CURR【NT RESEARC‖

川
MUSCULAR DYSTROPHY，JAPAN（CLINlCALRESEARCHES）

The Proceedings of theAnnual Meeting of Muscular
Dystrophy Research Group，
1980，Tokyo

PREFACE

Following thelast ProceedingsoftheAnnualMeetingofMuscularDystrophy
ResearchGroup，1978，1979，HCurrentResearchinMuscularDystrophy，1980

isPublished．
ThisvolumecontainsnotonlytheabstractsofthepaperspresentedattheAnnual
Meeting oftheClinicalResearchGroup ofMuscularDystrophyin1980，butalso
SOmeOftheresearchwo止sperformedduringpastthreeyears・
Meanwhile，arealprogresshas beenmadein the nelds ofthegenetic，Clinical，morphologiCalandmetabolicaspectsofthediseaseandthismayleadtotheunder−Standingofthefundamentaldefbcts．Still，Wehavetoemphasizethattheseandotherdiscoveries，Whiletheyaresignincantlyadvancedandcertainlyveryencouraging，arebynomeansfinal．
Lastly，I as the chairman of this Research Group，WOuldliketoexpressmy
Sincere appreciation to the Ministry ofHealth and Welfare，NationalCenter for
NervOuS，Mental and Muscular Disorders，and MuscularDystrophyAssociation ofJapanforpersistentnnancialsupporttothisResearchGroupforthreeyears．
MythanksarealsoduetoDr・H・Sugita，theco・editor，Whohasmadeitpossible
topublishthisreport．

KazuoMiyoshi

March，1981

CONTENTS

PREFACE[CHAPTER ONE] Experimental
Muscular Dystrophy
1) Early Morphological
Changes in Dystrophic

Yoshigoro Kuroiwa
Morphometric Investigationof Developing Muscles of Dystrophic
Chicken with Special Reference to the Posterior Latissimus Dorsi Muscle:
Haruomi Nakamura

2)3) Intracytoplasmic
4)
5)

6)

7)
8)

[CHAPTER

2)

3)

4)

12

Massive Fiber Necrosis - :Ikuya Nonaka
A Radioautographic
Study of the Activated Satellite Cell in Dystrophic
Chicken Muscle: Kimio Oguchi
Trophic Effects of Sympathetic Ganglia on Normal and Dystrophic Chick
Skeletal
Muscles in Tissue Culture (2) - Acetylcholinesterase
andCatecholamine
Staining - : Hiroshi Tsukagoshi
The Movement of "Filopodia"
to Normal and Dystrophic Chick Muscles:
Takao Nakanishi
Study on Neutral Lipid in the Muscle of Dystrophic
Chicken:
Masao Kinoshita
Gangliosides
in Dystrophic
Chicken Muscle: Tadashi Miyatake

TWO]

10

Vacuolation of ƒ¿WFibers in Chicken Muscular Dystrophy- Probable Early Pathological Event Initiates

1)in Chicken
Chicken Muscles:

14

16

18

20

22
24

Genetic and Clinical Studies of Progressive Muscular Dystrophy
and Related Disorders
Analysis of Amyotrophic Diseases - Duchenne Type Muscular

Genetic
Dystrophy- : Toshiyuki Furusho
Facioscapulohumeral
Syndrome: The Non-Hereditary Variety:Tetsuo Furukawa
Familial Oculocranioskeletal
Neuromuscular Disease with Abnormal
Muscle Mitochondria- AFollow Up Study: Shukuro Araki
Computerized Tomographic Findings of the Central Nervous System in
Myotonic Dystrophy: Hiroo Imura

26

28

30

34

5) An Unusual Case with Facio-Scapulo-Humeral Muscular atrophy:
6)

7)

Keishi Ueda
Centronuclear Myopathy - ADistal from with A Distal from with Juvenile
Onset and No Ocular Involvement: Keiichi Takahashi

A Case of Atypical Myopathy with Contractures
Involvement: Hiroshi Tsukagoshi

of Joints

36

38

and Cardiac
40

[CHAPTER
1)
2)
3)
4)

THREE]

Anatomical and PathologicalStudies of Progressive
Muscular Dystrophy and Related Disorders

Muscle Pathology of Distal Myopathy: Yasuo Toyokura
Distal Myopathy and Lysosomes: Nobuyoshi Fukuhara
Tubulomembranous Myopathy: Nobuyoshi Fukuhara
Debrancher Deficiency Myopathy: Three Adults Including

5)
6)

42
44

46

Two Siblings:

Mitsuhiro Osame
Transplantationof Human Dystrophic Muscle to Nude Mice:
A Morphologic Study: Yoshihiro Wakayama
A Morphological Study of Experimental Mitochondrial
Myopathy with48

2, 4-Dinitrophenol:

52

ShukuroAraki

7)

Myopathy

54

8)
9)

microscopic Studies -: Takeshi Sato
Study on Disuse Atrophy: Masao Kinoshita
Histochemical
Study on Lipid Metabolism in Motor Neurone Disease:

60

10)

Toshiji Mozai
HistologicalStudies
Toshiji[CHAPTER
1)

2)
3)[CHAPTER
a)
1)

2)

3)

4)

with Mitochondrial

Inclusion

Bodies50- Clinical and Electron-on Muscle in a Case with Schwartz-Jampel

Syndrome-:
62

Mozai

FOUR]

PhysiologicalStudies
Related Disordersof Progressive

Single Fiber EMG of Progressive
Hiroshi Nishitani

Muscular DystrophyandMuscular Dystrophies:
64

Experimental Myotonia in HumanExternal Intercostal
Muscles:
Teruyuki Kurihara
Contractile
and Chemosensitive Properties of Muscle Treated with66

Calcium Ionophore

68

FIVE]

(A23187)

Biochemical Studies
Related Disorders

Reds Cells
Phosphatidyl

Inositol: Masaharu

Takamoriof Progressive

Metabolism

Muscular Dystrophy

in Duchenne Erythrocyte

andMembranes:

Keiichi Takahashi
Concanavalin A Binding on the Erythrocytes
in Duchenne Muscular
Dystrophy: Masanori Uono
Calcium Flux of Erythrocytes
in Duchenne Muscular Dystrophy:

70

Shin'ichi

74

Shoji

Ca and ATP Contents of Erythrocytes

72

in Duchenne Muscular Dystrophy:
76

Mizuko Yoshida

5)58

ATPase and Adenylate Kinase of Erythrocyte
Myotonic Dystrophy: Shiro Mawatari

Membranes in Human
78

6)

b)
1)

2)
3)

4)
5)
6)

7)

c)
1)

2)
3)

Erythrocyte
Acetylcholinesterase
Yoshigoro Kuroiwa

in Myotonic Dystrophy:
80

Metabolism and Enzymes
Congenital
Myopathy and Abnormal Tryptophan Metabolism:
Masatoshi Yagita
Some Properties of Creatinase in Human Skeletal Muscle: Yasuo Natori
Abnormal Metabolism of Creatine in Skeletal Muscles of Muscular
Dystrophies:
Kazuo Miyoshi
Enzymatic Studies on the Mechanism ofCreatinuria
in Rats with
Experimental Thyrotoxic Myopathy: Keishi Ueda
Exertional Rhabdomyolysis
and Myoglobinuria:
A Case Report andMetabolic Study: Tadashi Miyatake
Turnover of Human Myoglobin as Studied by Radioimmunoassay:
Hisaomi Kawai
GOT Isozymes in Muscles of Patients with Ophthalmoplegia
Plus Syndrome
and Experimental Mitochondrial
Myopathy: Seiichiro Tarui

Contractile
Proteins and Proteases
Analysis of the Contractile
Protein of a Single Human Muscle Fiber:
Akio Takagi
Studies on the Structural Proteins in Experimental Serotonin Myopathy:
Mitsuo Iida
Calcium-Dependent
Degradation in Muscle Protein - Effect of
Ca Ionophore(A23187)4)

Degradationof Myofibrillard)1)

OthersImmunoglobulin
and the Immune Complex in Fukuyama Type CongenitalMuscular Dystrophy:
Kiyotaro Kondo
Isolationof Nerve Growth Factor from HumanPlacental Tissue:

2)
3)

and a CANP Inhibitor

Proteins

Kyozo Hayashi
Level of Nerve Growth Factor-Like
Kyozo Hayashi

THE LIST OF THE MEMBERSON MUSCULAR DYSTROPHY(E-64-c)by Cathepsin-: Hideo Sugita

H: Yukihiro

Sanada

82

84
86

88

92

94

96

100

102

104
106

108
110

Immunoreactivity

OF THE CLINICALIN JAPAN

in Human Serum:
112

RESEARCH

GROUP
115

The Proceedings of theAnnual Meeting of Muscular
Dystrophy Research Group，
1980，Tokyo〟MCl血′即∫けOp々yGr0叩

EARLYMORPHOLOGICALCHANGESINDYSTROPHICCHTCKENMUSCLES
YoshigoroKuroiwa，SllinjiIshimoto，IkuoGotoandMichiyaOhta＊

DepartmentofNeurologyandDepartmentofNeuropathology＊，NeurologiCalInstitute，FacultyofMedicine，

KyushuUniversity，Fukuoka

Wepreviouslyreportedl）thatearlymorphologiCalchangesappearedinPLDmusclesatlweekafter
hatchingandthesechangesbecamemoreprominentat3weeksafterhatching・Thistime，WeStudiedthe
CaVeOlae on the plasma membrane surface ofthe superficialpectoralmuscles at various stages after
hatchingincontrolanddystrophicchickens．
Materialsandmethods
Weusedthesuperficialpectoralmuscles，Whitefibers，Ofcontrol（line412）anddystrophicchickens（line413）atlday，1weekand3weeksafterhatching．Forthefreeze−fractureEMstudy，themuscle
specimens示ere routinely fixedin2．5％glutaraldehyde and cutinto1−mm Cubesfollowingfixation．
Then，theblockswereimmersedin25％glycerol．Thesespecimenswerefrozenbyimmersioninliquid
nitrogen・Theywerefracturedandshadowedwithplatinumandcarboninafreeze−fracturedeviceoper−
atedat−1150C andbeneathl xl0−6Torr．Replicaswerecleanedinbleachandwashedwithdistilled
Water and examined with an electron micro−
SCOpe．200−micron square areas representing a
totalsampling area were countedin order todetermineaveragecaveolardensity．
Resdts（Fig．1）︵．ュ︑N 冨一宕＞何リーOSLqq∈⊃∪

Atldayandlweekafterhatching，there
WaS a differencein caveolar density between
COntrOl and dystrophic muscles．The distriburtion，Shapeand size of caveolae were more

irregularin dystrophic chickens（Fig．2）．There
Were nO remarkable differencesin caveolar
densitybetweenl dayandl weekafterhatch−ing．However，thecaveolardistributionshowed
more random arrangement in dystrophic
muscles．At3weeks after hatching，therewas
anincrease of caveolar densityin dystrophic
muscles ascomparedwithcontrolmusclesandthe caveolardistributionwasmoreirregularand
randomin dystrophic muscles．In control
muscles，the caveolaewerearrangedinaregularpattern．There were no remarkable changesin
Jday J week

3weeksCaVeOlar density of controlmuscles atl day，lweekor3weeksafterhatching．

Stage of posthatching
C：ControI D：Dystrophy

Discussiom
The caveolae are considered to be either
PlnOCytOtic vesicles or T−SyStem OpenlngS．

Fig．1CHANGESOFCAVEOLARDENSITY（totdsamplingaIeaS：200〃m2）

There were no obvious changesin dystrophic−10−

muscles atl day after hatchinginour
previous study，While the changes of
CaVeOlar density，distribution and size
Were already seen atl day afterhatch−ingin dystrophicmuscles．Thisstudyof
CaVeOlaeis considered tobemoreusefulfor detecting the early morphologlCal
Changesofthedystrophicprocess．
Referencesl）Kuroiwa，Y．，lshimoto，S．，Goto，I．and
Ohta，M．：EarlymorphologiCalChangesin
dystrophicchickenmuscle．Proceedingsof
Ann．Meet．of Muscular Dystrophy Res．
Group．，P．12，1978and1979．

Fig．2（C）Pface ofmuscleplasmalemmafromaldayoldnormalchicken．
¶leCaVeOlardistributionhasaregularpattern．×21．000（D）E face of muscle plasmalemma from al day old dystrophic
Chicken．The caveolar distributionis more random and the
distribution and sizearemorevariablethanincontroIchickens．
×40．000

ー11−

〟〟∫C〟hr功∫打叩力γGro岬

MORPfIOMETRICmESTIGATIONOFDEVELOPINGMUSCLESOFDYSTROPHICCHICKENWITHSPECIALREFERENCETOTHEPOSTERIORLATISSIMUSDORSIMUSCLEHaruOmiNakamura，MamiKotani，KuniyasuTakadaandKenjiImaoka
DivisionofNeuropathology，InstituteofNeurologiCalSciences，TottoriUniversitySchoolofMedicine，Yonago
The posteriorlatissimus dorsimuscle（PLD）isawell−known affected musclein thedystrophiq
Chickenofline413，itisinstrikingcontrast・tOthehealthyappearanceoftheanteriorlatissimusdorsi
muscle（ALD），and hasvarious beneficialpoints forexperimentalprocedures．Thisinvestigation was
designedtomeasurethenumberanddiameterofmusclefibersinordertogalnSOmebasicmorphological
data forPLDin comparison with those forALD，Superficialpectoralis（PS），bicepsbrachii（BB）and
gastrocnemiusmuscle．
MaterialSandmethods
ThePLDandALDmuscleswereremovedasawholefromdystrophicsat3，5，8and16Weeksafter
hatching and smallstripsofthePS，BBandgastrocnemiusmuscleswereobtainedfromdystrophicsat
5，12，19，26and33daysafterhatching，andalsofromage−matChedcontroIs．Inordertocalculatethe
totalnumberoffibersinPLDandALD，theimagesofeachmuscletrunkwerereconstruCtedfrommany
Pictures of paraffin cross sections after fixingin paraformaldehyde solution．Thediametersofabout
500musclefibersweremeasuredatrandomonmagnifiedmicrophotographsofeachcase．ForthePS，
BBandgastrocnemiusmuscles，CryOStatSeCtionswereusedformeasurementoffiberdiameters．Thedata
WereeValuatedbystatisticalanalysis．
Results
Tablel shows the mean value and standard deviation of the totalnumber ofPLDmusclefibersin
eachgroup．Thetotalnumberat3weeksafterhatchingwasabout9，800indystrophics，COrreSpOnding
to2．5timesthevalueforALDmusclefibers（about3，700），butshowednosignificantdifferencefrom
thecontroIs．Thetotalnumberoffibershadadecreasingtendencyinthefollowingstagesofdystrophics
and had diminished definitely at16weeks，On the other hand，the totalnumbers offibersin thedystrophicALDmuscleswere3，772．0±712．5，3，410．3±506．8and3，356．0±140．6at3，5and8Weeks
Ofage，reSPeCtively，ShowingnoslgnificantdifferencefromcontroIs．
The mean muscle diameters ofPLDincreasedgraduallyindeveloplngStageSWithoutasignificant
differencebetweendystrophicsandcontroIsunti18Weeksofage，andthemeanvaluewasabout42pm
at16weeksin dystrophics，being higherthan that ofcontroIs（about30FLm）（P＜0．01）．Themeanmusclediameters ofPS andBBmusclesshowedhighervaluesindystrophicsthancontroIsafter12days
Ofage（P＜0．05）．Thedistributionofmusclefiberdiameterswascomparedusingmegahistogramsofthe
diametersofeachstage．Largefiberswereobserved at3weeksinPLDofdystrophics，Smallfiberswere
Seen additionally at5and8weeks，Tablel・TotalnumberofmusclefibersinPLD and manyhypertrophicfibersofover

聖、（Weeb）

C。ntmI Dys．r。，hic P

竺＿誓誓＿diameterappearedat16
Weeks．No slgnificant change was

3

11，797．0±1，098．7（n＝3）9，825．0±1，984．1（n＝3）

NS

510，496．7±

616．9（n＝3）8，042．0±6，112．4（n＝3）

NS

89，483．3±2，736．9（n＝3）6，194．0±1，186．6（n＝3）

NS

16

12，742．8±1，965．4（n＝9）7，164．8±2，465．8（n＝6）＜0．005

observedin the distribution of fiber
diametersin the ALD muscle．On the
Otherhand，manyfibersbecamelarger
in diameter than controIs after12days of agein PS and BBmusclesof

NS：nOtSignincant

dystrophics（Imaoka）．
ー12−SummaryMorphometricresultsobtainedforthePLDmuscleindevelopingdystrophicchickenscanbesum−
marizedasfollows．
l・Thetotalnumberofmusclefibersexhibitednosignificantdecreaseat3weeksafterhatchingin
COmparisonwiththe controIs，beingabout2・5timeshigherthan thevaluefortheALDmuscle，anddecreasedsignificantlyat16weeks．
2・Themeanvalueoffiberdiameterswasincreasedat16weeks，butnotdifferentsignificantlyfrom
COntrOIsunti18weeks・Thefiberdiametersshowedwidevariationafter3weeksofagecomparedwith
COntrOIs．

3．1n the PLD muscle theincreasein meanfiberdiameterand thevariationindiameter occurred
laterthanin thePS and BB muscles・ThePLDmuscleindeveloplngStageSWaSSuPpOSedtobeonly
mildlydamagedincomparisonwiththePSandBBmuscles．
Reference
Imaoka，K・：MorphometricandhistochemiCalobservationindevelopingmusclesofdystrophicchickens．YonagoActa
medica，23106・115，1979．

−13−

肋gC打払r功grrOpわ′Gro岬

INTRACYTOPLASMICVACUOLATIONOFαWFIBERSINCHICKENMUSCULARDYSTROPHY−PROBABLEEARLYPATHOLOGICALEVENTmTIATESMASSIVEFIBERNECROSIS−
IkuyaNonakaandSatomiOkada
DvisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMuscularDisorders，Tokyo
The main pathologicalchangesindystrophicchickenincludemusclefibernecrosiswithanactive
regenerating processwhichisalsoacharacteristicfindinginhumanmusculardystrophy・However，theevidence ofpreferentialdamage to the white muscle，intracytoplasmic vacuolation，alackofopaquefibersoverloadedbycalciumionsindystrophicchickenleadustoassumethatthepathophysiologyln
fiber necrosis might be different betweenhumanandchickenmusculardystrophy・Inadditiontothedegenerativechanges，adelayinfibertypetransformationfromtheimmatureαRtothematureαWfiber
一 ◆■ ￣ふ‥′ 凝in the dystrophic whitemusclehasbeen

疇r〉

shown by histochemical examination（1，2）．Theintracytoplasmic vacuoles
localized mainlyin theαW fibers havebeenthoughttoplayanimportantrolein
developingfibernecrosis（3）．Toelucidate

平言上禁書誓g軍票三雲．，豊。：eC：票，三：互一読 dystrophic white muscle，an electron
microscopIC Study was performed tocorrelate histochemical fiber type withvacuolarformation．

Materialsandmethods
遽妄

Toexamine theelectronmicroscoplC養≡characteristics of each fiber type，the準、塾 pect。raliss。P。rficialis（PS）andposterior

latissimus dorsi（PLD）muscles weredissected from bothnon−dystrophic（line㌻412）and、at2−4monthsafterhatching．Themuscles

ミ

dystrophic（line413）chickens

Wereimmediatelyimmersedin relaxing

solution at pH

6．8

containing EGTA，

k ATP，TRIS and maleate．Ateased single
J fiberwas dividedinto two segmentsin
the solution under a dissecting micro−
SCOpe；Oneforhistochemicalexamination
toidentifythemuscle fibertypeandthe
other for electron microscoplC eXamina−tion．Small pleCeS Of the specimens
obtained from dystrophic and non−
I：ig・］＾：Proliferativecaveolaewithahoneycombstructureform−
ingiLlargevacuole（arrowhead）andavacuolatedT−tubule
（V）in

anくlW

fiber

from

dvstrophic

PLD・Lanthanum

nitratestaining，×17，000．
1B：MyoribTillar degenerationandZ−bandlossinthevicinity
oriIlarge vacuole with a focalmembranedefect（arrowheads）．×15，300．
−14−

dystrophic white muscles were fixedin

cacodylate buffered2．8％glutaraldehyde
solution for2−4hours．After rinsingln
the same buffersolution，they were post

fixedin s−COllidine bufferedl．3％OsO4

COntaininglanthanumnitrate（RevelandKarnovsky）．
Results
IntheαWfibersfromthePSmuscle，thetransverse（T）tubulesranattheleveloftheZ−linewhichmeasured500−600Ain width，WhereastheT−tubuleswerelocatedatA−IjunctioninbothαRandαW
fibers from the PLDmuscle．Since theZ−lineinαRfibersmeasured800−1000AandthatinαWfibers

600−800A，tWOSubtypesofthemusclefibersinPLDweredistinguishablebyelectronmicroscopy．
WhenlanthanumnitratewasusedforelectronmicroscoplCeXamination，theT−tubuleswereclearly
demonstrated・TheT−tubulesinthedystrophicαWfibersappearedtobeproliferatedformlngahoney−
COmb to conglomerated struCture（Fig．lA）・The smallintracytoplasmic vacuoles were stained bylanthanumnitratesuggestingthattheyoriginatedfromtheproliferatedT−SyStem．Aconnectionbetween
thevacuolesandT−tubulesorcaveolaewasalSodemonstratedbylanthanumnitratestaining．
Inthevicinityofthevacuoleswithapartialdefectivemembrane，focalmyofibrillardegeneration，
Z−Streaming，lossofZ−lineandvacuolatedorganelleswererecognized（Fig．1B）．Ontheotherhand，the
T−SyStemandmyofibri1swerewellpreservedintheαRfibersinwhichthemitochondriawereenlarged
andremarkablyincreasedinnumber．
Discussion
The present study again confirmed preferentialdamage to white musclein dystrophic chicken（1，2）．Eveninthewhitemuscle，OnlytheαWfiberundergoesvacuolarchangeinassociationwithfocal
myofibrillardegeneration・Thereforethedegenerativeeventsseemtobeexpressedoncethemusclefiber
hasacquiredthehistochemicalcharacteristicsofanαWfiber・Thesarcotubularsystemproliferationmay

be the earliest pathological manifestationin the dystrophic processin chicken．The proliferated
T−tubulesandcaveolaesubsequentlyformlargevacuolesbytheirfusionand／orexpansionofunknown
CauSeS（3）．
Since the T−tubules are opened to the extracellular space，the vacuolesprobably containhigh
COnCentrations ofcalciumions・A focaldefect ofthe vacuolarmembrane，aSShowninFig．lB，may
Permitcalciuminfluxintothesarcoplasmwhichactivatescalciumdependentproteasessuchascalcium
activated neutralprotease（CANP）．Digestion of the Z−band by the proteaseinitiates myofibrillar
degeneration followed bymassivefibernecrosiswithphagocyticinvasion・Fromthepresentresults，it
Can be concluded that theproliferative eventinthesarcotubularsystemisapreludetothe：neCrOtic
PrOCeSSin dystrophic chicken・Understanding themechanismoftheproliferativeprocessmayhelpto
SOIvethepathophysiologyinfibernecrosisinchickenmusculardystrophy．
Referencesl）Ashmore，C．R．＆Doerr，L：Postnataldevelopmentoffibertypesinnormalanddystrophicskeletalmuscleofthe
Chick．Exp．Neuro1．，30：431，1971．
2）Nonaka，Ⅰ・，Sunohara，N・，Sugita，H．etal．：AhistologiCalandhistochemicalstlldyofchickdystrophicmuscle．

Neurol．Med．（Tokyo），11：259，1979．
3）Libelius，RリJirmanova，Ⅰ．，Lundquist，I．etal．：T−tubuleendocytosisindystrophicchickenmuscleanditsrelationto

musclefiberdegeneration．ActaNeuropathol．（Berl．），48：31，1979．

ー15−

肋gC〟血r功∫汁OpカyGr（フ岬

ARADIOAUTOGRAPHICSTUDYOFTHEACTⅣATEDSATELLITECELLNDYSTROPHICCHICKENMUSCLE
KimioOguchi＊andTetsttjiNagata＊＊＊DepartmentofMedicine（Neurology），ShinshuUniversitySchoolofMedicine，Matsumoto＊＊DepartmentofAnatomy，ShinshuUniversitySchoolofMedicine，Matsumoto

SatellitecellswhichwerefirstreportedbyMaurol）wereincreasedinnumberinmyopathies・They
undergomitosisandareincorporatedintomusclefibersandbecomemusclenuclei．2）Thussatellitecells
areconsideredtobethesourceofmusclefiberswhenthefibersareiruuredorshowdegeneration．
Inthepectoralismuscleofthedystrophicchicken，manySatellitecellsareobserved．Thepurposeof
this study was to distinguish the activated satellite cellfrom the quleSCent Satellite cellbymeansof
radioautographyusing3日−thymidineandtoperformultrastruCturalobservationoftheactivatedsatellite
Cell．

Materialsandmethods
4dystrophicchickens（line413）and4controIchickens（1ine412）ofeithersexagedonedayand
21daysafterhatchingwereused．Theyreceived4timesevery6hrintraperitonealinjectionsofdosesof
22−67uCigbodyweightof3H−thymidine．Thesuperficialpectoralismusclewasfixedbyimmersionin
3％glutaraldehyde for20min，POStfixedin OsO4，dehydratedin alcoholand propyleneoxide，and
embeddedin epon−araldite．l um sections and ultrathin（gold−Silverinterference color）Sectionswere
treatedwithSAKURANRM20rSAKURANRH2bythedippingorwire−loopmethod．3）Thespeci−
menswereexposedfor4to6WeeksintherefrigeratoranddevelopedbytheLIGOPmethod・4）Forlightradioautography the specimens were stained with toluidine blue and for electron microscopic radio−
autographytheywerestainedwithleadcitrate．
Inlight radioautography，We eStimated the number of activated satellite cells by the following
labelingindex．
numbersoflabelednuclei

×100

wholenumbersofnuclei

Using an oillens，100

different

visualfieldswere counted．
Resultsandcommemts
Alightmicroscoperadioauto一

graph showslabeled nuclei readily（Fig．1．A）．Onedayafterhatching，
the labeling index of controI
Chickensis

4．59，and that ofdystrophic chickensis

4．19．21

daysafterhatching，that ofcontroI
chickensis

3．86，and that ofdystrophic chickensis5．68．Atthis
Stage，thelabelingindex ofdystro−

I：ig．l A：Light microscope radioautogram of thepeCtOralismuscle one
day after hatching．The nuclei（arrowhead）are overlaid with
Silvergrains．

TI：Hlcctronmicroscoperadioautogram；thesate11itecellnuclei（AC）
iLrCOVerlaidwithsilvergrains．MF；muSClefiber．
−16−

Phic chickens washigherthanthat
ofthe controIchickens．
On electron microscope radio−
autography，thelabel was foundOnlyinsatellitecells（Fig．1，B），nOneOfthetruemusclenucleiwerelabeled．
Thelabelednucleiofsatellitecells（activated satellite cells）had a r？und，OValorirregularform．
Chromatininthenucleiofthe

activatedsatellitecells

showedvariationfrommuchtolittle．The

con＿tents ofthe activated satellite cellsalsoshowedvariation・Insomeactivatedsatellitecells，theplasma
membraneofthesatellitecellandthatofthemusclefibershowedtheHfootHstruCture．Thissuggested
thatthesatellitecellisincorporatedintothemusclefiber・きThelabelisalSofoundonthenucleiofthesatellitecellsundergoingmitosis．
The cytoarchitecturalcharacteristicscouldnotbeusedtodifferentiatetheactivatedsatellitecells
fromthequleSCentSatenitecellsintheearlystage．
Relerencesl）Maru0，G・：Satelliteceuofskeletalmusclefibers．J．Biophys．Biochem．Cyto1．，9：493，1961．
2）Moss，F・P・＆Leblond，C・P・：Satellitecellsasthesourceofnucleiinmusclesofgrowingrats．Anat．Recリ170：421，
1971．

3）Yoshida，Y・，Murata，F・，Ohno，S・，etal・：Amodifiedwire−loopmethodforquantitativeelectronmicroscopicradio−
autography．Histochemistry，57：93，1978．
4）Murata，F．＆Nagata，T．：LIGOPmethod：Ourexperienceofanewdevelopmentmethodfortheelectronmicro−
SCOpicradioautography．
IGAKUNOAYUMl．113：896，1980．

−17−

〃〟JC〟αrpyかOp妙GroIJP

TROPHICEFFECTSOFSYMPATHETICGANGLIAONNORMALANDDYSTROPHICCHICKSKELETALMUSCLESNTISSUECULTURE（2）一ACETYLCHOLINESTERASEANDCATECHOLAMINESTAlNNG−
HiroshiTsukagoshi＊，TakayoshiKobayashi＊＊，KatsuhikoKayanuma＊＊，YasunobuHanda＊＊＊andYoshifusaShimizu＊＊＊＊DepartmentofNeurology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo＊＊DepartmentofMedicine（Neurology），ShinShuUniversitySchoolofMedicine，Matsumoto＊＊＊DepartmentofAnatomy，ShinshuUniversitySchoolofMedicine，Matsumoto

