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)

The polymerase chain reaction (PCR) was used to develop a method for detection and relative quantification of the choline acetyltransferase (ChAT) mRNA in neonatal and adult rat CNS. Oligonucleotide primers derived from a porcine ChAT cDNA sequence were used in coupled reverse transcriptase (RT)-PCR to amplify a cDNA sequence of 206 bp which arises in a cycle- and RNA-dependent manner and which hybridizes with both an internal oligonucleotide and a ChAT cDNA probe. ChAT mRNA was detected in spinal cord, septal area, striatum, cortex and hippocampus but not in cerebellum and cardiac or skeletal muscle. In the septal area, relative quantitative evaluation of ChAT mRNA levels by RT-PCR indicates that this transcript is developmentally regulated and increased following intracerebral administration of nerve growth factor (NGF) to both neonatal and young adult rats. This suggests that the increases of ChAT activity observed in basal forebrain during development or after NGF administration are, at least in part, associated with an increase in corresponding levels of mRNA.
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PMID:Choline acetyltransferase messenger RNA expression in developing and adult rat brain: regulation by nerve growth factor. 164 35

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.
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PMID:Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function. 217 49

The repeated intracerebroventricular administration of nerve growth factor (5 micrograms/2.5 microliters) to neonatal rats induced the activation of choline acetyltransferase in forebrain cholinergic neurons that was paralleled by a concomitant change in the density of muscarinic cholinergic receptors in the cerebral cortex. The administration of nerve growth factor altered muscarinic binding sites in a biphasic fashion during postnatal development. A significant stimulation of the developmental increase in the density of muscarinic binding sites occurred in nerve growth factor-treated animals at days 2 and 3 after birth. Conversely, nerve growth factor induced a significant decrease in the receptor number at postnatal days 8 and 14. Muscarinic receptor number returned to control values after treatment, suggesting that nerve growth factor-induced changes to muscarinic cholinergic receptors are reversible. Nerve growth factor administration did not affect muscarinic cholinergic receptor density in striatal membranes and did not alter the relative content of cortical messenger RNAs encoding m1 and m3 muscarinic cholinergic receptor subtypes at postnatal day 14, as determined by reverse transcriptase-polymerase chain reaction. The up- and down-regulation of muscarinic cholinergic receptors induced by nerve growth factor during postnatal development may be temporally related events associated with concomitant changes in the activity of choline acetyltransferase.
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PMID:Intracerebroventricular administration of nerve growth factor affects muscarinic cholinergic receptors in the cerebral cortex of neonatal rats. 813 Jul 36

Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter-specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c-NOS), and the growth-associated protein GAP-43, three genes known to be expressed in central cholinergic neurons. A "nondestructive" method of screening cultured cells for their expression of c-NOS was established using depolarization with medium containing 50 mM potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E-115 (c-NOS-positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature-sensitive SV-40 transforming activity and neomycin resistance. Cell colonies surviving in G418-containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33 degrees C, differentiated by switching to a low-serum medium and growth at 39 degrees C, and screened for depolarization-induced accumulation of nitrite in the medium. The subset of putative c-NOS-positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.
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PMID:Use of capillary electrophoresis with laser-induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: applications in the cloning of cells. 937 66

Innervation of diencephalic neurons producing melanin-concentrating hormone by choline acetyltransferase-containing axons was examined using double immunohistochemistry. In the rostromedial zona incerta and perifornical regions of the lateral hypothalamic area, many choline acetyltransferase-positive fibers were detected in the immediate vicinity of melanin-concentrating hormone perikarya and their proximal dendrites. Putative contact sites were less abundant in the far lateral hypothalamus, and only scattered close to the third ventricle. After injections of the retrograde tracer FluoroGold, most of these projections appeared to originate in the pedunculopontine and laterodorsal tegmental nuclei. Finally, to determine the putative effect of acetylcholine on the melanin-concentrating hormone neuron population, the cholinergic agonist carbachol was added to the medium of hypothalamic slices in culture. Using competitive reverse transcriptase-polymerase chain reaction, carbachol was found to induce a rapid increase in the melanin-concentrating hormone messenger RNA expression. This response was abolished by both atropine, a muscarinic antagonist, and hexamethonium, a nicotinic antagonist. Thus, the bulk of these results indicates that the diencephalic melanin-concentrating hormone neurons are targeted by activating ascending cholinergic projections.
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PMID:Rat diencephalic neurons producing melanin-concentrating hormone are influenced by ascending cholinergic projections. 1039 86

