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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that is characterized by selective destruction of insulin-secreting beta-cells. Cytokines have been implicated as effector molecules that participate in both islet inflammation and beta-cell destruction during the development of IDDM. In this study, the effects of cytokines on the expression of inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) by human islets were examined. In combination, the cytokines, human recombinant interleukin-1 beta (IL-1 beta), human recombinant tumor necrosis factor-alpha (TNF-alpha), and human recombinant interferon-gamma (IFN-gamma), induce the time-dependent formation of nitrite and prostaglandin E2 (PGE2) by human islets. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) completely inhibits cytokine-induced nitrite formation and attenuates PGE2 production by human islets. L-NMMA does not inhibit cytokine-induced expression of COX-2 by human islets, suggesting that nitric oxide may directly activate cyclooxygenase, an effect that has been previously demonstrated for isolated rat islets. This combination of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) also induces the expression of iNOS mRNA by human islets as demonstrated by both reverse transcriptase-polymerase chain reaction and Northern blot analysis. We further show that the tyrosine kinase inhibitors genistein and herbimycin A prevent IL-1 beta plus IFN-gamma-induced expression of COX-2 and iNOS and the production of PGE2 and nitric oxide by human islets. These results demonstrate that cytokines induce the expression of iNOS and COX-2 by human islets and that cytokine-induced expression of both COX-2 and iNOS by human islets appears to require the activation of a tyrosine kinase(s).
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PMID:Tyrosine kinase inhibitors prevent cytokine-induced expression of iNOS and COX-2 by human islets. 876 39

The ability of prostaglandins to inhibit collagen synthesis and induce prostaglandin G/H synthase in bone cells appears to be mediated by the prostaglandin F2 alpha receptor (FPR). We have identified FPR mRNA in the osteoblastic cell lines, Py1a from rats and MC3T3-E1 from mice, as well as in the stem cell cultures, MN-7 and mouse marrow, using reverse transcriptase-polymerase chain reaction technology (RT-PCR). Expression of FPR mRNA was increased in Pyla, MN-7 and marrow cells with prolonged culture or dexamethasone treatment and decreased after treatment with fluprostenol, a selective FPR agonist.
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PMID:Expression and regulation of prostaglandin F receptor mRNA in rodent osteoblastic cells. 883 44

Prostaglandins are involved in mediating several important processes in mammalian reproduction, including the initiation of parturition. In the present study, we examined the expression in the rat uterus of two-rate limiting enzymes involved in prostaglandin production, cyclooxygenase (COX) 1 and 2. Expression of the COX-2 gene in the pregnant rat uterus gave rise to a single mRNA transcript of approximately 4.4 kb. COX-2 mRNA levels increased 3.5 fold between day 7 of pregnancy and the onset of parturition on day 22. In contrast, COX-1 mRNA levels remained constant during the same period. To investigate factors involved in mediating the regulation of COX-1 and COX-2 gene expression, rat endometrial stromal and epithelial cell lines, were used. In the stroma-derived cell line, CUS-V2, COX-2 gene expression was demonstrated by reverse transcriptase/polymerase chain reaction (RT-PCR) and by immunocytochemistry. In these cells, COX-2 gene expression was inducible by the cytokines interleukin-1 beta and tumor necrosis factor alpha, but not by interleukin-6. The two former cytokines also induced prostaglandin F2 alpha production. In contrast, COX-1 gene expression was constitutive in this cell line. In the endometrial epithelium-derived cell line, CUE-P both COX-1 and COX-2 genes were expressed in a constitutive fashion. In conclusion, the present in vivo and in vitro data indicate that decidual COX-2, but not COX-1, gene expression is regulated during pregnancy and implicate specific cytokines as possible inducers within the decidua.
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PMID:Regulation of COX-2 gene expression in rat uterus in vivo and in vitro. 897 7

The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.
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PMID:Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. 899 3

Expression of mRNAs encoding the two prostaglandin endoperoxide synthase (PGHS) isoenzymes (PGHS-1 and -2) was investigated in differentiating clonal Ob1771 mouse preadipocytes and in mouse adipose tissues. Northern analysis revealed that the expression level of PGHS-1 mRNA was reduced by 98+/-0.2% (P <0.01) during differentiation of Ob1771 cells, whereas PGHS-2 mRNA was not detected. By reverse transcriptase-polymerase chain reaction analysis, however, both PGHS-1 and -2 mRNA was detected in Ob1771 preadipose cells. In addition. mRNAs encoding both isoforms were markedly expressed in primary adipose precursor cells with considerably lower expression levels in mature adipocytes (56 75% reduction, P<0.01). Furthermore, exposure to dexamethasone (10 nM) for both 24 h (explants of adipose tissue) and 48 h (Ob1771 adipose cells) resulted in enhanced expression of PGHS-1 mRNA. whereas expression of PGHS-2 mRNA in explants of adipose tissue (24 h incubation) was reduced by 83 +/- 9% (P<0.05). In contrast, exposure to angiotensin II (100 nM) enhanced expression of PGHS-1 mRNA both in mature adipocytes (4 h incubation) and explants of adipose tissue (24 h incubation), and elevated PGHS-2 mRNA expression in mature adipocytes (4 h incubation). In conclusion, this report suggests a differential expression of PGHS mRNAs during adipose cell differentiation, and further suggests that the machinery for prostaglandin synthesis in mature adipocytes may be induced by various hormones.
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PMID:Expression of the two isoforms of prostaglandin endoperoxide synthase (PGHS-1 and PGHS-2) during adipose cell differentiation. 925 65

