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)

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein required, in peripheral cells, for the activation of 5-lipoxygenase (5-LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5-LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5-LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK-886 (0.25-1 microM) prevents the somatostatin-induced augmentation of the hippocampal K+ M-current. Our results provide necessary evidence for the presence and signaling role of 5-LO and FLAP in central neurons and strongly support their proposed participation in somatostatin-receptor transmembrane signaling.
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PMID:Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling. 852 47

1. The Ca2+ permeability of non-NMDA and NMDA receptor channels was studied using a fluorometric flux measurement approach in somata and dendrites of CA1 pyramidal neurones in rat hippocampal slices. For this purpose, the Ca2+ fraction of the total cation current (named 'fractional Ca2+ current') was measured directly from the change in the Ca(2+)-sensitive fura-2 fluorescence at 380 nm excitation wavelength. 2. The fractional Ca2+ current through the somatic NMDA receptor channels was 10.69 +/- 2.13% (mean +/- S.D.) and that through dendritic receptor channels was 10.70 +/- 1.96%. The fractional Ca2+ current was not dependent on the extracellular Mg2+ concentration and its voltage dependence was in agreement with the Goldman-Hodgkin-Katz current equation. 3. AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) or kainate applications produced small but significant Ca2+ entry. Fractional Ca2+ currents of 0.58 +/- 0.34% were measured for somatic AMPA applications, 0.68 +/- 0.20% for somatic kainate applications, 0.66 +/- 0.25% for dendritic AMPA applications and 0.61 +/- 0.16% for dendritic kainate applications. 4. The expression pattern of glutamate receptor subunits encoding messenger ribonucleic acids (mRNAs) was analysed with the single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) approach applied to CA1 pyramidal neurones. The AMPA receptor subunits GluR-A, GluR-B and GluR-C, and the NMDA receptor subunits NR2A and NR2B were found to be abundantly expressed in all CA1 pyramidal neurones tested. 5. This study establishes the fractional Ca2+ current through somatic and dendritic NMDA and non-NMDA receptor channels in CA1 pyramidal neurones. The dendritic, presumably synaptic, NMDA receptor channels are highly Ca2+ permeable and have a fractional Ca2+ current closely resembling that of somatic extrasynaptic NMDA receptor channels. Both somatic and dendritic non-NMDA receptor channels are of the 'low Ca2+ permeable' type and have a fractional Ca2+ current that is about twenty times smaller than that of NMDA receptor channels.
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PMID:Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones. 881 9

Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.
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PMID:Increase in mRNAs encoding neonatal II and III sodium channel alpha-isoforms during kainate-induced seizures in adult rat hippocampus. 907 59

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.
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PMID:Distribution of GABA receptor rho subunit transcripts in the rat brain. 975 43

Considerable evidence indicates a critical role for dopamine in the reinforcing effects of cocaine. Because dopamine has been shown to be a critical modulator of gap junction communication in both eye and brain, we sought to examine whether extended intravenous cocaine self-administration would affect the expression of gap junction channel-forming proteins (connexins). Using ELISA, Western analysis, immunohistochemistry, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and non-radioactive in situ hybridization, we demonstrate that withdrawal from chronic cocaine self-administration causes lasting changes in connexin32 (Cx32) expression in the nucleus accumbens and hippocampus at 2, 7 and 21 days after the last cocaine injection. A sustained decrease in Cx32 protein and mRNA levels is noted in areas that have been implicated in cocaine craving (i.e. nucleus accumbens and subfields of the hippocampal formation). A progressive increase in gap junction protein and mRNA expression is noted in areas that become hyperexcitable after chronic cocaine exposure (i.e. CA1 hippocampal neurons). We speculate that gap junction communication may be critically involved in reinforcement processes and neuroadaptive changes produced by drugs of abuse.
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PMID:Long-term changes in connexin32 gap junction protein and mRNA expression following cocaine self-administration in rats. 1051 Jan 98

