有關(guān)猴頭菇在大腦中促生ngf的研究v120en

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TheInduceroftheSynthesisofNerveGrowthFactorFromLion'sMane(Hericiumerinaceus)

ResearchReport

TheInduceroftheSynthesisofNerveGrowthFactorFromLion'sMane(Hericiumerinaceus)

?Copyright2002byHirokazuKawagishi,1Ph.D.,ShoeiFurukawa,2Ph.D.,CunZhuang,3Ph.D.,andRikaYunoki4;Japan&USA(ExploreIssue:Volume11,Number4)

Nervegrowthfactor(NGF)iscloselyrelatedtoAlzheimer'sdementia,andstudieshavesuggestedthatthediseasemaybepreventedoritssymptomsmaybeimprovedwhenNGFisgivenintothebraindirectly.However,sinceNGFisaproteinitusuallycannotpassthroughtheblood-brainbarrier.Recently,researchershavetargetedonthesubstancesthatcouldpassthroughtheblood-brainbarrierandinduceNGFsynthesisinthebrain.Somecompoundswithlowermolecularweighthavebeenfoundtohavesuchbioactivity.Amongthesebioactivecompounds,hericenonesanderinacines,whichwereisolatedfromanediblemushroomcalledasLion'sMane(Hericiumerinaceus),showedremarkableactivityofstimulatingthesynthesisofNGF.TheycouldbedevelopedasadietarysupplementormedicinetobeusedfortreatingAlzheimer'sdementia.Thisarticleoffersanintroductiontotheisolationmethod,bioactivityassayandchemicalstructureysisofhericenonesanderinacines.

SignificanceoftheStudyontheInduceroftheSynthesisofNerveGrowthFactor

Itisexpectedthattheapplicationofaninducerofsynthesisofnervegrowthfactor(NGF)wouldcontributetothemedicaltreatmentandthepreventionofdisordersrelatedtothecentralandtheperipheralnervoussystems.Forinstance,thecauseofAlzheimer'sdementiaisyettobeclarifiedandthewaytoitspreventionortreatmentisyettobeestablished.Thepatients,however,havesimilardamagesinthebraintothosewiththedisorderwithbasalforebraincholinergicneuron(),sufferingmalfunctionin associatedwithmemoryandlearning(suchascellloss,atrophyofnervecell,andneuritedegeneration).BecauseNGFexertsatrophicactionon ,lackofNGFisconsideredoneofthecausesofthisdisease.ResearcheshavebeentargetingonNGFasatreatmentofthedementia(Thoenen&Barde,1980;Yankner&Shooter,1982;Furukawaetal.,1984,1987;Furukawa&Furukawa,1988,1989;Furukawa&Kawagishi,1991).

However,NGFisaproteinandcannotpassthroughtheblood-brainbarrier.Itneedstobeinjecteddirectlyintothebraintobeeffective.Infact,aremarkablefindingwasreportedataninternationalmedicalconferencein1990.Accordingtothereport,awomanwithAlzhehimer'sdementiaimprovedhersymptoms,suchasenhancingmentalability,aftertheadministrationofNGFderivedfrommicedirectlyintoherbrainusingcatheter(Seigeretal,1993).However,suchtreatmentcannotbeacceptedasatherapyforthedisease.Ifwecouldtakeasubstancebyoraladministrationorinjection,whichpenetratesthemembraneandstimulatestheNGFsynthesis

insidethebrainandthenifthisinduced-NGFcouldrepairthedamagednervousfunctions,suchsubstancemaybeappliedasasafertherapytopreventthisdisease.Evenifthissubstancecouldnotgothroughthebarrier,itwouldbestillbeneficialfordisordersoftheperipheralnervoussystemsinceNGFhasasimilaractivityonneuronsattheperipheralnervoussystem.

