简介：ThegenomeoftheenterohemorrhagicEscherichiacoliO157:H7EDL933contains177“O”-islands(OIs).TostudytheirpotentialcontributiontotheO157-specificpathogenicity,wesurveyedthedistributionof22OIsbyPCRandDNAhybridizationin17isolatesofShigatoxinproducing(Stx-positive)E.coliO157:H7,andcomparedwiththeirdistributionin21isolatesofStx-negativeE.coliO157and21isolatesofnon-O157entericpathogens.Fourteenof22OIswerepresentinnon-O157entericpathogensanalyzed.Eightof22OIswerefoundonlyinthe17Shigatoxin-(Stx)positiveE.coliO157:H7isolates,buttheywereabsentfromthe21Stx-negativeE.coliO157:NMandO157Hundisolatestested.Amongthe8OIs,onlyOI43orOI48wereexclusivelydetectedinStx-positiveE.coliO157:H7,absentfromneitherofStx-negativeE.coliO157andnon-O157entericpathogens,suchasSalmonella,ShigeUa,Citrobacter,Vibriocholera,enteropathogen-icE.coli(EPEC),enteroadherentE.coli(EAEC),enteroinvasiveE.coli(E1EC)andenterotoxingenicE.coli(ETEC).TheOI43andOI48are83kbinsizeandidenticalinDNAsequences,whichencodegenesforurease,telluriteresistanceandadherence.ByanalyzingtheirjunctiongeneswithPCRandDNAhybridization,wefoundthat21ChineseisolateshaveOI48only.However,for7Japanesepatientisolates,4haveOI43and3haveOI48;forAmericanisolates,2havebothofO143andOI48,2haveOI48only.ThesedataconfirmedthehighlyplasticityofthepathogenicE.coligenome.TheuniquepresenceofOI43/OI48inStx-positiveE.coli0157:H7denotesitscriticalroleinthepathogenicityspecifictothispathogen.

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简介：ToconstructandexpressthefusionproteinStx2B-IntiminC300ofEHEC0157:H7,andtofurtherinvestigateitsimmunoprophyiacticpotential,thegeneofStx2B(stx2b)fromEHEC0157:H7chromosomewasclonedintopMD18-Tvector.Thereafter,theamplifiedgenewasclonedintoprokaryoticexpressionplasmidpET-28a(+)-eaeC300,whichwasconstructedpreviously.TherecombinantpasmidpET-28a(+)-stx2b-eaeC300wastransformedintoE.coliBL21(DE3).Afterinducement,theproteinStx2B-IntiminC300wassuccessfullyexpressedandanalyzedwithsodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE),WesternblottingandN-terminalaminoacidresidualsequencing.Toevaluateitsimmunoprophyiacticpotential,itwasprimarilypurifiedbyion-exchangechromatographyandinjectedinto30BALB/cmicewithAl(OH)3inthesubscapularregion.Tendaysafterthelastboostervaccination,20micewereattackedwithEHEC0157:H7lysateandtheprotectiveefficacywasobserved.Inthepresentstudy,thegeneofStx2B-IntiminC300wassuccessfullyclonedintopET-28a(+)vector.TheresultsofSDS-PAGEandWesternblottingassayshowedthatthefusionproteinwassuccessfullyexpressedintheinclusionbodyform,accountingfor25%oftotalexpressionproducts,anditsmolecularweightwasabout43kDa.TheresultoftheN-terminalaminoacidresidualsequencingshowedthatitwasidenticaltothatofthemoleculardesigned.Thepuritywasabout75%afterprimarypurification.AnimaltestsrevealedthatthefusionproteinStx2B-IntiminC300haselicitedhightiterofprotectiveantibodyrelatively.TheseresultsdemonstratethatthefusionproteinStx2B-IntiminC300issuccessfullyexpressedinprokaryoticexpressionsystemandshowscertainimmunoprophyiacticpotential.