We have reported that the muscles from dystrophic chick degenerated earlier than those fromnormalchickin single cultures．In co−Cultures ofthemuscles and the sympatheticganglia，themuscles，
eitherthenormalorthedystrophicones，Weremaintainedforalongertimethanthoseinsinglecultures・
Inco−Culturesofthem，abridgewasformedwithabundantmuscleandsympatheticnervefibersbetween
bothexplants．Some ofthenervefibersterminated onthemusclefiberswheretheacetylcholinesterase
activitypositivelyappeared（l）．Thepresentstudyaimedtoclarifytherelationshipbetweencholinergic
and adrenergic fibersin co−Culturesofvariouscombinationsofthemusclesandthesympatheticganglia
usinghistochemicaltechniques．
Materialsandmethods
The pectoralmuscles from normalanddystrophicchickembryosoflldaysoldandthecervicalsympatheticgangliafromthoseof13daysoldwereusedasmaterials・Fragmentsofthemusclesandthe
ganglia were explanted alone or togetherwithspecialseparationofabout2−3mmoncollagencoatedcoverslips．Combinationsofthetissueswereasfollows：A，nOrmalmuscle（NM）；B，dystrophicmuscle
Fig．1−A：Co−Cultureofthenormalmuscle（for7days）andthe normalsympathetic ganglion（for5days）・
AChEactivityappearsonthesarcolemma，form−
ing crescents（arrows show sympathetic nerve
fibers）AChE

stainingbyNakamura

Srubeanic

acidtechmique．×490．
B：Co−Culture of the normal muscle（for23days）
andthedystrophicsympatheticganglion（for21days）．A sympathetic nerve nberrunSalOngthe
musclenberandterminatesatthepositivesiteof
AOIEactivityonthesarcolemma．Bodian

ssilver

impregnationafterAChEstaiming・×1200．

C：Single culture of the dystrophic sympathetic
ganglion for13days．「nle CytOplasm ofsympa−
thetic neuronsin the explant shows brightfluorescenceofCA．Theglyoxylicacidtechnique．
×120．
D：Single culture of the dystrophic sympathetic
ganglion for13days．Sympathetic nerve fibers
radiating from the explant show brightfluores−
cenceofCA．Theglyoxylicacidtechnique．×60．
E：Co−Cultureofthedystrophicmuscle（for17days）
and the normal sympathetic ganglion（for15days）．Asympatheticnervefiberrunningparallel
withthemusclefibers（M）showsfluorescenceof

CA（arrows）．Theglyoxylicacidtechnique．
×260．
F：Co−Cultureofthedystrophicmuscle（for30days）andthenormalsympatheticganglion（for28days）・Sympathetic
nervefibersrunformlngadensebundlebetweenbothexplants．Bodian−18−

ssilverimpregnation・X210．

（DM）；C，nOrmalsympatheticganglion（NS）；D，dystrophicsympatheticganglion（DS）；E，NMandNS；
FINMand DS；G，DM andNS；H，DM and DS・Cultureswere maintainedin ahumidified air−tight
Chamberwhichwasgassedwith3％CO2and97％air．Thefluidmediumwaschangedthreetimesaweek．
Itconsistedof80％EaglesMEMwithHanks

salts，5％chickembryojuice，15％horseserumand200

u／mlofpenicillin．Fordemonstrationofacetylcholinesterase（AChE）activity，themodifiedKamOVSky−
Roots

andNakamura

Srubeanicacidtechniqueswereused．Fordemonstrationofcatecholamine（CA），

liquidformaldehydeandglyoxylicacidtechniqueswereused．
Resultsanddiscussion（AchEstaining）
In anycombination ofthe muscles and the sympatheticganglia，pOSitive sites ofAchE activity
appearedin the form ofdots orfilaments along the sarcolemma of the muscle fibersin the bridge
formedbetweenbothexplantsafteroneto twoweeks ofculture．Thiscouldbedemonstratedmorein
detai1byuseofanenhancementmediumcontainingrubeanicacidascomparedwithuseofthemodified
KarnOVSky−RootStechnique．Inthismethod，thepositivesitesappearedintheformofcrescents（Fig．

1−A）orsemicircles．
Afterthreetofourweeksofculture，theyappearedinthemuscleexplantandfurtheronthemuscle
fibersradiatingfromtheexplant・Amongthecombinationsinco−Cultures，nOprOminentdifferencewas
ObservedintheformandnumberofthepositivesitesofAchEactivity．
（BodianssilverimpregnationafterAchEstaining）

Sympatheticnerve fibersranalongorparallelwiththemusclefibersandsomeofthemterminatedatthepositivesitesofAchEactivityonthesarcolemma（Fig．1−B）．
（CAstaining）
Withtheliquidformaldehydemethod，thecytoplasmofbothlargeneuronsintheexplantandsmall
neuronsmigratingoutfromtheexplantwereshowntocontainfluorescentgranulesofCAinculturesof
thesympatheticganglia．Butfluorescencewasnotinducedinthesympatheticnervenbers．Theglyoxylic
acid technique ofFurness and Costayielded good results as comparedwiththeliquidformaldehyde
method・With this methodInOt Only the cytoplasm of both thelarge and smallneurons，but also
SympatheticnervefibersradiatingfromtheexplantexhibitedbrightfluorescenceofCA（Fig．1−C，D）．
Inco−Cultures of the muscles and the sympathetic ganglia，however，Only a few fibers showed
fluorescence ofCAalong the muscle fibersin the bridge betweenbothexplants（Fig．1−E），althoughmanysympatheticnervefiberswereshowntoexistonBodian

ssilverimpregnation（Fig．1−F）．

Patterson et al．Showed that thepresenceofcertainnonneuronalcellsorconditioned mediumof

themhad aprofoundeffectonthetypeoftransmitterchosenbythesympatheticneurons（2，3）．From
thispoint，itissuggestedthatthesympatheticadrenergicnervefiberschangedintothecholinerglCOneS
inco−Culturesoftheskeletalmusclesandthesympatheticganglia．
Rererencesl）Tsukagoshi，H・，Kobayashi，T．，Kayanuma，K．，Komashu，S．andShimi2：u，Y．：Trophiceffectsofsympatheticganglia
OnnOrmalanddystfOPhicchickskeletalmusclesintissue
Japan

culture，In

Currentresearchinmusculardystrophy，（TheproceedingsoftheannualmeetingoftheMuscularDystrophyResearchGroup）Tokyo，1978，1979，

p．41．
2）Patterson，P．H．andChun，L．LY．：Theinfluenceofnonneuronalcellsoncatecholamineandacetylcholinesynthesis
andaccumulationinculturesofdissocitatedsympatheticneurons．Proc．Nat．Acad．Sci．USA71：3607，1974．
3）Patterson，P．H．andOlun，LL．Y．：Theinductionofacetylcholinesynthesisinprimaryculturesofdissociatedrat
SymPatheticneuronsI．Effectsofconditionedmedium．Dev．Biol．56：263，1977．

ー19−

肋∫Cukr砂∫けOpわ，Gr0岬

THEMOVEMENTOF

FILOPODIAMTONORMALANDDYSTROPHICCHICKMUSCLES

TakaoNakanishiandKeisukeArasaki
Depa∫tmentOfNeurology，InstituteofClinicalMedicine，TheUniversityofTsukubaJbaraki
Ithasbeensuggestedthattherearemembraneabnormalitiesinthemusclesofthosewithprogres−sivemusculardystrophy．ThistheoryisbasedonmanyobservationssuchasanincreaseinserumCPK，depolarizationoftherestingmembranepotential，decreaseddurationoftheoutwardpotassiumcurrent
duringmuscleexcitation，andadecreaseinthenumberofintra−membraneparticles・Ontheotherhand，
filopodia−fibrous processes prqjecting from the growth cone ofadissociated neuron−are nOW
assumedtobeabletodiscriminateobjectsbymeretouch．1，2）Therefore，inthisstudy，anattemPt WaS
made to find out when these membrane abnormalities are recognized，by the filopodia of spinal
motoneurons．
We analysed dynamically the movement offilopodia and growthcones ofnormalsplnalmoto−neuronsin four different groups：（1）5−day cultured normalmuscle，（2）5−day cultured dystrophic
muscle，（3）morethan2−Weekculturednormalmuscle，and（4）morethan2−Weekcultureddystrophic
muscle．The superficialpectoralmuscle was excised froml1−12dayold embryos（stage37−38）of
normaland dystrophic chickens．Muscle tissue was dissociatedby O．25％trypsin，SuSPendedin thefluidmedium

formuscle（Eagle，sMEM：eXtraCtOfthechickembryo：Calfserum＝8：1＝1），andplated

oncollagen−COateddishes．Thesedisheswereincubatedforthreehoursat370C・inanatmosphereof
5％cO2and95％airsaturatedwithwatervapour・Then・thedisheswererinsedwithaCa2＋−andMg2＋−
free solution，and treated with O．05％trypsinfor15minutesatroomtemperature・Myogeniccells
removedfromthebottomofthedishesbytheseprocedureswereagainplatedoncollagen−COateddishes，
whichwereincubatedunderthesameconditionsdescribedabove．Cellsstillfloatingafterthetwo−hdurincubation were used for the selective cultureofthemuscle．Thecellswereseededinothercollagen−Coated dishesat aconcentrationofaboutlO，000cellsperlmlofculturemedium，andculturedfor
5daysto3weeks．Thecelldensitywaslowsothatanovergrowthofmusclewouldnotinterferewith

the observation of filopodia．Asforthecultureofmotoneurons，aCCOrdingtothemethodusedby
Masukoetal．（1979）3），theventralhalfofthespinalcordwasobtainedfromnorma160−66hourold
chickembryos（stage17）．Cordfragmentswereculturedat37Ocfor24hoursinapluggedtube，Whichwasrotated

at70rpmin

extractoftheagyratoryshakerandcontainedafluidmediumfornerves（EaglesMEM：

chickembryo：Calfserum＝8：0．5：1．5）with10pMofcytosinearabinoside・Thesefrag−mentswerethendissociatedbyO．25％trypsin，andthedensityofneuronswassetat60，000to80，000
cellsperlmlofculturemedium．Thesuspensionofneuronswasseededdirectlyintodishescontaining
themuscleculture，andincubatedfor2hoursunderthesameconditionsaspreviouslydescribed・The
fluidmediumcontainingmuchdebriswasthenreplacedbyacompletelynewone，afterobservingthe
establishmentofneuronsonthebottomofthedishes．Themovementoffilopodiawasthenrecordedby
phase−COntraStCinematographyforthreehoursperneuron・Afterthat，SOmeOfthedisheswerecultured
for one to three weeks for further observation ofmorphologicalchanges ofmotoneurons，Whereas
theotherswerefixedinFEAsolution（5％formalin，5％aceticacid，90％alcohol）andstainedbyasilver
impregnationtechniquetoconfirmthepresenceofmotoneurons．
1）Themovementoffilopodiatothenormalmuscle．Infourcasesoutofeachofthetwogroupsof
five5−dayculturedmusclesandfiveofthemorethan2−Weekculturedmuscles，thegrowthconemaking
contactwiththemuscle surfacehadatendencytoimmobilizeitselfandtoceaseprqjectingfilopodia
inanhour．However，1nOneCaSeOfeachgroup，thegrowthconeanditsfilopodiamovedawayfrom
the muscleinaboutanhouraftertheirinitialcontactwiththemuscle．2）Themovementoffilopodia・
tothedystrophicmuscle．Inthreecasesoutofthefive5・daycultureddystrophicmuscles，thegrowth−20−COneWaSimmobilizedonthemusclesurfacebythesameprocessasthatinthefourcasesofthenormal
muscle・In twocases，however，thegrowth conedidnotceasemovlng，and the filopodiacontinued
Palpating the muscle surface formore than an hourafterinitiallytouchingthemuscle．Ontheotherhand・infourcasesoutoffiveofthemorethan2−Weekcultureddystrophicmuscles，thegrowthcone，OnCehavingtouchedthemuscle，mOVedawayfromit・Inonecase，thegrowthconeofamotoneuron
attacheditselffirmlytothedystrophicmuscleanditsdetachmentwasnotobservedwithinthreehours．
Althoughitiscertainthatthereareotherkindsofwel1−differentiatedneuronsbesidesmotoneurons
in theposteriorhorn，aSWellas atthejunctionoftheanteriorandtheposteriorhornsofthesplnal
COrd4・5，6），Whicharetechnical1yhardtoeliminatecompletely，theobtainedsuspensionofneuronsis
believed tobe composed ofabout90％motoneuronsandlessthanlO％contaminants（personalcom−
municationfromDr・Shimada）・Itisalsotruethatthemovementoffilopodiashowsdiversevarieties，

andthe3−hourobservationmaynotbeenoughtoassertthatthefilopodiaofacertainmotoneuronstop
movlng・Consequently，inthisstudy，WePaidattentiontotheoverallrateofoccurrenceoftheremoval
Offilopodia，aSWellastryingtomakeasmanyobservationsaspossible．Fromtheseobservationsitmay
bepossibletosaythatthemoreseverethemusculardegenerationis，themorelikelyitistoobservethe
removalof the growth coneand filopodia from the muscle．Supposing that the filopodiaare theuprobingchemicaltouchsystem7）andthattheyareabletorecognizethesurfacestruCtureOfmuscle，

itmaybepostulatedthatthefrequencyofoccurrenceoftheremovaloffilopodiareflectsabnormalities
Of the surface struCture（the basement membrane or theplasmamembrane）ofdystrophicmuscle．
HoweverISinceourpresentobservationswerenotnumerousenoughtodrawanydefiniteconclusions，
itisabsolutelynecessarytoexaminemorecasesinthefuture．
Referemcesl）Nakai，J・，Kawasaki，Y．：Studiesonthemechanismdeterminingthecourseofnervefibersintissueculture．I．Tne
reactionofthegrowthconetovariousobstruCtions．Z．Zellforsch．，51：108，1959．
2）Nakai，J・：ThemechanismdetermimingthecourseofthespinalmotorneⅣetOthemyotome（abstr．）．DeY．Growth＆Dif知．，17：309，1975．
3）Masuko，S・，KuTOmi，H・，Shimada，Y・：Isolationandcultureofmotoneuronsfromembryonicchickenspinalcords．
Proc．Natl．Acad．Sci．USA，76：3537，1979．
4）Langman，J・，Haden，C・C・：Formationandmigrationofneuroblastsinthespinalcordofthechickembryo．J．Comp．
NeufO1．，138：419，1970．
5）Kanemitsu，A・：Cytoarchitecturedelamoelleipini占rechezlepouletenvisag占eaupointdevuededgveloppement．

moc．JapanAcad．，46：77，1970．
6）Kanemitsu，A・：Etudequantitativedelaneurogin占sedanslamoelle占piniGrechezlepouletparl，autoradiographie．

noc．JapanAcad．，48：758，1972．
7）Sperry，R．W．：Organogenesis（DeHaan，R．L．＆Ursprung，H．，ed．），Holt，Rinehart＆Winston，NewYork，1965．

−21−

肋∫C〟krの∫JrOpカツCro岬

STUDYONNEUTRALLIPIDINTHEMUSCLEOFDYSTROPHICCHICKEN
MasaoKinoshita，YukoUchino，YoAraki＆NobuoWakata¶leFourthDepartmentofInternalMedicine〉TohoUniversityITokyo
It haslong been recognized that thereis an excessive amount ofneutrallipidsinthemusclein
musculardystrophy．However，the truemechanismoftheaccumulationhasnotbeenclarified・Inthe
presentwork，theamountofneutrallipidsinthemuscleandthefattyacidcompositionswerecompared
betweendystrophicandcontroIchickens．
Theamountoftriglyceridewasl．94±0．93mg／100mgwetweightinthedystrophicmusclecom−
paredtoO．24±0．13mg／100mgw．W．inthecontrol．Diglyceridewasalsoincreasedindystrophy，butmonoglyceridecouldnotbedeterminedbecauseofthesmallamount・
Asshowninthefigure，thefattyacidcompositionsoftriglyceridedifferedbetweenthedystrophic
andcontrolmusclesinthatthereweredecreasesinC16palmiticacidandC18‥loleicacid，butincreases

inC18stearicacidandC18：21inoleicacidinthedystrophicchicken．Ontheotherhand，nOSignificant
changewasseeninthefattyacidcompositionofdiglyceride・Thesedecreasesorincreasesoffattyacidsubfractions of triglyceride were compared tothose ofsubcutaneousadiposetissue・The fatty acidcompositionsweresimilarinthedystrophicandcontrolbirds，andnoslgnificantdifferencewasseen・It
wasfoundthattheabovementionedchangesinC16，C18，C18：landC18：2inthemuscleofdystrophic
chickengrosslyresembledthoseoftheadiposetissueinbothdystrophicandcontrolbirds・TheseLfacts
suggestthattheaccumulationofneutrallipidsinthedystrophicmuscleisnotduetoaprlmarydefectin
intracellularlipidmetabolismbutsecondarytolossofmusclefibersofanobscureorigin・⊂18

P81両Ik ocid A

M
C18
Steく】パc qCid A

C18：loleic qcid

C18：2
日noleic cICid0

10址mL・〜Cle Hssue

20

30／ノ／／／／ノA‥OdiPSe−i55Ue

ConIroI▼dyshphy

Fig．FattyacidcompositionofTriglyceride

−22−

40

肋∬〟ねrJ坤封ナOpわ′Gm叩

GANGLIOSIDESNDYSTROPHICCHICKENMUSCLE
TetsushiAtsumi＊，ToshioAriga桝，KyokoNakamura＊＊andTadashiMiyatake＊・H

Depa＝tmentOfNeurology書，JichiMedicalSchool，TochigiandTokyoMetropohtan
hstituteofMedicalScience＊＊，Tokyo

Introduction

Abnormalitiesofmuscular gangliosideshave beenreportedindenervatedmuscles，themuscleof
dystrophicmouseandthemuscleofexperimentalrabbitmusculardystrophy・Recentlygangliosideshave
been found to belocated exclusivelyinthe cellmembrane and to play animportant roleinsuch
reactionsasreceptorinteraction，Cellularadhesionandcellularrecognition．WehaveanalyzedthestruC−turesofthemusculargangliosidesinchickensandcomparedthegangliosidesofdystrophicchickenandcontroIchicken．
MaterialsandMethods
l．DeterminationofstruCtureSOfmaingangliosidesinchickenskeletalmuscle
Totallibids were extracted from30kg of muscle of whiteleghorn chickenswith chloroform−
methanol（1：2andl：1，byvolume）atroomtemperature．TheextractswereappliedtoaDEAE−Sephadex
column．meneutrallipidswereelutedwithmethanolandthengangliosideswererecoveFedbyelution
with O．2M sodiumacetateinmethan01．Thegangliosidefractionswerefurtherseparatedandpurified

bythinlayerchromatography（TLC）．Eachpurifiedgangliosidewasanalyzedforsugarbygasliquid
Chromatography（GLC）．
2．Gangliosidecompositionofskeletalmuscleofdystrophicchicken
Gangliosides were extracted from3g ofsuperiorpectoralmuscles obtained fromlO dystrophic
chickens（1ine413）andlOcontroIchickens（line412），andanalyzedbythinlayerchromatographywith
twosoIventsystems（Ando，etal．1））．
ResultsandDiscussion
5gangliosides，A，B，C，DandE，Wereisolated．AhasthesameRfvalueasGM3，andBthatofGD3・TheothergangliosidesshoweduniquemovementonTLC，andtheRfvaluesofthesegangliosidesdonot
coincidewithanyknowngangliosides．Eachgangliosideappearedasadoublet・
The sugar compositions of the purified gangliosides were determined by gas chromatography・
GangliosidesA，Band Ccontainglucose，galactoseand N−aCetylneuraminicacid・Themolarratioof
glucose，galactoseandN−aCetylneuraminicacidisl：1：1inA，l：1：2inB，andl：l：3inC，andDcontains
glucose，galactose，N−aCetylglucosamine，N−aCetylneuraminicacid，inthemolarratioofl：3：2：1・Ganglio−
side Ewasrecoveredinatoosmallamounttobeanalyzed．GangliosidesA，BandCwerehydrolyzed
withClostridiumperfringensneuraminidase，andthehydralyzateswereexaminedbyTLC・Al1ganglio−
sides were converted tolactosylceramide．Theseresultsindicated that allgangliosideshavelactosyl−
ceramide asthe core struCture．To determine the sugarlinkagesinthesegangliosides，permethylation
studies wereperformed・Gasliquid chromatography ofpartially methylated neuraminicacidshowedthat fromgangliosideA，therewasonlyonepeak，andfromgangliosideB，thereweretwopeaks・The
ratioofthefirsttosecondpeakwasl：1，andfromgangliosideC，thereweretwopeaks，theratioofthe
first tosecond peak wasl：2．Eachpeak wasexaminedwithamassspectrometer・nefirstpeakwas
determinedtobel，2，4，7，8，9−0−methyl−N−aCetylneuraminicacid，andthesecondpeakwasdetermined
to bel，2，4，7，9−0−methy1−8−OTMS−N−aCetylneuraminicacid・From thesefindings，thestructuresof
gangliosides A，B and C were determined to be NANA−Gal−GIc−Cer，NANA−NANA−Gal−GIc−Cerand−24−NANA−NANA−NANA−Gal−GIc−Cer，reSPeCtively．A wasGM3，andBwasGD3．CwasGT3andwasavery
uniqueganglioside．Thisgangliosidehadbeenfoundonlyincodfishbrain2），andsohereisfirstdemon−
stratedinmuscle．GangliosideDcontainsN−aCetylneuraminicacid，galactose，N−aCetylglucosamineandglucoseinthemolarratioofl：3：2：1．mesugarlinkagesingangliosideDhavenotbeendetermined，but
consideringthesugarcompositionandthemovementonTLC，thestructureofgangliosideDwascon−sidered to be the same as NANA−Gal−GIcNA−Gal−GIcNA−Gal−GIc−Cer，Which was one of three mono−
sialongangliosidesisolatedfromchickenmusclesveryrecentlybyChienandHogan3）・Thestructureof
gangliosideEwasnotdetermined．
The ganglioside patternS OfthedystrophicchickenmusclesandcontroIchickenmuscleswereex−amined with both thealkaline andtheneutralsoIventsystems．The5gangliosides，A，B，C，Dand E，migrated，andmoreover，tWOadditionalgangliosideswerefound．GangliosideFc0−migratedwithsialyl−paraglobosidewhichwasisolatedfromhumanredcellmembrane．ThestruCtureOfthisgangliosidefrom
chickenmuscleswasdeterminedtobeNANA−Gal−GIcNA−Gal−GIc−CerbyChienandHogan3）andthe
struCture WaS thesameassialylparaglobosideinhumanredcellmembrane．Gwasanovelganglioside
anditsstruCtureWaSPartiauydeterminedtobeNANA−Gal−GalNA−Gal−Gal−GIc−CerbyChien＆Hogan3）・
Each ganglioside conteIJ WaS
measured by scanning th胱7 Table Gangliosidecompositionofnormalanddystrophicchickenmuscles
SPOtSOntWOdifferentplateswith
Ganglioside

Control（N＝10）Dystrophy（N＝10）Dystrophy／Control

a densitometer．In dystrophic

C（GT3）

0．3±0．2＊

0．6±0．4＊

3．7±2．3

16．8±13．6b0．7±0．8′l．1±0．60

1．7±0．9

G4

0．6土0．4

O．5±0．23

O．5±0．4

F

2

1

E

5

4

D

0

twopossiblecauses．Firstisanin−

5．3±3．02

thesegangliosidescouldbedueto

2．0士1．1

7

GT3alsoincreased．EandGwere
notchanged（Table）．
The increased amounts of

23．1±8．1

B（GD3）

2

increased．GangliosideF，GD3and

5．5±1．2

2

GM3，ganglioside D，WaS highly

48．7±22．5

A（GM3）

4

times．Especially，

0

3．6

13．5±4．2′

increased

JLgNANA／gprotein
Tota1

3

Chickenmuscles；totalgangliosides＊P＜0．025

CreaSe Of membrane components
indystrophicmuscletissuecontaininganincreaseofthesarcotubularsysteminnecroticmusclefibers・
Thesecondisincreasedbiosynthesisordecreasedcatabolismofthesegangliosidesindystrophicmuscles・
Atthistime，themetabolicpathwaysofthesegangliosidesarenotclarified，SOitisdifficulttodiscuss
the disturbanceofgangliosidemetabolismindystrophicmuscles．Furtherstudiesareneededtoclarify
仙eproblem．
Referencesl）Ando，S．，Chang，N．C．andYu，R．：HighperfomancethinJayerchromatographyanddensitometricdetermination
ofbraingangliosidecompositionsofseveralspecies・ATlalBiodlem・89：437−450，1978・
2）Yu，R．K．andAndo，S．：StruCtureSOfsomenewcomplexgangliosidesoffi血brainInstruCtureandfunctionof
gangliosides（editedbySvennerholm，etal．）PlenumPress，1979，p・33−46・
3）Chien，J．LandHogan，E．L．：01araCterizationoftwogangliosidesoftheparaglobosideseriesfromchicken永eletal
muscle．Biochhl．Biophys．Acta620，454−461，1980．

ー25−

肋∫C〟わr砂∫汁OpカブCro岬

GENETICANALYSISOFAMYOTROPICDISEASES−DUCHENNETYPEMUSCULARDYSTROPHY−

ToshiyukiFurusho＊，YoshihideKanehisa＊＊，HiroakiNakashima＊＊，MitsuhiroOsame＊＊＊，AkihiroIgata＊＊串，HachiroNakajima＊＊＊＊，HisaomiKawai＊＊＊＊＊andKazuoMiyoshi＊＊＊＊＊＊DepartmentofHygiene，FacultyofMedicine，KagoshimaUniversity，Kagoshima＊＊DepartmentofNeurology，NationalHospitalofMinamiKyushu，Kagoshima＊＊＊DepartmentofMedicine，FacultyofMedicine，KagoshimaUniveISity，Kagoshima＊＊＊＊DepartmentofLegalMedicine，FacultyofMedicine，TokyoMedicalandDentalUmiversity，Tokyo＊＊＊＊＊DepartmentofMedicine，FacultyofMedicine，TokushimaUniversity，Tokushima

For the analysis ofX−geneticlinkage betweenthelociforDuchenne typemusculardystrophy，
Xgabloodgroups，andcolourblindness，aStudywillbemadeusingasaprobandofDuchennetypemusculardystrophy（DMD）Kagoshimadata（about48families）andTokushimadata（about40families）
andtestsoflinkageandestimationofcrossovervalueswillbeconducted．Thisyearastudywasmadeon
lOfamiliesintheKagoshimadata・Theresultsofthisstudyaregiveninthefollowingtable．
Tablel，ResultsofXgabloodgroupandcolourblindnesstestsinDMDduring1980
T est co m p leted

f
1

2

3

N u m ber o f N u m ber o f N u m b er o fsibsh ip s
sibs
DM D

S
1

5

5

5

F ather

X ga b lo od colou r
grou p s blind ness

5

3

3

X ga b lo od colou r X ga b loo dcolou rgrou p s b lin d ness group s
blin dness
2

， 2

（
60 ．
0）
2

12

3

11

5

17

3

14

34

42

22

17

17

18

（
5 0．
0）

（
5 2．
9）

17

17

4

2

6

24

8

3

5

2

1

3

15

4

2

6

1

2

12

4

2

7

1

1

7

1

13 9

61

44

45

M ale：8 5

61

（
3 1．
7）

（
32 ．
4）

2

（
40．
0）
4

， 4

3

， 3

，

2

（
4 0．
0）
5

（
23．
5）

（
40．
5）
4

M o th er

，

5

（
3 1．
3）
3

（
23．
1）

，

3

（
2 1．
4）
ノ

3
T est com pleted
（
12 ．
5）
2
（
13 ．
3）

1

2
（
16 ．
7）

T otd

48

S：Numberofaffectedpersonsineachsibship．r：NumberofDMD

9

， 9
（
19．
1）

10

，

10

（
2 1．
3）

（）：％

Asallthedatawerecollectedbysingleascertainment，thesegregationratio（P）isestimatedfromthe
followlngequation

jI＝王ミ・−26−

whereRisthetotalnumberofDMDcases，Nthenumberofsibships，andTthetotalnumberofchildren
includedinthesibships．Usingtheforegointequationthesegregationratiowasestiinatedtobeasfollows：
P＝0．351±0．0785

Thedifferencebetweenthisestimatedvalueandtheexpectedvalue（0．5）isl．9timeslargerthanthe
error，Whichisnotstatisticallysignificantatthe5％level．Thus，DMDdoesnotconformwiththetheory
Of X−linked recessiveinheritance．Itis，however，a fact that the segregation ratioislower than the
expectedvalue，butheregeneselectionmaybeinvoIved．
Atanyrate，forthetestingofX−1inkedinheritance，itisaprerequisitethatthegeneticmechanism
Ofthisentitybeelucidated．Intheyearfollowingaftercompletionofthestudyonthecasesyettobe
examined，itisnecessarythatareviewbemaderegardingthispoint．
Rererencesl）ALANE．H．EMERY：Geneticlinkagebetweenthelociforcolourblindnessandduchennetypemusculardystrophy・
J．Med．Genet．3：92，1966．
2）HELEN BLYTH，C．0．CARTER，V．DUBOWITZ，A．E．H．EMERY，JUNE GAVIN，H・A・JOHNSTON，V・A・
McKUSICK，R．R．RACE，RUTHSANGER，andPATRICIATIPPETT：Duchenne

smusculardystrophyandtheXgbloodgroups：aSearChforlinkage．J．Med．Genet．2：157，1965．
3）MAYANAZATZ，SUELIB．ITSKAN，RUTHSANGER，0．FROTA−PESSOA，andP．H・SALDANHA＝Newlinkage

datafor the X−linkedtypeSOfmuscular dystrophyand G6PDvariants，COlourblindness，andXgbloodgroups，
J．Med．Genet．11：321．1974．