AssayMethod

Basedontheaboveconcept,thescreeningforasubstancethatstimulatesNGFsynthesishasbeencarriedoutusingprimaryastrogliaderivedfromratcerebralcortexinvitro.AmongtheprimarycellsthatfacilitateNGFsynthesis,includingfibroblasticL-Mcells,weelectedastroglialcellsduetothefollowingreasons:Withinthebrain,neuronandastrogliaareresponsibleforNGFproduction.NeuroncontrolsNGFsynthesistomaintainthefunctioninthematuredbrain,whileinthebrainatthegrowthperiodorwithsomedamage,astrogliaystheroleinstead.ItispossiblethatasubstancestimulatingNGFsynthesistomaintainthefunctioninthematuredbrain,whileinabrainduringthegrowthperiodorthatwithsomedamages,astrogliaarebelievedtoytheroleinstead.ItislikelythatasubstancestimulatingNGFsynthesisbyneurongeneratessomekindofunfavorableneuralactivityagainsttheindividual,sinceanexcitatoryneuralnetworkinducesNGFproductionbyneuron.Thisiswhyweelectedtouseastrogliacellsinthisscreening.Thisassayisoptimumforourpurpose.Thesubstanceshowsactivityinthisassaymustdemonstratethesimilaractivityinvivo.

LowMolecularWeightCompoundsIdentified

CatecholCompounds

Intheperipheralsystem,themoresympatheticinnervationincreases,themoreNGFsynthesisisactivated.Itishighlyactivewithinsubmandibulargland,heart,andbloodvesselinrats.WeassumedthatnoradrenalinemaybetheneuralsignaltoregulateNGFsynthesis.Consequently,catecholamine(adrenaline,noradrenaline,anddopamine)hasbeenfoundtoaccelerateNGFproductionsignificantlyatstationaryastrocyte(Furukawaetal.,1986a).Cholinergicagonistwasonlyslightlyeffectiveonthesynthesisamongthevariousneurotransmittersinthecentralnervoussystem.TheconcentrationofNGFsecretedintothemediaincreasesdependingoncatecholaminelevelsandmRNAconcentrationforintercellularNGFalsoincreases.ThesimilaractivityhasbeenobservedwithintheestablishedfibroblasticcelllineL-Maswellasintheastrocyte.Furtherstudyonthestructure-activitycorrelationhasindicatedthattheactivityisdependentonthecatecholstructureandthatthecompoundswithtwosaturatedcarbonsonthesidechainhasshownthehighesteffect.Therewasnosignificantdifferenceinthepresenceordifferenceoffunctionalgroupsonthesidechain.Anotherimportantfindingwasthatnoadrenergicreceptorhadbeenrequiredwiththesecatecholstoinducethereaction(Furukawaetal.,1986b,1989;Furukawa&Furukawa,1990).Inotherwords,itgoeswithouthormoneandneurotransmitteractivitiesmediatedbyanadrenergicreceptor.Thiscouldleadustoapossibledevelopmentofnewmedicinewithoutsideeffectsinthefuture.

Basedontheaboveexperimentsinvitro,theinductionofNGFwasexaminedusing4-methylcatechol[1](Fig.1)inrats,anditisfoundthattheconcentrationofNGFhasbeen

increasedintheheartandthesubmandibulargland(Kaechietal.,1993).

HericenonesDerivedFromFruitBodyofLion'sMane

Obviously,itisquiteimportanttoclarifytheinternalsubstancesthatareoriginallyresponsibleforthestimulationofNGFproductionforeachorgan.However,becausesuchinternallyactivesubstanceslikeadrenalinesarehormones,itsadministrationfromoutsidemaylosetheinternalhormonebalance,andtheirpracticalapplicationforpreventionortreatmentofAlzheimer'sdiseasemaybeharmful.Itispreferable,therefore,thatthesubstanceissafeand esactivespecifically.Withthisbackground,thecompounds,calledhericenoneC-H,wereisolatedfromLion'sManeafterthescreeningtestfortheactivestimulantofNGFsynthesistargetingtoconstituentsofmushrooms(Fig.2)(Furukawa&Kawagishi,1991;Kawagishietal.,1991,1993).

Thesecompoundsarethefirstactivesubstancesfoundinnaturalproducts,whichareaseffectiveasadrenaline.Lion'sManehasbeenappreciatedasanherbalmedicinein,andthecultivationhasrecentlystartedinJapan.Eachgroupofhericenones,C[2]-E[4]andF[5]-H[7],containsthealcoholsitepeculiartoeachgroupandeachhericenoneconsistsofoneofthreesimplefattyacids.HericenonD[3]demonstratedthestrongestactivityamongthesecompounds.Itmeansthattheactivitylevelvariesaccordingtothestructureoffattyacids.Theanimaltestiscurrentlyundergoing.