简介：ToclarifytheroleofAPOBEC3G(A3G)incellulardefenseagainsthepatitisBvirus(HBV),theexpressionofA3GinnormalhumanliverandtheregulationoftheA3Gexpressioninhep-atomacellline(HuH-7)wereinvestigated.ExpressionlevelofAPOBEC3smRNAinhumanliverwasdeterminedbyRT-PCR.HuH-7andHepG2cellsweretreatedwithvariousconcentrationsofIFN-α(0U/ml,100U/ml,500U/ml,1000U/ml)for12h.ThemRNAlevelsweremeasuredbyaquantitativeRT-PCR,theresultswerenormalizedrelativetothespecimenswithoutIFN-αstimulation.TotalproteinofHuH-7cellstreatedwithvariousconcentrationsofIFN-αfor48hwassubjectedtoWesternblotanalysis.Forreportergeneassay,HuH-7cellsweretransfectedwiththereporterplasmidscontainingIRF-Esitesanditsmutantswithdifferentlengths.Thenthecellsweretreatedwithorwithout1200U/mlIFN-aforadditional12h(1000U/ml)after24hoftransfection,andthecelllysatewaspreparedandassayedforluciferaseactivity.ItwasfoundthatnormalhumanliverexpressedthemRNAofA3G.A3GmRNAexpressioninHuH-7andHepG2cellswereup-regulatedbyIFN-αstimulationinadose-depen-dentmanner.WesternblotanalysisindicatedthatA3GproteinexpressionwasalsoenhancedbyIFN-αstimulation.SequenceanalysisshowedtheexistenceofputativesitesofIFNregulatoryfactorelement(IRF-E)in5'regionofA3Ggeneupstreamtheinitiationcodon.IFN-αstimulationresultsin6-to8-foldincreaseinluciferaseactivityincellstransfectedwiththeplasmidcontainingIRF-Esitesofthe5'upstreamsequences,whereasluciferaseactivitydidnotchangeincellstransfectedwiththeplasmidcontainingmutantIRF-EsitesorwithoutIRF-Esites.Asaconclusion,A3Gareexpressedinnormalhumanliver.A3Gexpressionwasup-regulatedbyIFN-αstimulationinhepatomacellsandcouldbeinvolvedinhostdefensemechanismsagainstHBV.ERF-Esitein5'regionofAP0BEC3Ggeneupstreamtheinitiationcodonplaysanimportantroleinthisp

简介：TheaimofthisstudyistoinvestigatetheeffectsofallitridinontheexpressionoftranscriptionfactorT-bet/GA-TA-3inmiceinfectedbymurinecytomegalovirus.BALB/cmicemodelsystemofmurinecytomegalovirus(MCMV)infectionwasestablished.Inwhich20modelmicewereallocatedrandomlyintoallitridintreatedgroup(n=10)andinfectedcontrolgroup(n=10).Allitridin(25mg·kg^-1·d^-1)wasusedintreatedgroupatthe24hbyintraperitonealroute(once/d×14d),andthesamevolumeofsalinesolutionwasinjectedcontrolmice.Normalcontrolmice(n=10),wereonlygivenwiththesamevolumeof0.89%sodiumchloride,withoutinfectionwithMCMV.TheexpressionlevelsoftranscriptionfactorT-bet/GATA-3mRNAweremeasuredbyRT-PCR,andtheexpressionlevelsofThelper1(Thl)cytokineIFN-γandTh2cytokineIL-10insupernatantofspleencellcultureweremeasuredbyELISA.ExperimentalresultsshowedMCMVinfectioncouldmarkedlydown-modulatetheexpressionofIFN-γandT-bet,andsignificantlyup-modulatetheexpressionofIL-10andGATA-3mRNA.AllitridincouldinduceincreasedexpressionoftranscriptionfactorT-betmRNAandThlcytokineIFN-γsignificantly(P<0.01),anddecreasedexpressionoftranscriptionfactorGATA-3mRNAandTh2cytokineIL-10markedly(P<0.01).ItisconcludedthatMCMVinfectionleadstodisequilibriumofThl/Th2cytokineexpression:thelevelofThlcytokineIFN-γdecreasessignificantlyandTh2cytokineIL-10overexpressesmarkedly.Allitridincanup-regulatetheexpressionofT-betandIFN-γ,andinhibittheexpressionofGATA-3mRNAandIL-10inMCMVinfectedmice,indicatingaTh1dominantstatewhichshouldenhancethespecificcellularimmunereactionsagainstCMVandbehelpfulforclearanceofthecytomegalovirusinhost.

