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)

The E-series prostaglandins (PGEs) are complex lipid regulators of B lymphocyte function. They inhibit the growth of certain B lymphoma lines. We report that heterogeneity with respect to PGE-induced growth inhibition correlates with the maturation state of the B cell lines. Specifically, the pre-B cell line 70Z/3 and the immature lymphoma CH31 are extremely sensitive to PGE2. To a lesser degree, other immature lymphomas (CH33, ECH408.1 and WEHI-231) are sensitive to PGE2. More mature lymphomas (BAL-17, CH12 and CH27) and fully differentiated myelomas (J558 and MOPC-315) are insensitive to PGE2. It is unknown what subtype of PGE receptor(s) (EPs) are expressed by B lymphocytes. It is also unknown if modulation of EP receptor expression could account for the differences in the sensitivity of these B cell lines to PGE2. To investigate these issues, reverse transcriptase polymerase chain reaction, Northern blot and DNA sequencing analyses were employed to obtain a definitive EP receptor subtype profile for these B cell lines, and for normal splenic B lymphocytes. Both normal and transformed B lymphocytes express mRNA encoding EP1, EP3beta and EP4 subtypes of PGE receptors. The B lineage cells do not express EP3alpha nor EP3gamma mRNA. The B cell lines are clonal, indicating that EP1, EP3beta and EP4 mRNA are coexpressed. Surprisingly, quantitative differences in basal EP1, EP3beta and EP4 expression were not observed between B cell lines despite their differing susceptibilities to PGE-induced growth inhibition. Conversely, the polyclonal activator LPS selectively upregulates EP4 mRNA expression in the mature B cell line CH12, but not in the LPS-sensitive pre-B cell line, 70Z/3. The activator LPS does not affect EP1 nor EP3beta mRNA expression. Treatment with dbcAMP, an analog of cAMP, mimics PGE-induced growth inhibition indicating that Gs-coupled EP2 and/or EP4 receptors mediate this inhibitory signal. Indeed, EP2 agonists mimic PGE2-induced growth inhibition unlike IP, EP1 and EP3-selective agonists. These data indicate that EP2 receptors are sufficient for mediating PGE-induced growth inhibition of susceptible B lineage cells.
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PMID:A molecular analysis of PGE receptor (EP) expression on normal and transformed B lymphocytes: coexpression of EP1, EP2, EP3beta and EP4. 860 22

The conditions necessary for evolution are amplification, mutagenesis and selection. Here we describe the evolutionary response of an in vitro replicating system to the selection pressure for fast growth and show what happens to the amplified molecules within this replication system. Our emphasis is on methodology, on the monitoring and the automation of experiments in molecular evolution. In order to perform in vitro studies on the evolution of RNA molecules, a modified self-sustained sequence replication (3SR) method was used. In the first step of the 3SR reaction, the RNA template is reversely transcribed by HIV-1 reverse transcriptase, followed by a second strand synthesis and the transcription of the resulting dsDNA by T7 RNA polymerase. The selection pressure (fast growth) was achieved by applying the principle of serial transfer pioneered in the laboratories of Sol Spiegelman and Leslie Orgel. At the end of the exponential growth phase of the 3SR reaction, an aliquot of the reaction mixture is transferred into a new sample containing only buffer, nucleotides and enzymes while RNA template molecules are provided by the transfer. The conditions in the exponential growth phase allow the RNA molecules to be amplified in a constant environment; all enzymes (HIV-1 reverse transcriptase and T7 RNA polymerase) and nucleotides are present in large excess. Therefore, transferring reproducibly within the exponential growth phase is equivalent to selecting for fast growth; those molecules which can replicate faster will displace others after several transfers. The experiments were performed using a serial transfer apparatus (STA) which allows the nucleic acid concentration to be monitored on-line by measuring the laser-induced fluorescence caused by intercalation of thiazole orange monomers into the RNA/DNA amplification products. The serial transfer experiments were carried out with an RNA template (220b RNA) that represents a 220-base segment of the HIV-1 genome and comprises the in vivo primer binding site (PBS) for the HIV-1 reverse transcriptase. It could be shown that after only two serial transfers two RNA species (EP1 and EP2) emerged that were much shorter. EP1 (48b) and EP2 (54b) were formed by deletion mutations within the original 220b RNA template in the very beginning of the serial transfer experiment; due to their higher replication rate (calculated from the growth curves derived on-line) these two deletion mutants displaced the original 220b RNA template in the course of the following thirty transfers. We assume that these two RNA species evolved independently of each other. Their formation was probably induced by a strand-transfer reaction of HIV-1 reverse transcriptase. Sequence analyses of these two evolution products seem to confirm such a presented pathway. 30 years after Spiegelman's experiment, the study described here is another answer to the question he posed: 'How do molecules evolve if the only demand is the biblical injunction: multiply?'. The answer, derived from a modified 3SR amplification system (mimicking a part of the HIV-1 replication cycle in vitro), is the same as thirty years ago: The RNA molecules adapt to the new conditions by throwing away any ballast not needed for fast replication. Clearly, this is only one aspect of molecular evolution; however, it shows that we should be careful in designating unidentified genetic material as 'junk DNA'.
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PMID:30 years later--a new approach to Sol Spiegelman's and Leslie Orgel's in vitro evolutionary studies. Dedicated to Leslie Orgel on the occasion of his 70th birthday. 939 69