The rate limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline by the high-affinity choline transporter (CHT1). Here, we investigated the distribution of CHT1 in the rat trachea. CHT1-mRNA was detected by reverse transcriptase-polymerase chain reaction in trachea without epithelium, abraded tracheal mucosa, and in epithelial cells obtained by laser-assisted cell-picking. Accordingly, CHT1-mRNA could also be detected in tracheal epithelial cells by in situ hybridization. Recently obtained polyclonal rabbit and guinea-pig antisera against a synthetic peptide corresponding to amino acid residues 29-40 of the rat CHT1 sequence localized CHT1 protein in combination with antisera against the vesicular acetylcholine transporter in cholinergic fibers innervating tracheal glands and the tracheal muscle. In case of the tracheal epithelium, CHT1 was restricted to the apical membrane of the ciliated cells, as demonstrated by confocal laser scanning and electron microscopy using an affinity-purified CHT1 antiserum. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favor of ACh synthesis being fueled by choline uptake via CHT1 after release and breakdown of ACh at the luminal surface. Accordingly, cholinergic regulation of tracheal epithelial function is governed by local release and recycling of ACh by ciliated cells.
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PMID:Expression of the high-affinity choline transporter, CHT1, in the rat trachea. 1265 36

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors (GluR1-4) are one of the most important ionotropic glutamate receptors in the striatum, a key region of the basal ganglia. The present study investigated the trend of developmental expression of AMPA receptor subunits in the striatum of rats in different developmental stages, i.e., at postnatal day 7 (PND7), PND21 and adult. Perfuse-fixed striatal sections were used. The expression of AMPA subunits was studied by immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR revealed that the levels of expression of the GluR1 and GluR3 mRNAs were the same among the age groups. The level of GluR2 mRNA was highest in PND21 rats and lowest in adult. The highest level of GluR4 mRNA was detected in rats at PND7. Similar trends of GluR1, GluR2 and GluR2/3 immunoreactivity expression were observed using commercially available specific antibodies. In addition, a large proportion of parvalbumin-immunoreactive GABAergic interneurons in the striatum were found to display GluR1 immunoreactivity in PND21 and adult rats. In contrast, most of the choline acetyltransferase-immunoreactive cholinergic interneurons were found to display GluR2 immunoreactivity but less GluR1 and no GluR2/3 immunoreactivity in PND21 and adult rats. The present study suggests that there is a distinct pattern of expression of AMPA-type receptor mRNAs and proteins in the rat striatum at different stages of development.
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PMID:Differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate glutamate receptors in the rat striatum during postnatal development. 1473 60

