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)

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.
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PMID:Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter. 130 41

The noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.
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PMID:Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. 149 20

The syndrome of hereditary resistance to 1,25-dihydroxyvitamin D3 is due to defective function of the vitamin D receptor (VDR). The recent cloning and nucleotide sequence determination of the human VDR chromosomal gene have enabled a direct evaluation of the genetic basis for this disease in affected patients. In this report we employed polymerase chain reaction techniques to amplify the gene exons that encode the DNA-binding domain of the VDR from two 1,25-dihydroxyvitamin D3-resistant patients whose receptors displayed defective binding to nonspecific DNA. Although their families were apparently unrelated, each patient displayed an identical homozygous point mutation within the third exon, a mutation that causes substitution of a glutamine for an arginine residue highly conserved within the entire steroid receptor superfamily. We introduced this base change into the normal VDR cDNA via site-directed mutagenesis, transfected an expression vector containing this cDNA into cells, and examined the functional properties of the resultant VDR expression product. The produced mutant receptor bound 1,25-dihydroxyvitamin D3 with normal affinity, but displayed weak affinity for the nuclear fraction and for heterologous DNA. More importantly, the protein was inactive in promoting transcription in a cotransfection assay employing a chloramphenicol acetyltransferase gene reporter fused down-stream of the VDR-inducible osteocalcin gene promoter-enhancer. These results provide the genetic and functional basis for the phenotype of rickets in this inherited disease.
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PMID:A unique point mutation in the human vitamin D receptor chromosomal gene confers hereditary resistance to 1,25-dihydroxyvitamin D3. 217 43

The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.
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PMID:Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression. 254 79

Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a human syndrome that arises as a result of heterogeneous molecular defects in the vitamin D3 receptor. Recent studies have identified single unique point mutations within the second or third exons that encode the DNA-binding domain of the vitamin D receptor (VDR) gene in two families with this syndrome. In the experiments reported here, these mutations were introduced into the normal VDR cDNA by site-directed mutagenesis and the mutant products evaluated for hormone, nuclear, and DNA-binding characteristics. Each mutant VDR was expressed in COS-1 cells at equivalent levels, and saturation analysis of cell cytosol revealed normal affinity for the 1,25-dihydroxyvitamin D3 hormone. Incubation of transfected cells with radiolabeled hormone followed by lysis and extraction suggests a lowered salt dependence for solubilization of the mutant VDR. Concomitantly, mutant receptors exhibited reduced affinity for immobilized calf thymus DNA. While cotransfection of the wild type receptor together with a vitamin D-inducible (osteocalcin) chloramphenicol acetyltransferase reporter gene construction in CV-1 cells resulted in strong induction by 1,25-dihydroxyvitamin D3, neither mutant receptor was capable of directing significant activity either as a function of receptor or hormone concentration. These data suggest that the unique point mutations identified in each of these two families are responsible not only for the phenotype originally ascribed to the abnormal receptor but also severely compromise each protein's ability to activate transcription.
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PMID:Mutant vitamin D receptors which confer hereditary resistance to 1,25-dihydroxyvitamin D3 in humans are transcriptionally inactive in vitro. 255 49

We have cloned the genomic DNA encoding rat osteocalcin and have isolated fragments in the 5' flanking region which mediate the effects of 1,25-(OH)2D3 (1,25-dihydroxyvitamin D3) on osteocalcin gene transcription. Approximately 3 kilobase pairs of the osteocalcin gene's 5' flanking region, including the promoter and transcription start site, were fused to the reporter gene chloramphenicol acetyltransferase. Transfection into ROS 17/2.8 rat osteosarcoma cells demonstrated low level basal expression of the chloramphenicol acetyltransferase gene. The expression increased markedly in the presence of 1,25-(OH)2D3; induction was observed at doses as low as 10(-11) M 1,25-(OH)2D3. Chloramphenicol acetyltransferase activity increased as early as 16 h after stimulation with 10(-9) M 1,25-(OH)2D3. Basal chloramphenicol acetyltransferase activity in ROS 24/1 and 25/1 cells was much lower than in ROS 17/2.8 cells. In these two cell lines, there was little induction of chloramphenicol acetyltransferase activity in the presence of 10(-9) M 1,25-(OH)2D3. Deletion studies of the 5' flanking region demonstrated two regions that contribute to the induction by 1,25-(OH)2D3. Deletion of a 650-base pair fragment ending 1.4 kilobase pairs upstream from the initiator ATG led to an 80% decrease in responsiveness. Removal of an additional 1.1 kilobase pairs, leaving a 300-base pair promoter containing fragment obliterated responsiveness to 1,25-(OH)2D3.
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PMID:Regions of the rat osteocalcin gene which mediate the effect of 1,25-dihydroxyvitamin D3 on gene transcription. 278 91