−27−

〟〟JC〟血′砂∫〝叩わ′Gr0岬

FACIOSCAPULOHUMERALSYNDROME；TTIENON−HEREDITARYVARIETY
TetsuoFurukawa＊andJamesB．Peter＊＊＊DepartmentofNeurology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo＊＊DepartmentofMedicine，UCLASchoolofMedicinq LosAngeles

Thefacioscapulohumeral（FSH）typeofmusculardystrophywasfirstrecognizedbyLandouzyand
Dderinenearlyacenturyago．BecausetheFSHdistributionofmusclewastingisnotlimitedtomuscular
dystrophy，the

term

FSH

syndrome

isusedtoinclude

myopathic

andneuropathicdiseases．Three

CaSeSWithcharacteriSticsdistinctfromtheusualFSHsyndromeweredescribed．
Case1−27−year−01dfemale．At18monthsofage，Shehadafebrileillnesswithatemperatureof
nearly400C・Onemonthlater，Paralysisofthechild，smouthandunclearspeechweredetected．Shewas
unabletorunaSfastasotherchildren・Atage4，theretrospectivediagnosiswaspolio．Atage27，Shewas
a tallwoman with myopathic facies・Muscle atrophy was apparentintheshoulder，hipandperoneal
regions．Scapulaalata was prominent．Serum CPK was237（normal＜100）．EMG showed a great
decreaseinthenumberofmotorunitsandfibri11ationpotentials．Familyhistorywasnegative．
Case2−15−year−01dfemale．Attheageof15months，discoveryofhorseshoekidneyledtoa4−hour
OPerationundergeneralanesthesia・Afterleavlngthehospital，Shehaddifficultypickingherselfupfrom
asittingposition．Shehadnofacialexpression．Atage4，theFSHtypeofmusclewastingwasobserved．

Musclebiopsyoftherighttibialisanteriorshowednoparticularchanges．Atage15，muSClewastingwas
Observedinthescapuloperonealregionwithslightweaknessoftheorbicularisoculi．SerumCPKwas29．
EMGfindingsofdenervationinthegastrocnemiusandtibialisanteriOrsuggestedaneurogenicprocess，
Familyhistorywasnegative．
Case3−49−year−01dfemale・Attheageof5yearsandlmonth，Shedevelopedhighfever，nauSea
and vomiting．Twomonthslater，hermothernoticedthatthechilddraggedherlegsandherbackwas

not straight．She could not stand upwithoutusingherhandsand couldnotsmile．A doctortold the
ParentS

that

the

child

had

had

infantile

paralysis．Atage49，herclinicalfeatureswerethoseofanadvancedFSHsyndrome．SerumCPKwas40．EMGrevealednodenervationpotentials．Familyhistory
WaSnegative．
TheonsetandprogressionofthemostFSHsyndromeareinsidiousandmayoccuranywherefrom
Childhood

tolateadulthood．TylerandStephens

analysisofthedystrophic

FSH

syndrome

showedthattheonsetwasfrom7to55．InouranalysisoftheneurogenicFSHsyndrome，theearliestonsetwas
7years．Thecasescitedinthepresentstudyhadonsetsof15months，18monthsand5years，reSpeC−
tively．Theonsetwassubacuteandinitialdiagnoseswere

polio

inallthecases．Generally，therewasno

Significant progression or slight partialrecoveryln muSCle weakness except for2episodeswith theSuddenappearanceofnewsymptomsinCasel．
In our patients symptoms were first obvious after an acute febri1eillness or surgicaloperation．
Occasionally，We haveseenambulantpatientswithneuromusculardisorderslosealotofstrengthaftera
few daysin bed due to a febrileillness．Thelack offamily historyinallourcaseswillruleoutthe
POSSibilitythattheirdiseasesareprematuremanifestationoftheusualFSHsyndrome．

Hanson and Rowland described3unrelatedpatientswhowereonglnallythoughttohaveM6bius
Syndromebecauseoffacialweaknessininfancy，butwerelaterdiagnosedtohaveFSHmusculardystro−
Phy．Characteristics oftheir caseswere：（l）earlyonsetofthedisease，（2）relativelyslowprogression，（3）asymmetryand（4）1ackoffamilyhistory．OurcaseshavesimilarcharacteristiQStOtheirs，butwere
notcongenital．
Asearchoftheliteraturerevealedsimilarpatients．In1914，Dejerinedescribeda17−year−01d boy−Withscapulohumeralmusclewasting，Whosediseaseappearedat2妬yearsofageafterpulmonaryinfec一一28−

tionofunknownorigin．Hisconditionwasstationaryafterage6．A16−year−Oldboywiththeneurogenic
FSHsyndromedevelopedhisdiseaseafterfebrileillness（possiblywhoopingcough）atage3（Kanbayashietal）．Thesecasesalsolackedafamilyhistory．
Polymyositis rarely representsFSHmusclewasting．Inourcasesnofindingsofpolymyositiswere

detected．However，it cannot beruled out thatinflammation might have played arolein thepath0−
genesisofthediseaseattheinitialstages．
Althoughlittle attentionhasbeenpaid tosuchcases，reCOgnitionofthepresence ofthisvarietyis
Ofimportanceinprognosticationandingeneticcounseling．

−29−

肋∫C〟kr砂JrrOpわ′Gro〟p

FAMILIAL OCULOCRANIOSKELETAL NEUROMUSCULAR DISEASE WITH ABNORMALMUSCLE MITOCHONDRIA−A FOLLOW UP STUDY
ShukuroArakilKazuoYazakiandYoshinoriYamamura
TheThirdDepartmentofInternalMedicine，MiyazakiMedicalCollege，Miyazaki
Therehavebeenmanycasereportsofprogressiveexternalophthalmoplegiaassociatedwithnumer−
Ousneurologicalsignsandsymptomsandabnormalityofmitochondria．Thepathogenesisandnatureof
thissyndromearestillcontroversial・Recentlywestudiedonesuchcasewhichhadbeenalreadyreported
by Tamura et alin1974underthe title offamilialoculocranioskeletalneuromusculardiseasewith
abnormal muscle mitochondria．The purpose ofthisreportis to describe the clinicalcourse ofthis
patientovertheperiodofthelast7yearsandtodiscusstheproblemsintheclassificationandrelation−
Shipofsimilardisorders．

A34−year−OldwomanwasseeninSeptember1980Withthechiefcomplaintofophthalmoplegia・
Shewas theproductofanuncomplicatedpregnancyanddelivery・Herparentswerefirstcousins・She
hadbeenslowinphysicalandmentalactivitiessinceschooldays・Bilateralblephaloptosisstartedat18
yearsofage・Thenophthalmoplegia，naSalvoice，dysphagia，hearingloss，generalizedmuscleatrophyand
weaknesssuccessivelydeveloped．At27yearsofageshewasevaluatedbyTamuraetall）・Shewasnot
followed over the next seven years，While her symptomswere slowly progressive・In1980She wasreferredtousbyanotorhinolaryngologistwhomshevisitedforherhearingloss・Fouroutof6siblings
includingherselfwereaffectedbythesamediseasetovariousdegrees・1）
ThephysicalfindingsandthelaboratorydataaresummarizedinTablelincomparisontothoseofTamuraetal．Thecharacteristicfeaturesweremildmentaldeficiency，markedexternalophthalmople−
giawithptosis，mOderatefacialandbulbarpalsy，neurOgenichearingloss，generalizedmusclewastingand
weakness predominantlyin the nuchaland proximalmuscles・Deep tendonreflexesweregenerally
hypoactive．Therewasnoplgmentarydegenerationoftheretinanorcerebellarsigns・Nomyotonianor
sensorylosswerenoted．Thegaitwasalmostnormal，thoughmildGowers，signwasobserved・Thecerebrospinalfluidshowedamildincreaseofproteincontent（50mg／dl）・Therewasnoconduc−tiondefectonECG．TheEEGdemonstrated5to6Hzgeneralizedthetaslowings・NeedleEMGrevealed
differentfindingsineachmuscletested，aSShowninTablel・＝Myopathicchanges，，includeshortdura−tion，low voltage NMU andfu11interference pattern・HNeurogenic changesMinclude high voltage，
polyphasicNMUaswellasgiantactionpotentials，anddecreasednumberofNMU・
Themusclebiopsyfromthelt．quadricepsmuscledisclosedmildvariationsinthesizeofthemuscle
fibers，mOSt Of which were angularfibers・Therewerealsomusclefibersshowinggranularchanges・Centralnucleiwereobservedin37％ofthefibers．Therewasamoderateincreaseofconnectivetissue
with cellinfiltration．ModifiedGomori−trichromestainingdemonstratedafewraggedredfibersinthe
areaofgranularchangesshownbytheH・E・Staining・DPNHstainingrevealeddarkstainedareasatthe
subsarcolemma oftheragged red fibers．Moth−eatenfiberswerealsoobserved・ElectronmicroscoplC
examination revealed abnormallylnCreaSed mitochondria，glycogenandlipidin thesubsarcolemma・However，theincreasedmitochondriawerealmostnormalinshapeandsize・Noparacrystallineinclu−
sionswereobservedinthemitochondria．
CTscanningdemonstrateddiffusesymmetriclowdensityinthewhitematter・
Discussion
AsshowninTablel，mOStOftheclinicalmanifestationsofthispatientwerecompleteby27years
ofage，andfurtherprogressionofherdiseasewasslowoverthenext7years・Therewereonlyafewnew
findingssuchasapparentmentaldisturbance，abnormalEEGanddiffuselowdensityofthewhitematter−30−

Tablel Acomparisonoftheclinicalfeaturesandlaboratory
dataofTamuraetal，1973，andoftheauthors，1980．

A ge （years）
B ody w eight（kg）
Ptosis ＆ophthal m oplegia
M uscle w eakness＆atrophy
h cialbulbarnuchalproxim al
distalM ental f
u nction
H ear
i ng loss（
neurogenic）
O ptic fu ndi
D TR sCerebe11ar signs
Sensory loss
ECG
EEG
CSF protein
Serum CPK ，
LD H
N CV
EM G
m yopathic
m ix edneurogenic
M uscle biopsy
SiteVar
i ationsin the size ofm uscle fibers
centralnuclei
許anular changes
connective tissue
angula∫丘bers
ragged red fibers
m itochondria
l changes
num berSizeparacrysta
l line inclusion
CT scan

TA M U R A （
1973）
27
34
m arkedA U TH O R S （
1980）
34
29
m arkedm oderatem oderatem oderatem oderatem ildnorm dm ildnofm aldecIeaSedm oderatem oderatem oderatem oderatem ild
fun scal e IQ 65 （W A IS）
Rt．
20 dB，
Lt．
30 dB ．
（
1000 H z）
norm aldecreased

nOne
nOne
nofm alnorm al
75 m g／dl
norm alnotdonenOne
nOne
norm d5 to 6 Hz diffu se theta slow lngSadm ixed．
50 m g／dl
norm al
Rt．
facia
l；
m ildly slow
Rt．
m edian，ulnar ＆peroneal；nOrm al

R t．
bicepsRt．
stem OCleidom astoid，
Rt．
deltoid ＆Lt．
bicepsRt・tr
i ceps＆R t．
thenarRt．
quadr
i cepsLt．
bicepsm ildnoti
n creased
PreSent
increasedabsentpresent

Lt．
quadr
i cepsm ildh creased
present
increased
present
less rem arb ble

h creased
e山a∫ged
PreSent
notdoneincreased
alm ostnorm alabsentdiffuselow density of the w hitem atterin the CT scan．There were also some different findingsin EMG and muscle biopsy．Itisnot certain
Whetherthesedifferencesreflectthechronologicalchangesortopographicvariations．
Thisis a unlque familialdisease whose clinicalmanifestations andlaboratory findingsincluding
mitochondrial abnormalities mimic those of Kearns−Sayre syndrome which has been considered as asporadic disease．2）since the originalreport of this case by Tamura etal，there have been some
reports3）・4）ofsimilarfamilialdiseaseswhosespectrumOfsymptomsandmodeofinheritancearenot
COmPletelyidenticalineachcase．Thenomenclatureand therelationship betweeneachcaseaswellas
Kearns−Sayresyndromeare stillcontroversial．AtpresentsolelyseminologicdiscussionwithoutetiologiC−31−

肋∬〟血r上）yJ打叩妙Gr0岬

consideration would be oflimitedvalue．Further etiologiCinvestigationsarerequiredtoresoIvethese
problems．
Referencesl）Tamura，K．，Santa，T．，＆Kuroiwa，Y．：Familialoculocramioskeletalneuromusculardiseasewithabnormalmuscle
mitochondria．Brain，97：665，1974．
2）Berenberg，R．A．，Pellock，J．M．，DiMauro，S．，etal．：Lumpingorsplitting？

Ophthalmoplegia−plusorKeams−Sayre

SyndrOme？AnnNeurol，1：37，1977．

3）Leshner，R．T．，Spector，R．H．，Seybold，M．etal．：Progressiveexternalophthalmoplegia（POE）withraggedredfibers：
AnintrafamiLalstudy．Neurology（Minneap．），28：364，1978．
4）Schnitzler，E．R．，＆Robertson，W．C．Jr．：FamilialKea∫nS−Sayresyndrome・Neurology（Minneap．），29：1172，1979・−32−

肋∫C〟血r砂∫打OP月γGro岬

COMPUTERIZEDTOMOGRAPHICFmDmGSOFTHECENTRALNERVOUSSYSTEMINMYOTONICDYSTROPHY

HirooImura＊，TakekazuOhi＊，HikaruOhta＊，YoshihisaTachioka＊，HiroshiNisl山ani＊＊andTerukuniImai＊＊＊＊TheSecondDepartmentofInternalMedicine，FacultyofMedicine，KyotoUniversity，Kyoto＊＊TheDepartmentofNeurology，UtanoNationalHospital，Kyoto＊＊＊TheDepaltmentOfNeurology，KitanoHospital，Osaka

htroduction
Patientswithmyotonicdystrophy（MD）oftenshowmentalimpairment，hypersomnia，abnormality
Ofsmooth pursuit movement，light−neardissociationandinclusionbodyofthethalamus，thatsuggest
CentralnervOuS SyStem（CNS）invoIvementinthisdisease．WeexaminedCNS ofMDbycomputerized
tomography（CT）．
Materialsandmethods
Acontrolgroup（C）andanMD−patientgroupwereusedforthisstudy．TheCgroupconsistedof16
malesand15femaleswhodidn

thaveanyorganicdiseases．TheMDgroupconsistedoflOmalesanda

female．TheexaminationswerecarriedwithCT−SCannerS，EMIlOlO（160×160matrix）andDELTA50
FS（256×256matriⅩ）．Theslicewidthswere10mmand8mm，reSPeCtively．TheangleofCTwas150

0Verthecanthomeatalline．Measurementsweremadeonthex−rayfilmsusingatranslucentmillimeterrule．IneachCT，thefollowlngdimensionsweremeasured：A．themaximumtransverseinnerdiameterof
the skull，B・the maximum width of cella media，C・the maximum distanCe between the tipsofthe
anterior horns，D．themaximumwidthofthe thirdventricle andE．themaximumwidthoftheinter−hemisphericfissure．Fromtheabsolutevaluesoftheabovedata，（1）thecellamediaindex（B／A），（2）theanteriorhornindex（C／A）and（3）thethirdventricleindex（D／A）werederived．
Results
Patients with MD generally showed dilatation of the ventricular system on CT．The cellamedia
indicesofthefifthandsixthdecadeswereslgnificantlygreaterthanthoseoftheCgroupsofcorrespond−
ing ages（Tablel）．Likewise，theanteriOrhornindicesweregreaterinpatients ofthefourthandsixth
decadesthan age−matChed controIs．ThethirdventricleindicesinMDweregreaterinallagegroupsandthedifferencesbetweenMDandCgroupsweremostprominent．Ontheotherhand，theinterhemisphe−riC fissures were only slightlylarger，thoughnotsignificantly，inMDgroups，PrObablybecauseofthe
difficultyinmeasurements．
Tablel IndicesoftheventricularsystemonCTAge
30 −39Y．
0．
40−49 Y．
0．
Sex
M ale
Fem aleM ale
Fem aleM D 24．
3±3．
4 ／
30．
8±
4．
6＊ 25．
6（1） C
18．
8 ±3 ．
820．
7 ± 1．
62 1．
5 ±2 ．
72 2．
6 ±2 ．
4
M D

3 0．
9 ±3 ．
0＊C

2 5．
5 ±2 ．
3

M D

4．
8 ±1 ．
7＊ ／C

2．
5 ±0 ．
5

（
2）

（
3）

2 1．
3 ±6 ．
82．
4 ±0 ．
1

M±SE％

50 −59 Y．
0．
Male
Female
29．
4±
3．
8＊／
2 1．
1 ±2 ．
230 ．
6 ±4 ．
7

28 ．
2

3 1．
4 ±2 ．
3＊

25 ．
9 ±3 ．
026 ．
6 ±1 ．
1

26 ．
5 ±1 ．
2

5．
7 ±2 ．
4＊3．
9

6．
6 ±1 ．
7＊2．
6 ±0 ．
52．
3 ±0 ．
93．
1 ±1 ．
0＊significant（P＜0．05）

−34−

25 ．
4 ±3 ．
725 ．
8 ±3 ．
1

3．
2 ±1 ．
1DisellSSion
The present study demonstrates the greater third ventricleindicesin MD patients thanthoseof
COntrOIs．Theenlargementofthethirdventricleisespeciallyremarkable．PatientswithMDareknobnto
have hypertrophicchangesoftheskulldue topossible abnormalsecretionofgrowthhormone．Inour
Studies〉howeverタthe

maximumtransverseinnerdiameteroftheskullin

MD

was

not

significantly

different fromthatintheCgroup．Thenweused thisdiametertocalculatevariousindiceswhichwere
alsoeasytomeasure．PneumoencephalographicstudiesinMDrevealedthedilatationofthethirdventri一

Cleandthebodyofthelateralventricle．TheseobservationsarecompatiblewithourCTfindings．The
enlargement of the third ventriCle seems particularlylmPOrtant，Since patients with MD often showmanyneuroendocrinologicalabnormalitieswhichcanbeascribedtothedysfunctionofthediencephalon．
Itispossiblethatthedilatationofthethirdventricleisofsignificance，SPeCifical1yforMD・However，CTnndingschangewithageandvariousstates．Itisimportant，therefore，tOStudythecorrelationbetween
thedilatationoftheventricularsystemanddurationofthediseaseorneuroendocrinologicalfindings．
Condusion
Patients with MD showed enlargement oftheventricularsystem，eSpeCiallythethirdventricleon
CT，SuggeStingtheorganicchangeoftheNCS．
Referencesl）Refsum，S・，Engest，A．＆L6nnum，A．：Pneumoencephalographicchangesindystrophiamyotonia．ActaPsychiatr．
Scand．（Suppl．137），34：98，1959．
2）Refsum，S．，L6nnum，A．，Siaastad，0．，etal．：Dystrophiamyotonia．Neurology，17：345，1967．
3）Haug，G・：AgeandsexdependenceofthesizeofnormalYentriclesoncomputerizedtomogTaPhy．Neuroradiology，
14：201．1977．

−35−

肋gC〟krqyg汁Op妙Gr0乙Jp

ANUNUSUALCASEWITHFACIO−SCAPULO−HUMERALMUSCULARATROPHY

UedaKeishi＊，HatanakaYoshio＊，NQjimaMotoo＊＊andInoueMasafumi＊＊＊HealthAdministrationCenter，EhimeUniversity，Matsuyama＊＊OrthopedicDepartment，EhimeUniversitySchoolofMedicine，Matsuyama

Introduction
Progressive muscular atrophy was regarded as a neurogenic ormyogenicdisorderandithasbeen
known that neurogenic atrophyis remarkablein the distalportions of extremities，While myogenic
atrophyis predominantin proximal portions．However，the relationship between neurogenic andmyogenicatrophyhasbeendiscussed on thebasisoftheageofonset，muSCleinvoIvements，prOgreSSion
andgenetics，andmanycaseshavebeenstudiedbyresearchersinordertocometoanagreementl，2）AsforcaseswithmuscularatrophyinvoIvingthe face，Shoulderand humerus，theyhavebeensub−
dividedinto three groups，i．e．facio−SCapulo−thigh type，facio−SCaPulo−PerOneal type and scapulo−
PerOnealtype，andfurthermorecasesofeachgroupwerealsodifferent．
Anunusualcasewithfacio−SCapulo−humeralmuscularatrophyispresentedhere．
Casereport
A30−year−Old manwascompletelynormalinchildhood．Attheageof15，hecouldnotliftaheavy
Object above his head and became gradually unable toelevatehis arms．At theage of17，hebecame
unable torunfast and had difficultyin dorso−flexion ofhis anklejointsand climbingsteps．Attheage
Of29，he fell frequently and had difficultylneXtendinghislefthand，and then showed facialmuscle
atrophy．
His past history was unremarkable．His parents were not consanguineous．His one elder brother
COmplained of gait disturbance．Generalphysicalexamination was normal．NeurologiCalexamination
revealed onlyfacialmuscularweaknessinthe cranialnerve territory and he couldnotclosehiseye−1ids
or whistle．Evaluation of motor function revealed weakness of thedeltoids，biceps，triceps，rightforearmextensors，hip flexorsand adduc−
tors，quadricepsandtibialextensors．Muscularatrophywasremarkablein
the proximal portions of the extremities except for tibial atrophy．
Fasciculation，SenSOry disturbance，ataXla，lnVOluntary movements andjoint contractures were not observed．All deep tendon reflexes weredecreased．Hehad awaddlinggait，hadtogetupfromthefl00rWiththe
helpofhishandsandcouldnotstandonhistoes．
LaboratoryexaminationsshowedanormalhemogramandurinalysIS．
The erythrocyte sedimentation rate was4mm／hr．Latex agglutinations
for rheumatoid arthritisandtheantinuclear antibody test were negative
andliver function，Serum electrolytes and endocrinologicalstudieswere
allnormal．SerumCPKwas154u．（normal＜70）andaldolasewas3．1u．
（normal＜2．2）．Daily urinary excretion of creatine was610mg／day．
Chest，CerVicalandlumbalX−raySandECGwerenormal．
Electromyography ofthe biceps，triceps，forearm extensors，quadri−

CepS，gaStrOCnemius and tibial anterior revealed no fibrillationsatrest，
andlarge action potentials and fullinterferencepatternswithpartiallylowamplitudeatmaximumvoluntarycontraction．

Frozen sectionsofbiopsied specimensfrom therightgastrocnemius）：ig．3Oycarsold，maleWere uSed forexaminationsincludingH．E．staining，mOdified trichrome−36−

Staining and SDH andATPase（pH9．4）staining．H．E．stainingshowedalmostanormalhistogram andOnlysporadicallyfloccularchange．ATPaseandmodifiedtrichromestainingwerealsonormal．
Comment
The characteristicfeaturesofourcasewereasfollows：（l）onset at15years−01d，（2）slowprogres−Sion，（3）slightelevationsofserumenZymelevels，（4）unusualmuscleinvoIvementsasdescribedabove，（5）electromyographicfindingsnotcompatibiらwithtypicalneurogenicormyogenicchanges，and（6）almostnormalhistologicalfindingswiththesocalled

floccularchange．

Benign muscular atrophyinvoIving the face，Shoulder and humeruS has been reported as facio−
SCapulo−humeralmuscular dystrophy，Kugelberg−Welanderdisease（K−Wdisease），Chronicpolymyositis，
scapul0−PerOnealatrophyandEmery−Dreifussdisease3）．Amongthesediseases，Chronicpolymyositiswould
Show skin rash，muSClepain，increasederythrocytesedimentationrateandpositivelatexagglutination
for rheumatoid antibody．Emery−Dreifuss disease would showjoint contractures anddisordersofthe
Cardialconductionsystem，however，thesefindingscouldnotbefoundinourcase．
Hischaracteristics，eSPeCially findings（2），（3），（5）and（6）described aboveleadustoincludehimWithinthecategoryofK−Wdisease，althoughhismuscleinvoIvementisapartfromtheclassicalformof
KJWdiseasewhichsimulateslimb−girdlemusculardystrophy．
Emery4）categorizedK−Wdiseaseasoneofthesubgroupsofchronicspinalmuscularatrophy，While
Furukawa5）tentativelydividedK−Wdiseaseinto4subgroupsontheformsofthemuscleinvoIvementsin
musculardystrophy．

Astomuscleatrophyofthefacio−SCaPulo−humeraltypeofthisorigin，thevariousformshavebeen
SubsequentlyreportedasmoredividedsubtypeswithorwithoutmuscleinvoIvementofface，Ofthigh，Or
Oflowerleg．OurcaseshoYedmuscleatrophyinproximalportionsoftheupperextremitiesatfirst，thenin thelowerlegs，thighS，face and forearmsin that order．Considering these points，facio−SCapulo−
humeralmuscularatrophymayhavetoberegardedasonecategory．
Therefore，itisnecessarythatwestudy morecasesoverlongertermsinordertodeterminewhich
facio−SCaPulo−humeralatrophyshouldbelumpedintoonecategoryordividedintomoresubgroups．
Referencesl）Walton，J．N．：DisordersofvoluntaTymuSCle，3rdEdition，Church止1，London，1974，P．870．
2）Furukawa，T．：ClassificationsofspinalprogressivemusculaJatrOPhiesakintoKugelberg−Welanderdisease．Neurol．
Med．，12：319，1980．
3）Rowland，L．P．，Fetell，M．，01arte，M，，etal．：Emery−Dreifussmusculardystrophy．Ann．Neuro1．，5：111，1979．
4）Fried，K．andEmery，A．E．H．：Spinalmuscularatrophytype2，aSeparategeneticandclinicalentityfromtype1（Werdnig・Hoffmandisease）andtype3（Kugelberg−Welanderdisease）．Clin．Genet．，2：203，1971．
5）Furukawa，T．，Tsukagoshi，H．，Sugita，H．，etal．：Neurogenicmuscularatrophysimultatingfacio−SCaPulo−humeral
musculardystrophy，withparticulaHeferencetotheheterogeneityofKugelbergJWelanderdisease．J．Neurol．Sci．，
9：389，1969．

−37−

肋gC〟kr功gfropカツGro乙Jp

CENTRONUCLEAR如YOPATTIYADISTALFORMWITHJUVENmEONSETANDNOOCULARINVOLVEMENT
KeiichiTakahashi，KazukoGoto，KertjiJinnai，FumikoShundo，YukihiroKomineandTakuoFujita
DepartmentofMedicine，KobeUniversity，Kobe
SincethefirstreportofmyotubularmyopathybySpiroandhiscolleaguesin19661），mOrethan
50patientswithmyotubularorcentronuclearmyopathyhavebeenreported・Theclinicalmanifestations
ofthismyopathyarequitevariable2），COmmOnlyconsistingofptosisandweaknessofextraocular，facial，palataland masticatory muscles・In this communication，We rePOrt the second casecharacterizedby
juvenileonset，distalatrophyandabsenceofocularinvoIvement．
Atwenty−SeVen yearOldmanstartedtoexperiencedifficultyindorsiflexionoftheleftankleatage
12，andnoticeddifficultylnClimbingastaircaseatage16andinachinningexerciseatage20・Heneverexperienced double vision or difficultyln SWallowing・Physical examination showed normalcranial
nerveS，mOderate atrophy ofbilateralforearmsandlegswithslightcontractureoftheleftanklejoint．
Onmanualmuscletesting，generalizedweakness（4）anddistinctdistalweakness（3）weredisclosedwith
hypoactive deeptendonreflexes．Neithercerebellarsignnorsensoryabnormalitywaspresent・Labora−
tory studies showednormalCK（43IU），Slightlyelevatedserumcreatine（1．0mg／dl），Creatinuria（145mg／day）and decreased urine creatinine（417mg／day）．Electromyography showed fibrillation voltage
intheleftanteriortibialmuscleandmyopathicpatternsintheothermuscles・Biopsywasperformedon
the right anterior tibial muscle．H＆E and Gomoris trichrome stainings disclosed central nuclei，

myotubularstruCture，Variablesizeofmusclefibersandfattydegeneration（Fig．1−a）．ATPasestainings
atpH9．4，4．6and4．3andNADHreactionrevealedsmallfiberswithcentralnuclei，COnSistedofboth
typeI and typeII fibers（Fig．1−b）．An electron microscopic study showed no abnormalitiesin
mitochondriaexceptfortheirdeformity，Withoutatendencyofperinuclearaccumulation．Moxleyetal▼

b：ATPase（PH9．4）×200

Fig．1a：Trichromestain X200
−38−

reportedin1978a case with a similar clinicalpicture，but they noticed granularmaterialandmito−

chondriaaroundthenucleus3）・Astothepathogenesis，functionaldenervationduringthemyogenesisis
POStulated．
Rererencesl）Spiro，A．J．，Shy，G．M．andGonatas，N．K．：Myotubularmyopathy．Arch．Neuro1．，14：1．1966
2）Raju，T・N・K・，Vidyasagar，D・，Rayes，M．G．et al．：CentronuclearmyopathyinthenewbornperiOdcausingsevere
respiratorydistress．Pediatrics59：29，1977
3）Moxley，R・T，，III，Griffs，R．C．，Markesbery，W．R．，etal．：Metabolicimplicationsofdistalatrophy．J．NeuroISci．
39：247，1978
Tllis study was supportedby Grant No．80−02−27from the NationalCenter for Nervous，Mentaland Muscular
Disorders（NCNMMD）oftheMinistryofHealthandWelfare．Japan−39−

