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)

Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor channels. We now show by reverse transcriptase-polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non-NMDA glutamate receptor genes (GluR1-7, Ka-1, and Ka-2) and that four of these genes (GluR2, GluR6, GluR7, and Ka-1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non-NMDA glutamate receptor channels are readily isolated from NT2-derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2-derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. We expect that the NT2-derived human neuronal culture system will facilitate studies of human neuronal non-NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.
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PMID:Expression of non-NMDA glutamate receptor channel genes by clonal human neurons. 751 97

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.
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PMID:Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage. 753 52

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

We evaluated expression of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor (GluR) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in nine established cell lines: rat CG-4 (oligodendroglial lineage) and RINm5F insulinoma cells; human CHP134, SMS-KCNR, SKNSH, and Nb69 neuroblastoma cells; and human D384Med, D425Med, and D458Med medulloblastoma cells. CG-4 expressed mRNAs encoding GluR2-7, KA-1, and KA-2 non-NMDA GluR (Yoshioka et al.: J Neurochem 64:2442-2448, 1995) and NR1 (NMDAR1) and NR2D NMDA GluR. After differentiation to oligodendrocyte-like cells, CG-4 also expressed NR2B mRNA. Rat insulinoma cells expressed GluR5 and KA-2 non-NMDA and NR1 and NR2D NMDA GluR mRNAs. The four human neuroblastoma lines all expressed mRNAs encoding GluR2-4, 6, 7 and KA-1 non-NMDA and NR1 NMDA GluR, and the three human medulloblastoma cell lines all expressed mRNAs encoding GluR1, 6 and KA-1, but none of the NMDA GluRs. Whereas CG-4 is susceptible to kainate excitotoxicity, treatment of insulinoma, neuroblastoma, and medulloblastoma lines with L-glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), or NMDA failed to cause cell damage or to augment 45Ca2+ influx. Thus, despite expressing a variety of non-NMDA and NMDA GluR genes, the human neuroblastoma and medulloblastoma and rat insulinoma lines failed to assemble Ca(2+)-permeable NMDA or non-NMDA GluR channels. This failure confers protection against excitotoxicity and may contribute to progression of tumors of these types.
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PMID:Expression of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor genes in neuroblastoma, medulloblastoma, and other cells lines. 891 93

The channel properties of the multimeric ionotropic glutamate receptors can be regulated by their subunit composition. The relationship between the structure and physiological functions of glutamate receptors, however, is difficult to study in the CNS because of the large number of these subunits, their widespread distribution, and neuronal heterogeneity. To avoid these difficulties, and to uncover possible novel functions of ionotropic glutamate receptors in sensory neurons, we examined the expression of non-N-methyl-D-aspartate glutamate receptor subunits in a simple neuronal system: the olfactory epithelium. It contains only one neuronal type, the olfactory receptor neuron, that receives no synaptic innervation within the epithelium and therefore should not require conventional postsynaptic glutamate receptors. The axons of these neurons, however, terminate and release glutamate in the glomerular region of the olfactory bulb, and may contain presynaptic glutamate receptors. By reverse transcriptase-polymerase chain reaction amplification and RNase protection assays, we showed that a subset of non-N-methyl-D-aspartate receptor subunits is expressed in the olfactory epithelium. The most abundant is KA2, which can form kainate-selective ion channels with GluR5 or GluR6. Messenger RNAs for GluR6, and for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate-type (AMPA/KA) GluR2 and GluR3 subunits, are also present, but at levels lower than that of KA2 by an order of magnitude. In situ hybridization and immunocytochemistry localized KA2 to only the olfactory receptor neurons, and not to any other cell type in the olfactory epithelium. Surprisingly, antibodies against KA2 or GluR5/6/7 primarily stained the olfactory neuron dendritic knobs that are specialized for odorant signalling at the sensory epithelial lumenal surface, and the olfactory neuron axon bundles that project to the olfactory bulb. The presence of a limited subset of non-N-methyl-D-aspartate receptor subunits in the olfactory epithelium, and the localization of a kainate-selective receptor to both the axons and specialized dendritic knobs of olfactory receptor neurons, which receive no known synaptic input, suggest that these non-N-methyl-D-aspartate receptor subtypes may mediate either novel non-synaptic functions in the olfactory neuron dendrites or presynaptic functions in the olfactory nerve terminals or axons. These data also suggest that the olfactory sensory system, possessing a relatively simple anatomical organization and a limited number of glutamate receptor subunits, may be useful for elucidating facets of the complex relationships between subunit composition and physiological function of ionotropic glutamate receptors.
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PMID:Expression of non-N-methyl-D-aspartate glutamate receptor subunits in the olfactory epithelium. 920 Jul 25

