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)

Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.
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PMID:Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists. 755 25

Previous studies from our laboratory have shown that messenger RNAs (mRNAs) coding for a cAMP-specific phosphodiesterase (PDE4A) are present in mature rat and mouse germ cells. However, no information is available about the properties of the expressed proteins. To determine their structure and regulation, the PDE4A isoforms expressed in the rat testis were identified and compared to the variants expressed in the brain. Western blot analysis using an antiserum specific for PDE4A demonstrated the presence in testis extracts of two distinct proteins with apparent masses of 98.8 and 86 kDa. The electrophoretic mobilities of these proteins differ from those of proteins detected in the brain extracts (113 and 76 kDa). Reverse transcriptase-PCR of the different splicing mRNA variants expressed in testis confirmed the presence of at least one novel PDE4A mRNA that is distinct from the PDE4A splicing variants identified in the brain and other tissues. Expression of the complementary DNA encoding this variant in a heterologous system resulted in an increase in PDE activity and the appearance of an immunoreactive protein with a mass of 98.8 kDa. No 86-kDa protein could be generated with this transfection. Upon fractionation of testis extracts by HPLC diethylaminoethyl-chromatography, a peak of cAMP-PDE activity coeluted with the two immunoreactive species. During testicular development, the 98.8-kDa protein is present in trace amounts at 10 days, and its level increases with the age of the animals, reaching a plateau at 40 days. The 86-kDa protein appears at 20 days of age and reaches its maximum at 40 days. Studies on the cellular site of expression demonstrated that the two polypeptides are most abundant in round spermatids and are expressed in trace amounts in pachytene spermatocytes, whereas they could not be detected in Sertoli or interstitial cells. The 98.8-kDa, but not the 86-kDa, protein was also expressed in epididymal spermatozoa. These data demonstrate the expression of novel cAMP-specific PDEs coded by the PDE4A gene. The expression of these isoforms is maximal in round spermatids and is maintained in mature spermatozoa. The genesis of the lower mol wt species remains to be determined.
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PMID:Developmental regulation of unique adenosine 3',5'-monophosphate-specific phosphodiesterase variants during rat spermatogenesis. 864 Dec

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.
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PMID:Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture. 1045 54

In vivo effects of milrinone, a selective phosphodiesterase 3 (PDE-3) inhibitor, on plasma free fatty acids (FFA), glucose, and insulin levels were examined in alert rats. In dose response studies, intravenous injection of 1, 5 or 25 micromol/kg of milrinone provoked an immediate increase in plasma concentrations of FFA and insulin, while glucose levels rose only in response to the 5- and 25-micromol/kg doses. During euglycemic-hyperinsulinemic (approximately 450 pmol/L) clamps, intravenous injection of milrinone (25 micromol/kg) completely inhibited insulin suppression of lipolysis and of endogenous glucose production, while having no effect on insulin-stimulated glucose uptake (ISGU). To explore the reason why ISGU was not affected, we performed reverse-transcriptase polymerase chain reaction (RT-PCR) with RNA from skeletal muscle, fat, and liver. The results showed that PDE-3B mRNA was expressed in adipose tissue and liver, but it was not detected in skeletal muscle. We conclude that PDE-3 plays a major role in the inhibitory action of insulin on lipolysis in fat and on glucose production in liver and, in addition, seems to be involved in insulin secretion in pancreatic beta cells.
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PMID:Milrinone, a selective phosphodiesterase 3 inhibitor, stimulates lipolysis, endogenous glucose production, and insulin secretion. 1462 13

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6beta, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase-PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3+/-1 microM; Km cAMP: 20.5+/-1.5 microM), PDE6 is a cGMP-specific PDE (Km cGMP: 37+/-13 microM) and PDE11 is a dual-specificity PDE (Km cGMP: 6+/-2 microM; Km cAMP: 18.5+/-5.5 microM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71+/-39 microM for PDE1, 0.65+/-0.015 microM for PDE6 and 1.6+/-0.5 microM for PDE11) and sildenafil (IC50 was 1.3+/-0.9 microM for PDE1, 0.025+/-0.005 microM for PDE6 and 0.12+/-0.06 microM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.
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PMID:Cyclic nucleotide phosphodiesterases in Drosophila melanogaster. 1567 86

Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.
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PMID:Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid. 1769 92