简介:ThekinematicsofturningmaneuversofstartledCrucianCarp(Carassiusauratus)arepresented.AllescaperesponsesobservedareC-typefast-starts.Thepositionofthecenterofmassandthemomentofinertiaofthefisharecalculated.Theresultsshowthatthepositionofthecenterofmassisalwaysat35%ofthelengthofthefishfromtheheadandthepositionofthecenterofmassandmomentofinertiacanbeconsideredunchangedduringC-startofCrucianCarp.HydrodynamicanalysisoftheC-startisgivenbasedonthekinematicsdatafromourexperiments.TheC-startconsistsofthreestages.Instage1,thetailfinoffishrapidlyflapsinonedirection,andalargemomentactsonthefish'sbody,whichrotatesaroundthecenterofmasswithanangularacceleration.Instage2,thetailfinflapsmoreslowlyintheoppositedirectionatslowerspeed,thefish'sbodyrotatesaroundthecenterofmasswithangulardecelerationandthecenterofmassofthefishmovesalonganarc.Instage3,themomentapproximatelyequalszero,thefish'sbodystopsrotatingandthecenterofmassthemovesalongastraightline.
简介:目的探索BALB/c和C57BL/6两个品系在有关实验中的不同作用。方法选取了结合随机测序与生物信息学分析设计合成的神经系统表达的一些基因的反义核酸(antisense)中的2个,用Hamilton微量注射器将其分别定量注射到BALB/c和C57BL/6小鼠的侧脑室,并分别设注射生理盐水和随机序列核酸(Scramble)的对照组。每一反义核酸实验组和对照组各注射10只小鼠,之后观察实验组与对照组在不同行为学实验中的差异。小鼠的行为学检测模型为:考察日常代谢能力的摄食量,考察Locomotionactivity(移动)的旷场行为,考察疼痛阈值的甩尾试验和考察记忆能力的步下法实验。结果注射No.1基因的反义核酸后,两品系的实验组均在测试记忆力的步下法(Step-downTest)试验中表现出记忆力减弱,且与对照组差异明显,说明No.1基因的功能确与记忆相关。注射No.2基因的反义核酸后,在测试移动能力的旷场行为(OpenFieldBehavior)试验中,BALB/c实验组跨格、直立行为均比对照组明显减少,说明受此反义核酸影响显著,而C57BL/6实验组则与对照组无大的差异。此外,在生理盐水对照组和随机序列核酸对照组的实验中以及其他行为学模型的实验中,两品系也存在着一定的差异。结论用遗传背景不同的多品系进行相关实验,可进一步建立新基因功能初筛中有显著结果的基因的复筛平台;同时,实验结果对于进一步研究什么样的品系适用于什么样的实验将具有较大的意义。
简介:ThemuscleproteinmyosinbindingproteinC(MyBPC)isalargemulti-domainproteinwhoseroleinthesarcomereiscomplexandnotyetfullyunderstood.MutationsinMyBPCarestronglyassociatedwiththeheartdiseasefamilialhypertrophiccardiomyopathy(FHC)andtheseexperimentsofnaturehaveprovidedsomeinsightintotheintricateworkingsofthisproteinintheheart.WhilesomeregionsoftheMyBPCmoleculehavebeenassignedafunctionintheregulationofmusclecontraction,theinteractionofotherregionswithvariouspartsofthemyosinmoleculeandthesarcomericproteins,actinandtitin,remainobscure.Inadditicn,severalintra-domaininteractionsbetweenadjacentMyBPCmoleculeshavebeenidentified.Althoughthebasicstructureofthemolecule(aseriesofimmunoglobulinandfibronectindomains)hasbeenelucidated,theassemblyofMyBPCinthesarcomereisatopicfordebate.ByanalysingtheMyBPCsequencewithrespecttoFHC-causingmutationsitispossibletoidentifyindividualresiduesorregionsofeachdomainthatmaybeimportanteitherforbindingorregulation.Thisreviewlooksatthecurrentliterature,inconcertwithalignmentsandthestructuralmodelsofMyBPC,inanattempttounderstandhowFHCmutationsmayleadtothediseasestate.
