Cell-penetratingpeptideandionchannelsblocker
Crotamine isa basicpeptide presentinthe venom ofthe SouthAmerican rattlesnakeCrotalusdurissusterrificus.MultipleBIOLOGicalfunctionshavebeenattributedtoCrotamine.Itisanaturalcell-penetratingpeptidewithselectivebiologicalactiontowardsactivelyproliferatingcelltypes.Moreover,ithasbeenreportedthatcrotamineisablockerofKv1.3(IC50around300nM)aswellasKv1.1andKv1.2.Ithasanalgesicpropertiesandmyonecroticeffects.Inaddition,crotaminebelongstothebeta-defensinpeptidesandassuchdemonstratesantibacterialpropertiesbyinteractingwithlipidmembranes.
Description:
AAsequence: YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG-OH
Disulfidebridges: Cys4-Cys36;Cys11-Cys30;Cys18-Cys37
Length(aa):41
Formula:C214H326N64O54S7
MolecularWeight:4883.82Da
Appearance:whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:
Source:synthetic
Purityrate:>95%
Reference:
CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus
DeLuccaFL.,etal.(1974)CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus(Ophidia,Reptilia)aftermanualextractionofthevenom.Studiesontemplateactivityandbasecomposition.BiochemJ.PMID:4463939
Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus
Crotamine,a4.88kDaneurotoxicprotein,hasbeenpurifiedtoapparenthomogeneityfromCrotalusdurissusvenombygelfiltrationonSephadexG-75.Wheninjected(i.p.ors.c.)inadultmaleSwissmice(20-25g),itinducedatime-dosedependentanalgesiceffectwhichwasinhibitedbynaloxone,thussuggestinganopioidactionmechanism.Whencomparedwithmorphine(4mg/kg),crotamine,eveninextremelylowdoses(133.4microg/kg,i.p.,about0.4%ofaLD50isapproximately30-foldmorepotentthanmorphine(w/w)asananalgesic.Onamolarbasisitismorethan500-foldmorepotentthanmorphine.Itisalsomuchmorepotentthanthelowermolecularweightcrudefractionsofthesamevenom.Theantinociceptiveeffectsofcrotamineandmorphinewereassayedbythehotplatetestandbytheaceticacid-inducedwrithingmethod.Therefore,bothcentralandperipheralmechanismsshouldbeinvolved.Histopathologicalanalysisofthebrain,liver,skeletalmuscles,stomach,lungs,spleen,heart,kidneysandsmallintestineofthecrotamineinjectedmicedidnotshowanyvisiblelesioninanyoftheseorgansbylightmicroscopy.Sincecrotamineaccountedfor22%(w/w)ofthedesiccatedvenom,itwasidentifiedasitsmajorantinociceptivelowmolecularweightpeptidecomponent.
MancinAC.,etal.(1998) Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus(SouthAmericanrattlesnake)venom:abiochemicalandpharmacologicalstudy.Toxicon.PMID:9839677
Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus
Hereinwereportthatcrotamine,asmalllysine-andcysteine-richproteinfromthevenomoftheSouthAmericanrattlesnake,canrapidlypenetrateintodifferentcelltypesandmouseblastocystsinvitro.Invivocrotaminestronglylabelscellsfrommousebonemarrowandspleenandfromperitonealliquid,asshownbyfluorescentconfocallaser-scanningmicroscopy.Nuclearlocalizationofcrotaminewasobservedinbothfixedandunfixedcells.Inthecytoplasm,crotaminespecificallyassociateswithcentrosomesandthusallowsustofollowtheprocessofcentrioleduplicationandseparation.Inthenucleus,itbindstothechromosomesatS/G2phase,whencentriolesstartdividing.Moreover,crotamineappearsasaMarkerofactivelyproliferatingcells,asshownby5-BrdUcell-proliferationassay.CrotamineinthemicromolarrangeprovednontoxictoanyofthecellculturestestedanddidnotaffectthepluripotencyofEScellsorthedevelopmentofmouseembryos.
KerkisA.,etal.(2004)Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus.FASEBJ.PMID:15231729
BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake
Moleculesisolatedfromanimals,insects,plantsormicroorganismscanprovideprototypesfordesignofbiopharmaceuticalproducts.Somevenomtoxinsandtheirderivativesareusedinmedicine,whileothersprovidetemplatesfordevelopmentofnewdrugs.Themildtoxin,crotamine,asmallbasiclow-molecular-weightpolypeptidepurifiedfromthevenomofaSouthAmericanrattlesnake,Crotalusdurissusterrificus.Crotaminewasdiscoveredmorethan50yearsagoandonlyinthepastsixyearshasitsexceptionalbiologicalversatilitybeendemonstrated.Particularly,itscell-penetratingABIlity,whichallowscrotaminetocrosscellmembranesandtoaccumulateinthenucleus;itsuseforintracellularvesicletrackingandasacellcyclemarkeranditscapabilityfordeliveringDNAintoreplicatingmammaliancells.Bothantimicrobialactionandpotentialselectiveantitumoractivityofcrotaminehavealsobeenfound.Multidisciplinaryapproachesandpathwaysofdiscoveryplacedcrotamineinararecategoryofversatilebiomolecules,inwhichconcentration,moleculartargetpreference,structuralancestryandspecificitytowardbiologicalmembranesplayanintegralrole.Crotamineisadruggablepeptidewithhighpotentialforuseasanimagingagentfordetectingdividingcells,forintracellulardeliveryofhydrophilicbiomolecules,andasanalternativechemotherapeuticcompoundagainstaggressivetypesofcancer.