1. The role of cyclo-oxygenase (COX) in the regulation of anion secretion (measured as short- circuit current, Isc) in cultured epididymal epithelia from immature rats was investigated. 2. COX inhibitors attenuated the increase of anion secretion caused by bradykinin (LBK) but had no effect on that caused by PGE2, suggesting that prostaglandin synthesis mediates the secretory response of the tissues to LBK. 3. The apparent IC50 values for indomethacin, piroxicam and L-745,337 in inhibiting the LBK-induced Isc were 0.14, 1.34 and 15.7 microM, respectively. This order of potency: indomethacin > piroxicam > L-745,337 >> DFU suggests the involvement of the COX-1 isozyme in the mediation of the secretory response to LBK. 4. Among the COX products (prostaglandins, thromboxane and prostacyclins) tested, only PGE2 and, to a much lesser extent, PGF2alpha stimulated anion secretion by cultured rat epididymal epithelia. 5. The effect of PGE2 was mimicked by 11-deoxyl PGE1, a specific prostaglandin E (EP)2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist, indicating that cyclic AMP-coupled EP2/4 receptors are involved in the LBK-stimulated anion secretion. 6. A reverse transcriptase-polymerase chain reaction study detected the expression of COX-1 and COX-2 mRNA in intact rat epididymis and in cultured epididymal epithelia. The expression of COX-1 mRNA was reduced by LBK by 44 %. 7. Immunohistochemical studies demonstrated the presence of COX-1 immunoreactivity in the basal cells of the intact rat epididymis. By comparison, COX-2 immunoreactivity was detected in the apical pole of the principal cells. 8. The role of COX in the formation of the epididymal microenvironment and the implication of long term administration of non-steroidal anti-inflammatory drugs (NSAIDs) on male fertility are discussed.
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PMID:Regulation of anion secretion by cyclo-oxygenase and prostanoids in cultured epididymal epithelia from the rat. 988 52

In the present study, we examined whether prostaglandin (PG) E2 and PGI2 regulated intercellular adhesion molecule-1 (ICAM-1) expression in human oral gingival epithelial cells stimulated with tumor necrosis factor alpha (TNF alpha). TNF alpha potently induced ICAM-1 expression in a dose- and time-dependent fashion. PGE2 and carbacyclin (a stable analogue of PGI2) significantly decreased ICAM-1 expression in TNF alpha-challenged oral gingival epithelial cells. Next, of the four subtypes of PGE2 receptors (EP1, EP2, EP3 and EP4), we examined which subtype(s) mediated inhibition of TNF alpha-induced ICAM-1 expression by PGE2. 11-deoxy-PGE2, an EP2/EP4 agonist, significantly suppressed TNF alpha-induced ICAM-1 expression, whereas butaprost, an EP2 agonist, sulprostone, an EP1/EP3 agonist, and ONO-AP-324, an EP3 agonist, caused no effect on it. By reverse transcriptase-polymerase chain reaction, expression of EP4 mRNA was detected in oral gingival epithelial cells. Dibutyryl cAMP, a cAMP analogue, and forskolin, a direct activator of adenylate cyclase, significantly inhibited TNF alpha-induced ICAM-1 expression in oral gingival epithelial cells. From these results, we suggest that PGE2 and PGI2 inhibit TNF alpha-elicited ICAM-1 expression by cAMP-dependent pathways via EP4 receptors and IP receptors, respectively.
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PMID:Prostaglandins E2 and I2 downregulate tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human oral gingival epithelial cells. 1115 20