简介：InordertoinvestigatewhetherlipoxinA4(LXA4)hasanantagonisticeffectonlipopolysaccharide(LPS)-inducedsynthesisofinterleukin(IL)-1β,IL-6andIL-8inratpulmonarymicrovascularendothelialcells(PMVEC),andtoexplorethemolecularmechanismsofsignalpathwayinLXA4actions,culturedPMVECweretreatedwithLPS,withorwithoutpreincubationwithLXA4.ProteinsofIL-1β,IL-6andIL-8insupernatantwereanalyzedbyenzyme-linkedimmunosorbentassay(ELISA).ExpressionsofmRNAofIL-1β,IL-6andIL-8weredeterminedbyRT-PCR.Expressionsofphosphorylationofphosphoinositide3-kinase(PI3-K)andmyeloiddifferentiationfactor88(MyD88)wereanalyzedbyWesternblot.ActivitiesofDNA-bindingofnuclearfactor-kappaB(NF-κB)andactivatorprotein-1(AP-1)weremeasuredbyelectrophoreticmobilityshiftassay(EMSA).TheresultsshowedthatLPSinducedproductionofIL-1β,IL-6andIL-8inratPMVECviaMyD88/PI3-K/NF-κBandAP-1pathway-dependentsignaltransduction.LPS-stimulatedexpressionofPI3-K,activitiesofNFκBandAP-1,secretionofproteinandexpressionofmRNAofIL-1β,IL-6andIL-8butnotMyD88expressioninPMVECwereinhibitedbyLXA4inadose-dependentmanner.Inconclusion,LXA4inhibitssynthesisofIL-1β,IL-6andIL-8bydown-regulationofPI3-K/NF-κBandAP-1signalpathwayinPMVEC.

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简介：Inthepresentstudy,theeffectofblockageofthecostimulatorysignalCD86attimeofimplantationontheexpressionsofTGF-β1,MMP-9,TIMP-3andPAI-1proteinsatthematernal-fetalinterfaceandtheoutcomeofpregnancyinmurineabortion-pronemodelwasinvestigated,inwhichtheCBA/JxDBA/2matingswereusedastheabortion-pronemodelandtheCBA/J×BALB/cmatingsusedasthenormalpregnantmodel.Thestudywasperformedinfollowingthreegroups:2groupsoftheabortion-pronemodel,whichwereexperimentalgroupandcontrolexperimentalgroup,and1groupofnormalpregnantmodel,andeachgrouphad10pregnantCBA/Jmiceexclusively.FemalepregnantCBA/Jmiceintheexperimentalgroupreceivedanintraperitoneal(i.p.)injectionof100μgofantimouseCD86mAbin200μlofPBSatday4.5ofgestation,andtheirrelevant-isotopematchedratIgG2bwasadministratedinthecontrolexperimentalgroupwiththesamedosageandatsametime.Forthenormalpregnantgroup,notreatmentwasgiven.ThepregnantCBA/Jmicewerekilledonday13.5ofgestation.Then,theembryoresorptionratewascalculatedandtheexpressionsofTGF-β1,MMP-9,TIMP3andPAI-1weredetectedbyusingimmunohistochemicalmethods.Itwasdemonstratedthattheembryoresorptionrateintheexperimentalgroupwassignificantlyreducedincomparisonwiththatinthecontrolexperimentalgroup(x2=7.441,P=0.006),buttherewasnosignificantdifferencewiththatinnormalpregnantgroup(x2=0.016,P=0.898).TheexpressionsofTGF-β1andPAI-1intheexperimentalgroupweresignificantlyincreasedincomparisonwiththatinthecontrolexperimentalgroup(P=0.010,P=0.003,respectively),withnosignificantdifferencefromthatinthenormalpregnantgroup(P=0.500).However,theexpressionofMMP-9intheexperimentalgroupwassignificantlyreducedincomparisonwiththatinthecontrolexperimentalgroup(P=0.012)withnosignificantdifferencefromthatinthenormalpregnantgroup(P=0.500).Theexpression

简介：ExposureofnaivemurineCD4^+TlymphocytestosuperantigensuchasstaphylococcalenterotoxinB(SEB)inducesastrongproliferativeresponse.ProlongedexposureorsubsequentrestimulationoftherespondingTcellpopulationwithSEBleadstotheapoptoticeventsofactivation-inducedcelldeath(AICD).ThesignalingmechanismresponsiblefortheAICDisatargetofintensiveinvestigation.However,theprecisedownstreamsignahngpathwaysofSEB-inducedAICDremainsunclear.Ourresultshereshowthatthesequentialactivationofcaspase-1/ICE-hkeandcaspase-3/CPP32-hkecysteineproteasesprobablyplaysaroleinthesignalingtransductionofSEB-inducedAICD,butcaspase-3/CPP32-hkeproteasesactivationdoesnotdependoncaspase-1-likeproteasesactivation.HerbimycinA,aspecificinhibitorofproteintyresinekinases,inhibitcaspase-3/CPP32-1ikecysteineproteasesactivation.However,itdoesnotpreventDNAfragmentationofCD4^+TcellsapoptosisinducedbySEB.TheseresultsindicatethatproteintyrosinekinasespathwayisprobablyinvolvedinthesignalingtransductionofCD4^+TcellsapoptosisinducedbySEBand“crosstalks”withthepathwayofcaspase-3/CPP32-1ikeproteasesactivation.