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 previous reports, we have shown that cAMP-specific phosphodiesterase (PDE4) is the major PDE in the rat parotid gland, and that PDE4 is activated by phosphorylation. In this study, we investigated the expression of PDE4 isoform genes and alternative splicing variants of PDE4D in the rat parotid gland using reverse transcriptase-polymerase chain reaction (RT-PCR). PDE4A, PDE4B, PDE4C and PDE4D of PDE4 subfamily were expressed. PDE4D was found to be the dominant PDE4 isoform. A weak band of PDE4C was detectable. Three alternative splicing variants (PDE4D1, PDE4D2 and PDE4D3) derived from the rat PDE4D gene were expressed in the parotid gland. These data suggested that the intracellular cAMP level is regulated by multiple response mechanisms through the activations of the PDE by phosphorylation and gene expression in the rat parotid gland.
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PMID:Expression of mRNA encoding cAMP-specific phosphodiesterase isoforms in rat parotid glands. 898 29

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398+/-50 nM and 252+/-16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16+/-0.01 in intact platelet enzyme, to 0.37+/-0.05 in cGI-PDEDelta1 and to 0.49+/-0.04 in cGI-PDEDelta2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [32P]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and [32P]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.
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PMID:Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets. 917 84

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.
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PMID:Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node. 959 96

The process of thymic selection is critical for the generation of the mature T-cell repertoire, yet the nature of the self-peptides that serve this function is not known. Several studies suggest that tissue-specific auto-antigens are expressed in the thymus. We initiated this study to examine the expression of a panel of auto-antigens related to several autoimmune diseases in the thymus, peripheral lymphoid organs, and various cell lines. We looked for the expression of these antigens by reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorter (FACS) analysis, immunoblotting, and immunoprecipitation. We found that in the thymus there is evidence for the expression of a wide variety of disease-related self-antigens including myelin antigens, insulin, cardiac myosin, and retinal S antigen. By FACS analysis, several monoclonal anti-myelin basic protein antibodies were found to bind to immune cells. In Western blotting, we could find in the thymus and other lymphoid organs the expression of myelin basic protein, proteolipid protein, and cyclic nucleotide phosphodiesterase; in contrast, the staining for myelin oligodendrocyte glycoprotein, microtubule-associated Tau protein, and insulin were negative in these organs. The results of these studies confirm that there is evidence for the expression of a variety of auto-antigens in the immune system, both at the mRNA and protein levels, potentially enabling them to participate in the process of thymic education.
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PMID:Expression of autoimmune disease-related antigens by cells of the immune system. 978 84

In the present study, for the first time, PDE4 subtypes were identified and semi-quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving beta-agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and beta-actin were identified by digoxigenin (DIG)-labelling reverse transcriptase-polymerase chain reaction and semi-quantified by DIG-detection enzyme-linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03 - 10 microM), rolipram (0.1 - 10 microM) and theophylline (10 microM - 1 mM) inhibited PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration-dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration-dependent manner by rolipram (0.01 - 100 microM) both before and after allergen challenge.
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PMID:Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects. 1142 97

In order to investigate the neuropathological effects on the developing rat brain after intrauterine infection, identification of glail fibrillary acidic protein (GFAP), 2', 3'-cyclic nucleotide phosphodiesterase (CNPase), and neurofilament (NF) was observed. Escherichia coli (E. coli) was inoculated into uterine horn of pregnant rats when gestation was 70% complete (15 days) and the control group was inoculated with normal saline. Immunohistochemistry was used for evaluation of GFAP, CNPase, and NF expression in pup brains at postnatal day 7 (P7) and reverse transcriptase-PCR (RT-PCR) to analyze macrophage inflammatory protein-1 alpha mRNA (MIP-1 alpha mRNA), macrophage inflammatory protein-1 beta mRNA (MIP-1beta mRNA), the regulated upon activation normal T expressed and secreted chemokine mRNA (RANTES mRNA) and Eotaxin mRNA expression in pup brains at P1, P3 and P7. The numbers of GFAP-positive cells of the E. coli-treated group pups were marked increased in periventricular white matter and hippocampus at P7 compared with the control group but no significant different levels of GFAP expression in corpus callosum were found between two groups. The integrate density (ID) of CNPase-positive staining of the Escherichia coli-treated group pups were marked decreased in periventricular white matter and corpus callosum at P7 compared with the control group. The ID of NF-positive staining of the Escherichia coli-treated group pups were marked decreased in periventricular white matter at P7 compared with the control group and no significant different levels of NF expression in corpus callosum were found between two groups. The expression of MIP-1 alpha mRNA and MIP-1 beta mRNA in brain of the E. coli-treated pup rat were higher than the control at P1, but the expression of MIP-1 alpha mRNA and MIP-1 beta mRNA in brain of the pup rat at P3 and P7 had no significant difference between two groups. The alteration of expression of GFAP, CNPase, and NF in the brain of neonatal rats after intrauterine infection suggested that intrauterine infection could cause neonatal white matter damage. Moreover, the transient increase in expression of chemokine such as MIP-1 alpha, MIP-1 beta in neonatal brain after intrauterine infection indicated that MIP-1 alpha, MIP-1 beta may be a mechanism mediating between the neonatal white matter damage and the intrauterine infection.
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PMID:White matter damage and chemokine induction in developing rat brain after intrauterine infection. 1623 36

To investigate whether antihemostatic function of vascular endothelial cells (VECs) is changed in type-II diabetic model rats, the mRNA expressions of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), thrombomodulin (TM), PA inhibitor type-1 (PAI-1), and phosphodiesterases (type 3A, 3B, and 4D PDEs) were quantitated by the method of comparative reverse transcriptase-polymerase chain reaction (RT-PCR). VECs from type-II diabetic model Otsuka Long-Evans Tokushima Fatty (OLETF) rats and from its normal counterpart (LETO) rats were cultured for 24 h with dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) or a type-3 PDE inhibitor, cilostazol. Intracellular cAMP concentration was determined by the chemiluminescent enzyme-linked immunosorbent assay (ELISA) system. In cultured VECs from OLETF rats, the basal mRNA expressions of u-PA and TM were significantly decreased as compared to those in cultured VECs from LETO rats. TM mRNA expression in cultured VECs from OLETF rats was increased 2.1-fold at 24 h after treatment with db-cAMP (3 mmol/L). Basal mRNA expressions of type 3A, 3B, and 4D PDEs were significantly higher in VECs from OLETF rats than those from LETO rats. After treatment with cilostazol (30 micromol/L), intracellular cAMP was significantly increased at 60 min and TM mRNA expression was increased 1.5-fold at 24 h. Therefore, elevation of intracellular cAMP by db-cAMP or cilostazol up-regulated TM mRNA expression in cultured VECs from OLETF rats.
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PMID:Elevation of intracellular cAMP up-regulated thrombomodulin mRNA in cultured vascular endothelial cells derived from spontaneous type-II diabetes mellitus model rat. 1709 Apr 5