Tutorial work 1 questions and answers apart from Part (L) PDF

Preview

Summary

Tutorial 1 Q's and A's Full apart from Part L)...

Description

Questions a)Whatarereactiveoxygensplecies? b)Whatiscatalase?Whatareexamplesofotherantioxidantenzymes,andwhatdotheydo? c)Whydotheseauthorsthinkthatoverexpressionofcatalasemightincreasemouselifespan? d)Whymightexpressionofcatalaseinmitochondriainparticularleadtoincreasedlifespan? f)Figure1:whichofthetwofounderMCATmouselines,4033or4403,showsbetterover‐expressionofcatalase? g)InFigure2,whatcolourarethemitochondriastainingwithcatalase?Canyouseethemitochondria?Howare themitochondriastained?Whichlineisbeingexaminedhere,4033or4403? h)InFigure3C,whichline(4033or4403)wouldyoupredictshouldshowalargerincreaseinlifespan?Isthisisthe case? i)DidMCATexpressionslowtherateofaccelerationofage‐specificmortality? j)WhywastheenzymeaconitasechosentomeasureH2O2‐inducedinactivation? k)DescribethetwomethodsusedtomeasuretheeffectofMCATonlevelsofDNAdamage. l)DoMCATshowreduceddamagetomitochondrialDNA? m)Figure4:WhichMCATlineisbeingstudiedhere‐4033or4403? n)Howdoaconitaseactivitylevelschangewithage?Whatmechanismscanyouthinkofthatmightexplainthis change? o)Bywhatmechanismsmightincreasedmitochondrialcatalaseexpressionincreaselifespan? p)Lastparagraphofthispaper:Whywasbackcrossingthemiceimportant?Whathappenedtolifeextensionafter backcrossing?Howcantheauthorsarguethatthisdoesnotnegatetheirconclusions?Doyouthinkthisissound? WhatmighthavechangedduringthebackcrossingtoalterthelifespanoftheMCATmice?   A) ReactiveOxygenspeciesareacollectivegroupconsistingofsuperoxideO2 .‐,Hydrogen peroxideandthehydroxylradicalOH..Superoxideisformedwhenelectronsareleakedfrom therespiratorychain,andtheleakedelectronreducesoxygen.Thissuperoxidecanthen entertheROScascade,firstbyspontaneousdismutationtohydrogenperoxide,whichin turnproducesthehydroxylradicalwhencatalysedbyironandcopperions. B) Catalaseisanaturallyoccurringenzymethatcatalysesthedecompositionffofhydrogen peroxidetowaterandoxygen.Anotherenzymewithanti‐oxidantpropertiesismentionedin thepaperitself,Glutathioneperoxidasewhichisalsoabletocatalysetheconversionof hydrogenperoxidetowater.Itisalsoabletoreducelipidhydroperoxidestocorresponding alcohols.Anotherantioxidantenzymewithasimilarroleissuperoxidedismutasewhich catalysesthedismutationofsuperoxideintooxygenandhydrogenperoxide,partoftheROS cascade.Oneofthefastestenzymes.Superoxidedismutasefastestenzymeknown.Catalase occursnaturallyintheperoxisomeandthecytosol. C) Catalaseisresponsiblefor‘removing’hydrogenperoxide.Thismeansthatitcannotbe convertedtotheROShydroxylradical,oractasanROSitself.TheauthorproposesthatROS playacausativeroleintheageingprocessby attackingbiologicalsystemsandcausinga functionaldeclineinorgansystems.Accumulationofsuchdamageovertimeisthoughtto lead tothemanyconditions,suchasarteriosclerosis,associatedwithageing.Morecatalase removesmoreofthehydrogenperoxidepresent,andthusretardstherateofROSdamage. Tryingtoprovefree‐radicaltheoryofageing.bmm D) ROSareproducedinthemitochondria,wheretherespiratorychainislocated.TheROS superoxideisformedwhenelectronsleakfromthisrespiratorychainandreducefree oxygen.Thissuperoxidethengoesontoformhydrogenperoxideandhydroxylradicalvia theROScascade.ThusthemitochondriaisthesourceoftheROSandbyexpressingcatalase inthemitochondriaithasabetterchanceofremovingthehydrogenperoxideandthus limitingthedamagecaused. E) Founderline4033showsthebetteroverexpressionofcatalaseinalmostallcases.Inthe heart,muscleandbraintissuefounderline4033showsafargreaterexpressionofcatalase

