Amajorroleforproteolyticactivityandproteinase-activated.pptVIP

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis Kristina K. Hansen, Philip M. Sherman, Laurie Cellars, Patricia Andrade-Gordon, Zhengying Pan, Amos Baruch,John L. Wallace, Morley D. Hollenberg, and Nathalie Vergnolle PNAS June 7, 2005 vol. 102 no. 23 8363–8368 生物科技學系四年級 邱英哲 Escherichia coli Pathogen Citrobacter rodentium ( C. rod. ) Human Host Murine Abstract 1. Proteinase-activated receptor ( PAR ) Family PAR1~4 2. PAR2’s activation induces acute inflammation Experimental Procedures 1.mice : six-week-old C57BL/6 wild-type mice、 PAR2-deficient ( - / - )C57BL/6 background 2.C. rodentium 107 CUF in 0.1ml or equal volume of broth (Control) 3.soybean trypsin inhibitor ( STI ) treatment : orally gavaged(100μl) with 40mg/day STI 4.Colon tissue : flushed with 0.5 ml PBS pH 7.4 1. Reference : a. C. rodentium release the trypsin-like proteinase b. Using the substrate, Boc-Gln-Ala-Arg-AMC for testing the proteinase activity 2. Hydrolysis rate over 20 min at RT luminal fluid 5μl substrate solution 200 μl 3. Determined by using a Fluoroskan Ascent microplate fluorometer (螢冷光儀) 1. Kirsten virus-transformed rat kidney (KNRK) cell vector-transfected with rat PAR2 (KNRK-PAR2) ≒106 cell/ml 2. Homogenates : 30-40 mg of colonic tissue in 1.5 ml 3. Using calcium ionophore A23187 (2μl) as a standard ( activated PAR2 ) 4. Desensitization of PAR2 ：by using SLIGRL-NH2 ( activated PAR2 ) Identified the activating proteinase(s) Using fluorescent in-gel assays To identify the molecular masses and substrate cleavage by proteinase with trypsin-like Purify of C. rodentium-infected luminal fluid : By affinity chromatography an