-erbA gene. 824 69

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.
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PMID:Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control. 827 54

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription probably play an important role in initiation and maintenance of virus replication. We have identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]) at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. The basal promoter strength of constructs that contained deletions in the R/U5 region of the HTLV-I long terminal repeat were analyzed by chloramphenicol acetyltransferase assays following transfection of Jurkat T cells. We consistently observed a 10-fold decrease in basal promoter activity when sequences between +202 to +246 were deleted. By reverse transcriptase polymerase chain reaction RNA analysis, we confirmed that the drop in chloramphenicol acetyltransferase activity was paralleled by a decrease in the level of steady-state RNA. DRE 1 did not affect the level of Tax1 transactivation. Using a gel shift assay, we have purified a highly enriched fraction that could specifically bind DRE 1. This DNA affinity column fraction contained four detectable proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: p37, p50, p60, and p100. The affinity column fraction stimulated HTLV-I transcription approximately 12-fold in vitro. No effect was observed with the human immunodeficiency virus or adenovirus major late promoters. Following renaturation of the proteins isolated from an SDS-containing gel, p37, but not the other protein fractions, was able to specifically bind to DRE 1.
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PMID:Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression. 847 78

The objectives of this study were (i) to characterize the plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene (pCAT) complexed with cationic liposomes (Lipofectin and LipofectACE) in terms of particle size and zeta potential, (ii) to compare pharmacokinetic characteristics, and (iii) to study the hepatic uptake mechanisms. DNA/LipofectACE complexes showed a negative zeta potential of -36 mV at 1:5 w/w ratio, but a positive zeta potential of (19 mV at 1:10 w/w ratio. Lipofectin samples showed a positive zeta potential) of 21-28 mV at these ratios. These preparations showed a wide particle size distribution ranging from 600 to 1200 nm. Following intravenous injection of 1:10 w/w ratio [32P]pCAT/liposome complexes at a dose of 0.1 mg DNA/kg into the tail vein of mice, radioactivity was rapidly eliminated from the plasma and almost 50-60% of the dose was taken up by the liver within 5 min after administration. Plasmid DNA/liposome complexes were predominantly taken up by the liver nonparenchymal cells. The hepatic uptake was inhibited by preceding administration of dextran sulfate (DS), but not by polycytidic acid (poly[C]) and polyinosinic acid (poly[I]), suggesting the involvement of a phagocytic process. We suggest that these complexes are preferentially taken up by the liver nonparenchymal cells mainly via Kupffer cell phagocytosis.
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PMID:Physicochemical and pharmacokinetic characteristics of plasmid DNA/cationic liposome complexes. 858 40


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