F)

G)

H)

I)

J)

K)

L) M)

N)

O)

thanline4403.Inliverandkidneytissuetheexpressionstaysfairlyconstant,andisinline withwildtypepopulations. Themitochondriaarestaininggreenwiththecatalase.CytochromeC,acomponentofthe electrontransferchain,isstainedred,andthenucleusisstainedblue.Youcannotseethe individualmitochondria,simplycomponentsofthemitochondria.Thisisfounderline4033. Youwouldexpectthe4033linetoshowalargerincreaseinlifespanduetotheincreased expressionofcatalase.However,thisisnotthecase,and infactline4403seemstohavethe slightlyhigherlifespan. TheevidenceshowsthatMCATdidslowtherateofaccelerationofagerelatedmortality. Thiscanbeseenaspotentiallyfatalconditionsrelatedtoageing,suchasarteriosclerosis, appearconsiderablylesssevereafter20‐25monthsinMCATmiceasopposedtothewild typemice(1.29to1.85). Aconitaseisrapidlyinactivatedbyhydrogen peroxidewithinthemitrochondria.Therefore byusingaconitaseyoucancomparewhetherover‐expressionofcatalaseisremovingmore hydrogenperoxideintheMCATmitothaninthewildtype.Iftheaconitaseisinactivatedless rapidly,thenthereislesshydrogenperoxidepresent,suggestingthatthemitochondriaare moreeffectiveatlimitingtheoxidativedamage. Thetwomethodsweretomeasurethelevelsof8‐hydroxydeoxyguanosine,aproductof DNAoxidation,inDNAfromskeletalmuscleandtheheart.Thiswastomeasureoxidative damagetototalDNA.Mitochondrialdeletionsassociatedwithmitochondrialdamagewere seenbyusinglong‐extensionpolymerasechainreaction.Thisallowedustodetectthe deletionsaslowmolecularweightproducts. InskeletalmuscletheMCATdoseemstoshowreduceddamagetothemitochondrialDNA. AsignificantfewernumberofdeletionproductswasfoundintheskeletalmuscleofMCATas opposedtowildtype.Asregardstolevelsof8‐hydroxydeoxyguanosine,anagerelated increasedwasfoundintheskeletalmuscleofwildtypethatwasnotshowntohave increasedintheskeletalmuscleofMCAT. ? Aconitaselevelsdecreasewithage.Thisisduetothebuild‐upofROS,inthiscasespecifically hydrogenperoxide.AsyouagethisROSbuildsupandhydrogenperoxideisableto deactivatetheaconitase.Themechanismsthatexplainthisarethefactthathydrogen peroxideisreleasedduringtheROScascadewhenitisdismutasedfromsuperoxide. Electronsthathaveleakedoutfromtheelectrontransportchainreduceoxygentoformthis superoxide. Increasedmitochondrialcatalasewillleadtomoreofthehydrogenperoxide,producedfrom thesuperoxideionwhichisinturnproducedwhenelectronsleakfromtheETC,being brokendowntoharmlessbyproductsinsteadofformingthehydroxylion.Thereforeless ROSwillbepresentinthecelltocauseoxidativedamagetotissues.Thislesseningof damagecouldin turnbethereasonforareductioninrateofageingandsubsequent increaseinlifespan.Anotherwayitmayincreaselifespanisagainduetoreducedlevelsof hydrogenperoxide,whichinturncanleadtolessintracellularperoxidedependentsignalling andlesssecondarystressresponseasaresult. Backcrossingisimportant,matingmixedbackgroundhybrids,withfavourablenontumouro forminganimal.Itcouldhavecausedsilencingofspecificpromoterorenhancergenes,thus modifyinganddecreasingtheamount ofMCATexpressed.Infact,aftermultiple generationsofback‐crossing,thephenotypeofageprotectionseemstohavebeenlost entirely.Duetothefactthatthislastgenerationcontainedover99%ofb6hybrids,thisdoes notseemtobeanencouragingresult.Howeverpreviousevidenceseemstohaveshown thatitdoeshaveaneffectonageing....