CURRENTRESEARCHⅠN

MUSCULARDYSTROPHY，JAPAN（BIOMEDICALRESEARCHES）
Vol．2

TheProceedingsofthe

AnnualMeetingofMuscular
DystrophyResearchGroup，
1980，Tokyo

Acid-induced

Myotonia

57
9

Latent Muscular Dystrophy in Genotypically
Dystrophic-dwarf
Mice
Autoradiographic
Studies on the Regeneration of Transplanted Muscles
Muscle Transplantationin the Etiological
Elucidationof Avian Muscular Dystrophy
The Ectopic Differentiationof Striated Muscles in Rat Pineal Body Cell Culture
Tissue Culture Studies of Dystrophic Muscles
Mitotic Activities
of Mononucleate Cells Cultured In vitro Originated from Regenerating
Muscles of Normal (+/+)
and Dystrophic (dy/dy) Mice
A Factor Extracted from the Brain Affecting Development of Tetrodotoxin-sensitive
Spike
Potential
in Cultured Chick Skeletal Muscle
Class Specificity of Sera and Muscle Trophic Factor for Muscle Cell Growth In vitro
A Freeze Fracture Study of the Plasma Membrane of Dystrophic Chick Muscles
Structural Diversity in Chicken Skeletal Muscles
Troponin in Cardiac and Skeletal Muscles in Chicken Embryos Studied by Immunofluorescence
Microscopy
Immunoelectronmicroscopic
Observations of the Localizationof Troponin Along the Thin
Filament of Dystrophic Chicken Breast Muscle
Developmental Studies of the Membrane Properties of Striated Muscle Fibers
Shortening Velocity of Skinned Muscle Fibers from Dystrophic Chicken Muscles
Study of the Electrophysiological,
Morphological,
and Metabolic Aspects of Dystrophic Chick
Muscle Membrane in Culture
Propertiesof Muscle Fiber and Neuro-muscular Transmission in Anthracene-9-carboxylic

3

1

CONTENTS

11

13

15
17
19

21

23
25
27

29

31

Mechanism of the Increase in Protein Synthetic Activity in Chicken Dystrophic Muscle
The Proportion of Monomeric and Polymeric Actins in Embryonic and Dystrophic Chicken
Skeletal Muscles
Neonatal Denervation of Rat Sciatic Nerve. I. The Effect of Denervation on Myosin
Differentiation
Connectin, an Elastic Protein of Muscle
Membrane Composition and Ca-uptake Ability of Sarcoplasmic Reticulum from Dystrophic Chicken
Studies of Myelin-associated
Enzymes in the Central Nervous System of Murine Muscular
Dystrophy
Mode of Proteolysis of Myofibrillar
Structural Proteins by Cathepsin-B
Enzymatic Relations in Dystrophic Mice and Chickens
iii

33
35

37
39
41
43
45
47

Studies on Neutral Proteases in Muscle
The Effect of Calcium Ions on Calcium-activatedEffect49
51

Neutral Proteinaseof N-[N-(L-trans-3-Carboxyoxirane-2-carbonyl)-L-leucyl]-3-methyl-butylamine,

Inhibitor,

on Protein

Degradationin Intact

a Protease53

Muscle Cells

The List of Members the Research Group on Muscular Dystrophyiv

in Japan (Biomedical

Sciences)55腑†∫C〟血r勒∫什即妙Gr（）岬IJATENTMUSCUI．ARDYSTROPIIYINGENOTYPICALLYDYSTROPHIC−DWARFMICE
TsuyoshiTotsuka，KimiWatanabe，andSigehiroKiyono
DepartmentofPhysiology，InstituteforDevelopmentalResearch，AichiPreftcturalColony，Aichi
Wehaveproposedaworkinghypothesisfbrthedevelopmentofmurinemusculardystrophy，aSSum一

marizedinFig・1・Thisisbasedonthefbllowingexperimentalresults．Inmusculardystrophicmice（dy／dy；
StrainC57BL／6J−dy），thefbrelegsseemedtofunctionalmostnormallyevenat3monthsofage，Whereas
thehindlegsshowedcharacteristicsignsofthediseaseatanearlyage（about2weeks），SuggeStingthat
COmparativestudiesonthefbre−andhindlegmusclesmightyieldcluesclarifyingthemechanisminvoIved
inthedevelopmentofmurinemusculardystrophy・However，thefbrelegsofdystrophicmicewerealso
foundtobeafrtctedbythediseaseatleastbylmonthofage・Thus，theenduranceofthefbrelegswas
muchlessthanthatinnormalmicel）・Moreover，itwasfbundthatthefbrelegmuscles，aSWellasthehindleg
muscles2，3），Wereabnormalincollagencontent4，，inthelevelofproteaseactivities5），andinthehistologicalimages（unpublishedresultsfromourlaboratory）．AIso，thegrowthofthefbre−andhindlegmuscleswas
arrestedataboutlmonthofage・Fromabiochemicalstudyofthehindlegmuscles，thedrasticinCrease
intheapparentvolumepermusclecellwhichoccursonandaflerlOdaysofageinnormaldevelopment
WaSnOtObservableindystrophicmice6）．Findingsthusfarshowedthattherewasnoremarkabledi晩rence
toexplainthevariationinthedegreeofapparentseverltyOfthefbre−andhindleginvoIvementinanyfta−
tureofthemusclesbutratherthedifferencewasinthegrowthofthebones7）．Thegrowth（lengthening）
OfthefbrelegboneswasmuchsmallerandslowerthanthatofthehindlegbonesduringtheperiodffomlOto20daysofageinmusculardystrophicmiceaswellasinnormalmice・Ttisnoteworthythattheonset
OfthediseaseobservableinthehindIegs（about14daysofage）occurredinthisperiod．
Accordingtoourworkinghypothesis，theclinicalsignsofthediseasemustbealleviatedindystrophic
miceinwhichgrowth，includingbone−lengthening，WaSarreSted．Toinvestigatethis，aftwe鮎ctivemethods
areavailableatpresent，SuChashypophysectomyandgeneticmethods．Genesbm（brachymorphic），bp

45m。。ths。fage聖も5m。。ths

Genedy」？−arreSb

RhVthofskekblmuscle

Choksterd？

0き紬でaSeh椰劇輌Hom叩R叩t叫Cdkgeへ

聖ヒ芦．∈∈コU．UNきγ十dy／dy0Time（min）

No・BodyWt・

80ne−Muscletmbabnce：7

Time（min）

BodyWt・HirdLegMuscte

H星亨g H：…g S：；3g′leg

Pmg貯SSd thedb的甜蜘Symptomtic下血雨？
叫輌CtOmy？（gene塾brRbm？）
Fig．1

3m

8．3

4t

8．3

−−

さ11．9

0．21

；10．9
Dystrophic

0．22

Fig．2

Fig・1・Aworkinghypothesisforthedevelopmentofmurinemusculardystrophy．
Fig・2・LatentmuscuIar dystrophyingenotypica11y dystrophic−dwarfmice．The genotypicallydystrophic・dwarf
mice（Nos・3and4）WerenotdystrophictilltheyweretreatedwithGHandT．．f，m：fema］e，maZe，reSpeCtively．

−1−

（brachypod），brp（another brachypod），and dw（pituitary dwarf）may be suitable fbr the purpose．We
havealreadysucceededinproducinggenotypicallydystrophic−brachymorphic（dy／dy・bm／bm），dystrophic−brachypod（dy／dy・brp／brp），anddystrophic−dwarf（dy／dy・dw／dw）mice．In thepresent study，reSults fordystrophic−dwarfmicearereported．In orderto obtain dystrophic−dwarfmice，CrOSSeSWere Performed
between Fl hybrid carriers（dy／＋・dw／＋），Whichwere produced bycrossing C57BL／6J−dystrophiccar−riers（dy／＋・＋／＋）withDW／J−dwarf8）carriers（＋／＋・dw／＋；PurChased from theJackson Laboratory）．

IntheF2hybridmice，phenotypicallynormal，dystrophic，anddwarfmicewereobservedbutnotthedys−
trophic−dwarftype．Allthemicetestedinourwire−net−Climbing（WNC）apparatus9），eXCeptforthedys−
trophiconeswhichcontinuallydraggedtheirhindlegsafterabout3monthsofage，Showedalmostnormal

locomotiveactivitiesevenat4．5monthsofage（Fig．2）．Someofthephenotypicallydwarfmice，however，begantomaniftstanabnormalsignintheireyeswithage，Whichisacharacteristicofmurinemuscular
dystrophy．Fromthisandthetheoreticalexpectation（i．e．，One−SixteenthoftheF2hybridmiceshouldbe
genotypicallydystrophic−dwarfmice），itwassuggestedthatthedystrophic−dwarfmicewerelatentinthe
phenotyplCallydwarfones．Infact，thiswasdemonstratedbythefbllowingexperiments．
F2hybriddwarfmicewereinjectedintraperitoneallywithbovinegrowthhormone（GH）andthyro−xine（T4）．AsthephenotypicallydwarfmicegrewwiththeGH−T4−treatment（100FLgGHandlOFLgT4／daylO），
everyotherdayfbrabout15days），SOmeOfthembegantoexhibitthecharacteristicclinicalsignsofmus−
culardystrophy（sometimesdraggingtheirhindlegsandrevealingdepressedactivityintheWNCapparatus）．
Thus，genOtypicallydystrophic−dwarfmicecouldbeidentified．Thesedystrophic−dwarfmicehadlessmuscle

intheirhindlegsthanthenondystrophicdwarfmicewhichhadbeenraisedwithGH−T4−treatment．Five
dystrophic−dwarfmicehavebeenobservedthusfar．
Fromthepresentfindings，ltWaSCOnCludedthatmusculardystrophywaslatentingenotyplCallydys−
trophic−dwarf（dy／dy・dw／dw）miceinwhichgrowthhadbeenarrestedbythegenedw．Thissuggeststhat
hypophysectomymaybeanefrbctivesymptomatictreatmentfbrmurinemusculardystrophy・︶ ︶ ︶ ︶ ︶ ︶ ︶ ︶ ︶ ︶l つ ▲ つ J 4 5 ′ 0 7 8 q ノ 0
1

Watanabe，K．andTotsuka，T．
Totsuka，T．andWatanabe，K．
Totsuka，T．andWatanabe，K．
Watanabe，K．andTotsuka，T．
Totsuka，T．andWatanabe，K．
Watanabe，K．andTotsuka，T，肋J．βわJリ99， 171−173，1979．
肋d月わJり94， 327−329，1977．
肋J．月わ7．，97， 235−237，1978．
〟g正月わ7．，98， 257−260，1979．
〟eJ．βわ7．，98， 149−152，1979．
肋J．βわ7．，93， 203−206，1976．
Totsuka，T．andWatanabe，K．：Med．Biol．，99，1−5，1979．
Snell，G．D．：frtフC．肋JJ．dc。d．∫cf．比丘A，15，733−734，1929．
Totsuka，T，，Watanabe，KりandSano，M．：CurrentResearchinMuscularDystrophy，Japan，pp．74−75，1977・

Totsuka，T．andWatanabe，K．：Med．Biol．，99，335L337，1979．

−2−

ノ仇J∫rJJ肋r功′∫什qPわ・Gr（フ叩AUTORADIOGRAPHICSTUDIESONTHEREGENERATIONOFTRANSPIJANTEDMUSCLES
ToshioTeraoandNakaakiOhsawa＊

TheFirstDepartmentofInternalMedicine，TeikyoUniversity，Tokyo＊TheThirdDepartmentofInternalMedicine，FacultyofMedicine，UniversltyOfTokyo，Tokyo

Ourstudiesaimatananalysisoftheregenerationofhumandystrophiedmusclesheterotransplanted
intonudemice．PreviOusstudiesrevealedthatregeneratedmusclesofdystrophiedmuscleoriginandcon−
trolmuscleorlglnareSimilarunderlightmicroscopyintheirstainlngCharacteristics，theirstructuralfea−
tures，SuChasthelocationofthenucleus，andtheirhistochemicalcharacteristics．
In order to elucidatethemechanism oftheregeneration ofdystrophiedmusclesin nudemice，We
havebeentryingtoanalyzetheabilityofthesatellitecells，Whichareknowntobetheoriginofregenerated
muscles・Thepresentreport describesthedevelopment ofanautoradiographictechniquetoanalyzethe
abilityofthesatellitecellsduringmuscleregeneration．

Musclesfromnormalddymicewereused．TheM．extensordigitorumlongus（EDL）fromddymice
Werehomotransplantedinto otherddymice．Two daysand7daysaftertransplantation，5FLCi／gbody
Weightof3H−thymidinewaslrUeCtedintraperitoneally．Sixhourslater，themiceweresacrificedandtrans−
plantedmusclesweresubiectedtoautoradiography．
Two days after transplantation，nO uptake of3H−thymidinewas observed・Seven days after trans−
plantation，ampleuptakeof3H−thymidinewasobserved．Atthistime，thegraftedmuscleswereableto
beseparatedintothreezones：theouter，middle，andinnerzones．

1．3H−thymidineuptakeofEDL7daysaftertransplantation．
−3−

Intheouterzone，manynuCleitakingup3H−thymidinewereobserved．Inthiszone，manyneWlyre−
generatedmyotubeswerealsoseen．Thenucleiofthosemyotubes，however，didnottakeup3H−thymidine，Whichindicatedthatthosenucleiatthisperiodhavenotendencytodivide．
Themiddlezone contained manymyofibrils whosenucleisometimes took up3H−thymidine．These
aremyonucleiorsatellitecellswhicharedimculttodifrerentiateunderlightmicroscopy．
Theinnerzoneconsistedofdegeneratedmuscleswhichhadno3H−thymidineuptake．
Weare plannlngtO analyzethe diffbrencein3H−thymidineuptakeinsate11itecells between dystr0−
phiedandcontrolmusclesheterotransplantedintonudemice．

1）Snow，M．H．：Ce〃恥．ReJ．，186，535−540，1978．
2）Hsu，LandTrupin，G．L．：MuscleRegeneration，RavenPress，pP．115−120，1978．
3）Wakayama，Y．andSchotland，D．L．：MuscleRegeneration，RavenPress，pp．121−129，1978．
4）Hironaka，T．andMiyata，T．：Exp．Neurol．，47，1−15，1975．

−4−肋∫C〟hrqy∫Jr呼力γGro叩MUSCLETRANSPLANTATIONINTHEETIOLOGICAI，ELUCIDATIONOFAVIANMUSCULARDYSTROPHY
Tets可iHironaka，YoshioIkari，YuheiMiyata＊andMasanoriOtsuka＊
DepartmentofPharmacology，TeikyoUniversitySchoolofMedicine，Tokyo＊DepartmentofPharmacology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo

Wehavepreviouslydevelopedatechniquefbrtransplantinganentiremusclewithoutpretreatment
fromoneanimaltoanotherl）・Inthepresentstudyweappliedthistechniquetoavianmusculardystrophy
toexaminethequestionofwhetherdystrophicdisorderoriginatesinmuscleornervetissues，withspecial
refbrencetoneuralinfluenceonmusclegrowth．Theextensorcarpiradialislongus（ECRL）musclewas
Chosen．ThischoiceinVoIvedavarietyoffactorswhichshouldbenoted：Thismuscleissmallsothatits
reinnervationcanbeestablishedwithinseveraldaysIJ，prOvidedthattherateofaxonregenerationhas
avelocityofaboutlmm／day2）．Thesmallsizeofthemusclefurthermadethetransplantationofanentire
musclepossiblewithoutmincingl），Whichofftrsanexperimentalsystemthatcanbeanalyzedmorequanti−tatively・Thissurgicalproceduregavenoregenerationata11whentheECRLmusclewassimplyremoved
withoutbeingreplacedbyatransplant（acontrolfbrthezero−tranSpIant），althoughthehostmuscle

budM

regeneratedtogetherwiththedonormuscleinthemincedgraftexperimentuslngPeCtOralmuscle3）．The
relativelyisolatedlocationoftheECRLmusclefacilitatedtheavoidanceofiqiurjestothetransplanted
andsurroundingmusclesduringsurgery，andconsequentlytheregeneratedmusclescouldbeclearlydis−
tjnguishedfromtheothermuscles．
Normal（line412）anddystrophic（1ine413）chickenswereused．Twoto3daysexovo，Chickenswere
anesthetizedwithether，andtheECRLmuscleswerecross−tranSplantedbetweentwochickens．Fourtypes
Oftransplantationwerecarriedout：nOrmalmuscletransplantedintonormalordystrophicchicken（NnOrDn）anddystrophicmuscletransplantedintonormalordystrophicchicken（NdorDd）．Thedetails
Ofthesurgicalprocedurehavebeendescribedinapreviousreport4）．AnimalswerekeptinanaseptlCCOn−ditionthroughouttheoperationandwerenotglVenanyantibiotic・Thetransplantedmusclewasallowed
toregenerateandwasexamined60−65daysaftertransplantationwhentissuereconstitutionwasvirtuallyCOmplete・ReinnervationofthetransplantedmuscleswasconfirmqdbyobservlngamuSClecontraction
inducedbynervestimulationorbyhistochemicalstainingofacetylcholineesterase5）．
Tablel・Cross−tranSp】antationofECRLmusclesinnormalanddystrophicchickens

TypeoftranSplantation Numberofoperation

Survivalrateofmuscle Muscleweightfor successful
60−65daysafteroperation transplant（％）（mg）
444±99．8JJ＝11344±72．9〝＝15241±81．0
JI＝9235±62．7
JI＝7

Mean±S．E．
NorD：nOrmalhostordystrophichost；nOrd：nOrmalmuscleordystrophicmuscJe．
−5−

Thesurvivalratesofthetransplantedmuscleswere58，60，36，and37％fbrNn，Nd，Dn，andDd，respectively（Tablel）．Thedystrophichostshadworseconditionsthanthenormalhostsfbrmusclesur−
vival，butthetypeofmuscle，Whethernormalordystrophic，didnotinfluencethesurvivalrate．
Sincenotallofthefburtypesoftransplantssurvived60−65daysaflertheoperation，themuscleweight
wascomparedamongthesurvIVlngmuSClesinthefburtypesoftransplantation，andthuscontamination
byapossiblerqiectlngreSPOnSeWaSaVOided．TheresultsaresummarizedinTablel．

Despitetherebeingnostatisticallysignificantdifftrenceinthemuscleweightofnon−OperatedECRL
musclesbetweennormalanddystrophicchickens，1，040±28．8mg（mean±S．E．，n＝21）andl，104±36．8mg（n＝33），reSpeCtively，thenormalmuscletransplantedintothenormalhost（Nn）attainedaslightly

largermuscleweight thanthe dystrophicmuscle transplantedinto the dystrophichost（Dd）．However，
thestatisticalsignificancelevelwasP＜0．2（Tablel）．
ThegrowthinweightofthedystrophicECRLmuscletransplantedintothenormalhost（Nd）wasas
goodasthatofthenormalECRLmuscletransplantedintothenormalhost（Nn）．Ontheotherhand，boththenormalECRLmuscletransplantedinto thedystrophichost（Dn）andthedystrophicECRLmuscle
transplantedintothedystrophichost（Dd）werehamperedingrowth．Theseresultsstronglysuggestan
importantroleofextramuscularfactor（S）inmusclegrowth，andthatthefactor（S）maybe deftctivein
dystrophicchickens．Thepresentresultsareconsistentwiththoseobtainedinmurinemusculardystro−
phy6㌧butnotwiththoseobtainedinavianmusculardystrophyusingmincedgraflsofpectoralmuscles3）・
TheseexperimentsaredifferentatleastinthethreefbllowlngreSpeCtS：kindsofmusclesandnervesused，
sizesofthemuscles，andpretreatmentofthetransplantedmuscles．
Tablelincludestheresultsoftransplantationofnormalmuscleintothenormalhost（Nn）anddys−
trophicmuscleintothedystrophichost（Dd）．Therefbre，eVenifsomeproblemsinhistocompatibilityex−
isted，theseeffectsshould beannulledbycomparingNdandDnwithNnandDdcontroIs．The results
showedthatthee鮎ctofthehostsismoreimportantthantheeffectofthedonorsandthat dystrophic
muscleshaveanabilitytorespondtothehost−Sideinfluence（qf：refi．3，7）．
1）Hironaka，T．andMiyata，Y．：Experienfia，29，1584−1586，1973．
2）Gutmann，E．：AnnlL．Rev．PhysEol．，38，177−216，1976．
3）Cosmos，E．andButler，J．：hlResearchinMuscleDevelopmentandtheMuscleSpindle（Banker，B・Q．，Przybylski，
R．J．，VanDerMeulen，J．PりandVictor，M．，eds．），Pp．149−162，ExerptaMedica，Amsterdam，TheNetherlands，

1972．
4）Hironaka，T．，Morimoto，S．，Tsunoo，A．，Miyata，Y．，andOtsuka，M．：LnCurrentReseユrChinMuscularDys−
trophy，Japan，pp．14−15，1977．
5）Karnovsky，M．J．andRoots，L．：J．Hisfochem．q′tOChem．，12，219−221，1964．

6）Hironaka，T．andMiyata，Y．：Nat〟re，244，221−223，1973．

7）Linkhart，T．A．，Yee，G．W．，Nieberg，P．S．，andWilson，B．W．：Deveわp．Biol．，48，447−157，1976．

−6−肋∫C〟kr勒∫Jr叩妙Gr（7岬

THEECTOPICDIFFERENTIATIONOFSTRIATEDMUSCLESINRATPINEAI，BODY
CELI．CUI，TURE
T．S．Okada，KenjiWatanabe，＊HirohikoAoyama，andYoshiakiNQjyo＊

DepartmentofBiophysics，FacultyofScience，UniversityofKyoto，Kyoto＊DepartmentofAnatomy，FukuiUniversityofMedicine，Fukui

Mammalianplnealbodiesareanendocrineorgan．Theirancestraloriginasavisualorgan，however，
stillseemstobeevidencedbythepresenceofstructuressuggestlngaphotoreceptorfunctionduringfbtal
StageS．Thus，itseemedinterestlngtOuStOeXarninetheposSibilityofmultipotentialitylnCelldi能rentiation
Ofpinealcellsincellculture．Mostunexpectedly，thedifferentiationofwel1−developedmyofibersoccurred
COnSistentlyinsuchculturesl）．
Pinealbodieswere taken fromnewborn rats．Coverlngtissues were carefully removedunder adis−
SeCtionmicroscopeandveryclean丘agmentsconsistingofhomogeneousplnealcellswere dissociatedby
treatmentwith trypsin and collagenase．Ce11s wereinoculated onto Falcon plasticculture dishes uslng
Eagle

sMEMsupplementedwith6％fetalcalfserumasacuIturemedium．Cultureplateswereincubatedat370Cunder5％CO2−95％airforabout20days．Thecellularchangesoccurringwithinthisperiodwere
Observeddaily．