Osteocalcin is an abundant noncollagenous protein in bone, and its synthesis is stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, the rat osteocalcin gene was isolated, sequenced, and found to be a single-copy gene that is highly conserved between human and rat. Northern blot analysis of RNAs from a number of rat tissues revealed osteocalcin mRNA only in calvariae, consistent with bone-specific expression of osteocalcin. In order to investigate promoter activity and its modulation by 1,25(OH)2D3, plasmids containing the osteocalcin promoter region linked to the reporter enzyme bacterial chloramphenicol acetyltransferase (CAT) were used to transfect rat osteosarcoma ROS 17/2.8 cells, which express osteocalcin endogenously, and UMR 106 cells, which lack osteocalcin expression. Transfected ROS 17/2.8 cells exhibited a higher basal CAT activity than UMR 106 cells. Moreover, 1,25(OH)2D3 stimulated the CAT expression 5-10-fold only in ROS 17/2.8 cells and not in UMR 106 cells. By use of unidirectional deletion analysis, a domain strongly responsive to 1,25(OH)2D3 was identified between bases -1035 and -871 upstream from the site of transcription initiation, while a weakly responsive region was found further downstream.
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PMID:Characterization of the rat osteocalcin gene: stimulation of promoter activity by 1,25-dihydroxyvitamin D3. 326 36

The effects of two vitamin D analogs, 1,25-dihydroxyvitamin D-2 and 24-epi-1,25-dihydroxyvitamin D-2, were examined on osteocalcin gene expression in the rat osteosarcoma cell line ROS 17/28. Our results indicate that these analogs are more transcriptionally active than 1,25-dihydroxyvitamin D-3, particularly the 24-epimer. Assessment of reporter gene chloramphenicol acetyltransferase (CAT) activity, using the vitamin D responsive element (VDRE) derived from the human osteocalcin gene promoter. revealed that both analogs stimulated CAT activity 5- to 10-fold. 1,25-Dihydroxyvitamin D-2 was slightly more active than 1,25-dihydroxyvitamin D-3, while the 24-epimer was twice as effective. 1,25-Dihydroxyvitamin D-3 also stimulated osteocalcin mRNA accumulation by 2-fold over vehicle-treated cells, 1,25-dihydroxyvitamin D-2 by 2.5-fold, and 24-epi-1,25-dihydroxyvitamin D-2 by 4-fold. Electrophoretic mobility shift assays using the osteocalcin vitamin D responsive element revealed no increase in DNA binding with either analog when compared to 1,25-(OH)2D3. Examination of CAT activity using the rat 24-hydroxylase VDRE indicated no significant difference in transcription with these compounds, suggesting that the vitamin D-2 analogs preferentially activate osteocalcin gene expression.
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PMID:Transcriptional control of the osteocalcin gene by 1,25-dihydroxyvitamin D-2 and its 24-epimer in rat osteosarcoma cells. 764 Mar 5

Although steroid hormone receptor activation has been known to be dependent on ligand binding, we report here ligand-independent transcriptional activation of the vitamin D receptor and retinoid receptors. In these studies, CV1 cells were transiently transfected with a human vitamin D receptor (VDR) expression vector and a reporter plasmid that contains multiple copies of the rat osteocalcin vitamin D response element up-stream of the bacterial chloramphenicol acetyltransferase (CAT) gene [osteocalcin (OC)VDREtkCAT]. Treatment of cells with 10(-8) M 1,25-dihydroxyvitamin D3 resulted in a 25-fold induction of CAT activity. When cells were treated with 5-50 nM okadaic acid (OA), an inhibitor of protein phosphatase-1 and -2A, significant inductions of CAT activity (18- to 57-fold) were observed. As VDR and dopamine receptors are colocalized in certain brain regions, we also examined whether VDR-mediated transcription can be activated by dopamine. VDR was found to activate CAT gene expression in cells treated with 200-500 microM dopamine (3- to 11-fold induction) or the selective D1 agonist SKF38393 (20-fold induction). Cells were also transfected with retinoic acid receptor (RAR) or retinoid-X receptor (RXR) expression vectors and reporter plasmids that contain either a retinoic acid response element or an RXR-specific response element. OA alone induced chloramphenicol acetyltransferase (CAT) activity in cells transfected with RAR alpha, RAR beta, RXR alpha, RXR beta, or RXR gamma (3- to 18-fold induction). However, OA did not affect transcription by RAR gamma, suggesting specificity of activation by OA among the retinoid receptors. Although the retinoid receptors have been detected in brain, maximum stimulation of transcription was not greater than 1.6-fold in the presence of 100-500 microM dopamine or 100 microM SKF38393 treatment. These data suggest specificity for dopamine activation among steroid hormone receptors and that phosphorylation alone, in the absence of ligand, can activate VDR- and retinoid receptor-mediated transcription.
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PMID:Ligand occupancy is not required for vitamin D receptor and retinoid receptor-mediated transcriptional activation. 777 73

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
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PMID:Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells. 789 Aug 7


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