肋gC〟肋r砂JrrOP妙Gro岬

ACASEOFATYPICALMYOPATHYWITHCONTRACTURESOFJOINTSANDCARDIAC
INVOLVEMENT

HiroshiTsukagoshi＊，KatsuhikoKayanuma＊＊，KimioOguchi＊＊，NobuoYanagisawa＊＊
andMorieSekiguchi＊＊＊＊DepartmentofNeurology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo＊＊DepartmentofMedicine（Neurology），ShinshuUniversitySchoolofMedicine，Matsumoto＊＊＊DepartmentofMedicine，TheHeartInstituteofJapan，TokyoWomensMedicalCollege，Tokyo

AcaseofatyplCalmyopathywithcardiacconductionabnormalities，myOCardialdiseaseandtricus−
Pidregurgitation（TR）wasreported，a27−year−01dfemale．
Sheshowedunsteadygaitatthe ageofthree・Atagefive，itwasnoticedshehadmusclewastingof
theextremities・Whenshewas15yearsold，WaStingofthefacialmusclesdeveloped．Atage22，Shewas
admitted to ahospitalbecauseofpalpitationandgeneraledema．Atthistime，aChestX−ray disclosed
marked cardiomegaly and creatinephosphokinase（CK）was38U（0−25）．Shewasreferred to ourclinic
forfurtherevaluationofmuscularweaknessandcardiacdiseaseatage24，OnMay26，1977，An Adams−
Stokesattackoccuredandapermanentpacemakerwasinserted，atage26・Attheageof27，Shediedof
heart failure．

No consanguinitywasfoundinherparents，bothofwhomhadmarkedcardiomegaly；hermotherhadessentialhypertensionandherfatherischemicheartdiseasedisclosedbyECG．CK forhermother
WaS27U（0−25）．They had neithermuscularweaknessnorEMG abnormalities．Hertwobrotherswere
healthy．
NeurologlCal examination at age

24revealed moderate to severe wasting and weakness of themusclesofthelimbgirdlesandfourextremities，Predominantlylntheproximalmuscles，mildwastingof

facialmuscles，and contracturesofneck，elbow，hip andknee
joints（Fig．1）．There was no hypertrophy of the calves．All
deep tendon reflexes were absent．Pathologicalreflexes and
SenSOry disturbance were not observed．CK was127IU（30−230）．EMG showed short duration andlow amplitude
POtentials．Biopsy of the biceps brachii muscle revealed
moderate non−SpeCificmyopathicchange，derangementoftheinterfibrillar network onNADH stainingand typeIpredomi−nance onATPasestaining，SuggeStingmyopathy．Theheredity，Clinicalcourse，distribution ofmuscularatrophyandCKvalue
are not compatible with any type of congenital myopathy，
arthrogryposis multiplex congenita，myOtOnic muscular
dystrophy，Duchenne，facioscapulohumeral orlimb−girdle
muscular dystrophy，Or Cardiopathic muscular dystrophy（Norris et al．）1）．Our case resembles malignantlimb−girdle
musculardystrophy described byMiyoshiet al．2）．However，
theCKvalue，facialwastingandheartdiseasearenotcompati−
blewiththistype．

Cardiac examination revealed right−Sided heart failure，
tricuspid regurgitant murmur and globular cardiomegaly

Fig．l PllOtOgraphs ofpatientshowingcon−
tractures of elbow，hip and knee
JOints，and wastingoffacialmuscles，
limb一girdlesandextremities．

（CTR63％）．ECGshowedatrialflutter，COmPleteatrioventric−ular block，idioventricular rhythm with QRS patterns of
COmPleteleft

一40−

bundlebranchblock

andventricularex一trasystoles・Right heart catheterization and ventriculography disclosedisolated TR．Rightventricularendomyocardialbiopsyrevealed hypertrophy and degeneration ofmyocytes，derangement ofmuscle
bundles，interstitial fibrosis and endomyocardialthickening，and electron microscopIC eXamination

disclosedsparsityofmyofibrils，wideningofintercalateddiscs，1nCreaSeOfglycogendeposition，mito−
Chondrialchange，anddilatationandincreaseofthesarcotubularsystem，SuggeStingmyocardialdisease．
Thus・her cardiac disease was regarded as asecondarymyocardialdiseaseaccompaniedbyatypicalmyopathy・SeveralCases of degenerative neuromuscular disease withmitralregurgitation have beenreported3），butisolatedTRisextremelyrare．
ClinicalcharacteristicsofourcaseresembleEmery−Dreifussmusculardystrophy4・5），Whichconsists
Ofthefollowingcomponents：（1）X−linkedrecessiveinheritance，（2）contracturesatelbows，anklesandneckappearingearlyinchildhood，（3）thedistributionofweakness，affectingarmmusclesmoreseverely
than scapular musclesin the upperlimbs，in thelowerlimbs distalmuscles are affectedearlierthan
proximalmuscles，（4）veryslowlyprogressivecourse，and（5）cardiacconductionabnormalitiesappear
universallyinaffectedindividualsleadingultimatelytopersistentatrialarrest．Thissyndromeisregarded
as a form ofX−linked musculardystrophy，althoughhistologicalandEMGexaminationsrevealeither
myopathic orneurogenicpatternsormixturesofthe two・Ourcase differsfromitin thefollowlng
POints：（1）ShewasasporadiccaseandnotTurnerssyndrome，（2）contracturesathipandkneejoints

WereprOminentandanklejointswereintact，and（3）complicationofTR，Ourcaseisconsideredatypicalmyopathywithcontracturesofjointsand cardiacinvoIvement，nOtbeingEmery−Dreifusssyndrome，althoughthepossibilitythateitherhermotherisacarrierorherfatherhasanincompleteformofthis
Syndromecannotberuledout．
Rererence5
1）NorriS，F・H・，Moss，A・J・＆Yu，P・N・：Onthepossibilitythatatypeofhumanmusculardystrophycommencesin
myocardium．Ann．N．Y．Acad．Sci．，138：342，1966．
2）Miyoshi，K・，Tada，YりNakano，M・，etal．‥Malignantlimb−girdletypeofprogressivemusculardyitrophy．nle

existenceofthistypeandthesignificanceintheclassincationofPMD・TheproceedingsoftheMuscularDystrophy
ResearchGroupinJapan．（II）．1974，p．39．
3）Perloff，J・K・，Roberts，W・C・，DeLeon，A．C．，etal．‥rrhedistinctiveelectrocardiogramofDuchenne

sprogressive

muscula∫dystrophy．Am．J．Med．，42：179，1967．
4）Emery，A・E・IL＆Dreifuss，F・E・：UnusualtypeofbenignX−Linkedmusculardystrophy．J．Neurol．Neurosurg．
Psychiatfy，29：338，1966．
5）Rowland，LP・，Fete11，M・，01arte，M．，etal．：Emery・Dreifussmusculardystrophy．Ann．Neuro1．，5：111，1979．

ー41−

肋∫C〟krの∫rrOp力γGro岬

MUSCLE PATHOLOGY OF DISTAL MYOPATHY

YasuoToyokura，HiroshiKurisaki＊andMasamiTakatsu

DepartmentofNeurology，InstituteofBrainResearch，FacultyofMedicine，UniversityofTokyo・Tokyo＊DepartmentofNeurology，TokyoNationalHospital，Tokyo

Introduction

DistalmyopathywasestablishedasadistinctentitybyWelanderin1951・Therefollowedreports
ofthediseasevarylnginheredity，ageOfonsetanditsclinicalcourse，indicatingtheclinicalheter0−
geneityofthisdisorder・Westudieddistalmyopathytoseewhetherthereareanycommonfeaturesor
notfromtheviewpointofmusclepathology・
Materialsandmethods
Thirteenmuscleswereobtainedfromninepatientswithclinicaldiagnosisofdistalmyopathy・They
werefrom23to37yearsold．Twoofthemweremaleandsevenwerefemale・Onlyoneofthecases
hadafamilyhistoryofautosomalrecessiveinheritance・ThesitesofbiopsywereM・bicepsbrachii，M・
quadricepsfemorisorM・gaStrOCnemius・In4cases，muSCleswereobtainedfromtwodifferentsites・
Onepartofeachbiopsiedmusclewasfreshlyfrozenandstudiedhistologicallyandhistochemically・
The otherpart was fixedin formalin andinglutaraldehyde，andstudiedbylightmicroscopyand
electronmicroscopy．
Results
Allthemusclesexamined showed essentiallyidenticalchanges，althoughtheyvariedindegreeofchanges．Inseverercases，almostallmusclefiberswerereplacedbyfattissues・Thedistalmuscleswere
more severely affected than proximalmuscleswhen different muscles were examinedinthesame
patient．Nonspecificdystrophicchangesobservedinallthemusclesincludedmarkedvariationinfibersize，fibersplittingandnecroticfibers・Centralnuclei，Pyknoticcellclumpsandphagocytosisofmuscle
CASES（％）
VacuoleMUSCLES（％）

9／9（100）13／13（100）NADH⊥TRhyperactive
fibers9Myeloidbody（100）136（67）Fibersplitting

99（100）11

（100）
（69）
（85）

FibernecroSis
9（100）10（77）

8（89）10

（77）

7

（78）

9

（69）

8

（89）11

（85）

3

（33）

（23）

Phagocytosis
Centralnuclei
Pyknoticcellclumps
Ringfiber
Cellinfiltration

2（22）Jr十十四十Fig●□±［∃EMstudynotperformed
Musclepathologyofdistalmyopathy

−42−

3
2

（15）

fiberswerepresentinmostcases・Phagocytosedmusclefibersshowedacidphosphataseactivity．Ringfiberswerepresentinthreecases，andmildcellinfiltrationwasobservedintwocases，Whichdidnot
respondtosteroidtherapy．
The characteristicchangesinourcases werethepresenceofvacuolesandNADH−TRhyperactive
fibers・Vacuolesvariedinsizeandweresituatedeithercentrallyorperipherally．Theywerenegativefor
PASandOilRedOstain・TherimofvacuoleswasstainedbasophilicwithH＆E，andredwithmodified
Gomoritrichrome・Thevacuoleswereacidphosphatasepositive・Theywerepresentineithertypelor
type2fibers・Onelectronmicroscopy，thesevacuoleswereorwerenotboundbyasinglemembrane．
They contained myeloid bodies，degraded mitochondria or other membranousstruCtureS，degradedfibrousbodiesandothercytoplasmicdegradedproducts・Somemyeloidbodieswerepresentindepend−
entlyofvacuoles．
TheothercharacteristicchangewasNADH−TRhyperactivefibersresemblingsmal1angulatedfibers．
However，theydifferedfromthoseappearingondenervationintherespectthatmostofthemcontained
VaCuOles・AIso，manyOfthesefibersseemedtobederivedfromtheneighboringfibersbysplitting．
Discussionandconclusion
RoutinehistopathologiCalstudiesinbothsporadicandinheritedcasesofdistalmyopathyrevealed
thatthecommonmusclepathologywasdystrophicchanges・Vacuoleswerepresentinsomecases，but
Wereabsentinothers・Forexample，Welanderdescribedthepresenceofvacuolesinsomecasesin1951，
but Edstr6m failed to find vacuolesin his histologicalandhistochemicalstudiesofWelandertype
distalmyopathyin Sweden，andheconcludedthatthecharacteristicmusclepathologywastypel
atrophyresemblingmyotonicdystrophy・TheJapanese，autOSOmalrecessivedistalmyopathydescribedbyMiyoshialsohasnovacuoles．
In contrast，OurCaSeSWereCharacterizedbyvacuolesandNADH−TRhyperactivefibers．Thesame
histochemicalandultrastruCturalchangesofdistalmyopathyweredescribedbyMarkesberyandSuno−hara・Thesevacuoleswere characteriZed byacidphosphataseactivityandbythecontentofdegraded
SubstancesrevealedbyelectronmicroscopICStudies，andthoughttobeautophagicvacuoles

NADH−TRhyperactive fiberswere also thought to beexocytoseddegradationproductsfromtheirfibersplitting
nature・Thesedegeneratingprocessesofdistalmyopathyshouldbedifferentiatedfromtheothertypes
Ofmusculardystrophy．
Inconclusion，itisouroplnlOnthattherearetwotypesofdistalmyopathyinviewofthemuSCle
Pathology，1・e・，Withorwithoutvacuoles．However，nOdistinctcorrelationhassofarbeenfoundbetween
thedifferentmusclepathologyandclinicalheterogeneityofthedisease．

ー43−

肋gC〟kr勒∫rrOp妙Gr（フ岬DISTALMYOPATHYANDLYSOSOMES
ToshihideKumamotoandNobuyoshiFukuhara
DepartmentofNeurology，BminResearchInstitute，NiigataUniversity・Niigata
Thelysosome−SyStemPlaysanimportantroleintheproteincatabolismormetabolicprocessinthe
cytoplasmofthecel1，0ntheotherhand，thissystemissuspectedtoparticIPateincellinjuryorfocal
cytoplasmicdegradationincells，includingmusclefibers（deDuveandWattiaux，19661）；Pitt，19752））・
Thepurposeofthisstudywastoinvestigatetheparticipationofthelysosome−SySteminthemuscle
fibersofvariousmyopathies，mainlyofdistalmyopathy，inviewofHdegradation

・Materialsandmethods
17musclespecimensfrompatientswithmusculardystrophieswerestudied・TheylnCluded70fthe
Duchennetype，20ftheBeckertype，30fthefacioscapulohumeral（FSH）type，30fdistalmyopathy，
and20fmyotonicdystrophy（MD）．Eachpatientshowedalmosttypicalclinicalfeatures，reSPeCtively・Oneofthe3Caseswithdistalmyopathywassporadic，butthedther2Werefamilial，andtheirparents
were first cousinsand three oftheir five siblingshad asimilarneuromusculardisease・Ina113cases，
muscleweaknessandwastingstartedinthelegswithsubsequentinvoIvementofarmsataround23to
30yearsorage．
BiopsleSWerePerformedunderlocalanesthesiaontherectusfemorisoronthebicepsbrachii・Specimenswerefrozeninisopentanecoo11edindry−ice・Transversecryostatsectionswereprocessedfor
routinestaining（DubowitzandBrooke，19733））・Forelectronmicroscopicalobservation，tissueslices
werefixedin2．5％glutaraldehydeinO．lMphosphatebuffer（pH7・3），POStfixedinl％OsO4SOlutionin
O．lMphosphatebuffer，dehydratedingradedalcohol・andembeddedinepon−araldite・Ultrathinsec−
tionswerestainedwithuranylacetateandleadcitrate．AIso，aCidphosphatase（ACPase）preparations，amarkerenzymeoflysosome，WereStained（BarkaandAnderson，19624））・
Results

Histopathologicalfindingsin themusclesoftheDuchenne，BeckerandFSHtypesofmuscular
dystrophyandMDshowedcharacteristicfindingsofeachdisease，aSrePOrtedbymanyauthors・Indistalmyopathy，lightmicroscopyshowedmildormoderatevariationofthefibersize，anda
numberofsmallangulatedfiberssporadically・Therewasamoderateincreaseofcentrallyplacednuclei
in the muscle fibers．Simple necrosis and fiber splittingwereoftenshown・Therewasnoevidence
suggestinganinflammatoryreaction・Macrophageswerescarcelyrecognized・Themoststrikingfinding
forthemusclefibersofallthreecaseswasrimmedvacuoles（DubowitzandBrooke，19733）；Fukuharaetal．，19805）），Whichwerelocatedinthecenterorperipheryofmanysmallornormal−Sizedfibers・These
vacuoleswereringedbymaterialthatwasintenselybasophilicinH・E・PreParationsandredinGomori
trichrome．TheATPaseandDPNHpreparationsshowedthatbothfibertypesinvoIvedvacuoles，butthey
weremorefrequentintypeIthantypeIIfibers・Thefiberswithvacuoleswereatrophicandstained
darklybyDPNHoccasionally．
ACPasepreparationsshowed almostnegativestaininginsideofthevacuoles・ApositiveACPase
reactionwasseeninthemarglnOfsomevacuoles〉butrarely・Underanelectronmicroscopeinthesevacuolescouldbeseen．membranouslamellarstruCtureSOfvarioussizesandshapesandvariousheter0−
genousmaterials，Whichwereenclosedbyalimitingmembrane・Frequencies ofrimmedvacuolesand ACPasepositive fibersineachmusclestudiedaregivenin
Tablel．
IntheDuchenne，BeckerandFSHtypesofmusculardystrophy，rimmedvacuoleswerenotfound，−44−

Tablel Frequency（％）ofrimmedvacuolesandacidphosphatase（ACPase）positivefibers
M aterials

N o ．or cases

Progressive m u scular dy stroph y（
except fo r dista
l m yop ath y ）R im m ed V acu oles
m ea
n ±S D （m in．
一m aX ．
）（％）

A C P ase p ositive Lib ers
m ean ±SD （m in ．
−m aX ．
）
（
％）

12

0

0．
01 ±0 ．
14 （
0−
0．
3）

D uchenn e typ e

7

0

0．
13 ±0 ．
16 （
0−
0．
3）

B eck er typ e

2

0

0

F S H ty pe

3

0

0．
0 8 ±0 ．
15 （
0−
0．
3）

D istal m yo pathy

35．
10 ±8．
05 （
0．
4−
14 ．
4）

15．
6 0 ±7 ．
00 （
8．
5−
22．
5）

M y o tonic d y stro ph y

2

0

4．
6 5 ±0 ．
49 （4 ふ 5．
0）andACPasepositivefiberswereseeninonlyO．1％ofthefibers．AlthoughACPaseactivitywasslightly，
presentandwasrecognizedintheperisarcolemmalregions．Ofcourse，markedactivitywasshowninthe
CytOPlasmofnumerousmacrophages・MDalsoshowednorimmedvacuoles，butshowedACPasepositive
fibersin4・65±0・43％・ACPaseactivitywasalmostallfoundinthe degeneratingfibers，particularlyaroundtheinternalnucleiorinthemacrophagesinvadingthemusclefibers・tndistalmyopathyrimmed
VaCuOles were seenin5．10±8．05％and muscle fiberswith ACPase positivegranuleswerenotedin
15・60±7・00％・ACPasepreparationsshowedpositivegranulesinsideofthealmostnorma1−100kingfibers
aswellasiIithefiberswithrimmedvacuoles．
Discussion
In thepresentstudy，numerOuSrimmedvacuoleswerefoundindistalmyopathy．ACPaseactivity
WaSrarelyfoundinthemarginsoftherimmedvacuoles，butelectronmicroscopicalstudiesshowedsuch
VaCuOleswhichwerepresumedtobeautophagicinnature・Therefore，rimmedvacuoleswerepresumed
to be autophagiC vacuoles or secondarylysosome，Without or with ACPase activity（deDuve andWattiaux，19661））・Ontheotherhand，numerOuSmuSClefibersinourcaseshadACPasepositive
granules・ACPaseisoneoftheratherspecificlysosomalenzymesandworksasagoodmarkerforlysos0−malactivity（EssnerandNovikoff，19616）；HoltandHicks，19617））．Inadditiontonumerousautophagic
VaCuOlesandtheincreasedactivityofACPase，fewmacrophageswerefoundindistalmyopathy．Inothermusculardystrophiesrimmedvacuoleswereseldomseenexceptinoculopharyngealmusculardystrophy（DubowitzandBrooke，19733））andACPasewasalsonotincreasedexceptinthenecroticfibersinvaded
bymacrophages．
ConclllSions
The degenerative processin distalmyopathy appeared to progress mainlythroughthelysosomal
SyStemandmightdifferfromothermusculardystrophies．
Referencesl）deDuve，C．＆Wattiaux，R．：Functionsoflysosomes，Annu．Rev．Physiolリ28：435，1966．
2）Pitt，D・：LysosomesandCellFunction，Longman，London，1975．
3）Dubowitz，V・＆Brooke，M・H．：MuscleBiopsy−AModernApproach，Saunders，London，1973．
4）Barka，T・＆Anderson，P・J・：Histochemicalmethodsforacidphosphataseusinghexazoniumpararosanilinascoupler，
J．Histochem．Cytochem．，10；741，1962．
5）Fukuhara，N・，Kumamoto，T．＆Tubaki，T．：RimmedVacuoles，ActaNeuropathol．（Berl．），51：229，1980．
6）Essner，E・＆Novikoff，A．B．：LocalizationofacidphosphataseactivityinhepaticLysosomesbymeansofelectron
microscopy，J．biophys．biochem．Cyto1．，9：773，1961．
7）Holt，S・J・＆Hicks，R．M．：Thelocalizationofacidphosphataseinfatlivercellasrevealed．bycombinedcytochemi−
Calstainingandelectronmicroscopy，J．biophys．biochemCyto1．，11：47，1961．

ー45−

肋JC乙

加勒∫汁Op妙Groェ岬

TUBULOMEMBRANOUS MYOPATHY

NobuyoshiFukuhara＊，ToshihideKumamoto＊，HirooHirahara＊andTadaoTsubaki＊，＊＊＊DepartmentofNeurology，BrainResearchInstitute，NiigataUniversity，Niigata＊＊TokyoMetropolitanNeurologicalHospital，Tokyo

Recent histochemical and ultrastructural studiesonskeletalmuscle revealed manyneuromusculardiseasesasclinico−Pathologicalentities．We

have

reported

anew

progressive

myopathywith

tubulo一

membranousinclusionsl）．Theoriginandnatureoftheinclusionsareobscure，butitwouldbeworth−whiletoreportthemorphologicalfeaturesoftheskeletalmuscle・Casehistory
Aboy，bornin1961，WaSadmittedtoNiigataUniversityHospitalfordisturbanceofgaitattheageof16．Hisparentswerefirstcousinsonceremoved・HewastheslowestrunneratSChool，buthisparents
didnotregardhimasabnormalbecausetheyalsohadpoorexerciseabilities・Sincetheageof15，heranlesseasilythanbefore，Walkedwithawaddlinggait，andgottiredveryeasily・Mildweaknessandwasting
were notedin the proximallimbmusclesandfacialmuscles・Laboratorydatawerenormalexceptfor
slightlyincreased serum CPK・Needle EMG showed myopathic featuresin severalmusclest Nerve
conduction velocities were normal．Leukocytelysosomal enzyme analysIS reVealed normallevels ofarylsulfataseAand B，α−andβ−galactosidase，andN−aCetyl−β−D−glucosaminidase・Ultrastructuralstudies
onperipheralbloodlymphocytesandskinalsorevealednoabnormalinclusions・Atrialofprednisolone
had・nOSignificanteffectonmusclestrength・Furtherprogressionofthemusclewastingwasnotedinthe
lowerextremitiesafterthattime．Hewalkedwithamarkedwaddlinggaitandneededsupport・Amarked
increaseofthelumbarlordosisandaprominentGowers

signwerenoted．SerumCPKwas1236mU／ml（normal：belowlOO）．
MorphologiCalstudies
A muscle biopsywasperformed on theleftquadricepsfemoris・LightmicroscopICalobservations
showed moderate variation of fiber diameters，eXtenSive fiber−Splitting and centralnucleiwithmild
endomysialfibrosis．Numerous rimmed vacuoles2，3）were seen・Insemi−thinepoxysectionsstained
withtoluidine blue，lntraSarCOPlasmicinclusionsstained faintly，meaSuring2・5pm bylOpm at thegreatest diameter，Were nOtedin40−50％offibers ofwhichthearchitecturewasotherwisenormal・PAS preparations did not show any
abnormal accumulation ofglycogen．The
most remarkable findings under an elec−tron miscroscope were numerous abnor−
malinclusions consisting oflaminatedtubulomembranous structures（TMS）（Fig．1）．Thelame11ae were regularly
spaced with a periodicity of8・5−9nm・Theselaminated TMS were sometimes
curvedalittleandinplacestheyappeared
as concentriclamellae according to theplane ofsectioning・The second type ofinclusion consisted of curvifilamentous
materials abutting on to the typeI
inclusions．The diameter ofeachfilament
Fig・l Tubulomembranousstructures X18，000−46−

WaS3−6nm・The3rdtypeofinclusionwasmoderatelyelectron−densegranularmaterialsurrOundedby
a single umit membrane wllich sometimesincludedlamellarstructuresidenticalwiththoseinthefirst
typeofinclusions・Glycogengranulesaggregatingwithinamembranewasalsoobservedalthoughrarely．
Discussiom
ThepatternofthemuscleinvoIvementinthispatientwassimilartolimbgirdledystrophy，eXCept
forthefacialinvoIvementandrapidprogressionofmuscularwasting・TheTMSinthepresentpatient
SOmeWhatresemblestheinclusionsofFabrydisease，4）butclinicalandbiochemicaldataweredifferent．
Theperiodicityandformofparallelmembranesoftheinclusionswerealsodifferent．4）TypeIIinclu−
Sionsinthispatientvaguelyresembledcurvilinealprofilesinceroidlipofuscinosis，buthisclinicalpicture
WaSdifferent・TypeIIIinclusionshave some similaritiestotheHdensegranulofilamentousmassesHin
thedominantlyinheritedmyopathydescribedbyFardeauetal・5）Therefore，ultrastruCturalfindingsof
themusclebiopsyspecimeninthepresentcasedidnotconformwithanyoftheneuromusculardiseases
hithertodescribed．
Inconclusion，WeSuSPeCtthatthispatientsuffersfromahithertounrecognizedprogressivemuscu−
1ardiseaseandthedescriPtivetermHtubulomembranousmyopathyMisproposed．
Refereneesl）Fukuhara，N・，Kumamoto，T．，Hirahara，H．etal．：Anewmyopathywithtubulomembranousinclusions．J．Neur。1．
Sci．，50：95，1981．
2）Dubowitz，V・＆Brooke，M・H・：MusclebiopsyTAmodernapproach・Saunders，London，1973，
3）Fukuhara，N・，Kumamoto，T・＆Tsubaki，T・＝Rimmedvacuoles・ActaNeuropathol．（Berl．），51：229，1980．
4）Fukuhara，N・，Suzuki，M・，Fujita，N・etal・＝Fabry

sdisease．OnthemechanismoftheperipheralnerveinvoIvement．

ActaNeuropathol．（Berl．），33：9，1975．
5）Fardeau・M・・Godet−Guillain，J・・Tome・F・M・S・etal・：Unenouvelleaffectionmusculairefamiliale，dennieparlaccumulationintra−SarCO−plsmlqued，unmaterielgTanulo−nlamentairedenseenmicroscopleelectronlque・Rev・

Neurol．（PariS），134：411，1978．

一47−

肋∫C〟ねr砂∫rr0タカγGr0岬I）EBRANCHERDEFICIENCYMYOPATHY：THREEADULTSINCLUDINGTWOSIBLINGS
MitsuhiroOsame＊，TeruOIwamasa＊＊，HidetoshiFukunaga＊，MasanoriNakagawa＊andAkihiroIgata＊＊DepartmentofInternalMedicine，FacultyofMedicine，KagoshimaUmiversity，Kagoshima＊＊DepartmentofPathology，SchoolofMedicine，KumamotoUniversity，Kumamoto

A34yearoldfemale（casel），her27yearoldbrother（case2）anda32yearoldfemale（case3）
hadslowlyprogressivemuscleweaknessandatrophywhichbeganinthethirddecadeoflife・Theweak−ness was greater distally than proximally，but sternomastoid and thigh adductormuscleswere also
severelylnVOIved．Parentsoftheaffectedsiblingsweredoublecousins・Therewasnoconsanguinityintheotherfamily．
Liver function test results were normaland no hepatomegaly was found even on a technetium
scintigram．SerumCPKlevelswereslightlyelevated．
The abnormalaccumulation ofglycogen was observedin subsarcolemmalareasinPAS stained
frozensectionsofbiopsied muscles．On electron microscopicobservation，manyglycogencontaining
vacuoleswerefoundinthemuscle．
Biochemicalanalysesofmusclewere

doneincases2and3（Tablel）‥（1）Glycogencontentwas

slightlyincreased．（2）Debranchingenzymeactivityforphosphorylaselimitdextrinwasreduced・This
became more markedwhenα−glucosidase wasinhibitedwithcadmiumorantibodiestoyeastα一gluco−

Tab！el ResultsofBiochemicalAnaysesofBiopsiedMuscle

Case2

0

7
4
1

7．30mg／g．W．W．ormuscle
6

4
0

U

7

0

1
0

0

3
0

0

0．70pM／mgprotein／min．
0．40llM／mgprotein／min．

1

0

′
b

3

つ ▲
つ ▲

0

7

4

0

8
0

0

B

Control

L

0
3

A

′

0

mospI10けlase Total

3

0
1

つ一

′ 0
0 0

3

Glycogencontent

ControI Case3

0．30FIM／mgprotein／min．

Debranchingenzymeactivity（1）withphosphorylaselimitdextrinas
Substrate（a）withinhibitorforα−glucosidase
2

