Mutations in the cystic fibrosis transmembrane conductance regulator （CFTR） gene have been implicated in the onset of cystic fibrosis and other clinical respiratory disorders. In the present study, we investigated t...Mutations in the cystic fibrosis transmembrane conductance regulator （CFTR） gene have been implicated in the onset of cystic fibrosis and other clinical respiratory disorders. In the present study, we investigated the role of CFTR variations, poly-T, TG-repeats, and M470V in susceptibility to bronchial asthma and chronic bronchitis in a Chinese population in Jiangsu province, China. A total of 72 bronchial asthma patients, 68 chronic bronchitis pa- tients, and 117 healthy subjects were included in this study. The Tn-TGm haplotype was sequenced and the CFTR variant M470V was detected using restriction fragment length polymorphism （RFLP）. We found that the fre- quency of TS-TG12-V470 in chronic bronchitis patients was 0.07%, which was notably higher than that in healthy subjects （0.01%） and bronchial asthma patients （0.04%）. Thus, the presence of the TS-TG12 haplotype of the CFTR gene is likely to play a role in the development and progression of respiratory conditions, such as chronic bronchitis.展开更多
A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of...A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.展开更多
Pancreas divisum is a kind of congenital anatomic abnormality;its diagnostic basis depends mainly on imaging examination. With the development of medical science, imaging technology has been improved and added, and a ...Pancreas divisum is a kind of congenital anatomic abnormality;its diagnostic basis depends mainly on imaging examination. With the development of medical science, imaging technology has been improved and added, and a complete examination system including ERCP, B-Ultrasound, MRCP, S-MRCP and CT, etc. has been formed. There are even researcher, through further analysis of pancreas divisum on the level of genes, found that CFTR is a risk factor causing such disease. This paper is focused on the value of these examination methods in the diagnosis of pancreas divisum.展开更多
The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFT...The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane(BCO) G551D-CFTR activators(G551D_ BCOs) with K_d down to 1 μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551D_ BCOs by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551D_ BCOs-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551D_ BCOs; (2) G551D_ BCOs and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.展开更多
Previous studies reported that capsaicin potentiates AF508 mutant cystic fibrosis transmembrane conductance regulator（CFTR） channel gating defect by transfected cell-based assays. It has been postulated that orally ...Previous studies reported that capsaicin potentiates AF508 mutant cystic fibrosis transmembrane conductance regulator（CFTR） channel gating defect by transfected cell-based assays. It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients. We tried to reproduce and extend those pre-clinical data of previous studies. Cell-based fluorescence func- tional measurements in Fischer thyroid epithelial cells（FRT） expressing CFTR showed no effect of capsaicin on potentiating AF508-CFTR, while genistein showed a strongly positive activity. Studies show that capsaicin and dihy- drocapsaicin activated cAMP-prestimulated wild-type CFTR in a dose-dependent manner with a maximal response of 70% of that activated by genistein, thus gave an apparent EC50 of （40.4±6.8）μmol/L and （150.2±7.4） μtmol/L respectively. Preliminary study shows that the binding sites for capsaicin and dihydrocapsaicin may be probably partially overlapped with that for genistein because the maximal activation of wild-type CFTR with genistein is partially blocked by caosaicin and dihydrocapsaicin.展开更多
Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A...Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.展开更多
基金supported by the Biotechnology and Biological Sciences Research Council and the Cystic Fibrosis Trust.H Li was supported by EuroCareCF(LSHM-CT-2005-018932)while J-H Chen and Z Xu were supported by scholarships from the University of Bristol and ORS awards from Universities UK.
基金supported by the Key Talent's Subsidy Project in Science and Education of the Jiangsu Province (No. 2007158)
文摘Mutations in the cystic fibrosis transmembrane conductance regulator （CFTR） gene have been implicated in the onset of cystic fibrosis and other clinical respiratory disorders. In the present study, we investigated the role of CFTR variations, poly-T, TG-repeats, and M470V in susceptibility to bronchial asthma and chronic bronchitis in a Chinese population in Jiangsu province, China. A total of 72 bronchial asthma patients, 68 chronic bronchitis pa- tients, and 117 healthy subjects were included in this study. The Tn-TGm haplotype was sequenced and the CFTR variant M470V was detected using restriction fragment length polymorphism （RFLP）. We found that the fre- quency of TS-TG12-V470 in chronic bronchitis patients was 0.07%, which was notably higher than that in healthy subjects （0.01%） and bronchial asthma patients （0.04%）. Thus, the presence of the TS-TG12 haplotype of the CFTR gene is likely to play a role in the development and progression of respiratory conditions, such as chronic bronchitis.
文摘A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.
文摘Pancreas divisum is a kind of congenital anatomic abnormality;its diagnostic basis depends mainly on imaging examination. With the development of medical science, imaging technology has been improved and added, and a complete examination system including ERCP, B-Ultrasound, MRCP, S-MRCP and CT, etc. has been formed. There are even researcher, through further analysis of pancreas divisum on the level of genes, found that CFTR is a risk factor causing such disease. This paper is focused on the value of these examination methods in the diagnosis of pancreas divisum.
文摘The G551D-CFTR mutation causing cystic fibrosis(CF) results from a missense mutation at codon 551(G551D) in the gene encoding of the cystic fibrosis transmembrane conductance regulator(CFTR). The G551D mutation in CFTR results in a reduced functional channel but G551D-CFTR is appropriately inserted in the apical membrane. In previous studies we discovered a class of high-affinity bicyclooctane(BCO) G551D-CFTR activators(G551D_ BCOs) with K_d down to 1 μmol/L. In this study, we analyzed the pharmacological activation of G551D-CFTR by the G551D_ BCOs by means of short circuit current analysis and cell-based fluorescence quenching assay. The G551D_ BCOs-induced G551D-CFTR activation is cAMP-dependent and is less sensitive to thiazolidinone CFTR inhibitor CFTRinh-172. These data suggest that (1) the phosphorylation of G551D-CFTR by protein kinase A is required for the activation by G551D_ BCOs; (2) G551D_ BCOs and CFTRinh-172 may act at the same site on the G551D-CFTR molecule.
基金Supported by New Century Excellent Talents Program in University(No.NCET-07-0406)Liaoning Excellent Talents Pro-gram in University(No.2006R33)+1 种基金Dalian Municipal Science and Technology Fund(No.2006J23JH018)Science and Tech-nology Fund from the Education Department of Liaoning Province, China(No.20060492).
文摘Previous studies reported that capsaicin potentiates AF508 mutant cystic fibrosis transmembrane conductance regulator（CFTR） channel gating defect by transfected cell-based assays. It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients. We tried to reproduce and extend those pre-clinical data of previous studies. Cell-based fluorescence func- tional measurements in Fischer thyroid epithelial cells（FRT） expressing CFTR showed no effect of capsaicin on potentiating AF508-CFTR, while genistein showed a strongly positive activity. Studies show that capsaicin and dihy- drocapsaicin activated cAMP-prestimulated wild-type CFTR in a dose-dependent manner with a maximal response of 70% of that activated by genistein, thus gave an apparent EC50 of （40.4±6.8）μmol/L and （150.2±7.4） μtmol/L respectively. Preliminary study shows that the binding sites for capsaicin and dihydrocapsaicin may be probably partially overlapped with that for genistein because the maximal activation of wild-type CFTR with genistein is partially blocked by caosaicin and dihydrocapsaicin.
基金Supported by the National Natural Science Foundation of China(Nos.30670477, 30973577 and 30770493)the National Basic Research Program of China(No.2009CB521908)
文摘Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.
