Table Of Content

THERELATIONSHIPOFCYSTEINE-RICHINTESTINALPROTEINTO CELLULARHOSTDEFENSE By LORRAINELANNINGHAM-FOSTER ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1999 ToRandal,forencouragingmethroughoutthisworkandformaking laugh. ACKNOWLEDGMENTS Manypeoplehavehelpedmethroughoutmygraduateworkatthe UniversityofFlorida. IammostgratefultoDr.RobertJ.Cousinsfor allowingmetoworkinhislabandtobeapartofhisexciting,innovative researchprogram. IhavelearnedseveralresearchtechniquesandI appreciatehisinvolvementandsupportduringmyproject. Iwouldalsolike tothankthemembersofofmysupervisorycommitte(Drs.Langkamp- Henken,Neu,Percival,andSamuelson)fortheirencouragementand participation. InadditiontotheworkIhaveperformedformydissertation,Ihave alsoworkedwithotherindividualsattheuniversitywhohavehelpedmeto expandmyteachingexperiences. IwasinspiredbyMrs.BrendaOwensin theLactationCenteratShandsteachinghospitaltoreachouttoandhelp mycommunity. IwouldalsoliketothankDrs. Langkamp-Henkenand Percivalforgivingmetheopportunitytoteachintheircourses. SomeofthepeoplewhodeservemorethanksthanIcouldeveroffer aremylabmatesandco-workers. Ilearnedagreatdealduringthestartof myprojectfromChristinaKhoo,BarbaraDavis,VickiSullivan,Ray Blanchard,andBobMcMahon. Additionally,SteveDavis,JenniferMoore, LeahCoy,andJayCaohavetoleratedmemostrecentlyandallofthem deserveawardsfortheirpatienceattheendofmywork. Iobtainedagreat dealofhelpwithcellculturetechniquesfromKellyHerrlinger-Garciaand BrandonMarion. WarmestthanksgotoVirginiaMauldinandWarrenClark forbothtechnicaland,mostimportantly,emotionalsupport. OneofthemainreasonsthatIamatthispointinmylifeandcareeris myfamily. Myfather,mother,andstepmotherhavetaughtmethathard workanddedication arethekeystoarewardinglife,andIstriveeachday tofollowtheirexample. Mysistersandbrotherhavealwaysencouragedme andhelpedmealongtheway. Iwouldalsoliketothankseveralauntsand unclesandmyhusband'sparentsfortheirconstantguidanceandinterestin myeducation. Moreover,Iwouldliketodedicatethisworktothememory ofmymother,PatriciaCatoLanningham. ThepersonwhomIcouldneverthankenoughforhisloveandhelp duringmyentiregraduatecareerandmostofmyundergraduateworkismy wonderfulhusbandandpartnerinlife,RandalFoster. WhenIcomplained, helistenedandsoothed. WhenIrejoiced,hecelebratedbymyside. He pushedmewhenIwasstubbornandlazy. IhopeIcanreciprocatehishelp ashenowbeginshisgraduatework. iv TABLEOFCONTENTS page ACKNOWLEDGMENTS iii ABBREVIATIONS vii ABSTRACT viii CHAPTERS 1 INTRODUCTIONANDLITERATUREREVIEW 1 Introduction 1 Objectives 4 Hypothesis 4 LiteratureReview 4 2 DEVELOPMENTOFCRIPELISA 32 Introduction 32 MaterialsandMethods 38 Results/Discussion 40 3 HUMANMILKMONONUCLEARCELLS 45 Introduction 45 MaterialsandMethods 46 Results 48 Discussion 55 4 FURTHERCHARACTERIZATIONOFCRIP TRANSGENICMICE 59 ReviewofInitialCharacterization 59 BreedingStrategy 64 V BodyandOrganWeightAnalysis 67 Results 68 Discussion 70 5 SENSITIVITYOFCRIPTRANSGENICMICE TOENVIRONMENTANDENDOTOXIN 73 Introduction 73 MaterialsandMethods 74 Results 77 Discussion 92 6 IMMUNECHALLENGE 98 InfluenzaVirusChallenge 98 MaterialsandMethods 100 Results 103 Discussion 110 7 ROLEOFDIETARYZINCANDMETALLOTHIONEIN PRODUCTIONONCRIPEXPRESSION 113 Introduction 113 MaterialsandMethods 114 Results 117 Discussion 132 8 SUMMARY,SPECULATIONS,ANDCONCLUSIONS 136 Summary/Speculations 136 Conclusions 142 LITERATURECITED 145 BIOGRAPHICALSKETCH 163 vi ABBREVIATIONS RDCoAA Dovineserumaiuumin PLLAUAoNCTV1 ccyocnlviecntaimoplniaflicationandselectionoftargets [Iht,i,rrj\lP^RnIlPr (numan,raT/cysieine-ncninxesiinaiprotein Lnlr-1g CRIPtransgenicmouse Lnr cysteine-richprotein CutILIlInCoAA aeenizayymeea--ntnyKpeeanyipmermsuemnosistoirvoiieyntassay rOo fetalcalfserum rl1 fluoresceinisothiocyanate unL1 