By2−3daysafterinOculationtwotypesofcellswererecognized：（1）fJattenedcellsattacheddirectly
totheculturesubstrateand（2）smallerroundcellswhichwerenotdirectlyattachedtothe substratebut

WereSuperimposedontopofthecellsofthefirsttype．Flattenedcellswereprovidedwith piliformpro−
CeSSeS，theshapeofwhichchangedconstantly．

Byabout2weeks，thedi能rentiationoftwo otherdistinctcelltypestookplace．Onewas neuronal
Cellswhichwereprovidedwithelongatedaxon−likeprocesses．Thesecellswereoftenassembled orinter−
COnneCtedwitheachotherbytheprocesses．Theneuronalcellspossiblyoriginatedfromroundcells．The
Othertypewas striated muscles which，aCCOrding to daily observations oflivingcultures，Seemed to be
derivedfrom thecells directly attached to the culturesubstrate（Fig．A）．Musclefiberswere assembled
intobundlesandcontracted，thoughirregularly，inresponsetoexternalmechanicalstimuli（Fig．B）．
This musclefiber difrtrentiation occurredin all di仔もrentinocula so far cultured．On each culture
plate，SeVeralmusclecellfbciwerefbrmed．Thus，itseemstobeveryunlikelythatmuscle di斤もrentiation

isduetoaninadvertentinclusionofmusclecelltissuesintheoriginalinocula．Weassurnethatthepineal
body containS either myoblasts or multipotentialceIIs which can di能rentiateinto both endocrine andmusclecelldirections．
Indeed，therehavebeenseveralreports

indicatingmusclefibersinh7Situplnealbodiesinsomever−tebratespecies．Anotherexampleofmuscledifrtrentiationffomunexpectedoriginisinmammalianthymus
Cultures2J．Tosumupthesituation，thepresentfindingofmuscledifkrentiationinpinealbodyculturesisnotasporadic，butrather，aCOnSistenteventwhichmayoccurinthecourseofnormaldevelopmentof
this organinsit〟．The developmentalorJgln Ofthis differentiationis not yet known．The physiologicalmeaningoftheectopicoccurrenceofmuscledifftrentiationremainstobesoIved．

1）Watanabe，K．，Aoyama，H．，NQjyo，N．，andOkada，T．S．：Devek）P．GrowfhD伊r．，23，inpress．
2）Wekerle，H．，Patterson，B．，Ketelsen，U．−P．，andFeldman，M．：NaflLre，256，493−194，1975．

−7−

A：anearlystepinthemuscledifferentiationinculturesofplnealcellsafter
2weekscultivationin

vitYO．Arrowsindicate

early

myotubeswith

severalnucleieach．A microphotograph ofaliving culture with a phase contrast
microscope．ca．×250．

B：thedifrerentiationof well−Striated muscle斤bersin a4LWeek culture ofp）nealceHs．Amicrophotograph of afixed culture with aphase contrast
microscope．（・a．×500．

−8−

肋∫C〟血r上）γ∫Jr叩わ・Gr（）叩TISSUECULTtJRESTUDIESOFDYSTROPHICMUSCLES
TakeshiYonezawaandHidetoshiOkabe
DepartmentofPathology，KyotoPrefecturalUniversityofMedicinC，Kyoto
Forobservationofthesequences ofdevelopment ofdiseaseprocesses，tissuecultureis oneadvan−
tageous and preftrable technique．Musclefibers from Duchenne−tyPe muSCular dystrophy，and froma
CarrierandhealthyindividualSwereintroducedintoanin vitrosystemand dystrophicalterationswere
Studied・Aquantitativeanalysisoftheproliftrationactivityofmyogenicce11swasalsoperfbrmedusing
Cellculturetechniques．
ExperimentI．Developmentofdystrophicchangesinvitro．
MaterialsandMetI10ds：Fragmentsofmuscletissueobtainedfrom6patientswithDuchennedys−
trophy，1carrier，andhealthyindividualswithmatchedageswerecultivatedoncollagen−COatedcoverslips．
Oneto3daysafterexplantationcross−SeCtionsofspinalcordfromfttalmiceorrats，140r15daysinutero，
Were

added

to

the

culturesand

WereCOmPOSedofequalpartsofGey

maintained

with

sBSS，Eagle

Maximowsdouble−COVerSlip

method．Feeding

media

sMEM，horseserum，Chickembryoextract，andglucose

toproduceafinalconcentrationof600mg％．
Results：Whenthemusclefibersbecameattachedtothegrowingmotoraxonffomthesplnalcord，
rapid deterioration ofthe muscle occurred．Spindle−Shaped myoblasts may grow around the explants．

Whenthemusclefiberswerenotinnervated，theoriginalcross−Striationsremainedunchangedandmyo−
blastic prolifbrationwas markedly retarded．Around2weeks or so，OutgrOWingspindlecells began tofusewitheachother．LongitudinalfibrillarstruCtureSWereOftenobserved．Around4Weekssomeofthe
fused myotubes developed cross−Striation．Throughout the experimentalcourse twitchingwas not seen
ineitherstriatedornonstriatedmultinucleatedcells．Inthesemyotubes，VaCuOlationwasoftenobserved．
InDuchennemusclefibers，aSCOmparedtohealthymuscle，myOtubefbrmationseemedtobedisturbed
andvacuolarchangesweremorefrequentlyobserved．Someofthefibers developedsegmentalgranular
Changesinthesarcoplasmwhichisidenticaltosegmentalnecrosis．Thesealterations，VaCuOlationofthe
SarCOPlasmandsegmentalgranulardegeneration，Wereinterpretedasdystrophicchangesinvitro．
ExperimentIL ProlifbrativeactlVltyOfdystrophicmusclecellsinvitro．

Method：MusclefragmentsfromDuchennedystrophyandanonclinicalcarriersiblingweretrans−
ftrredto plastic且asksandPetridishes，andmaintainedinaCO2incubator．Theftedingmediumwas
COmpOSedof85％L15and15％fbtalbovineserum．Subcultivationwascarriedoutfburtimesas showninthefbllowingtable．
TrypslnlZationlWashandcentrifugelFilterthroughlenspaperlIncubationinftedingmediurr），40min．
lCollectionofnoatingcells
I

CultivationinCO2incubator
5×105cellsweresubculturedineachtime．
−9−Fig．1・Cross−Striationoflivingdystrophicmusclegrowninvitro．
Fig．2．VacuolizationofIivingdystrophicmuscle．
Results：IntheprlmaryCulture，OutgrOWthwasvisibleonthe4thdayandmyotubeswithmorethan
lOnucleiwereseenafterthe17thdayinbothdystrophicandcarriergroups・Inthefirstsubculturesmyo−
blasticcellsinthecarriergroupwere2・5timesmorenumerousthanindystrophiccultures．Nodifrbrence
WaSSeenintheactivitiesofmyotubeformationbetweencarrieranddystrophicmusclecultures．ThatlS，
myotubeswithmorethanlOnucleiwerefirstobservedonthe17thdayafterexplantationinbothgroups．
However，inthefollowJngSubcultivation，dystrophicmuscleproducedaloweractivltyOfmyoblasticpr0−
1ifbration，and consequently retarded myotube formation・These differences were more emphasizedin
thesucceedingsubcultures．
Comments：Thoughmaturationanddifferentiationofhumanmuscleinvitroareatpresentincom−
plete，SeVeraldifferencesin the developmentalprocesses among Duchenne−typedystrophy，Carrier，and
COntrOlhealthymusclewereobtained・First，regenerativeactivltyWaSlessprominentinDuchennedys−
trophy・Thismightbeadirectexpressionofagenedefectin thisdisease・MorphologlCalfeatures，SuChasvacuo】ationofthesarcoplasmandsegmentaldegeneration，Observedindystrophicmusclewereclosely
Si■一Iulatedtothefindingsindystrophicchickmusclesfoundin bothil

ivoandinvitrosystems．Byim−

l一一・OV．ngtheculturetechniquesforhunlanmuSCle，mOredetailsofalterationscanbeanalyzed．

1日山一叩、A・，Gallup，B・，Skeate，Y・，andDubowitz，V∴J・NeLLrOl．St・I．，13，333−350，1971．
二日iHll叩．l・andDubowitz，V．：Neuropatho／．Appl．Ne〟rObiol．．1，205−229，1975．
1）Wilkow、ki，J・A・1Durbidge，M・，andDubowitz，V∴Jn Tnlr0，12，98−106，1976．
4）Witk（−WゝkiHl・A∴βわ／・Revリ52，431−476，1977．

一10−

ルねでC〟hr勒∫Jr叩わ′Gr（7岬MITOTICACTIVITIESOFMONONUCI．EATECEIJIJSCULTUREDJJV17rR0
0RIGINATEDFROMREGENERATINGMUSCLESOFNORMAL（＋／＋）AND

DYSTROPHIC（dy／dy）MICE
TsutomuKagawa，EiiiChikata，andRiichiroHamano
LaboratoryofCellBiology，ToneyamaNationalHospital，5−1−1，Toneyama−Cho，Toyonaka−City，Osaka，Japan
Themitoticactivitiesofmononucleatecel】S，Culturedinvilro，OrlglnatedfromregeneratlngmuSCles
fromeithernormalordystrophicmicewerestudied．
Adultnormal（C57Bl／6＋／＋）and dystrophics（C57Bl／6Jdy／dy）micewereusedthroughout．The
proceduresfbrthepreparationofregeneratingmusclesandtheliberationofmononucleatecellsfromeither
SOurCeWeredescribedpreviouslybyKagawaetal．1￣3）．Inourstandardclonalcultures（Experimentsland

2）4I，Cultureswereestablishedbyplating500cellsfromeithersourcein5mlofthe

freshmedium（1），，OntOa60−mmFalconplasticpetridishcoatedwithO．2％gelatin5）．Incubationwascontinuedat370Cin
ahumidifiedatmospheregasedwith5％CO2and95％air．Eachculturewasftdwiti15mlofthe
medium（1）growthldayafterplating．Mononucleatecellsfromeitheroriginweresuccessfullyculturedunderthepresentcultureconditionsandtheyyieldedcoloniesofmyogeniccellsandoffibroblasts・At6days
aftertheinitiationofcultures，allcultureswerefixedwithethanolandstainedwithhematoxylin．Mitotic
activitywasshownbythenumberofcelldivisionswhichoccurredduringthecultureperiod．Thenumber
Ofcelldivisions（X）wascalculatedfromthenumberofnucleiofcells（N）fbrmingeachcolonyaccording
tothefbllowingformula：X＝logN／log2．

ExperimentlandExperiment2：Tennormalandlldystrophicculturesjnexp．landll normal
andlldystrophiconesinexp．2werecarriedout（Tablel）．Theaveragenumberofcoloniesformedper
dishandtheplatingefBciencylneaChseriesofculturesareshowninTablel・Inexp．1，thenumberofce11
divisions（NCD）ofnormalmyogeniccellswas6．93±1．21andthatofdystrophicones was5．38±1．13．
Thedi騰rencebetweenthemwassignincant（P＜0．001）（Table2）．TheNCDofnormalfibroblastsand
thatofdystrophiconeswere4．50±0．90and4．23±0．84，reSPeCtively．Thedi耽rencebetween themwas
muchsmallerthanthatobservedbetweennormalanddystrophicmyogeniccells，butitwasalsosignificant（P＜0・02）・Inexp．2，theNCDofnormalmyogeniccellswas6．14±1．39，thatofdystrophiconeswas5．16±1・15，andthedi能rencebetweenthemwassignificant（P＜0．001）（Table2）．TheNCDofnormalfibro−
blastsandthatofdystrophiconeswere4・44±1・12and4・22±1・05，reSpeCtively，andthedifrerencebetween
themwasnotsignificant．

Experiment3：AsshowninTablel，mOreCOloniesperdishwerefbrmedinnormalculturesthanin
dystrophiconesbothinexp・1andinexp・2・Thesefindingssuggestedthatpopulationefrectmayhave
enhanCedthemitoticactivityofmyogeniccellsinnormalcultures，inwhichmorecolobieswereformedper
dishthanindystrophicones・Totestthispossibility，mOnOnuCleatecellsffomeithernormalordystrophic
SOurCeWereCulturedunderthesamecultureconditionsatvaryingcelldensitiesof500，250，and125per
dishandtheNCDsofmyogeniccellsandfibrobIastsfromeithersourceinculturesateachcelldensity
WereObtained・Intheseculturesinwhichcelldensitiesweresuccessivelyalteredasdescribed above，theaveragenumberofcoloniesfbrmedperdishwasfbundtobe18．0，12．1，and6．7innormaland33．8，16．1，
andlO・0indystrophiccultures，reSpeCtively（Tablel）．Thenumberofculturesateachcelldensityffom
eithersourceisshowninTablel．InthecuIturesatthedifrtrentcelldensitiesdescribedabove，theNCDs
Ofnormalmyogeniccellswere6・99±1・29，6・89±1・21，and7・00±1・53andthoseofdystrophiconeswere
4・98±1・20，5・22±1．34，and4．89±1．19，reSpeCtively（Table2）．NosigniBcantdifftrencesamongtheNCDs−11−

Tablel．Platinge伍Ciencyandnumberofcoloniesformedperdishineachseriesofexperiments
Seriesofexp．

Exp．l Exp．2

Sourceofcells Norm．Dystr．

Norm．Dystr．
2

5
0

L
U
1

5

3

7

0

′

q

1

0

8

3

0

5

1

2

1

′

4

L

5

′

0

′

12．3

U

ノ

2

3

21．5

0

1

4

00

5．1

q

12．9

14．4

00

4．7

11．5

b

13．2

11．5

′

13．9

14．6

3

35．6

0

Myoblast
Fibrob】ast

6．8
33．8

1

18．0

′

19．1

1

24．7

U

25．4

4

50．2

12

∠

No．OrcolJpl．

0

3．6

1

3．8

0

4．9

5
2

5．1

0

PLefBciency（％）

10．0

500

0

10

5

500

11

つ−

500

11

0

500

10

1

500

11

0

500

No．orcultures5
2

Inoculumsize

Table2．NumberofCellDivisionsofMyoblasts

Seriesofexp．Inoculumsize Normal（斎n）
Exp．1

500

Dystr．（斉d）

Xn−Xd d．f t−Stat．
1．55

510

タ＜0．001（373）

5．38±1．13（139）

6．93±1．21

Exp．2

500

6．14±1．39（124）

5．16±1．15
（51）

0．98

173

f，＜0．001

Exp．3

500

4．98±1．20

2．01

254

タ＜0．001250

6．99±1．29（116）
6．89±1．21（140）
5．22±1．34

1．67

139

上，＜0．001125（83）
7．00±1．53（58）
4．89±1．19

2．11

5l f，＜0．001

（27）

（26）

n：nOrmal；d：dystrophy；扁：aVeragenumberofcelldivision；1−Stat．：1−Statistics；d．f：degreeof丘eedom；（）：numberofcoloniesusedforthecountofnumberofcelldivisions．
Ofmyogenicce11sinculturesat differentcelldensitiesineithernormalordystrophicculturescouldbe
fbund（Table2）．Inaddition，theNCDsofnormalmyogeniccellswerealwayssignificantlylargerthan
thoseofdystrophiconesinculturesatanyce11density，atleastunderthepresentcultureconditions（Table
2）．TheNCDsoffibroblastsculturedatdifferentcelldensitieswerenotsigniBcantlydi爪汀ent丘omone
anotherinculturesfromeithersourceandtheNCDofnormalfibroblastswasnotsignificantlydifferent
fromthatofdystrophiconesinculturesatanyofthecelldensitiesdescribedabove．Thesefindingsshowed
thatmyogeniccellsfromnormaloriginshowedhighermitoticactivitythanthosefromdystrophicorlgln，regardlessofcelldensityorofthenumberofcoloniesofculturesinwhichtheywerecultured．Thepre−
sentresultssuggestthethreefbllowingquestions：（a）Isthedecreaseinthenumberofcelldivisionsof
dystrophicmyogeniccellsduetoaprolongationofgenerationtimeortoalimitednumberofcelldivi−
sions（Celllineagetheory6））？（b）Isitduetotheexpressionofadystrophicgene（dy／dy）？（C）Coulditbe
oneofthecausalfactorsofvariOusdystrophicchangesobservedindystrophicmuscles（dy／dy）bZVEvo？

Ananalysisoftheseproblemsisnowinprogressinourlaboratory・
1）Kagawa，T．，Chikata，E andTani，J．：Deveわp．BioI．，55，402，1977．
2）Kagawa，T．，Chikata，E．，Tani，J．，andTsutamune，T．：Devekp．Biol・，65，526，1978．
3）Kagawa，T．，Chikata，E．，Tani，JリandTsutamune，T．：30thAnn．MeetingofJapanSoc．ofCellBioLAbst．（A−28），

1977．

4）Kagawa，T．，Chikata，E．，andHamano，R．：CurrentRes．MuscularDyst．Japan，13，1979・
5）Richler，C．andYafre，D．：Deveわク．BわJ．，23，1，1970．
6）Kligman，D．andNameroff，M．：Exp．CdlRes．，127，237，1980・−12−肋．†C〟肋r如け叩わ′Gr（フ岬

AFACTOREXTRACTEDFROMTHEBRAINAFFECTINGDEVEI．OPMENTOF
TETRODOTOXIN−SENSITIVESPIKEPOTENTIALINCULTUREDCHICKSKELETALMUSCI．E
MasaakiraKano，SuteoYamazaki，TeikichiSatoh，andMitsueYamamoto
DepartmentofPhysiology，SchooJofMedicine，KitasatoUniversity，Kanagawa
The action potentialofadultinnervated vertebrateskeletalmuscZefibersis generated by tetrodo−
toxin（TTX）−SenSitive sodium channels．The TTX−SenSitive sodium channelsinthe skeletalmuscle cell
membraneofnewbornratsincreaseindensityaflerbirthinrelationtotheestablishmentofnormalinner−
VationlJ・Conversely，denervationofadultratskeletalmusclediminishesthedensityoftheTTX−SenSitive
SOdiumchannelsinthemusclecellmembrane2）．ThedevelopmentandmaintenanceoftheTTX−SenSitive
SOdiumchannelsinthemusclecellmembranecouldthenbeconsideredasoneofthemembraneproper−
tieswhichareunderthetrophiccontrolofinnervation．
Usingchickskeletalmusclece11cultures，itwasshownthatce11−freenerveextractscouldduplicate
theefrectofinnervationonthedevelopmentoftheTTX−SenSitivesodiumchannelsinnoninnervatedskele−
talmusclecells3−5）．Thus，theresultsindicatethattrophiccontroloftheTTX−SenSitivesodiumchannels
Ofmusclecellsmightwellbemediatedbyasolublesubstanceproducedbynervoustissue，aSSuggeSted
byHarris6）andGutmann7J．Inthepresentstudy，WerePOrtthataproteinffactionisolatedfromchickbrainextracthasaneurotrophicactivityinthatitenhancesthedevelopmentoftheTTX−SenSitivesodium
Channelsinculturedchickskeletalmusclecells．
Musclecultureswerepreparedfromthebreastmuscleofll−day−01dWhiteLeghornchickembryos
accordingtothemethoddescribedpreviousIy3）．ThebasicculturemediumwasEagle，Sminimalessential
mediumsupplementedwithhorseserum（10％by volume），nerVe−freechickembryo extract（2−5％byVOlume），glutamine（1mM），penicillin（50U／ml），andstreptomycin（50jLg／ml）．Thenerve−ffeechickembryo
extractwasmadefromlO−day−01dchickembryosdeprivedofthebrainandspinalcord，byhoIT）OgenlZlng
theembryo bodiesin salineandcentrifugingthehomogenateat2，000×gfbrlOmin．MusClecultures
Werealsomaintainedinculturemediumcontainingbrainextract．Thebrainextractwaspreparedfrom
Wholebrainsof19−day−Oldchickembryosbycentrifugingal：2homogenateinsalineat2，000×gforlO
min．

WhenmuscIecellsweregrowninthepresenceofbrainextractfbr2weeks，themaximumrateofrise
OfTTX−SenSitivespikepotentialofthecellswassign摘CantlygreaterthaninthecontroIculturesmain−tainedinthebasicculturemedia・Thissuggeststhatthebrainextracthasatrophicactivitythatenhances
thedevelopmentoftheTTX−SenSitivespikepotentialoftheculturedmusclecells．
Thebrainextractwascentrifugedagainat20，000×gfbr30min．TrophicactlVltyCOuldbedetected

inthesupernatant，butnotintheresuspendedprecIPltate．Thesameresultwasobtainedaftercentrifugation
atlOO，000×gfbr60min，t．e．，thesoluble丘actionmayberesponsiblefbrtheactivity．
Theactivecomponentinthe20，000×gSuPernatantWaSisolatedbyconsecutiveammoniumsulfate

precipitation，andretainedaflerremovaloflowmolecularweightcompoundsbydialysisagainstsaline．
Tablel summarizestheseresults．
Theproteinnatureoftheactivecomponentinthebrainextractwasfurthersuggestedbytheresults
Ofenzymedegradation．ThelOO，000×gsupernatantwastreatedwithenzymefor60minat37OC．The

trophicactivitywasabolishedbytrypsinandprotease，butwasresistanttoDNaseandRNase（Table2）．
Thesusceptibilitytotrypslnandproteasesuggeststhattheactivecomponentisaprotein．
−13−

Tablel．E鮎ctofbrainextractonthemaximumrateofriseorTTX−SenSitivespikepotentialofmusclecellsin
2−Week−Oldcultures

Fractionadded Protein（FLg／ml）

Maximumrateofrise（V／sec）（59／4）73．8±3．2

Contro1
2000×gSup．

300（56／4）84．3±3．0＊

20Kxgppt．

40（31／2）59．8±2．5

260（62／4）96．8±3．4＊

20Kxgsup．
Saltrated（Amm．sulL）
0−4095100（63／4）96．4±3．1＊

40−609も50（61／4）87．5±3．2＊

Dialyzed
InnersoL240（35／2）91．1±4．2＊

OutersoI．

10（61／4）65．3±3．0

Mean±S．E．＊indicatesastatisticallysignifiCantincreasefromthecontrol．

Table2．E鮎ctofenzyme−treatedbrainextractonthemaximumrateofriseof
TTX−SenSitivespikepotentialofmusClecellsin2−Week−Oldcultures

Treatment Maximumrateofrise（V／sec）
Control（8）70．9±5．0

lOOgxgSup．
Non−incubated（16）113．8±3．5＊

Incubated（16）99．8±3．7＊

DNase−treated（16）93．9±5．4＊

RNase−treated（12）88．7±2．9＊

Trypsin−treatedProtease−treated（16）68．6±4．3（14）67．3±4．6

Mean±S．E．＊indicatesastatisticallysigni丘cantincreasefromthecontrol．

1）Harris，J．B．andMarshall，M．W．：Nat〟reNewBiol．，243，191−192，1973・
2）Redftrn，P．andThesleff，S．：AcfaPhysiol．Scand．，＄2，70−78，1971．

