简介:Glucosetransporter4(GLUT4)isresponsibleforinsulin-stimulatedglucosetransportingintotheinsulin-sensitivefatandmusclecells.ThedynamicsofGLUT4storagevesicles(GSVs)remainstobeexploredanditisunclearhowGSVsarearrangedbasedontheirmobility.Weexaminedthisissuein3T3-L1cellsviainvestigatingthethree-dimensionalmobilityofsingleGSVlabeledwithEGFP-fusedGLUT4.Athinlayerofcytosolrightadjacenttotheplasmamembranewasilluminatedandsuccessivelyimagedat5Hzunderatotalinternalreflectionfluorescencemicroscopewithapenetrationdepthof136nm.Employingsingleparticletracking,thethree-dimensionalsubpixeldisplacementofsingleGSVwastrackedataspatialprecisionof22nm.Boththemeansquaredisplacementandthediffusioncoefficientwerecalculatedforeachvesicle.Trackingresultsrevealedthatvesiclesmovedasifrestrictedwithinacagethathasameanradiusof160nm,suggestingthepresenceofsomeintracellulartetheringmatrix.ByconstructingthehistogramofthediffusioncoefficientsofGSVs,weobservedasmoothdistributioninsteadoftheexistenceofdistinctgroups.TheresultindicatesthatGSVsaredynamicallyretainedinacontinuousandwiderangeofmobilityratherthanintoseparateclasses.
简介:TheE(envelope)proteinisthesmalleststructuralproteininallcoronavirusesandistheonlyviralstructuralproteininwhichnovariationhasbeendetected.WeconductedgenomesequencingandphylogeneticanalysesofSARS-CoV.Basedongenomesequencing,wepredictedtheEproteinisatransmembrane(TM)pro-teincharacterizedbyaTMregionwithstronghydrophobicityandα-helixcon-formation.Weidentifiedasegment(NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH)inthecarboxyl-terminalregionoftheEproteinthatappearstoformthreedisulfidebondswithanothersegmentofcorrespondingcysteinesinthecarboxyl-terminusoftheS(spike)protein.ThesebondspointtoapossiblestructuralassociationbetweentheEandSproteins.OurphylogeneticanalysesoftheEproteinsequencesinallpub-lishedcoronavirusesplaceSARS-CoVinanindependentgroupinCoronaviridaeandsuggestanon-humananimalorigin.
简介:WestudiedstructuralandimmunologicalpropertiesoftheSARS-CoVM(mem-brane)protein,basedoncomparativeanalysesofsequencefeatures,phylogeneticinvestigation,andexperimentalresults.TheMproteinispredictedtocontainatriple-spanningtransmembrane(TM)region,asingleN-glycosylationsitenearitsN-terminusthatisintheexteriorofthevirion,andalongC-terminalregionintheinterior.TheMproteinharborsahighersubstitutionrate(0.6%correlatedtoitssize)amongviralopenreadingframes(ORFs)frompublisheddata.ThefoursubstitutionsdetectedintheMprotein,whichcausenon-synonymouschanges,canbeclassifiedintothreetypes.OneofthemresultsinchangesofpI(isoelectricpoint)andcharge,affectingantigenicity.ThesecondchangeshydrophobicityoftheTMregion,andthethirdonerelatestohydrophilicityoftheinteriorstructure.PhylogenetictreebuildingbasedonthevariationsoftheMproteinappearstosupportthenon-humanoriginofSARS-CoV.Toinvestigateitsimmunogenicity,wesynthesizedeightoligopeptidescovering69.2%oftheentireORFandscreenedthembyusingELISA(enzyme-linkedimmunosorbentassay)withserafromSARSpatients.Theresultsconfirmedourpredictionsonantigenicsites.
简介:利用构建的犬2型腺病毒E3区缺失质粒pBE3L分别构建了含有和不含外源性启动子的绿色荧光蛋白(GFP)基因和狂犬病病毒糖蛋白(Rgp)基因的重组表达质粒pBE3LGFP、pBE3LCGFP、pBE3LRgp和pBE3LCRgp,并分别对DK细胞进行了转染实验,以检测其表达,结果显示,pBE3LCGFP质粒在转染DK细胞后,于36h即可观察到荧光,72~96h无明显差别,传3代后仍可见表达荧光的细胞,pBE3LCRgp质粒转染DK细胞后,于48~96h用间接免疫荧光染色可检测到糖蛋白的表达,而pBE3LGFP和pBE3Rgp质粒转染DK细胞后经检测均无表达,表明构建的E3区缺失性载体不能利用E3区自身的启动子进行目的基因的表达,但利用外源性启动子可使外源基因获得良好表达.
