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)

Tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor-alpha mediates induction of other cytokines, COX-2, PGs, and metalloproteinases, which leads to cartilage degradation. We developed an in vitro human synoviocyte assay system for screening inhibitors of proinflammatory mediators in herbal extracts. Synoviocytes (5 x 10(5) cells/well) obtained during primary knee replacement from osteoarthritic patients were incubated with: control media alone or ginger extract (hydroxy-methoxy-phenyl compounds [HAPC]: EV.EXT 77), 1 h before activation with 1 ng/ml TNF-alpha, 10 ng/ml interleukin-1beta, or control media alone at 5% carbon dioxide, 37 degrees C. Cell viability, TNF-alpha, COX-2, PGE-2, nuclear factor kappaB (NF-kappaB), and inhibitory subunit I kappa B-alpha (IkappaB-alpha) expression were analyzed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 microg/ml) significantly inhibited the activation of TNF-alpha and COX-2 expression in human synoviocytes as well as suppressed production of TNF-alpha and PGE-2. Inhibition of TNF-alpha and COX-2 activation was accompanied by suppression of NF-kappaB and IkappaB-alpha induction. Using our in vitro assay, we discovered that the ginger extract blocks activation of proinflammatory mediators and its transcriptional regulator suggesting its mode of action. These observations indicate that ginger extract-HAPC offers a complementary and alternative approach to modulate the inflammatory process involved in arthritis.
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PMID:An in vitro screening assay for inhibitors of proinflammatory mediators in herbal extracts using human synoviocyte cultures. 1531 68

Prostaglandins (PGs) formed via the cyclooxygenase (COX) pathway mediate hyperalgesia in sensory nerve endings. To investigate the role of the COX isoforms in pain transmission we recently studied nociception in COX-isozyme-deficient mice using models of "sharp" rapidly transmitted pain (hot-plate) and slowly developing, diffuse pain (writhing) [Ballou L, Botting RM, Goorha S, Zhang J, Vane JR. Nociception in cyclooxygenase isozyme-deficient mice. Proc Natl Acad Sci USA 2000;97:10272]. Our results demonstrated that COX-1 (and not COX-2) was the primary isoform involved in nociception in both model systems. Given the importance of dorsal root ganglia (DRG) in pain transmission we examined the expression patterns of COX-1, -2 and the recently described variant of COX-1 retaining intron-1, originally referred to as "COX-3" but hereafter referred to as COX-1 variant (COX-1v), in mouse L4 or L5 DRG taken from normal and COX-isozyme-deficient mice. Messenger RNA and protein for COX isoforms from DRG, spinal cord as well as, heart, brain, kidney, spleen and skin of adult mice were isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Patterns of COX-isoform expression were determined using immunohistochemical techniques. We found that COX-1 and COX-1v were both expressed in neurons while COX-2 expression was completely undetectable in the DRG. Immunohistochemical analysis of COX expression in DRG of mice exhibiting the chronic pain and inflammation associated with collagen-induced arthritis (CIA) expressed COX-1 and COX-1v while no COX-2 could be detected. For purposes of comparison, COX-1v mRNA was also expressed in heart, brain, spinal cord, kidney, spleen and skin. Together, these data support a role for COX-1 and perhaps COX-1v, not COX-2, as the primary producers of PGs in mouse DRG in normal and in mice subject to chronic pain and inflammation. These data also suggest potential alternative analgesic mechanisms of action for the newly developed, COX-2 selective inhibitors and the nonsteroidal anti-inflammatory drugs (NSAIDs) in pain transmission in the peripheral nervous system.
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PMID:Nociception and the differential expression of cyclooxygenase-1 (COX-1), the COX-1 variant retaining intron-1 (COX-1v), and COX-2 in mouse dorsal root ganglia (DRG). 1556 Jan 14