简介:Amurinemacrophage-likecelllineJ774,acquired,inresponsetoLPS,anabilitytokilltumornecrosisfactor(TNF)-insensitivetargetP815mastocytomacellswhereasanothercellline,P388D1didnot,LPStriggeredsignalingmechanismsbetweenthetwocelllineswerecomparedwithanaimtoinquireaboutthepossiblenatureoftheabove-mentioneddifference,TheresultswhowedthattwocelllinesrespondtoLPS-treatmentbyparallelactivationofbothphospholipasesCandA2(PLCandPLA2)toapproximatelythesameextent.ThemaximumresponseoftothenzymesofJ774cellswasnotedwithin10minthetreatmentwhereasthatofP388D1cellsrequiredmorethan20min,TheotherpropertiesofLPS-responsiveenzymesstudiedweresimilarbetweentwocelllines,includingActivationofPLCandPLA2andPKCinmacrophagesbyLPS.Ca2+augmentationofenzymeactivation,participationofguaninenucleotidebinding(G)proteinsintheinitialactivationpreocesses,andinhibitionofenzymeactivationbythepriortreatmentofcellswithcholeraorpertussistoxinsetc.Moreover,LPS-triggeredactivationofPLCandPLA2wasfoundtobefollowedbytheincreaseofPKCactivitiesinbothcelllines.Inspiteofthesesimilarities.J774cellspossessedbothbasicandacidicformsofPKCactivities,whileP388D1cellsownedonlyPKCofbasicform,Nevertheless,thequestionwhyJ774cellsbutnotP388D1cells,canacquirethetumoricidalactivity,aganistP815,cellsfollowingLPStreatmentrematinstobeanswered.
简介:InordertodevelopclinicaldiagnostictoolsforrapiddetectionofSARS-CoV(severeacuterespiratorysyndrome-associatedcoronavirus)andtoidentifycandidateproteinsforvaccinedevelopment,theC-terminalportionofthenucleocapsid(NC)genewasamplifiedusingRT-PCRfromtheSARS-CoVgenome,clonedintoayeastexpressionvector(pEGH),andexpressedasaglutathioneS-transferase(GST)andHisx6double-taggedfusionproteinunderthecontrolofaninduciblepromoter.WesternanalysisonthepurifiedproteinconfirmedtheexpressionandpurificationoftheNCfusionproteinsfromyeast.Todetermineitsantigenicity,thefusionproteinwaschallengedwithserumsamplesfromSARSpatientsandnormalcontrols.TheNCfusionproteindemonstratedhighantigenicitywithhighspecificity,andtherefore,itshouldhavegreatpotentialindesigningclinicaldiagnostictoolsandprovideusefulinformationforvaccinedevelopment.
简介:Thenorepinephrinetransporter(NET)isamemberoftheNa^+/Cl^-dependentneurotransmittertransporterfamilyandconstitutesthetargetofseveralclinicallyimportantantidepressants.TodelineatethecriticalaminoacidresiduesandthefunctionofC-terminalinregulatingtransportactivityofNET,hereweconstructedtwositemutants(V70F,F72V;V70I,F72V)andoneC-terminaltruncatedmutant(Δ611-617).ThewildtypeandmutantsofNETwereexpressedinXenopusoocytesbyinjectionoftheircRNA.Wefoundthatallofthesemutantslosttheirtransportactivity.TheseresultsindicatethattheaminoacidresiduesofV70andF72,andthelastsevenaminoacidsofC-terminalareessentialtothetransportactivityofNET.
简介:Thec-erbB-2proto-oncogeneencodesa185kDaproteinp185,whichbelongstoepidermalgrowthfactorreceptorfamily.Amplificationofthisgenehasbeenshowntocorrelatewithpoorclinicalprognosisforcertaincancerpatients.ThemonoclonalantibodyA21whichdirectedagainstp185specificallyinhibitsproliferationoftumorcellsoverexpressingp185,henceallowsittobeacandidatefortargetedtherapy.InordertoovercomeseveraldrawbacksofmurineMAb,wecloneditsVHandVLgenesandconstructedthesingle-chainFv(scFv)throughapeptidelinker.TherecombinantscFvA21wasexpressedinEscherichiacoliandpurifiedbytheaffinitycolumn.SubsequentlyitwascharacterizedbyELISA,Westernblot,cellimmunohistochemistryandFACS.Alltheseassaysshowedthebindingactivitytoextracellulardomain(ECD)ofp185.BasedonthosepropertiesofscFvA21,wefurtherconstructedthescFv-Fcfusionmoleculewithahomodimerformandtherecombinantproductwasexpressedinmammaliancells.Inaseriesofsubsequentanalysisthisfusionproteinshowedidenticalantigenbindingsiteandactivitywiththeparentantibody.Theseanti-p185engineeredantibodieshavepromisedtobefurthermodifiedasatumortargetingdrugs,withaviewofapplicationinthediagnosisandtreatmentofhumanbreastcancer.
