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OBJECTIVE To compare the effect of four 5-hydroxytryptamine type 4 (5-HT4) receptor agonists:cisapride,zacopride,macopride and 2-[1-(4-piperonyl) piperazinyl]-benzothiazole (BZTZ),on rat cardiac inward rectifier potassium channel (IK1)and heart rhythm.METHODS The whole-cell configuration of patch-clamp technique was used to record effects of 5-HT4 receptor agonists onIk1 in enzymatic dissociated rat ventricular myocytes or Kir2.1 transfected HEK 293 cells.Western blotting was used to observe the expression of Kir2.1 channel exposed 24 h to agents in ventricular myocytes.Langendorff-perfused hearts were perfused with four agents respectively for 30 min.The electrocardiogram was recorded simultaneously.RESULTS BZTZ,cisapride and mosapride 0.1-10 μmol· L-1 decreasedIk1 in a concentrationdependent manner.At the same concentration (1 μmol· L-1),BZTZ showed the most potent inhibition onIκ1 (P＜0.01),followed by cisapride.Mosapride showed slight inhibition efficiency.However,zacopride enhanced Iκ1 (P＜0.01).In Kir2.1 heterologous expression systems,zacopride activated Kir2.1 current (P＜0.01) while mosapride had no effect.In ex vivo Langendorff-perfused hearts,BZTZ and cisapride 1μmol· L-1 elicited singnificant rhythm disturbances,and the total of premature ventricular beats (PVB) were 159±28 and 61±13.50％ (4/8) (P＜0.05) and 25％ (1/8) of the hearts exhibited ventricular tachycardia (VT),while 37.5％ (3/8) and 12.5％ (1/8) of the hearts exhibited ventricular fibrillation (VF),respectively.Mosapride and zacopride had no side effects on heart rhythm.Zacopride also suppressed BZTZ-or cisapride-induced arrhythmias.BZFZ had the strongest proarryhthmic potency among the 5-HT4 agonists,followed by cisapride,mosapride and zacopride.CONCLUSION Iκ1 might be an independent risk factor for arrhythmogenesis and a new target for screening safe 5-HT4 receptor agonists and gastrointestinal prokinetic agents.
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Objective:To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris.Methods:The α-facor-hirudin gene was amplified from pPIC9-hirudin by PCR and sub-cloned into PA0815.The multi-copy recombinant plasmid pA0815-(α-Hirudin)n was constructed.The recombinant was transformed into P.pastoris strain GS115 for induction expression and then the activity of secreted products was identified.Results:A new multi-copy vector pA0815-(αHirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently,which was confirmed respectively by PCR and SDS-PAGE.The products possessed the activity of thrombin inhibitor.Conclusion:This result offers efficient P.pastoris stains for mass production of biological active hirudin.
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Objective:To construct pGEX-3X/hTSHa Escherichia coli(E.coli)expression system and prepare purified recombinant GST-recombinant human thyroid stimulating hormone(rhTSH)α protein.Methods:The complete coding sequence of hTSHα was obtained by RT-PCR with total RNA extracted from fresh chorial tissue as the template,and thereafter cloned into expression vector pGEX-3X by EcoRl and BamHI digestion.The recombinant plasmid was transformed into E.coli Mach1-T1 and then induced expression by WrG.The GST-rhTSHα fusion protein was identified by SDS-PAGE and its antigenicity was verified by a modified competitive ELISA.Results:A specific protein band of 36 ku,in accordance with predicted molecular weight,could be visualized in SDS-PAGE.As the result of ELISA,the recombinant GST-hTSHα protein can inhibit the intact TSH molecular binding with anti-TSHα antibody in a dose dependent manner.Conclusion:The cDNA of hTSHα was cloned and the recombinant expression vector pGEX-3X/hTSHα was constructed successfully.The recombinant GST-rhTSHα protein could be highly expressed in E.coli Machl-T1 and was approved of possessing antigenicity.
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Objective To study the validity of RAND-UCLA (Rand Corporation and University of California at Los Angeles) consensus panel method in developing guidelines of referral indications for low back pain (LBP).Methods Evidence-based clinical guidelines for LBP management at community level and its referral guidelines published since 2001 and other tools were retrieved with varied tools.All clinical guidelines met inclusion criteria were evaluated with clinical studies and evaluation tools (AGREE).An pool of indication items was established based on evidence for developing referral indications for LBP, which were added by RAND-UCLA consensus panel method, and alternative referral indications were selected and clinical guidelines for LBP referral were established.Results A total of 15 copies of clinical guidelines from nine countries or regions were included in it after critical appraisal.Four copies of referral guidelines from two countries were included.Referral indications for LBP were derived directly from the RAND-UCLA consensus panel process, consisting of 44 referral indications for three groups (immediate, urgent and routine referral).Conclusions The RAND-UCLA consensus panel method is a more useful and practical tool in developing clinical guidelines, referral guidelines, which is worthwhile being recommended and spread.