Melatonin attenuates acute gastric lesions induced by topical strong irritants because of scavenging of free radicals, but its role in the pathogenesis of stress-induced gastric lesions has been sparingly investigated. In this study we compared the effects of intragastric (i.g.) or intracerebroventricular (i.c.v.) administration of melatonin and its precursor, L-tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on gastric lesions induced by water immersion and restraint stress (WRS). The involvement of pineal gland, endogenous prostaglandins (PG) and sensory nerves in gastroprotective action of melatonin and L-tryptophan against WRS was studied in intact or pinealectomized rats or those treated with indomethacin or rofecoxib to suppress cyclooxygenase (COX)-1 and COX-2, respectively, and with capsaicin to induce functional ablation of the sensory nerves. In addition, the influence of i.c.v. and i.g. melatonin on gastric secretion was tested in a separate group of rats equipped with gastric fistulas. At 3.5 hr after the end of WRS, the number of gastric lesions was counted, the gastric blood flow (GBF) was determined by H2-gas clearance technique and plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for determination of expression of mRNA for COX-1 and COX-2 by reverse transcriptase-polymerase chain reaction (RT-PCR) and of the mucosal generation of prostaglandin E2 (PGE2) by RIA. Melatonin applied i.g. (1.25-10 mg/kg) or i.c.v. (1.25-10 microg/kg) dose-dependently inhibited gastric acid secretion and significantly attenuated the WRS-induced gastric damage. This protective effect of melatonin was accompanied by a significant rise in the GBF and plasma melatonin and gastrin levels and in mucosal generation of PGE2. Pinealectomy, which suppressed plasma melatonin levels, aggravated the gastric lesions induced by WRS and these effects were counteracted by i.g. or i.c.v. application of melatonin. Luzindole abolished completely the gastroprotective effects of melatonin and L-tryptophan and attenuated significantly the rise in GBF evoked by the indoleamine and its precursor. Indomethacin and rofecoxib, which diminished PGE2 biosynthesis by c. 90 and 75% or capsaicin denervation, attenuated significantly melatonin- and L-tryptophan-induced protection and the rise in the GBF. Both the protection and the hyperemia were restored by addition of exogenous CGRP to capsaicin-denervated animals. COX-1 mRNA was detected by RT-PCR in the intact and melatonin-treated gastric mucosa, while COX-2 mRNA, which was undetectable in the intact gastric mucosa, appeared in WRS-exposed mucosa, especially in the melatonin-treated animals and this was accompanied by increased generation of PGE2 in gastric mucosa. Pinealectomy downregulated COX-2 mRNA and this effect was reversed by supplementation of pinealectomized animals with melatonin. We conclude that, (a) exogenous melatonin and its precursor, L-tryptophan, attenuates WRS-induced gastric lesions via interaction with MT2 receptors, (b) this protective action of melatonin is because of an enhancement of gastric microcirculation, probably mediated by PGE2 derived from COX-2 overexpression and activity, the activation of brain-gut axis involving CGRP released from sensory nerves, and the release of gastrin and (c) the pineal plays an important role in the limitation of WRS-induced gastric lesions via releasing melatonin, which exerts gastroprotective and hyperemic activities against stress ulcerogenesis.
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PMID:Importance of the pineal gland, endogenous prostaglandins and sensory nerves in the gastroprotective actions of central and peripheral melatonin against stress-induced damage. 1620 93

Considering possible tumorigenic activity of cyclooxygenase (COX) isozymes in myeloma, we examined expression levels of COX-1 and -2 in seven human myeloma cell lines (ARH-77, IM-9, RPMI-8226, HPC, HS-Sultan, TSPC-1, and U-266). As analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), all the cell lines constitutively expressed COX-1, while COX-2 levels markedly varied among different cell lines. Induction of COX-2 by phorbol ester was observed in RPMI-8226 and HPC cells. In contrast, COX-2 was constitutively expressed in ARH-77 and IM-9 cells. Moreover, the high expression level of COX-2 protein in ARH-77 cells was verified by Western blotting. Intact cells of ARH-77 converted 14C-labeled arachidonic acid to prostaglandin E2, F2alpha, and D2, and this activity was dose-dependently inhibited by selective COX-2 inhibitors (SC-58125 and NS-398), a non-selective COX inhibitor (indomethacin), and relatively high concentrations of a selective COX-1 inhibitor (SC-560). These COX inhibitors also suppressed the proliferation of ARH-77 cells, but significant suppression was seen only at 100 microM, a much higher concentration than those sufficient for the COX inhibition. Moreover, proliferation of the myeloma cells lacking COX-2 was also suppressed by 100 microM of SC-58125. These results suggested that the anti-proliferative effect of the COX inhibitors is independent of the inhibition of COX-2.
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PMID:Cyclooxygenase isozymes are expressed in human myeloma cells but not involved in anti-proliferative effect of cyclooxygenase inhibitors. 1638 88

