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)

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.
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PMID:Elevation of cAMP is required for down-regulation, but not agonist-induced desensitization, of endogenous dopamine D1 receptors in opossum kidney cells. Studies in cells that stably express a rat cAMP phosphodiesterase (rPDE3) cDNA. 839 59

Dopamine (DA) is known to modulate the post-synaptic response of the excitatory amino acid (EAA) neurotransmitters in the striatum. Thus the intrinsic neurons in this nucleus are potential sites of cross-interaction between these two systems. The recent isolation of 5 different DA receptor subtypes and more than 20 EAA subunits argues for a complicated functional role for the protein products encoded by these transcripts. The simultaneous detection of cellular mRNA distributions and translated protein products was an initial step to determine differences in post-translational expression at the cellular level of resolution for two of these receptors. The cloned D2 DA receptor subtype and the ionotropic GluR1 EAA receptor subunit were examined by fluorescence in situ transcription (FIST) following hybridization of specific cDNA primers, complementary to the mRNA transcripts encoding these receptors. Nascent extension of the annealed primer using reverse transcriptase was detected after incorporation of fluorescently labeled dUTP. Protein products were visualized by standard immunofluorescence after incubation with anti-peptide antisera that were selective for each receptor protein. The experimental data corroborate previous work describing the regional expression of ligand binding and in situ hybridization detected with radiolabeled probes for the DA and EAA receptor systems in the striatum. The dual fluorescence method can be completed within 2 days and may be adapted to cellular localization of many novel mRNA/protein combinations to examine post-translational processing within thin tissue slices.
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PMID:Co-expression of receptor mRNA and protein: striatal dopamine and excitatory amino acid subtypes. 879 41

Dopamine acts, under appropriate conditions, as a selective neurotoxin. This toxicity is attributed to the autoxidation of the neurotransmitter into a reactive quinone that covalently modifies cellular macromolecules (i.e. proteins and nucleic acids). The oxidation of the catecholamine to a quinone is greatly accelerated by the enzyme tyrosinase. There is controversy, however, as to whether or not tyrosinase is expressed in human brain. In the present study, RT-PCR was utilized to demonstrate the presence of tyrosinase mRNA in post-mortem human brain tissues. Using gene-specific amplification primers, specific tyrosinase amplicons were detected following analysis of RNA from substantia nigra of four individuals. Analysis of cerebellar RNA from the same individuals produced no amplification products. Control reactions performed in the absence of reverse transcriptase failed to generate PCR products for any tissue tested. Three amplicons were subjected to direct DNA sequencing and all proved to be identical with tyrosinase sequences, thus obviating the possibility of amplification of a related gene. It is clear, therefore, that the tyrosinase gene is expressed in the human substantia nigra, lending support to previous studies describing tyrosinase-like activity and immunoreactive protein in the brain. This enzyme could be central to dopamine neurotoxicity as well as contribute to the neurodegeneration associated with Parkinson's disease.
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PMID:Tyrosinase mRNA is expressed in human substantia nigra. 910 85

The reverse transcriptase-polymerase chain reaction (RT-PCR), in combination with 5' and 3' rapid amplification of cDNA ends (RACE), was used to clone a G protein-coupled receptor from turkey brain mRNA. This cDNA clone has an open reading frame of 1,311 base pairs encoding a 436-residue protein with seven transmembrane-spanning domains and exhibits high homology with previously cloned mammalian D2 dopamine receptors. Northern blot analysis of turkey brain mRNA detected an approximate 2.4-kb transcript. RT-PCR and subsequent nucleotide sequence analysis of turkey brain and peripheral tissue mRNA also demonstrated the presence of an alternatively spliced mRNA corresponding to the predicted D2 short isoform. RT-PCR experiments demonstrated a widespread distribution of alternatively spliced D2 dopamine receptor transcripts throughout the turkey brain and in select peripheral tissues as well. In situ hybridization experiments detected strong autoradiographic signals over much of the turkey telencephalon, diencephalon, mesencephalon, cerebellum, pituitary, and pineal gland. Dopamine has several important functions as a neurotransmitter and hormone in mammals and may have similar actions in avian species. The cloning and tissue distribution of the D2 receptor subtype should enable the investigation of any functional role dopamine and dopamine receptors exert on the physiology and behavior of birds.
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PMID:Molecular cloning and tissue distribution of an avian D2 dopamine receptor mRNA from the domestic turkey (Maleagris gallopavo). 1023 44