3）Kano，M．andYamamoto，M．：J．CbIl．Physiol．，90，439−444，1977．
4）Kano，M．，Suzuki，N．，andOjima，H．：J．Ce〟．PhysEol．，99，327−332，1979．
5）Yamazaki，S．andKano，M．：Eq．Neurol．，Submitted．
6）Harris，A．J．：A7mLL．Rev．朗ysiol．，36，251−305，1974．
7）Gutmann，E．：AtmLI．Ret7．Physiolり38，177−216，1976・−14−ルh∫CJ血rqy∫什qp砂Gro岬

CLASSSPECIFICITYOFSERAANDMUSCIJETROPHICFACTORFORMUSCI．ECELLGROWTH劇Ⅴγ∫mO
EijiroOzawa，IchiroKimura，andYasukoHagiwara
DivisionofCellBiology，NationalCenterfbrNervous，Mental，andMuscularDisorders，Tokyo

ChickmyogeniccelIsdonotgrowinamediumcomposedofEagle，Sminimumessentialmedium（MEM）andhorseserum，butgrowwellwhenasmal1amountofchickembryoextract（EE）isaddedto
themedium・EEcanbereplacedbychickorduckserum，andbychickmuscletrophicfactor（MTF），Whichisacomponentofchickseruml・2）・ThecellsalsogrowinamediumcomposedofMEMandchickserum3｝．
Thusitisclearthatchickserumisdifferentfromhorseseruminregardtochickmyogeniccellgrowth，
anditispossiblethatthisdiffbrenceismainlybasedonthedi能renceofMTFwhichcouldbealsocon＿
tainedinmammalianserum・Thepresentworkwasconcernedwithwhetherthereisspeciesorclassspeci−
Gcityamongsera，andwhethertheabovepossibilityisavalidone．

Chickcellswereculturedwithamediumcomposedof15％horseserumandvariousconcentrations
OfvariousseraandMEM・After4daysofincubation，thecellswereharvestedfbrcreatinekinase（CK）
assay，Whichwasinterpretedasshowingthegrowthrate・Figurelshowstherelationshipbetweenthe
COnCentrationofseraandCKactivity・Inthecasesofchickandducksera，CKactivitybegantoincrease
atO・1andO・3％，reSpeCtively．Thenitincreasedgraduallywiththeincreaseinconcentration．Inthecases
Ofdoveandfbtalbovinesera，itbegantoincreaseat3％，andincreasedfairlysharply．Tnthecaseofadultbovineserum，itremainedatanalmostunassayablelevel，althoughsomemyotubescouldbeseenunderamicroscope．

Inordertocomparetheeffectsofvariousseraonchickmyogeniccellgrowth，thecellswereincubatedwithmediacomposedof82％MEM，14％horseserum，and4％variouskindsofsera．Thegrowthrates
areshownsemlquantitativelyinTablel・Avianseraweremoreorlessefftctive，butmammalianseraex−
Ceptfbrfttalbovineserumwerenoteffective・Seran・OmGahsandCbrt〟m玩wereverye能ctive，but
thoseofmasiamLS・Numih・andMekLqa血werenotsoeHbctive，althoughallofthembelongtoGalli．
Ontheotherhand，SeraffomAnser的rmeswerefairlyefftctive．

︵ S至︒盲＼−︒喜己︶音＞叫︼Uく鍼U

Fig・1・E能ctsofvariousseraonchickmyogenicce11growth．
−15−

Tablel．EffectsofvariousSeraOnChickmyogeniccellgrowthinvitro
Birds

Mammals

Bantam（十十十）Calr
BovineJapanesequail

（＋＋＋）
（＋）

Goldenpheas年nt
Koreanpheasant
TurkeyGuineafowI
Duck

Kite

RabbitSwine
MonkeyHuman︶

Herringgull
Dove

Dog
Rat

（＋）
（＋）
（＋）
（＋＋）
（＋＋）
（＋）
（＋）
（＋）

︵

Goose

︶
︶
︶
︶
︶
一 i寧■≡■；■
︵
︵
︵
︵
︵

Fetalbovine

（＋＋＋）

︶
︶
一 一
︵
︵

（十十十）

Cochin−Chinafowl

︶
＋
︵

Chick

Table2．E鮎ctsofvariousseraonratmyogeniccellgrowthinvitro
Birds
Chickembryoextract

Chick

一

Fetalbovine（＋＋）
Bovine

（＋＋）

︶

︵
l

︶
︶
︶
︶
︶
︶

︶

︵

一

︵

︵

︵

一

︶

︵

︵

︵

Dove

︵

一

︶

︵

Goose

︵

一

︶

MonkeyHuman

Duck＋ ＋ ＋ ＋ ＋ ＋＋ ＋ ＋ ＋ ＋ ＋

一

︶

Horse

︵

Rabbit
Dog
Swine

一

︶

︵

TurkeyGuineafowI

（十十）

（−）

︶

Koreanpheasant

Rat

（＋）

︵

JapanesequailGoldenpheasant

Mammals

MTFswerepreparedfromchickandfetalandadultbovinesera・ChickMTFwaseffectiveat3jLg／
ml，butfttalandadultbovineMTFwereonlyslightlyeffectiveat300FLg／ml・
Newbornratmyogenicce11sdidnotgrowinamediumcomposedofchickserumandMEM，but
grewwellwhenasmallamountofratserawasaddedtothemedium・Inordertocomparevariouskinds
ofsera，ratCe11swereincubatedwithmediacomposedof77％MEM，19％Chickserum，and4％various
kindsofserafbr5days．ThegrowthratesareshownsemlquantitativelyinTable2・Mammalianserawere
efkctivebutavianserawerenot；EEwasslightlyeffective・Inthistable，theeffectsofallthemammalian
serawereexpressedby（＋＋），althoughtheywerenotnecessarilythesame・RabbitMTFwase鮎ctiveonratcellgrowthat8FEg／ml，butchickMTFwasnote鮎ctiveat240jLg／
ml．
Conclusion：ThereisaclassspecifiCityamongserafrommammalsandavesinregardtomyogenic
cellgrowth，althoughtherearesomeexceptlOnS，andthisspecificitylSmainlybasedonMTF・1）Ozawa，E．andKohama，K．：Proc．JqpanAcad・Sci・，49，852，1973・
2）Ozawa，E．andKohama，K．：M〟SCkNerye，1，230，1978・

3）Iiagiwara，Y．，Kimura，1．，andOzawa，E・：Deve／・GrowthD脾retzt・，inpress・−16−

ルル∫C〟ねr上）γ∫什（汐カタGr（）岬AFREEZEFRACTURESTUI）YOFTHEPLASMAMEMBRANEOFDYSTROPHICCHICKMUSCI．ES
YuzoIwasakiandHirokoIshii
NationalCenterforNervous，MentalandMuscularDisorders，Kodaira，Tokyo
Toelucidatetheroleoftheplasmamembraneinthedevelopmentofmusclece11degenerationinmus−
Culardystrophy，WeCOnductedafreezefracturestudyoftheskeletalmuscleofdystrophic（Jine413）andCOntrOl（1ine412）chickens ofvarious agesstartingat dayO．SuperfiCialpectoralis andlateraladductor
muscleswerechosenforthisstudysimplybecausethepost−nataldevelopmentofthesemuscleshasbeen
wellcharacterizedl）．
Smallpiecesofmuscletissuewereobtainedunderpentobarbitalanesthesiaandtheywereimmediately
fixedwith2・5％glutaraldehydeinphosphate−bufreredsaline（pH7．4，285mOsm）forafewhoursat40C．
Thefixedtissueswereglycerinatedin25％glycerininPBSandquenchedinliquidnitrogen−COOledFreon
12．Thetissuewasfracturedat−1150Cunderavacuumof1−2×10−7TorrwithanEikofreezeetchdevice．
Thefracturesurfacewasimmediatelyshadowedwithplatinumandcarbon・The replicas wereretrieved
afterdigestionwithcommercialbleachsolutionandwashingwithseveralchangesofdistilledwater．Phot0−
graphsweretakenwithaHitachiH−600electronmicroscopeoperatlngatlOOkV．
ThemostremarkablefindingintheplasmamembraneofdystrophicchickmuscleswasthepreserlCe
Ofalargenumberofcaveolae，Whichhadlosttheorderlydistributionseenincontrolmuscles（Fig．1）．
SuchachangewasevidentinbothsuperfiCialpectoralisandlateraladductormusclesof5−Week−01dchickens
butitwasapparentonlyinthesuperfiCialpectoralismuscleinchickens2Weeksofage．OndayO，how−ever，the denslty and distributjon pattern ofthe caveolaein dystrophic chickens wereindistinguishable

from thoseincontroIs・ForafurtherassessmentofthedensltyOfcaveolae，apPrOXimately300pm20f

Fig．1．Freezefractureprofilesoflateraladductormusclesof5−Week−01dchickens．A：COntrOl；B：dystrophic；
magnification：×10，000．

−17−−

Tablel．Densitiesofcaveoles（perlOjLmB）
Breastmuscle
2weeksdayO

5weeksControl l08．6±3．6

128．1±14．8140．1±4．0

Dystrophic l11．5±5．3

199．7±17．3241．5±5．2

タ＜0．5

タ＜0．01

Legmuscle
Control lO6．4±1．1

140．0±9．2139．7±8．0

Dystrophic lO5．3±2．9131．1±9．3

261．8±11．7

タ＜0．5

ア＜0．001

タ＜0．5

theplasmamembranewithanapparentcontinultyWiththecytoplasmaofmusclecellswas chosenfor
eachmuscleandthe densityofcaveolaewasmeasuredwiththeaidofaKontronImage Analyzer．The
resultsofthemeasurementsaresummarizedinTablel．Theresultsindicatethatanincreaseinthe number
Ofcaveolaecoincideswithadisorderlycaveolaedistribution．
Althoughnoprecisecorrelationhasbeenestablishedbetweenthefreezefractureprofi1eandthetrans−missionimageofcaveolae，1tlSgenerallyassumedthatthecaveolaeonthecellmembranerepresent plnO−
CytOticvesiclesand，inmusclecells，OPenlngSOftheT−SyStemaSWe11．Ifthisisthecase，anincreasednum−
berofcaveolaeonthecellmembranecanbeindicativeofanelevatedtransmembranetransport actlVlty
andmaybeamorphologicalexpressionofa

leakymembrane

inmusculardystrophysinceaconcomitantObservationofthesemuscletissueswithatransmissionelectronmicroscoperevealednodetectableabnor−
malityintheplasmamembrane．AhistopathologiCalstudyofthesemuscletissuesdisclosedgrosslynormal
tissueexceptfbrtheoccasionalpresenceoflipiddropletsinthebreastmuscleof5−Week−01ddystrophic
Chickens．Therefbre，itcanbeassumedthatthealterationintheplasmamembraneisanearlyeventinthe
degenerativeprocessofmuscletissues．
1）Ashmore，C．R．andDoerr，L．：Exper．Neurolリ30，431446，1971．

−18−

九才ぬぐ〟肋rかJ，∫什qp妙GrP〟p

STRUCTURALDIVERSITYINCHICKENSKEI．ETALMUSCI．ES
HarunOriIshikawa，ShoichiroTsukita，andSachikoTsukita
DepartmentofAnatomy，FacultyofMedicine，UniversityofTokyo，Tokyo
Thestructuraldetailsofmusclefibersvarytoagreatextentindifferentmuscles．Carefulanalysesof
SuChstructuraldiversltyrnayleadtoabetterunderstandingofnotonlyfunctionalcorrelates butalsoof
localfactorsfbrtheselectivityofaffectedmusclesinmusculardystrophy．Fromthisviewpoint，Weare
Studyingthevariationsinthecellstructures，eSpeCiallyinthemembranoussystem，amOngdifferentmuscles
inthesamespeciesofanimalS．Inthepresentstudythefinestructuralvariationsinthesarcoplasmicretic−
ulumandT−SySteminvariousmusclesofthechickenwereexaminedunderanelectronmicroscope．
Adultchickens（male，WhiteLeghorn），1−1．8kginbodyweight，WereuSedfbrthisstudy．Underin−
traperitonealanesthesia，Variousmusclesweredissectedoutandfixedinastretchedstatewith2．5％glu−tara］dehyde−2％fbrmaldehydeinO．lMSOdiumcacodylatebufrtr，PH7．3，followedbypost−fixationwith

1％OsO4inthesamebufftr．TissuesweredehydratedinethanolandembeddedinEpon812．Thinsections
WereeXaminedunderaHitachiHU−12electronmicroscope．
The following chicken muscles were examinedinthis study：PeCtOralis mqior muscles（Superficial
layer），anterior and posteriorlatissimus dorsimuscles，anterior tibialis，eXtenSOr digitorum COmmunis，
SartOrius，ischio−peCtineusmuscles，andextrinsic（rectusmedialis）andintrinsic（ciliaris）eyemuscles．
ThinSectionsofmuscletissuescutlongitudinallyandtransverselywereexaminedtoanalyzetheover−alldistribution and regionaldifftrentiation ofthesarcoplasmiCreticulum（SR）and T−SyStem．Chicken

muscleswereclassifiedintotwomajortypesaccordingtothedistributionlevelsoftheT−SyStem：（1）muscle
COmPrisedofmusclefibersinwhichtheT−tubulesrunattheleveloftheZdisc（Z−1eveltype）；and（2）mus−ClecomprisedofmusclefibersinwhichtheT−tubulestaketheircourseattheleveloftheA−Ijunction（A−I−leveltype）．Ofthemusclesweexamined，OnlythepectoralismqiormusclebeIongstotheZ−leveltype（Fig・1）・Therestofthemusclesfal1undertheA−I−leveltype（Fig．2）．Observationsthatthepectoralis
musclescontainT−tubulesattheZ−levelhavebeenreportednotonlyinchickensl・2〉butalsoinpigeons3）．
Sinceourexaminationoffinchbreastmusclesaddsanotherexample，thesetwodifrerentpositionlngSOf
theT−tubulesseemtobecommontomanyotheravianskeletalmuscles．Thisalsomaybe emphasized
inrelationtothefactthatthepectoralismuscleisthemajormuscleaffectedinchickenmusculardystrophy．
TheoveralldevelopmentandregularityoftheT−SyStemVaryamOngdifferentmuscles．Inthepecto−
ralis，leg，andposteriorlatissimusdorsimuscles，theT−SyStemWaSdistributedveryregularlywithrespect
tothesarcomerepattern，WhereastheT−SyStemintheextrinsicandciliarymuscleswasmuchlessregularinitsarrangement．Inanteriorlatissimusdorsimuscles，theT−SyStemWaSSeentObedistributedinavery
irregular pattern．However，high−VOltageelectronmicroscopy ofthick sections combinedwith selective
Stainingrevealedthat transverselyrunningT−tubules werepositioned almostexclusivelyat thelevelof
theA−Ijunction．Furthermore，triadswerefbrmedevenalonglongitudinallyrunningT−tubules，Predomi−
nantlyattheA一日evel．
Theoveralldevelopment andreg10naldifferentiation oftheSRwithrespect tothesarcomerealso
Variedamongdifrtrentmusclesofthechicken．ELjaceviewsoftheSRwereonlyseeninlongitudinalsec−
tionswheremyofibrilswerecuttangentia11y．CarefulcomparisonbetweentheimagesoftheSRinbothlongitudinalandtransversesectionsshowedthatthereglOnaldifftrentiationoftheSRcouldbeimaged
andquantitatedbasedonthemembraneprofilesseenintransversesections．
ThepatternoftheSRnetworkcanbecategorizedintotwotypescorrespondingtothetypesinthe
T−SyStemdistribution．Inpectoralismuscles，triadswerefbrmedattheZ−level．TheSRextendedfromthe−19−Fig・1・Electronmicrographofalongitudinallycutmusclefiberfromchickenpectoralismajormuscle．TheT−
tubulesruntransverselyformingtriadsattheleveloftheZ−band（arrows）．ThesarcoplasmiCreticulum（SR）extends
fromtheI−band（I）tothemiddleoftheA−band（A）．

∵㌫■▲・∴、志
享妻要撃葦

言草享て

チ∵二￣二＝∴ご芋∴Fig・2・Electronmicrographsofmusclefibersfromchickensartoriusmuscle．a：Longitudinalsection．TheT−tubules（a，

rOIl，S）runattheIeveloftheA−Ijunction・Thesarcoplasmicreticulum（SR）isweHdeveloped，COnneCtingadjacenttriadsjtllheI−band（I）butnotintheA−band（A）．b：Transversesection．Thesarcop）asmicreticulumprofileswellreflecHhcjmagesseeninthelongitudinalsection．

terminalcislernae at the Z−leveltoward themiddle ofthe sarcomere，Where the SR formed ftnestrated
COllars（Fig・1）・InlegnluSCles，Ontheotherhand，WhenthetriadswereformedatthelevelofA−Ijunction，
theSRwasdeveIoped．11uCh morein thel−bandconnectlngbetweenadjacenttriadsthanintheA−band（Fig・2）・Such ftatures wcre we日renected on themembraneprofilesin transversesections．TheamountOf theSRin the A−band varied amongdifferent muscles・Incertainlegmuscles，theSRnetworks werepoorlyformedat the）eveloftheA−band so that theboundarybetweenindividualmyofibriJsmight bebarelydiscernibleintransversesections（Fjg．2）．
1）Mendell，J・R・：J・Ultraslruct．Re＞S．，37，383，1971．2）MacNaughton，A．F．：J．Anatリ118，171，1974，
3）Ashhust，D．E．：TYss〟eandCel／，1，485，1969．

−20−

ルJJ∫C∫血rpγ∫Jr叩んyGr（）岬TROPONININCARDIACANDSKELETALMUSCLESINCHICKENEMBRYOSSTUDIEDBYIMMUNOFLUORESCENCEMICROSCOPY
YutakaShimadaandNaqjiToyota
DepartmentofAnatomy，Sごhっ01ofMedi＝ine，ChibaUniverslty，Chiba

Thediirerentiationoftroponin（TN）incardiacandskeletalmusclesofchjckenembryoswasstudied
byindirectimmunonuorescencemicroscopy・Serialsectionsofembryoswerestainedwithantibodiesspeci−
fictoTNcomponents（TN−T，−Ⅰ，and−C）n・Omadultchickencardiacandskeletalmusc）es．Cardiacmuscle

begantobestainedwithantibodiesraisedagainstcardiacTNcomponentsinembryosafterstagelOof
HamburgerandHamiltonl）（Fig・1）・Itreactedalsowithanti−SkeletalTN−IfromstagelOtohatching．
SkeletalmusclewasstainedwithantibodiesraisedagainstskeletalTNcomponentsafterstage14．Italso
reactedwithanti−CardiacTN−Tand−Cfromstage−4tohatching・Immunoelectronmicroscopyrevealed
thatthenewlysynthesjzedregulatoryproteinsareassembledattheircharacteristicpositionfromtheinitial
phasesofmyofibrillogenesis．TheresultsaresummarizedinTablel．

Fig・1・Sagittalsectionofawholechickenembryoatstage20stainedwithantibodyagainst
CardiacTN−T・Skeletalandcardiacmuscleswerestained・×37．Bar，0．5mm．
−21−

Tablel．DifrtrenliationofTNincardiacandskeletalmusclesofchickenembryos

Stages（Hamburger−Hamilton）ofchickenembryos
TissueAntibody
against＋＋＋

十 一 一 一 一 一 一 十

＋

十

一

＋ ＋

一

＋

十

一

＋

一

＋ ＋ 一 一 一 ± 一 ± ＋

一

＋

一

SkeletalTN−r
SkeletalTN−C

一

CardiacTN−C
SkeletalTN−T

十CardiacTN−I

一

SkeletalTN−C
Skeletalmuscle CardiacTN−T＋

SkeletalTN−I＋ ＋ ＋ 一 十 一 ＋ 一 ＋ ＋＋CardiacTN−C
SkeletalTN−T＋ ＋ ＋ 一 十 一 ＋ 一 十 ＋＋

Cardiacmuscle CardiacTN−T
CardiacTN−I

8
10
14
20
45
（26−29hr）（33−38hr）（50−53hr）（70−72hr）19−20days）

ThepresentimmunonuorescencemiCroscoplCStudydemonstratedthatTNcomponentsincardiac
musclebegintoappearataboutstagelO・Itisatthisstageinchickenembryosthattheheartbeginsto
beat2）andthatmyofibrillarstructuresbegintobeobservedbyelectronmicroscopyofthemyocardium3）・
Thus，thefirstappearanceofTNcomponentscorrespondswellwiththetimeofinitiationoffunctional
andstructuralintegrityformationincardiacmuscle・TheobservationthatTNcomponentsofcardiac
musclearesynthesizedmuchearlierthanthoseofskeletalmuscleprobablyreflectsthefactthattheheart
beginStofunctionatanearlystagewhentheskeletalmusculatureisstillimmature・
Duringembryonicdevelopment，CardiacmusclesynthesizesTN−Tand−Cpossessingcardiac−tyPeantigenicity，andTN−Iwithantigenicdeterminantssimilartothosepresentincardiacaswellasinskeletal
muscles．EmbryonicskeletalmusclesynthesizesTN−Ipossessingantigenicityfbrskeletalmuscle・and
TN−Tand−Cwhichshareantlgenicitiesfbrbothcardiacandskeletalmuscles・Thus，jnthedevelopment
ofcardiacandskeletalmuscle，itisapparentthataprocessoccursinwhichthefiberchangesitsgenomic
programming：itceasessynthesisoftheTNcomponentswhichareimmunologlCallyindistinguishable
fromeachother，andsynthesizesonlytissuetype−SpeCificproteinsaflerhatching・Theproblemofwhether
thesedevelopmentalchangesinthegeneexpressionfbrTNcomponentsareprogrammedendogenously。rareduet。theinfluenceofanexogenousfactor（S）requiresfurtherinvestigation・However，ifanex0−
genousfactor（S）doesa能ctthemuscles，theroleofinnervatingnervescannotberuledout，Sinceithas
beenknownthatinnervationleadstothedifferentiationofindividualmusclefiberstospecifictypes4）・
1）Hamburger，V．andMamilton，H・L・：J・Morphol・，88，49−92，1951・
2）DeHaan，R．L・：MorphogenesisoftheVertebrateHeart・h：Organogenesis（DeHaan，R・LandUrsprung，IL・eds．），pp．377−419，Holt，Reinhart＆Winston，NewYork，1965・
3）Manasek，F．J．：dm．J．C。r（肋7．，25，149−168，1970・
4）Buller，A．，Eccles，J．，andEccles，R・：J・PjlySiol・，150，417−139，1960・−22−