KerkisI.,etal.(2010)BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake.ExpertOpinInvestigDrugs.PMID:21062230
Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake
Animalvenomscompriseanaturallyselectedcocktailofbioactivepeptides/proteinsandothermolecules,eachofwhichplayingadefinedrolethankstothehighlyspecificinteractionswithdiversemoleculartargetsfoundintheprey.Researchfocusedonisolation,structural,andfunctionalcharacterizationsofnovelnaturalbiologics(bioactivepeptides/proteinsfromnaturalsources)hasalongwaytogothroughfromthebasicsciencetoclinicalapplications.Herein,weoverviewthestructuralandfunctionalcharacteristicsofthemyoneurotoxincrotamine,firstlyisolatedfromtheSouthAmericanrattlesnakevenom.Crotamineisthefirstvenompeptideclassifiedasanaturalcellpenetratingandantimicrobialpeptide(CPPandAMP)withamorepronouncedantifungalactivity.IncontrasttootherknownnaturalCPPsandAMPs,crotaminedemonstratesawidespectrumofbiologicalactivitieswithpotentialbiotechnologicalandtherapeuticvalues.Morerecentstudieshavedemonstratedtheselectiveinvitroanticanceractivityofcrotamine.Invivo,usingamurinemelanomamodel,itwasshownthatcrotaminedelaystumorimplantation,inhibitstumorcellsproliferation,andalsoincreasesthesurvivalofmiceengraftedwithsubcutaneousmelanoma.Thestructuralandfunctionalpropertiesandalsothepossiblebiotechnologicalapplicationsofminimizedmoleculesderivedfromcrotaminearealsodiscussed.
KerkisI.,etal.(2014)Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake.BiomedResInt.PMID:24551848
Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.
Ourgoalwastodemonstratetheinvivotumorspecificaccumulationofcrotamine,anaturalpeptidefromthevenomoftheSouthAmericanrattlesnakeCrotalusdurissusterrificus,whichhasbeencharacterizedbyourgroupasacellpenetratingpeptidewithahighspecificityforactivelyproliferatingcellsandwithaconcentration-dependentcytotoxiceffect.Crotaminecytotoxicityhasbeenshowntobedependentonthedisruptionoflysosomesandsubsequentactivationofintracellularproteases.Inthiswork,weshowthatthecytotoxiceffectofcrotaminealsoinvolvesrapidintracellularcalciumreleaseandlossofmitochondrialmembranepotentialasobservedinrealtimebyconfocalmicroscopy.Theintracellularcalciumoverloadinducedbycrotaminewasalmostcompletelyblockedbythapsigargin.Microfluorimetryassaysconfirmedtheimportanceofinternalorganelles,suchaslysosomesandtheendoplasmicreticulum,ascontributorsfortheintracellularcalciumincrease,aswellastheextracellularmedium.Finally,wedemonstrateherethatcrotamineinjectedintraperitoneallycanefficientlytargetremotesubcutaneoustumorsengraftedinnudemice,asdemonstratedbyanoninvasiveopticalimagingprocedurethatpermitsinvivoreal-timemonitoringofcrotamineuptakeintotumortissue.Takentogether,ourdataindicatethatthecytotoxicpeptidecrotaminecanbeusedpotentiallyforadualpurpose:totargetanddetectgrowingtumortissuesandtoselectivelytriggertumorcelldeath.
NascimentoFD(2012)Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.MolPharm.PMID:22142367
Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.
Crotamineisabasic,42-residuepolypeptidederivedfromsnakevenomthathasbeenshowntopossesscell-penetratingproperties.CrotamineformsnanoparticleswithavarietyofDNAandRNAmolecules,andcrotamine-plasmidDNAnanoparticlesareselectivelydeliveredintoactivelyproliferatingcellsincultureorinmice.Assuch,thesenanoparticlescouldformthebasisforanucleicaciddrug-deliverysystem.Herewedescribethepreparation,purification,andbiochemicalandbiophysicalanalysisofvenom-derived,recombinant,chemicallysynthesized,andfluorescent-labeledcrotamine;theformationandcharacterizationofcrotamine-DNAand-RNAnanoparticles;andthedeliveryofthesenanoparticlesintocellsandanimals.
HayashiMA.,etal.(2012)Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.MethodsMolBiol.PMID:22791447
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常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
:)
我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
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