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

Prolactin (PRL) secretion is regulated by both inhibitory and stimulatory factors. Dopamine is the primary inhibitor, but multiple factors stimulate PRL gene expression and release. These can be divided into two categories: those that rapidly stimulate PRL release and those that induce the PRL gene followed by increased release. The pituitary intermediate lobe (IL) contains a PRL-releasing factor (PRF) that rapidly stimulates PRL release. From a mouse IL tumor, we established a non-melanotroph cell line, mIL5, which secretes a factor that stimulated PRL gene expression and release in vitro. This PRF activity did not rapidly stimulate PRL release and bound to heparin. Our objective was to examine the regulation of PRL by heparin-binding proteins and characterize the PRF activity produced by mIL5 cells. PRL gene expression and release was determined using GH3 cells, stably transfected with a PRL promoter/luciferase reporter (GH(3)/luc). After screening mIL5 cells by reverse transcriptase polymerase chain reaction, we found that they expressed two heparin-binding growth factors basic fibroblast growth factor (FGF-2) and heparin-binding epidermal growth factor (HB-EGF) which were considered strong candidates for PRL transcriptional regulatory activity. To determine whether the activity produced by mIL5 cells is attributed to FGF-2 or HB-EGF, three approaches were used: heparin-affinity chromatography, Western blotting, and immunoneutralization. The PRF activity in conditioned media eluted from heparin with 1 M NaCl whereas both FGF- 2 and HB-EGF eluted with >1 M NaCl. Neither growth factor was detectable in mIL5 cells by Western blotting. Antibodies directed against FGF-2 and HB-EGF, alone or together, did not abolish this activity from mIL5 cells. In conclusion, FGF-2 and HB-EGF are potent stimulators of PRL gene expression and release but do not account for most of the endogenous PRL gene activity in mIL5 cells. The distinct heparin-binding factor that stimulates PRL gene transcription remains to be identified.
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PMID:Prolactin regulation by heparin-binding growth factors expressed in mouse pituitary cell lines. 1266 66

Dopamine (DA) neurons located in the mammalian midbrain have been generally implicated in reward and drug reinforcement and more specifically in nicotine dependence. However, roles played by nicotinic acetylcholine receptors, including those composed of alpha7-subunits [alpha7-nicotinic acetylcholine receptors (nAChRs)], in modulation of DA signaling and in nicotine dependence are not clearly understood. Although midbrain slice recording has been used previously to identify functional alpha7-nAChRs, these preparations are not optimally designed for extremely rapid and reproducible drug application, and rapidly desensitized, alpha7-nAChR-mediated currents may have been underestimated or not detected. Here, we use patch-clamp, whole-cell current recordings from single neurons acutely dissociated from midbrain nuclei and having features of DA neurons to characterize acetylcholine-induced, inward currents that rapidly activate and desensitize, are mimicked by the alpha7-nAChR-selective agonist, choline, blocked by the alpha7-nAChR-selective antagonists, methyllycaconitine and alpha-bungarotoxin, and are similar to those of heterologously expressed, human alpha7-nAChRs. We also use reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemical staining to demonstrate nAChR alpha7 subunit gene expression as message and protein in the rat substantia nigra pars compacta and ventral tegmental area. Expression of alpha7 subunit message and of alpha7-nAChR-mediated responses is developmentally regulated, with both being absent in samples taken from rats at postnatal day 7, but later becoming present and increasing over the next 2 weeks. Collectively, this electrophysiological, pharmacological, and molecular evidence indicates that nAChR alpha7 subunits and functional alpha7-nAChRs are expressed somatodendritically by midbrain DA neurons, where they may play important physiological roles and contribute to nicotine reinforcement and dependence.
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PMID:Electrophysiological, pharmacological, and molecular evidence for alpha7-nicotinic acetylcholine receptors in rat midbrain dopamine neurons. 1517 98