肋∫C〝ねrpp／rq如γGro叩IMMUNOELECTRONMICROSCOPICOBSERVATIONSOFTHELOCAI．IZATIONOF
TROPONINALONGTHETHINFILAMENTOFDYSTROPHICCHICXENBREASTMUSCI．E
IwaoOhtsuki
DepartmentofPharmacoIogy，FacultyofMedicine，KyushuUniversity
Previousimmunoelectronmicroscopyshowedthattroponindistributesalongthewholelengthof
thethinfilamentofchickenskeletalrnuscleatregularintervalsof38nm・Thenumberofanti−trOpOnin
Striationsfbrmedalongthethin別amentbundlewasalwaysfbundtobetwenty−fbur．
RecentlyanimmunocytologicalprocedurecalledthesaponinmethodHwasdevelopedthathasmade

itpossibletoobservedevelopingorimmaturemyofibrilsinsituwithoutdamaglngtheoverallstructure
Ofmusclefibers・Applicationofthistechnlquetotroponinlocalizationinchickembryonlcbreastmuscle
revealedthatthenumberoftroponinStriationsalongthethinfi1amentwasdefinitelymorethanthatin
adultthinfilament・Thisfindingstronglysuggeststhatthefinalmaturationofthethinfilamentconsists
Ofashorteningofthelongerfi1amentsintoaconstant】engthanerhatching．
Inthepresentinvestigation・Weundertooktofbllowtheprocessofthinfilamentshorteningindys−
trophic（413）andintact（412）Chickenbreastmusclesaflerhatching．
Thethinbundleswereseparatedfrombreastmusclelto5weeksafterhatching．Thesefiberswere
eitheruseddirectlyorimmersedinglyceroIsolutiontomakeglycerjnatedmuscles．Twomethodswere
usedfbranti−trOpOninstaining・Thefirstwasasaponintechniqueinwhichmusclefibersweretreatedwith
lO￣4g／mlsaponinandthenwithanti−trOpOninantibody，inarelaxingsolutioncontainingO．lMKCl，10

mMPhosphate（pH7・0），10mMMgCl2，5mMATP，andO・5mMGEDTA・Then，thespecimenswere鮎ed
andembeddedinEpoxyresinfbrthinSectionlng・Inthesecondmethod，thethinfi1amentsweredissociated
丘omthickBlamentsbyhomogenizationinthere】axlngSOlution，treatedwithantibodysolution，andstain−
ednegativelywithuranylacetate・Thesespecimenswereobservedunderanelectronmicroscopewithan
accelerationvoltageoflOOkV．
Figurelshowsalongitudinalsectionofthinmamentregionsinmyofibrilsofdystrophicchicke亡
breastmuscle7daysaflerhatching・Anti−trOpOninformedtwenty一Rvetotwenty−eightstriationsalong

thethinfilamentbundle・NothinAlamentswithtwenty−fburanti−trOpOninstriationswereobserved．Thesematuredthinfilamentswithtwenty−fburstriationsfirstappeared4weeksafterhatching，butlongerfila−
mentswithmorethantwenty−fburstriationscouldbeobservedevenat3monthsaflerhatching．
Anti−trOPOninantibodyfbrmedtwenty−fivetothirty−threeperiodicstriationsalongthethinRlaments
inintactchickenbreastmusclelweekafterhatching・butfbrmedtwenty−fburstriationsalongallthinmamentbundles3weeksafterhatchingsofarobserved．
Theabovefindingsclearlyindicatethattheprocessofshorteningofthinfilamentsintoaconstantlengthindystrophicchickenbreastmuscleismuchslowerthaninintactmusclefibers．
1）Ohtsuki・I・，Masaki，T・，Nonomura，Y・，andEbashi，S・：J・Btoche肌，61，817−819，1967．
2）Ohtsuki，Ⅰ・：エ瓜，Cカビ肌，75，755−765，1974．
3）Ohtsuki，I・，Manzi，R・M・，Palade，G・E・，andJamieson，J・D・：Biol・Cbll，31，119−126，1978．
4）Ohtsuki，Ⅰ∴上月わCお肌，85，1377−1378，1979．

−23−≡二・二・三・業苦章慧歪

Fig．1．Anti−trOPOninstainingof thinmamentsof dystrophic chicken breast muscle7daysafter hatching・
×40，000．Anti−trOPOninformedregulartransversestriationsonthethinfilamentswhichextendedinbothdirections

fromtheZ−linesituatedatthecenterofeachfigure．Numbersonbothsidesofthefigureindicatethenumberofanti−trOpOninstriationsalongeachthin創amentbundle・ー24−

肋C〟brpγ∫什qpわ′Gr（）叩DEVELOPMENTALSTUDIESOFTHEMEMBRANEPROPERTIESOFSTRIATED
MUSCI．EFIBERS
KunitaroTakahashi
LaboratoryofNeurobiology，TnstituteofBrainResearch，MedicalSchool，TokyoUniversity，Tokyo
CalCiumactionpotentialshavebeenobservedinvariousexcitablemembranes，SuChasincrustacean
Striatedmuscles，heartmuscles，SmOOthmuscles，neurOendocrinecells，andespecia71yaxonterminalswhere
neurotransmittershavebeenconsideredtobereleasedlJ・Inaddition，theeggcellmembranesfromwhere
Ce11ulardi能rentiationstartsshowacalciumactionpotentialinmanyspecies，SuChasascidians，eChynoderms，
andmammals2）．

Ontheotherhand，intracellularionizedcalciumfacilitatesmusclecontraction3），tranSmitterrelease，
eggce11ftrtilizationchanges4），andcellcleavage・Ithasbeenreportedthatthecalciumionophorewhich
enhancesthepermeabilityofthemembranetocalciumhasconsiderableeffectsuponvariouskindsof
CytOkineticphenomena・ItisreasonabletosuspectthattheCa−Channels，thenativecalciumionophores，
areessentialconstituentsofmusclecellmembranewhichhasaspecializedcytokineticapparatus．Since
especiaHyinthestriatedmusclecellofascidiantadpolelarvathemyofibrilsarelocatedjustbeneaththe

plasmamembrane，thecalciumin伽xthroughCa−Channelsfromthesurfacemayplayanimportantrole
inthecontroIofthecontractileapparatus・Thus，thepresentstudyaimstoanalyzethedifftrentiation

PrOCeSSOfthestriatedmuscleoftheascidiantadpolelarvawithspecialregardtotheCacurrentthroughCa−Channelsinthemembrane．

1・Methods：Adult ascidianHalocynfhiaroretziwhichhadmaturedeggs andspermwerekept
inacoldaquariumat40C・Theeggsandspermwereobtainedffomthespawninganimalsafter24hrin
awarmbathat120C・TheeggswereftrtilizedbyspermfromheteronymOuSanimalS．Thedevelopmental
timewasmeasuredfromthisftrtilizationperiodinanincubationbathataconstanttemperatureoflOOC．
Atthe8−Or16−Cellstage，theembryosweretransftrredtoanotherbathcontaininglpg／mlcytochalasin
Binordertoarresttheirce11cleavageandculturedfurtheruntilthecontrolintacttadpolelarvaehatched．
AflerremovingthechorionmembranebydigestionwithlOmg／mlpronasethecleavage−arreStedembryos
Werekeptinahigh−PrOteinousexternalsolutionandwerepenetratedwithtwomicroelectrodes．Theex＿

perimentswerecarriedoutwitheitherconstantcurrentstimulationorvoltage−Clamp．Theexternalsolution
WaSuSuallylOOmMSrC12，400mMNaCl，10mMKCl，and5mMPIPES−Na（pH7．0）．Whennecessary，SOdiumwasreplacedwithcholineorstrontiumwithcalcium．Thereasonfortheuseofstr。ntiums。Iuti。nWaStOincreasethecurrentthroughtheCa−Channels．
2．Results：
1）DevekpmentqrCbcILrrefltincleavLqe−arreSted16−Ce／lembwos．IthasbeenreportedthatNa
andCaactionpotentialsdevelopincleavage−arreStedembryosasintheexcitablecellsintheintacttadpole
larvae5）・Sincetheblastomeresin16−Cellembryoswerelargeenoughtobepenetratedwithtwomicro−
electrodes，theCacurrentwasanalyzedbythevoltage−Clamptechniqueatallstagesofdevelopment．Unti1
50hrafterftrtilizationtheintercellularcommunicationbetweentheblastomereswassotightthatthe
embryobehavedasacellandthesumofmembranecurrentswasobservedontheembryonicsurface．The
totalcalciumcurrentinanembryowasinitiallydecreasedunti120hrofdevelopmentandwasenhanced
againafler35hr・Afler50hrmostoftheintercellularcommunicationdisappearedandthemembrane
CurrentSinindividualblastomerescouldbeanalyzed・Thepresumptivefateofeachblastomereinthe16−−25−

cellembryohasbeendescribedclassical1yandindividualblastomereshavebeennamed・Theenhancement
oftheCacurrentappearedinallobservedB5−1blastomereswhichcontainedthepresumptlVemuSClereglOnandwerelocatedinthevegetalhemisphere・ThisCacurrentwasalwaysfollowedbyadelayedKcurrent・IntheblastomereaCaspikewhichwasidenticaltothatinthemusclecellsofintacttadpolelarvaewas
evokedbyconstantcurrentstimulation（muscletype）．However，theB5−2blastomerewhichalsocontained
thepresumptivemusclereg10nSdidnotshowanycalciumcurrent・BlastomeresA5−1andA5−2didnot
showanyvoltage−dependentinwardcurrents（non−eXCitabletype）・Thegroupofsmallblastomeres，b573
andbH，intheanimalhemisphereshowedapureCacurrentwithoutafollowingdelayedKcurrent・TheseblastomereshavebeenassignedtothepresumptlVeePidermalreg10nSandtheyproducedextremelylong−lastingactionpotentialsunderconstantcurrentstimulation（epidermaltype）・Anothergroupof
smallblastomeres，a5＿3anda5＿4，Showedin60％ofcasesaNacurrentfbllowedbyadelayedKcurrent（neurontype）andtheyhavebeenknownasthepresumptivebrainregionoftadpolelarvae・WhentheseblastomeresdidnotshowtheNacurrent，theybecametheepidermaltype・
2）DevelqpmentdCbc〟rrentincleavage−arreSted8−CellembfyOS・Evenincleavage−arreSted8−Cellembryos，thelargeblastomereintheposteriorportionofthevegetalhemisphere・B4−1，ShowedaCacurrent
followedbyadelayedKcurrentin30％ofthecasesobserved・ThesameblastomereB4−1，WaSelectrical1ycoupledwiththegroupofsmallblastomeresintheanimalhemisphereanddifferentiatedintotheepidermal
type40％ofthetime．Sometimes，theblastomerewasisolatedasinthecasefbrthemuscletype，butdid
notshowanyexcitability・InafbwcasesasmallbIastomere，a4−1，WaSisolatedandshowedneuron−typedifferentiation．Asdescribedabove，blastomereB4−lShowedthreetypesofdifferentiation：muSCle type，
epidermaltype，andnon−eXCitabletype・TheacetylcholinesterasewasstainedbytheKarnowskymethod
andthet。talsurfaceareaoftheblastomerewasfbundtoincrease5−foldwithmeasurementofthetotal
capacitanceonlyinthecaseofthemuscletype・Itwassuggestedthatthedifferentiationintothemuscle
typeinducedinfbldingorexpansionofthesurfaceareaoftheblastomere・Theepiderma1−typedifferentia−
tionalsoinducedanincreaseinthetotalcapacitanceatthepenetratedblastomere・However，thiswasnot
causedbyrealexpansionofthesurfaceareabutbyelectricalcouplingwiththegroupofsma11blastomeres
intheanimalhemisphere．
Discussiom；The

B5＿1blastomereincleavage−arreSted16−Cellembryos

orB4−1in8−Cellembryos

showedthemuscle−typedifferentiationwithaCaspikeandacetylcholinesteraseactivlty・Intheseblast0−
meres，Preliminaryobservationsunderanelectronmicroscopeshowedmyofibrilsunderneaththeplasma
membraneandanaccumulationofthemitochondria，Whichischaracteristicofthemuscle cellsinintact
tadpolelarvae・Thus，theentirelargeblastomereseemedtohaveonlymusclecharacteristicsanddidnot

showanymosaicpropertiesmixedwithothercellulartypes・SincetheB5−1blastomerecontainsthepre−
sumptiveendodermalreglOnaSWell，itisconcludedthattheblastomereselectsonlyonecellulartypefrom
morethantwopossibilities・Durther，inthecaseof8−Cellembryos，theB4−1blastomereactuallychoseeitheroneofthreecellulartypesequallyandtheexpressionofthedifferentiationseemedtobealwaysof
OnetyPeinoneblastomere．
1）Reuter，H∴frqg．βわ〆yJ．〟oJ．βわJリ25，3−13，1973・
2）Okamoto，HリTakahashi，K．，andYamashita，N・：J・Physiol・，267，465−195，1977・

3）Ebashi，S．andEndo，M．：Prog．Biqphys・Mol・Biol・，18，123−183，1968・
4）Epel，D∴C〟r．物．加γ．βわLl2，185−249，1978・
5）Takahashi，K．andYoshii，M．：J．P毎sEol．，1981，inpress・−26−

肋∫CJ血rpy∫Jr呼わ′Gro叩SHORTENINGVELOCrrYOFSKINNEDMUSCLEFIBERSFROMDYSTROPHICCHICKENMUSCLES

MakotoEndo，ShinobuYagi，YasunoriKakuta，TohruIshizuka，andKeisukeHoriuchi
DepartmentofPharmacoIogy，TohokuUniversitySchooIofMedicine，Sendai
Usinganti−trOpOninantibody，Ohtsukietal・1）Lbundthatthinfilamentsinbreastmusclesfromdystroph−icchickensarelongerthanthosefromnormalchickens，andinthisrespect，reSemblethosefromembry−
Onicmuscles・Ontheotherhand，We2）haveshownthatintheregionofsarcomerelengthsbelowthepolnt
atwhichthinfilamentsfrombothsidesmeeteachotherinthemiddleofA−bands，0nlyverysmalltension
COuldbeproducedbyaloweractivatingrangeoffreeCa−ionconcentrationsascomparedwiththatat
longersarcomerelengths，incontrasttothefactthatthetensionproducedbyahigherorsaturatingrange
OffreeCa−ionconcentrationsatsuchshortsarcomerelengthsisnotmuchdifferentfromthatatlonger
SarCOmerelengths・Aphenomenonprobablyrelatedtothisisthefact2）thatthemaximumshortening
VelocItyundernoexternaHoad，VmaⅩ，OfmusclefibersactivatedbyaloweractivatingrangeoffreeCa−

ionconcentrations，againunlikethatbyhigherfteeCa−ionconcentrations，becomesverysma11atsuch
Shortsarcomerelengths・AlthoughitisnotyetcertainataIlthatthismechanicalbehavioratshortsar−
COmerelengthsisreallyduetomeetlngandoverlapofthinfilamentsfrombothsides，itisconsideredquitepossiblethatfibersfromdystrophicmuscIeswithlongerthinGlamentsmightstartshowingsuchmechanicalbehaviOratsarcomerelengthslongerthanthoseatwhichnormalfibersstarttoshowit・Therefbre，inthis
StudytheVmaxofnormalanddystrophicfibershasbeenmeasuredatvarioussarcomerelengths．
Posteriorlatissimusdorsimuscleswereisolatedfromadult（24months）dystrophic（line413）andnormal（line412）chickensandimmersedinarelaxingsoIutioncontaininglOOFLg／mIsaponintoskinthe
fibers3）・Singlefiberswereisolatedltomthemusclesintherelaxingsolution，andabout3−mmJongparts
Ofthefiberswithunifbrmsarcomerelengthswereused・Oneendofthepartswasconnectedtoastrain−
gaugetransducerandtheothertotheleverofamovlng−COilmiCroammeter・Byftedingasufncientmagni−

tudeofcurrenttothemicroammeterinadirectiontostretchthefiberbutpreventingthelever丘ommovlng
withastop，anisometricconditionwasobtained・Vmax，OrStrictlyspeakingVo，WaSmeaSuredinthefb1−
10wingway・WhentheisometrictensionreachedasteadylevelafteranapplicationofCa−COntainingsolu−
tion，thecurrentftdtothemicroammeterwassuddenlyswitchedtothatofthereversedirection，Whichmadethefibershortenquicklytoapre丘xedlevel・Sincethemovementoftheleverofthemicroammeter

WaSrnuChfhsterthanVmax，fibersslackenedandmyofibrilsthenstartedshorteningwiththespeedof
Vmax，andaftertakinguptheslack，StartedredevelopJngtenSion．Thetimefromreleaseuntilthestartof
redevelopmentoftensionwasusedasanindicatorofVmaX・Experimentswereperfbrmedat200C．
Anactivatingsolutioncontaining3×10−6MfreeCa−ionconcentrationwasappliedtoskinnedfibers
fromnormalanddystrophicmuscles，andquick−releaseexperimentswereperfbrmedasdescribedabove．
ThetimefbrtheBbertotakeupthesIackproducedbyquickshortenlngOf O・9FLmperSarCOmereWaS

measuredatthreediffbrentsarcomerelengths・TheresultsareshowninTablel・Asshowninthetable，
thetimefbrshorteningofnormalfibersbecamelongeratsarcomererangesfrom2・9to2・OFLmCOmparedtothetimerequiredbylongersarcomeres・Indystrophicfibers，Whilethetimefbrshorteningofsarcomeres
ffom3・3to2・4FEmWaSnOtSignificantIydifftrentfromthatofnormalfibers，thatftom3・1to2・2FEmWaS
definitelylongerthanthatofnormalfibersatthesameJengthandthatofdystrophicfibersfrom3．3to
2・4FLm・ThefiguresinTablelareacollectionofdataofonlythefirstcontractionofeachfibertoavoid
anyinnuenceofdeteriOrationduringthecourseofexperiments・However，Whentheexperimentswere−27−

Tablel．TimerequiredforO．9FLmShortenlngperSarCOmere
Normal（msec）
From3．3to2．4Jm

Dystrophy（msec）

127±15（〝＝9）153±11（〃＝7）227±12

From3．1to2．2JJm

128±8
（〃＝4）

（〃＝3）

From2．9to2・OJ

163±39343±69

（柁＝3）

（〝＝4）

m

repeatedonthesame丘bersatdifferentsarcomeres，qualitativelysimilarresultswereobtained・

Theresultsobtainedhereareasweexpectedoriginally．However，1tisnowunlikelythatthepresent
resultsareduetolongerthinfilamentsindystrophicfibers，SinceOhtsuki（personalcommunication）has
shownthatthefrequencyofencounteringlonger−than−nOrmalthinfilamentsisrathersmallinadultdys−
trophicmusclesoftheageweusedhere・OnepossibilityisthattheCasensitivityofdystrophicfibersmight
beslightlylessthanthatofnormalfibers・Thisandotherpossibilities，aSWe11astheproblemofwhyVmaⅩ

atloweractivatingfreeCa−ionconcentrationssharplydecreasesatshortsarcomeres，areCurrentlyunderinvestlgation．

1）Ohtsuki，I．etal．：CurrentResearchinMuscularDystrophy，Japan，p・40，1977・
2）Endo，M．：Seibuts〟b〟tSuri，15，247−259，1975（inJapanese）・
3）Endo，M．andIino，M．：J．MuscleRes．CellMotiIり1，89−100，1980・−28−

JW〟∫川hrかγ∫／rqpわ・GJ・0岬

STUDYOFTHEELECTROPHYSIOLOGICAL，MORPHOLOGICAL，ANDMETABOLICASPECTSOFDYSTROPIIICCHICKMUSCLEMEMBRANEINCULTURE

KenHotta，NobukoTomita，andHiroyukiSugiyama＊

DepartmentofPhysiology，NagoyaCityUniversity，MedicalSchool，Nagoya＊DivisionofBiologicalMembranes，NationalInstituteforPhysiologicalScience，OkazakiWereportedpreviously

that

the

membranepotentialofdystrophic

musclecel1s

wasslgniBcar］tly

lowerthanthatofnormalcontroIsl・2Inthisstudy，thedevelopmentofspecifiCfeaturesofdystrophic
musclewasfbllowedbymeansoftissueculturefromtheveryearlystagesofgenesis．
Thematerialusedwasthemyotomesobtainedfrom chickembryos（N．H．413）which had beenin−
Cubatedat370Cfor6days・Themyoblastsattachedtothemyotomesfusedandgrewrapidly，fbrming
myotubuleswithinl or2daysafterexplantation，aSShowninFig・1・Striations begantoappearinside
Ofthemyotubulesinaboutlweekandtheywerecompletedasmusclefibersinabout2weeks，cultivation．
However，therewasnonoticeabledifferencebetweendystrophicandnormalmusclefibersasfaraslight
miCroscopicobservationswereconcerned．
1）Membranepotential：Microelectrodes could penetrateinto the tubules2days after explanta−tion・AsShowninFig・2，themembranepotentialofthecellbecamedeeperanddeeper from−20mV（2days）t0−50mV（8days）andfurthertoabout−70mVafter15days．Thecellsofthedystrophicstrain（413）alwaysexhibitedalower membranepotential：the difftrencewassmallbut statisticallysignificant
fromthatofcontroIs（412）・Therewasnodi斤もrencebetweenthecontrolandtheBroilerstrain（Fig．2）．
2）Ultraslr〟Cture：Soonafterthemyotubuleswereformed，manyfilamentousstructuresappeared

insidethecells（Fig．3）．Thesestructuresgrewintomyofibrilsasthedaysincultureproceeded．However，
thestructuralintegrltyOfcultivatedcellswasweakcomparlngwiththatofcellsgrowninvivo．Theultra−
StruCturalcharacteristicsofdystrophicmusclewhichwereobservedinadultmuscle3）couldnotbeseen．
jノ ルreJ〟ろoJ由椚q／αCe−／／J．1イ（・／J（，／／WJ・tでll／）J（け り（ゾトR）：Breast muscle cells

Obtainedh■OmChickembryos

after4daysinculturewere
used for this experiment．
3H−labeled Erab−tOXin

b，a

SpeCific blocker of Ach−R，
wasabletobindmoretothe
normal muscle membranethan to dystrophic muscle
membrane．This means thereceptor contentislessin
dystrophic muscle．
metabolic

rate

of

The

Ach＿R

measuredbytheappearance
Of metabolic products of
Erab−tOXin b was higherin
dystrophic muscle although

Fig・1・Phasecontrastmicroscopicobservationofamyotome，1dayafter
explantation（N．H．412，×200）．
−29−

this resultwasapreliminary
10

4ノかねC〟∫∫わ〃J The

resultsshownhereconfirmed
thatthemembranepotential
Of dystrophic muscle cells
was lower than that oftion．The

turnoverofmem＿

brane proteins as reflectedbythemetabolismofAch−R，
alsoseemstobedifferentin

0Stage Of myotubule forma−

一再コuヱOd茎2qEむ≡

normal controIs from thedystrophic musclein earlyStageSOfdevelopment．These
facts mayindicate that adeftct in the membrane
SyStem eXists

indystrophic

muscle ftomits genesis
although morphologlCallyit
appearsnodifferent．

10

15

Daysin culture

Fig．2．DevelopmentofmembranepotentialincuIture．［＝］：N．H．412（control）；−：N．H．413（dystrophy）；J≡巨broiler（control）．
Numbersindicatethenumbers ofmeasurements．＊Pく0．05．