Metabotropic glutamate receptor subtype 6 (mGluR6) is restrictedly expressed in the retinal ON bipolar cells and ablation of mouse mGluR6 by gene targeting results in a loss of ON responses to light stimulus and impairs the detection of visual contrasts. We have isolated genomic clones containing the human mGluR6 gene and determined the whole nucleotide sequence of the mGluR6 gene. The transcription initiation site of the human mGluR6 gene has been identified using primer extension analysis in combination with reverse transcriptase-mediated polymerase chain reaction analysis of human retinal RNA, while the termination of the mGluR6 mRNA has been assigned by the analysis of rapid amplification of 3'-cDNA ends. The human mGluR6 gene consists of 16,742 base pairs with 10 exons separated by nine introns. The human mGluR6 is composed of 877 amino acid residues with a signal peptide of 24 amino acid residues and the mature protein shows a 94.6% homology with the rat counterpart. A CpG-rich island is present at exon 1 and its preceding putative promoter region and this unusual sequence, like several tissue-specific genes, may be important for a specific expression of the mGluR6 gene in the retinal bipolar cells. The human mGluR6 gene has been mapped to chromosome 5q35 by the analyses of blot hybridization of a DNA panel of human/mouse/hamster somatic cell hybrids and fluorescence in situ hybridization of human chromosomes. This study should provide the genetic basis for not only better understanding the molecular mechanism underlying a tissue-specific expression of the mGluR6 gene but also exploring a potential defect in human mGluR6 in a certain inherited eye disease.
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PMID:The whole nucleotide sequence and chromosomal localization of the gene for human metabotropic glutamate receptor subtype 6. 921 6

To determine the subunit composition of high-affinity kainate receptors in native neurons is a challenging problem because of the expression of more than one GluR subunit. In the present study the question of whether GluR5 and/or GluR6 subunits combine with KA-1 or KA-2 subunits in vivo is addressed by performing detailed physiological, pharmacological, and molecular characterization of functional kainate receptor channels in acutely dissociated trigeminal ganglion (TG) neurons. The results show that (1) smaller diameter TG neurons (<30 microm) respond to L-glutamate and kainate, and the currents gated by kainate desensitize with prolonged agonist exposure; (2) all kainate receptor subunits are detected to some extent by reverse transcriptase-PCR, whereas glutamate receptor subunits GluR5 and KA-2 are expressed at high levels in the TG; (3) there is an obvious similarity between the features of native kainate receptor channels in TG neurons and of heteromeric recombinant GluR5(R)/KA-2 channels in pharmacological properties, desensitization, rectification, ion permeability, and mean channel conductance; and (4) the age-dependent increase in GluR5 and KA-2 RNA levels in the TG is correlated well with an increased number of kainate-sensitive cells during postnatal development. Our data suggest that the heteromeric GluR5/KA2 combination actually occurs in TG neurons and give a clue as to the subunit composition of native kainate receptor channels.
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PMID:Glutamate receptor subunits GluR5 and KA-2 are coexpressed in rat trigeminal ganglion neurons. 925 73

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.
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PMID:Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells. 953 5

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long-term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase-polymerase chain reaction, we showed that primary cultures of rat CECs express N-methyl-D-aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A-C subunits), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl-propionate (AMPA) receptors (GLUR1-4 subunits), and metabotropic receptors (mGLUR). Exposure of the cultures to 2 mM glutamate, a well-established mediator of ischemic damage, for 30 min increased significantly the phosphorylation of calcium/calmodulin-dependent protein kinase II even after 10- and 60-min recovery times. This effect could be prevented by the NMDA blocker MK-801. The presence of multiple glutamate receptor types may confer a finely tuned responsiveness of the cerebral endothelium to glutamate in physiological and pathological conditions.
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PMID:Expression of glutamate receptors on cultured cerebral endothelial cells. 985 65

In order to investigate the process of gene expression of N-methyl-D-aspartate glutamate receptor (NMDAR) subunits in the rat neostriatum and how this relates to motor behaviors, a single dose of antisense phosphodiester oligodeoxynucleotide specific for NMDAR1 was unilaterally applied in the neostriatum in a stereotaxic apparatus. After one day of antisense treatment, ipsilateral rotation behaviors that were induced by apomorphine were found in the treated animals. Reductions in the levels of expression of NMDAR1 and NMDAR2A messenger RNAs (NMDAR1: 20.6%; NMDAR2A: 19.7%) were found in the antisense-treated striatal tissues by reverse transcriptase-polymerase chain reaction. There was no change in the levels of NMDAR2B, NMDAR2C and NMDAR2D messenger RNAs. After two days, western blotting experiments showed that there were decreases in the levels of expression of NMDAR1 (decreased 27.6%) and NMDAR2A (decreased 19.2%) proteins in the NMDAR1 antisense-treated striatal tissues. In addition, NMDAR1 immunoreactivity was found to decrease in intensity in the NMDAR1 antisense-treated neostriatum. At the cellular level, the intensity of NMDAR1 immunoreactivity in perikarya of presumed medium spiny neurons was found to decrease. These results indicate that a single dose of NMDAR1 antisense modifies the expression of NMDAR1 messenger RNA and protein in neurons in the neostriatum. The modification in the expression of NMDAR1 has differential effects in the expression of NMDAR2 subunits. Gene expression of the native NMDAR subunits is likely to be a dynamic process. The change in gene expression of the NMDAR subunits in the neostriatum may have a profound effect on the motor behaviors of rats.
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PMID:Changes in expression of N-methyl-D-aspartate receptor subunits in the rat neostriatum after a single dose of antisense oligonucleotide specific for N-methyl-D-aspartate receptor 1 subunit. 1086 43


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