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)

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 regulation of murine mammary tumor virus (MuMTV) production by mammotropic hormones, hormonomimetic substances, and cyclic nucleotides was investigated. The virus produced in control and treated mammary tumor cell cultures was quantitated by measuring the supernatant reverse transcriptase activity in exogenous reaction using poly(rC).oligo(dG) as template-primer. Two days after exposure, the synthetic glucocorticoid, dexamethasone (DXMT), increased spontaneous MuMTV production at optimal concentration (0.1 mumol) up to ten times. Dibutyryl derivative of cyclic AMP had no effect on spontaneous MuMTV production, whereas the drug potentiated suboptimal concentrations of the glucocorticoid. Natural prostaglandins, potent agonists of adenylate cyclase catalyzing intracellular synthesis of cyclic AMP, enhanced both basal (up to five times) and DXMT-stimulated (up to 1.6 times) MuMTV replication. The MuMTV-stimulating activity of prostaglandins decreased in the order of PGA1 greater than PGE1 greater than PGB1 greater than PGF2 alpha. Prostaglandins can be replaced partially by norepinephrine and isoproterenol by enhancing the DXMT-mediated MuMTV stimulation, whereas these drugs remained without effect on spontaneous MuMTV production. Theophylline, an antagonist of cAMP-phosphodiesterase converting cAMP to AMP, enhanced the virus-stimulating activity of DXMT as well as of prostaglandins. The enhancement of MuMTV production by adenylate cyclase agonists do not correlate absolutely with the estimates of intracellular cAMP levels, since the highest amounts of cAMP has been repeatedly observed in cells treated with PGE1 and norepinephrine. The results indicate that besides hormones, other hormone-like substances and cyclic nucleotides may be involved in the complex mechanism of hormone-regulated MuMTV genome expression.
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PMID:Role of natural prostaglandins in the control of murine mammary tumor virus expression. 628 Dec 84

The capacity of brain tumor samples to synthesize pituitary adenylate cyclase activating polypeptide (PACAP) was evaluated by the reverse transcriptase-polymerase chain reaction technique (RT-PCR). The expression of PACAP receptors was assessed by a combination of RT-PCR techniques, conventional binding techniques, and also by the ability of PACAP to stimulate adenylate cyclase activity. A weak PACAP mRNA and PACAP receptor mRNA expression was detected in only 3 of 16 meningiomas. A weak PACAP-stimulated adenylate cyclase activity (+20%) was detected in 10 of the 16 samples but binding of labeled PACAP was never observed. In the 16 gliomas studied (including two oligodendrogliomas and two ependymomas), PACAP mRNA was identified in 13 samples and PACAP receptor mRNA in 15 samples. PACAP receptors were identified in all the samples by binding studies and/or by PACAP stimulation of the adenylate cyclase activity. PACAP mRNA was never detected in pituitary adenomas (three prolactinomas, two mixed PRL-GH-producing tumors, three GH-secreting tumors, three gonadotrophinomas, one ACTH-producing tumor, two nonsecreting tumors) whereas PACAP receptor mRNA was highly expressed in all the tumors except prolactinomas, where it was at the limit of detection, confirming the binding and adenylate cyclase activation results. Thus, it is unlikely that the neuropeptide PACAP could influence meningioma's cell growth; PACAP secreted from extratumoral areas may influence pituitary tumors and PACAP could participate to gliomas development.
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PMID:Expression of pituitary adenylate cyclase activating polypeptide and receptors in human brain tumors. 747 7

A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 microM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 microM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 mM led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 microM). The membrane permeable 8-bromo-cyclic GMP (100 microM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor. Moreover, in these acute experiments there appears to be no obligatory link between the secretion and the expression of renin, suggesting that both parameters are separately regulated.
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PMID:Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells. 763 56

Calcitonin inhibits both osteoclast formation and bone resorption, and is a primary treatment for patients with hypercalcemia and increased bone turnover. However, the clinical utility of calcitonin is limited because patients become refractory to calcitonin after several days (the calcitonin "escape phenomenon"). The molecular basis for calcitonin "escape" is unclear. To determine the regulatory mechanisms controlling calcitonin receptor (CTR) expression in osteoclasts and their precursors, we treated immature mononuclear precursors for human osteoclast-like multinucleated cells (MNC) formed in vitro with 1,25-(OH)2D3, to induce their differentiation to committed mononuclear precursors, and mature multinucleated osteoclasts, and used reverse transcriptase (RT)-PCR to assess expression of CTR mRNA in both committed mononuclear precursors and MNC. The PCR fragment produced was cloned and sequenced to confirm that it was derived from CTR mRNA. CTR mRNA expression was detected in mononuclear MNC precursors after 7 d of 1,25-(OH)2D3 treatment. It was also present in osteoclast-like MNC and highly purified giant cells from osteoclastomas, but not in monocytes or macrophage polykaryons formed in vitro. Calcitonin markedly decreased CTR but not actin mRNA expression in giant cells and MNC after 12 h, and removal of calcitonin restored CTR mRNA expression. Similarly, calcitonin decreased calcitonin-induced adenylate cyclase activity. These data suggest: (a) downregulation of CTR gene expression by calcitonin may in part explain the calcitonin "escape phenomenon"; and (b) expression of CTR mRNA occurs in mononuclear osteoclast precursors within 7 d after exposure to 1,25-(OH)2D3.
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PMID:Downregulation of calcitonin receptor mRNA expression by calcitonin during human osteoclast-like cell differentiation. 781 11

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with approximately 70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk- cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensin > or = rat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.
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PMID:Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. 784 62