Fig．3．Electron micrograph offilamentous structures appearinginside myotubulesin a3−day−Cultured sample（N．H．412，×44，000）．

］）Oba，T．，Kanie，R．，Watari，N．，andHotta，K．：Eh）．Ne〟rOl．，62，214−229，1978．

2）Hotta，K．，Ojika，Y．，andMurase，M．：CurrentResearchinMuscularDystrophy，PP．17L18，1979．
．1）Holta，K．，Oba，T．，Koshita，M．，Nishihara，M．，andOjika，Y．：Ann．ReporI，53−58，1979．

−30【

肋∫CJ血rqy∫什叩カyGrO岬

PROPERTIESOFMUSCI．EFIBERANDNEURO−MUSCULARTRANSMISSIONINANTHRACENE−9−CARBOXYI．ICACID−INDUCEDMYOTONIA
HirosiKuriyama，SusumuKorenaga，Ken−ichiroOda，andYushiIto
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity，Fukuoka
Controversyexistsastotheroleofnervousfactorsintheinductionofexperimentalmyotonia．Caccia
andassociatesl）reportedthat theinitiation ofmyotoniawith20，25−diazacholesterol did not occurindenervatedskeletalmuscle；Eyzaguirreeta／・2）demonstratedthegenerationofmyotoniabyapplication
Of2，4−DtomuscledenervatedfbrlOdays；however，Iyeretal・3）andEbersteinandGoodgold4，claimed
thattheseverltyOfexperimentalmyotoniaisdependentontimeaflerdenervationasmyotoniacouldnot
beinducedby2，4−DlOdaysaflerdenervation．
Weattemptedtoclarifywhetherneuralfactorsinfluencethegenerationofmyotoniainducedbyan−
thracene，uSingthemiCroelectrodeandelectromyogrammethods．
YoungadultWistarKingratsofeithersexweregivenanthraceneintraperitoneally（250mgperday／kg）br2−3days．
Myotoniawasalsoinducedbyaddinganthracenetothemusclechamberinconcentrationsranging
betweenlO￣2tolOT6M・Theneuromuscularpreparationsoftherathemidiaphragmorextensordigitorum

longus（EDL）wereusedintheseinvltrostudies．
SpontaneousdischargesrecordedbyEMGproceduresinvivocouId beclassifiedintothreetypes，
i・e・，（i）regularburstsoftrainsofrepetitivedischargesalternatingwithsilentperiodsordischargesatvery
lowffequency；（ii）waxingandwaningdischargesathigh丘equency；and（iii）continuousandregulardis−
Chargefrequencies・Thedischargepatternsmainlyappearedinthepresentexperimentsastype（i）or（ii）．
Theamplitudeofovershootandthemaximumrateofriseoftheactionpotentialdeclinedconcomitantly，
inproportiontothedepolarjzationofthemembraneduringthelaststageofburstdischarges，andabor−
tiveactionpotentialswereobserved・Inthemyotonicanimals，burstdischargeswerealsoinducedbydirect
musclestimulation・Myotonicactivitylastingfbrftwhundredmsecwastriggeredaftersciaticnervestimu−
lationwithabriefrectangularpuIseduringthesilentperiodbetweenthetraindischarges・Thisrepetitive
firingwasnotobservedinthecontrolrats．
Toobservetheeffbctsofnervousfactorsonburstdischarges，neurOmuSCulartransmissionwasblocked
bysurgiCalsectioningofthenerveoranintravenouslnJeCtionofcurare．Neitheroftheseapproacheshad
anyeffectonthemyotonicburstdischarges・However，aftercurarization，theirregularandintermittent
dischargeswhichoccurredbetweenthemainburstdischargeswereblocked．
Therestingmembranepotentialremainedunafrected

aftertreatmentwithanthraceneinbothdener＿

Vatedandcurarizedpreparations・Afterapplicationofanthracene，SpOntaneOuSSpikedischargeswere
recordedfromboththediaphragmandEDL・Full−amplitudeactionpotentialswithafrequencyofl0−30

HzonenappearedjustaRerinsertionofthemiCroelectrodeandthesespikessubsequentlydecreasedin
丘equencyandceasedaflerseveralsec．
Tablelshowsthevariouspassivemembraneparametersmeasuredfromcontrolandmyotonicdia−
phragmandEDLmuscles・Inbothmuscles，theinputresistance，SpeCificmembraneresistance，andlength
andtimeconstantsofthemembranewereincreasedduringtheapplicationofanthracene．However，thepassiveparameters measuredonspecifiCinternalresistance or specificmembranecapacitanceand thefiberradiusremainedthesameinbothcasesofthediaphragmorEDLinnormalandmyotonicprepara−tions・ThismeanSthattheactionofanthraceneappearsmainlyonthemembraneresistanceofmuscle−31−

Tablel．PassivemembraneparametersmeasuredfromthediaphragmandEDLinthepresenceorabsenceof
anthracene（10￣4M）

2232
0±
0±
0±
0
±
U475
1111

cm（FLF）

800U
05
1
2
±±±±
80
23
42
4
1
3333

T（msec）Rm（L2cm2）Ri（L2cm）
00
02
0300
′
3
2
1
7
±±±±
21
つ2
︼つ
00
7
0
03
8﹂
1410 138′hV3
0001
±±±±
7130
15つ︼5

p（FLm）

4′b72
4±
4±
3±
3
±
5232
く1
J2
72
43
1

l（mm）

70
90
′1
Oqノ
0
0000
±±±±
00
7′
0O
／q
7ノ
5
0101

001
02
40ノ
4
7
00±
0±
0
±
±
4q
2′
52Uq′
2
l101

0ノll1

CAIhn
CAnIh

Dia註EDLn Rin（ML2）

CandAnth：COntrOlandanthracene（10，4M），reSpeCtively；Rin：inputresistanceofmembrane；l：lengthconstant；p：radiusofmusclefiber；T：timeconstantofmembrane；Rm：SpeCificmembraneresistance；Ri：longitudinalmyo−
plasmicresistance（internalresistance）；cm：SPeCificmembraneCapaCitance．
負bers．

Miniatureend−PlatepotentialS（m．e．p．p．S）wererecordedbefbreandduringtreatmentwithanthracene
fromthesameend−Plateineachexperiment．With application ofanthracene，the mean amplitude ofm．e．p．p．swasincreased，thedurationwasprolonged，andthemeanamplitudewasfurtherincreasedby
theadditionofprostigmine．Withtheadditionofcurare，theamplitudeofm．e．p．p．wasmarkedlyreduced．
Theseresultsindicatethatthepotentiatingactionofanthraceneontheamplitudeofm．e．p．p．sisnotdue
toinhibitortyefftctsoncholinesteraseactivity．
Afterapplicationofanthracene，thebell−Shapeddistributionofm．e．p．p．Sintheamplitudehistogram
shiftedtotherightalongthehorizontalline，and

compositem．e．p．p．S

oftheamplitudeoverlOmVwere

recorded．Asthethresholddepolarizationrequiredfbrthegenerationoftheactionpotentialwasdecreased
aftertheapplicationofanthracene，thecompositem．e．p．p．S

trlggeredtheactionpotentialofthemuscle

membrane．ThehumpobservedduringtheriSingphaseofthespontaneousactionpotentialwasprobably
generatedbythe

compositem．e．p．p．S

duetoar）increaseintheamplitudeofm．e．p．p．sinthepresence

ofanthracene．
Toelucidatetheroleofthenervoussystemonmyotonicdischargesinthepresenceofanthracene，eX−
perimentswereperfbrmed using denervatedmuscles・Ratsweresurgically denervated by excision ofa
l0−mmSegmentOftherightsciaticnerve，JuStPrOXimaltothebifurcationofthetibialandcommonper−
onealIlerVeS．Thetreatedanimalswereseparatedintothreegroups，aCCOrdingtothedurationofthede−

nervation．Onegroupwasgiven20r3daysintravenousadministrationsofanthracene5to7daysafter
denervation，andthesecondgroupwasgivensimi1arinjections20to30daysafterdenervation・ThethirdgroupservedasthecontroIs（5to30daysafterdenervation）．AllofthedenervatedanimalsshowedclinicalslgnSOfstiffnessoraprominentresponse，tOmeChanicalstimulationofthemuscle・Aftertheanthracene
treatment，remarkableincreasesinthefrequencyofspontaneouselectricalactivities wereobserved・The
dischargeswerecontinuousandregular−tyPeWithahighftequency，butdive−bomberpatternswithapre−
potentialwerenotobserved．Themicroelectrodestudiesdonein vivoalsodemonstratedthegeneration

Ofactionpotentialswitharegularamplitudeandhighfrequency・
Thepresentresultssupporttheconceptthatchemical1y−inducedmyotoniaismainlyduetoaltera−tionsoftheelectriCalpropertiesofthemusclemembrane．
1）Caccia，M．R．：Exp．NeLLrOl．，56，628，1977・
2）Eyzaguirre，C．，Folk，B．P．，Zierler，K．L．，andLilienthal，J．LリJr・：Am・JIPhysiolリ155，69，1948・
3）Iyer，V．，Whiting，M．B．AリandFenichel，G．：Ne〟rOky，27，73，1977・

4）Eberstein，A．，andGoodgold，J．：肋sckNerve，2，364，1979．

−32−

肋∫C〟血r勒∫／rqpわ′Gr（）岬MECHANISMOFTHEINCREASEINPROTEINSYNTHETICACTIVITYINCHICXENDYSTROPHICMUSCLE
TomohMasaki＊，＊＊andAkiraYoshikawa＊＊＊InstituteofBasicMedicalSciences，TheUniversityofTsukuba＋＊SectionofContractileProteins，TokyoMetropoIitanInstituteofMedicalScience

Oneofthemoststrikingfbaturesofdystrophicmuscleisthedecayanddisappearanceofmusclecells
aftertheonsetofthedisease．Thisphenomenonisessentiallytheresultofthebalancebetweentherates
Ofsynthesisanddegradation・Therefbre，itisinterestingtoknowwhichisthemoreimportantreason，
proteinsynthesisordegradation，CauSingthiscatastrophetothemuscle．Toclarifythisproblem，theprotein
Syntheticactivityinchickendystrophicmuscleandthatinthenormalmusclewerecompared．
Dystrophicchickens（NewHampshire，line413）andnormalchickens（1ine412）wereused．Poly−ribosomesandthe160，（X旧×gSupernatantWerePreParedfrom13−daychickembryosbytheusualmethod．
Theresultsofexperimentsontheincorporationofanimoacidsbythesefactorsshowthatthesynthesis
Ofpolypeptidesbythefhctorsfromdystrophicbreastmuscleincreasedl．35±0．10（average±S．E．，n＝10）timesmorethanthatoffactorsffornnormalmuscle，Whilethatbythecardiacmuscleisalmostthesame．
ThesedataclearlyshowthattheproteinsyntheticactlVltyOfdystrophicmuscleisactivatedeveninan
invitrosystem・Asshowninapreviouspaper，uSlngthecrudeelongationfactor丘actionpreparedn・Om
thedystrophicandnormal160，000×gSupernatantS，itwasshownthatpolyphenylalaninesynthesisffom

phenylalanyltRNAwasactivatedbythisdystrophiccrudeelongationfactor．Thisdifrbrencebetweenthe︵？OT∈dU︶

S20SOqて0−⁝苧Sチリゴー0号pu岩

0

01

2

3
Crude EF1

5

（mg／mlI

Fig・1・Bindingofl14C］−1ysyl−tRNAtopuromycin−treatedribosomesinthepresenceofguanylyl（β，γ−methylene）

diphosphonate（GMP−PCP）andcrudeelongationfactorsfromnormalanddystrophicchickenmuscles4weeks
arterhatching．
−33−

dystrophicandthenormalfactorsrnaybeduetothedi鮎renceintheamountoftheelongationfactors
containedinbothfractionsratherthantoaqualitativedifftrencebetweenthetwofactors・

Toclarifythisd騰rence，thebindingof14C−lysyl−tRNAtopuromycin−treatedribosomeswasmeasured
inthepresenceofthisfactorandguanylyl（β，γ−methylene）−diphosphonate（GMP−PCP）・Onemoleof14C−

lysyl−tRNAbindstolmoleofribosome，andproteinsynthesisdoesnotproceedtofurtherstepsunderthiscondition．AsshowninFig．1，14C−lysineboundtoribosomesincreasesasanincrementoftheelonga−
tionfactorffaction．Abovea2mg／mlconcentrationoftheelongationfactorfraction，thebindingof14C−
lysyl−tRNAtoribosomesincreaseslinearlyasanincrementofthecrudeelongationfactorfraction・The
linesofthedystrophicandthenormalfactorsformparallellines・Therefore，fromthisdata，theamounts
ofboththedystrophicandthenormalproteinwhichshowthesameefftctonthebindingoflysyltRNA
toribosomescanbecompared．Approximatelythreetimesmoreproteinofthenormalfractionisneededtoinducethesameeffectasthatofthedystrophicfraction・Asimilarexperimentcanbedoneusingdiph−theriatoxin．Theresultobtainedbythisexperimentcorrespondsverywelltotheresultsmentionedabove・
Theratiooftheproteinconcentrationofthe160，000×gSupernatantOfthedystrophicmuscletothatof
normalmuscleatvariousdevelopmentalstageswereasfollows：1．48±0・39（n＝4）for13−dayembryos；
0．95±0．11（n＝2）forthe2ndand3rddaysaflerhatching；andO・68±0・04（n＝5）forthe7−8thweekafter
hatching．mhesedatashowthatdystrophicmusclehasadifferentturnovermechanismfromthatofnormal
muscle，andalsosuggestthattheincreaseintheamountofproteinfactornecessaryforproteinsynthesis
indystrophicmuscleisnotduetotheapparentincreaseinmuscleatrophy・
Thesedatastronglysuggestthattheincreaseintheproteinsynthesisinchickendystrophicmuscle

isdueatleastpartlytotheincrementintheamountofelongationfactorlindystrophicmuscle・
Otherdataalsosuggestanincreaseinthesolubleproteinfactors・Kohamaetal・Showedadifferential
increaseintheturnoverrateofeachmyofibrillarproteinindystrophicmusclewhencomparedwiththat
innormalmuscle．Thepresentresultstogetherwiththeirresultssuggestthatindystrophicmusclethe
synthesisofthesomesolubleproteinswereactivated・Thisproblemisnowunderfurtherinvestigation・
1）Weinstock，Ⅰ．M，andMarkiewiez，L：BiocjzEm．Biqphys・Acta，374，197，1974・

2）Morrissey，J．J．andKerwar，S．S．：L的Sci．，20，1091，1977・

3）Ilattelle，B．andFlorini，J．R．：BiochemistYy，12，635，1973・
4）Baieve，D．andFlorini，J．R．：Arch．Biochem．Biqphys・，139，393，1970・
5）Petryskyn，R．andNucholis，D．M・＝BEochim・Biophys・Acta，435，391，1976・

6）Masaki，T．，Yoshikawa，A．，Harada，N．，andShinozaki，H．：Curr．Res．MuscJgLb，St・Jpn・，22，1977・
7）Masaki，T．andYoshikawa，A．：Curr．Res．M〟SCleDyst・々n・，37，1979・
8）Saleem，M．andNichollis，D．M．：Biochem．J．，180，51，1979・
9）Masaki，T．andYoshikawa，A．：ProceedingofSymposiumonMuscularDystrophy，1981，inpress・10）Masaki，T．andYoshikawa，A．：inpreparation・−34−

肋∫C〟ねr勒∫JrqpカyGro岬

THEPROPORTIONOFMONOMERICANDPOLYMERICACTINSINEMBRYONICAND
DYSTROPHICCHICKENSXELETALMUSCLES
TakashiObinata＊，＊＊andNorikoShimizu＊＊DepartmentofBiology，FacultyofScience，ChibaUniversity，Chiba＊＊TheTokyoMetropolitanInstituteofMedicalScience，Tokyo

IndeveloplngmuSClecells，COntraCtileandregulatoryproteinsaresynthesizedandpolymerizedinto
fi1amentousforms，andthenassembledintomyofibrillarstructures・ManylnVeStlgationshavebeendone
tocharacterizethemyofibriHarproteinsinembryonicanddystrophicmuscletissues，andithasbeendem−
OnStratedthatthetypesofmyofibri11arproteinspresentinembryonicl・2｝anddystrophic3，4，skeletalmus−

Clesaresomewhatdi恥rentfromthoseinnormaladulttissues・However，theassemblyofmyofibrillarpro−
teinsinembryonicmusclecellshasscarcelybeeninvestigated．
Inthepresentinvestigation，theamountofmonomericandpolymericactinsinembryonicanddys−
trophicchickenskeletalmuscleswasexaminedsoastounderstandthepolymerizationofnascentactin
moleculesintothefi1amentousfbrmsinthedevelopingmuscleandthedegradationofactinfilamentsin
thedystrophicmuscle．
ThebreastmusclefromchickenembryosofvariousagesorthesuperfiCialpectoralismuscleofdys−
trophic（N・H・，line413）ornormal（N．H．，line412）post−hatchedchickenswasusedasmaterials．Theis。＿

latedmuscletissueswerehomogenizedin3to5vo】umesofphosphate−bufrtredsalinecontainingO．9％
NaCl，10mMPhosphatebufrer（pH7・0），1mMMgC12，0．5mMATP，0．1％TritonX−100，andO．1mMPMSF．
ThehomogenateswerecentrifugedatlOO，000×gfor2hrandtheresultantsupernatantwasusedfbrthe

determinationoftheamountofmonomericactininthetissue・Analiquotofthetotalhomogenatewas
treatedwithO・6MKItodepolymerizealloftheactinfilamentsintomonomericformandusedfbrthe
determinationoftotalactinconcentration・InthepresenceofO・6MKI，bothsyntheticF−aCtinandactin

Fig・1・ChangesinG−aCtinconcentration，tOtalactinconcentration，andthepercentof
G−aCtininthetotalactinpresentinchickenbreastmusCledmingdevelopment．
−35−

Tablel．ComparisonofG−aCtinconcentration，tOtalactinconcentration，andthepercentofG−aCtininthetotal
actinbetweendystrophic（D）andnormal（N）chickens

20−Dayembryo

2−MonthN−Chicken

2−MonthD−Chicken

Totalprotein（mg／gwetweight）

81．3

222

172

G−aCtin（mg／gwetweight）

0．36

0．14

0．43

Totalactin（mg／gwetweight）

10．4

34．8

24．8

G−aCtin／totalactin（％）
Actintypeα（β，γ）

1．73

0．4

4．3
α

α

Totalembryobreastmuscleandsuper丘Cialpectoralischickenmusclewereexamined・Nodifferenceinactintype
wasdetectedbetweennormalanddystrophicchickens，aSeXaminedbyisoelectricfocusingelectrophoresis・
filamentsinmyofibrilsaIaproteinconcentrationrangeofupto2mg／mlwerealmostcompletelydepoly−merizedwithinahalfLhour．Actinwas quantified by the DNaseIinhibition assay method devisedby
Blikstadetal．5）withminormodifications．
FigurelshowsthechangesinG−aCtinconcentration，tOtalactinconcentration・andpercentofG−
actininthetotalactininchickenbreastmuscleduringdevelopment．Theembryonicchickenskeletalmuscle
atearlydevelopmentalagescontainsalargeramountofG−aCtinincomparisonwithwell−developedmuscle・Inthebreastmuscleof10−dayembryos，aboutlmg／mlofG−aCtinwaspresent，andabout40％ofthe
totalactinwaspresentinthemonomericform・Asmuscledevelopmentprogresses，theamountoftotal
actinpresentintheskeletalmusclemarkedlyincreases，WhiletheconcentrationofG−aCtinremarkably
decreases．At2monthsafterhatching，0nlyaboutO．14mg／mlofG−aCtin，WhichconstitutesaboutO・4％
ofthetotalactin，WaSdetectedinthebreastmuscle・ItiswellknownthatifpurifiedG−aCtinisputinphys−iologiCalsaltsolutionitispromptlypolymerizedtoformF−aCtin，andG−aCtincoexistsonlyatalevelofaboutO．05mg／ml．Incomparisonwiththisvalue，theconcentrationofG−aCtininembryonicmuscle
seemstobeextremelyhigh・WhenthemonomericactininthesolublefractionofembryonicmusClewas
purifiedbyion−eXChangecolumnchromatographyandammoniumsulfatefractionation，theactinbecame
polymerizable・Theseobservationssuggestthatpolymerizationofactinissuppressedbysomefactorin
youngembryonicmuscletissue・
Next，theG−aCtinconcentrationandthepercentofG−aCtininthetotalactinpresentindystrophic
chickens2monthsaflerhatchingwerecomparedwiththoseinnormalchickensatthesameage・AsshowninTablel，thebreastmuscleofdystrophicchickenscontainsahigherconcentrationofG−aCtinthanthe
breastmuscleofnormalchickens．Itissuggestedthatthedepressionofactitl−pOlymerizationand／Orthe
stimulationofthedegradationofactinfilamentsmaybestimulated・
1）Masaki，T．andYoshizaki，C・：J・Biochem・，76，123−131，1974・
2）Matsuda，R．，Obinata，T．，andShimada，Y・：Devehu・Bk，［り82，11L19，1981・

3）Takeda，S．andNonomura，Y・：BEomed・Res・，1，176，1980・
4）Obinata，T．，Takan0−Ohmuro，H・，andMatsuda，R・：EEBSLeft・，120，195，1980・
5）Blikstad，I．，Markey，F．，Carlson，L．，Persson，TリandLindberg，U・：Ce〃，15，935，1978・−36−

〃JJj・C〟血rJ）γ∫／r叩わ，Gr（フ叩NEONATALDENERVATIONOFRATSCIATICNERVE
I．THEEFFECTOFDENERVATIONONMYOSINDIFFERENTIATION
HideoSugita，ShoichiIshiura，TakashiMikawa，＊andIkuyaNonaka

DivisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMusCularDisorders，Kodaira，Tokyo＊DepartmentofPharmacology，FacultyofMedicine，UniversityofTokyo，Tokyo

Myosinlightchainsinnormalandneonataldenervatedratmusclewerestudiedtoexaminetheneural
effectonthedifrerentiationofmyosinmolecules．Thoseoffastorslowtwitchmusclewereidentifiedby
Slnglefibergelelectrophoresis．