ErinacinesFoundinMyceliaofLion'sMane

Activesubstancesinvitromaybefurtherinvestigatedinvivo,butanimaltestsrequirealargetyofsample.Thecultivationofmushrooms(fruitbody)needswell-controlledlight,temperature,andhumidity.Sowetriedtoproduceactivecompoundsfrommycelium,thepre-stageofafruitbodyandeasiertogrow,inordertoobtainconsiderableamountofhericenone.Thisexperimenthasnotbeencompletedyet,butwewereabletoobtainaseriesofditerpenoidsnamedaserinacineA-I,whichhavepowerfulactivitieswithdifferentchemicalstructuresfromtheseofhericenones.(Fig.3)(Kawagishietal.,1994,1996a,1996b;Leeetal.,2000).

Thesesubstancesaresostrongthattheycanberecognizedasthemostpowerfulonesamongallidentifiedsubstancesatpresent(Fig.4).

OtherCompounds

Asotheractivecompounds,propentofylline[11]asaxanthinederivative,1,4-benzoquinones[12,13],PQQ[14],andOPQ[15]areknown(Shinodaetal.,1990;Yamaguchietal.,1993a).Additionally,phertamideA[16]hasbeenreportedasthesecondactivesubstancederivedfromnaturalproductssincehericenones(Yamaguchietal.,1993b).Accordingtothecomparativestudyontheactivesubstancesmentionedabove,thefindingswithrespecttothestimulationofNGFsynthesisareasfollows;1.theypossessmorethanoneactivesiteandmechanisms,

thespecificityseemsratherweak,3.thesecompoundsprobablyhaveanunknowncommonfactor,4.theremustbeotheractivegroupsofcompoundsyettobefoundthatcanfacilitatethefunction.Asfor1.,above,thefollowingresultisreportedinthestudyoftheNGFsynthesiswithfibroblasticcellL929inmice(Carswelletal.,1992).Underco-existenceofpropranolol4-methylcatechol[1]demonstratedthestimulationofNGFsynthesis,whileisoproterenol[17]havingcatecholinitsstructurelike4-methylcatecholdidnot.Thisisbecausepropranololisanantagonistforadrenergicreceptor,andisoproterenolisanagonistforit.Thisindicatesthat,althoughtheogousstructureisrecognizedincatechols,someofthemstimulateNGFsynthesiswithadrenergicreceptorwhiletheotherscanbeactivewithoutit.Thesimilaractivitywasobservedonganglioside,themaincomponentofcellmembranes,thatwasonlyatSchwanncellthatformsmyelinsheathintheperipheralnerve.Noactivitywasconfirmedatastrocytesorfibroblasticcells.

Experiments

AssayInVivo

(Fig.5,Furukawaetal.,1983;Saito&Furukawa,1987;Matsuietal.,1990)

IncubationandAssayforAstroglialCell

RatsfromWistaronthefifteenthdayofgestationhavebeenobtained.Thecerebrumwastakenonacleanbenchafterremovingthecerebellumandlowerpartsofcerebrumfromtheinfantrats(within3daysfromthebirth).Meningeswereremovedusingps(thethinmembranewouldbepeeledoffthesurfaceofthecerebrum)tocollectapuresamplesinceevenalittlemeninges,ifremained,wouldmaketheglialcellslosethepuritybeingmixedwithfibroblasticcellsthathavehighproliferationpotency.Also,itshouldbeavoidedtousethesubcultureatlowcelldensitytokeepthesamplepurityhigh.Theobtainedcerebrumwasputinadisposablecentrifugetube(15ml),mincedfinelywithadissectorknife(ordissectorscissorsorps),andwashedinPBS(10mMphosphate-bufferedsaline,pH7.4)bypipetting.Thenthissolutionwascentrifuged(3,000xg,10min)andthesupernatantwasdiscarded.Repeatedthesestepstwice.Addedabout2mlportionof0.25%trypsinandincubateditat37°Cfor15~30min.Added5mlofPBStostopthereaction,centrifuged(3,000xg,10min),andremovedthesupernatant.RinsedthepelletwithPBSbypipetting,centrifuged(3,000xg,10min),anddiscardedthesupernatant.Repeatedthisprocedureonemoretime.Afterpipettingthepelletin5mlFCS-MEM(minimumessentialmediacontaining10%fetalcalfserum),pouredanother5mlofFCS-MEM,transferredittogetherwiththepelletintoa10cmlaboratorydish,andincubateditin5%CO2at37°Cfor2~3days(onecerebrum/dish).Itiseasytodistinguishbetweenastrocytesandmeninges-derivedcells,sincetheformerhasanicestonewallshapeandthelatterhasastalkyshape.ChangedFCS-MEMandtheincubation.Continuouslychangedthemediaonceineverythreedaysuntiltheconfluentwas plished.Then,removedthemedia,performedtrypsintreatment,andloadedthecellsineachwellona