简介:Mammaliancelltotipotencyisasubjectthathasfascinatedscientistsforgenerations.AlonglastingquestionwhethersomeofthesomaticcellsretainstotipotencywasansweredbythecloningofDollyattheendofthe20thcentury.Thedawnofthe218thasbroughtforwardgreatexpectationsinharnessingthepoweroftotipotentcyinmedicine.Throughstemcellbiology,itispossibletogenerateanypartsofthehumanbodybystemcellengineering.Considerableresourceswillbedevotedtoharnesstheuntappedpotentialsofstemcellsintheforeseeablefuturewhichmaytransformmedicineasweknowtoday.Atthemolecularlevel,totipotencyhasbeenlinkedtoasingulartranscriptionfactoranditsexpressionappearstodefinewhetheracellshouldbetotipotent.NamedOct4,itcanactivateorrepresstheexpressionofvariousgenes.Curiously,verylittleisknownaboutOct4beyonditsabilitytoregulategeneexpression.ThemechanismbywhichOct4specifiestotipotencyremainsentirelyunresolved.Inthisreview,wesummarizerethestructureandfunctionofOct4andaddresstoOct4functioninmaintainingtotipotencyorpluripotencyofembryonicstemcels.
简介:InteractionbetweencytotoxicTlymphocyte-associatedantigen-4(CTLA4,CD152)andB7molecules(B7-1andB7-2)isofimportanceinthecellulareventsoflymphocyte,includingantigen-specificT-cellactivationandinductionofautoreactiveT-cell.WedescribehaerethefirstintroductionofamurinesolubleCTLA4gene,CTLA4Ig,toMm1cells,amacrophagiccellline.CTLA4IgwassuccessfullyexpressedonMm1cellsandtheexpressedCTLA4IgwasfoundtobefunctionallyactiveintheirbindingtoB7moleculesbyflowcytometryandimmunofluorescencestudies.ThebiologicalactivityofCTLA4IgfromthetransfectedMm1cellswasstudiedandshowedinhibitoryactivityonmixedlymphocyteculture.AhighCTLA4Igproducingmacrophagiccelllinewasobtained.AsMm1cellswereregardedasdifficultforgenetransfectionandtherehassofarbeennoreportonexpressionofCTLA4IggeneonMm1cells,theseresultssuggestedthattheCELA4IgexpressingMm1cellscouldbeusefulforanalysisofCTLA4andB8moleculeinteractioninbothmacrophageandT-cell.
简介:Trichosanthin(TCS)isapotentallergentomice.Accordingtoourpreviousexperiments,itcouldbringouttheIgEresponsetoovabumin(OVA)ifTCSwasgivenonedaybeforeOVAimmunization,whileOVAalonecouldnotinduceIgEtoit.Inthiswork,thekineticsofinterleukin4(IL-4)andinterferonγ(IFN-γ)geneexpressioninthemesentericlymphnode(MLN)ofTCS-immunizedmicewasinvestigatedusingasemi-quantitativeRT-PCRmethod.ItindicatedthatTCSinducedsignificantIL-4geneexpressionandthepeaksofIL4geneexpressionwereondayoneafterTCSimmunizationinbothprimaryandsecondaryresponse.Incontrast,theIFN-γgeneexpressionwassuppressed.Furthermor,theIL-4geneexpressioninthesecondaryresponsewaslowerthanthatintheprimaryresponse.ThusthepresenceofIgEmemoryBcellswerestudied.ResultsshowedthattheamountofmatureIgEmRNAarosesignificantlyandrapidlyonedayafterTCSrestimulation,whileintheMLNofthemiceprimed30daysbeforeandwithoutboost,itwasalmostasthesameamountoftheunimmunizedcontrol.ThesefindingssuggesttheexistenceoftheIgEmemoryBcellsinthemiceaftertheprimaryTCSimmunization.