In pre-eclampsia, the ratio of prostacyclin:thromboxane production rate is decreased favouring the vasoconstrictive thromboxane. One of the rate-limiting steps in prostaglandin synthesis is cyclooxygenase (COX) activity. Therefore, we investigated the expression of COX-1 and COX-2 in human placenta and placental bed. Tissue specimens from the 29th to 40th week of pregnancy were obtained from Caesarean sections after uncomplicated and pre-eclamptic pregnancies before the onset of labour. COX-1 and COX-2 were localized immunohistochemically with the identification of positive cells by double immunofluorescence staining. The protein and mRNA levels were analysed by immunoblotting and quantitative reverse transcriptase-polymerase chain reaction. Expression of both COX-1 and COX-2 could be observed in placenta and placental bed. COX-1-like immunoreactivity was observed in most cell types with strongest staining in macrophages. Only macrophages, endothelium, vascular leiomyocytes and fibroblasts stained positively for COX-2. In placenta, COX-1 and -2 expression was unchanged after pre-eclampsia. In placental bed, protein and mRNA levels of COX-1 were increased in the pre-eclamptic group (P < 0.05), whereas COX-2 expression did not differ significantly from normal pregnancies. An increased expression of COX-1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed. A therapy with drugs inhibiting COX-1 might be beneficial in this condition.
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PMID:Cyclooxygenase-1 and -2 in human placenta and placental bed after normal and pre-eclamptic pregnancies. 940 2

Chronic inflammation is a recognized risk factor for human cancer, but the causal mechanisms are poorly understood. We previously demonstrated that platelet activating factor (PAF) can induce alterations in the in vitro growth properties of primary rat fibroblasts. In the study reported here, exposure of primary human skin fibroblasts to PAF for 1 h in serum-free medium was shown to cause sustained proliferation over 50 d in medium containing low serum and anchorage-independent growth in soft agarose. Both properties could be inhibited by pretreatment with a PAF receptor antagonist, CV3988 (10 microM); a tyrosine-kinase inhibitor, genistein (1 microgram/mL); or a protein kinase C (PKC) inhibitor, staurosporine (50 nM) but not with a cyclooxygenase inhibitor, indomethacin (200 nM-20 microM). PAF had no effect on doubling time, saturation density, or cell viability under normal monolayer growth conditions in complete medium. Treatment with lyso-PAF, an inactive metabolite of PAF, had no effect in either of the assays. Control and PAF-induced cell proliferation in low-serum medium was inhibited by PAF receptor antagonists present during the extended growth period. The presence of PAF receptor mRNA in human skin fibroblasts was demonstrated by reverse transcriptase-polymerase chain reaction. The presence of a functional receptor was indicated by an early (2 min) transient increase in PKC activity and an increase in fos mRNA after PAF treatment. PAF-induced PKC activity was blocked by pretreatment with either staurosporine (50 nM) or CV3988 (1 microM). These results suggest that PAF is a mitogenic factor that contributes to the known increase in risk of malignancy associated with chronic inflammatory conditions.
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PMID:Receptor-mediated and protein kinase-dependent growth enhancement of primary human fibroblasts by platelet activating factor. 972 13

Recent studies have shown that cyclooxygenase exists in two isozyme forms. Since differences in the pharmacological profiles of nonsteroidal anti-inflammatory drugs (NSAIDs) might be accounted for by varying degrees of selectivity for these isozymes, cyclooxygenase-1 and -2, the relative potency of various NSAIDs in inhibiting their activities was examined in intact human cells. We used human platelets cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 for measuring cyclooxygenase selectivity. The presence of the enzymes was confirmed by immunoblotting and immunoprecipitation analysis, and by the reverse transcriptase-polymerase chain reaction. Mean IC50 values (microM) for human platelet cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 and cyclooxygenase-1/-2 IC50 ratio of various NSAIDs were as follows: aspirin, 3.2, 26, 0.12; diclofenac, 0.037, 0.00097, 38; etodolac, 122, 0.68, 179; ibuprofen, 3.0, 3.5, 0.86; indomethacin, 0.013, 0.044, 0.30; loxoprofen (active metabolite), 0.38, 0.12, 3.2; NS-398, 12, 0.0095, 1263; oxaprozin, 2.2, 36, 0.061; zaltoprofen, 1.3, 0.34, 3.8; respectively. Our bioassay system employing intact human cells to assess the cyclooxygenase selectivity of NSAIDs may provide clinically useful information.
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PMID:Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal anti-inflammatory drugs using human platelets and synovial cells. 965 Aug 52

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.
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PMID:ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist. 972 36

In vitro, prostaglandins (PG) have strong inhibitory effects on T cell activation and proliferation and inhibitors of PG synthesis (NSAID) increase proliferation and activation of T cells. Although most studies have failed to demonstrate cyclooxygenase (COX) activity in lymphocytes, there is contradictory evidence on the synthesis of different PG. We have studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot the expression of COX-1 and -2 mRNA and protein in resting and activated peripheral blood or Jurkat T cells. Cells were activated by T cell receptor triggering with OKT3 antibodies and activation confirmed by flow cytometric analysis of surface CD69. COX enzymatic activity was measured by determination of arachidonic acid (AA)-induced PG synthesis. Both peripheral blood and Jurkat T cells expressed COX-1 and -2 mRNA and protein. COX-1 was constitutively expressed and did not change after OKT3 stimulation. COX-2 was inducible upon OKT3-induced activation. In spite of the presence of COX mRNA and immunoreactive protein, AA-induced PG synthesis was not detected at the EIA detection (pM) level. The potential role of cyclooxygenases in T cells deserves further study, since no PG of the studied series seem to be synthesized by T cells.
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PMID:Cyclooxygenase-1 and -2 are expressed by human T cells. 993 24


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