gui-associaieulympnoiaTissue note hemotoxylinandeosin 'g immunoglobulin ii mieneuKin- IPM inteneron i.p. intraperitoneal IS.U KnOCKOUt LIVIU LiM-oniy 1LDrCb lipopolysaccharide MadinDarbycaninekidney AM>lL1PPI muscleLIMprotein MMNC milkmononuclearcell MRE metalresponseelement MT metallothionein NF nuclearfactor Non-Tg nontransgeniccontrolmouse PBS phosphate-bufferedsaline PCR polymerasechainreaction PE phycoerythrin PHA phytohemaglutinin PBMC peripheralbloodmononuclearcell SPF specificpathogenfree TBS tris-bufferedsaline TNF tumornecrosisfactor vii AbstractofThesisPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy THERELATIONSHIPOFCYSTEINE-RICHINTESTINALPROTEINTO CELLULARHOSTDEFENSE By LorraineLanningham-Foster August1999 Chairperson:RobertJ.Cousins MajorDepartment:FoodScienceandHumanNutrition Cysteine-richintestinalprotein(CRIP)isazinc-fingerproteinwhich hasbeenidentifiedinseveraltissuesandcells. CRIPmRNAexpressionis developmentallyregulatedintheratintestine,withlevelsincreasingtothat oftheadultbythetimeofweaning. Thegoalofmydissertationproject wastoutilizetwoavailableresearchmodels(humansandtransgenicmice) inordertolearnmoreaboutthefunctionofCRIP. Asensitive,enzyme- linkedimmunosorbentassay(ELISA)wasdevelopedwhichallowedthe quantificationofCRIPproteininthesemodels. CRIPmRNAandproteinexpressionwasevaluatedoverthefirst6 weeksoflactationinhumanmilkmononuclearcells. Therewasatrend viii towardadecreaseinCRIPmRNAexpressionovertheperiodoflactation examined. Thistrendwasalsotrueforsubjectsexaminedlongitudinallyfor CRIPproteinexpression. Theothermodelwhichwasutilizedinthisprojectwasatransgenic mousemodelwhichoverexpressestheratCRIPgene. Twoofthemain areaswhichwereexaminedwiththetransgenicmiceweretheeffectof environmentandacuteimmunechallenges(endotoxinandinfluenzavirus) onCRIPexpression. CRIPexpressionwashigherwhenmicewereplacedin aconventionalenvironmentascomparedtoaspecificpathogenfree(SPF) environment. TheCRIPtransgenicmiceweremoresensitivetoboth endotoxinandinfluenzavirus. Therewasalsoashiftincytokineproduction instimulatedmice. CRIP-TgmiceproducedmoreIL-10andIL-6andlessIL- 2andIFN-yascomparedtoNon-Tgmice. Additionally,theeffectofzincdeficiencyonCRIPexpressioninCRIP transgenicandnon-transgeniccontrolmicewasexaminedsincethezinc fingerscouldbetargetsofdietaryzincrestriction. Zincdeficiencydidnot haveamarkedeffectonCRIPlevelsinthetransgenicmice. However,when CRIPexpressionwasexaminedinmetallothionein-nullmice,therewasan effectofmetallothioneinlevelsonCRIPprotein. Theabilitytoexpress increasedamountsofCRIPisapparentlyrelatedtothemetallothioneinpool inthemouse. ix CHAPTER 1 INTRODUCTIONANDLITERATUREREVIEW Introduction Cysteine-richintestinalprotein(CRIP)isazinc-fingerproteinwhich hasbeenidentifiedinseveraltissuesandcells. CRIPmRNAexpressionis developmentallyregulatedintherat,withlevelsincreasingtothatofan adultratbythetimeofweaning(BirkenmeierandGordon, 1986;Levenson etal., 1993). CRIPisamemberoftheLIM-onlyfamilyofproteins(Dawid etal., 1998)andassuchhasaLIMdomain.Thisfeatureconsistsoftwo zinc-fingermotifsthathaveahighlyconservedCCHCplusCCCCsequence. BeingthemostelementaryoftheLIM-onlyproteins,CRIPhasasingleLIM motif.AlthoughnoclearbiologicalroleforCRIPhasbeendefined, experimentalevidencesuggeststhatCRIPmayfunctioninimmunecell activation(Hallquistetal., 1996)and/orincellularproliferationor differentiation(Dawidetal., 1993). Thegoalofthisdissertationprojectwastoutilizetwoavailable researchmodelstolearnmoreaboutthefunctionofCRIP. Additionally,a sensitive,immunochemicalassaywasdevelopedwhichallowedfor quantificationofCRIPproteininthesemodels. Thedevelopmentofthis

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174 Pages·1999·5.1 MB·English
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