Tostudytheroleofnervesinthedeterminationoffibertype，WeinvestIgatedtlleefrtctofsciaticde−
nervationinnewbornratsonthepostnataldevelopmentoffibertypeandth〇Changeinmyosinmolecules．

Whentheleftsciaticnervewasseveredatbirth，thelefthindlimbinallratsbecameparalytic．Thediameter
Ofallmusclefiberswasdecreasedexceptforaftwhypertrophiedfibersintheextensordigitorumlongus（EDL）andwecouldnotdetectanyevidenceoffibertypegroupingornormal−Sized負bers．Furthermore，
Whentheperipheralnervesintheatrophiclimbwereexamined，al1myelinatedfibersweresmallindiameter
andpoorlymyelinated，SuggeStingapreviOuscompletedenervationintheproximalportionofthenerve．
Figurel shows theprotein patterns ofdenervatedrat soleus andEDLmuscles．The myofibrillar pro−
teinpatternofadultsoleusfiberbundlesshowedthatmorethan70％ofthefibershaveaslow−typemyOSin
lightchain．Sincetwo−dimensionalgelelectrophoresis ofaslnglefiberin the adult rat soleus and EDL
Showedonlyonetypeoflightchaininonefiber，theheterogeneltyOfLlinlanesland2resultedfrom a
mixtureoftheslowandfastfibersinthebundle．Ontheotherhand，theadultratEDLmusclehasonly
fast−tyPefibersasfarasweobserved（Fig．1，1anes3，4）．TheblurreddissolutionofL2inFig．1wasat−
tributedtothedifferenceintheconcentrationofacrylamide．Llisthoughttobeoneofthemosttype−discriminatingproteins onthegeloverthewiderangeofacrylamideconcentrations（13−16％）．Weob−
SerVedanunexpectedchangeinLlaflerneonataldenervation．Almostallimmaturefibersintheneonatal
SOleusmuscleturnedintothematurefast−tyPebiochemically（Fig．1，lane5）andhistochemically（Fig．
2B）asdescribedbyRubinsteinetal．Anembryoniclightchainwasnotfoundinthesemusclesupontwo−
dimensionalgelelectrophoresis．Ontheotherhand，anunuSualthickbandwhichcorrespondedtoslow−
typeLl（Fig．1，1ane6）wasobservedinthedenervatedEDLmuscle．ThemyosinATPasestainrevealed
thatasmal1numberofintermediatefibersintheEDLmusclewerehypertrophied，Whereastheremalnlng
type−2fiberswerefairlyatrophied（Fig．2D）．Thereforethebandofslow−typeLIwaspossiblyderived
fromthehypertrophiedandhistochemica11yintermediatefiber．Onthebasisofthesefindings，itcanbe
COnCludedthatimmatureneonatalmusclefiberscanbecomethefasttypeafterdenervationwhetherthey
arepresumptivefastorslow．
Neonatalsciaticdenervationcausedadevelopmentalarrestinthesoleusmuscle．Almostallthefibers
begantosynthesizeafast−typemyOSin．Sinceanewlysynthesizedfast−tyPemyOSinhasL31ightchain，thedenervatedsoleusmusclemightnotdedifferentiated，butdifrtrentiateintothefast−tyPe．Fromtheseresults
itisconcludedthatinthesoleusmuscleneonatalsciaticdenervationpreventsthesynthesisofslow−typemyosinlightchainswithoutafrectingthesynthesisoffast−typelightchains．TheresuItssuggestthatthe
Synthesisofslow−typemyOSindependsonaspecifiCmotorinnervationatthisstageofdevelopment．

Ontheotherhand，aftwhypertrophiedfibers（5％ofthetotalmusclefibers）wereobservedinEDL
muscleaflersciaticdenervation．Thesefibers，Whichmayhaveoncebeendestinedtobecomeslowfibers，Showedintermediatepropertiesongelelectrophoresis（Fig．1）andATPasestaining（Fig．2）．Wecannot
explaintheappearanceofsuchfibersonlyinEDLmuscle．Weobservedthatsuchintermediate負bersatr0−
−37−

2料掻マ

∴一﹄亨雷−

！

−−−三

−

−一一

1欝毎ざト
！
、

−−

−→■

−

一−
−

一−

，≒ 」・｛∵㌦
Fig．1．SDS gelelectrophoresis（14％）of denervated rat muscle．Lanesl，2：COntrOI
adult soleusfiber bundle；lanes3，4：COntrOl adult EDLfiber bundle；lane5：
denervatedsoleus，Singlefiber；lane6：denervatedEDL，hypertrophiedsing）e斤ber．

Fig．2・39−day−01drat．Thetype−1（1）and−2（2）fibers aremixed with a type−1fiber
predominancein the controI soleus（A），Whereas alImusclefibers behaved as
type2withnumerousmyotubesiりthesoleus with neonataldenervation（B）・

In the controI EDL（C），apprOXlmately97％of thefibers are type−2fibers．
Note hypertrophicintermediatefiberswith atrophic type−2爺bersintheEDL
afterneonataldenervation（D）．A−D：rOutineATPase．
Phiedafter60daysofneonataldenervation．

Whetherimmaturemusclecellsareprogrammedtobefast orslow，they havethepotentialtosyn−
lheSizebothtypesofmyosin・Wehavedirectlydemonstrated thecoexistenceofbothtypesofmyosinin
denervated EDL musclefiber using single−fiber gelelectrophoresis（Fig．1，1ane6）．The sameresult was
rcportedbyPetteetal．withanexperimentaleffectinducedbye］ectricalstimulation．Therearetwohypoth−

Cn：S SuggeSted byRubinsteinetal．toexplaintheseevents．First，a Changein muscle Bber type would
invoIve atrophy ofonefiberaccompanied by theappearanceofthe other type．Alternatively，amuSCle
HbcI

ilscJrcouIdbechangedfromonetypetotheother．Wefavorthelatterpossibilityonthebasisofthe

resL山HCPOrtCd here．
−38−

肋∫C〟hrかγ∫Jrqp毎Gro岬

CONNECTIN，ANELASTICPROTEINOFMUSCLE
KoscakMaruyama
DepartmentofBiology，Facu】tyofScience，ChibaUniversity，Chiba
Thereare two kinds ofmajorcytoskeleton structuresinvertebrateskeletalmuscle．Oneis theso−
Calledintermediatefi1amentsurroundingtheZdiscandlinkingittransverselytotheneighboringZlinesl｝．
Theotheris theconnectin netlongitudinallylinkingneighboringZlines2）．Theintermediatefilaments
COnSistofdesmln，a55，000−daltonprotein，andtheconnectinnetsconsistofconnectin，Veryhighmolecular
Weightprotein（probablymorethanonemi11iondaltons）．
Thepresentreportisconcernedwiththelocalizationofconnectinwithinamyofibril，andtheefftcts
Ofmuscleproteases．

1）Pr印arationdconnec血：Connectinwaspurifiedfromchickenbreastmuscleaccordingtothe
methodofWangetal．3）．WehavealreadyreportedthatWang，stitinisidenticalwithconnectin4）．Chicken
breastmusclewashomogenizedandwellwashedwith50mMKClcontaining5mMEGTAandlmMNa−
HCO3・In3％polyacrylamidegelelectrophoresis，theconnectinbandwithadoubletwasveryimmovable；itwasonthetopofthegel，aSSeeninFig・1・WhenEGTAwasomittedinthepreparationofmyofibrils，
theconnectinbandwassomewhatdeteriorated（Fig．1A，1），andtheadditionoflmMCaCl2reSultedin
adecreaseintheamountofoneofthedoublets（Fig．1A，2）．Thiswasprobablyduetotheactionofcal−
Cium−aCtivated proteaseduringpreparationofmyofibrils，aSShownbelow・Theconnectinbandslowly
disappearedduetospontaneousbreakdownin1％SDSatroomtemperature．Thus，afterlweek，sstorage，
itcompletelydisappeared（Fig・1B）．Therefbre，COnneCtinShouldbepreparedasquicklyaspossible，and
mustbestoredatOOCeveninthepresenceofO．1％SDS．ThemyofibrilsweredissolvedinSDS，andafter
removalofinsolublecollagen，thesolutionwassubiectedtogelfiItrationusingaBiogelA50−mCOlumn3）．
Connectinwasrecoveredjnthefirstseveralfractjonsimmediatelyfbllowlngtheelutionofvoidvolume．
2）LocalizationqrcoflneCtininmyq斤briLS：Antiserum againstisolatedconnectinwas raisedin arabbitbyinjectlngitwithconnectinfromchickenbreastmuscle．Theimmunoglobinswereobtainedby
amnitychromatography・ConnectinwasmixedwithlO％polyacrylamideandtheresultantgelsus−pen−
SionwaspackedontoacolumnandtheadsorbedproteinswereelutedbyconcentratedMgCl2．Thein−
directimmundm10reSCenCeteChniquewasappliedto revealthelocalizationofconnectininmyofibrils．
ConnectinwasmostconcentratedintheA−IjunctionreglOn，andthemiddlepartoftheAbandandthe
WholeIbandwereweaklystainedwiththeantibodies・TheMlinesandZlineswerenotstainedatall，COntradictorytotheresultsofWangefal．3）．Afterremovalofmyosin，thefreeendregiOnoftheactin創a−mentswasstrongly幻uorescent・FurtherextractionofactinwithO．6MKIresultedintheappearanceof
月uorescentfibersbetweentheZlinesofthesarcomere，buttheZlineswerenotstained・Itappearsthat
theconnectinnetsareinvoIvedinlocatingmyosinfi1amentsatthecenterofasarcomerebyattachingto
bothendsofmyosinfilaments・ThisviewisingoodagreementwithLocker，sgapfilamenttheory5）．AftermyosinandactinaredissoIvedaway，theconnectinnetsareprobablyassociatedwitheachothertoform
bundlesthatcanbeseenunderamicroscope．
3）聯ctdmuscleproteasesonco177leCtinh

itu：Connectinnetsinmyofibrilsareverysensitive

to chymotrypsin or trypsin・However，Calcium−aCtivated neutralprotease（CANP）6）did not hydrolyze
COnneCtinrapidly・Withaweightratioofl：100tomyofibri11arproteins，CANPslowlysplittheupperbandoftheconnectindoublet，andthelowerbandwasbrokendownevenslowly．Thisisofsomeinterest，Sinceinthebreastmuscleofdystrophicchickentheconnectincontentisappreciablydecreasedandthe
−39−

（B）

（A）

1

2

1

3

2

3

欄
．
ニ
ー
転

曽
宰十

儀

∵十二■

1

噂−雷電一I

∴

■

表滞積⁚

恥転

；
磯

Fig．1．SDSgelelectrophoresispatternsofconnectinofchickenbreastmusCle・A＝myOfibrilspreparedin50mM

KClandlmMNaHCO3（1），preparedinthepresenceoflmMCaC12（2），andpreparedinthepresenceof5mMEGTA（3）．B：effectofstoragein1％SDSatroomtemperature．Freshlyprepared（1），StOredfor2days（2），andfor4days（3）．

amountoftheupperbandproteinrelativetotheloweroneisdecreased・Indystrophicmuscleithasbeen
shownthatCANPactivltylSenhanced・Ontheotherhand，Serineproteaseofmastcellorlgin7）veryquicklyhydrolyzedconnectinintosmallpeptides，buttheywerestilllargerthanthemyosinheavychain・
1）Lazarides，E．：Nature，283，249−438，1980．
2）Maruyama，K．：InMuscleContraction．ltsRegu】atoryMechanisms（Ebashi，S・，Maruyama，K・，andEndo，M・，eds．），PP．485−496，JapanSci．Soc．Press，Tokyo／Springer−Verlag，Berlin，1980・
3）Wang，K．，McCure，J，，andTu，A．：Proc．Natl．Acad．Sci．U．S．Aリ76，3698−3702，1979・

4）Maruyama，K．，Kimura，S．，Ohashi，K．，andKuwano，Y・：J・Biochem・，89，701−709，1981・
5）Locker，R．H．andDaines，C．J．：FibrousProteins（Parry，D．A．D．，ed．），Vol．II，pp・43−56，AcademicPress，London，1980．

6）Tshiura，S．，Murorushi，H．，Suzuki，K．，andImahori，K．：J．Biochem．，84，225−230，1978・
7）Sanada，Y．，Yasogawa，N．，andKatunuma，N．：Biochem．Biqphys・Res．CbmmLLn・，82，108−113，1978・−40−

ルれ∫C〟ねr勒∫押印々yGrの叩

MEMBRANECOMPOSITIONANDCA−UPTAKEABILITYOFSARCOPI．ASMIC
RETICULUMFROMI）YSTROPHICCIIICKEN
ToshioSakai，HiroakiNishuima，SuketuneIwagaki，TakayukHmai，andMasayukiWatanab。
DepartmentofPhysiology，TheJikeikaiUniversity，SchoolofMedicine，Tokyo
IsolationofthesarcoplasmiCreticulum（SR）丘omskeletalmuscleofdystrophicchickenhasbeend。nebyseveralinvestigators・WealsoattemptedtopreparethefragrnentedsarcoplasmiCreticulum（FSR）from
thepectoralismusclesofdystrophicchicken（line412and413）usingthemodifiedmethodofNish斬ma
etal・1）at8，000q365，000×gwithTris−buhratpH6・8（H−fraction）・Inthispreparation，themaximum
ValueofCa−uPtakeonFSRfromdystrophicmusclewas180−198×10−9mol／mgprotein／60sec．Thisval。eShowednosignificantdi鮎rencewithcomparingthenormalmuscle，190−210×10−9mol／mgprotein／30sec．
ButtheCa−uptakeabilitydecreaseda15％ascomparedtothatofthenorrnalmuscles．Theresultsasmen＿

tionedabovemightsuggesttheCa−aCCumulatingcapacltyOftheSRfromdystrophicmuscleisalmost
Similartothatofnormalmuscle．
TtissuggestedthattheCa−aCCumulatingcapacltyOftheSRseemstobesmallerindystrophicmuscle2J，
andalso，destructionoftheT−SRjunctionandespeciallylongtubularpartshavebeenobservedbyelectron
microscopicstudy・ThereexistsapossibilitythattheFSRfractionalonekeepingthenormalfunctionwas
preparedfromdystrophicmuscleinaboveexperiment・Then，WeeXamineda】ighterfraction（L−FSR）
than36，500×ginwhichapartoflongtubularonewouldbeincluded・Inthepresentexperiment，WeObtain−

edtwofactsasfbllows：1）theyieldofthisfractionjndystrophicmuscleincreasedascomparedwithnormal
muscle；2）theCa−uptakeabilityoftheL−FSRfromnormalmusclesshowed120×10−9mol／60sec，withmaximumvalueof135×1079mol／120sec，WhileintheL−FSRextractedffomdystrophicmuscleitshowed
to65×10−9mol／60sec，withamaximumvalueof77×10￣9mol／120sec．NamelytheCa−uptakeabilityof

thisfractionwasmarkedlyinhibited．

Intheexperimentalprocedure，WereCOgnizedthatthedystrophicmuscleinbu能rsolutionbyWarr−
ingblenderwasmoreeasilyhomogenizedthanthenormalmuscle・Thisindicatesthatdystrophicmuscle
hasaweakerphysicalstructurethannormalmuscle・FromtheseresultsweconsideredthattheinCrease
intheyieldoftheFSRwasnotbecauseoftheproliftratedmembraneasspeculatedbyScaIesetal．3）andGideonelal・4），butratherwasduetotheweakenedmembraneoflongtubularsystem．
Innext・Wetriedtofractionatetheproteininthelight−andheavy−fractionsfromnormalanddystroph−icmuscles・Thesecompositeproteins were preparedwith polyacrylamide gelelectrophoresis carried
OutOndiscgelusingtheTris／GlycinesystemofLaemmii（1970）7），aSdescribedbyWeberandOsborn8）．
TheamountofCa−PumpPrOtein5）oflOO，000LllO，000daltonsintheH−LtactionoftheFSRfromdystroph−icmusclewasshowntobealmostequaltothatoftheH−fractionfromnormalmuscle・Thisprotein
hasbeenreportedbyMarkandBaskin6）tobethe（Ca−Mg）ATPaseprotein，themainproteinintheFSR＿

membrane・IntheL−n・aCtionoftheFSRfromdystrophicmuscle，the（Ca−Mg）ATPaseproteinwasmark−edlydecreasedincomparisonwiththatfromnormalmuscle．
ThegelsoftheH−andL−fractionsoftheFSRshowedtwoadditionalmajorbandsinwhichonewas
aCa−bindingprotein（mol・Wt・2365，000）andanotherwasprotein2355（mol．wt．R355，000）．IntheH−frac＿tionofFSRfromdystrophicmuscle，thequantityofbothproteinswasthesameorsmallerthanthatin
normalmuscle・However，thequantltyOfbothproteinsintheL−fhctionoftheFSRffomdystrophic
musclewasmarkedlydecreased．

Itiswellknownthatactivityofthe（Ca−Mg）ATPaseisobviouslyrelatedtothelipidenvironrnent，−41−

Tablel．Yieldofmicrosomefromdystrophicmuscle
ControI

Dystrophy（mg／gwetwtmuscle）
H＿丘action O．46

0．98

L＿丘action O．23

0．41

︵U彗OJdPヒ︑S茎∈もこ旦8号8Timein Seconds
Fig．1．The Ca−uptake activlty Of H− and L−FSR・○△COntrOl；●▲dystrophicmuscle；Circules：heavyfractionofFSR（H−FSR）；triangles：1ightfractlOnOfFSR（L−FSR）・

andparticularlytochangesincholesterollevels9）・

WehavealreadyreportedthatthecholesterolandFFAcontentsintheFSRfromdystrophicmuscle
wereincreased2−fold，andtheT−GcontentwasincreasedmorethanlO−fbld・IntheL−fractionoftheFSR
fromdystrophicmuscle，bothcontentsweremarkedlyincreasedandespeciallytheT−Gcontentwasincreas−ed20−fbld．Buttheprotein／lipidratiointheL−fractionoftheFSRfromdystrophicmusclewasdecreased・
Therefore，theincreaseintheyieldofthelongtubularmembranecouldbeexplainedbyincreasedbuoyancy
withthedecreaseoftheprotein／lipidratioandthephysicallydestructiblemembranestructure・Inatenta−
tivesummary，WeCOnSideredthatthemajorfactorsinchangeofthebiochemicalactivitieshaveacIose
relationtotheincreaseofT−G，FFAandcholesterolintheL−FSRofdystrophicmuscle・
1）Nishijima，H．etal．：Proc．々n．Acad・，49，367−371，1973・
2）Endo，M．etal．：CurrentResearchinMuscularDystrophy，Japan，pP・25−26，1980・
3）Scale，D．gJd∴βわで鋸肌βゆ毎∫．dcね，465，535−549，1977・
4）Gideon，A．etal．：Ann．N．Y．Acad．Sci．，31，670−691，1979・
5）Meissner，G．：Biochim．Biqphys．Acta，389，51−68，1975・
6）Mark，R．E．andBaskin，R．J．：Biochem・Med・，19，277−293，1978・
7）Laemmli，K．：〟が〟rg，227，680−685，1970・

8）Weber，K．andOsborn，M．：J．BioZ・Chem・，244，4406，1969・
9）Warren，G．B．etal．：Nature，255，684−687，1975・−42−

〃JJ∫CJ血rqy∫Jr叩毎Gro叩

STUDIESOFMYELIN−ASSOCIATEDENZYMESINTHECENTRAI．NERVOUSSYSTEM
OFMURINEMUSCULARDYSTROPHY
ShigekatsuTsujiandHiroshjMatsushita

DepartmentofPhysiology，WakayamaMedicalCo］lege，Wakayama

PreviousbiochemicalinvestigationsshowedaconsistentpatternofmyeJindeficiencyinthecentral
nervoussystemofdystrophicmice・Apronouncedreductioninthetotalamountofmyelinrecoveredn・om
thespinalcordofdystrophicmiceatonemonthofagebydifrtrentialuItracentrifugationhasbeendem−
OnStratedl）・Aseveredecreaseinthegalactolipidconcentrationandaconsiderableincreaseinthech0−

1esteroIconcentration，WhicharethemincomponentsofmyelinStruCture，amOngtOtallipidshavealso
beenshowninthebrainandspinalcordofdystrophicmice2，・Thefindingsofreducedactivitiesof2′，3′−

CyClicnucleotide3′−Phosphohydrolase（CNP）andcholesterolesterhydrolase（CEH），bothenzymeSknown

tobecloselyassociatedwiththemyelinfraction，inthebrainandspinalcordofdystrophicmicealsopro−videadditionalevidenceforhypomyeIinationinthecentralnervoussystemofdystrophicmice3）．Inorder
toelucidatemoreclearlythedisturbancesofmyelinmetaboIisminthecentralnervoussystemofdystrophic

miceandtoclarifytherelationshipbetweentheneuralabnormalityandthedevelopmentofmuscular
dystrophy，WeinvestigatedthedevelopmentalchangesinCNPandCEHactivitiesinthepurifiedrnyelin
丘actionofthebrainandspinalcordofdystrophicmice．
C57BL／6J−dystrainmicecarryingdystrophiamuscularis（dy）werebredinourJaboratory．Homo−ZygOuSa恥cted（dy／dy）andunaFbctedlittermateanimals（＋／？）werestudiedatseveraldiffbrentages．
MutantswereidentiGedbyobservationofperiodicdraggingoftherearftetandcJasp】ngOfthehindlimbs
Whentheanimalisliftedbythetailbeginnlng12−14daysafterbirth・Thebrain，includingthecerebellum，
brainstem，andmedullaoblongatawasremovedandweighed・Thespinalcordwasremovedbyopenlng
thesplnalcolumn・ThetissueswerehomogenizedinlOvo】umesoficecoldO・32MSuCrOSeinatenonhomog−
enizer・Thepurifiedmyelinn・aCtionwasiso！atedwithaconventionalsubcelluIarfractionationprocedure，essentiallyaccordingtoNortonandPoduslo4）・Thehomogenatewascentrifugedat900×gfbrlOmin

jnarefrigeratedcentrifuge・Theprecipitate（Pl）waswashedtwotimeswithO・32MSuCrOSe．Thesuperna−tant（Sl）andwashingsolutionwerecombinedandlayeredoverO・85MSuCrOSeinatransparenttube．The
tubeswerecentrifugedwithaswingbucketrotorat75，000×gfbr30min・Thelayersofcrudemyelinat

theinterfaceofthetwosucrosesolutionswerecollectedwithaPasteur−Pipetteandsuspendedjnwater
byhomogenization・Aflerl−hrdispersidnforosmoticshock，thissuspensionwascentrifugedat75，000×g
for15min・ThecrudemyelinpelJet（P2）wasagaindispersedinwaterandcentrifugedat12，000×gfor
lOmin・Theresultingsupernatant（S2）wascentr汀ugedtwotimesinthesamemanner．Eachpe）1etwas
COmbinedwiththeoriginalmyelinpellet（P3）andresuspendedin0・32MSuCrOSe・Thissuspensionwas
layeredoverO・85MSuCrOSeinatransparenttubeandeentrifugedwiththeswingbucketrotorat75，000×g

for30min・Thepur摘edmyelinwasremovedfromtheinterLbcewithaPasteur−PIPetteandusedLbrdeter−
minationofenzymeactlVlty．
ForthedeterminationofCNPactlVlty，themethodofOlafsonetal・5）with2′，3′−CyClicAMPassub−