96-wellte.AfterswitchingtheFCS-MEMthreetimesinninedaysataboutthree-dayintervals,themediawasconvertedtoserum-media[containing0.5%bovineserumalbumin(BSA)]anditwasrepeatedthreetimesinninedaysonceineverythreedays.Duringthisprocedure,theastrocytewasinastationaryphaseandNGFproductionsloweddown.Thymidinemaybeaddedtoconfirmthehaltofthepropagation.Also,itwouldbeevenbettertomakesurethedecreaseofNGFlevelinnutrientmediaovertime.Themediawasexchangedwith100μlofnewoneonthepreviousdayofthesampleaddition.Dissolvedthewaterinsolublesamplesinethanol,methanol,ordimethylsulfoxide(DMSO)anddilutedthemwiththemediasoasnottoexceed1%ofthesolventvolume.Othersampleshadbeenpreparedonanothertebeforehandwithaseriesofdilutions,usingamultichannelpipetteandincubateditfor24hoursafteradding5μloftheexperiments.MeasuredtheNGFlevelsbyanenzymaticimmunoassay.

StorageofAstrogliacells

WashedthecellstreatedwithtrypsininFCS-MEMandcentrifugedit(3,000xg,10min).

Afteradding0.6mlFSC-MEMtotheprecipitatewhilecooling,put0.4mlof50%DMSO(inFSC-MEM).

Afterrefrigeratingitfor2-4hoursinanultra-deepzer(-80°C),storeditinliquidnitrogen.

Resuspensionofthecell

Letthefrozencellsbacktotheroomtemperaturerapidly.Putitinacentrifugetube,addedabout9mlofFCS-MEM,andincubateditat37°Cfor20~30min.

Aftercentrifugation,incubatedthepelletagainintheFCS-MEM.

EnzymeimmunoassayforNGF

PreparationofAntibodyte

Anti-NGFantibody,IgG(producedbypurificationofanti-NGF,rabbitantiserum,byproteinAcolumn)wasdilubedin0.1Mtris-hydrochloricacidbuffer(pH7.6)tillitsconcentrationof100μg/mlwasobtained.Put5μlofthissolutioninadropatatimeinthecenterofeachwellona96-wellELISAte(U-bottom).Sealedthetewithparafilmandletitforanhouratroomtemperature.Removedtheantibody(reusable)andfilledeachwellwith150μlbufferA(0.1Mtris-HClbuffer,pH7.6containing0.1%BSA,0.4MNaCl,1mMMgCl2,and0.02%NaN3).Afterapproximay30min,thebufferwasremovedbyanaspirationwith8channelmanifold,add150μlofbufferAcontaining1%milkwasaddedinordertoblockeachwell,andleftitforanhour.Thetewascarefullyhandlednottobeevaporatedduringtheprocedures.

ReactionWiththeSamples

Themilksolutionwasaspiratedandthen20μlofsamplesolutionswasimmediaycedineachwell.Itmustbeperformedquicklytoavoidevaporationofthesamplesanddrynessofwells.Itis mendedthatthesamplesolutionswerepreparedbeforehandonanotherteandfilledthewellsusingamultichannelpipetteallatonce.Sealedittightwithparafilmandletit

reactinashakerwithmildmovementfor2hoursatroomtemperature.Afteraspiratingthesamplesolutions,150mlofbufferAwaspouredandthenremovedbyaspiration.Thisprocedurewasrepeatedtwicetowashthete.

AdditionofLabeledAntibody

Biotinylatedanti-NGFantibody(biotinylatedafterthepurificationusinganaffinitychromatography,10μg/ml)wasdiluted1,000-foldinbufferA(with1%serumofahealthyrabbit)and20μlofthesolutionswereaddedtoeachwell.Putlidswithparafilmtightlyandletthemstandfor12~18hoursat4°C.

