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)

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.
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PMID:Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech. 1271 4

Large-conductance, voltage- and calcium-activated potassium (MaxiK) channels play a key role in cell excitability. MaxiK channels are composed of a pore-forming alpha-subunit and a regulatory beta-subunit, of which four (beta1-4) genes have been identified. Previous findings suggested that MaxiK channel activity is regulated by estradiol. However, the underlying mechanisms have remained incompletely documented. Therefore, we used reverse transcriptase polymerase chain reaction to clone four cDNA fragments that were specific to the guinea pig alpha, beta1, beta2, and beta4 genes. Using a sensitive ribonuclease protection assay, we found that the alpha and beta4 mRNAs were the most abundant mRNAs in the brain and pituitary, whereas in the aorta, the alpha-subunit was coexpressed with the beta1-subunit. Moreover, there was a significant upregulation of the alpha- but not the beta1-subunit mRNA and the alpha-subunit protein in the aorta of the estrogenvs oil-treated ovariectomized animals. In specific brain areas including preoptic area, ventral hypothalamus, hippocampus, and amygdala, and in the pituitary, neither the alpha- nor beta4-subunit mRNAs were affected by estrogen. These findings suggest that estrogen may not affect the mRNA expression of MaxiK channels in the brain and pituitary. However, estrogen causes increased expression of MaxiK alpha in the aorta, which may explain some of the cardioprotective effects of estrogen in women.
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PMID:Effect of 17beta-estradiol on mRNA expression of large- conductance, voltage-dependent, and calcium-activated potassium channel alpha and beta subunits in guinea pig. 1272 1

The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.
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PMID:Latent herpes simplex virus infection of sensory neurons alters neuronal gene expression. 1291 67

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36-EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36-EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.
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PMID:Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina. 1505 12

Five viral RNA transcripts have recently been detected in purified virions of human cytomegalovirus (HCMV) strain AD169, a well-characterized member of the family Herpesviridae [Science 288 (2000) 2373]. While the function of these transcripts and/or the proteins they encode remains to be elucidated, it is not known whether these transcripts are unique to strain AD169 or are present in other HCMV strains. The objective of this study was to determine if these RNAs are present in other HCMV laboratory strains (Towne and Davis), and a low passage clinical isolate (CL203). These strains of CMV were purified by sequential ultracentrifugation through 20% D-sorbitol and glycerol-potassium tartarate gradients and the morphology and infectivity of the virions confirmed by electron microscopy and inoculation into cell culture. When RNA extracted from the purified virions was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) the UL 21.5 and TRL/IRL 2-5 transcripts were detected in virions of HCMV strains AD169, Davis, Towne and CL203. The presence of the UL 21.5 and TRL/IRL 2-5 RNA transcripts in all strains tested demonstrates that the packaged transcripts occurs in all strains of HCMV suggesting that they may have a relevant role in the biology of this virus.
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PMID:Detection of RNA in purified cytomegalovirus virions. 1524 50

Discovery of "regulators of G-protein signaling" (RGS) as GTPase-activating proteins for heterotrimeric G proteins has provided a highly sought "missing link," reconciling past discrepancies between the in vitro GTPase activity of purified G proteins and the kinetics of physiological responses mediated by G-protein signaling in vivo. With the number of RGS genes in the mammalian genome at more than 30, associating specific RGS proteins to specific G-protein-coupled receptor (GPCR) signaling events has become a focus of RGS investigators. The ubiquitous expression of multiple RGS proteins has complicated this effort, yet the outlook has been encouraged with the identification of RGS9 as the determinant mediating rapid recovery of the transducin-dependent photoresponse. G-protein-gated inwardly rectifying potassium (GIRK) channels that mediate inhibitory synaptic transmission via GPCR activation of pertussis toxin-sensitive G proteins are similarly accelerated by RGS proteins when reconstituted in heterologous cell expression systems and fully reproduce the gating properties of native GIRK channels in neurons and cardiomyocytes. The endogenous neuronal and cardiac RGS protein(s) that accelerate GPCR-->GIRK channel-gating kinetics are currently not known. This article describes methods used to measure the receptor-dependent GIRK channel-gating parameters reconstituted in Chinese hamster ovary (CHO-K1) cells and Xenopus oocytes, as well as rat atrial myocytes and rat cerebellar granule neurons as model cells with native GPCR-->GIRK channel signaling. Applications of these methods for structure-function-based studies of RGS proteins, G proteins, and GPCRs are discussed. We also describe single cell reverse transcriptase polymerase chain reaction methods developed to profile atrial myocyte and neuronal RGS expression to identify specific RGS proteins for targeted knockdown or knockout.
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PMID:Measuring the modulatory effects of RGS proteins on GIRK channels. 1531 64