StrateandthemethodofSogin6）with2′，3′−CyClicNADPassubstratewereusedwiththepurifiedmyelin

PretreatedwithO・3mg／mlTritonX−100Solutionusedasanenzymesource．
CEHactivityinthepurj鮎dmyelinfractionwasdeterminedbythecolorimetricprocedurepreviously
described7）．

ThetotalactivityofmyelinCNPinboththebrainandspinalcordofcontrolmicewasextremelylow−43−duringthefirst8post−nataldays，afterwhichtimetherewasarapidincreaselastlnguntilday28・Aslightandgradualdeclineintheactivityappearedtooccurfromthisperiodtoadulthood・Inthespinalcordof
dystrophicmouse，theenzymeactivitywasextremelydepressed，Showingapproximately60％ofthenormal
levelduringthemaximumperiod，Whiletheactivityshowedasimi1arageresponse，peakingataround30daysafterbirth，indystrophicaswellasincontrolmice・At14daysofageimmediatelyaftertheap−
pearanceofsymptoms，theactivityinspinalcordofdystrophicmicealreadyshowedonly70％ofthe
activityofnormallittermates・Inthebrainofdystrophicmice，thepatternofincreaseintotalmyelinCNPactivitywasidentical
tothatofthecontroIsduringthefirst28post−nataldays・Afterthisperiod，aSuddendecreaseintheactivity。CCWredinthebrainofdystrophicmouse，reaChingapproximately60％ofthecontrollevelataround40daysofage・Expressedonthebasisoftheproteincontent・however，theactivityofmyelinCNPinboththebrainandspinalcordofdystrophicmicewasnota能cted・Thesefindingsindicatethatthereduction
inthetotalactivityofmyelinCNPinthecentralnervoussystemofdystrophicmicecorrelateswithare−
ductioninthetotalamountinthemyelinsheathratherthanwithaspecificlackinthesynthesisofCNP
enzymeprotein．
ArapidincreaseinthespecificactivityofmyelinCEHinthedeveloplngandmaturingspinalcord
ofcontrolmiceoccurredduringtheperiodfrom8−20daystoreachalmosta20−fbldincreaseoverthe
newbornlevel．Afterthisperiod，theactlVltyremainedconstantorpossiblydeclinedslightlytowardthe
ad。ltl。Vel．Theactivityindystrophicmicewasextremelydepressed，Showingapproximately60％ofthe
controllevelimmediatelyaftertheappearanceofthesymptoms，andreachedonlyabouthalfofthenormal
levelatadulthood．ThedevelopmentalpatternofmyelinCEHinthebrainwasdilTerentfromthatinthe
Spinalcord．
Inthebrainofcontrolmice，arapidincreaseintheactivityofmyelinCEHoccurredduringdays
8−30．Afterthisperiod，agradualbutsteadyincreaseappearedtocontinuetowardtheadultlevel・Inthebrainofdystrophicmice，thepatternofincreaseofspecifiCactivityofmyelinCEHwasidenticaltothat
ofnormallittermatesduringthefirst20post−nataldays，afterwhichtimeasuddendecreaseoccurred
lastingunti130daystoreachapproximately50％ofthecontrolleveL
ThedevelopmentalchangesinCNPandCEHactivitiesindicatethattheseenzymesareexclusively
localizedinthemyelinfractionofthecentralnervoussystem・NotonlydoesthesharplnCreaSeinactlVlty
coincidewiththeperiodofactivemyelination，butthechangesalsoappeartorenectpreciselythedifrtrencesindevelopmentalcharacteristicsofthebrainandspinalcord・Theresultsobtainedinthisexperimentclearly
polntOutthattheprocessofmyelinfbrmationinthecentralnervoussystemofdystrophicmicewasafl

fectedmoreseverelyinthesplnalcordthaninthebrainandthattheremightbesomed舵rencesinthe
causesofmyelindeficiencyinthesetwoorgans・Thepresent触dingscorrelatingenzymeaCtivitywith
thereducedsynthesisofmyelinsuggestedthatmyelinCEHactivityplaysanimportantroleinthedevelop−
mentofhypomyelinationinthecentralnervoussystemofdystrophicmice・EarlierworkbyRabinowitz8）
andKabara9IhasshownanincreasedrateofcholesteroIsynthesisandadecreasedconcentrationofch0−
1estero1−eSterSinthebrainofdystrophicmice・This，incombination＿withourobservations，SuggeStSthat

thecentralnervoussystemofdystrophicmiceisunabletobringaboutthecombinationofcholesterolwith
theothermyelinconstituentstofbrmamaturemyelinmembrane・
1）Tsuji，S．andMatsushita，H・：CurrentResearchinMuscularDystrophy，Japan・pp・60L61，1977・
2）Tsuji，S．，Nokami，H．，andMatsushita，H・：J・Physiol・Soc・JLPan，40，273，1978・
3）Tsuji，S．andMatsushita，H．：J．Physiol・Soc・Jqpan，42，282，1980・
4）Norton，W．T．andPoduslo，E．：J・Ne〟rOChem・，21，749−757，1973・

5）01afson，R．W．，Drummond，Gエ，andLee，J・F・‥Canad・J・Bioche肌，47，961−966，1969・
6）Sogin，D．C．：J．Ne〟rOChem．，27，1333−1337，1976・

7）Tsuji，S．andMatsushita，H・：CurrentResearchinMuscularDystrophy，Japan，pp・47−48，1979・
8）Rabinowitz，J．L．：Biochim．Biqphys・Acfa，43，337−338，1960・
9）Kabara，J．J．：TbxasR甲．Biol・Med・，22（1），143−151，1964・−44−

八九∫C 血r功∫／r（pわ′Grβ叩

MODEOFPROTEOLYSISOFMYOFIBRILLARSTRUCTURALPROTEINSBYCATHEPSIN＿B

NobuhikoKatunuma，KazuhideIsogai，andTakashiNoda
DepartmentofEnzymeChemistry，TnstituteforEnzymeResearch，SchoolofMedicine，TokushimaUniversity，

TheintracellularconcentrationsofmyofibrilJarproteinsareregulatedbytheirratesofsynthesisanddegradation・Inatrophicmuscle，thecontentsofmyofibrillarstructuralproteinsandtheintensityofmus−
Cularcontractionsaremarkedlydecreased・Wehavereportedthatarnarkedincreaseincathepsin−Bactivities
WaSObservedinatrophicmusclesofDMDpatientsl），dystrophicmice（C57BL／6J−dy／dy）2），anddystrophic
Chickens（1ine413）3）・Itispossiblethatcathepsin−Bmayplaysomero】einboththedecreasesinthec。n＿tentsofstructuralproteinsandintheintensltyOfmuscularcontractions・Somemyonbrillarproteinsare
degradedbycathepsin−B・Atamolarratioofcathepsin−Btosubstrateofl：100，theorderofsusceptibilities
WaSmyOSin≫troponin＞tropomyosin≫actin・α−Actininwasnotdegraded．Themyosinheavychainwas

degradedintoseveralfragmentswithmolecularweightsof175，000，170，000，160，000，and145，000，Whereas
thelightchainswerescarcelydegraded・Cathepsin−Bdegradedtroponin−Trapidly，andtroponin−Imore
Slowly，butdidnotdegradetroponin−C・Troponln−Tandtroponin−Iweredegradedjntothreefragments
withmolecularweightsof30，000，18，000，and12，800・Tropomyosinwasdegradedslightlyanditsproducthadamolecularweightof32，000・Actinwasalsodegradedonlyslowly，andnoliberatedproductcouldbe
detected・MorphoIogicalchangesinmyofibrilspreparedffomglycerinatedrabbitpsoasmuscle during
incubationwithcathepsin−Bwereobserved・Threenotablephenomenawereobservedunderphase−COn−trastmicroscopy：1）adisappearanceoftheZ−bandintheearlystageofincubation：2）adecreaseinthe
densityoftheA−band；and3）anapparentenlargementofthewidthoftheH−band．ElectronmiCroscopic
Observationsandliberatedproductsfrommyofibrilsduringincubationwithcathepsin−Bgaveapproprlate
explanationsfbrthesephenomena・α−Actinin，thechiefsubstanceconstitutingtheZ−band，WaSreleased

fromthemyofibrilsbycathepsin−Btreatment・TheZ−bandmaybepeeledoff丘omthethinfilamentsthroughthedegradationofsomeproteincontributingtotheattachment・TheA−bandlostitsdensitybutthecross−bridgepartmightbenotdestroyedduringincubationwithcathepsin−B・Myosinmaybedegradedatthe
edgeoftherodportion．

WeweresurprisedtofindthattheM−linewasreleasedfrommyofibrilsintothesupernatantsoftheincubationmediumwithoutanychangeinantigenecityagainsttheantibodytoM−PrOtein．NoproteasehasbeenreportedthatcanstriptheM−linen・OmmyOfibrils・ThedisappearanceoftheM−1inemayresult
intheenlargementinthebreadthoftheH−bandunderphase−COntraStmicroscopy．
Wehavealreadyreportedthatcathepsin−Bdegradesaldo】aseprefbrentiallyandtheincreaseinthe
activityofcathepsin−Biscorrelatedwiththedecreaseintheactivityofa．dolase・Therefbreitispossible
thatthecontentsofmyofibrillarstructuralproteinsindystrophicanimalsdecreaseandthattheirquali−
tativefunctionchangesduetoattackbycathepsin−B，andthattheanaerobicATPsupplyfbrmuscular
COntraCtionisa150reduced・Forthesereasons，thewholemuscularfunctionmaybestronglydamaged
bycathepsin−B．
1）Katunuma，N・・Yasogawa，N・，Kito，K・，Sanada，Y・，Kawai，H・，andMiyoshi，K．：J．Biochem．，83，625，1978．
2）Sanada，Y・，Yasogawa，N．，andKatunuma，N．：J．Biochem．，83，27，1978．
3）Noda，T・，Tsogai，KリandKatunuma，N．：J．Biochem．，89，1981，inpress．

−45−

l：ig．1，Morph0］0gicalchangesinmyofibrilsduringincubationwithcathepsin−B・Myo甫brilswereincubatedwith5（hllM KPB，PH6．0，5mM2−merCaptOethanol，andl mM EDTA with cathepsin−B・Reaction was ter−nlinこIle（ttlbyaddingE−64andcentrifugedat3，000rpmfor5min・Theresultantpelletwasappliedtoelec−

川1111icl−OSCOPy．

ー46−肱J∫C〟肋r勒∫JrqpカγGro岬

ENZYMATICREIJATIONSINDYSTROPHICMICEANDCHICl（ENS
TakaakiAoyagiandTakaoWada
InstituteofMicrobialChemistry，Tokyoln previOus papers we demonstrated that the activities ofaminopeptidases，endopeptidases，glyco−
Sidases，phosphatase，eSteraSe，andribonucleaseareabnorma11yelevatedinvariousorgansofdystrophic
miceandchickenswhencomparedtothecontroIsl・2）．Thissuggestedthepotentialusefulnessofseveral
enzymeinhibitors foundin ourlaboratories3・4）．The purpose ofthepresent studywas to elucidate therelationshipofenzymaticchangesindystrophicmicetothoseindystrophicchickens．
Normalanddystrophicmice（C57BL／6N）as wellasnormaland dystrophicchickens（1ine412andline413）Wereusedasexperimentalanimals．TheanimalswerekilIedbycervicaldislocationandhomog−
enatesoforgansandmuscleswerepreparedin10timestheirvolumeofphosphate−bu斤もredsaline（PBS，
PH7・2）byusinganUltraturraxatthemaximalspeedforlmin．Thehomogenatewascentrifuged（5，000
rev．／min，20min），andthesupernatantwaswithdrawnand usedfbrenzymesources．The methods for
enzymeassayshavebeenpresentedindetailelsewherel）．
Inmusclefromdystrophicmice，themostsignificantdi斤もrencewasseeninactivitiesofglycineamino−

peptidase（Gly−AP），Ala−AP，Leu−AP，Met−AP，Phe−AP，Trp−AP，Gly−His−Lys−AP，N−Ac−β−D−gluco−
Saminidase，and free RNase（P＜0．001）．A highly significant di馳rencewas seenin aminopeptidase A（AP−A），Cathepsin−C，α−D−glucosidase，and total RNase activities（P＜0．01）．A significant di仔もrence
WaS Seenin Ser−AP，Gly−Pro−Leu−AP，Cathepsin−D，β−D−galactosidase，α−D−mannOSidase，and esterase（タく0．05）．
Indystrophicchickens，highlysignifiCantincreaseswereseenintheactivitiesofAP−A，AP−B，Gly−
AP，Ala−AP，Ser−AP，Pro−AP，Leu−AP，Met−AP，Trp−AP，Cathepsin−D，N−Ac−β−D一glucosaminidase，eSter−ase，and totalRNase（P＜0．01）．Significantincreases wereseeninPhe−AP，Chymotrypsin−like，elastase−like，and phosphataseactivities（P＜0．05），andpossiblysignificantincreasesin Gly−His−Lys−AP，N−Ac−
Tablel．ComparisonsbetweencontroIsanddystrophicmiceofaminopeptidasesinmuscle
Activities±S．D．（n＝4）

1−Value

EnzymeControI Dystrophy

0．73±0．08

1．68±0．32

AP−B

5．02±1．11

5．38±1．14

Gly−AP
Ala−AP

0．65±0．17

2．10±0，275．77±0．96

14．13±0．50

Ser−AP

0．51±0．18

0．85±0．17
1．08±0．43

Leu−Ap1．10±0．50
3．59±0．31

7．70±0．20

Met−AP

4．64±0．51

11．80±0．60

rMet−AP4．58±0．34

Phe−AP

5．32±1．31
4．97±0．70

Trp−AP

0．96±0．15

12．40±0．80
3．18±0．05

Gly−Pro−AP
Gly−Pro−Leu−AP0．52±0．03

0．85±0．13

0．50±0．14

0．80±0．14

Gly−His−Lys−AP

0．09±0．04

0．40±0．00

Pro＿AP

Enzymeactivitiesareexpressedasnmol／min／mgprotein．
−47−′b5727一′b′b′bOqノ24400
5．0．9．10
52
．2
21
．8
一一
1
1
3274．3．14．

AトA

Significanceof
difference

ア＜0．01NS
ア＜0．001
ア＜0．001♪＜0．05NS
P＜0．001
ア＜0．001NS
P＜0．001
ア＜0．001
P＜0．01
ア＜0．05
P＜0．001

Table2．Comparisonsbetweencontrolanddystrophicmiceofvariousenzymesinmuscle
Activities＊S．D．±（n＝4）

トValueEnzymeControI Dystrophy

cathepsin−D＊＊

0．01± 0．00

α一D−glucosidase

0．20± 0．01

0．43± 0．05

β−D−galactosidase

0．04± 0．05

0．20± 0．08

a−D−mannOSidase

0．08± 0．09

0．20± 0．00

N−Ac−β−D−glucosaminidase

0．17± 0，05

0．98± 0．10

Phosphatase1．25± 0．30

Esterase

14．10±16．00

1．78± 0．53
80．10±45．40

RNaSe（什ee）＊＊＊

1．08± 0．17

5．53± 0．70

′
b
3

0
2

5

0 0
2

3
2

7
2
2
0 0
7

1．53±0．36

0．00± 0．00

1

15．60± 6．90

0．41± 0．09

っ J
つ ︼
7
3
0
つ ▲

8．12± 8．95

Cathepsin一c2

Elastase−likeq ／3

18．60±2．40

18．40± 3．74

12．20± 4．80

l517．00±10．70

Chy−try−like1

Trypsin−like

Significanceof
dif托rence

NS
P＜0．1
NS
P＜0．01
P＜0．05
タ＜0．01
タ＜0．05
P＞0．054
7
4

ア＜0．001
NS

0
7
3

1

P＜0．05

1
2

タ＜0．001

2＊Enzymeactivities．are．expressedasnmol／min／mgprotein・＊＊Cbthepsin−PaCtlVltylSeXpreSSedasabsorbance（at280nm）／min／mgprotein・＊＊＊RNaseactlvitiesareexpressedasunit／mgprotein．1unitisequivalenttotheactivityoflngstandardRNase

frombovinepancreas．
β−D−glucosaminidase，andfreeRNase（P＜0・1）・α一D−glucosidase，β−D−galactosidase，andα−D−mannOSidase

activitiesweregreatlyandsignlficantlydecreasedindystrophicchickens（P＜0・01）・

Inordertocomparetheseenzymaticchangesindystrophicchickenswiththoseindystrophicmice，
anotherstatisticalapproachwasadopted．Thet−Valuesobtained

fromStudent

st−teSt（Tablesland2）

Wereconsideredto beusefulindicestoexpressthestatisticaldifrerencebetweenthedystrophicanimals
andtheircontroIs．Thet−Valuesfor14kindsofaminopeptidasesshowedasignificantcorrelationbetween
thetwokindsofmodelanimals（γ＝0．55，P＜0．05）．Thismaymeanthattheincreasesintheseenzymes

arenotindependentofeachother，butthattheycollectivelyperformsomesequenceofevents・Incontrast，
nosignifiCantcorrelationwasseenintheotherenzymes．
Theseresultsindicatethattheintramuscularmetabolicabnormalitiesindystrophicchickensaregener−

allydi鮎rentfrombutarepartlycorrelatedwiththoseindystrophicmice・
1）Aoyagi，T．，Wada，TリKqjima，F．，Nagaj，M．，andUmezawa，H．：J．Clin．D7VeSf．，67，51−59，1981・

2）Wada，T・，Kqjima，F・，Harada，S・，Aoyagi，T・，andUm？ZaWa，H・：J・Pharm・勒n・，4，184−190，1981・
3）Aoyagi，T．andUmezawa，H．：hProteasesandBiologlCalControl（Reich，E．，Rifkin，D・B・，andShaw，E・，eds・），
p．429．ColdSpringHarborLaboratory，NewYork，1975．
4）Aoyagi，T．andUmezawa，H．：DzIndustrialandC】inicalEnzymology61（VitaleL・J．，andSimeon，V・，eds・），p・89・
FederationofEuropeanI】iochemicalSocieties，1979．

ー48−

・lJJハ川晶・JllVI叩／リイJ…〃／I

STUI）IESONNEUTRAIJPROTEASESINMUSCI．E
KertiiTakahashi，YoshikazuIchihara，andKazuhiroSogawa
DepartmentofIiochemistry，PrimateResearchInstitute，KyotoUniversity，Inuyama
Previouslywereportedtheoccurrenceandsomepropertiesofneutralprotease（S）firmlyboundto
themiCrosomalmembranesofratskeletalmusclel，aswellasrat2）andmonkeylivers3），Whichcouldbe
SOlubilizedonlyinthepresenceofcertaindetergentssuchassodiumcholateanddeoxycholate．Asacon−tinuationofthesestudies，WehaveinvestigatedinmoredetailtheeLrtctsofvariOusproteaseinhibitors
OnthecaseinolyticactivityandcoexistingaminopeptidaseactivityofapartiaIlypurifiedenzymeprepara−tionobtainedasdescribedpreviouslyl）・WehavealsoinvestigatedthesubstratespecifiCityoftheenzyme
preparationtowardvariousurea−denaturedproteinSandaminopeptidasesubstrates・Inaddition，Wehave
COmParedtheactivitiesofsuchneutralproteasesinthemicrosomaland／orsupernatantfractionsofthebreastmuscleofnormalanddystrophicadultchickens．

Aspartlyreportedpreviouslyllthecaseinolyticactivity（pH8・0，250C）2〉wasinhibitedsignifiCantly
by diisopropylphosphorohoridate（DFP），phenylmethanesulfbnyrnuoride（PMSF），Na−tOSyl−L−lysine
Chloromethylketone（TLCK）・Na−tOSy1−L−phenylalaninechloromethylketone（TPCK），P−Chloromercuri−
phenylsulfbnate（pCMPS），Chymostatin，antipain，EDTA，EGTA・ando−Phenanthroline・Otherreagents
tested（dithiothreitol，PePStatin，leupeptin，elastatinal，bestatin，SOybeantrypsininhibirot，adzuki−beanproteinaseinhibitorI，heparin，CaC12，andMgCl2）weremuchlessefftctiveorwithoutanyeffect．Onthe
Otherhand，theaminopeptidaseactivity（measuredwithL−leucinep−nitroanilideassubstrateatpH7．O
and370C）3）wasinhibitedbyo−Phenanthrolinemorestrong．ythanthecaseinolyticactivity，WhereasDFP，
PMSF・TLCK・andTPCKweremuchlessinhibitory・Theseresultsclearlyshowthattheenzymeprepara−tionisamixtureofneutralendopeptidase（S）andaminopeptidases．
Themicrosomalmembrane−boundenzymepreparationhydrolyzedvariousurea−denaturedproteins
andtherelativeratesofhydroIysiswereintheorder：histone（793）＞PrOtamine（181）＞hemoglobin（175）＞CaSein（100）＞myOSinB（37）＞fibrinogen（25）＞γ−globulin（21）＞bovineserumalbumin（19）＞OValbumin（16）＞myosinA（3）・Sincehistoneandprotaminearegoodsubstrates，theenzyme（S）appearstobetrypsin−
like・Ontheotherhand，thespecificactivities（nmol／hr／mgprotein）towardvariousaminopeptidasesub−
StrateSWereintheorder：Gly−Pro−MCA（616）＞Ala−2NA（169）＞Leu−2NA（108）＞Arg−2NA（57）＞Gly−
Phe−2NA（41）＞Cys−d1−2NA（13）＞Pr0−2NA（12）（MCA：methylcoumarineamide；2NA：2−naphthylamide）．
ThehighactivitytowardGly−Pro−MCAmaybeduemainlytomembrane−bounddipeptidylaminopeptidase
ThemiCrosomalfractionofchickenbreastmusclewasalsoshowntocontainmembrane−boundneutral
prOteaSeSincludingaminopeptidases・ThecaseinolyticactivitiesbothperproteinWeightandtissueweight
Werefbundtobehigherindystrophicchickenmusclethaninnormalchickenmuscle，butthedifftrences
Werewithin2−仙d・Thediffbrencesweremuchgreaterinaminopeptidaseactivities・Thespecificactivities（nmol／hr／mgprotein）ofthemicrosomalmembrane−boundaminopeptidasesofnormalchickenmuscle
tOWardsyntheticsubstrateswereintheorder：Leu−2NA（153）＞Ala−2NA（125）＞Gly−Phe−2NA（83）＞Arg−2NA（67）＞Cys−di−2NA（25）＞Pr0−2NA（21）＞GJy−Pro−MCA（16）・Thisresultisquitedimrentffom
thatobtainedfbrthemiCrosomalaminopeptidasesofratskeletalmuscleandappearstorenectaspecies
difrerence・Thedystrophytonormalratios（DP）ofthespeC泊cactivitieswereArg−2NA（7・9，11．5），Gly−
Pro−MCA（4・1，6・0），Leu−2NA（3・2・4・7），AIa−2NA（3・1，4・6），Pr0−2NA（2・0，3・0），Gly−Phe−2NA（1．4，2．0），
andCys−di−2NA（1・2，1・7）（thevaluesinparenthesesaretheD′NratiosofthespeciBcactivitiesperprotein