The number of older patients admitted to peritoneal dialysis (PD) programmes is growing. At the same time, there is increasing data about the role of mesothelial cells in determining the functional alteration of the peritoneum during PD. However, little is known about the functional changes accompanying the ageing process in mesothelial cells. We aimed to evaluate whether the aging process is accompanied by changes in some functional characteristic of the human peritoneal mesothelial cells (HPMC), which could account for the poor prognosis observed in old patients with PD. HPMCs were isolated from patients undergoing a nonurgent, nonseptic abdominal surgical procedure, without renal, vascular or inflammatory disease. Cytokine levels (by enzyme-linked immunosorbent assay (ELISA)), nitrates+nitrites, and cyclooxygenase (COX) activity (by a chemiluminescence assay), cytokines, COX, nitric oxide synthase (NOS), and nuclear factor (NF)-kappaB1, two messenger ribonucleic acid (mRNA) gene expressions (by reverse transcriptase (RT)-Multiplex PCR), COX, and NOS promoter gene activities, and NF-kappaB-dependent transcription (by transient transfection assays) were determined. Our data show a significant increase in cytokines, COX, and NOS activities, and mRNA expression of cytokines, COX-2, inducible nitric oxide synthase (iNOS) and precursors of NF-kappaB in HPMCs from old people. This was also the case for COX-2 and iNOS promoter gene activities and NF-kappaB-dependent transcription. There was a positive correlation between the age of the donor's cell and the proinflammatory profile of the HPMCs. Such age-dependent increase (around two-three times) is partially abolished by different antioxidant or free-radical scavengers. Thus, aging is accompanied by the presence of an inflammatory state in HPMCs, which involves the participation of different reactive oxygen species.
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PMID:Changes in the human peritoneal mesothelial cells during aging. 1640 21

Obstruction and stretch induce cyclooxygenase (COX)-2 expression and prostanoid synthesis in urinary tissues, causing pain, inflammation, hypercontractility, and cell proliferation. Our objective was to characterize acute COX-2 induction during in vivo ureteral obstruction, establish a cell culture model of urothelial stretch-induced COX-2 expression, and evaluate whether mechanotransduction could alter transcriptional and post-transcriptional regulation of COX-2. We performed laparoscopic unilateral ureteral ligation in pigs and allowed progression for 1, 2, 6, 24, or 48 h. We evaluated COX-2 expression with reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunoblotting. We cultured primary human urothelial cells on stretch plates, applied stretch for up to 48 h, and measured COX-2 expression by RT-PCR and immunoblotting, transcription with run-on assays, and mRNA stability with actinomycin mRNA decay assays. In vivo ureteral obstruction induced COX-2 expression 4-fold within 6 h, maintaining induction for 24 h. In cell culture, stretch induced COX-2 steady-state mRNA and protein within the first 3 h of stretch, maintaining this induction for over 6 h. Three hours of stretch doubled COX-2 transcription relative to unstretched controls and increased COX-2 mRNA half-life 3-fold. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in the urothelium and establish a primary urothelial cell culture model for the study of stretch-induced COX-2 mechanisms. This is also the first report to identify alterations in steady-state COX-2 mRNA having components of both transcriptional and post-transcriptional regulation of stretch-regulated COX-2. Future elucidation of COX-2 signaling may identify novel therapeutic targets for treating stretch and distension of urinary tissues.
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PMID:Evaluation of urothelial stretch-induced cyclooxygenase-2 expression in novel human cell culture and porcine in vivo ureteral obstruction models. 1650 12

In this study we determined the effect of cholinoceptor agonist pilocarpine on the stimulation of nitric oxide synthase (NOS) and on prostaglandin E2 (PGE2) generation upon rat dental pulp. By reverse transcriptase/polymerase chain reaction (RT-PCR) we identified several products corresponding to m1, m2, m3, and m4 muscarinic acetylcholine receptors (mAChRs). The stimulation of M1, M2, M3, and M4 mAChRs by pilocarpine increases NOS activity and PGE2 generation. There is a correlation (correlation coefficient=0.05) between NOS activity and PGE2 generation through the activation of phosphoinositide by phospholipase C (PLC), phospholipase A2 (PLA2), and cyclooxygenase 1 (COX-1). Exogenous PGE2 restored NOS activity inhibited by indomenthacin (INDO), whereas nitric oxide (NO) donor restored PGE2 generation inhibited by NG-methyl-L-arginine acetate salt (L-NMMA). These data indicate that both NO and PGE2 interact with their own respective biosynthetic pathways modulating NOS and COX activities. Results could contribute to understanding the involvement of NO and PGE2 in healthy dental pulp given that cellular signals through the parasympathetic system modulate the function of the dentin-pulp complex.
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PMID:Nitric oxide synthase and PGE2 reciprocal interactions in rat dental pulp: cholinoceptor modulation. 1725 32

