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)

The mitogen-activated protein kinases (MAP kinases) p42mapk and p44mapk are serine/threonine kinases rapidly activated in cells stimulated with various extracellular signals by dual phosphorylation of tyrosine and threonine residues. They are thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Here we demonstrate that activation of these ubiquitously expressed MAP kinases is essential for growth. To specifically suppress MAP kinase activation in fibroblasts, we transiently expressed either the entire p44mapk antisense RNA or p44mapk kinase-deficient mutants (T192A or Y194F). As expected, and through independent mechanisms, both approaches strongly inhibited MAP kinase activation. The antisense reduced the expression of endogenous p42mapk and p44mapk by 90%, whereas overexpression of the T192A mutant inhibited growth factor activation of both endogenous MAP kinases by up to 70%. As a consequence, we found that the antisense as well as the T192A mutant of p44mapk inhibited growth factor-stimulated gene transcription (collagenase promoter assay with chloramphenicol acetyltransferase reporter) and cell growth. These effects were proportional to the extent of MAP kinase inhibition and reversed by coexpression of the wild-type p44mapk. Therefore we conclude that growth factor activation of p42mapk and p44mapk is an absolute requirement for triggering the proliferative response.
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PMID:Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. 839 1

The mechanisms controlling the proliferation of astrocytes are of great interest but are not well defined. We have previously shown that the endogenous neuropeptides, endothelin-3 (ET-3), and atrial natriuretic peptide (ANP), modulate the proliferation of astrocytes through positively and negatively regulating the transcription of the immediate-early gene egr-1 which transactivates basic fibroblast growth factor (bFGF) by unknown mechanisms. In these studies, we determined the involvement of MAP kinase (Erk) activation by ET-3 in the transcription of egr-1, and the molecular determinants by which Egr-1 transactivates bFGF. Transfection of astrocytes with a mitogen-activated protein (MAP) kinase (MAPK) expression vector increased the transcription of a cotransfected egr-chloramphenicol acetyltransferase (CAT) construct 3-fold. This induction was totally abolished by a dominant negative MAPK mutant. A 3-fold induction of egr-CAT expression by ET-3 was significantly reduced by treatment with ANP, or a cotransfected dominant negative MAPK plasmid. Using mobility shift assays, we showed that ET-3 induced the expression of Egr-1 protein which bound specifically to several early growth-related protein (Egr-1) binding sites on the bFGF promoter, and that this effect was significantly reversed by treatment with ANP. We also found that the Sp1 transcriptional factor was bound at these same sites, but was not stimulated by ET-3. Deletion experiments indicated that only the site at -160 bp of the bFGF promoter was significant for bFGF transactivation by Egr-1. We conclude that the astrocyte mitogen, ET-3, stimulates egr-1 transcription through a MAP kinase (Erk) related mechanism, and that Egr-1 transactivates bFGF through a specific noncanonical, Egr-1 site on the promoter. ANP inhibits each of these steps, providing a pathway for its anti-proliferative action.
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PMID:Egr-1 activates basic fibroblast growth factor transcription. Mechanistic implications for astrocyte proliferation. 870 7

Green tea polyphenols, major constituents of green tea, are potent chemopreventive agents in a number of experimental models of cancer in animals. The mechanisms of cancer protection by these agents are not clear, but may involve modulation of the enzyme systems responsible for the detoxification of chemical carcinogens. The present studies show that a green tea polyphenol extract (GTP) induces chloramphenicol acetyltransferase (CAT) activity in human heptoma HepG2 cells transfected with a plasmid construct which contains an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene. This indicates that GTP stimulates the transcription of Phase II detoxifying enzymes through the ARE. To explore the upstream signaling pathways leading to gene expression, we studied the involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1). Potent activation of ERK2 was seen following treatment of HepG2 cells with different concentrations of GTP. Similar to ERK2, JNK1 was also strongly activated by treatment with GTP, although to a lesser extent and in a different dose-dependent fashion. Kinetic studies revealed that GTP activation of JNK1 was delayed and sustained, whereas ERK2 activation was rapid and transient. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos, as determined by reverse transcriptase-coupled polymerase chain reaction. Taken together, these studies provide insights into the action of GTP and suggest that the stimulation MAPKs may be the potential signaling pathways utilized by GTP to activate ARE-dependent genes.
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PMID:Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression. 905 42

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.
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PMID:Signaling from G protein-coupled receptors to the c-jun promoter involves the MEF2 transcription factor. Evidence for a novel c-jun amino-terminal kinase-independent pathway. 925 89