The expression of the messenger RNAs coding for glucagon-like peptide-I (GLP-I) receptor, VIP receptor, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptor as well as the expression of the receptor proteins were demonstrated in the rat medullary carcinoma of thyroid cell line 6/23 by the following experiments: 1) RNA extraction, reverse transcriptase, and polymerase chain reaction with specific primers; 2) binding of the radiolabeled ligands [125I]GLP-I-(7-36)-NH2, [125I]PACAP-(1-27), and [125I]VIP and inhibition by, respectively, unlabeled GLP-I-(7-36)-NH2, PACAP-(1-27), and VIP; and 3) study of adenylate cyclase activation by the peptides and selective inhibition of the VIP/PACAP response by the antagonist [D-Phe2]VIP. Besides the highly selective GLP-I receptor, PACAP receptors of types I and II were present on the cell line and coupled to adenylate cyclase. PACAP stimulated the adenylate cyclase through type I and II receptors, whereas VIP interacted with type II receptors only. Messenger RNA analysis indicated that at least three splice variants of the PACAP type I receptor may be expressed in 6/23 cells.
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PMID:Pituitary adenylate cyclase-activating polypeptide receptors of types I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23. 792 14

The effects of adenosine 3',5'-cyclic monophosphate (cAMP) on cardiac myocyte nitric oxide (NO) production were studied. Maximal nitrite (NO2(-)) production by cultured neonatal rat cardiac myocytes was achieved with 500 U/ml interleukin-1 beta (IL-1 beta) for 48 h (4.6 +/- 0.3 nmol/1.25 x 10(5) cells; n = 12). Cardiac myocytes exposed to 500 U/ml IL-1 beta for 48 h stained positively for inducible nitric oxide synthase (iNOS) by immunohistochemistry. Forskolin (FSK; adenylate cyclase stimulator) or dibutyryl cAMP (DBcAMP; membrane-permeable cAMP analogue) administration alone had no effect on NO2(-) production. The addition of FSK or DBcAMP to IL-1 beta significantly increased NO2-) levels vs. IL-1 beta alone (9.7 +/- 0.6 and 10.9 +/- 0.8 vs. 4.6 +/- 0.3 nmol/1.25 x 10(5) cells per 48 h, respectively; P < 0.01; n = 12). Semiquantitative reverse transcriptase-polymerase chain reaction revealed increased iNOS mRNA in myocytes treated with FSK+IL-1 beta or DBcAMP+IL-1 beta vs. those treated with IL-1 beta alone. The addition of FSK or DBcAMP to IL-1 beta increased iNOS mRNA half-life over IL-1 beta treatment alone (10.6, 11.7 vs. 2.4 h, respectively). Cardiac myocytes do not express iNOS in response to cAMP alone. Rather, cAMP enhances iNOS mRNA stability following cytokine exposure.
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PMID:cAMP enhances inducible nitric oxide synthase mRNA stability in cardiac myocytes. 859 15

The capability of rat pituitary cells to express receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and VIP was evaluated by binding studies and measurement of adenylate cyclase activity on whole gland preparations and by reverse transcriptase-polymerase chain reaction (TR-PCR) using specific primers on preparations from isolated cell populations enriched in PRL- and GH-producing cells. Data obtained on whole gland preparations indicated that selective PACAP receptors (PACAP Type I) predominated. The mRNA coding for PACAP Type I and for the non-selective PACAP receptors Type II VIP2 (but not VIP1) were identified. The mRNA coding for four different spliced variants of the PACAP Type I receptor were detected. In PRL producing cells, three variants and the VIP2 mRNA were detected, whereas in GH-producing cells the mRNA coding for the variant having a 28-amino acid insert (termed HOP) in the third intracellular loop was the only present.
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PMID:Differential alternative splicing of PACAP receptor in pituitary cell subpopulations. 867 20

Vasoactive intestinal peptide (VIP) has been considered as an autocrine growth factor in neuroblastomas. Pituitary adenylate cyclase activating polypeptides (PACAPs) are newly recognized members of the VIP family of neurohormones. As compared to VIP, PACAP has been reported to be biologically more potent and more efficient in tissues expressing selective PACAP receptors rather than common VIP/PACAP receptors. PACAPs and VIP interact with the same affinity and stimulate adenylate cyclase activity with the same efficacy and potency on the VIP receptors, but PACAPs act also on a more selective PACAP receptor that also recognizes VIP but with a 100- to 1,000-fold lower affinity. Thus, depending on the type of receptors expressed at a cell surface, PACAP may be more potent and efficient than VIP. The capacity of 22 surgical specimens of neuroblastomas and of 5 established cell lines to synthesize PACAP and VIP and to synthesize and express PACAP receptors and VIP receptors was studied. Using the reverse transcriptase-polymerase chain (RT-PCR) method with specific primers, we detected the mRNAs coding for PACAP and VIP in 19 and 3 out of 22 samples, respectively. PACAP mRNA was expressed in 3 of the 5 cell lines studied and VIP mRNA in 4. Using the same techniques, PACAP and VIP receptors mRNA were detected in 21, and 13 of the 22 tumor samples and in 5 and 1 of the cell lines studied, respectively. The expression of the PACAP receptor was demonstrated by direct binding studies and/or by the relative potency of PACAPs and VIP to stimulate adenylate cyclase activity in 16 of the 22 tumors and in all the cell lines. In addition, there was no correlation between tumor stage and the expression of mRNA coding for the peptides and the receptors. The present results demonstrated that PACAP could also be a candidate as an autocrine regulator of neuroblastoma which a higher activity than VIP.
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PMID:Pituitary adenylate cyclase activating peptide and its receptors are expressed in human neuroblastomas. 869 38


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