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

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.
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PMID:Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes. 876 74

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexamethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method. Dexamethasone treatment increased Y1 mRNA in Y-1 cells, whereas the cAMP and ACTH decreased it. We also observed that the amount of Y1 receptor RNA was unaffected by phorbol 12-myristate 13-acetate, a protein kinase C stimulator, but was abolished in a cell line expressing apolipoprotein E (apoE). The results indicated that NPY-Y1 receptor mRNA in Y-1 cells is highly regulated by several intracellular messengers. The role of apoE in such regulation is of particular interest in view of evidence that the isoform of the molecule is highly correlated to the age of onset of Alzheimer's disease. The effect observed in the Y-1 cell line which expresses apoE may implicate a possible role of this protein in the process of neuronal death that occurred in the Alzheimer's disease.
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PMID:Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells. 879 89

Although adrenomedullin (AM) previously has been identified in human tumors, its role has remained elusive. Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed AM mRNA in 18 of 20 human normal tissues representing major organs, and 55 of 58 (95%) malignant cell lines. Western blot and high performance liquid chromatography analysis showed immunoreactive AM species of 18, 14, and 6 kDa that are consistent with the precursor, intermediate product, and active peptide, respectively. Immunohistochemistry and in situ RT-PCR performed on paraffin-embedded tumor cell lines of various tissue origins exhibited AM cytoplasmic staining. Neutralizing monoclonal antibody to AM inhibits tumor cell growth in a concentration-dependent manner, an effect that was reversed with the addition of exogenous AM. Responding tumor cells were shown to have approximately 50,000 AM receptors per cell by Scatchard analysis with 125I-AM and expressed AM receptor mRNA by RT-PCR. Our data showed 36 of 48 (75%) tumor cell lines expressed AM receptor mRNA by RT-PCR assessment, all of them also expressed AM. In the presence of AM, cAMP levels were shown to increase in tumor cells. Our collective data demonstrate that AM and AM receptor are expressed in numerous human cancer cell lines of diverse origin and constitute a potential autocrine growth mechanism that could drive neoplastic proliferation.
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PMID:Adrenomedullin expression in human tumor cell lines. Its potential role as an autocrine growth factor. 879 36

Beta 1- and beta 3-adrenoceptor mRNA and protein expression, and contribution of each subtype to the catecholamine-sensitive adenylyl cyclase system were studied during the adipose conversion of the murine 3T3-F442A cell line. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that emergence of beta 3-adrenoceptor transcripts was concomittant with that of the gene encoding adipsin, a very late marker of adipose differentiation. Conversely, the induction of the beta 1-adrenoceptor mRNA occurred early after cell commitment towards adipose conversion. Changes in beta-subtype gene expression were accompanied by parallel modifications in receptor expression and function. 125I-cyanopindolol saturation and competition binding experiments showed a 3-fold increase in beta 1-adrenoceptor density in day 3 post-confluent cells. The beta 3-subtype population became detectable later and represented approximately 95% of total beta-adrenoceptors in day 8 and day 12 post-confluent cells. Adenylyl cyclase activity in response to the beta 3-adrenoceptor-selective agonists CGP12177 (4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one), ICI201651 ([(R)-4-(2 hydroxy-3-phenoxypropylamino-ethoxy)-N-(2- methoxyethyl)phenoxy-acetamide]) and cyanopindolol was virtually absent in young adipocytes, but dramatically increased in mature cells. The respective contributions of the beta 1- and the beta 3-subtypes to the production of cAMP were resolved by an Eadie-Hofstee computer analysis of isoproterenol and norepinephrine concentration-response curve of adenylyl cyclase activity. Agonist response curves in the presence of beta 1- and beta 2-adrenoceptor antagonist indicated that the beta 1-subtype accounted for the totality of beta-adrenoceptor-mediated adenylyl cyclase activation in young adipocytes. In mature adipose cells approximately 90% of this response was due to an activation of the beta 3-adrenoceptor. In addition, approximately 84% of the maximal norepinephrine-stimulated lipolysis was mediated by the beta 3-adrenoceptor in fully differentiated adipocytes. The differentiation-dependent expression of beta-subtypes in adipocytes is a biphasic process involving an initial and moderate induction of beta 1-adrenoceptors followed by the emergence of a prominent beta 3-adrenoceptor population. Compared analysis of both receptor occupancy and cAMP production shows that the beta 3-subtype is more efficiently coupled to the adenylyl cyclase system than the beta 1-adrenoceptor. Thus in mature adipose cells this receptor subtype represents the core of cAMP-dependent regulation of the lipolytic, antilipogenic and thermogenic effects of catecholamines.
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PMID:Developmental expression and functional activity of beta 1- and beta 3-adrenoceptors in murine 3T3-F442A differentiating adipocytes. 885 Nov 74

