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

Studies from our laboratory concerning regulation of calbindin include regulation by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], receptor regulation as a possible mechanism for modulating calbindin's response to hormone, tissue specific regulation and regulation by factors other than 1,25(OH)2D3. With regard to receptor regulation, we found that the induction of calbindin mRNA in intestine and kidney by 1,25(OH)2D3 is not accompanied by a corresponding alteration in vitamin D receptor (VDR) mRNA in the vitamin D-deficient, low calcium rat. However, in the vitamin D-replete rat, administration of 1,25(OH)2D3 results in an induction of both calbindin and VDR mRNA in these tissues. These results suggest the presence of an inhibitor of 1,25(OH)2D3-mediated receptor up-regulation in the vitamin D-deficient, low calcium animal. Glucocorticoids can also regulate calbindin gene expression. Dexamethasone treatment (50 micrograms.100 g body weight-1.d-1 for 4 d) results in a 75% decrease in rat intestinal calbindin-D9k mRNA. This decrease may be related to the inhibition of intestinal calcium absorption previously observed after glucocorticoid administration. Kidney calbindin-D28k mRNA is unaffected by glucocorticoid treatment, indicating tissue specificity of the glucocorticoid response. To evaluate more precisely the means whereby 1,25(OH)2D3 and other modulators can influence calbindin gene expression, we isolated the chromosomal gene for calbindin-D28k by screening a mouse genomic library in cosmid. Ros 17/2.8 cells were transfected with recombinant plasmids in which the mouse calbindin promoter is fused to the reporter gene encoding chloramphenicol acetyltransferase. Deletion studies have enabled us to identify sequence elements in the mouse calbindin-D28k gene that confer basal activation and a hormone inducible response.
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PMID:Molecular aspects of the calbindins. 154 30

Expression of genes encoding transferrin and the vitamin D-binding protein is described in a cell line, U-2 OS, derived from a human osteogenic sarcoma. The mRNA transcripts of transferrin and vitamin D-binding protein were shown to be the lengths of those found in normal human liver. The cells synthesize and secrete the transferrin and vitamin D-binding proteins, in addition to human albumin and ceruloplasmin. The U-2 OS cells were successfully transfected with chimeric genes carrying 670 bp of the 5' regulatory sequence of the human transferrin gene fused to a reporter chloramphenicol acetyltransferase gene. These data indicate that the appropriate transcriptional factors required for expression of four plasma proteins are produced by U-2 OS nuclei and that the U-2 OS cell line will be useful for studies analyzing regulation of these genes.
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PMID:Expression of transferrin and vitamin D-binding protein genes in an osteogenic sarcoma cell line. 229 48

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

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.
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PMID:Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3. 747 23

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

A major metabolite of the vitamin D analogue 1 alpha-hydroxyvitamin D2 in human liver cells in culture has been identified as 1 alpha,24(S)-dihydroxyvitamin D2 [1 alpha,24(S)-(OH)2D2]. 1 alpha-Hydroxyvitamin D3 incubated with the same cells gives rise to predominantly 25- and 27-hydroxylated products. Our identification of 1 alpha,24(S)-dihydroxyvitamin D2 is based on comparisons of the liver cell metabolite with chemically synthesized 1 alpha,24(S)-(OH)2D2 and 1 alpha,24(R)-(OH)2D2 by using HPLC, GC and GC-MS techniques. The stereochemical orientation of the 24-hydroxyl group was inferred after X-ray-crystallographic analysis of the 24(R)-OH epimer. 1 alpha,24(S)-Dihydroxyvitamin D2 binds strongly to the vitamin D receptor and is biologically active in growth hormone and chloramphenicol acetyltransferase reporter gene expression systems in vitro, but binds poorly to rat vitamin D-binding globulin, DBP. We suggest that this metabolite, 1 alpha,24(S)-(OH)2D2, possesses the spectrum of biological properties to be useful as a drug in the treatment of psoriasis, metabolic bone disease and cancer.
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PMID:1 alpha,24(S)-dihydroxyvitamin D2: a biologically active product of 1 alpha-hydroxyvitamin D2 made in the human hepatoma, Hep3B. 764 51

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

The human placenta synthesizes 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and expresses the vitamin D receptor (VDR), but the roles of 1,25-(OH)2D3 and the VDR in placental physiology are poorly understood. In this study, we have demonstrated that 1,25-(OH)2D3 stimulates the synthesis and release of human placental lactogen (hPL), one of the major secretory products of syncytiotrophoblast cells. Enzymatically dispersed trophoblast cells from term placentas exposed continuously to 1,25-(OH)2D3 (0.1, 6, and 37 microM) for 5 days released significantly more hPL than control cells after the third day of exposure. On days 4 and 5, the amounts of hPL released by cells exposed to 1,25-(OH)2D3 were 2.54- and 4.14-fold that of control cells (P < 0.001 in each instance). The stimulation by 1,25-(OH)2D3 was dose dependent and was accompanied by stimulation of hPL messenger RNA levels. Transient transfection studies of BeWo choriocarcinoma cells transfected with hPL promoter constructs coupled to the chloramphenicol acetyltransferase reporter gene indicated that the stimulation of hPL expression is due at least in part to stimulation of hPL gene expression. Deletion analysis studies of the hPL promoter indicated that a region between -500 to -1200 basepairs is necessary for 1,25-(OH)2D3 responsiveness. Analysis of this region shows a consensus vitamin D response element (VDRE) DNA-binding site of a direct repeat motif separated by three bases. Ligation of this placental VDRE site into a heterologous chloramphenicol acetyltransferase vector caused 1,25-(OH)2D3 responsiveness. Moreover, mobility shift assays demonstrated binding of VDR to placental VDRE. These results indicate that 1,25-(OH)2D3 stimulates the synthesis and release of hPL by a mechanism involving hPL gene transcription and support a role for vitamin D and the VDR in placental function.
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PMID:Regulation of human placental lactogen expression by 1,25-dihydroxyvitamin D3. 798 55


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