Functional ATP-sensitive potassium (K(ATP)) channels can be reconstituted by expression of various combinations of different pore-forming subunits (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR) subunits. Using dominant negative and gene knockout approaches, Kir6.2 subunits have been identified as required pore-forming components of plasmalemmal K(ATP) channels in ventricular myocytes. Previous data obtained in heterologous expression systems suggest that Kir6.1 and Kir6.2 subunits are capable of forming a functional heteromultimeric channel complex. However, until now the existence of such heteromultimeric Kir6.1/Kir6.2 complexes has not been demonstrated for native K(ATP) channels. The primary aim of this study was to identify the molecular composition of native K(ATP) channels in primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) from human origin. We specifically investigated the potential that heteromultimeric Kir6.1/Kir6.2 channels exist in these cells. Using reverse transcriptase-polymerase chain reaction, we detected the expression of Kir6.1, Kir6.2, and SUR2B in both cell types. Western blotting and immunoprecipitation assays demonstrated the presence of Kir6.1 protein in both HCAEC and HCASMC; however, Kir6.2 protein was only expressed in HCAEC. Interaction between Kir6.1 and Kir6.2 subunits was demonstrated by reciprocal co-immunoprecipitation of these two subunits in HCAEC. Furthermore, Kir6.1 and Kir6.2 were detected in the immunoprecipitate when using an anti-SUR2 antibody. Confocal microscopy imaging demonstrated Kir6.1 and Kir6.2 subunits to co-localize at the cell surface membrane in HCAEC. In conclusion, our data characterize the molecular composition of primary human coronary smooth muscle and endothelial cells. We demonstrate that human coronary endothelial K(ATP) channels consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits.
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PMID:K ATP channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits. 1538 Jun 76

After intracerebral hemorrhage (ICH), many changes of gene transcription occur that may be important because they will contribute to understanding mechanisms of injury and recovery. Therefore, gene expression was assessed using Affymetrix microarrays in the striatum and the overlying cortex at 24 h after intracranial infusions of blood into the striatum of adult rats. Intracerebral hemorrhage regulated 369 of 8,740 transcripts as compared with saline-injected controls, with 104 regulated genes shared by the striatum and cortex. There were 108 upregulated and 126 downregulated genes in striatum, and 170 upregulated and 69 downregulated genes in the cortex. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed upregulation of IL-1-beta, Lipcortin 1 (annexin) and metallothionein 1,2, and downregulation of potassium voltage-gated channel, shaker-related subfamily, beta member 2 (Kcnab2). Of the functional groups of genes modulated by ICH, many metabolism and signal-transduction-related genes decreased in striatum but increased in adjacent cortex. In contrast, most enzyme, cytokine, chemokine, and immune response genes were upregulated in both striatum and in the cortex after ICH, likely in response to foreign proteins from the blood. A number of these genes may contribute to brain edema and cellular apoptosis caused by ICH. In addition, downregulation of growth factor pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway could also contribute to perihematoma cell death/apoptosis. Intracerebral hemorrhage-related downregulation of GABA-related genes and potassium channels might contribute to perihematoma cellular excitability and increased risk of post-ICH seizures. These genomic responses to ICH potentially provide new therapeutic targets for treatment.
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PMID:Brain genomics of intracerebral hemorrhage. 1603 71

The role of histamine in regulating excitability of sympathetic preganglionic neurons (SPNs) and the expression of histamine receptor mRNA in SPNs was investigated using whole-cell patch-clamp electrophysiological recording techniques combined with single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in transverse neonatal rat spinal cord slices. Bath application of histamine (100 microM) or the H1 receptor agonist histamine trifluoromethyl toluidide dimaleate (HTMT; 10 microM) induced membrane depolarization associated with a decrease in membrane conductance in the majority (70%) of SPNs tested, via activation of postsynaptic H1 receptors negatively coupled to one or more unidentified K+ conductances. Histamine and HTMT application also induced or increased the amplitude and/or frequency of membrane potential oscillations in electrotonically coupled SPNs. The H2 receptor agonist dimaprit (10 microM) or the H3 receptor agonist imetit (100 nM) were without significant effect on the membrane properties of SPNs. Histamine responses were sensitive to the H1 receptor antagonist triprolidine (10 microM) and the nonselective potassium channel blocker barium (1 mM) but were unaffected by the H2 receptor antagonist tiotidine (10 microM) and the H3 receptor antagonist, clobenpropit (5 microM). Single cell RT-PCR revealed mRNA expression for H1 receptors in 75% of SPNs tested, with no expression of mRNA for H2, H3, or H4 receptors. These data represent the first demonstration of H1 receptor expression in SPNs and suggest that histamine acts to regulate excitability of these neurons via a direct postsynaptic effect on H1 receptors.
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PMID:Histamine excites neonatal rat sympathetic preganglionic neurons in vitro via activation of H1 receptors. 1635 29

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(-)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(-) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(-) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(-) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(-) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling.
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PMID:Mini-dystrophin expression down-regulates overactivation of G protein-mediated IP3 signaling pathway in dystrophin-deficient muscle cells. 1644 5


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