Weightandtissueweight，reSPeCtively）・TheelevationofactivitiestowardArg−2NAandGly−Pro・MCA−49−

indystrophicmuscleisespeciallynotable・

Theaminopeptidaseactivitiesinthesupernatantfractionofchickenbreastmusclehadaspectrum
differentfromthatofthemicrosomalmembrane−boundaminopeptidaseactivities・Thespecificactivities（nmol／hr／mgprotein）wereintheorder‥Ala−2NA（1220）＞Arg−2NA（766）＞Leu−2NA（560）＞Pr0−2NA（186）＞Gly−Phe−2NA（93）＞Cys−di−2NA（30）＞Gly−Pro−MCA（2）・Theseaminopeptidaseactivitieswere
alsofoundtoincreasemarkedlyindystrophicmuscle・TheD／NratioswereGly−Pro−MCA（6・5，2・5），Pr0−
2NA（4．7，1．8），Gly−Phe−2NA（4・3，1・7），Ala−2NA（3・7，1・4），Arg−2NA（3・6，1・4），Leu−2NA（3・6，1・4），and
cys−di−2NA（2・6，1・0）（thevaluesinparenthesesarethoseperproteinweightandtissueweight，reSpeC−
tively）．

Increasedlevelsofcalcium−aCtivatedneutralproteaseandcathepsin−DactivitiesinthesupernatantfractionwerealsoobservedfordystrophicchickenmusCle・Whenthesupernatantfractionwaspassed
throughacolumn（1・7×150cm）ofSepharoseCL−6B，theaminopeptidaseactivitiestowardGly−Phe−2NA・

Arg−2NA，andLeurp−nitroanilidewereelutedatapositioncorrespondingtoanapparentmOlecularweightof63，000，andcathepsin−DatapositioncorrespondingtoanapparentmOlecularweightof30・000−35，000・
Nodifrtrenceinelutionpositionwasobservedbetweennormalanddystrophicmuscle・Ontheotherhand，
theelutionpatternSOfthecalcium−aCtivatedneutralproteasewereapparentlydifferentbetweennormal
anddystrophicmuscle・InthecaseofnormalmusCle，aSlngleproteasepeakwaselutedatapositioncor−
respondingtoanapparentmOlecularweightof47，000，Whileinthecaseofdystrophicmuscle，themaJOr

peakwaselutedatapositioncorrespondingtoanapparentmolecularweightoflOO，000，withashoulder
correspondingtoanapparentmOlecularweightof47，000・Themolecularweightofthecalcium−aCtivatedneutralproteaseofchickenmusclehasbeenreportedtobe80，0004）・Thereasonforthisapparentdis−
crepancyremainstobeelucidated・Itmaybeduetothepresenceofaninhibitorofthisproteaseinthe
Inordertoelucidatetherolesofthemembrane−boundneutralproteasesinthemetabolismofmuscle

proteins，itisnecessarytofurtherpurifythemtohomogeneityandinvestigatetheirpropertiesincluding
theirmodeofactiononmuscleproteinS・Furtherstudiesarealsonecessaryontheirlocalizationinmus−
culartissues．Ontheotherhand，fbrcomparisonofproteaseactivitiesinnormalanddystrophicmuscle，
itseemsimportanttodosimi1arcomparativestudieswithmammalianspeciessuchasmice・Itwi11bealso
necessarytoinvestigateandcomparethelevelsofproteaseinhibitorsinmuscletissues・
1）Takahashi，K・，Ichihara，Y・，andSogawa，K＝Proc・OftheAnnualMeetingofMuscularDystrophyResearchGroup，Tokyo，P・51，1979・
2）Sogawa，K．andTakahashi，K＝J・Biochem・，84，763，1978・

3）Sogawa，K・andTakahashi，K・：J・Biochem・，86・1313，1979・
4）Ishiura，S・，Murofushi，H・，Suzuki，K・，andImahori，K・：J・BEoch狐・84，225，1978・−50−

九九∫C〟kr勒∫Jr（如γGrP岬

THEEFFECTOFCALCIUMIONSONCAI・CIUM−ACTIVATEDNEUTRAI．PROTEINASE
KazutomoImahori

DepartmentofIiochemistry，FacultyofMedicine，UniversityofTokyo，Tokyo
Weconsiderthatcalcium−aCtivatedneutralproteinase（CANP）hasaveryimportantroleintheinitial
StepOfmyofibriJlarproteindegradationbecause（i）CANPspeci丘calIyremovestheZ−band，Whichisim−
POrtantinmaintainingthemyofibrillarstructurel｝；and（ii）CANPdoesnotlocateitselfinlysozomesbut
intheZ−bandasweIlasinthesolublefraction21・OurstudiesonCANPwereinitiatedundertheassump−tionthatitplaysanimportantroIeinthedegradationofmyofibrillarproteininmusculardystrophy，
Whereseriousmuscleatrophyisobserved．
RecentIy，WePuriBedCANPtoahomogeneousstateforthefirsttime丘omchickenbreastmuscle3）．
WealsopuriBeditfromhuman4），mOnkey，andrabbitskeletalmuscle・TheCANPsthusfarpurifiedfrom
Varioussourcesresembleeachotherasfarastheirenzymaticpropertiesareconcerned，thoughvarying
resultshavebeenreportedonthemolecularweightandthesubunitcompositionofthisproteinase．This
enZymeClearlyhasanessentialthiolgroupatitscata．yticsite，butalsohasanabsoluterequlrementfbr
Ca2＋fbritsactivity・Theroleplayedbythemetalioninthereactionmechanismhasnotbeenelucidated，

butitisveryimportant・Theresultsobtainedconcemingthisproblemwillbereportedhere．
WeusedpurifiedrabbitskeletalmuscleCANP・Judgingfromdiscgelelectrophoresis，thisproteinase
WaSpurifiedtoahomogeneousstatebyamethodsimilartothatappliedfbrthepreparationofhumanmuscleCANP4）・TheproteinasegavetwobandsuponSDSgelelectrophoresis・ThemoIecularweights
Ofthesetwosubunitswereestimatedtobe80，000（80K）and31，000（30K），reSPeCtively，andtheiramountratiowasl・0：0・8−1・2bytraclngthegels・Theactivesitewaslocatedonthe80Ksubunit．
Itisveryd潤cuIttoexaminetheroleofCa2＋onCANP，Sincetheproteinaseiseasilyinactivatedby
autolysisinthepresenceofCa2十・Wefbund，however，thatautolysiswasfuIJysuppressedwhenE−64waspresentintheenzymesolution・Inthisconditjon，thepresenceandabsenceofCa2＋madeCANPbehave
difftrentlyupondiscgelelectrophoresisorontheDEAEcellulosecolumnchromatography．Further一more，itdissociatedintoitssubunitsonlyinthepresenceofCa2＋・ThesephenomenaarespecificfbrCa2＋，

andarealsoobservedincarboxymethylatedCANP（CM−CANP），WhichwasmodifiedwithIAAandwas
COmpletelyinactivated・CANPwascarboxymethylatedasfbllows・AflertheadditionofIAA（molarratio：
100／oneenzyme），theCANPsolutionwasinCubatedfbr4hrat300C・Caseinolyticactivitywaslostbythis
jncubation・ThemixturewasdialyzedagainstIAA−freebufftr・Bythisprocedure，IAAmodifiedaboutl・4cysteinresiduresoftheenzyme（the80Ksubunit）・Weusedthisrnodifiedenzymeinfurtherexperi−ments・Manypropertiestestedsofar，eXCeptfbrthecatalyticactivityofCM−CANP，areindistinguishable
ffomthoseofnativeCANP・AsStatedaboveitwasindicatedthatthepIoftheCANPchangedfrom4．3
to4・9whenCa2＋（5mM）wasadded，aSSeenintheeIectrophoresisoncelluloseacetatestrips．Thisobserva−
tionsuggestedthat：（i）Ca2＋ionscancelledsomeofnegativechargesonthemoleculeofCANP，thustheaPParentpZchanged；Or（ii）Ca2＋inducedaconformationalchangeinCANP・Althoughtheformerpos−

SibiJitycannotberuledout，evidencewhichsupportstheconfbrmationalchangeswasobtajned．TheCD
SpeCtradrasticallychangedwhenCa2＋wasaddedtotheCANPinthewavelengthrangefrom260to340

nm・ThiswavelengthrangeusuallygaveusinfbrmationonthestructureinthevicinityoftheTrp，Tyr，
Phe，andCys−Cysresidues・Inthiscase，theseCDspectralchangeswereattributedtoachangeintheen−
vironmentofsomeTrpand／orTyrresidues・ThesespectralchangeswerespecはctoCa2＋andshoweddependencyontheCa鈷concentration・Furthermore，thisstructuralchangeandtheappearanceofactivity−51−

ofthenativeenzymegavethesameCa2＋dependency・SimilarresultswereobtainedfbrtheUVdifrtrence

speCtra．Ontheotherhand，WeCOuldhardlyobservetheCDspeclralchangeinthewavelengthrange（200−250nm）whichrenectsthesecondarystructure・Thatis，grOSSStruCturalchangesinthepolypeptideback−bonewi11nottakeplacethroughCa2＋binding・Aspreviouslystated，CANPdissociatedintoitssubunits
whenCa2十wasadded．OnemightthinkthattheCDandUVspectralchangesmaybeduetothedissocia−

tionofthe30Kfromthe80subunit，butthisprobabilitywasruledoutsincetheisolated80Ksubunit
itselfshowedthesamestructuralchangesasthoseofCANP・ThusCa2＋inducesrearrangementsofsome

specificaminoresiduresofCANPwhicharenecessaryfortheappearanceoftheactivity・
TheseresultssuggestedthatCANPcouldhavetwoforms‥aninactivatedformandanactiveform・
TheroleofCa2＋Ontheenzymeistochangethestructurefromtheinactivefbrmtotheactivefbrm，and

todissociatetheproteinaseintoitssubunits・Themetalionconcentrationthatisrequiredforthesestruc−turalchangesis，however，muChhigherthanthatofintracellularCa2＋・AfactorthatenablesCANPtochangeitsstructureatthephysiologicalconcentrationofCa2＋mayexisth

ivo・Wearenowexamining

thispossibility・Moreover，anOthertypeofCANP，WhichoperatesundertheFLMOrderconcentrationof
ca2＋wasdiscoveredbyMellgren5）・TherelationshipbetweenourCANPandthenewCANPisalsonow

underinvestigation．

1）Dayton，W．R．，Goll，D・E，Stromer，M・H・，Reville，W・J・，Zeece，M・G・，andRobson・R・H・：ProteasesandBi0−
logicalControl（Reich，E・，Rifkin，D・B・，andShoe，E・，eds・），Pp・551，ColdSpringHarborLaboratory，1975・
2）Ishiura，S．，Sugita，H・，Nonaka，I・，andImahori，K・：J・Biochem・，87・343，1980・
3）Ishiura，S．，Murofushi，H・，Suzuki，K・，andImahori・K・：J・Biochem・，84，225，1980・
4）Suzuki，KJshiura，S・，Tsl払S・，Katamoto，T・，Sugita，H・，andImahori，K・：FEBSLetf・・104，355・1979・
5）Mellgren，R．L．：FEβ∫上gJJ・，109，129，1980・−52−

漉J∫C〟血′勒∫Jrqp毎Gr（）岬

EFFECTOFNllNl（L−TRANSL3−CARBOXYOXIRANE−2−CARBONYL）−L−LEUCYL］−3−

METHYL−BUrYIJAMINEIAPROTEASEINHIBITOR，ONPROTEINI）EGRADATIONININTACTMUSCI．ECELI．S
KazuhiroKohama，TomoakiShirao，ShoheiMitani，andSetsuroEbashi
DepartmentofPharmacology，FacultyofMedicine，UniversityofTokyo，Tokyo
Thee蝕ctofalowconcentrationofN−lN−（L−tranS−3−Carboxyoxirane−2−Carbonyl）−L−leucyl］−3−methyl−
butylamine（E64C）uponproteindegradationinisolatedorculturedmuscleswasexamined．
1）Bbldb好qr戯対Ctomuscle：Theamount ofE64Cretainedinadultrat soleusmuscle was6．6
timeslargerthantheamountofinulin（Fig・1），WhenitwasincubatedinKrebs−Ringersolution（KR）lI
COntaining12．6FLCi／mlof3H−E64C（3．4×10−5g／ml）or3H−inulin．TheamountofE64C boundtothe
musclewas2．3FLg／gwetweightofmuscle．

2）聯cJdE64Con毎radbtiondmuscJeproteinsasawhole：Therateofdegradationofproteins
inmuscleasawholewasestimatedfromtheamountoftyrosinereleasedinKRcontainingcycloheximide（KR−CH）after2hrs

incubationl，2）．Largeramountsoftyrosineindicatehigherratesofproteindegradation．

Theamountoftyrosineintheabsence（TyrC）orpresence（TyrEC）of2．5×10−6g／mlI≡64Cwas235．8j＝

91・5（n＝13）or196・2士74．8（n＝13）（nmol／gwetweightofmuscle，土S．D．），reSpeCtively（Tablel）．The
difrtrencesbetweenTyrCandTyrECweresignificant（P＜0．05）．
TherewasatendencyfbrtheefrbctofE64CtobemorepronouncedinmusclesforwhichtheTyrC
Valuewashigher・Ifwetakethevalueshigherthan250intoconsideration，itishighlysignificant（P＜0．005）．
E64Cwaseffectiveevenataconcentrationaslowas2．5×1078g／mlinKR−CH．E64Cwasmoreefrtc−
tivewhenlO￣5g／mlofionophoreA23187coexistedinKR−CH；thevalueoflTyrC−TyrEC］／TyrCinthe

×102cpM／mg

Fig．1．Acouple ofadult rat soleus musces wereisolated from tendon to tendon．After
Weighing，OneWaSincubatedfor2hrinKRcontaining12．6FLCi／mlof3H−inulinand
O・17FLCi／mlof14C−inulin，andtheotherinKRcontaining12．6FLCi／mlof3H・E64C（3・4×10￣5g／mI）andO．17FLCi／mlof14C−inulin．The musclesweretakenoutofthe

KRandhomogenizedinO．3MKCIcontainingO．2NNaOH．Theradioactivityorthe
homogenatewascountedwithascintillationcounter．SoIvent spacewascalculated
bytheradioactivityof14C，and3Hradioactivityinthespacewassubtractedfromthe
tota13Hradioactivity．Barsshow3Hradioactivityremalnlnginthemuscles．
−53−

Tablel，AmountsoftyrosinereleasedinKR−CH（TyrC）orKR−CHcontaining
E64C（TyrEC）（nmol／gwetweightofmuscle）
TyrC

3

8

︹

2
5

8

つ ム

5

2

l

1
つ

ノ

J

2

q

3

O

1

0

O

4

4

7

8

5

4

0

っ

1

2

／ L U

つ

2

J

J

7

q

2

2

8

9

ノ

U

2

0
0

O

1

q ′
1
1

2
L

0

1

′

0
1

1

U

1
1

1

2

235．8
7

3

Mean

TyrEC

03
29
9 9 840682414 6 1 7 2 8
36
5
6
60
1つんl11132212111

No．ofexperiment

196．2

Procedures forisolation，Weighing andincubationof thesoleusmuscle were thesameasdescribedinthelegendtoFig．1exceptthat themusClewasincubatedin
KR−CHorKR−CHcontaining5×10￣6g／mlE64C．

absenceorpresenceoftheionophorewas16．8％（Tablel）or18．6％，reSpeCtively（d：ref・2）・
3）餅ctq／郎イCondqgraddtionq／myqFbrilh7rPrOte血Sasawhole：Thesamenumbersofmyo−
blastsfromchickembryonicbreastmusclewereinoculatedoncultureplates3）and cultured fbr3days

underthesameconditions．Theplateswerepulsedwith3H−1abeledleucinefor24hranddividedintotwo
groups．Onegroup（G−EC）waschasedinthepresenceofl0−6g／mlE64Candtheother（G−C）initsabsence・After2days

Chasing，Culturedcellswereharvestedfromeachplateandhomogenizedin0．1MKCL

Myo−

fibrillarproteinswererecoveredas theO．l MKC1−insolublefraction4J．TheradioactlVltyOfthisfraction
WaSdeterminedbyliquidscintillationcounting．
Theradioactivity（×103cpm±S．D．）ofmyofibrillar proteins perplatein G−C was814j＝28（n＝5）（Exp．1），826土23（n＝6）（Exp．2），and910士78（n＝5）（Exp．3）．ThatinG−ECwas841士38（n＝5）（Exp・1），912j＝37（］7＝6）（Exp．2），and996土43（n＝3）（Exp．3）．ThedifferenceinradioactivitybetweenG−Cand

G−ECwassignificant（P＜0．01）accordingtoblockdesignanalysis．
4）騨ctd上古4Co〃dbgradbtionqrfWOS血heavychai17：Musclecells，Cultured，pulsed，andchased
asdescribedabove，Wereharvested．Myosin−BwasextractedfrommyofibrillarproteinspreparedasdescribedaboveandrunonSDSpolyacrylamidegelelectrophoresis5）．AfterstainingwithCoomassieBrilliantBlue，
thebandofthemyosinheavychainwascutoutofthegel．Theradioactivity4）andtheamountofprotein6）
inthebandweredetermined．Lowspecificactivitiesmeanhigherratesofproteindegradation．
Thespecificactivity（Cpmperarbitraryunitofprotein±S．D．）ofthemyosinheavychaininthepres−enceof5×10￣6g／mlE64Cwas26・5±2・9（n＝6）andthatinitsabsenFe22・0土2・6（〃＝6）・Thedi耽rence
inthespecificactivitieswassignificant（P＜0．05）．
1）Fulks，R．M．，Li，J．B．，andGoldberg，A．L．：J．Biol．Chem．，250，292，1975・
2）Kameyama，T．andEtlinger，J．D．：Nature，279，344，1979．
3）Kohama，K．andOzawa，E．：Deveわク．GrowlhD解r．，19，139，1977．
4）Kohama，K．andHoltzer，H．：J．Biochem．，89，341，1981．
5）Laemmli，U．K∴∧仏力〟■β，227，680，1970．
6）Fenner，C．，Traut，R．RリMason，D．T．，andWikman−Coffelt，J．：Anal．Biochem．，63，595，1975・−54−

MEMBERS OF THE RESEARCH GROUP ON
MUSCULAR DYSTROPHYINJAPAN
PiomedicalSciences）
Chairman
Ebashi，Setsuro
DepartmentofPharmacology，FacultyofMedicine，UniversltyOfTokyo，Tokyo
Members
Aoyagi，Takaaki

InstituteofMicrobialChemistry，Tokyo
Endo，Makoto
DepartmentofPharmacology，TohokuUniversity，SchoolofMedicine，Sendai
Hotta，Ken
DepartmentofPhysiology，NagoyaCityUniversity，MedicalSchool，Nagoya
Imahori，Kazutomo
DepartmentofBiochemistry，FacultyofMedicine，UniversltyOfTokyo，Tokyo
Ishikawa，Harunori
DepartmentofAnatomy，FacultyofMedicine，UniversityofTokyo，Tokyo
Iwasaki，Yuzo
DivisionofUltrastructureResearch，NationalCenterfbrNervous，MentalandMuscularDisorders，Tokyo
Kagawa，Tsutomu
LaboratoryofCellBiology，ToneyamaNationalHospital，OsakaKano，Masaakira
DepartmentofPhysiology，SchoolofMedicine，KitasatoUniversity，Kanagawa
Katunuma，NobuhikoDepartmentofEnzymeChemistry，InstituteforEnzymeResearch，SchoolofMedicine，TokushimaUniver−Sity，Tokushima
Kuriyama，Hiroshi
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity，Fukuoka
Maruyama，Koscak
DepartmentofBiology，FacultyofScience，ChibaUniversity，Chiba
Masaki，Tomoh
DepartmentofPharmacology，InstituteofBasicMedicalSciences，TheUniversltyOfTsukuba，Ibaraki，andTheTokyoMetropolitanInstituteofMedicalScience，Tokyo
MatsⅥShita，Hiroshi

DepartmentofPhysiology，WakayamaMedicalCollege，Wakayama
No110mura，Yoshiaki
DepartmentofPharmacology，FacuItyofMedicine，UniversityofTokyo，Toky0−55−

Obimata，TakasIli
DepartmentofBiology，FacultyofScience，ChibaUniversity，Chiba
Ohts山d，Iwao
DepartmentofPharmacology，FacultyofMedicine，KyushuUniversity・Fukuoka
Okada，T．S．
DepartmentofBiophysics，FacultyofScience，UniversityofKyoto，Kyoto
Okada，Yoshio
ResearchInstituteforMicrobialDiseases，OsakaUniversity，Osaka
Otsuka，Masanori
DepartmentofPharmacology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo
Ozawa，Eijiro
DivisionofCellBiology，NationalCenterfbrNervous，MentalandMuscularDisorders，Tokyo
Sakai，Toshio
DepartmentofPhysiology，TheJikeiUniversity，SchoolofMedicine，Tokyo
Shimada，Yutaka
DepartmentofAnatomyandBiology，FacultyofMedicine，ChibaUniversity・Chiba
Sugita，Hideo
DivisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMuscularDisorders，Tokyo・andDepartmentofNeurology，InstituteofBrainResearch，FacultyofMedicine，UniversityofTokyo，Tokyo
TakahasIli，Kenji
DepartmentofBiochemistry，PrimateResearchInstitute，KyotoUniverslty・Inuyama
Takahashi，Kumitaro
DepartmentofNeurophysiology，InstituteofBrainResearch，FacultyofMedicine，UniversltyOfTokyo・Tokyo
Terao，Toshio
TheFirstDepartmentofInternalMedicine，FacultyofMedicine，TeikyoUniversity，Tokyo

Totsuka，Tsuyoshi
DepartmentofPhysiology，InstituteforDevelopmentalResearch，AichiPreftcturelColony，Aichi
Yomezawa，Takeshi
DepartmentofPathology，KyotoPrefecturalUniversltyOfMedicine・Kyoto−56−