The precise role of insulin-like growth factor (IGF)-1 in gastric ulcer healing is unknown. In experimental rat gastric ulcers, we examined expression of IGF-1 mRNA and protein by reverse transcriptase-polymerase chain reaction or enzyme-linked immunosorbent assay and immunostaining, respectively. In cultured rat gastric epithelial RGM1 cells, we examined effects of exogenous IGF-1 on cell migration, re-epithelialization, and proliferation-essential components of ulcer healing. We also examined whether IGF-1 induces cyclooxygenase (COX)-2 expression and determined the role of phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways in mediating IGF-1 actions. Gastric ulceration triggered an approximately threefold increase in IGF-1 expression in epithelial cells of the ulcer margins (P < 0.001 versus control), especially in cells re-epithelizing granulation tissue and in mucosa in proximity to the ulcer margin. Treatment of RGM1 cells with IGF-1 caused a dramatic increase in actin polymerization, an eightfold increase in cell migration (P < 0.001), a 195% increase in cell proliferation (P < 0.05), and a sixfold increase in COX-2 expression (P < 0.01). Inhibitor of phosphatidylinositol 3-kinase abolished IGF-1-induced RGM1 cell migration and proliferation, actin polymerization, and COX-2 expression. The up-regulation of IGF-1 in gastric ulcer margin accelerates gastric ulcer healing by promoting cell re-epithelization, proliferation, and COX-2 expression via the phosphatidylinositol 3-kinase pathway.
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PMID:Novel roles of local insulin-like growth factor-1 activation in gastric ulcer healing: promotes actin polymerization, cell proliferation, re-epithelialization, and induces cyclooxygenase-2 in a phosphatidylinositol 3-kinase-dependent manner. 1739 62

Sulindac exerts its antitumorigenic effects in oral squamous cell carcinoma (SCC) cells by modulating survivin in a Stat3-dependent manner. Immunohistochemistry was used to detect the protein levels of phosphorylated-tyrosine Stat3 (p-tyr Stat3) and survivin in SCC tissues. Western blot, reverse transcriptase polymerase chain reaction, Annexin-V and cell proliferation assays were used to determine p-tyr Stat3 and survivin protein and mRNA expression, and cell viability following treatment with cyclooxygenase (COX) inhibitors, Stat3 siRNA, or the forced expression of Stat3 or survivin. Immunohistochemical analysis revealed an overexpression of p-tyr Stat3 in T1 SCCs. The importance of constitutive Stat3 activation in tumourigenesis was confirmed by siRNA inhibition of Stat3, resulting in cell growth inhibition and apoptosis, via a downregulation of survivin mRNA and protein expression. The forced expression of survivin partially reversed these effects of Stat3 inhibition. Sulindac, but not other COX inhibitors, downregulated Stat3, which correlated to an inhibition of cell proliferation, survival and survivin expression. Transfection of constitutively active Stat3 restored survivin expression and partially rescued SCC cells from sulindac-induced antitumorigenic effects. These data indicate that survivin is a downstream target and effector of oncogenic Stat3 signalling in SCC, which is targeted by sulindac in a COX-2-independent manner.
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PMID:Survivin is a downstream target and effector of sulindac-sensitive oncogenic Stat3 signalling in head and neck cancer. 1756 5

Docking tools created for structure-based design and virtual screening have also been used to automate ligand alignment for comparative molecular field analysis (CoMFA). Models based on such alignments have been compared with those obtained based solely on shared ligand substructures, but such comparisons have generally failed to distinguish between conformational specification (alignment in the internal coordinate space) and embedding in a shared external frame of reference (Cartesian alignment). Here, large sets of inhibitors were docked into two cyclooxygenase and two reverse transcriptase crystal structures, and the poses generated were evaluated in terms of the CoMFA models they produced. Realigning the conformers obtained by docking by rigid-body rotation and translation to overlay their common substructures improved model statistics and interpretability, provided the protein structure used for docking was reasonably appropriate to the ligands being considered.
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PMID:A ligand's-eye view of protein binding. 1821 15


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