简介:Asimplemethodtocreateachromosome-specificDNAlibrqaryofrice,includingmicrodissection,amplification,charterizationandcloning,isdescribed.Ricechromosome4fromametaphasecellhasbeenisolatedandamplifiedbytheLinkerAdapterPCR(LA-PCR).ThePCRproductswerelabeledasprobeswithDIG-11-dUTPusingtherandomprimingmethod.SouthernblotanalysiswithricegenomicDNAandspecificRFLPmarkersdemonstratedthatthePCRproductswerederivedfromricechromosome4.Alargelibrarycomprisingover100,000recombinantplasmidmicroclonesfromricechromosome4wasconstructed.Colonyhybridizationshowedthat58%oftheclonescontainedsingleorlow-copysequencesand42%containedrepetitivesequences.ThesizeofinsertsgeneratedbyPCRrangedfrom140bpto500bp.ThismethodwillfacilitatecloningofthespecificchromosomeDNAmarkersandimportantgenesofrice.
简介:Changesinthedistributionof1P1-antigeninthedevelopingchickretinahavebeenexaminedbyindriectimmunofluorescencestainingtechniqueusingthenovelmonoclonalantibody(MAb)1P1.Expressionofthe1P1antigenwasfoundtoberegulatedinradialaswellasintangentialdimensionoftheretina,beingpreferentiallyorexclusivelylocatedintheinnerandouterplexiformlayersoftheneuralretinadependingonthestagesofdevelopment,Withtheonsetoftheformationoftheinnerplexiformlayer1P1antigenbecomesexpressedintheretina.Withprogressingdifferentiationoftheinnerplexiformlayer1P1immunofluorescencerevealed2subbandsatE9and6subandsatE18,Atpostnatalstages(afterP3)immunoreactivitywasreducedinaninside-outsidesequenceleadingtothecompleteabsenceofthe1P1antigeninadulthood.1P1antigenexpressionintheouterplexiformlayerwasalsosubjecttodevelopmentalregulation.Thespation-temporalpatternof1P1antigenexpressionwascorrelatedwiththetimecourseofhistologicaldifferentationofchickretina,namelythesynapserichplexiformlayers.Whetherthe1P1antigenwasfunctionallyinvolvedindendriteextensionandsynapseformationwasdiscussed.
简介:CTLA4Ig是人CTLA4胞外区与人免疫球蛋白铰链区、CH2区、CH3区组成的融合蛋白,可以与B7结合,通过阻断B7与CD28的结合,从而阻断B7介导的T细胞活化必需的共刺激信号,可作为免疫抑制剂用于器官移植。将CTLA4Ig融合分子克隆到真核表达载体pCI-dhfr,并用脂质体方法转染到COS7和CHO-dhfr-细胞中,用氨甲喋呤筛选转染的CHO-dhfr-细胞。用RT-PCR、ELISA、细胞免疫荧光染色和Western-blot鉴定重组蛋白的表达。采用A蛋白纯化重组蛋白。
简介:Interleukin-4isacytokineproducedbyactivatedTcells,mastcells,andbasophilsthatelicitsmanyimportantbiologicalresponses[1](seeTab1).TheseresponsesrangefromtheregulationofhelperTcelldifferentiation[2]andtheproductionofIgE[3]totheregulationoftheadhesivepropertiesofendothelialcellsviaVCAM-1[4],Inkeepingwiththesediversebiologicaleffects,high-affinitybindingsitesforIL-4(Kd20to300pM)havebeendetectedonmanyhematopoieticandnon-hematopoieticcelltypesatlevelsrangingfrom50to5000sitespercell[5].ThisreviewwillfocusonthediscretesignaltransductionpathwaysactivatedbytheIL-4recxeptorandthecoordinationoftheseindividualpathwaysintheregulationofafinalbiologicaloutcome.
简介:Arabidopsisthaliana嘘一deacetylase1(AtHD1或AtHDA19),酵母RPD3的一个相当或相同的事物,是在植物的许多生理、发展的过程的一个全球管理者。尽管有为在植物基因规定和开发的AtHD1的一个角色的基因证据,AtHD1的生物化学、细胞的性质糟糕被理解。这里,我们在vivo报导AtHD1的细胞的本地化模式并且嘘在vitro的一项deacetylase活动。绿荧光灯的蛋白质(GFP)的短暂、稳定的表示在洋葱房间标注了AtHD1并且分别地,在转基因的Arabidopsis的根,种子和叶子表明AtHD1在euchromatic区域大概在原子核是局部性的并且从核排除。AtHD1的本地化模式与涉及核形成和transgenes的silencing并且分别地重复了DNA元素的AtHD2和AtHDA6的那些不同。另外,一hist一deacetylase活动试金证明在细菌生产的recombinantAtHD1示威了一特定嘘在vitro的一项deacetylase活动。数据建议AtHD1是原子蛋白质并且拥有嘘为对植物生长和开发重要的全球transcriptional规定负责的一项deacetylase活动。