′

′

h

L
U

Y

β−Amylolysis

3（b）withoutinhibitorforα−glucosidase（2）incorporationof14C−glucoseinto
glycogen0

cadmium

3anti−yeaStα−glucosidaseserum

253．8

583．8

11．0

34．3

，PM／mgprotein／min．
0．09

5．13〃M／mgpIOtein／min．

0．18

6．60FLM／mgprotein／min．
，−

U／mgprotein／min．
％digestion

α−Glucosidasewithglycogenassubstrate
pH4．O

0．08

0．03

0．07

0．03FLM／mgprotein／min．

pH7．0

0．11

0．10

0．11

0．09FLM／mgprotein／min．

0．21

0．12

2．9

0．1pM／mgprotein／min．

Acidphosphatase

Table2

DebrancherActivityinErythrocytes

Case2

Case3

Control

0．36

2．29

2．45units／gHb／min．

−48−

Sidase・Incorporationof14C一如coseintoglycogenwasalsoreduced（case2）．（3）Glycogenhadshort
OuterChainsonβ−amylolysis（case2）．（4）α−GlucosidaseactivitywasincreasedatpH4．0andnormalat
PH7．0．（5）Acidphosphataseactivitywasincreased．
Biochemicalanalysesoferythrocyteswerealsodoneincases2and3（Table2）：Incorporationof
14C−glucoseintoglycogenwasreducedincase2butnormalincase3．
BeforeDiMauroetal1）reported5patientsandwe2）riportedthese3patients，Only3patientswith
debrancherdeficiencymyopathyhadbeenreported3・4）・Inourwork，debrancheractivitywasmeasured

bytwodifferentassaymethods・Oneisbasedonreversalofthephysiologicalreaction，withincorpora−tionoflabeledglucoseintoglycogen・Theothermethodisbasedonthephosphorolyticdegradationof
PLD，Whichdependsonthepresenceofdebrancher・Thedebrancheractivitywasfoundtobereducedby
bothmethodsincase2，thispatienthadamyl0−1，6−glucosidasedeficiencymyopathy・Incase3，thedebrancheractivitywasdefinitelyreducedwiththeformermethodinmuscle，butふasnormalWiththelattermethodinerythrocytes・Thisresultsuggeststhatcase3maybelongtosubgroupIIIDofVanHoof
andHers5）・Inthesepatients，thetransferaseactivityismissingwhiletheamyl0−1，6−glucosidasefunction
Oftheenzymeispreserved5）・Patient3inDiMauro，sstudyl）wasalsosupposedtobelongtothissub−
grOup．

OurthanksareduetoDr．A．G．EngelandDr．S．DiMaurofortheirkindcomments．
Referencesl）DiMaru0，S・，Hartwig，G．B．，Hays，A．，etal．：Debrancherdeficiency：Neuromusculardisorderin5adults．Ann．
Neuro1．，5：422，1979．
2）Osame，M・，Iwamasa，T・，Kawabuchi，M・，etal．：Familialmuscletypeamyl0−1，6−glucosidasedenciency．IVInter−nationalCongressonNeuromuscularDiseases．207（Abstract）1978．
3）Brunberg，J・A・，McComick，W．F．，andSchochet，S．S．：TypeIIIglycogenosis：anadultwithdiffuseweaknessandmusclewasting．Arch．Neurol．25：171，1971．
4）Murase，T・，Ikeda，H・，Muro，T．，etal．：MyopathyassociatedwithtypeIIIglycogenosisJ．Neurol．Sci．20：287，1973．
5）VanHoof，F・，Hers，H・G・：ThesubgroupsoftypeIIIglycogenosis．Eur．J．Biochem．2：265，1967．

ー49−

肋gC〟Jαr功JJrop妙Gro岬

TRANSPLANTATIONOFHUMANDYSTROPHICMUSCLETONUDEMICE：AMORPHOLOGICSTUDYYoshihiroWakayama
DivisionofNeurology，DepartmentofMedicine，ShowaUniversityFujigaokaHospital，Yokohama
Itisunknownwhethertheregenerationofhumandystrophicmusclesisnormal，althoughthisisa
Veryimportant subject．Someinvestigatorshave suggested that themuscleregenerationisineffectivein
thisgeneticdisorder（1，2）・However，Whythemuscleregenerationisineffectiveisunknown．Therefore，Iconductedanexperimentonhumandystrophicmuscletransplantationintonudemiceandcomparedtheseresultsmorphologicallywiththoseofnormalhumanmuscletransplantationintonudemice（3）．
Biopsiedmusclesusedinthisstudywereobtainedfrom7patientswithDuchennemusculardystro−phy（DMD）and five histochemicallynormalmuscleswereobtainedfromorthopedicpatientswithout
any neuromuscular diseases・SmallpleCeSOfmuscleswereinsertedintothesubcutaneousspaceofthebackofnudemiceandtheincisionswereclosedbycottonsutures・Themiceweresacrificedl，2，3，4Weeksaftertransplantation・EachtransplantwascutintosmallpleCeS，fixed，dehydratedandembedded
inplastic，andprocessed forlightandelectronmicroscopy．Theresultsofnormalhumanmuscletrans−
plantationhavealreadybeenreportedelsewhere（3）．Inthisreport，Iwouldliketopresenttheresultsof
DMDmuscletransplantationupto2weeks．
Light microscopICally，afterl week oftrallSPlantation，regeneratingmyoblastsand multinucleatedmyotubes were seen，eSPeCiallyattheperipheryofthetransplant（Figure）．After2weeks，thegrowth
Ofmyotubeswasobserved．
Electron microscoplCally，afterl week，myOblasts and multinucleated myotubes were observed
Within the basallaminaofthedegeneratingmusclefibersofDMDandthesece11Swereusuallycloselyalignedwithinthebasallamina，WraPPingthecelldebrisofdegeneratingmusclefibers．Moreover，these
Cells were not always associated withthe degenerating muscle fibers，andWere also present within the basal
lamina of theinterstitialspace，being
packed compactly with each other
With theintertwined cell processes．
The basal1amina of these fibers was
duplicated and partly pleated．The
myoblasts and myotubes contained
randoinlydistributedactinandmyosin
filaments，Z−linematerial，intermediate
Sized filaments，microtubules andabundant ribosomes and polysomes
Which are also present along nascent
myofilaments．In addition，Clusters
Of glycogen granules with and／or

Fig．Thclightmicrographshowsdegeneratingmusclefiberandaregen−erating myotube（arrow）afterl week oftransplantationin a DMD
transplant．Thedegeneratingmuscle董iberusuallyoccupiesthecenterof

the transplantandshowsthegranularcytoplasmicfeature；Whereasthe
regenerating myotube which usually occupleS the periphery ofthetransplantisthinner，andcontainsafinecytoplasmicmatrixandseveral
euchromaticnucleiwithprominentnucleoli．（Toluidineblue x500）−50−

Without membrane materials andlipid
dropletswereobserved．After2weeks，electron microscopIC eXamination of
the regenerating myotubesin thetransplanted DMD muscles revealed
the partial cross striation of themyofilaments，leptomeresin the subsarcolemmal and perinuclear regions and tangle formation ofT−tubulesinadditiontothefindingsnotedinmyotubesofDMDtransplantsafterlweek．
The plasma membrane ofthe regeneratingmyotubeswas continuous andits disruption，SuChas
reportedin DMD muscle plasmamembrane（4），WaSnOt Observedinthisstudy．Noconspicuousdif−
ferencesofthemorphologyoftheregeneratingmyotubesbetweencontrolandDMDmuscletransplants
Werequalitativelyobservedupto2weeks．Afurtherstudyupto4weeksaftertransplantationwillbe
COmPletedsoon．
Schotland et al．（5）have reported the alteration of theintramembranous struCture OftheDMD
muscle plasma membrane utilizing the freeze fracture technique．So a freeze fracture study of theregenerating muscle fibers onlong term transplantation may provide furtherinformation about the
Pathogenesisofhumanmusculardystrophy，Referencesl）Mastaglia，F．L．＆Kakulas，B．A．：RegenerationinDuchennemusculardystrophy：AhistologiCalandhistochemical
Study．Brah92：809，1969．
2）Wakayama，Y．，Schotland，D．L．，Bonilla，E．＆Orecchio，E．：QuantitativeultrastruCturalstudyofmusclesatellite
CellsinDuchennedystrophy．Neurology（Minneap．）29：401，1979．
3）Wakayama，Y．，Schotland，D，L．＆Boni11a，E．：Transplantationofhumanskeletalmuscletonudemice：Asequential
morphologicstudy．Neurology（Minneap．）30：740，1980．
4）Mokri，B．＆Engel，A．G．：Duchennedystrophy：Electronmicroscopicfindingspointingtoabasicorearlyabnormal−
ityintheplasmamembraneofthemusclefiber．Neurology（Minneap．）25：1111，1975．
5）Schotland，D．L．，Bonnla，E．＆Wakayama，Y．：Applicationofthefreezefracturetechniquetothestudyofhumanneuromusculardiseases．Muscle＆Nerve3：21，1980．

−51−

〟〟gCukr砂∫打Op力yGr0叩

A MORPHOLOGICAL STUDY OF EXPERIMENTAL MITOCHONDRIAL MYOPATHY WITH
2，4−DINITROPHENOL
ShukuroAraki，YoshinoriYamamuraandEiichiKato¶le¶liTdDepartmentofInternalMedicine，MiyazakiMedicalCouege，Miyazaki

HMitochondrialmyopathy

maybedefinedasamusclediseaseinwhichthemitochondrialchanges（aggregation，enlargement，Cristal alterations，paraCryStallineinclusions）are the m毎Or pathologiCalfeature，butits pathogenesishasnotyetbeenclarified・Insomecasesofmitochondrialmyopathytheloose coupling ofoxidative phosphorylation ofmitochondriawasdemonstrated・Thesemitochondrialchangeshavebeendemonstratedinratskeletalmuscleswhichwerekeptundertheischemicconditionor
followingintra−arterialinfusion of2，4−dinitrophenol（DNP），an unCOuPler ofoxidativephosphoryla−tion．1，2，3）

Wedemonstratedtheappearanceofmorphologicalchangessimilartomitochondrialmyopathiesby
intra−arterialinfusionofDNP，andbyintra−muSCularirdectionofDNPwhichhasnotyetbeentried．We
confirmed that thelatterproduced the same morphologicalchanges ofthe musclesasinthecaseof
intra−arterialinfusion．
Materialsandmethods
MaleSprague−Dawleyratsweighing250−300gwereused．Theratswereanesthetizedbyi．p・injectionofNembutal，and the abdomen was opened・Theiliac artery on one sidewasligated，andtheaortic
artery was cannulated with a sma11−borepolyethylene catheter・Fifteen mg ofDNPwasintroduced
throughtheintracatheteroveraperiodof3−5min・Themusclebiopsiesfrombilateralfemoralmuscles
weretaken15min，30min，45minand60minaftertheildectionofDNP．Thebiopsiedmuscleonthe
dde ofwhich theiliac arterywas notligated was theintra−arteriallyinfused group，and the muscle
specimenontheoppositesidewasthecontrolgroup・Inthethirdgrouptheratswereanesthetizedby
l．P．injectionofNembutal，andfifteenmgofDNPwasinjectedintramusculary，Themusclespecimens
wereobtainedfromthefemoralmuscles15min，45minand60minaftertheinJeCtionofDNP・Eachspecimenwasdividedintotwoparts・Oneofthepartswasimmediatelyfrozeninisopentane，andabout
10FLmthicktransversecryostatsectionswerepreparedandserialsectionswerestainedwithhematoxylin
andeosin，mOdifiedGomoritrichromeandNADHtetrazoliumreductase・Thesecondpartwasfixedin
OsO4following fixationin2．5％glutaraldehyde and embeddedinEpon812．UltrathinsectionswereviewedunderaJEMlOOBelectronmicroscope．
Results
The H．E．Stainshowednoremarkablechangesineachgroup．From15minaftertheintra−arterial
infusion of DNP themodifiedGomoritriChrome stainshowed distinctraggedred fibers（RRF），being
mostmarkedinthebiopsiedmusclesof30minaftertheinfusion・Inthethirdgrouptheentirebody
musculature becamerigid between45and60min afterintramuscularinjectionofDNP，andtherats
diedin a few minutes．Marked RRF were demonstratedinthenecropsiedmusclesimmediatelyafter
death．

Only one ofthecontrolgroupsshowed distinctRRF・Thiscouldbecausedbytheeffectoftheischemiaand／orinfusionofDNP．
ThepercentageofRRFeachtimewasestimatedasfollows・Fifteenminafteri・a・infusionshowedl0−17％（mean12．1％）；30min，8−45％（26．9％）；45min，18−35％（27．0％）；and60min，21％，reSpeCtively・Inthecontrolgrouponlyonesample，inwhichdistinctRRFwereseen，Showedhighvalues，9％ofRRF
atfifteenminafterirdection，19％at30min，and16％at45min，Whiletheothersshowedlowvalues，−52−

0−10％（4．4％）．
NADHtetrazoliumreductasereactionsshowedhighactivitiesinthefiberswhichwereseenasRRF
WithmodifiedGomoritrichromemethods，andtheseactivitiesweremostmarkedinthesubsarcolemmalareas・Thesefiberswereconsideredtobetypelfibers・Therewerenohighactivitiesexceptforinone
SamplewhichshowedRRFinthecontrolgroup．
An electronmicroscoplCStudyrevealednomitochondrialabnormalitiesinthecontrolmuscles．In

the musclesinfusedwithDNPintraarterially，markedmarkedaggregationofmitochondriawasseenin
thesubsarcolemmalspaceandintermyofibri1larspace，andenlargementofthemitochondriaandcristal
alterationwereseen，butparacrysta11ineinclusionswerenotdemonstrated．Inthemusclesinjectedwith
DNPintramuscularly，thesamechangesofthemitochondriawerenotedasintheintra−arteriallyinjected
grOup．
Diseussiom

Althoughmanypatientswithmitochondrialmyopathieshavebeenreported，thepathogenesishas
notyet beenclarified・To study thepathogenesisofthis disease，Melmedet a12）andSahgaleta13）
experimentally reproduced RRF and mitochondrialchangessimilarto mitochondrialmyopathiesby
intra−arterialinfusion ofDNP・InthefirstplacewealsoinfusedDNPintra−arterially，andfromfifteen
minafterinfusionRRFwereseenandthepeakwasseenatthirtyminafterinfusion．
Becauseofthetechnicaldifficultiesofintra−arterialinfusionandofkeepingtherataliveduringthe
longperiodtherepetitiveadministrationofDNPisalmostimpossible，thereforethechroniceffectcould
not be studied．Thei．p．injection ofDNP failed to reproduce the mitochondrialchanges．．Forthesereasons we triedintramuscularinjection of DNP，and confirmed the appearance of mitochondrial
Changesasinintra−arterialinfusion．
From the clinical aspect，the experimental models of mitochondrial myopathiqs which werereportedinthepastwerenotidenticaltohumanmyopathies．Moreappropriatemodelshavetobemade
infuture experiments・We wouldlike to make experimental models which areidenticalto human
myopathiesin both symptoms and morphologicalchanges by repetitiveintramuscularadministration
OfsmalldosesofDNP．
Referencesl）Karpati，G・・Carpenter，S・，Melmed・C・，etal・：ExPerimentalischemicmyopathy・J・Neurol・Sci・，23：129，1974・
2）Melmed，C・，Karpati，G・＆Carpenter，S．：Expenmentalmitochondrialmyopathyproducedbyinvivouncoupling
Ofoxidativephosphorylation．J．Neurol．Sciリ26：305，1975．

3）Sahgal，V・，Subramami，V・，Hughes，R・，etal・：Onthepathogenesisofmitochondrialmyopathies．Anexperimental
Study．ActaNeuropathol．，（Berl．），46：177，1979．

−53−

肋∫CUねr砂∫けOp々γGro岬

MYOPATtWWITHMITOCHONDRIALNCLUSIONBODIES−CLmCALANDELECTRONMICROSCOPICSTUDIES−
TakeshiSato，MidoriAnnoandKatsunoriNishi
Depa∫tmentOfNeurology，JuntendoUniversitySchoolofMedicine，Tokyo
Introduction
Abnormalities of skeletalmuscle mitochondria，including paracrystalline−likeinclusions，have
beenreportedinagreatnumberofvariOusmuscledisorderswithusuallyobscureaetiologyl）・ItiswellknownthatprominentabnormalmitochondriaarepresentinpatientswithocularmyopathyorKearn−
Sayresyndrome．Suchabnormalmitochondriaarefoundnotonlyinskeletalmusclesbutalsoinotherorgans．Inthispaper，mitochondrialinclusionbodiesinthreecaseswithocularmyopathy，aCaSeWith
humeroperonealmuscularatrophy，andacasewithquadricepsmyopathyarereported・
Casereport
Casel（Ocularmyopathy）．Thepatientwasa48−year−01dfemalewhohadbeenhealthyuntilabout

33yearsof age，Whenshe started tonoticerightblephaloptosis．Thesymptomprogressedgradually，
andattheageof461eft−Sideptosisappeared，tOO・Physicalexaminationrevealedbilateralophthalmople一gla，andEMGshowedslightmyopathicchange・CPKlevelwas29I・U・
Case2（Ocularmyopathy＋slightlygeneralizedmyopathy）．Thepatientwasa46−year■）ldfemale

whohad been healthyuntilabout30yearsofage，Whenshestartedtonoticebilateralblephaloptosis
afterthethirddelivery．Sinceshehadanoperationforagoiterattheageof33，thesymptomprogres−
sed．Physicalexaminationsrevealed bilateralophthalmoplegla，and slightweaknessofface，neCkand
shouldermuscles．EMG showed a myogenic change and EEGindicated a dysrhythmic basicpattem・
CPKlevelwas170I．U．，andslightelevationofTTT，ZTTandCCFindicatedchronichepatitis・Thyroid
functionandBMRwerenormal．

Case3（Kearns−Sayre syndrome）．Thepatientwasa39−year−01dmalewhoseeye−lidswereslightly
ptoticsincechildhood，Afteradolescencebilateralblephaloptosisprogressedrapidlyandattheageof
30，hebegantolosehisvisualacuityslightly・Physicalexaminationsrevealedbilateralophthalmople一

gia，andslightweaknessandatrophyofneckanddistalextremities・Thecerebellarsignwasquestiona−
ble．Laboratory examinationsrevealeddysrhythmiainEEG，myOgenicchangeinEMG，elevation of
CSFprotein（101mg／dl），CalcificationofthebasalgangliaandcorticalatrophyinCT，Slightmental
retardation（WAIS75），Slightdeafness，CPK45Ⅰ．U．，andslightelevationoflipids・Liverbiopsyindicatedfattydegeneration．

Case4（Humeroperonealmuscular atrophy）．Thepatient，a48−year−01d male，WaSSlenderfrom
childhood andunskillfulinchinningexercisesandrunning・Attheageof27，A−Vblockwasdetected
byECG，andhehascomplainedofpalpitationanddyspneaforthepast10years・Fromaboutthesame time he noticed weakness of the extremities．He received an operation for apacemakeratthe
ageof48．Physicalexaminationsrevealedweaknessandatrophyofhumeroperonealmuscle，andcon−
tracture ofcervicalvertebrae and spine．Laboratoryexaminationsrevealed a completeA−Vblockin
EEG，myOgenicandslightlyneurogenicchangesinEMG，andCPKwas54I・U・
Case5（Quadriceps myopathy）．The patient wasa31−year−01d malewhohadhaddifficultyin
runningfastsincechildhood．Attheageof18，hehaddifficultyingolnguPanddownstairsandjump−ing，andhestartedtonoticeatrophyofthethigh・Physicalexaminationsshowedlocalizedatrophyand
weaknessinthethigh．LaboratoryfindingsshowedmyogenicchangeinEMG，CPK490I・U・，andslightelevationofGOT，GPTandLDH・CRP，RAtestandLEtesthadnegativeresults・

一54−

Histochemicalstlldy
The deltoid musclein Cases2and3showed ragged−red fibers（20−30％）and type2atrophy．The
frontalmuscle andexternalocularmuscle ofCasesl and2revealedscatteredmusclefiberatrophyand
anincreaseofcollagenfibers．AsthebicepsinCase4revealedcollagenfibersonly，WereeXaminedthe
pectoralis major and found scattered ragged−red fibers and typeIfiberatrophy・The quadricepsin
Case5revealedvariabilityln fibersize，SCattered atrophicfibers，tyPeIfiberatrophyandragged−redfibers，butcellinfiltrationwasnotrecognized・UltrastruCturalstudy
Inallcases，WereCOgnizedaggregatedandmodifiedmitochondria，andparacrystallineinclusionsin
mitochondria．The abnormalitiesin mitochondria were moresevereinCasesl，2and3thaninCases
4and5．InCase3，Wefoundparacrystallineinclusionsinthelivermitochondria，tOO・Weexaminedthe
fine struCture Of the paracrystallineinclusionswithvariousprqjections，and we concluded that thisinclusionbodyhadabox−likeshapecontainingfilamentoussubunitsinthreedimensions（Fig・）

tもこ

B

衝け㍉

Fig．Electronmicrographsshowaportionofaparacrystallineintramitochondrialinclusionwithavarietyof
projectionswithpresumedside（A），tOp（B）andvertical−Cut（C）sections・
A：×160，000，B：×120，000，C：×150，000

Discussiom
Inadditiontotheocularmyopathy，Cases2and3showedslightmyopathicchangesinextremities・
Case3，SO−CalledKearnS−Sayresyndrome，hadinvoIvementofotherorgans，1iver，CentralnervOuSSyStem
andretina．Case4isalmostthesamecategoryashumeroperonealmuscularatrophyorscapulo−PerOneal
dystrophy，OrEmery−Dreifussyndrome，WhichinvoIvedneurogenicand myogenicchangesandrevealedaut。SOmal recessiveinheritance．Case5is so−Called quadriceps myopathy，Whichis thought to be a
syndrome，SuChaspolymyositis，muSCulardystrophyandneurogenicatrophy・Case5wastreatedwith
predonisolone，40mgdaily・AftertwoweekstreatmenttherewasmarkedimprovementinhisCPK
level．Therefore，thispatientwasdiagnosedaspolymyositissyndrome・
Abnormalmitochondria containing paracrystallineinclusions are foundin various disorders ofmuscle，eSpeCiallyln OCular myopathy・Butlittleisknown about theirchemicalabnormalities・As−55−

MulcularDystrophyGroup

DiMauro2）mentioned，manymOreHmitochondrialmyopathies，，（inarestrictivebiochemicalsense）Will
berecognizedassystematic，biochemicalstudiesofmitochondrialfunctionarecarriedout．
Referencesl）hnd，J．M．＆Clark，J．B．：Mitochondrialmyopathies．Biochem．Soc．Trans．7：231，1979．
2）DiMauro，S．：Mitochondrialmyopathies：Whichandhowmany？in（Ed．byA．T．Milhorat）ExcerptaMedica，Amsterdam，1974，P．506．

ー56−

Exploratoryconceptsinmusculardystrophy肋∫α血r功∫けOp力yGro叩STUDY ON DISUSEATROPHY

MasaoKinoshita串，KazuhiroMitzutani＊拳，MasayukiYokota＊＊andMitsuoMotegi＊＊＊TheFourthDepartmentofInternalMedicine，TohoUniveISity，Tokyo＊＊DepartmentofOrthopedics，TohoUniversity，Tokyo

In order toinvestigate the mechanism of disuse atrophy，a CaStimmobilization experiment was

performedonadultalbinorabbits・Immobilizationwasperformedbythreedifferentmethods：theknee
wasflexed and the ankle wasdorsi−flexedingroupl，thekneewasflexedandtheanklewasplanter−
flexedingroup2，andthekneewasextendedandtheanklewasdorsLflexedingroup3・
Ingroupl，theanteriortibialmuscleshowedatrophyoftypeIIAfibers4weeksafterthestartof
immobilization，and subclassification of typeIIwasdifficultinthe8thweek・Inthisgroupinthe

gastrocnemiusmuscle，bothtypeIandtypeIIAfiberswereatrophied・meChangeofdiameteroftype
IfiberswasmostmarkedingroupIandleastsignificantingroup3．
Thistendency was absolutely parallelto thegrade ofstretchofthemuscle・Ontheotherhand，
atrophy oftypeIIAfiberswas observedinallgroupsregardlessofthegradeofstretch・Thereforeit
was considered that the true effect of disuse was seenintypeIIAfibersmostsignificantly，andthe
decreaseindiameteroftypeIfiberswasprobablyduetolossofpropermusculartonus・Themost severehistologicalchange was seeninthe anterior tibialmuscleingroup3whichwas
significantlyrelaxed．Twoweeksafterthestartofimmobilization，themusclebegantOloseitsnormal
colourandelasticityshowingtumor−likeswellingwithinthemuscle・
Therewere manyatrophied fiberswithcentralareasofwhichtheintensityofstainingwithSDH
orroutinemyosinATPasewaseithertoodarkortoopalecomparedtothatofusualareas・Alsocentral
nucleiwerefrequentlyseeninthesefibers・Furtherinvestigationisnecessaryforclassification・Contra−
dictingsomepreviousstudiesintheliterature，theratiosofthenumberoffibersofeachhistochemical
typedidnotshowanysignificantchangeaftercontinuousimmobilization・Intheanteriortibialmuscle，
theratiosoftypesI，ⅠIAandIIBwere7・9，34・7and57・4％，reSpeCtively，inthecontrolrabbit，andwere
nearlyequa14weeksafterthestart．

−58−

ChangeOffibersizeafter4weeksimmobilizationintheknee−flexed，ankle−dorsi−flexed
position．Diameterofintermittent（typeIIA）fiberswasdecreased．
RoutinemyosinATPase．xlOO．

−59−

肋gC〟ね′pγJ打Pp力yGro岬−HISTOCHEMICALSTUDYONLIPIDMETABOLISMINMOTORNEURONE DISEASE−
ToshiiiMozai，MasazumiTakenaka，ShingoKonishi，
YoshikazuMasuiandKedchiShinoda
TheFirstDivision，DepartmentofInternalMedicine，OsakaMedicalCo11ege，Osaka
Inthepreviouspaper，WhichreportedtheclinicalandlaboratoryfindingsintwocasesofKennedy，sdiseasefoundintwo differentfamilies，itwaspointedoutthatabundantdepositionofdropletsstained
byoilredOinmusclefibers，fattydegenerationoftheliverandhypertropICCardiomyopathywerecom−
monlyseen・Inonecase，electronmicroscopICeXaminationofbiopsiedheartmusclerevealeddeposition
Oflipidbodiesincardiocytes・Anincreaseofplasma triglyceridelevelwithincreased pre−B−lipoproteinfractionwasalsonoted
inbothcases．ThenaquantitativestudyonthedepositionoffatgranulesinmusclefibersinKennedysdiseasewasperformed．
MaterialsandMethods
Muscle specimens were obtained from quadriceps femoris muscleintwocaseswithKennedy，sdisease（CaSeSland2）aswellastwocaseswithtypicalamyotrophiclateralsclerosis（cases3and4）・
SerialcryostatsectionsweresubmittedtomyosinATPasestainingatpH9・4andoilredOstaining・Thendepositionoffatgranulesstainedineachtypelfiberandtype2fiberwasdetermined・Forquantitative
determination，thelineinterceptmethodreportedbyLucasandSeberl）wasemployed・Photographic
prints（1120magnification）ofeachfiberweremadeandthenumberofdepositedgranulesin40fibers
eachoftypelandtype2Selectedatrandomwascalculated・
Lipidcoverageperunit（＝C）ofeachfiberwasestimatedbythelineintercepttechniqueasfollows・

C＝右面
Ml，Ms＝numberofdropletsonlong，Shortdiameter
Ll，Ls＝lengthoflong，Shortdiameter
W＝aVeragedropletsizeforthegroup

Inaddition，thefibertypehistogramwasalsostudied・
Resultsl）nefibertypehistogramshowedamarkedincreaseinthenumberofhypertrophicfibersof
morethanlOOmicr．inmusclesobtainedfromcasesland2．
2）Incasel，perCentlipidcoveragewas5to15percent（averagelO・76％）intypelfibersand
about5percent（average5．12％）intype2fibers．
Incase2，perCentlipidcoverageintypelfiberswas3to5percent（average3・99％）inmanyfibers，thoughinthesame fibersitreachedlOpercent．Intype2fibers，thefindingswerealmostthesame（average4．64％）asintypelfibers．
Inbothcasesland2，therewasnodifferenceinpercentlipidcoveragebetweenhypertrophicfibers
andnon−hypertrophicfibersofbothtypesland2・
InmusclesobtainedfrompatientswithALS，PerCentlipidcoveragewaslessthan2percent（averagel．35and2．04％intypelfibers，andO．67andl．69％intype2fibers）inmanyfibersofbothtypes・一60−

Discussion
The distribution and quantityin muscle fibers of neuromuscular disorders have rarely beenreported．Theincidence oflipid dropletsin human skeletal musclein various abnormal states was
examined by Harriman and Reed．2）They examined13cases ofmotorneuron disease andfoundmoderatedepositionoffatinlOcases・However，theydidnotdoaquantitativestudy．Wefoundmore
abundant depositionoffatdropletsinbothfibersoftypeland2inKennedy，sdiseaseascompared
WiththedepositioninALS．
WhnethepercentlipidcoverageseeninmusclesfromourpatientswithKennedy，sdiseasewaslower
thanthatinneutrallipidstoragediseasedemonstratedbyMcKeranetal・3），thisfindingsuggeststhat
theremaybeametabolicdisturbanceoflipidasthebasisofKennedy，sdisease，Whichmayalsocause
Cardiomyopathy，fattyliverandhyperlipidemia．
However，itisknownthattheamountoflipidinmuscleisinfluencedbyage，SeXandnutritional
State・FurtherhistochemicalStudiesonthedepositionoffatinmusclefibersaredesired．
Referencesl）Lucas，H・A・＆Seber，G・A・F・：EstimatingcoYerageandparticledensityusingthelineinterceptmethod．Biometrika，
64（3）：618−622．
2）Harriman，D．G．F．＆Reed，R．：Theincidenceoflipiddropletsinhumanskeletalmuscleinneuromusculardisorders：
Ahistochemical，electron−microscopicandfreeze−etChstudy．J．Path．，106：1，1972．
3）McKeran・R・0・，Royston，J・P・，Ward，P・，etal・：Twopopulationsoftypelfibersinstriatedmuscle鱒OmaCaSeOf
neutralhpidstoragedisease．J．Neurol．Sci．，43：1，1979．

