论文部分内容阅读
AIM:To clone and characterize the porcine aquaporins(AQPs)in the gastrointestinal system.METHODS:A PCR-based cloning strategy and RACEwere used to clone full-length AQP coding sequencefrom reversely transcribed pig liver cDNA.Stopped-flowlight scattering and a YFP-based fluorescence methodwere used to measure the osmotic water permeabilityof erythrocytes and the stably transfected CHO cells.RT-PCR,Northern blot,and immunohistochemistrywere used to determine the gastrointestinal expressionand localization of cloned AQPs.Protein expression intransfected cells and red blood cells was analyzed byWestern blot.RESULTS:An 813 bp cDNA encoding a 271 aminoacid porcine aquaporin(designated pAQP1)was clonedfrom liver mRNA(pAQP1 has a 93% identity withhuman AQP1 and contains two NPA motifs conservedin AQP family,one consensus sequence for N-linkedglycosylation,and one mercury-sensitive site at cysteine191).RT-PCR analysis revealed extensive expressionof pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript inselected digestive organs,pAQP1 protein was localizedat central lacteals of the small intestine,microvesslesof salivary glands,as well as epithelium of intrahepaticbile ducts by immunoperoxydase.High osmotic water permeability that is inhibitable by HgCl_2 was detected inporcine erythrocytes and CHO cells stably transfectedwith pAQP1 cDNA.Immunoblot analysis of porcineerythrocytes and pAQP-transfected CHO cells revealedan unglycosylated 28 ku band and larger glycosylatedproteins.CONCLUSION:pAQP1 is the first porcine aquaporinthat can be molecularly identified so far.The broaddistribution of pAQP1 in epithelium and endotheliumof porcine digestive organs may suggest an importantrole of channel-mediated water transport in fluidsecretion/absorption as well as in digestive function andpathophysiology of the gastrointestinal system.
AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system. METHODS: A PCR-based cloning strategy and RACE web for clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Topped-flowlight scattering and a YFP- based fluorescence method used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells. RT-PCR, Northern blot, and immunohistochemistry used to determine the gastrointestinal expression and localization of cloned AQPs.Protein expression intransfected cells and red blood cells were analyzed by Western blot .RESULTS: An 813 bp cDNA encoding a 271 aminoacid porcine aquaporin (designated pAQP1) was clonedfrom liver mRNA (pAQP1 has a 93% identity withhuman AQP1 and contains two NPA motifs conservedin AQP family, one consensus sequence for N-linkedglycosylation, and one mercury -sensitive site at cysteine 191). RT-PCR analysis confirmed extensive expressionof pAQP1 mRNA in porcine digestive glands and g ut.Northern blot showed a single 3.0 kb transcript inselected digestive organs, pAQP1 protein was localizedat central lacteals of the small intestine, microvesslesof salivary glands, as well as epithelium of intrahepaticbile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl_2 was detected inporcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Immunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins. CONCLUSION: pAQP1 is the first porcine aquaporin can be molecularly identified so far. The broad discription of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an importantrole of channel-mediated water transport in fluid secretion / absorption as well as in digestive function andpathophysiology of the gastrointestinal system.