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)

Na+ and Cl- conductances in the apical membrane of respiratory epithelial cells are essential for electrolyte and water transport in the airways. Apart from the well described defect in adenosine 3' : 5' cyclic monophosphate-(cAMP-) dependent activation of Cl- conductances in cystic fibrosis (CF), an increased Na+ conductance has also been reported from transepithelial measurements. In the present experiments we tried to identify these conductances in nasal epithelial cells using patch-clamp and microelectrode techniques. With these methods we found identical and relatively low membrane voltages of about -36 mV in both freshly isolated and primary cultured normal and CF nasal epithelial cells. A Cl- conductance could be activated by cAMP in normal (deltaG = 0.3 +/- 0.8 nS, n = 10) but not in CF (deltaG = 0.3 +/- 0.1 nS, n = 11) cells, whereas Ca2+-dependent Cl- currents activated by adenosine 5'-triphosphate (ATP) and bradykinin were present in both types of cells. Cell-attached membrane patches from stimulated cells did not reveal discernible single-channel events when activated with any of the agonists. A Na+ conductance was also detected in freshly isolated ciliated respiratory cells in impalement studies, as evidenced by the hyperpolarization induced by 10 micromol/l amiloride (deltaV = -5.2 +/- 0.6 mV, n = 56) and when Na+ was replaced in the bath by N-methyl-D-glucamine (NMDG) (deltaV = -5.7 +/- 0.9 mV, n = 14). In whole-cell patch-clamp experiments, the amiloride-induced hyperpolarization was significantly larger in CF (deltaV = 9.7 +/- 2.4 mV, n = 22) when compared to normal (deltaV = -3.3 +/- 0.9 mV, n = 27) cells in short-term culture. Reverse transcriptase polymerase chain reaction analysis of normal respiratory cells identified messenger RNA of both the cystic fibrosis transmembrane conductance regulator (CFTR) as well as the human epithelial Na+ channel (hNaCh). The present experiments confirm the absence of a cAMP-dependent Cl- conductance in CF respiratory epithelial cells and support previous findings obtained in transepithelial and microelectrode studies which indicate an increased Na+ conductance in respiratory epithelial cells from CF patients.
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PMID:Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductance in cystic fibrosis. 858 4

The splicing variant, 5T allele, in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was shown to be associated with partial penetrance of the clinical expression. This splicing variant leads to two possible transcripts: one normal and the other aberrantly spliced that lacks exon 9. The aim of this study was to analyze the molecular basis of the partial penetrance in individuals carrying the 5T allele. We analyzed the level of the correctly spliced RNA transcribed from the 5T allele in nasal and epididymal epithelium and correlated it with disease expression. Semiquantitative nondifferential reverse-transcriptase-PCR showed a considerable variability (6%-37%) in the total level of correctly spliced RNA transcribed from the 5T allele in nasal epithelium from 11 patients. A significant nonlinear correlation (r = .82, P = .002) between the level of the normal CFTR transcripts and the severity of lung disease was shown. No individuals with normal lung function and minimal or no lung disease (FEV1 >80% predicted) had <25% of normal transcripts, and individuals with <15% of normal transcripts did not have FEV1 >80%. The level of normal transcripts in epididymal epithelial cells from four infertile males with congenital bilateral absence of the vas deferens was low (6%-24%). In infertile males with normal lung function the level of correctly spliced transcripts in the nasal epithelium was higher than the level in the epididymal epithelium. These results indicate that there is variability in the efficiency of the splicing mechanism, among different individuals and between different organs of the same individual. This variability provides the molecular basis of the partial penetrance of cystic fibrosis disease in patients carrying the 5T allele.
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PMID:The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. 898 51