一61−〟〟∫仰山r勒gr和戸々yGro叩−HISTOLOGICALSTUDIESONMUSCLEINACASEWITH
SCHWARTZJAMPELSYNDROME−
ToshijiMozai，ShingoKonishi，YoshikazuMasui，MasazumiTakenaka
andIchizoFukuda
TheFirstDivision，DepartmentofIntemalMedicine，OsakaMedicalCollege，Osaka
TwentysixcasesofSchwartzJampelsyndromecharacterizedbyblepharophimosis，Shortstature
andabnormalitiesofbonesandjointshavebeenreported．However，0nlyafewhistochemicalstudieson
musclehavebeenperformed・Westudiedbiopsiedmusclehistochemicallyinacasewiththesyndrome
andfoundtypelfiberpredominance・
Caserecord
Aseven・year−01dboywasadmittedinAugust，1978，tOOsakaRosaihospitalbecauseofabnormality
ingaitandotherbodyactions・Hisparentswerefirstcousins，andhismaternalgrandfatherandgrand−
motherwerealsoconsangulneOuS．Hewasbornatfullterm，3，100gramsbodyweight，afteranuncom−
plicatedpregnancy・HebegantOWalkatthe14thmonth，andsinceage4progressiveclumsinessingait
andotherbodyactionshasbeennotedinadditiontoshortnessofstature・

Findings on admission：His height was103．4cm（−3．36SD），While skeletalmuscles were welldevelopedandintelligencewasnormal・Hisfacewaspinchedwithblepharophimosisandhelookedsad・

Hehadmarkedlumbarlordosiswithslightscoliosisandpigeon−breast．Hisgaitwasstiffandclumsylike
amarionette，Gripmyotonia，unaffectedbycold，WaSdemonstrated・Percussionmyotoniawasnotde−
tected．Muscleswerestiffandfirmonpalpationwhiletherewasnoweakness・Thefirmnessofmuscle

decreased only slightlyduringgeneralanesthesiabeingunaffectedbywarmthorcold・Deeptendon
reflexes werealmost absent，and Babinski，s toe slgnWaSnOtelicited・Otherphysicalfindingswere
negative．

Laboratory findings：Routinelaboratory examinations revealed no abnormality exceptaslightincreaseofserumCPKandIgAdeficiency，lessthanlOmg／dl．Urinaryaminoacidanalysisrevealedno
abnormality．Theresultsofendocrinologicalandchromosomestudieswereallnormal・Radiographic
studies revealed maldevelopment of the facialskeleton，and deformities of skull，Chest，Splne andacetabulum．

Electrophysiologicalstudies：EMG findings duringvoluntary contraction were normal・While amyotonicresponsewasevokedbyinsertionofaneedleelectrode・Motornerveconductionvelocitiesin
uharandtibialnerveswerewithinnormalrange．

Musclebiopsystudies：Biopsyoftherightquadricepsfemorismusclewasperformed・
1．Lightmicroscopicstudiesrevealedthefollowing・
Onlyafewfiberswithslightnon−SPeCificmyogenicchangeswerefoundinHEstainedsections・Dia−
metersofmusclefibersdeterminedwithfrozentissuestainedforATP−aSeatPH9・4variedfromlOto
60micr．withaveragesof30fortypelfibersand31・8fortype2fibers・However，therewasmarked
typelfiberpredominance，74％ofallfibers・Almostallend−platesdemonstratedbycholinesterasestain−
ingshowedelongation．
2．Electron microscopIC Studies revealed swelling，destruction andvacuolization of the mito−
chondria，andpartialdisappearanceofZ−1ines・However，Onthewhole，themyofibrillararchitecturewas
Wellpreserved．
Comment：
ThepresentcasewasdiagnosedasSchwartzJampelsyndromefromtheclinicalfindings・Themuscle−62−

Stiffnesswasgeneralized，COntinuousandnotdecreasedbymovement．Sothestiffnesswasmoresimilar
tothatinthesyndromeofcontinuousmuscle−fiberactivityaccordingtoIsaacs，1）orneuromyotonia
thanthatincongenitalmyotoniaandmyotonicdystrophy．
NoteworthyinninecasesofSchwartzJampelsyndromeintheliteraturewasthespontaneouscon−tinuous electrical activity，reSembling electrophysiological findingsin the syndrome of continuous
muscle−fiberactivityorneuromyotonia．
InourstudiesタtyPelfiberpredominancewasclearlydemonstrated．Thepathogenesisofthisfind−ingisnotknown・However，itisinterestingtopointoutthespeculatedcorrelationbetweentypelpre−
dominanceandcontinuousmuscleactivityinthissyndrome．
Salmonseta12）andRomanuletal3）reportedthattheycouldchangethemusclefibertypenot
Onlybycrossinnervationbutalsobycontinuouselectricalstimulationwithaparticularpatternuslng
rabbits・Accordingtothem・WhenanervetOaHfast
the

fast

musclefibersconvertedinto

slow

musclewascontinuouslystimulatedataslowrate，

fibershistochemicallyandphysiologiCallywithin4L8

Weeks．

Thisinteresting experiment shows that the electrophysiologiCalstateofmuscleplaysarolein
maintainingthefibertype・ThusitissuspectedthatthetypelfiberpredominanceinOurcasemaybe
CauSedbycontinuousstiffnessonthebasisoftheclumsymovement．
Referencesl）Isaacs，H・：Asyndromeofcontinuousmusclefiberactivity．J．Neurol．Neurosurg．Psychiatry．，24：319，1961．
2）Salmons，S・andVrebova，G．：TneinfluenceofactivityonsomecontractilecharacteriSticsofmammalianfastandSlowmuscles．J．Physiol．（Lond．），201：535，1969．

3）Romanul，F・C・A・，Sreter，F・A．，Salmons，S．andGergely，J．：neeffectofchangedpattemofactivityonhisto−
Chemicalcharacteristicsofmusclefibers．In

ExploratoryconceptsinMuscularDystrophyIl，，（InternationalCon−

gressSeries，No・333）（editedbyMnhorat，A．T．）ExceptaMedica，Amsterdam．1974．p．344．

−63−

肋JC〟ね′叫ぶ打叩わ′Gro叩SINGLEFIBEREMGOFPROGRESSIVEMUSCULARDYSTROPHIES
HiroshiNishitamiandTetsuroKonishi
Depa∫tmentOfNeurology，UtanoNationalHospital，Kyoto

In。rdertoinvestigateelectrophysiologicalcharacteristics，theauthorscomparedsinglefiberelec−
tromyographic findingsofmusculardystrophieswith thoseofotherneuromusculardisorders・For
varioussinglefiberelectromyographicparameters，WeStudiedthemeaninterspikeinterval（MISI）and
thevelocityrecoveryfunction（VRF）whichreflectsvariabnityoftheinterpotentialinterval（IPI）of
twomusclefibersbelongingtothesamemotorunit．
MethodandMaterials
We measured the meaninterspikeintervalandthejitterinthevoluntarilycontractedextensor
digitorumCOmmunis（EDC）muscleinpatientswithvarioustypesofprogressivemusculardystrophies
and。therneuromusculardisordersusingasinglefiberelectrode（MedelecSF−25）・
Themeaninterspikeinterval（MISI）isthemeanintervalofearlyactionpotentialsofmusclefibers
belongingtothesamemotorunit・Ineachpatient，MISIwasmeasuredasthemeanvalueofmorethan
fiveMISIsfromeachEDCmuscleusingasuperimposedmethod．
Thejitterwasmeasuredwithasignalprocessor（Sanei7TO8）andcalculatedasthemeanconsecutive
difference．Forvoluntarilyirregularinnervationrates，theactionpotentialswererecordedwithadata，eC。rder（TEACR410）．Inordertoexaminetherelationshipbetweentheinterpotentialinterval（IPI）
and the precedinginterdischargeinterval（IDI），the
Slgnalprocessorplotted theIPIinahorizontalline
andtheprecedingIDIinaverticallineofonetotwo
hundred potential pairs of the same muscle fibers
l

t

l

．。．「」

中 一

1

旧卜＿

（Figurel）．

l

Results
In patients with progressive muscular dystro−

ト一一一一一b一一」，

phies，themeanvalueoftheMISIswasslgnificantly

higherthanthatofcontroIsubjects・Inpatientswith
other myopathic disorders and chronic polyneur0−
pathy，the mean value of the MISIs tended tobe

一一一■一一■■−●1higherthanthatofcontroIsubjects・1
1

InpotentialpairsfromnormalEDCmuscle，the1
1

IPIonly slightly，Ordidnotfluctuatewithirregular11

innervation rates．In potentiallPl
tm ec）＼一一、／一J

Fig・lTheIehtionshipbetweenIPIandlDL
Theinterpotentialinterval（IPl）andthe
preceding interdischargeinterval（IDI）
weremeasuresas

a

，and

b

inthisfigure

wIdch wefe de丘ned as htervals or eachcrosslng POint using alevel technique・
Eachintervalwas mappedinthemanner
h仙elower ngure．For each potential
palr，One tO tWO hundred points wereplotted．

palrS from patientswith progressivemusculardisordersand othermyo−
pathic disorders，thelongIPI，mOre than5msec，tended to be shorter during strong voluntary con−tractions than during weak contractions・Among
three action potentialsbelonglngtOthesamemotor
unit，theIPIofapotentialpalrWithalongerIPIwas
moreremarkablyinfluencedbytheirregularinnerva−
tion rates than that with a shorterIPI．On rare oc−
Casions，theIPIwithashortinterval，lessthanlmsec，tendedtobelongerduringstrongvoluntarycontrac一一64−

tions・TheIPIofacertaindouble discharge tended to belonger duringstrong contractions andthe
doubledischargedisappearedoveracertaininnervationrate．
InmyastheniagravisandperipheralneⅣOuSdisorders，themostpotentialpairswithabnormaljitter
did not showanapparentrelationship between theIPIand theIDI．Inpatients withGunlain−Barrg
SyndromeandHirayamadisease，thelongIPIalsotendedtobecomeshortduringstrongvoluntarycon−
tractions．

Discussion
The tendency oflong MISIofmultLaction potentials belonglngtOthesamemotorunitinvoIves
SOme factorssuch asvariance ofthelengthofnervetWigs，eCtOPicmotorend−Platesandmusclefiber
diametersormusclepropagationvelocities・Inmusculardisorders，thevarianceofmusclenberdiameters，Whichis a characteristicfeatureofmorphologicalchangesinprogressive musculardystrophies，may
reflect thetendencyoflong MISI．Butthesinglefibertechniquecouldnotdefinetherelationshipof
eachfactorwiththeMISI．
ConcernlngtherelationshipbetweentheIPIandtheIDI，thedegreeofthevelocityrecoveryfunc−tion（VRF）1）ofeachmusclefiberparticipatesinthefluctuationoftheIPI．Asthemusclefiberpropaga−
tionvelocitiesoflatercomponentstendedtobeslowerthanthoseofearliercomponents2）andtheIPI
tended to becomelong duringstrongcontractions，themusclefiberswithslowpropagationvelocities
must have more pronounced VRF than those with fast propagation velocities・This tendency was
Observedinboth■myopathicmusclenbersandneurogenicmuscle点bers3）．Weconcludedthatthemuscle

fiberswithslowpropagationvelocitiesshowedpronouncedVRFduetochangesofmusclefibermem−
branecharactersinprlmarymyOPathicdisordersandsecondarymusclefiberchangesinneurogenicdis−
orders．
Referencesl）E−St丘lberg：Propagationvelocityinhumanmusclefibresinsitu．ActaPhysiol．Scand．，70：SuPPl．287，1−112，1966．
2）E・St星lberg：Electrogenesisinhumandystrophicmuscle．InPathogenesisofHumanMuscularDystrophies（ed．by

LP，Rowland）pp．570−587，ExceptaMedica，Amsterdam−OxLord，1977．
3）E・Stilberg，M・S・SchwaftZandJ・V・TronteU：Singlefibreelectromyographyinvariousprocessesaffectingthe
anteriofhorncell．J．Neurol．ScL，24：403−415，1975．

一65−

肋∫C〟Jdr砂∫〝opわ，G和岬

EXPERmlENTALMYOTONIAINHUMANEXTERNALINTERCOSTALMUSCLES

TeruyukiKurihara＋，MasayukiTanaka＊，KiyotakaToshimori＊＊，
MasaoTomita＊＊＊andKoichiroShibata＊＊＊＊TheThirdDepartmentofMedicinC，＊＊TheFirstDepartmentofAnatomy，＊＊＊TheSecondDepartmentofSurgery，MiyazakiMedicalConege，MiyaZaki

InordertoapplythemethodsofExcitation−Contraction（E−C）Uncouplingdevelopedbytheauthors
andothersl）formammalianmusclestohumanskeletalmuscles，eXPerimentswereperfomedonhumanexternalintercostalmuscles obtained during thoracotomies．The purpose of thisstudywasasfollows：（1）torecordtherestingmembranepotentialsofnormalhumanexternalintercostalmuscles，（2）toapply
E−C uncoupling tohuman skeletalmuscle specimensforthepurposeofmakingintracellularrecordings

during muscle activities，andtoobservethemorphologicalchangesofthehumanmusclesafterE−CUn−
COuPlingbyelectronmicroscopy，and（3）toinducemyotoniainthehumanmusclespecimensinvitroby
exposuretoanthracene−9−Carboxylicacid2）．
Methods
Experiments wereperformedonhumanexternalintercostalmusclespecimenswhichwereobtained
from two cases of thoracotomies under generAlaneSthesia：One forlung cancer，and the other fortraumatictrachealstenosis．These twopatientsdidnothaveanymuscledisorders．Greatcarewastaken
nottoirdurethemusclefibers，andintactmusclespecimensfromtendontotendonwereused．
Excitation

andcontractionwas

uncoupledbyexposlng

SPeCimenst0400mMglycerolinaliterofmammalianRinger

the

human

externalintercostal

muscle！

ssolutionfor60minutes，andthenwashing

SOlutionfor20minutes．Thelattercontained5mMcalciumand5mMmagnesiuminmammalianRingerssolutionforbettermaintenanceoftherestingmembranepotentials．3）Themusclespecimenswerethen
exposedto5mg／Ldantrolenesodiumsolutionforanother20minutes．
Myotonia wasinducedin the human muscle specimens by adding anthracene−9−Carboxylicacid tothemusclechamberattheconcentrationofl0−4M／L．4）Duringtheexperimentsthemusclespecimens
wereconstantlyoxygenatedwitha95％02and5％CO2mixturethatpassedthroughthemusclechamber・Resting membrane potentials，myOtOnic bursts，WerereCOrdedintracellularly by conventionaltech−niquesusingglassmicroelectrodesfilledwith3MKCl．
Results（1）The mean resting membranepotentialsofthenormalhumanexternalintercostalmusclespeci−
menswere−84．4±2．9（S．D．）mV，and−81．2±2．9（S．D．）mV．meseweretheresultsfortwospecimens
intheoxygenatedTyrodeSolution．
（2）Myotonia wasinducedinthe human externalintercostal muscle specimens by anthracene−9−
Carboxylicacid withoutreductionoftherestingmembranepotentials：，83．1±1．6（S．D．）mV，and−81．5±2．2（S．D．）mV．
（3）Myotonia wassuccessfullyinducedafter E−CUncouplingaswell，andmyotonicburstswerere−COrdedintracellularlywithoutmovementinducedartifacts．Duringmyotonicburststherewas9tollmV
initialdepolarizationbeforeeachactionpotentialstarted．
（4）Electron microscopic observation ofsemithin（0．2FLm）sections ofthe human externalinter−
COStalmuscle specimens before and aftersubjectingto thepresentmethodofE−CUncouplingrevealedthatvesicleswere

produced

near

the

triad

aft

er

E−C

ー66−

Uncoupling・Themyofibril，myOfnament，andtheZ−bandwereallintact・Highpower（x72，000）observationofthevesiclessuggestedthecontinuati。n。fthevesiclestothet−tubules・Furtherinvestigationsarerequiredtoconfirmwhethertheterminalcistem
Orthet−tubulesaredisruPtedinassociationwiththeproductionofthevesiclesnearthetriad．Someex−
ternaltracerwouldberequiredtoclarifythispoint．
DiseuSSion
Inordertomaketheintraceuularrecordingsfortheskeletalmusclefibersduringrepetitiveactivity
Eisenberg and others developed atechniqueofE−CUncouplingforfrogskeletalmuscle．neauthors
andothersdevelopedamethodofE−CUncouplingwhichisapplicabletomammalianmuscleusinga
hyperosmolarglyceroIsolution，WaShingsolution，anddantrolenesodiumsolution．Thepresentinvesti−
gationrevealedthatthelattertechniquecould besuccessfullyappliedtohumanexternalintercostal
muscle specimensinvitro・Myotoniawasinducedinthehumanmusclespecimensbyanthracene−9−
CarboxylicacidandtheintracellularrecordingsweremadeduringmyotonicburstswiththehelpofE−C
Uncoupling as described above・ElectronmiCroscopicobservationofthehumanexternalintercostal

musclespecimensbeforeandaftersubjectingtothepresentmethod ofE−CUncouplingrevealedthe
formationofvesiclesnearthetriadwithoutproducinganychangesofthemyofibri1，myOfilament，OrZ−band・Withthistechnique，PrOlongedintracellularrecordingswithhumanskeletalmusclespecimens
duringactivitiesarepossible．
Sincethe present experimentsinvoIved myotoniainduced by anthracene−9−Carboxylic acidin
normalhumanmusclespecimens，furthercorrelationofhumanmyotonicdisorderstotheresultsofthe
PreSenteXperimentshastobeperformed．
Acknowledgement
ProfessorShukuroArakikindlyreviewedthemanuscript．
Rererencesl）Kurihara，T・＆Brooks・J・E・‥Excitation・COntraCtionuncoupling‥TheeffectofhyperosmolarglyceroIsolutionand

dantrolenesodiumonmamma鮎nmuscleinvitro・Arch．Neuro1．，32：92，1975．
2）Bryant，S・H・＆Morales−Agdera，A・＝ChlorideconductanceinnormalandmyotonicfibeTSandtheactionofmono−
CarboxylicaLOmaticacids．J．Physio1．，219：367，1971．
3）Eisenberg，R・S・・Howell・J・N・＆Vaughan，P・C・‥Themaintenanceofrestingpotentialsinglycerol−treatedmuscle
nbeIS．J．PIlySio1．，215：95，1971．
4）Kurihara，T・：Experimentalmyotonia．FouaPsychiatr．Neurol．Jpn．，31：653，1977．

−67−

肋JC〟kr功∫〝op力γGro岬

CONTRACTILEANDCHEMOSENSITIVEPROPERTIESOFMUSCLETREATEDWITHCALCIUMIONOPHORE（A23187）
Masal1aruTakamori，YoshhikoIdeandMitsuhiroTsujmata
FirstDepaItmentOfIntemalMedicine，NagasakiUniversitySdlOOlofMedicine・Nagasaki
Muscle membrane abnormalities have been suggested for the pathogenesis ofhuman muscular
dystrophy．Severalofthetheoriesdependonanabnormalinfluxofcalcium・Anexcessiveintracellular

calciumconcentrationmayaccountfortheincreasedproteindegradation，andthroughenhancementof
ca＋＋−aCtivated neutralproteases or ATP depletion，lead tomuscle necrosisandweakness・Calcium

ionophore，A23187，Whichraisestheintracellularcalciumconcentration，lSauSefultoolforstudying
thiscalcium−relatedtheoryofmusclecellnecrosis・Inthepresentinvestigation，COntraCt止eproperties
ofA23187−treatedmusclewerestudiedandcomparedwiththosewhichwepreviouslyobtainedfor
humandystrophicmuscle．

Diaphragms（63．7−70．8mgwetweight）weredissectedfromyoungadultIもaleratsoftheWistar
strain．Controlmusclewasequilibratedfor60minutesat37OcinmammalianRingersolutioncontaining
ethanol（10pl／ml），gaSSedwith5％CO2inoxygen・Testmusclewasimmersedfor60minutesat370cin
Ringersolutionwhichcontainedethanol−dissoIvedA23187（10pg／ml）andwasaeratedwithO2＋CO2（95：5）．Eachmusclewasthenplacedintheoxygenatedsolutioninachambercontainingplatinumplateelectrodes for maSSive stimulation，and was connected to a straingauge and camier amplifier foris。metriCrecording．Directstimulationwasassuredbycurarizationofthemuscle（d−TC，6FLg／ml）・Using
twitch（Pt），tetanuS（Po），and their derivatives，COntraCtileanalysesweredoneonthebasisofthe
conceptofthe

activestate，Whichwassubdividedintothetimecomponent（Tl／2R；TdPt／dt）・andthe

intensitycomponent（d2pt／dt2；dPo／dt）．
ResultsamdDiscussion
A23187−treated muscleshowedprolongationofTdPt／dt（timefromonsettopointofmaximum
velocityoftwitchdevelopment）andTl／2R（timefromonsettothehalf−relaxationleveloftwitch）・The
former，thedurationoftheactivestate，WaSmeaSuredasafactorexpresslngtheamountofcalcium
liberated from the sarcoplasmicreticulum andalSo the rate ofthereaction between calcium andtroponin・Thelatterfactor，thedecayoftheactivestate，reflectstheremovalofcalciumfromtroponinandtheuptakeofcalciumbythesarcoplasmiCreticulum，andmayalsoberelatedtoarelationshipbe−
tweencalcium−bindingbytroponinandATP−bindingbymyosin・Abnormalitiesoftheseindiceswhich
relate to subceuular events werealso foundindystrophicmuscle・Post−tetanicprolongation ofthe
relaxationtimeoftwitch（Tl／2R）wasalsoabnormallyincreasedinbothA23187−treatedmuscleanddystrophicmuscle．Themaximumaccelerationoftwitchdevelopment（d2pt′dt2；aCtivestateintensity

ofshortening）wasmeasuredtoexpresstheamOuntOfcalCiumreleasedfromthesarcoplasmicreticulum
attheinitiationofexcitation−COntraCtioncoupling・niswasnormalinA23187−treatedmuscle・W旭eit
was reducedin dystrophic muscle．The maximum tetanic force（Po；aCtivestateintensityofload−
bearing），attributedtothenumberandintrinsicstrengthoftheactin−myOSincross−bridges，WaSdecreased
inbothA23187−treatedmuscleanddystrophicmuscle・AIsodecreasedinbothmuscleswasthemaxi−
mumvelocityoftetanusdevelopment（dPo／dt）whichrelatestotheforce＞Velocityrelationofthecon−
tract止ematerialifnotcontaminatedqyabnormalseries−elasticcomponents・Thetwitch／tetanusratio（Pt／Po）wasmarkedlyincreasedreflectinggreaterreductionofthetetanicforceandtheprotractedtime
courseoftheactivestate，Whileitwasdecreasedindystrophicmuscle・−68−

Sensitivity to epinephrine of muscle contraction，mediatingthrough beta−reCePtOrSinthecAMP−dependent or cAMP−independent pathway，WaS PreViously found byustobeusefulforestimationof
membrane／submembrane calcium−related abnormalitiesin dystrophic muscle andin experimentally
denervated muscle・Immersionoftherat diaphragmsinasolutioncontainingisoproterenol（2FLg／ml）
CauSedthetwitchtoincreasewithprolongationoftheactivestatedurationanddecay．Thesamechange
intwitch asin the controlafterisoproterenoltreatment was seenin A23187−treated muscle，WhⅡedystrophicmusclerespondedinamannerdifferentfromthecontrol．
Conclusion
ThetreatmentofskeletalmusclewithA23187inducedmarkedchangesintetanusparametersthat
mainly relate to abnormalitiesin contractile proteinsandpartoftheactivationprocess・Twitchpara−
metersincludingits response to beta−adrenergic stimulants were not remarkably altered by A23187
except parameters expresslng the time course of the active state．Our previous study demonstrated
that，COmPared with changesin tetanus，abnormalities of twitch，Particularlyin the acceleration of
twitch development and response of twitch to beta−adrenergic stimulants，tend toappearinanearlyStageOfmuscledystrophy．Thetwitch／tetanusratiowasincreasedintheA23187−treatedmuscle，Whileit was decreasedindystrophic muscle．merefore，theA23187−treatedmuscleisnotwhollysimilarto
dystrophicmuscle．

−69−

肋∫CIJねr砂∫れ〉p力yGr0岬

PHOSPHATIDYLINOSITOLMETABOLISMINDUCHENNEERYTHROCYTEMEMBRANES
KeiichiTakahashi，KikukoNanba，KazukoGoto，FumikoShundo，KeltiiJinnal，
YukihiroKomineandTakuoFujita
Depa∫tmentOfMedicine，KobeUniversity，Kobe
It stillremains obscure whethererythrocytemembranesareprimarilyinvoIvedinDuchennemus−
culardystrophy，despitethefairlylargenumberofbiochemicalandmorphologicalabnormalitiesofthe
membranesalreadyreported．1）DiseetalreportedmorepronouncedfacilitationofKeffluxbycalcium
ionophoreA23187inDuchenneerythrocytesthanincontroIs．2）Increasedfragilityandechinocytosis
inDuchenne erythrocytes werealSo reported・Phosphatidylinositol metabolismis coupled withphysiologicalresponses of cellmembranes．3）AllanandMichellreported thatmicrovesiculationwas
accompanied by activation of polyphosphoinositide phosphodiesterase with the formation ofl，2−diacylglycerolandphosphatidate・4）nesereportspromptedustostudyphosphatidylinositolmetab0−
1isminDuchenneerythrocytes，includingtheeffectofcalciumions・Methods
BloodspecimensweretakenintoheparinizedsyrlngeSfrompatientswithDuchennedystrophyand
controIsincludingnormalsubjectsandpatientswithspinalprogressivemuscularatrophy．Erythrocytes
wereseparatedbytheuseofdextran，followedbywashingtwicewithO．9％NaCl，・hemolysisandwashingwithseveralkindsofhypotonicbuffers・Thefollowingconditionsgavethemaximumincorporationof
3H・inositolintothemembranes：20mMTris−HClbuffer（pH7．5），0．5mMATP，0．25mMCTP，20mM
MgCl2，0．05mM3H−inositol，100plery−throcytemembranes（aroundO．lmgprotein）
ina total volume of200Jll，incubated at
370c for30min．The reaction was termi−
nated with5％TCA，followed byextraction
Ofphospholipids with a mixture ofchloro−
form，methanoland conc．HCl（200：100：1，
V／V）and partitioningwith O．l N HCl・The
bottomlayer was subjected to thin−layer
Chromatography or radioactivity measure−ment．
Results：
Calciumions markedly reduced thein−
corporation of3H−inositolinto the mem−branes．EGTApreventedthisinhibition，Sug−
gesting aCa−SPeCificinhibition・Noessential
difference，however，WaS Seen between thepatients

and controIs．A thin−layer chr0−

matogram on Silica Gel H disclosedalmost
completeincorporation of3H−inositolinto
diphosphoinositide under these conditions・Fig・lIncorporation of3H−inositolinto erythrocyte
membraneslysedinlOmMTriS−HClbufferbH
7．4）containingl mM EDTAandlmMdithio−

Thehighestincorporationof3H−inositoIwas
achieved by the use of membranes，Pre−
paredwithlOmMTris−HCl，PH7．5，includ−

thfeito1．
−70−

inglmMEDTAandlmMdithiothreitol・The3H−inositolincorporationintoerythrocytemembranes
fromthepatientsandcontroIssimultaneouslypreparedwiththisbuffershowedasignificantdifference
asshowninFig．1．
Simultaneously measured adenylate cyclaseactivitiesofthemembranepreparationsalsofai1edto
revealany difference betweenthe two groupsand noactivationbyepinephrinewasdemonstratedat
all，unliketheresultsreportedbyWacholtzeta1．5）
These results did not support systemicinvoIvement of erythrocyte membranesin Duchenne
musculardystrophy．
ThisstudywassupportedbyGrantNo・80−02−27fromtheNationalCenterforNervous，MentalandMuscularDisorders（NCNMMD）oftheMinistryofHealthandWelfare，Japan．
Referencesl）Rowland，LP∴BiochemistryofmusclemembraneSinDuchennemusculardystrophy．Muscle＆Nerve3：3，1980．
2）Dise，C・A・，Goodman，D・B・P・，Lake，W・C・etal・：EnhancedsensitiYitytocalciuminDuchennemusculardystrophy．
Biochem．Biophys．Res．Commun．79：1288，1977．
3）Michell，R・H・：InositoIphosphohpidsandcensurfacereceptorfunction．Biochim．Biophys．Acta415：81，1975．
4）Al1an，D・＆Michell，R・H・：TYlerelationshipbetweenCa2＋−mediatedpolyphosphoinositidephosphodiesteraseactivi・

ty，1，2−diacylglycerolaccumulation，andmicrovesiculationineIythrocytes．Inprogressinclinicalandbiologicalresearch（editedbyLux，S・E・，Marchesi，V・T・＆Fox，C・F．）AlanR．Liss，Inc．NewYork1979．Vol．30，p．523．
5）Wacholtz，M．C．，Raible，D．G．，Jackowski，S．etal．：AdenylatecyclaseandATPaseactivitiesinredcellmembraneSOf
PatientsandgeneticcarriersofDuchennemusculardystrophy．Clh．Chim．Acta．96：255，1979．

ー71−

肋∫C〟krpygけOPカタGr0叩

CONCANAVALINABINDINGONTHEERYTHROCYTESINDUCHENNEMUSCULARDYSTROPHY
MasanoriUono＊，HirokumiBeppu＊二MotowoNakajima＊＊，FumiakiNishiyama＊＊＊andHiroshiHirano＊＊＊＊DepartmentofNeurologyITokyoMetropolitanNeurologicalHospital，Tokyo＊＊DivisionofChemicalToxicologyandlmmunochemistry，FacultyofPharmaceuticalSciences，