简介:小道,肿瘤坏死因素相关的导致apoptosisligand,是一个新奇有势力通过房间表面死亡受体Trail-R1和Trail-R2的激活的房间死亡小径的内长的使活跃之物。它的角色象在导致激活的房间死亡(AICD)的FasL一样,在免疫系统被表明了。然而,小道的机制导致了apoptosis遗体不清楚。在这份报告,重组体小道蛋白质被表示并且净化。导致apoptosis活动和JurkatT房间上的重组体小道的规定机制是探索试管内。Trypan蓝排除试金证明重组体小道蛋白质活跃地以一种剂量依赖者方式杀死了JurkatT房间。在JurkatT房间的导致小道的apoptosis被Bcl-2显著地在Bcl-2基因transfected房间在表示上减少。有PMA(phorbol12十四酸盐13醋酸盐)的处理,PKC使活跃之物,在JurkatT房间的压制的导致小道的apoptosis。由PMA的apoptosis的抑制被预告的处理与二度废除,一个PKC禁止者。总起来说,Bcl-2在表示上和PMA激活PKC,这被建议活跃地下面调整在JurkatT的调停小道的apoptosis房间。
简介:为了研究禽流感H5N1病毒在各个器官的增殖和病理变化,在生物安全实验室,我们将禽流感H5N1病毒通过尾静脉接种BALB/C小鼠。结果小鼠在不经过适应的情况下,直接感染发病,甚至死亡。在观察的7天内,感染小鼠临床症状主要表现呼吸急促,体温、体重下降。尸检表现肺出血,心外膜坏死以及肝脏的坏死。组织病理检查表现心、肝、肺等多器官的病变。肺的病变伴有纤维化的弥漫性肺泡损伤;心肌外膜大量淋巴细胞浸润、坏死;肝细胞大量坏死,淋巴细胞浸润。心、肝的坏死病变在H5N1禽流感病毒相关的研究中未见报道。经过对各个组织器官的病毒载量的检测,未发现病毒在各个病变组织中的复制。免疫组化的检测,各个组织中也未检出阳性的细胞反应。因此,我们认为H5N1禽流感病毒感染小鼠引起多个器官组织的损伤,甚至死亡,不是病毒在器官的复制,而可能是病毒感染小鼠,产生炎症细胞因子的高度表达,损伤多个器官组织所致。
简介:为了研究通过功能筛选得到的一个新的红细胞分化相关的全长cDNA(命名为EDRFI)的功能,选择K562细胞作为模型细胞来观察反义EDRFI表达对细胞功能的影响.通过快速构建,同时得到了正向和反向插入片段的真核表达载体pcDNA3-EDRFI-S(sense)及peDNA3-AS(antisense),并通过改进的LipofectAMINE细胞转染和G418筛选,得到了稳定表达的细胞株,进行基因组PCR鉴定,证明了转染的高效性.NorrhemB10t试验的结果表明,反义载体转染的K562细胞中,EDRF1在mRNA表达水平上受到下调,提示EDRF1反义DNA的表达可能抑制了EDRF1mRNA的正常转录.进一步,γ-珠蛋白和PKC-α的表达在反义构建质粒转染K562细胞中的表达均受到下调,这可能传递了红细胞分化的负反馈信号,从而抑制了珠蛋白的合成.
简介:Amurinemacrophage-likecellline,J774,acquried,inresponsetoLPS,anabilitytokilltumornecrosisfactor(TNF)-insensitivetargetP815mastocytomacells,whereasanothercellline,P388D1,didnot.LPS-triggeredsignalingmechanismsbetweenthetwocelllineswerecomparedwithanaimtoinquireaboutthepossiblenatureoftheabove-mentioneddifference.TheresultsshowedthattwocelllinesrespondtoLPS-treatmentbyparallelactivationofbothphospholipasesCandA2(PLCandPLA2)toapproximatelythesameextent.ThemaximumresponseofbothenzymesofJ774cellswasnotedwithin10minofthetreatment,whereasthatofP388D1cellsrequiredmorethan20min.TheotherpropertiesofLPS-responsiveenzymesstudiedweresimilarbetweentwocelllines,ineludingActivationofPLCandPLA2andPKCinmacrophagesbyLPSCa2+augmentationofenzymeactivation,participationofguaninenucleotidebinding(G)proteinsintheinitialactivationprocesses,andinhibitionofenzymeactivationbythepriortreatmentofcellswithcholeraorpartussistoxinsetc.Moreover,LPS-triggeredactivationofPLCandPLA2wasfoundtobefollowedbytheincreaseofPKCactivitiesinbothcelllines.Inspiteofthesesimilarities,J774cellspossessedbothbasicandacidicformsofPKCactivities,whileP388D1cellsownedonlyPKCofbasicform.Nevertheless,thequestionwhyJ774cells,butnotP388D1cells,canacquirethetumoricidalactiyity,aganistP815cellsfollowingLPS-treatmentremainstobeanswered.