Recent studies suggest the participation of cholinergic neurons in the brain processes underlying reinforcement. The involvement of cholinergic neurons in cocaine self-administration has been recently demonstrated in studies using muscarinic and nicotinic agonists and antagonists, microdialysis, assessment of choline acetyltransferase activity and acetylcholine (ACh) turnover rates. The present experiment was initiated to identify subsets of cholinergic neurons involved in the brain processes that underlie cocaine self-administration by lesioning discrete populations with a selective neurotoxin. Rats were trained to self-administer cocaine and the cholinergic neurotoxin 192-IgG-saporin or vehicle was then bilaterally administered into the posterior nucleus accumbens (NAcc)-ventral pallidum (VP). The 192-IgG-saporin induced lesions resulted in a pattern of drug-intake consistent with either a shift in the dose intake relationship to the left or downward compared to sham-treated controls. A second experiment used a self-administration threshold procedure that demonstrated this lesion shifted the dose intake relationship to the left compared to the sham-vehicle treated rats. The magnitude and extent of the lesion was assessed by measuring the expression of p75 (the target for 192-IgG-saporin) and choline acetyltransferase (ChAT) in the NAcc, VP, caudate nucleus-putamen (CP) and vertical limb of the medial septal nucleus-diagonal band (MS-DB) of these rats using real time reverse transcriptase-polymerase chain reaction. Significant reductions in gene expression for p75 (a selective marker for basal forebrain cholinergic neurons) and ChAT were seen in the MS-DB and VP while only small decreases were seen in the NAcc and CP of the 192-IgG-saporin treated rats. These data indicate that the overall influence of cholinergic neurons in the MS-DB and VP are inhibitory to the processes underlying cocaine self-administration and suggest that agonists directed toward subclasses of cholinergic receptors may have efficacy as pharmacotherapeutic adjuncts for the treatment of cocaine abuse.
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PMID:Involvement of cholinergic neuronal systems in intravenous cocaine self-administration. 1501 33

Opioid peptides have demonstrated modulatory effects on the vestibular afferent discharge and are putative vestibular efferent neuromodulators. The distribution of their receptors in the mammalian vestibular epithelia is not known. We used reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, Western blots and immunohistochemistry to study the expression of mu opioid receptor (MOR) in the Scarpa's ganglia and cristae ampullares of rats. MOR transcript was only detected in the somata of the vestibular afferent neurons. MOR-like immunoreactivity was observed in the somata of vestibular afferents and in nerve terminals in the cristae ampullares epithelia both in the center and peripheral regions. Double labeling of cristae sections with the MOR1 antibody in combination with antibodies against calretinin (a marker for vestibular afferents terminating in calices) and peripherin (a marker for afferents terminating in boutons), respectively showed that MOR1 immunoreactivity was in calyx, dimorphic and bouton vestibular afferents. MOR immunoreactivity was not detected in vestibular efferent fibers identified with choline acetyltransferase immunohistochemistry. These results indicate that MOR may mediate effects of vestibular efferents on afferents.
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PMID:Expression and distribution of mu opioid receptors in the inner ear of the rat. 1548 44

In non-neuronal contexts, ACh (acetylcholine) is thought to be involved in the regulation of vital cell functions, such as proliferation, differentiation, apoptosis and cell-cell interaction. In airways, most cells express the non-neuronal cholinergic system, each containing a specific set of components required for synthesis, signal transduction and ACh hydrolysis. The aim of the present study was determine the expression of cholinergic system components in bronchial aspirates from control subjects and patients with lung cancer. We conducted an analysis of cholinergic components in the stored soluble and cellular fraction of bronchial aspirates from non-cancerous patients and patients diagnosed with lung cancer. The results show that the fluid secreted by human lung cells contains enough AChE (acetylcholinesterase) activity to control ACh levels. Thus these findings demonstrate that: (i) AChE activity is significantly lower in aspirates from squamous cell carcinomas; (ii) the molecular distribution of AChE in both bronchial cells and fluids consisted of amphiphilic monomers and dimers; and (iii) choline acetyltransferase, nicotinic receptors and cholinesterases are expressed in cultured human lung cells, as demonstrated by RT-PCR (reverse transcriptase-PCR). It appears that the non-neuronal cholinergic system is involved in lung physiology and lung cancer. The physiological consequences of the presence of non-neuronal ACh will depend on the particular cholinergic signalling network in each cell type. Clarifying the pathophysiological actions of ACh remains an essential task and warrants further investigation.
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PMID:Cancer-associated differences in acetylcholinesterase activity in bronchial aspirates from patients with lung cancer. 1821 Dec 61


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