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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.
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PMID:Simultaneous screening for 11 mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex amplification and reverse dot-blot. 137 93

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to defective Cl- conductance in epithelial cells. While the CFTR gene product has been detected in the plasma membrane, its presence and functional role in the membranes of intracellular compartments remain to be established. The purpose of the present experiments was to functionally localize CFTR in the endosomal membrane and to test the role of the associated Cl- conductance in the regulation of endosomal pH (pH(en)). When using conductive protonophores, the net H+ flux across the endosomal membrane of Chinese hamster ovary (CHO) cells is limited by the movement of counterions. Thus, ionic permeability could be estimated indirectly, from the changes in pH(en) determined fluorimetrically. Measurements in situ and in a cell-free microsomal preparation indicate the presence of a protein kinase A (PKA)-activated anion conductance in endosomes from CHO cells transfected with CFTR, but not in endosomes from wild-type or mock-transfected cells. In endosomes isolated from CFTR-expressing cells, the stimulatory effect of PKA was diminished by a specific peptide inhibitor of PKA, by alkaline phosphatase treatment or by a monoclonal antibody against the second nucleotide binding fold of CFTR. Increasing counterion permeability by phosphorylation of CFTR or by addition of valinomycin failed to alter the rate or extent of endosomal acidification in situ. Our observations indicate that functional CFTR, susceptible to activation by PKA, is present in endosomes of transfected CHO cells. More importantly, the data suggest that factors other than counterion permeability are the major determinants of pH(en).
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PMID:The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH. 137 35

The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine. The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting. The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low. Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR. The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation). We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis. These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo.
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PMID:Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells. 750 92

Inside-out apical membrane vesicles were isolated from bovine tracheal epithelium. They were enriched 13- and 18-fold in two apical membrane markers, alkaline phosphatase and gamma-glutamyltransferase, respectively, and presented a low level of contamination by basolateral and intracellular membranes. These apical membrane vesicles of homogeneous inside-out orientation were used to measure 36Cl- influx. The 36Cl- influx was found to be (i) voltage-insensitive (ii) diphenylcarboxylic acid-insensitive, and (iii) from 55 to 100% activated by cAMP-dependent protein kinase according to initial rates and accumulation capacities. This rapid and ATP-dependent activation was associated with phosphorylation of a 170-180-kDa protein but was not observed with a nonhydrolyzable nucleotide like adenosine 5'-O-(3-thiotriphosphate). Immunodetection experiments showed that the mature form of bovine cystic fibrosis transmembrane conductance regulator (CFTR) was only present in the apical membranes. As compared with the previously described characteristics of CFTR, the 36Cl- uptakes detected here are the in vitro manifestation of the functional form of bovine CFTR located at the apical level in these tracheal epithelial cells. Inside-out apical membrane vesicles, with freely accessible cytoplasmic sides and functional CFTR, offer a new model system to study CFTR.
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PMID:Functional characterization of cystic fibrosis transmembrane conductance regulator (CFTR) in apical membranes purified from bovine tracheal epithelium. 751 Feb 90

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.
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PMID:Phosphatase inhibitors activate normal and defective CFTR chloride channels. 752 29

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
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PMID:cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. 914 51

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.
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PMID:Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo. 937 79

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice. No induction of Cl- secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means +/- SE: jejunum, 1.8 +/- 0.9 microA/cm2, n = 7; cecum, 3.7 +/- 0.8 microA/cm2, n = 7; rectum (in vivo), 1.9 +/- 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice (cftrUNC). In the trachea, IBMX produced a transient Cl- secretory response (37.3 +/- 14.7 microA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 +/- 4.7 microA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl- secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl- secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.
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PMID:Effect of IBMX and alkaline phosphatase inhibitors on Cl- secretion in G551D cystic fibrosis mutant mice. 948 40

1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.
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PMID:Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells. 976 19

We determined optimum conditions for delivering DNA to transformed human bronchial epithelial cells expressing wild-type (BEAS) or abnormal (2CF) cystic fibrosis transmembrane conductance regulator (CFTR) using cationic liposomes (Lipofectin, [N-(N,N-dimethylaminoethane)carbamyl] cholesterol[DC-Chol]/dioleoylphosphatidylethanolamine[DOPE], or LipofectAMINE) and reporter genes which measured overall transgene expression (luciferase) or the fraction of cells transfected (heat-stable alkaline phosphatase). All liposomes showed dose-related toxicity. Optimal liposome and lipid: DNA ratios were different for BEAS than for 2CF cells. For all 3 liposome preparations, small particle size and net cationic charge related to transfection efficiency. Both LipofectAMINE and DC-Chol/DOPE transfected a maximum of 3% of BEAS cells, but luciferase expression could be increased without increasing the fraction of cells transfected. LipofectAMINE transfected a maximum of 6% of 2CF cells, and luciferase expression could be increased with no further increase in fraction of transfected cells. DC-Chol/DOPE transfected over 12% of 2CF cells with relatively small increases in luciferase expression. We conclude that an optimal cationic liposome and lipid: DNA ratio for transfecting bronchial epithelial cells depends on: (1) small particle size and net cationic charge, (2) whether the cells have the cystic fibrosis defect, and (3) whether the desired outcome is transfection of the maximum fraction of the cells or maximum total expression of the transgene.
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PMID:Optimization of cationic liposome-mediated gene transfer to human bronchial epithelial cells expressing wild-type or abnormal cystic fibrosis transmembrane conductance regulator (CFTR). 1035 50


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