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)

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the growth and differentiation of human hematopoietic progenitor cells by activating transcription of specific genes. The mechanism by which binding of GM-CSF to its receptor stimulates gene expression remains unknown. To examine this process in more detail, we have transfected human monocytic leukemia cells U937 with a plasmid containing an AP-1 enhancer element and a chloramphenicol acetyltransferase recorder gene and treated them with GM-CSF. We find that GM-CSF stimulates a 2-3-fold increase in chloramphenicol acetyltransferase activity over a concentration range 1-1,000 units/ml. Northern and Western blot analysis demonstrates that the mechanism by which GM-CSF stimulates AP-1 enhancer activity involves increases in c-jun and c-fos mRNA levels, and increases in Jun protein. In a similar fashion the treatment of normal human monocytes with GM-CSF also induced increases in total cellular c-jun. Because protein kinase C plays a crucial role in activating c-jun transcription we examined the role of this enzyme in mediating the effects of GM-CSF. Treatment of U937 cells with inhibitors of protein kinase C including staurosporine 10 nM and H-7 50 microM, or down-regulation of protein kinase C by phorbol ester pretreatment blocks the induction of c-jun by GM-CSF. However, HA which does not block protein kinase C had no effect on GM-CSF stimulation of c-jun RNA levels. In addition, GM-CSF treatment causes the rapid translocation of protein kinase C to the particulate fraction which was maximal by 5 min and returned to base line by 80 min. These data suggest that the binding of GM-CSF to its receptor stimulates increases in c-jun mRNA and protein and activates AP-1 enhancer activity. These effects may be at least in part mediated by activation of protein kinase C.
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PMID:Regulation of c-jun expression and AP-1 enhancer activity by granulocyte-macrophage colony-stimulating factor. 190 Aug 39

Systemic gene transfer provides new opportunities for the analysis of gene function and gene regulation in vivo, as well as for human gene therapy. We used the chloramphenicol acetyltransferase reporter gene to examine several parameters important for the development of efficient, cationic liposome-mediated, intravenous (IV) gene transfer in mice. We then demonstrated that this approach can produce high level expression of biologically important genes. Specifically, we assessed the relationship of expression vector design to the level of systemic gene expression produced, and compared transfection levels produced by intravenously injecting DNA alone versus DNA-liposome complexes. We found that both the position of the heterologous intron, and the promoter element used in the expression plasmid, significantly affected the level of systemic gene expression produced. Although intravenous injection of plasmid DNA alone transfected every tissue analyzed, liposome-mediated delivery was much more efficient. We also established that repeated i.v. injection of DNA-liposome complexes produced high level systemic transfection. The second injection of DNA-liposome complexes produced levels of gene expression at least as high as those following a single i.v. injection. Thus, unlike some viral vectors, a neutralizing host-immune response does not limit re-expression, following reinjection of DNA-liposome complexes. Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, i.v. injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophage-colony stimulating factor cDNA or the human granulocyte-CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant.
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PMID:Cationic liposome-mediated intravenous gene delivery. 755 9

Minimally modified LDL (MM-LDL), obtained by mild iron oxidation or prolonged storage at 4 degrees C, has been shown to induce the expression of macrophage-colony stimulating factor (M-CSF) in cultured aortic endothelial cells. To examine whether other cell types also respond to MM-LDL, we investigated its effect on the expression of M-CSF mRNA in mouse L-cells and human aortic smooth muscle cells. Both L-cells and human aortic smooth muscle cells showed increased levels of M-CSF mRNA in response to 10 to 200 micrograms/mL MM-LDL in a dose-dependent manner. This allowed us to use mouse L-cells as a model to study the mechanism involved in MM-LDL-mediated increase in M-CSF mRNA. Nuclear runon assays showed that M-CSF gene transcription was activated by MM-LDL. In the present study, we identified specific elements that conferred MM-LDL-mediated transcriptional activation of the human M-CSF gene. Chimeric constructs containing sequential deletions in the 5'-promoter region of the M-CSF gene linked to a reporter chloramphenicol acetyltransferase (CAT) gene were transfected into mouse L-cells. The human M-CSF promoter region extending upstream from the transcription start site to nucleotide -406 showed maximum induction of CAT activity by MM-LDL. Induction of CAT activity was drastically reduced, with a deletion plasmid lacking the promoter region -406 to -344. A functional nuclear factor (NF)-kappa B binding site present in this critical region was required for MM-LDL-mediated induction of CAT activity since an internal deletion construct lacking this element showed significant loss of transcriptional activation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcriptional activation of the macrophage-colony stimulating factor gene by minimally modified LDL. Involvement of nuclear factor-kappa B. 758 32

Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-alpha (TNF alpha) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNF alpha-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrated that activation of NF-kappa B by L-NMA, ox-LDL, and TNF alpha was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-kappa B activation, the ability of NO to inhibit NF-kappa B activation and M-CSF expression may contribute to some of NO's antiatherogenic properties.
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PMID:Nitric oxide inhibits macrophage-colony stimulating factor gene transcription in vascular endothelial cells. 762 26

Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to 208-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription 20-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-IL6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and IL-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals.
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PMID:Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat. 773 95

Interferon-tau (oIFNtau), the major secretory product of ovine conceptuses between days 13 and 21 (day 0=day of estrus) of pregnancy, is implicated in the process of maternal recognition of pregnancy. Culturing of day-14 and day-16 conceptus tissues in the presence of human granulocyte macrophage-colony stimulating factor (hGM-CSF) or interleukin-3 (IL-3) produces a marked increase in oIFNtau mRNA and protein expression. Since GM-CSF and IL-3 are localized at the luminal and glandular epithelia of the ovine endometrium, maternally derived GM-CSF and IL-3 may affect conceptus production of oIFNtau in a paracrine manner. However, the molecular mechanisms by which endometrial GM-CSF and IL-3 up-regulate oIFNtau production have not been defined. As an initial investigation of the signaling pathway regulating the GM-CSF induction of the oIFNtau gene, day-16 conceptuses were treated with an inducer, phorbol 12-myristate 13-acetate (PMA) and an inhibitor, calphostin C of the protein kinase C (PKC) pathway. Treatment with either 150 units/ml hGM-CSF (P<0.01) or 10 nM PMA (P<0.05) resulted in a significant increase in oIFNtau mRNA expression. Pretreatment of conceptuses with 1 microM PMA for 12 h to produce PKC-deficient tissues or treatment with 50 mM calphostin C abolished the hGM-CSF-induced increase in oIFNtau mRNA. An in vitro expression system was established for the analysis of oIFNtau gene regulatory sequences. The oIFNtau010 gene has been isolated previously and found to be the principal oIFNtau gene up-regulated during the preimplantation period. 5'-Flanking regions of the oIFNtau010 gene, 2 kb and 0.8 kb, were cloned into a basic chloramphenicol acetyltransferase reporter plasmid. These oIFNtau010 promoter constructs, along with expression controls, were transfected into human choriocarcinoma cells (JAR and JEG3) and their responsiveness to hGM-CSF and second messenger system activators including PMA, calcium ionophore (A23187) and 8-bromo-cAMP were characterized. The oIFNtau010 promoter constructs were up-regulated by hGM-CSF and PMA treatments (P<0.01). Combined treatment with PMA and A23187 prevented the promoter activation seen with PMA alone. The conceptus culture data, along with the results from the transfection experiments, suggest that the stimulatory effect of GM-CSF on oIFNtau is mediated through the PKC second messenger system.
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PMID:Enhancement of ovine trophoblast interferon by granulocyte macrophage-colony stimulating factor: possible involvement of protein kinase C. 934 4

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.
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PMID:Gene expression along the cerebral-spinal axis after regional gene delivery. 1056 97

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.
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PMID:Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes. 1058 16

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