Polycyclic aromatic hydrocarbons (PAH) contained in fossil fuel combustion particles enhance the allergic response to common environmental Ags. A key question is: what are molecular pathways in the immune system by which PAH and conversion products drive allergic inflammation? Circumstantial evidence suggests that macrophages are involved in PAH-induced responses. We demonstrate that a representative PAH, beta-napthoflavone (BNF), and a representative quinone metabolite, tert-butylhydroxyquinone (tBHQ), induce Jun kinase and p38 mitogen-activated protein kinase activities in parallel with the generation of activator protein-1 (AP-1) mobility shift complexes in THP-1 and RAW264.7 macrophage cell lines. Activation of mitogen-activated protein kinases was dependent on generation of oxidative stress, and could be inhibited by N-acetylcysteine. Another genetic response pathway linked to PAH is the antioxidant response element (ARE), which regulates expression of detoxifying enzymes. BNF and tBHQ activated a human ARE (hARE) reporter gene in RAW264.7 cells. Interestingly, bacterial lipopolysaccharide also induced hARE/chloramphenicol acetyltransferase activity. While the hARE core, GTGACTCAGC, contains a consensus AP-1 sequence (underlined), AP-1 was not required for hARE activation. This suggests that PAH and their conversion products operate via ARE-specific transcription factors in the immune system. BNF and tBHQ did, however, induce AP-1 binding to the hARE, while constitutively active Jun kinase interfered in hARE/chloramphenicol acetyltransferase activation. This suggests that AP-1 proteins negatively regulate the hARE. These data establish important activation pathways for PAH in the immune system and provide us with targets to modulate the effect of environmental pollutants on allergic inflammation.
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PMID:Macrophage activation by polycyclic aromatic hydrocarbons: evidence for the involvement of stress-activated protein kinases, activator protein-1, and antioxidant response elements. 967 Sep 73

Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.
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PMID:The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. 991 9

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1beta induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1beta-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85alpha, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-kappaB in LPS- or IL-1beta-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1beta alone to induce the expression of iNOS, both LPS and IL-1beta individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-kappaB. Wortmannin alone was unable to activate MAP kinase and NF-kappaB. Moreover, wortmannin had no effect on LPS- or IL-1beta-mediated activation of MAP kinase and NF-kappaB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1beta-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-kappaB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-kappaB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-kappaB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.
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PMID:Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells. 1006 20

In the present study, we have examined the insulin-signaling pathways involved in myogenesis in mouse C2C12 skeletal muscle cell line, a cellular system that expresses high number of high affinity insulin receptors. Insulin (50 nM) rapidly (5 min) stimulated beta-chain insulin receptor, activated the phosphatidylinositol (PI) 3-kinase/Akt/p70S6-kinase signaling pathway, as well as phosphorylated both p44/p42- and p38-mitogen-activated protein kinases (MAPKs). Preconfluent cells were differentiated in a serum-free medium in response to 50 nM insulin for 72 h, as revealed by the formation of multinucleated myotubes and the induction of the creatine kinase activity. This differentiation process was also monitored by the inhibition of the PCNA content and induction of the cell cycle inhibitor p21. Furthermore, insulin induced nuclear factor-kappaB (NF-kappaB) DNA binding activity and down-regulated activating protein-1 (AP-1) DNA binding activity throughout the differentiation process. The use of specific inhibitors of the insulin-signaling pathways indicated that myogenesis was precluded by treatment for 72 h with LY294002 (an inhibitor of PI 3-kinase), rapamycin (a p70S6-kinase blocker), and SB203580 or PD169316 (p38-MAPK inhibitors). These inhibitors abolished insulin induction of NF-kappaB DNA binding activity and kappaB-chloramphenicol acetyltransferase (CAT) promoter activity, maintaining expressed cytosolic IkappaB-alpha protein, and increased AP-1 DNA binding activity and TRE-CAT promoter activity. These data suggest that insulin induces myogenesis in C2C12 through PI 3-kinase/ p70S6-kinase and p38-MAPK pathways, the signaling through p44/p42-MAPK being inhibited.
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PMID:Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities. 1114 17

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestin-dependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.
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PMID:Heregulin induces transcriptional activation of the progesterone receptor by a mechanism that requires functional ErbB-2 and mitogen-activated protein kinase activation in breast cancer cells. 1252 13

Antidepressant drugs are thought to counteract effects of hypercortisolemia, frequently associated with depression, by lowering cortisol level and by modifying the function of glucocorticoid receptors (GR). Indeed, classical antidepressants inhibit corticosteroid-induced gene transcription in cell cultures. The aim of the present study was to investigate effects of new generation antidepressant drugs on GR function in mouse fibroblast cells (L929), stably transfected with mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) plasmid (LMCAT cells). It has been found that reboxetine (at 10 and 30 microM), venlafaxine, citalopram and mirtazapine (at 30 microM), but not milnacipran, in statistically significant manner inhibited corticosterone-induced gene transcription. However, the effects of new generation antidepressant drugs were weaker than those evoked by imipramine, which was active already at 3 microM concentration. Further studies on the mechanism of antidepressant action on GR function revealed that protein kinase C, but not mitogen-activated protein kinases (MAPK), glycogen synthase kinase (GSK-3) and protein kinase B (PKB, Akt) play a role in this phenomenon.
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PMID:Effects of some new antidepressant drugs on the glucocorticoid receptor-mediated gene transcription in fibroblast cells. 1638 95


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