Replication-defective adenovirus (Ad) vectors have been used for gene transfer to the respiratory epithelium of experimental animals and individuals with cystic fibrosis. Studies from several laboratories have suggested that administration of first-generation Ad vectors results only in transient gene expression in the lung, due at least in part to destruction of vector-transduced cells by host cellular immune responses directed against viral proteins and/or immunogenic transgene products. We have constructed new Ad2-based, E1-deleted vectors encoding a weakly immunogenic transgene, the human cystic fibrosis transmembrane conductance regulator (hCFTR) under the control of the cytomegalovirus enhancer-promoter. These vectors contain wild-type E2 and E4 regions. These new Ad/CFTR vectors were instilled into the lungs of immunocompetent C57BL/6, BALB/c, and C3H mice. In vitro cytotoxic T lymphocyte (CTL) analysis indicated the presence of Ad-specific CTLs in treated mice. However, we were not able to demonstrate a CTL response specific for hCFTR. Reverse transcriptase PCR analysis demonstrated that hCFTR mRNA expression continued in all three strains of mice for at least 70 days, the last time point analyzed. The E3 region did not play a significant role in persistence of the Ad/CFTR vectors in the mouse lung. Functional hCFTR expression was also observed in the nasal epithelia of CF mutant mice. These results suggest that long-term expression of hCFTR is possible in the airway epithelia of immunocompetent mice without radical modification of Ad vector and in spite of the presence of CTLs.
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PMID:Adenovirus-mediated persistent cystic fibrosis transmembrane conductance regulator expression in mouse airway epithelium. 969 26

Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E(1) (PGE(1)). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF.
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PMID:Platelet activation in cystic fibrosis. 1570 96

Cystic fibrosis (CF) is a severely life-shortening genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Impaired bicarbonate secretion is a key component of CF-related pancreatic disease, but the role of impaired bicarbonate secretion in CF lung disease is less well understood. The submucosal glands of the conducting airways produce and secrete a complex airway surface liquid that lines the airway epithelium and plays a significant role in mucociliary clearance. The serous cell is the predominant cell type of the submucosal gland and a predominant site of CFTR expression. Calu-3 cells are a model of airway submucosal gland serous cells that demonstrates vectorial bicarbonate secretion in response to elevations in cAMP. Based on previously published measurements of unidirectional ion flux, pharmacological inhibition of short-circuit current and ion substitution studies, one can hypothesize the existence of an electrogenic sodium bicarbonate cotransporter (NBC) in the basolateral membrane of Calu-3 cells that mediates bicarbonate entry from the interstitium. To test this hypothesis, we performed reverse-transcriptase PCR, western blotting, and surface biotinylation to identify and localize electrogenic NBCs in Calu-3 cells. Our data demonstrate that both pNBC1 and NBC4 mRNAs can be identified and that their protein products are expressed at the basolateral membrane of polarized Calu-3 cells. These data suggest that these transporters contribute to regulated bicarbonate secretion across Calu-3 cells and perhaps human airway submucosal glands.
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PMID:Identification and membrane localization of electrogenic sodium bicarbonate cotransporters in Calu-3 cells. 1685 49

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.
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PMID:Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. 1722 Mar 10

Previously, we have shown that pancreatic acini release adenosine triphosphate (ATP) and ATP-handling enzymes, and pancreatic ducts express various purinergic P2 receptors. The aim of the present study was to establish whether pancreatic ducts also express adenosine receptors and whether these could be involved in secretory processes, which involve cystic fibrosis transmembrane regulator (CFTR) Cl- channels or Ca2+-activated Cl- channels and H(+)/HCO(-)(3) transporters. Reverse transcriptase polymerase chain reaction analysis on rat pancreatic ducts and human duct cell adenocarcinoma lines showed that they express A1, A2A, A2B, and A3 receptors. Real-time PCR revealed relatively low messenger RNA levels of adenosine receptors compared to beta-actin; the rank order for the receptors was A2A>A2B>or=A3>>A1 for rat pancreas and A2B>A2A>>A3>or=A1 for duct cell lines. Whole-cell patch-clamp recordings on rat pancreatic ducts showed that, in about half of the recordings, adenosine depolarized the membrane voltage, and this was because of the opening of Cl- channels. Using a Cl--sensitive fluorophore and single-cell imaging on duct cell lines, it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca2+ signals only in a few rat and human duct cells, which did not seem to correlate with Cl- signals. A2A receptors were localized in the luminal membranes of rat pancreatic ducts, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A2A receptors open Cl- channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini-to-duct signaling.
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PMID:Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport. 1805 56