TreatmentwithStreptoavidin-?-D-Galactosidase

Streptoavidin-?-D-glactosidasewaspurchasedfromthemarketanddiluted6,000-foldwithbufferA.Took20μltoeachwell.Sealedittightwithparafilmandincubatedforanhouratroomtemperature.Aftertheincubation,rinsedeachwellthreetimeswithbufferAandadded20mlof4-methylumbelleferyl-?-D-galactoside(10μg/mlinbufferA).Sealedwithparafilmandallowedittoreactfor5hoursatroomtemperatureshuttingthelightinthedark.Terminatedthereactionbypouring100μlof0.1Mglycine-NaOHbuffersolution(pH10.3).Measuredthefluorescenceintensitywithanexcitationwavelengthof360nmandadetectorwavelengthof450nm.TheconcentrationofNGFwascalculatedfromthefluorescenceintensityofthesamplesbyplottingtheintensityofstandardNGFonalogloggraph.Afluorophotometerwithflowcellsisemployedformoreaccurateevaluation,insteadofamicrotereader,whichisusedintheregularassaysbutnotreliableduetoinconsistentdata.

AssayInVivo

(DeterminationoftheNGFConcentrationatEachOrganByAddingSamples)AdministrationtoAnimals

Wistarrats(male)wereemployed.Appliedtheappropriateamountofthesamplessoastoobtainitsbloodconcentrationsof100μg/ml,10μg/ml,1μg/ml,and0.1μg/mlrespectively(theaverageweightofWistarratsis131gat6weeksofageand160.6gat7weeks,andthebloodweighs5.2~6.2%ofthebodyweight).Administered0.5mlofeachconcentrationofthesamplesintraperitoneally(Thewater-solublesamplesweredissolvedinPBS.Thefat-solublesamplesweredissolvedinethanol,then,PBSwasaddedtodilutethemtomake20%ethanolsolution.)intotheabdominaltwiceadayat12-hourintervals.Theratsweresacrificedwithchloroform4hoursafterthefifthinjectionandremovedtheheart,submandibulargland(rightandleft),sciaticnerve(rightandleft),andcerebrum.

ExtractionProcess

Heart,SubmandibularGland,andCerebrum

Homogenizedthemintheextractbuffer(1MNaCl,2%BSA,2mMEDTA,1Mtris-HCl

containing0.08unit/mlaprotinin,pH7.6.ThefinalpHneedstobeexact.)tomake2or5%(w/v)solution.Ultracentrifugedthehomogenate(300,000xg,15min)andfrozethesupernatantfortheenzymeimmunoassay.

SciaticNerve

Cutthesciaticnerve(2cm)thathadbeendeep-frozenimmediayafteritsremovalinto15pieceswithapproximay1.3mmlengtheach.Puteachpieceina96-wellmicroteandpoured100μlextractbuffer.Theextractionwascarriedoutbyrepeatingthezingandthawingmethod.Theextractwascentrifuged(3,000xg,30min),andeachsupernatantwastransferredtoa96-wellmicrote,thenfrozenandstoredfortheenzymeimmunoassay.

HericenonesIsolatedFromaFruitBodyofLion'sMane(Fig.6)

Thefruitbodyofthemushroomwascrashedinacetonebyablenderandleftfor1~2daysfortheextraction.Theliquidextractwasprocessedwithvacuumfiltrationandthemushroomfruitbodywasfurtherextractedbyacetonetwice.Theextractwasconcentratedusinganevaporatortill2lofthevolumeisobtainedandfractionatedwithchloroform.Ethylacetatewasaddedtotheaqueousphaseformoreextraction.Thefractionationoftheextractwouldbeanessentialstepforapplyingthecompoundstotheassay,becausethereisanoptimumconcentrationfortheactivationofNGFsynthesisandalsomostofthefractionsatthisstageexhibitcytotoxicactivity.Therefore,silicagelchromatographyandpreparativethinlayerchromatography(TLC)wereemployedandobtainedtwotypesoffractions,onewithhericenonesC-EandtheotherwithhericenonesF-H.BothfractionswerespottedatalmostthesamedistanceonthesilicagelTLCandtheseparationwasonlypossiblebyhighperformanceliquidchromatography(HPLC)usingODScolumn.