To evaluate the role of the prostaglandin E receptor (EP) subtypes in the development of inflammatory synovitis, we examined EP subtype mRNA distribution in the synovial tissue of rats with adjuvant arthritis and the effect of selective EP agonists on cytokine production by cultured rat synovial cells. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization to measure the level of EP subtype (EP1, EP2, EP3, and EP4) mRNA expression in synovial tissues and cultured synovial cells from the arthritic joints of rats. RT-PCR and ELISA were used to analyse the effects of two selective EP agonists on IL-6 production by cultured rat synovial cells. EP2 and EP4 mRNA expression in inflamed synovial tissues was up-regulated. EP2 and EP4 mRNA were co-expressed in synovial macrophages and fibroblasts in inflamed tissues. EP4 and EP2 agonists both inhibited IL-1-induced IL-6 production. Our results suggest that prostaglandin E2 regulates the functions of synovial macrophages and fibroblasts through EP2 and EP4, which are induced by inflammatory stimuli in rats with adjuvant arthritis.
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PMID:Up-regulation of prostaglandin E receptor EP2 and EP4 subtypes in rat synovial tissues with adjuvant arthritis. 1120 65

Several prostaglandin analogues used for glaucoma treatment have been shown to cause increased iridial pigmentation as side-effect. In the present study we identified the types of prostanoid receptors and cyclooxygenase (COX) enzymes that are expressed in human iridial melanocytes isolated from eyes of different colours. Iris specimens were obtained during trabeculectomy surgery, or from enucleated eyes, and the iridial melanocytes were isolated and cultivated. The transcription of the DP, EP1, EP2, EP3, EP4, FP, IP and TP prostanoid receptor genes as well as the COX-1 and COX-2 enzyme genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). Of the prostanoid receptors the FP receptor gene was found to be most consistently transcribed in the melanocytes isolated from both blue- and hazel-coloured eyes. No RNA of the DP, EP2 and TP receptor genes could be detected, whereas the EP1, EP3, EP4 and IP receptor genes were found to be transcribed in melanocytes from some eyes. The COX-2 gene was found to be transcribed, but the COX-1 gene less consistently. There was no difference in gene transcription pattern between melanocytes originating from eyes treated with latanoprost, and eyes not previously treated with the prostaglandin. These results indicate that the FP prostanoid receptor gene is transcribed in cultivated human iridial melanocytes of both blue and hazel eyes, whereas the other prostanoid receptor genes seem to be transcribed much less frequently, or not at all. Surprisingly, the COX-2 rather than the COX-1 gene, was found to be transcribed in the melanocytes.
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PMID:Transcription of prostanoid receptor genes and cyclooxygenase enzyme genes in cultivated human iridial melanocytes from eyes of different colours. 1251 24

We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+-imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1-4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1), ONO-AE1-259 (EP2) and ONO-AE1-329 (EP4), had little or no effect on [Ca2+]i. All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.
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PMID:Mechanisms of cytosolic Ca2+ suppression by prostaglandin E2 receptors in rat melanotrophs. 1253 67

Prostaglandin E(2) (PGE(2)) can have pro- or anti-inflammatory effects, depending on engagement of different PGE(2) receptor (EP) subtypes. The role of EPs in regulating autoimmune inflammation was studied in the murine arthritis/lupus model induced by pristane. Peritoneal macrophages were isolated (biomagnetic beads) from BALB/c, DBA/1, or C57BL/6 mice treated with pristane (intraperitoneally, 3 months earlier) or thioglycolate (3 days earlier) or with untreated controls. EPs, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells were cultured unstimulated or stimulated with lipopolysaccharide (LPS) or LPS + interferon-gamma in combination with EP subtype-specific agonists. Tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 production was tested by enzyme-linked immunosorbent assay (culture supernatant) and flow cytometry. TNF-alpha mRNA levels also were examined. High levels of EPs (EP4/2>EP1>EP3), iNOS, and COX-2 mRNA were expressed in peritoneal macrophages from pristane-treated but not untreated or thioglycolate-treated mice (RT-PCR). TNF-alpha production was inhibited 50-70% at 2-24 h by EP4/2 agonists, whereas IL-6 was enhanced up to approximately 220%. TNF-alpha inhibition is mediated partly via the protein kinase A pathway and partly via IL-6. Intracellular TNF-alpha staining was inhibited 20% by EP4/2 agonists. TNF-alpha mRNA levels were inhibited 50-70% at 2-24 h, indicating that TNF-alpha inhibition was partly at the level of transcription. EP1/3 agonists had little effect. Synovial cells from mice with pristane-induced arthritis (DBA/1) also expressed EP2/4, and the EP2/4 agonist inhibited TNF-alpha production. PGE(2) can modulate inflammatory reactions via the EP2/4 receptor through its regulation of TNF-alpha and IL-6. Modification of EP signaling may be a new therapeutic strategy in inflammatory/autoimmune diseases.
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PMID:Prostaglandin E2 receptors EP2 and EP4 are up-regulated in peritoneal macrophages and joints of pristane-treated mice and modulate TNF-alpha and IL-6 production. 1507 56