UniversityofTokyo，Tpkyo＊＊＊DepartmentofAnatomyIKyo血UniversitySchoolofMedicine，Tokyo

Aswaspreviously reported，red blood cells（RBCs）from geneticallydystrophicchickensshowa
20to40％reductionofConAbindingsitescomparedtonormalcontroIs・1）Usingthesametechniques，
wehaveinvestigatedtheConAbindingonRBCsfromDuchennemusculardystrophy（DMD）patients・
RBCswereobtainedfromsixDMDpatients，Of8to23yearsofage，andsixnormalcontroIs，Of17
to19yearsofage．UnfixedRBCs（lx106／ml）WereincubatedwithferritinconjugatedconcanavalinA（Fer−ConA；30−60pg／mlofConA）at370cfor30min．RBCslabeledwithFer−ConAwerewashedwithphosphatebufferedsaline（PBS；PH7．2）andsuspendedataconcentrationof50volume％・Asmall
volume ofthis solution was dropped onto the surface ofdistilled water・Some ofthese RBCs wereruPturedattheair−Waterinterface・TheRBCmembraneSSPreadflatbysurfacetension，Werepickedup
ona grid with asupportingfilm，WaShedwithdistnledwater，andprocessedfortransmissionelectron

microscopy．AscontroIs，RBCswereincubatedwithfreeferritin，OrWithFer−ConAinthepresenceof
O．2Mα−methyl−D・mannOSide．Inbothcases，nOferritinwasfoundonthecellsurface．
RBCmembranespreparedbylysisattheair−Waterinterfaceshowedthetopologicaldistributionof
Fer−Con A on the membrane surface and showedstrikinglythedifferencebetweennormaland DMD
patients．Numerousferritinparticles，Singlyorinsmallclusters，WeretaggedontothesurfaceofRBCs
obtainedfromnormalcontroIs．While，inthecaseofDMDpatients，thenumberofferritinparticleson
the membrane wasless．On the electron microscopic photographs（enlarged，X60，000），four regularsquaresofl，000nmsidelengthwerechosenrandomly，andweresubdividedintolOOnmxlOOnmunit
squares．Thenumbersofparticlesperunitsquareineachcasewerel・07±1・45，1・18±l・70，1・29±1・95，
1．30±1．68，1．47±1．68andl．63±1．68forDMD，andl．48±2．07，1．75±2．52，2・11±2・69，3・32±3・27，
4．04±3．28and4．15±3．68forthecontroIs．medifferencebetweenthemwassignificantatp＜0．05
1evel．

Inordertoassessthedistributionpatternofferritinparticles，Irimuraetal2）introduced，aSapara−
meterofclustering，theratioo（k）／0，and。m（k）．ora．Id。m（k）isthestandarddeviationofthenumberof
particlescontainedinunitsquareswithasidelengthof250xkÅrandomiydistributedwhichisobtain−
edtheoretically fromatableofrandomnumbers．AccordingtoIri血uraetal，thevalueofo，and。m（k）
isafunctionofvariablekandofthetotalnumberofparticles（TN＝thenumberofparticlesinl，296

unitsquares），aSShownbytheequation：

Crand。m（k）＝0・029TN／k（k＜6）0（k）is thestandard deviationofthenumberofferritinparticlesinunitsquareswithasidelengthof
250x kÅ，COuntedonelectronmicrographs．or（4）／Or，and。m（4）ratiosforDMDwerelessthanthoseof
normalcontroIsasshowninFig．1（p＜0．01）．
TheevidencepresentedhereindicatesthatRBCmembranesfromDMDpatientshaveslgnificantly
fewerreceptors for Con A，aSShowninthepreviousreportfordystrophicchickenRBCs・Ithasbeen
suggestedthatband3proteinsand／orotherglycoproteinsmakeuptheintramembraneparticles（IMPs）＿72＿

revealed by freeze−fracture techniques．3）Band3proteins are
Said to be majorbindingsitesforConAinhumanRBCs．Tnere−fore，theresultsofourpresentstudyseemtobeconsistentwith
thedescriptionofWakayamaeta14）withregardstoDMD−RBC
membranes andIMPs．Itis also suggestedthat ConAreceptors
may participatein the modulation of membrane fluidity，and
enzymes，5）Takingallthesereportsintoconsideration，Wemay
find

some

plausible

explanations

for

the

functionalab

normali−tiesinthemembraneofdystrophicRBCs．
Referemces

重∈︒PU円卓宣b

COnSequently，may COntribute to the regulation of membrane

1）Uono，M．，Beppu，H．，Nakajima，M．，Nishiyama，F．and Hirano，H．：
Concanavalin Abinding ontheerythrOCytePlasmamembraneofdys・
trophic chickens．Current Researchin Muscular Dystrophy，Japan：
The Proceedings ofthe AnnualMeeting ofthe Muscular Dystrophy
ResearchGroup，1978，1979，Tokyo：92−93，1980．
2）Ⅰrimura，T．，Nakajima，M．，Hiran0，H．andOsawa，T．：Distributionof

femitin・COnJugatedlectins on sialidase＞treated membranes of humanerythrocytes．Biochin．Biophys．Acta，413：192−201，1975．
3）Pinto da Silva，P．，Branton，D．and Douglas，．S．A．：Localization of

N

D

Fig．1

ConAantkensitesonhumanerythrocytesghosts．Natule，232：194−196，1971

4）Wakayama，Y．，Hodson，A．，Pleasure，D．，Bonina，E．and Schotland，D．L．：Alterationin erythrocyte membrane
structureinDuchennemusculardystrophy．Ann．Neuro1．，14：253−256，1978．
5）Nakajima，M．，Tamura，E．，ITimura，T．，Toyoshima，S．，Hirano，H．andOsawa，T．：MechanismoftheconcanavalinA
hducedchangeSOfmembranefluidityofchickenerythrocytes．J．Biol：hem．（Tokyo），89：665−676，1981．

ー73−

肋∫α血rJ抄JかOpカツGro岬

CALCIUM FLUXOFERYTHROCYTESINDUCHENNE MUSCULARDYSTROPHY

Sin

ichiShqji

TheDepartmentofMedicine，ShinshuUniversitySchoolofMedicine，Matsumoto
The calciumflux of erythrocytes from patients with Duchenne muscular dystrophy（DMD）was
measured．Passiveinflux was determined using erythrocytesincubated withlanthanum chloride as a
SPeCificinhibitor for calcium−aCtivated magnesiumJependentATPase．Therewasnosignificantdiffer−encein calciuminnuxincytosolandin membranefractionsbetweenDMDandcontrolage−matChedboys．Influx of calcium wasalSo determined using ATPJepleted erythrocytesincubated withoutthe
inhibitor．AgaintherewasnosignificantdifferencebetweenDMDandcontroIs．Effluxofcalcium wasmeasured using erythrocytesloaded with calciumionophore A23187．The calcium efflux speed was
related tointracellular calcium concentration andit reached a plateau at aboutlmMconcentration．
There was no significant differencein calcium efflux．rate as a function ofintracellularcalciumcon−
CentrationbetweenDMDandcontroIs．
Refefence
Shqji，S．：CalciumfluxoferythrocytesinDuchennemuscuhrdystrophy・J・Neurol・Sci・，inpress・−74−

肋∫mbr砂丘加タカノG和岬

CaANDATPCONTENTSOFERYTHROCYTESmDUCHENNEMUSCULARDYSTROPHY
MizukoYoshidaandKazuyaAndo
NationalCenterforNerYOuS，MentalandMuscularDisorders，Tokyo

Recently，highCalciumaccumulationinmusclecellsofpatientswithDuchenneMuscularDystrophy（DMD）hasbeenreported．Thepresentstudydealswiththemechanismofthisphenomenon．
hst year，We rePOrted that there was no appreciable differencein the occurrence of deformed
erythrocytes between DMD and controIs whenthe erythrocytes werein plasma or were storedina
buffersolutioncontainingdextrosewithoutCa2＋．−Ontheotherhand，WhentheerythrocytesfromDMD
andcontroIswerestoredinPhosphateBufferedSalinecontainingCa2＋（PBS（＋））withnodextrose，alargenumberofechinocytesinDMDerythrocytesoccurred．ItisgenerallyacceptedthattheechinOcytes
OCCurbyeithercalciumaccumulationorbyATPdepletionintheerythrocytes．
The present study attempted to clarify the cause of the occurrence of the echinocytesinDMD
erythrocytesbyusingabuffersolutioncontainingLCa2＋withdextroseforpreventingtheATPdepletion．

AIsocalciumandATPcontentsofDMDerythrocyteswerecomparedwiththoseofcontroIs．
Su叫ectsandMethods
ThebuffersolutionusedinthisstudywasPhosphateBufferedSalinecontainingCa2＋andMg2＋，

With5mM−dextrose added（PBSD（＋），PH7．4281mOsm／1．）Ileparinized venousblood was obtained
from DMD patients and age−matChed healthy boys as controIs．Erythrocytes separated from plasma
together with the buffy coat were washed three timeswithPBSD（＋）．Aftereachwashing，thebuffy
COat WaS remOVed．Then，they were suspendedin PBSD（＋），inl0−15％hematocritproportions，andstored・at40cfor72hours，beingstirredoccasionally．Thestorederythrocyteswerecentrifugedandthen
Were adjustedin PBSD（十）to45％hematocrit proportions，and were subjected to calciumandATP
levelmeasurements．me ATPin the fresh erythrocytesjustafterwashingthreetimeswithPBSD（＋）
WaSalSomeasured．
Ca determination：Erythrocyte calcium was determined by qjecting the calciumin the external

SOlution（TrappedPlasma）thatremainsinthespacesbetweenthepackedcells．meTrappedPlasmawas
measuredwithIndocyaninegreen（ICG：giftfrom DaiichiSeiyaku Co．，Ltd．）．mesamplesfortrapped
PlasmameasurementwerepreparedbycentrifugationatthesametimeasforthesampleforCadetermi−
nation．Calciumwasmeasuredbyatomicabsorptionspectrophotometry．SamplesforCadetermination，
boththepackedcellsandtheexternalsolutionweretreatedwithHNO3−H2SO4．
ATPdetermination：ATPinerythrocytesextractedwithO．6Nperchloricacid，StOredat40C，WaS
measuredwithin24hoursbytheenzymeassaymethod（ATP−teSt；Boehringer）
Hemoglobin（Hb）determination：Hbwasmeasured bytheazide−methemoglObinmethod（Hemokit−S：Nihonshoji）to evaluate the ATP content relative to Hb．Al1solutions used were preparedjust
beroreuse．
ResultsandDiscussion
Erythrocyte calcium：Tnecalcium contentoftheerythrocytesfromsevenDMDpatients（average
19．3years old）was2．5times higherthanthatofsevenhealthyboys（average19．7yearsold）．Itwas

COnfirmed that the ATP oftheerythrocyteswasnotdepletedunderthepresentconditions．Thehigh
CalCium contentin DMD erythrocytesascompared withcontroIs，issurmisedtobecaused bytheac−
cumulationofcalciumintheerythrocytesand

ATPaseactivity

bytheeasy

occurrenceofCainflux．BecauseCa2＋−

fortheCaeffluxpumpofDMDerythrocytesstoredat40cmightbelowsinceCa2＋一一76−

ATPaseactivityoftheerythrocytesstoredinthecoldisinhibitedorlow・Theseproposalsarealsosup−
portedbyotherreportsl）thatconfirmedhigherCa2＋−ATPaseactivityofDMDerythrocytesat370C・

Accordingly，the occurrenceofalarge numberofechinOcytesin DMD erythrocyteswhen they
are storedinPBS（＋）solutioncontainingCa2

Withoutdextrosewouldbeduetoaccumulationof

calCiumintheDMDerythrocytesbasedondepletionofATP・
ErythrocytesATP：The ATPcontentoferythrocytesfromtenDMDpatients（average16・4yearsold），afterstoringat40cfor72hoursandjustafterwashing，WaS12−15％higherthanthatoffourteen
healthyboys（average16．8yearsold）．ThisresultcoinCideswiththeresultsreportedbyDanon2）・One
ofthereasonsforthehighATPlevelinDMDerythrocytesmightbethehighCalciumcontentofDMD
erythrocytes，becausetheATPintheerythrocytescontroIsCabindingtomembranes・Conclusiomsl）CacontentofDMDerythrocytesstoredinPBSD（＋）at40cfor72hourswas2・5timeshigher
thanthatofcontroIs．
2）ATPcontentsofDMDerythrocytesafterstoringinPBSD（＋）at40cfor72hoursandjustafter

washingthreetimeswere12−15％higherthanthoseofcontroIs・3）TheoccurrenceofalargenumberofechinocytesinDMD erythrocytesinPBS（十）solution
wouldbeduetotheaccumulationofCaintheerythrocytesbasedonATPdepletionincells・
Referencesl）Ruitenbeek，W．：Membranel）Oundenzymesoferythrocytesinhumanmuscula∫dystrophy・J・NeuIOl・Sci・，41‥71，

1979．
2）Danon，M．J．etal．：Erythrocytemetabolisminmusculardystrophy・ATCh・Neurol・，35：592，1978・−77−肋∬〟kr砂∫佃戸力yGr0岬

ATPaseANDADENYLATEKlNASEOFERYTHROCYTEMEMBRANESINHUMANMYOTONICDYSTROPHY
ShiroMawatari＊，YasunobuAntoku＊串，HidekiIgisu＊＊＊，IkuoGoto＊＊＊andYo血短oroKuroiwa＊＊＊＊HealthAdministrationCenter，KyushuInstituteofTechnology，KitakytlShu＊＊NationalAkasakaHo呼ital，ChikugoCity＊＊＊DepartmentofNeurology，NeurologiCalInstitute，FacultyofMedicine，KyushuUniversity，Fukuoka

Variohsabnormalitiesoferythrocytes（or erythrocytemembrane）have beenreportedinhuman
myotonic dystrophy．Theyincluded（Na＋K）−ATPase，（Ca＋Mg）−ATPase，Cation exchange，PrOtein

kinase，adenineuptaketo ATPandADP，electronspinresonancepattern，membranedeformabnityand
erythrocyteshape．However，therearecontradictoryreportsonthesereportedabnormalities．Therefore，We re−eXamined ATPase activityoferythrocytemembranesinhumanmyotonicdystrophy．AdenylatekinasewasalSo examined，becausemostofthereportedabnormalitieswererelated directlytoadenine
nucleotides．

Patientswithmyotonicdystrophy（MD）andage−matChedcontroIs（patientswithlungtuberculosis）
Wereinthesameinstitutionformore thanonemonth．BloodfromthesubjectsandcontroIswastaken
intoheparinizedtubesonthesamedayatalmostthesametime．Thereafter，Preparationoferythrocyte
membranesandassayofenzymesweresimultaneouslydoneforMDandcontroIpatients．Aftersepara−tionofplasmaandbuffycoat，erythrocyteswerewashedthreetimeswith8．5％NaClandthebuffycoat
WaSremOVedateachwashing．Theerythrocyteswerehemolyzedwith5mMTris−HCl（pH7．4）andmem−
branes were washed four times with the samesolutionby centrifugationat25，000g for20min．The
whitemembraneswerefinallyresuspendedinthesamesolutionandstoredat−350cuntiluse・ATPase
activities

were

assayed

by

using

a

coupledenzymatic

method．Theassaymixturefor

totalATPase

COnSistedof50mMTris−HCl，pH7．5，5mMMgC12，100mMNaCl，15mMKCl，lmMATP，lmMPEP，0．25mM NADH，PyruVate kinase，LDH and erythrocyte membranes（300to400FLg）．The ouabain
concentrationwasO．1mM，Whenitwasused・Afterequuibrationat370C，theactivitywasfollowedby
recording the absorbanceat340nm．AdenylatekinaseactivityinthedirectionATP＋AMP→2ADP

WaSdetermined byaddingl mMAMPtotheassaymixtureofATPaseincludingO．1mMouabain，and
adenylate kinase activity was calculated by subtraction ofthe ATPaseactivityfromtheactivitywith
AMP．Adenylate kinase activityinthe direction2ADP→ATP十AMPwasalso assayedinareaction

mixturecontaining100mMtris−aCetate，pH7．0，10mMMg−aCetate，20mMglucose，0．6mMNADP，llmMADP，hexokinase，G−6−PDHandmembranes，andtheactivityat370cwasdeterminedbyrecord−
ingat340nm．Thesamemethodwasusedfortheassayofadenylatekinaseactivityinplasma．
TheenzymeactivitiesoferythrocytemembraneSareShownintheTable，andno differenceofthenbleErytbocytemembraneATPaseandadenylate aCtivitiesbetweenMDandcontroIpatientswas
kinaseactivityinmyotonicdystrophyandcontroIs．
observed．Adenylate kinase activity of theControI Myotonic
（n＝6）

（n＝5）

erythrocyte membranes was muchhigherthantheATPaseactivityinthepresentstudy・An

TotalATPase9．9±2．6

9．4±2．3

apparentincreaseofadenylatekinaseofplasma（Na＋K）−ATPase

2．8±0．8

2．7±0．7

WaSnOtedintwo out offivepatientswithMD，
but the meanaCtivity ofthe enzyme wasnot

Adenylatekinase
ATP＋AMP→2ADP

39．1j：8．8

41．1土16．0

2ADP−ナATP＋AMP

26．1±4．728．2±9．1

Enzymeactivities；mFlmOlesmin／mg．membraneproteh（at370C）

SignificantlydifferentbetweenMDandcontroI
Patients．
Our results forouabainsensitiveATPaseof
erythrocyte membranes of MD contradicted−78−thoseofthepreviousreports．Therearealsocontradictionsamongthereportedabnormalities．Brownet
al．1）reportedthat（Na＋K）−ATPasefromtwopatientswithMDwasstimulatedbyO．lmMouabain，and

theresultsofDobosz2）ShowedlessinhibitionoftheenzymebyO．lmMouabainthanthatofnormal
erythrocytemembranes・Mishraetal，3）whousedasimilarassaymethodforATPaseactivitytothatin
thepresentstudy，rePOrtedthatl mMouabaininhibitedtheenzymefromMDgreaterthanthatfrom
normalcontroIs．Theexactcause（S）ofthedicrepancyamongthereportsisnotknown．Slightdifferences
inpreparationoferythrocytemembranesmayhavecausedinconsistencyofthepropertiesofthemem−
branesorinconsistentresultsamongthereportedabnormalities・Inthepresentstudy，thepatientswith
MDandcontroIswereinthesameinstitution，andsamplesfrompatientswithMDandcontroIswereob−
tainedandprocessedsimultaneously．
Referencesl）Brown，H．D．，Chattopadhyay，S．K．，＆Patel．A．B．：Erythrocyteabnormalityinhumanmyopathy．Science，157：
1577，1968．
2）Dobosz，LN・：Erythrocyteghost（Na＋K）ATPaseactivityinDuchenne

sdystrophyandmyotomia．J．Neuro1．，214：

61，1976．
3）Mishra，S・K・，Hobson，M．，＆Desaiah，D．：ErythLOCytemembraneabnormalitiesinhumanmyotonicdystrophy．J．
Neurol．Sci．，46：333，1980．

ー79−

肋∫助かり∫加タカノGrO叩ERYTHROCYTEACETYLCHOLINESTERASEINMYOTONICDYSTROPHY

YoshigoroKuroiwa＊，HidekiIgisu＊，YasunObuAntoku＊，ShiroMawatari＊＊andIkuoGoto＊＊DepartmentofNeur0logy，FacultyofMedicine，KyushuUniYerSity，Fukuoka＊＊HealthAdministrationCenter，KyuShuInstituteofTeclm0logyandDepaTtmentOfNeurology，FacultyofMedicine，KyushuUniversity，Fukuoka
We studied the erythrocyte membranein myotonic dystrophy（MyD）with acetylcholinesterase（AchE）．PatientswithMyDandcontroIs（patientswithmildpulmonarytuberculosis）Wereinthesame
institutionformorethan6monthsandwerematchedbyageandsex．Experimentsl（Studyonhemoly−
SateS）and2（study on ghosts）were done on separate days but for each experiment samplesfrom
PatientsandcontroIswereobtainedatalmostthesametimeonthesamedayandprocessedsimultane−
OuSly．
Hemolysateswerepreparedalmostidenticallytoasbeforel）．ⅠムExperiment2，theerythrocytes

were washed three timeswithisotonicsalineaftertheplasmaandbuffycoatwereremovedcarefuuy．
The erythrocyteswere hemolyzed withlOvolumes of10mM tris−HCl，PH7．4，and the membranes
were sedimented at20，000g for20min．The membraneSWereWaShed5timeswiththesamebuffer
andcollectedbythesamecentrifugation．TheAchEactivitywasmeasuredbythemethodofEllmanetal2）slightlymodified．neactivationenergy，tranSitiontemperatureandHi11coefficientfortheinhibi−tionoftheenzymeactivitybyfluoridewereobtainedgraphicallyasdescribedpreviouslyl）．
TherewasnodifferencebetweenMyDpatientsandcontroIsintheenzymeactivityinhemolysatesoringhostswithinthetemperaturerangestudied（7−140cinExperimentl，and25−4lOcinExperi−
ment2）．No significant difference was noted between MyD patients and controIsin the transition
temperature（Tc）orintheactivationenergyeitheraboveorbelowTcinExperimentl．InExperiment2，
the activation energy did not differ between MyD patients and controIs．TheHiucoefficientforthe

inhibition byfluoridewasapproximatelylinMyDpatientsandcontroIsat13，25and370C・me
fluoride dose for50％inhibition oftheenzymeactivitywasdifferentbetweendifferenttemperatures
buttherewasnodifferencebetweenMyDpatientsandcontroIs．
The activation energy，tranSition temperature and Hill coefficient for theinhibition of AchE
activitybyfluoridewereconsideredtoreflectpropertiesofthemembrane．Thus，thepresentresultssug・
gestthatthereisnogrossabnormalityinAchEoritsenvironmentintheerythrocytemembraneinMyD．
Referencesl）Ⅰgisu，H．，Mawatari，S．，KufOiwa，Y．，etal．：ErythrocyteacetylcholinesteraseinDuchennemllSCulardystrophy．Clh．

Chim．Acta105：241，1980．
2）E山nan，G．L．，Courtney，K．D．，AndeIS，V．，etal．：Anewandrapidcolorimetricdeterminationofacetylcholinesterase
activity．Biochem．PhaTmaCOl．7：88，1961．

ー80−

肋∫α血′勒∫打叩カツGr フ岬

CONGENITALMYOPATHYANDABNORMALTRYPTOPHANMETABOLISM
MasatoshiYagita，HaruhitoNakahira，MichioTakuma，AkiraTaira，KazutoKameyama，ManabuYoshikawa，KenzoYoshida，KyokoKatayama，KatsuyaTamura，KanaeKusaka，HisaomiKawaiandKazuoMiyoshi
TlleFirstDepartmentoflnternalMedicine，SchoolofMedicine，TheUniversityofTokushimaITokush血a
We previously found andreported abnormalfindingsofthetryptophan−kynureninepathwayinpatientswithnemalinemyopathyandrelatedmyopathies・Herewedescribeabnormalmetabolismofthe
pathwayintwocaseswithso−Calledarthrogryposismultiplexcongenita（AMC）fromtwopedigrees，Who
werenewlyfoundbyus，andfurthermoresummarizetheresultsobtainedtodate・MaterialsandMethods
Twocaseswithso−CalledAMC，theirparentsandthegrandmotherfrom2pedigreeswereincluded
inthestudy．Tryptophanmetabolismwasinvestigatedbythemethodreportedpreviously・Briefly，after

theloadingofI；tryPtOPhanatadoseoflOOmg／kg（5g，insubjectsweighingmorethan50kg），24−hoururinary excretionofkynurenicacid（KA）andxanthurenicacid（XA）wasassayedbytheSatoh−Pricemethod．
Resultsl．Cases：Subiectswere2casesfrom2pedigrees；CaSel（M．T．，17−yr−01dmale）andcase2（Y・U・，
14−yr−01dfemale）．Theirparentswereapparentlyhealthyandwerenotconsanguineous・
Cardinal manifestationsin both cases were ankylosis ofmultiplejoints at birth or postnatally，
markedscoliosis，dislocationofhipjoints，high−arChedpalateandgeneralizedmuscularatrophy・Serum Creatine kinase（CK）activity was normalinboth．HistologiCalexaminations ofbiopsiedmusclerevealedmarkedtype2fiberpredominanceaswellasdiffuse，mildatrophyofmusclefibersin
thegastrocnemiusmuscleofcasel，andvariationoffibersize，markedtypelfiberpredominanceand
multicore・likefindingsinthelatissimusdorsimuscleofcase2．
2．Tryptophanmetabolism：UrinaryexcretionofKAandXAbeforetheloadingwasnormalinboth，lessthanO．5FLmOles／kg／day．Aftertheloading，however，24−hrurinaryeXCretionsofbothKAand
XAwereabnormallyincreased，i．e．，CaSel，KA18．7，XA2．4；CaSe2，KAll・7，XA2・7（normalvalues；
KA2．075．0，XAO．7−2．OJlmOles／kg／day）．Theincrease wasnotcorrected by theadministrationof
vitaminB6．1ncreasedexcretionofKAandnormalexcretionofXAwereobservedinthemotherofcase
landthematernalgrandmotherofcase2．FathersgavenormalValues・Discu∬ion
AbnormalfindingsobservedinthetwopatientswithAMCledtothepresumptionthatadeficiency
or decreased activity of3−HA−OXygenaSein the tryptophan−kynureninepathwayisthebasisofthe
disorder．IncreasedexcretionofKAobservedinthemotherwassupposedlyattributabletoexpressionof
thesamebasicabnormalityasinthepatients．
With the same method，We foundand reported various，differentabnormalitiesoftryptophan
metabolisminpatientswithnemalinemyopathy，inpatientswithcongenitalfibertypedisproportion，in
onecasewithunclassifiedmyopathywithtypelfiberatrophyandinpatientswithHartnup−likedisease・
Fourpatientsfrom4pedigreeswithnemalinemyopathyshowedmarkedlyincreasedexcretionof
KAand normalXA excretion after theloadingoftryptophan．Similarabnormalitieswerefoundalso
intheirmothersandthematernalgrandmotherofonecase・Wepresumedthepresenceofadeficiencyor
decreasedactivityofkynurenine−3−hydroxylaseofthetryptophan−kynureninepathway・X−1inkeddomi−−82−hmoles／kg／day）

nantinheritance ofthediseaseaswellasthis
abnormalmetabolismwasalsosuggested．
Both ofthe two casesinafamily（the

0

5

10

15

20

NormalPrOband and his father）affectedwith con−
NemaHnegenital fiber type disproportion showed amyopathymarkedinCrease of KA and amildincrease Congenitalfiber
OfXA，Which wasnotcorrectedbythead−ministration ofvitaminB6．Adeficiencyor

type
disproportion
AMC

decreased activity of3−hydroxyanthranilate
OXygenaSe Of the pathway wasindicatedin
thisdisorder．

Hartnup−likediseasenepatientwithunclassifiedmyopathy

UnclassifiedWithtypel fiber atrophy gave remarkably

myopathy

highValuesofKAandXA，eSPeCiallyofthe
latter．The

remarkableincrease

of

XA

DMD

ex＿

Cretionwasreducedbytheadministrationof
ahighdoseofPALP（300mg／day）．Similarfindings were also foundinthe daughter．

Myotonic
dvstrophy
CMT

Vitamin B6 dependency of kynureninase
WaSSuggeStedinthisdisorder．

SPMA

Urinary excretionsofbothKAandXA

仙・Hdisease

after theloading of tryptophan were ab−
normallylowintwo brothers affected by
Hartnup−like disease・Despite the similarity
OfclinicalmanifestationswithHartnup−likedisease，both patients demonstrated no
aminoaciduria and normalabsorption and

Lipidstorage
mYOPathy

Fig・TheUrinaryExcretionofKAandXAinNeuromus−
Cular Disorders（afterloadingofL・tryPtOphanata
doseoflOOmg／kg）

urinaryexcretionofL−tryPtOPhan・Similarfindingswereobservedintheapparentlyhealthyparents．A
deficiencyordecreasedactivityofformamidaseandanautOSOmalrecessivepatternofinheritancewere
SuggeStedinthisdisease．
No abnormalfindingsofthemetabolismwerefoundinDMD，myOtOnicdystrophy，CMT，W−H
disease，SPMAetc．
InthisreporttwocaseswithAMCwerenewlyincluded．
Summary
Disorderswithabnormalfindingsinthetryptophan−kynureninepathwaywererestrictedtorare，SO−

Calledcongenitalmyopathies・Abnormalmetabolismofthepathwaymightberelatedtotheetiologyof
thesedisorders．
Referencesl）Miyoshi，K・，Yagita，M・，Takuma，M・，etal・：Tryptophanmetabolisminpatientswithnemalinemyopathy．Annual
Report（NANBYO）1977，TheMinistryofEducationofJapan．1978，P．241．
2）Miyoshi・K・，Yagita，M・・Takuma，M・，etal・：Abnomaltryptophanmetabolisminpatientswithnemalinemyopathy．
Oh．Neuro1．，18：842，1978．
3）Miyoshi，K・，Yagita，M・，Taira，A・，etal・：Nemalinemyopathyandrelatedmyopathiesfromastandpointof
abnormalmetabolismintryptophankynureninepathway・The76thAnmalMeetingoftheJapaneseSocietyof
htemalMedicine．Tokyo，Aprn，1979．
4）Miyoshi，K・，Taira，A・，Yagita，M・etal・：TwocasesofcerebeuarataxiawithabnormaltryPtOPhanmetabolismina
Eamny・The24thAnnualMeetingoftheJapanSocietyofHumanGenetics．Tokyo，Oct．4，1979．
5）Yagita，M・，Nakahira，H・，Taira，A・，etal・‥AbnormaltryptophanmetabolismobservedinaJthEOgrypOSismultiplex