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BK(Ca)) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch-clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca(2+) entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Cell-attached and whole-cell patch-clamp of HBE cells exposed to 4alphaPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BK(Ca) channels subsequent to extracellular Ca(2+) influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BK(Ca) modulation after TRPV4 activation showed that the Ca(2+) signal can be generated away from the BK(Ca) location at the plasma membrane, and it is not mediated by intracellular Ca(2+) release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BK(Ca) in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BK(Ca) in response to high-viscous solutions.
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PMID:Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines. 1845 41

Ca(2+)-activated Cl(-) channels (CaCCs) contribute to airway Cl(-) and fluid secretion, and were implicated in the modulation of disease severity and as a therapeutic target in cystic fibrosis (CF). Previous in vitro studies suggested that members of the CLCA gene family, including the murine mCLCA3, contribute to CaCCs. However, the role of mCLCA3 in ion transport in native airway epithelia has not been studied, to the best of our knowledge. In this study, we used mCLCA3-deficient mice and determined bioelectric properties in freshly excised tracheal tissue, airway morphology, and gene expression studies, to determine the role of mCLCA3 in airway ion transport and airway structure. Bioelectric measurements did not detect any differences in basal short-circuit current, amiloride-sensitive Na(+) absorption, cyclic adenosine monophosphate-dependent Cl(-) secretion, and activation of Ca(2+)-activated (uridine-5'-triphosphate-mediated) Cl(-) secretion in mCLCA3-deficient mice compared with wild-type mice. Moreover, no histological changes were observed in the respiratory tract or any other tissues of mCLCA3-deficient mice when compared with wild-type control mice. The intratracheal instillation of IL-13 produced an approximately 30-fold up-regulation of mCLCA3 transcripts without inducing CaCC activity in wild-type airways, and induced goblet-cell hyperplasia and mucin gene expression to similar levels in both genotypes. Further, multiple specific reverse-transcriptase quantitative PCR assays for other CaCC candidates, including mCLCA1, mCLCA2, mCLCA4, mCLCA5, mCLCA6, mCLCA7, mBEST1, mBEST2, mCLC4, mTTYH3, and mTMEM16A, failed to identify the differential expression of genes in the respiratory tract that may compensate for a lack of mCLCA3 function. Together, these findings argue against a role of mCLCA3 in CaCC-mediated Cl(-) secretion in murine respiratory epithelia.
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PMID:mCLCA3 does not contribute to calcium-activated chloride conductance in murine airways. 2236 87

Cystic Fibrosis is a genetic disease and it affects the respiratory and digestive systems. Pseudomonas aeruginosa infections in Cystic Fibrosis are presented as the main cause for high mortality and morbidity rates. Pseudomonas aeruginosa populations can regulate their virulence gene expressions via the bacterial communication system: quorum sensing. Inhibition of quorum sensing by employing quorum sensing inhibitors can leave the bacteria vulnerable. Therefore, determining natural sources to obtain potential quorum sensing inhibitors is essential. Lichens have ethnobotanical value for their medicinal properties and it is possible that their secondary metabolites have quorum sensing inhibitor properties. This study aims to investigate an alternative treatment approach by utilizing lichen secondary metabolite evernic acid to reduce the expressions of Pseudomonas aeruginosa virulence factors by inhibiting quorum sensing. For this purpose, fluorescent monitor strains were utilized for quorum sensing inhibitor screens and quantitative reverse-transcriptase PCR analyses were conducted for comparison. Results indicate that evernic acid is capable of inhibiting Pseudomonas aeruginosa quorum sensing systems.
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PMID:Lichen secondary metabolite evernic acid as potential quorum sensing inhibitor against Pseudomonas aeruginosa. 2746 50


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