IsolationandStructuralDeterminationofErinacinesDerivedfromMyceliaofLion's

ManeIsolation(Fig.7)

Centrifugedthemyceliafollowingthe4weeksofshakecultureandseparatedthemyceliafromtheculturefiltrate.Theculturefiltratewasconcentratedbyanevaporatorandfractionatedwithethylacetateandwater.Themyceliawasputin85%ethanolforextraction,concentratedbyanevaporator,andthenfractionatedwithethylacetateandwater.Sincethefractionsintheculturefiltratedidnotdemonstrateanyactivity,werepeatedextractionofmyceliausingsilicagelcolumnchromatographyandpreparativeTLCandobtainedthepurifiederinacines.

StructuralDetermination

ErinacineA

ErinacineA[8]hasthemolecularformulaofC25H36O6detectedbyahigh-resolutionfast-atombombardmentmassspectrometry(FAB-MS)andisapentoseglycosideinditerpeneidentifiedusing1H-NMRand13C-NMR.Theexistenceofaconjugatedaldehydegroup(*9.31,*194.3)isindicatedinthissubstance.Theacetylationbyaceticanhydrideinpyridinewascarriedouttodetermineitssugartypeandthelinkageofthesugarandaglycone,asthesignalsforthesugarswereoverlappedby1H-NMRandcouldnotbeidentified.Allsignalsforthesugarswereyzedbythetriacetylcompoundsthathadbeenproducedduringthisacetylation[*4.56(d,J=6.23,H-1'),4.89(dd,J=6.23,8.06,H-2'),5.07(dd,J=

8.06,8.06,H-3'),4.86(m,H-4'),3.95(dd,J=12.09,4.76,H-5'),3.31(dd,J=12.09,7.70,

H-5')].Thisresultsuggeststhatitssugartypeisxyloseandthelinkageismadeby?-bond.Also,thecompound8wasproventohave?-D-xylosidoduetothefactthatitwashydrolyzedby?-glucosidase.ThestructureofthisisolatedaglyconehasbeenidenticalwithAllocyathineB2ineverydataincludingitsspecificrotation,whoseabsoluteconfigurationhadbeenpreviouslydeterminedbyAyeretal.afteritsisolationfromCyathusearlei.Thisfactdeterminedthestructureofthecompound8withitsspecificrotation(Ayer&Lee,1979).

ErinacineB

WepresumedthaterinacineB[9]was?-xylosidejustlikecompound8becauseofthemolecularformulaofC25H36O6asin8aswellasfromysisbyNMRspectrum.However,sincecompound9haslessdoublebondbyonecomparedtothatofcompound8anditgavediacetateafteritsacetylation,weconcludedthatonemorebondexistsbetweensugarandaglyconein9formingaring.Theysisoftheheteronuclearmultiplebondcorrelation(HMBC)spectrumhasconfirmedthelocationofthebond(Fig.8).

Itsstereochemistryhasdeterminedtheaspectsthatthe1H-NMRspectrumprovidedthecouplingconstantbetweenH-13andH-149.71Hzsuggestingtheconfigurationoftrans-diaxial,andthatthecrosspeaksappearedfromH-14toH-16,H-5toH-17,andH-5toH-13,respectively,ontheNOESYspectrum.

ErinacineC

TheresultsprovidedbytheNMRerinacineC[10]wereverysimilartothoseby9.However,

compound10didnothavetheformylgroup,whichhadobservedin9,anditstillhadthemolecularformulaofC25H38O6.,Therefore,thepresenceofhydroxymethylgroupissuggestedinsteadofformylgroup.Thischemicalstructurehasbeenconfirmedbythefactthattheoxidizationofcompound10with2,3-dichloro-5,6-dicyanobenzoquinone(DDQ)producederinacineB[9].

AbouttheAuthors

HirokazuKawagishi,Ph.D.,isaprofessorinthedepartmentofappliedbiologicalchemistry,FacultyofAgriculture,ShizuokaUniversity,836Ohya,Shizuoka422-8529,Japan.SyoeiFurukawa,Ph.D.,isaprofessorinthedepartmentofmolecularbiology,GifuPharmaceuticalUniversity,5-6-1Mitahora-Higashi,Gifu502-8585,Japan.CunZhuang,Ph.D.,isaseniorscientistinBioResearchInstitute,P.O.Box1354,Paramus,N.J.07653,USA,biore ,andRikaYunokiisanassistantscientistatMaitakeProducts,Inc.,

222BergenTurnpike,RidgefieldPark,NewJersey07660,USA,customers ,(201)229-0101.

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