Cyclooxygenases (COX) are associated with complex alteration in many pathologies of the central nervous system (CNS). Increased expression of COX-2 has been shown in injured or degenerated neurons, thus suggesting that COX-2 may contribute to neuronal damage. In this study, we present the expression of COX-1 and COX-2 mRNA and protein in striatum following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration to mice. MPTP causes an acute damage of dopaminergic neurons especially in the nigrostriatal dopaminergic system, thus diminishing dopamine (DA) content in striatum and decreasing the number of dopaminergic cells in the pars compacta of the substantia nigra (SN). C57Bl mice have received 60 mg/kg of MPTP introperitoneally. A group of mice received also rofecoxib 10 mg/kg from the 1st day following MPTP administration. Dopamine content in striatum (high-performance liquid chromatography-HPLC), mRNA expression of COX-1 and -2 (reverse transcriptase-polymerase chain reaction technique-RT-PCR), COX-1 and -2 protein content (immunoblotting) have been measured on day 1st, 3rd, 7th, 14th and 21st after the injury. We have found that COX-1 mRNA expression is not changed following MPTP administration, but COX-2 gene and protein expression in striatum increases from the 3rd to the 7th and 14th days, and diminishes on the 21st day. Production of prostaglandins is augmented only briefly after MPTP treatment and did not correlate with increased COX-2 mRNA and COX-2 protein production. Thus, the increase of COX-2 expression does not follow the acute stage of cell death but rather the recovery period after the injury. We also demonstrate that COX-2 activity inhibition by rofecoxib (10 mg/kg), which has been started 1 day after the injury, has not neuroprotective effect. Our study suggests that COX-2 does not contribute to neurons death following MPTP administration and that the inhibition of COX-2 activity is not beneficial to neurons injured by MPTP. However, COX-2 mRNA and protein expressions increase after MPTP injury; the role of these findings remains obscure.
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PMID:Cyclooxygenases mRNA and protein expression in striata in the experimental mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration to mouse. 1530 48

Dopaminergic cell loss in the mesencephalic substantia nigra is the hallmark of Parkinson's disease and may be associated with abnormal oxidative metabolic activity. However, the delicate balance underlying dopamine decline and oxidative stress is still a matter of debate. The aim of this study was to analyze the possible modulation of D2 agonists and antagonists on MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion) -induced cellular death in differentiated and undifferentiated PC12 cells. Using colorimetric assays, western blots and reverse transcriptase-PCR, we demonstrated that two D2 agonists, bromocriptine and quinpirole, consistently increased MPP+ -induced cytotoxicity in both differentiated and undifferentiated PC12 cells, whereas D2 antagonists do not modulate cell death. However, this increase in cellular death was reversed when bromocriptine or quinpirole were used in presence of D2 antagonists. On the other hand, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909), a potent inhibitor of the dopamine transporter, partially reversed MPP+ -induced cellular death and completely abolished the increase of cellular death induced by bromocriptine. Dopamine agonists and antagonists also modulate the expression of the dopamine transporter in PC12 cells; in particular, bromocriptine may alter MPP+ uptake by increasing DAT expression We also show that, in our cellular paradigm, D2 receptor mRNA levels are more abundant that D3 mRNA levels and MPP+ and /or bromocriptine could not modulate D2 gene expression while D3 gene expression clearly decrease after MPP+ and /or bromocriptine treatment.
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PMID:Dopamine D2 agonists, bromocriptine and quinpirole, increase MPP+ -induced toxicity in PC12 cells. 1700 Apr 68

Epidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-alpha and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection.
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PMID:Methamphetamine enhances HIV infection of macrophages. 1845 93


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