L-type voltage-sensitive Ca(2+) channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its alpha(1D) pore-forming subunit. If so, alpha(1D) mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated ("zipper") hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase-PCR analysis of alpha(1D) and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.
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PMID:Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices. 1075 53

Our laboratory has developed a competitive reverse transcriptase polymerase chain reaction (RT-PCR) procedure to analyse the mRNA expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in single cells. By the use of an internal RNA standard competing equally with the four subunit's mRNA, we have analysed 283 whole single hippocampus CA1 cells from adult rat brain. The cells were sampled from three groups of animals: one control group, one group subjected to preconditioning ischemia, and one group subjected to global cerebral ischemia. After reverse-transcription and PCR-amplification of mRNA in the cells, the PCR product was digested using subunit specific endonucleases and quantified by Cy-5 fluorescence. The median mRNA copy numbers achieved from control rats were 290, 247, 207, and 16 GluR1-4, respectively.
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PMID:Competitive quantitative measurement of the AMPA receptor gene expression at the single cell level. 1211 75

Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.
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PMID:In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons. 1248 Jan 75

Recent studies indicate that bone morphogenetic protein-4 (BMP4) and Noggin not only play an important role in the early development of the nervous system, but may also play a role in postnatal central nervous system (CNS) development. In this study, we examined the relative levels and localization of Noggin and BMP4 mRNA in the hippocampus of rats of different developmental stages with reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). RT-PCR showed that the temporal changes in the levels of expression of Noggin and BMP4 were different. The peak level of expression of Noggin mRNA was observed at embryonic day 13 (E13), subsequently gradually declined at 1-3 months (P1-3M) postnatal, and was detected only at a low level at P18M. In contrast, the expression of BMP4 mRNA increased gradually during embryonic development, reached a maximal level at 3 weeks (W) postnatal, and declined only slightly through P18M. In situ hybridization revealed that at embryonic stages, Noggin mRNA was localized throughout all hippocampal regions, whereas at early postnatal ages, Noggin mRNA was primarily localized in the anterior subiculum. At embryonic and early postnatal stages, no significant BMP4 mRNA expression was detectable in the hippocampus. At later postnatal ages, however, Noggin and BMP4 mRNA expression was observed in similar patterns. At 1-3 months postnatal, expression of BMP4 was observed mainly in the dentate gyrus and in the CA1-CA3 pyramidal cell layers. Lower hybridization signals were observed in the hilus and subiculum. Taken together, our results demonstrate that Noggin and BMP4 are expressed in embryonic and postnatal hippocampus, and that the temporal and spatial patterns of their expression are developmentally regulated. These data suggest that Noggin and BMP4 play important roles in hippocampal development.
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PMID:Spatial and temporal patterns of expression of Noggin and BMP4 in embryonic and postnatal rat hippocampus. 1464 11

Recent studies indicate that Noggin not only plays an important role in the early development of the nervous system, but may also plays a role in postnatal central nervous system (CNS) development. In this study, we examined the relative levels and localization of Noggin mRNA in the hippocampus of rats of different developmental stages with reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR showed that the peak level of expression of Noggin mRNA was observed at embryonic day 13 (E13), subsequently gradually declined at 1-3 months (P1-3M) postnatal, and was detected only at a low level at P18M. In situ hybridization revealed that at embryonic stages, Noggin mRNA was localized throughout all hippocampal regions, whereas at early postnatal ages, Noggin mRNA was primarily localized in the anterior subiculum. At later postnatal ages, Noggin mRNA expression was obviously observed in the dentate gyrus and in the CA1-CA3 pyramidal cell layers. Taken together, our results demonstrate that Noggin is expressed in embryonic and postnatal hippocampus, and that the temporal and spatial patterns of its expression is developmentally regulated.
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PMID:A combined in situ hybridization and RT-PCR method to detect spatial and temporal patterns of Noggin gene expression in embryonic and postnatal rat hippocampus. 1517 92


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