The activation of glutamate receptors, particularly N-methyl-D-aspartate (NMDA) receptors, initiates ischemic cascade in the early stages of cerebral ischemia. Postischemia, cerebral ischemia is also associated with an inflammatory reaction that contributes to tissue damage. The up-regulation of neuronal cyclooxygenase-2 (COX-2) and elevation of prostaglandin E2 (PGE2) have been reported to occur after cerebral ischemic insult. We therefore studied whether the COX-2 reaction product PGE2 affects glutamate receptor-mediated cell death in cultured rat cortical cells. PGE2 was found to augment NMDA-mediated cell death. The transcription of EP1, EP2, EP3 and EP4 PGE2 receptor genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). EP1, EP2 and EP3 receptor genes were found in cortical cells. Butaprost (an EP2 agonist) markedly enhanced NMDA-mediated cell death, whereas 17-phenyl trinor-PGE2 (an EP1 agonist) and sulprostone (an EP3 agonist) had little effect. Both PGE2 and butaprost elevated cAMP intracellular levels in the cortical cells; moreover, forskolin, an activator of adenylate cyclase, enhanced NMDA-mediated cell death. These results suggest that PGE2, acting via EP2 receptors, aggravates excitotoxic neurodegeneration by a cAMP-dependent mechanism.
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PMID:Prostaglandin E2 deteriorates N-methyl-D-aspartate receptor-mediated cytotoxicity possibly by activating EP2 receptors in cultured cortical neurons. 1630 9

The aim of this study was to explore the effect of 0NO-54918-07, a stable prostacyclin analogue, on the current-voltage (IV) curve and the intracellular Ca2+ concentration [Ca2+]i of NG108-15 neuroblastoma x glioma hybrid cells. The IV curve was measured with ramp pulses from -70 to 0 mV, and [Ca2+]i was determined with Fura 2. Bath application of 0.2 muM ONO-54918-07 reversibly increased the holding current at -70 mV by -81.1 +/- 14.8 pA (mean +/- SEM, n = 35) and the slope of the IV curve between -70 and -50 mV by the factor 2.24 +/- 0.24. The effect of 0.2 microM prostaglandin PGE1 was similar (DeltaI (hold) = -96.1 +/- 29.9 pA, g/g (control) = 2.72 +/- 0.44, n = 9). ONO-54918-07 concentrations of 0.04, 2 and 6 microM were also effective. From the dose-response curve, the concentration for the half maximal effect was obtained as 0.054 microM. When cells did not respond to ONO-54918-07, an effect could sometimes be elicited by a ramp pulse or by a second ONO-54918-07 application 30-50 min after the first. The effect of ONO-54918-07 was not affected by pre-treatment with the EP1 antagonists ONO-8713 or SC-51089. However, a 14-40 min pre-treatment with 1 microM RO3244794, a selective prostacyclin receptor (IP) antagonist, abolished the effect of 0.2 microM PGE1. The effect of 0.2 microM ONO-54918-07 vanished completely in the presence of 5 microM RO32446794. ONO-54918-07 and PGE1 produced a slow increase in [Ca2+]i that lasted at least 6 min. Delta[Ca2+]i induced by both substances reached approximately 12% of the peak Delta[Ca2+]i induced by application of bradykinin. In only a few cells, PGE1 produced a brief, transient rise of [Ca2+]i. Using reverse transcriptase polymerase chain reaction, a prominent expression of the IP was detected in NG108-15 cells. It is concluded that ONO-54918-07 mimics the effect of PGE1, supporting the notion that the PGE1 effect on NG108-15 cells is mediated by IP receptors.
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PMID:ONO-54918-07, a stable prostacyclin analogue, mimics the effect of prostaglandin PGE1 on NG108-15 cells. 1795 10


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