Expression and function of the beta 2-adrenergic receptor (beta 2-AR), a critical modulator of motor function, is altered in ischemic tissues. However, the mechanism by which ischemia influences gene expression remains controversial, in part because of the conflicting results reported by numerous investigators. To determine the relative importance of hypoxia and acidosis on beta 2-AR expression and function, steady-state mRNA levels and receptor function were measured in DDT1MF-2 hamster smooth muscle cells grown in 10% serum and 3 nM epinephrine in 5% CO2 (pH 7.50) and then exposed for 48 h to either combined hypoxia with acidosis (through incubation in 2% O2, 10% CO2, mean pH 7.14 at 48 h), hypoxia alone (2% O2, 2.5% CO2, pH 7.36), normoxia-acidosis (21% O2, 10% CO2, pH 7.12) or continued normoxia (21% O2, 2.5% CO2, pH 7.49). Combined hypoxia-acidosis downregulated the beta 2-AR membrane density by 50% compared to hypoxia alone and normoxia alone at 48 h. beta 2-AR coupling in these cells, as measured by cellular cAMP production in response to 10(-4) M isoproterenol, was decreased by hypoxia but increased by acidosis. The effect of hypoxia-acidosis on Bmax was abolished by inhibiting transcription with 1.0 microgram/ml actinomycin D. A quantitative reverse transcriptase polymerase chain reaction assay demonstrated a decrease in steady-state mRNA concentration with hypoxia-acidosis. Our experiments demonstrate an important distinction between the effects of modeled hypoxia and ischemia on beta 2-AR gene expression.
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PMID:pH is critical to the regulation of expression of the beta 2-adrenergic receptor gene in hypoxia. 897 14

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.
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PMID:Induction of guanosine triphosphate-cyclohydrolase by follicle-stimulating hormone enhances interleukin-1 beta-stimulated nitric oxide synthase activity in granulosa cells. 897

PGF2 alpha is a metabolite of arachidonic acid that triggers regression of the corpus luteum. Recent animal studies have indicated that PGF2 alpha (FP) receptor messenger ribonucleic acid (mRNA) is expressed in the corpus luteum. To understand the regulation of the FP receptor in the ovary we have cloned a partial complementary DNA (cDNA) sequence of the FP receptor from human granulosa cells obtained from women undergoing in vitro fertilization. The sequence of this cDNA is identical to the previously reported FP receptor sequences obtained from human uterine and placental cDNA libraries. Low levels of the FP receptor mRNA were observed in freshly isolated granulosa cells or in cultured granulosa-luteal (GL) cells, as detected by reverse transcriptase-PCR. hCG and 8-bromo-cAMP increased the steady state levels of the FP receptor mRNAs after incubation for 24-48 h, as detected by Northern blot hybridization. The stimulatory effect of hCG was concentration and culture stage dependent. Further, hCG and 8-bromo-cAMP increased binding of radiolabeled PGF2 alpha to intact GL cells. In contrast, phorbol 12-myristate 13-acetate inhibited basal as well as hCG- and 8-bromo-cAMP-induced FP receptor mRNA expression and binding of the radiolabeled ligand. In summary, hCG, 8-bromo-cAMP, and phorbol 12-myristate 13-acetate modulate the expression of the FP receptor in human GL cells, which may represent a mechanism to regulate the responsiveness of the ovary to PGF2 alpha.
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PMID:Regulation of prostaglandin F2 alpha receptor expression in cultured human granulosa-luteal cells. 897 3

In previous reports, we have shown that cAMP-specific phosphodiesterase (PDE4) is the major PDE in the rat parotid gland, and that PDE4 is activated by phosphorylation. In this study, we investigated the expression of PDE4 isoform genes and alternative splicing variants of PDE4D in the rat parotid gland using reverse transcriptase-polymerase chain reaction (RT-PCR). PDE4A, PDE4B, PDE4C and PDE4D of PDE4 subfamily were expressed. PDE4D was found to be the dominant PDE4 isoform. A weak band of PDE4C was detectable. Three alternative splicing variants (PDE4D1, PDE4D2 and PDE4D3) derived from the rat PDE4D gene were expressed in the parotid gland. These data suggested that the intracellular cAMP level is regulated by multiple response mechanisms through the activations of the PDE by phosphorylation and gene expression in the rat parotid gland.
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PMID:Expression of mRNA encoding cAMP-specific phosphodiesterase isoforms in rat parotid glands. 898 29

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.
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PMID:Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. 899 2


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