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)

Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells myosin V is expressed and treatment of melanocytes with the cyclic AMP-inducer 3-isobutyl-1-methylxanthine causes an induction of the myosin V message. In all cells myosin V colocalizes with actin bundles, concentrating in the subcortical cell zone. In the melanocyte it is closely associated with melanosomes. Quantitative analysis of myosin V labeling in melanocytes reveals a significantly higher (p < 0.005) presence of myosin V in the periphery of dendrites. These results suggest that myosin V is important in melanosome transport in human melanocytes. Possible roles in the other skin cells remain to be elucidated.
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PMID:Myosin V colocalizes with melanosomes and subcortical actin bundles not associated with stress fibers in human epidermal melanocytes. 980 47

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

From brain, heart and muscle tissue of the tree shrew (Tupaia belangeri), a higher order mammal, cDNA clones were isolated that encoded two functional splice variants of the corticotropin-releasing factor (CRF) type 2 receptor (CRF-R2). The first, full-length splice variant, amplified from brain and heart tissue, encoded a CRF receptor protein that is 410 amino acids in length and approximately 96% homologous to human CRF-R2alpha. The second, full-length splice variant, derived from skeletal muscle tissue, encoded a 437-amino acid CRF receptor protein that is approximately 92% homologous to human CRF-R2beta. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) amplifications and RNase protection analyses, showed that tree shrew CRF-R2alpha (tCRF-R2alpha) and tree shrew CRF-R2beta (tCRF-R2beta) were coexpressed in brain tissue but not in heart and skeletal muscle tissue. Finally, human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R2alpha and tCRF-R2beta were used to demonstrate that the CRF analogs urocortin and sauvagine bind with significantly greater affinity (21- to 140-fold) to these two CRF-R2 splice variants than do human/rat and ovine CRF analogs. In keeping with these results of our CRF binding studies, EC50 values were substantially lower for urocortin-and sauvagine-stimulated than for h/rCRF-and oCRF-stimulated cyclic AMP accumulation in HEK293 cells stably transfected with tCRF-R2alpha or tCRF-R2beta cDNAs. The tree shrew therefore constitutes an important animal model in which to investigate the role of CRF receptor subtypes in the stress response.
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PMID:Isolation and pharmacological characterization of two functional splice variants of corticotropin-releasing factor type 2 receptor from Tupaia belangeri. 1033 22

The alpha2-adrenoceptor mediating inhibition of forskolin-stimulated cyclic AMP accumulation in human neuroblastoma SH-SY5Y cells was further characterized. The alpha2-adrenoceptor agonists, UK 14,304 (5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline), oxymetazoline, guanfacine, (-)-noradrenaline and clonidine concentration-dependently decreased cyclic AMP accumulation in this cell line (Emax ca. 50% inhibition). Agonist pEC50 values ranged between 6.7 and 7.8. Clonidine was a partial agonist. The effects of UK 14,304 were blocked after a pertussis toxin treatment. The concentration-response curves of UK 14,304 were shifted to the right in a parallel manner by the following antagonists (mean pK(B) values): yohimbine (8.17), idazoxan (7.63), prazosin (6.66), 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2 H,4H) isoquinolindione (ARC 239; 7.12) and 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB-4101; 8.12). The relatively high pKB values of prazosin and ARC 239 point to a non-alpha2A-adrenoceptor-mediated effect. The relatively high pK(B) value of WB-4101 further characterizes the alpha2-adrenoceptor in SH-SY5Y cells as being of the alpha2C subtype. The analysis of the expression of alpha2-adrenoceptor subtypes by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the exclusive presence of alpha2C-adrenoceptor mRNA in SH-SY5Y cells. We propose that inhibition of forskolin-stimulated cAMP accumulation in SH-SY5Y cells be used as a functional model of human, native alpha2C-adrenoceptors.
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PMID:Functional alpha2C-adrenoceptors in human neuroblastoma SH-SY5Y cells. 1037 21

Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
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PMID:Glucose metabolism in Chlamydia trachomatis: the 'energy parasite' hypothesis revisited. 1041 34

The hydrolysis of the second messenger cyclic AMP (cAMP) by phosphodiesterase 3 (PDE3) is known to play an important regulatory role in the context of relaxation of cavernous smooth muscle of the penis. Thus, we investigated the PDE3A isoform from penile cavernous tissues of male patients with and without symptoms of erectile dysfunction at the molecular biological level. As revealed by reverse transcriptase polymerase chain reaction, of all tissues of the urogenital tract analyzed the expression of the PDE3A gene was highest in the corpus cavernosum. However, significant differences in the levels of gene expression were not found between the two subgroups of patients. Also, the determined nucleotide sequences of the cloned penile PDE3A cDNAs of all patients were absolutely identical. Surprisingly, some deviations could be detected in the cDNA sequences of PDE3A from human myocard and platelets. The data obtained indicate that neither the expression levels nor the sequence deviations of PDE3A are the main reasons for erectile dysfunction in men.
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PMID:Molecular biological characterization of phosphodiesterase 3A from human corpus cavernosum. 1042 99

1. In this study reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. 2. Using RT-PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. 3. At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70+/-0.31 (control) vs 8.73+/-0.16, n=6) nor 60 nM 2-benzyl-NV-pentanoyltryptamine (pEC50= 8.53+/-0.20 (control) vs 8.83+/-0.38, n = 6) significantly altered the potency of melatonin in the rat tail artery. 4. At concentrations non-selective for mt1 and MT2 receptors. 4-P-PDOT (3 microM) and 2-benzyl-N-pentanoyltryptamine (5 microM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17+/-0.16, n=8) is similar to the binding affinity for mt1 receptor. 5. Pre-incubation with 1 microM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95+/-0.19% conversion (control) vs 0.92+/-0.19%, n=4) or following exposure to 30 microM forskolin (5.20+/-1.30% conversion (control) vs 5.35+/-0.90%, n=4). 6. Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.
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PMID:Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats. 1043 7

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.
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PMID:Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors. 1068 1

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.
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PMID:P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells. 1069 14

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca(2+) signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line. BzATP- and ATP-induced [Ca(2+)](i) responses had EC(50) values of 18 and 651 microM, respectively. The maximal response to ATP was only 59+/-8% of that for BzATP. The BzATP-induced [Ca(2+)](i) increase was dependent upon the extracellular Ca(2+) concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP- and BzATP-induced [Ca(2+)](i) increases. Both BzATP and ATP induced depolarization in GH3 cells, with EC(50) values of 31 microM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152+/-21 and 146+/-16% of that elicited by 30 mM KCl. The rank order of agonist potency for [Ca(2+)](i) and depolarization responses was BzATP > > ATP >2-MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor alpha, beta-methylene ATP showed any effect. In low-divalent conditions BzATP evoked non-desensitizing inward currents, which were reversed at approximately 0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X(7) mRNA on GH3 cells by using reverse transcriptase (RT)-polymerase chain reaction (PCR). These results suggest that the GH3 cells have an endogenous P2X(7) receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells.
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PMID:ATP-induced [Ca(2+)](i) changes and depolarization in GH3 cells. 1095 73


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