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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human beta-amyloid protein may play an important, possibly primary, role in the pathogenesis of Alzheimer's disease (AD), and it appears to potentiate the susceptibility of neurons to excitotoxicity. AD is associated with alterations in the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtypes of glutamate receptors, and it has been suggested that excitotoxicity may play a role in neuronal damage in AD. In this study, we have used quantitative receptor autoradiography to examine NMDA and AMPA receptors in transgenic mice that contain the gene for the carboxyl-terminal 100 amino acids of the human amyloid precursor protein, beginning with the beta-amyloid region, which is under the control of the JC viral early region promoter. Reverse transcriptase-polymerase chain reaction confirmed that the brains of transgenic mice expressed beta-amyloid mRNA and that control mice did not. NMDA receptors, assessed with [3H]MK-801, were unchanged in the transgenic compared with the control mice. In the transgenic mice, there were no significant changes in [3H]AMPA receptor binding compared with controls. This study represents the first attempt to evaluate in transgenic mice the in vivo interaction between beta-amyloid expression and excitatory amino acid receptors.
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PMID:NMDA and AMPA receptors in transgenic mice expressing human beta-amyloid protein. 750 89

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
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PMID:Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase. 777 23

In this study, the relative abundance of splicing variants of Oreochromis non-NMDA subtype glutamate receptors was studied by quantitative reverse-transcriptase PCR (RT-PCR). The relative expression level between the flip and flop transcripts of fGluR2 alpha determined by quantitative RT-PCR is apparently much higher than that estimated by sequence analysis of the cloned RT-PCR products. Control studies were performed to demonstrate the accuracy of the application of quantitative RT-PCR analysis in studying the relative abundance between the flip and flop transcripts of glutamate receptors.
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PMID:Determination of relative abundance of splicing variants of Oreochromis glutamate receptors by quantitative reverse-transcriptase PCR. 870 49

Using oligonucleotide primers, we have amplified and sequenced the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors from the brain of 17-day-old chick embryos. Both flip and flop isoforms of each of these glutamate receptors (GluR) were identified and cloned. Nucleotide comparisons showed that the two isoforms for each chick receptor subtype were 71-78% identical, whereas homologous chick and rat isoforms were 94-98% identical. Reverse transcriptase-polymerase chain reaction and restriction enzyme analysis were employed to identify regional variation in flip and flop levels of each AMPA receptor. Flip isoforms of GluR 1-3 predominated in forebrain, while flop variants of GluR 1-4 were more prevalent in the cerebellum. This differential regional expression suggests that alternative splicing of AMPA receptor subunits contributes importantly to synaptic diversity in chick central nervous systems.
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PMID:Flip and flop isoforms of chick brain AMPA receptor subunits: cloning and analysis of expression patterns. 898 52

By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.
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PMID:mGluR5 metabotropic glutamate receptor distribution in rat and human spinal cord: a developmental study. 917 80

The RNA-dependent RNA polymerase (RdRp) of potato virus X (PVX) contains a glycine-lysine-serine (GKS) motif. This motif is present in the replication enzyme of many RNA viruses and is thought to be required for nucleoside triphosphate-binding. Three single amino acid changes, glycine to alanine (AKS), lysine to asparagine (GNS) and lysine to glutamate (GES) within the GKS motif of the PVX RdRp were tested for their effect on PVX accumulation. The GNS and GES mutations rendered the virus unable to accumulate in either tobacco plants or protoplasts, whereas substitution of glycine with alanine had only a minor effect on accumulation of PVX. The glycine to alanine mutation reverted to wild-type after passage on Nicotiana clevelandii plants. These findings suggest that the GKS motif is required for PVX replication and that strong selection pressures are active to maintain necessary sequences of the viral RdRp.
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PMID:Mutation of the GKS motif of the RNA-dependent RNA polymerase from potato virus X disables or eliminates virus replication. 919 15

Here we report the cloning and functional analysis of a cDNA encoding a functional glutamate receptor subunit of Oreochromis sp., a freshwater teleost fish. The deduced amino acid sequence of this cDNA clone, fGluR3 alpha, displays the highest sequence identity to that of the mammalian GluR3 subunit. Results of quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis indicated that the expression level of fGluR3 alpha in the cerebellum was much less than that in the telencephalon and optical lobe. Similar to its mammalian counterpart, variants of fGluR3 alpha were created by alternative splicing and RNA editing at the R/G site. The channel properties of homomeric fGluR3 alpha expressed in Xenopus oocytes were similar to those of the mammalian alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring receptors. The rank order of agonist potency of the expressed fGluR3 alpha is AMPA > or = glutamate > or = quisqualate > domoate > or = kainate. This is the first functional glutamate receptor of teleost fish being demonstrated to be sensitive to AMPA. Furthermore, this study suggested a strong functional conservation of AMPA-preferring receptors in vertebrates.
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PMID:Molecular and electrophysiological characterizations of fGluR3 alpha, an ionotropic glutamate receptor subunit of a teleost fish. 967 19

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised 'neuronal' serotonin transporter.
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PMID:Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles. 969 37

Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.
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PMID:Expression of a functional N-methyl-D-aspartate-type glutamate receptor by bone marrow megakaryocytes. 1021 82

The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L-glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1-aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype.
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PMID:Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones. 1072 83


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