COngenita・The25thAnnualMeetingoftheJapanSocietyofHumanGenetics・Sendai，Nov．7，1980．
−83−

肋∫αkrの∫〝op砂伽〟pSOMEPROPERTIESOFCREATINASEINHUMANSKELETALMUSCLE
YasuoNatoriandJoonPyoHan
TbeDepaJtmentOfNutritionalChemistry，SchoolofMedicine・TheUniversityofTokush血a，Tokudlin1a
Recently，thepresenceofcreatinase，Whichcatalyzesthehydrolysisofcreatinetosarcosineandurea，inhumanskeletalmusclewas reportedl）・Itwasalsoreportedthatthekineticbehaviorof
creatinasewasalteredinskeletalmuscleofpatientswithDuchennemusculardystrophy2）・
Thepresentstudywasundertakentoconfirmthepresenceofcreatinaseandtoexamineafewpro−
Pertiesoftheenzyme．
MaterialsandMethods
Human skeletalmuscle was obtained fromnon−myOPathypatientsatautopsy・Musclewashom0−
genizedinphysiologiCalsalinewithaPolytronandthehomogenatewascentrifugedat18，000xgfor30
min．ThesupernatantWaSthensubjectedtogelfiltrationonaSephadexG−150column（2・5x50cm）・
Creatinase activitywasassayedby creatineconsumption：theenzymereactionwascarriedoutat
370cfor30minandtheremainingcreatinewasdeterminedbythediacetylα−naPhtholmethod・

Re飢llts

Fig．lshowstheelutionprofileofcreatinaseactivityonaSephadexG−158Column・Twopeaksof
creatinaseactivitywereobserved；OneatamOlecularWeightof50，000andtheotherat25，000・
Each eluate fraction was
treated with trichloroacetic acid（fina15％），Centrifuged，and theconcentration of creatineinthe
SuPernatantWaSalsodetermined・Creatine was found to be eluted

inthelowrmolecular−Weightfrac−tion，nOtaSSOCiatedwithcreatin−
aseactivities．

Discussiom
Ourresultsconfirm thepre−
Sence of creatinase activityin
human skeletalmuscle reportedby Miyoshi and his cowork−ersl）・2），butdifferfromtheirre−
sults in that we detected two
peaks of creatinase activity on
Sephadex G−150gel filtration・
WhetherornotthepeakatM．W．
50，000representsadimerofthe
M．W．25，000speciesremainsto
Fraction No．

beelucidated．

Fig・lElution profile of A280・absorbing material（PrOteinsandlow−
molecular−Weight substances），Creatinase activityand creatineon

Thegelfiltrationstudyalso

Sephadex G−150Column（2．5x50cm）futration・Eightmlfrac−indicatesthat creatinein muscle

tionswereco11ected．

isapparentlynotassociatedwith−84一Creatinaseoranyotherbinding−PrOteinsbutexistsinafreestate．
Referencesl）Miyoshi，K・，Taira，A・，Yoshida，K・，Tamura，K・andUga，S・：Presenceofcreatinaseandsarcosinedehydrogenasein
humanskeletalmuscle−Proposalforcreatine−ureaPathway．Proc．JapanAcad．，56B：95，1980．
2）Miyoshi，K．，Tbira，A．，Yoshida，K．，Tamura，K．andUga，S．：Abnormalitiesofcreatinaseinskeletalmuscle。fPatientswithDuchennemusculaTdystrophy．Proc．JapanAcad．，56B：99，1980．

−85−

肋∫Cエ血r上砂∫〝opカタGr0叩

ABNORMALMETABOLISMOFCREATINEINSKELETALMUSCLESOF
MUSCULAR DYSTROPHIES

KazuoMiyoshi＊，AkiraTaira，KenzoYoshida，KatsuyaTamura，ShinjiMitsuhashi，ShigetoshiUga，KohjiKanazawa，KohzoImai，KanaeKusaka，HiroshiNishin0，
HiroshiIse，HisaomiKawaiandMasatoshiYagita
TheFirstDepaJtmentOflntermiMedicine，ScllOOlofMedicine，TheUniversityofTokushima，Tokushha＊OkinakaMemorialInstituteforMedicalResearch，Toranomon，Tokyo，and

TheFirstDepartmentofInternalMedicine，¶leUniYerSityofTokushima，Tokushima
Inpreviouspapers，WerePOrtedtheexistenceofcreatinaseactivityinhumanskeletalmusclesandits
abnormalkineticbehaviorforcreatineinDMDmuscles．
Inthispaper，WePreSentreSultsoffurtherexperiments・However，WeWOuldliketomakeacorrec−
tioninthispaper，Whatwehavehithertocalledmerelycreatinasewenowcallacreatinase−likeactivity
inabroadsenseincludingcreatinase．ThereasonsarethatourpreviousresultswereobtainedwithcrudematerialSand that stoichiometricalmeasurements were difficult．me true nature ofthisactivitywill
bedisclosedafterfurtherexperimentshaveconfirmedit．
Herewedescribetheresultsshowingthatthisactivityinskeletalmusclesoffetusesandofpatients
withanothertypeofmusculardystroplly，autOSOmalrecessivedistalmusculardystrophy（ARDMD），
whichwaspreviouslyreportedbyusinJapan，differsfromthatinadultskeletalmuscles・
MaterialsandMethods
Subjects were（1）healthy adultsornon−myOpathypatients，（2）humanfetuses（19th and32nd
weekofgestation）anda5−mOnth−01dinfant，and（3）Patientswithautosomalrecessivedistalmuscular
dystrophy（ARDMD）．Skeletalmuscles obtainedbyautopsyin（1）and（2）andbybiopsyin（3）were
usedfortheexperiments．
Skeletalmusclesstoredat−600cwerehomogemiZedandcentrifugedtoobtainsupernatants・The
activefractionobtainedbygelfntrationofeachsupernatantWaSuSedasthematerial・
Assuming the existence of creatine degradative activity，the decrease of creatine was assayed・Namely，lnareaCtionmixtureofthematerialandcreatine，theamOuntOfcreatinewasmeasuredbythe
diacetylcE・naPhtholmethodbeforeandafterthereactionandthedifferencewascalculated・
Re飢11ts

Resultsforskeletalmusclesofhumanfetusesandaninfant．
Inmuscles offetusesat the32nd week，thepeakactivitywasfoundinthesamefractionongel
filtrationasinhealthyadults．Themolecularweightoftheactivesubstancewasthesameasthatof
healthyadults，about50，000．
The effect ofincreasingthe concentrationofthe substrate creatineonthereactionvelocitywas
studiedusingthe fractioncollected・Ahyperboliccurvewasobtainedinfetalandinfantilemusclesas
inmusclesofhealthyadults．Alinearcurvewasobtainedineithermuscleondoublereciprocalplotting
ofthiscurve（Lineweaver−Burkplot）．However，theKmvalueofskeletalmusclesofa19th−Weekfetus
was3．8xl0−4M，COmParedwiththevalueof8・Oxl0−5Minmusclesofhealthyadults・A32nd−Weekfetusandthe5−mOnth−01dinfantshowedintermediatevalues．

Thus，thepresence ofadifferenttypeofthisactivityinfetalmuscles，Whichmightbecalledthe
fetaltype，WaSSuggeSted．
ResultsforskeletalmusclesofpatientswithARDMD．
InskeletalmusclesofpatientswithARDMD，thepeakactivitywasfoundinthefractionscorres−−86−

POndingtoamolecularweightofabout50，000asinthoseofhealthyadultsandDMDpatients．
However，kinetic analysis ofthe activity against theincreasing creatine concentrationgaveasig−
moidalcurveinARDMDmusclesasinDMDmuscles．TheconcentrationofcreatineglVlngthehalfvalueofVmaxwas3．6x10−4MinDMD，andl．Oxl0−3MinARDMD．
Vmax measuredinDMD and ARDMD muscleswiththesameproteincontentwas2．7timesand
morethan4timesthatofhealthyadults，reSPeCtively．
Summary
Itwasfoundthatthecreatinase−likeactivityinhumanskeletalmusclesvariedamongadults，fetuses，
andtypesofmusculardystrophies，DMDandARDMD，initskineticbehavior．
Thealteredcharacteristicsofthecreatinase−likeactivityfoundinskeletalmusclesofpatientswith

muscular dystrophiesmightbeintimatelyrelatedwiththeabnormalcreatinemetabolismand，further−more，Withtheetiologyofthedisease．
Rerereneesl）Miyoshi，K・，Taira，A．，Yosllida，K．，Tamura，K．，Uga，S．，Kanazawa，K．，Ilnai，K．，Kusaka，K．，Ise，H．，Kawai，H．and
Yagita，M．：AbnormalitiesofcreatinemetabolisminDuchennemusculardystrophy．CurrentReseardlinMusculaf
Dystrophy，Japan・TheProceedingsoftheAnnualMeetingoftheMuscularDystrophyResearchGroup，1978，1979，
Tokyo，p．100．
2）Miyoshi，K．，Tbira，A．，Yoshida，K．，Tamura，K．andUga，S．：Abnormalitiesofcreatinaseinskeletalmuscle ofpatientswithDuchennemusculardystrophy．Proc．JapanAcad．，56B：99，1980．
3）Miyoshi，K．，Iwasa，M．，Kawai，H．，Sasaki，N．，Kusaka，K．，Yagita，M．，Hiasa，M．andTada，Y．：Autosomalrecessive
distalmusculardystrophy・AnewtypeofmusculardystrophyobservedcharacteristicanyinJapan・Jap・J．Clin．
Med．，41：3922，1977．

ー87−

肋∬Ukr砂∫打Op妙Gro叩ENZYMATICSTUDIESONTHEMECHANISMOFCREATINURIAINRATSWITH
EXPERIMENTALTHYROTOXICMYOPATHY
KeishiUeda事，YoshioHatanaka靡，ChiakiHamaguchi＊事，MotooNojima＊＊
andToshioNakata＋＊華＊HealthAdministrationCenter，UniversityofEhime，Matsuyama＊＊DepartmentofOrthopedicSufgery，UniYerSityofEhime，Matsuyama＊＊＊CentralClinicalLaboratory，OhtemaeHospital，Osaka

Introduction
Thyrotoxicmyopathyiscommoninmiddle−agedmalepatientswithhyperthyroidism・Theweakness
and wastingoftheproximalmusclesoftheextremitiesarerecognizedpriortothesignsofthyrotoxi−
cosisanddisappearwiththetreatmentforhyperthyroidism・nereisnochangeofserumcreatinekinase
inspiteoftheincreasedurinaryexcretionofcreatine・In this work，We tried to clarifyenzymatically the mechanism of creatinuria on thyrotoxic
myopathywiththeuseofanimalstudies．
MaterialsandMethods（1）Materials：MalealbinoratsoftheWisterstrainweighingllO，130g，malealbinorabbitsweigh−ing3kg，and mice oftheC57BL／6Jstrainweighing30−40gwereused．AllanimalSweresacrifiedby
decapitation and tissue wasremoved as quickly as possiblein each animal・The human tissues wereobtainedbyautopsyofapatientwithosteosarcomawhohaddiedlOhoursbefore・（2）FoodforanimalS：Foodwasgiventoanindividualanimaladlibitum．nefollowingbasaldiet
wasused；CaSein（20．0％），dextrin（55．9％），SuCrOSe（15．0％），Cellulose（1．0％），vitaminmixture（1・0％），
Saltmixture（4．0％），Cholinechloride（0．1％），Oil（2．0％）andwater・（3）Injectionoftriiodothyronine（T3）：T3（fromSigmaCo．Ltd）wasdissoIvedinsalinewithO・02％

NaOH to50gamma／m1．missolutionwassubcutaneOuSlyinjectedinto eachratattherateof50
gamma／100gbodyweight．SalinecontainingO．02％NaOHwithoutT3WaSgiventothecontrolrats・（4）Enzyme preparation：Tne tissues were homogenized with a Potter homogenizer with10
volumesof50mMTris−HClbuffer（pli7．4）fortheassayofcreatinekinase（CPK），With10volumesof
lOOmM phosphate buffer withl mM of EDTA and O．15％of deoxycholate（pH7・4）for glycine
amidinotransferase（GAT），With20volumesofdistnledwaterforarginase，andwithlOvolumesofdis−
tilledwaterforcreatine．Eachhomogenatewascentrifugedat8000xgforlOminutes・Thesupernatants
wereusedasthematerialsfortheassay．Allproceduresdescribedabovewereperformedat40C・（5）Estimationofenzymeactivitiesandcreatine：CPK，GATandcreatineweredeterminedbythe
methodsofNodal），Walker2）andFolin3），reSPeCtively・Theactivityofarginasewasmeasuredaccording
tothemethodofSchimke4）exceptthatl／80Mglycine−NaOHbuffer（pH9．4）wasusedintheassay・
Resu1tsandDiscussiom（1）TherelationshipbetweenthecontentsofcreatineandtheactivitiesofGATintissues
Inthebmin，heart，liver，panCreaS，kidneyandskeletalmuscleofrats，thecontentsofcreatineandtheactivitiesofGATweredetermined．metissuesinwhichthecontentsofcreatinewerehighshowed
lowGATactivity．TheywerealsofoundtobehighinCPKactivity．Inaddition，thesecharacteristicsof
thedistributionsofthese threesubstancesinthetissueswerealsodemonstratedinthetissuesofrabbit，mouseandhuman．
The tissues for the synthesisaredifferent from those forthe storage ofcreatine・Therefore an−88−

umotes／min／
W．VV．g．
300

Sむ岬lち；U＜￠∈＞NUu1

Creatine
KinasetmuscleI

umoIes／hr／

Ft 圭
300

W．W．9．
20

10
GIYCineamidinotransferase
lkidneYI∵三二￣…二十−，DaYSaftertheinjection

Fig．l EffectsofT，Ontheactivitiesofboththecreatinekinaseofskeletalmuscleandthe
glycineamidinotransferaseofkidney．
Malerats were dividedinto threegroups．TTleratSOfAgroupwerefedonthe
basaldiet，BgrouponthebasaldietincludingO．25％ofcreatine，andCgrouponthe
basaldietinclildingO．5％ofcreatineforaboutoneweek二Afterwardsfiftygammaof

T，PerlOOgbodyweightwassubcutaneouslyirdectedinto theratsofeachgroup
everydaywiththedietdescribedaboveineach．EadlCircleshowstheaverageof3−6
animals．

imbalance betweensynthesisandstorage，aSareSult ofthe metabolicabnormalityineachtissueorof
hormonal disturbance，lSmOStPrObablythereasonforthecreatinuria．
（2）PhysiologicalchangesintheactivitiesofkidneyGAT
Food forrats waschangedfromthebasaldiettoaO．5％creatineaddedbasaldiet，andthentoa
Creatine free basaldiet．Asa result，after creatinewasaddedtothediet，theactivityofkidneyGAT
graduallydecreased．Onthecontrary，aftercreatinewasremovedfromthediet，thelevelincreasedagain．
Thesefindingsindicatethat，Whenthebodyis

full

ofcreatine，theactivityofthisenzymeTtherate

limitingenzymeincreatinebiosynthesis−isinhibitedbythecreatineitself．

Maleratscontinuedonthebasaldietgrewtol10gbodyweight．Afterwards，OnlywaterwasglVen
totheseanimalsforfourdays．TYLereSultwasth、てtheactivityofkidneyGATremarkablydecreased．

Over these four days，the body weight ofthe rats decreased t070g．There wastheexpectedlarge
amounts of muscle wasting．However，anincrease of creatine could not be detectedin theirurine．
Creatinebiosynthesisissupposedtobeinhibitedbythecreatinedrivenfromthemuscle．
（3）Theactivitiesofmuscle．CPKandkidneyGATinratswithexperimentalthyrotoxicmyopathy
Theconditionedratsweredividedintothreegroups．TheratsofAgroupwerefedonthebasaldiet，BgrouponthebasaldietincludingO．25％ofcreatine，andCgrouponthebasaldietincludingO．5％of
Creatine for about one week untilthe bodyweightreachedll0−150g．FiftygammaofT3PerlOOg
bodyweightwasinjectedintotheratsofeachgroupeveryday．
Asa result，the activities of muscle CPKdecreasedday bydayto87％after2days，85％after4−89−

肋∫C〟ねr勒ぶ什Opわ・Gro叩

days，and60％after6days of theirdections．These decreases of muscle CPK wereindependent ofCreatinecontentsinthedietandinducedonlybytheinjectionofT3．
0ntheotherhand，theactivitiesofkidneyGATinratsfedonthebasaldietdecreaseddaybyday
to78％after2days，58％after4days，and26％after6daysoftheirtjections．Onthecontrary，inrats
fed onthabasaldietincludingO．25％ofcreatine，thelevelsincreasedto112％after2days，143％after4days，and120％after6days．Furthermore，inratsfedonthebasaldietincludingO．5％ofcreatine，thelevelsincreasedalsoto150％after2days，200％after4days，and160％after6daysoftheinjections．
With theinjections of T3，the activity of kidney GAT decreased．That may bedue to feed backinhibitionbythe creatinedrivenfrommuscleasthatoffastingratissupposedso．Ifso，Withcreatine
inthe diet，thelowerlevelsofthisenzymehave tobeexpected withcreatinefrombothmusclesanddiet．However，anunreaSOnableresponseoccurred．
Conclusion（1）Tissuesforsynthesisofcreatineweredifferentfromthoseforstorage．AnimbalanCebetween
Synthesisandstorageofcreatineismostprobablythereasonforcreatinuria．
（2）Theactivity ofkidney GATwasreduced easilyby creatineinthediet，OrWithwastingmuscle
inthefastingstate．However，WiththeinjectionsofT3，inspiteofthedecreaseofmuscleCPKactivity，
aninverse（decreasingandincreasing）responseoccurredbetweenthelevelsofkidney GAToftherats
fed on the diet without and with creatine．Creatinein the diet has to overflow throughthyrotoxic
myopathyintotheurine．
Reierencesl）Noda，L．，Kuby，S．＆Lardy，H．：Creatinephosphokinase，MethodsinEnzymology，2，Acad．PressInc．，NewYork，
1955，2，p．605．
2）Wa比er，J．lL：StudiesonthemechanismofactionofkidneytransamidirLaSe．J．Biol．Chem．，224：57，1957．
3）Folin，0．：BeitragzurChemiedesKreatinsundKreatininsimHarne．Z．Physiol．Chem．，41：223，1904．
4）Schhlke，R．T．：AdaptiYeCharactcristicsofureacycleenzymesintherat．J．Biol．Chem．，237：459，1962．

ー90−〟恥蝕ね′勒封ナOpカタGr0叩

EXERTIONALRHABDOMYOLYSISANDMYOGLOBINURIA：
ACASEREPORTANDMETABOLICSTUDY
MasatoyoNishizawa，TomoyasuYahagi，TetsushiAtsumiandTadashiMiyatake
DepartmentofNeurology，JichiMedicalSchool，Toch短i

Introduction
Skeletalmuscleutilizescarbohydrateandlipidasenergysourcesanddisturbancesoftheirmetab0−
1ismmightcausetheacutedestruCtionofmuscles．Wedescribeinthisreportacaseofacuteexertional
rhabdomyolysis and myoglobinuna，and biochemicalstudiesonlipidandcarbohydratemetabolismin
thepatient．
Casereport
Thepatientwasatwentyyearoldmale．Hehadsufferedfromswelling，tenderness，andweaknessof muscles after strenuous exercise for fifteen years，and had recovered spontaneously after atleast
one hour ofrest．Musclegroupswhichwereused to excesswereinvoIved．Hehadexperiencedseveral
episodes of pigmenturia since thirteenyears old．These symptoms wereinduced by short strenuous
exerciseratherthanthatoflongdurationandmoderatestrength．Therewerenorelationsbetweentheir
OCCurrenCeand

temperature，mOisture，COntentSOffood，andfasting．Theso−Calledsecondwindpheno一

menonwasoncenotedduringmarathon．Therewasnohistoryofasimilarillnessinanymemberofthefam址y．Thepasthistorywasuncontributary．Hehadnotbeenexposedtoanytoxinsandhadtakenno
medications．
Physicalexamination revealed a we11−developed and well−nOurished male．No muscle tenderness，
weakness，atrOphyorhypertrophywasdemonstratedbyneurologicalexamination．
Laboratory dataincluding blood ce11counts，Serum electrolytes，blood ureanitrogen，Creatinine，
GOT，GPT，bnirubin，Plasmanon−eSterifiedfattyacid（NEFA），triglyceride（TG），andthyroidhormones
wereall normal．Glucose tolerance test results were normal．But even at rest，the values for serum

Creatinephosphokinase（CPK）andaldolasewereslightlyelevated．Electromyographyshowedthenormal
Pattern．
Abiopsy specimen ofthe quadriceps femorismusclewasreportedasshowingasmallnumberof
scattered muscle fibersundergoing degeneration and minimalmyopathic changes．Glycogenandlipid
droplets were not accumulatedinthe muscles．Electron microscopically muscle mitochondria hadnormalstruCtureS．
An exercise test was performed by climbing up stairs untilswelling，tenderness，and weakness
appearedinmusclesofthelowerextremities．ThevaluesofserumCPKandaldolaserosemarkedlyto
15，420IU／land49．1U，reSPeCtively．Twentyminutesafterthe exercise，he noticed extremelydark
urine，inwhichmyoglobinwasdemonstratedwithaspectrophotometer．
MethodS
Anischemic forearm exercisetestwasperformed andvenousbloodlactatewasmeasured．Biopsy
Samplesofmuscleswereobtainedunderprocaineanesthesiaandstoredinliquidnitrogenimmediately
after they were excised．Muscle enzyme activities oftheglycolyticpathwayincludingphosphorylase，
phosphoglucomutase，Phosphohexoseisomerase，and phosphofruCtOkinase were determined by theStandard assay methods，uSing the supernatant fraction of10，000xgcentrifugationofhomogenizedmuscles．
A seventy−tWO hour fasting test was performed and plasma NEFA，TG，Serum CPK，and urine−92−ketonebodiesweremeasured．Fivenormaltwentyyearoldmalevolunteersservedasthecontrolgroup．
The fatty acid composition ofplasma NEFAwasanalysedasfollows．Totallipidswereextracted
fromtwomloflyophnizedplasmawith chloroform−methanol（2：1，V／V）．NEFAwasseparatedbythin
layerchromatographyusingadevelopmentsystemofnJleXane−ethylether−aCeticacid（90：10：1，V／V／V），
COnVertedtomethylesterswith2．5％methanol−HClandanalysedbygasliquidchromatography．
Carnitine palmityltransferase（CPT）was assayed by two different methods：the hydroxamate
method（Crabtree and Newsholme，1972）and theisotope exchange method（Norum，1964）．Muscle
Carnitinecontentwasassayedbytheenzymaticmethodusingcarnitineacetyltransferase．
Results
Theischemicforearmexercisetestproducednomusclecramps．Thevenouslactatevaluerosefour−
foldpromptlyafterexercise．
In the assay oftheglycolytic enzymes，the activities of phosphorylaseandphosphoglucomutase
WereShowntobenormalandthoseofphosphohexoseisomeraseandphosphofructokinasewereslightly

higherthanthoseofnormalcontroIs．
During the fasting study，myOglobinuria was notinduced．Plasma TG rose normally and urine
ketone production wasnormal，but the elevationofplasmaNEFAwaslowerthanthatofthecontrol

group．PalmiticandstearicacidinplasmaNEFAwereslightlyhigherinthepatient．Inthecontrolgroup，
01eicacidwasincreasedafterfasting，butinthepatientitsincreasewassmaller．
The activity of CPTassayedbytwodifferentmethodswasshowntobenormal．Musclecarnitine
COntentWaSalsointhenormalrange．
Comments
Myoglobinuriaisarelativelyraresyndrome，Whichisknowntobecausedbyvariousconditionsl）2）3）・
Becauseabnormalmuscleenergymetabolismcouldexistinthiscase，metabolicstudieswereundertaken
toinvestigatethe two main ATP−PrOducing systems of musclesinexercise：One WaS the−anaerObic
glycolyticpathwaywhichplaysanimportantroleintheinitialstageofexercise，andtheotherwasthe

aerobiconeutilizingplasmafreefattyacidasanenergysource．
nleClinicalsymptomsofthiscasesuggestedmyoglobinuriaduetodefectsintheanaerobicglycoly−tic pathway．However，thebiopsiedmusclerevealednoaccumulationofglycogen．Venouslactatewas
PrOduced promptly underischemic exercise．Theactivitiesofmuscleglycolyticenzymesstudiedwere
Showntobenormal，SOthenwell−knowndiseasessuchasmyophosphorylasedeficiency（McArdle）and
PhosphofruCtOkinasedeficiency（Tarui）andlactic dehydrogenase M−typeSubunitdeficiency，Whichwas
recentlyfoundinapatientwithmyoglobinurla，COuldallberuledout．
Inordertobeutilizedasanenergysourceinmuscle，NEFAmustfirstreactwithCoA，andtheacyl
CoAmust thenbetransportedintothemitochondria withthe aid ofcarnitine−CPTsystemslocatedat
theinsideandoutsidesurfacesofthemitochondrialinnermenbrane．
Inthiscase，long−ChainsaturatedfattyacidsC16：OandC18：0，the・substratesofCPT，rOSenOrmallyafterfasting．TheactivityofCPTwasshowntobenormal．Musclecarnitinecontentwasalsonormal．
nle above results revealedthat this case did not correspond to any myoglobinuricsyndromein
Which the underlying metabolic disturbanCe WaS demonstratedinterms of enzyme defects，andSuggeStedthatthismightbeanew，nOtyetknowntypeofmyoglobinuria．Theunderlyingpathophysi0−
10gicalmechanismremainstobeelucidated．
Referencesl）Penn，A．S．：Myoglobinand myoglobinuria．hHandbookofclinicalneurology（editedbyVinken，P．J．＆Bruyn，
G．W．）North−Houand，Amsterdam，1979，Vol．41，p．259．
2）DiMauro，S．：Metabolicmyopathies．ibid．p．175．
3）DiMauro，S．＆DiMauro，P．M．：Muscle carnitine palmityltransferase deficiencyand myoglobinuria．Science182：929，1973．
−93−〟Ugαkr叫ぶ打Opわ′Gr。叩TURNOVEROFHUMANMYOGLOBINASSTUDIEDBYRADIOIMMUNOASSAY
HisaomiKawai，MasaruIwasa，KenjiroMasuda，KozoImai，AkiraKondo，KanaeKusaka，HiroshiNishinOandKazuoMiyoshi
TheFirstDepartmentofInternalMedicine，SchoolofMedicine，TheUmiversityofTokushima，Tokushima
Toelucidatetheprocessesofmetabolismofmyoglobin（Mb）inhumans，WeStudiedhalfllives（rLl／2）
ofserumMbinvariousclinicalcaseswithhypermyoglobinemiaandalsoinvestigatedtheTl／2inhealthy
volunteers andpatientswithnephropathy，hepatopathyormyopathyafterintravenousloadingofMb・MaterialsandMethods
Subjects were clinicalcaseswithacuteortransienthypermyoglobinemiasuchasmalignanthyper−
thermia，aCutemyOCardialinfarction，LDHM−Subunitdeficiency（HamamatsuMedicalCollege，Nishimuraetal）andsports（baseball，longdistancerunning）．SerumMbconcentrationsweremeasuredseriallyand
Tl／2Weredeterminedinthesecases・Ontheotherhand，humanMbpreparedbyuswasadministeredintravenously（i．V．）asasingledose（10mg／60kg）tohealthyvolunteers，Patientswithrenalinsufficien−
cy，Cirrhosis of theliver，Duchenne type musculardystrophy（DMD），myOtOnic dystrophyand
neurogenicmuscularatrophy・ThenserumMbconcentrationandTl／20fthesecasesweredetermined・
UrinaryMbwasalsoassayed．Mbwasmeasuredbyaradioimmunoassaymethoddevelopedbyus（sensi−
tivity，lng／ml）．
Resultsl）TII20fserumMbinclinicalcases：Tl／20fserumMbwere5hto9hinmalignanthyperthermia，
4hto9h30mininacutemyocardialinfarCtion，4hlOmininLDHM−Subunitdeficiency，4h20minto
13h30mininbasebal1，and4h40minto5h30mininlongdistancerunning．Amongthem，theshortest
Tl／2Were4hto4hlOmin，andwasclosetotheTl／20fhumanMbinserum，prObablybecauseMb
wasreleasedfromthemuscleonceandduringashorttime．

2）Correlation ofinitialconcentration of serum Mb（maximalValue）withTl／2inclinicalcases：Caseswithremark−ablehypermyoglobinemiasuchasmalignanthyperthermia（l xlO4−1xlO5ng／mi）andthosewithrelativelymildmyogl0−
binemiasuchassports（5xl0−1x103ng／ml）didnotdifferin
Tl／2Yalues・
3）Tl／2aSdeterminedbyi・V・loadingofMbinhealthy
individualsandsomeclinicalcases：Tl／20fserumMbintwo
healthy volunteers were 3h 20min（Fig．）and3h30min．
Tl／20fsomecaseswithrenalinsufficiencywereprolongedto
5h・Tl／2WaSprOlongedto5hinonecasewithcirrhosisoftheliver．Two cases with DMD gaveremarkablyprolongedvalues；
6hlOminand8h20min・Tl／2Were3h55minand3hlO
minintwo caseswithmyotonicdystrophy，and3h50minand
3h40minintwocaseswithneurogenicmuscularatrophy．
4）Urinary excretion of Mb afteri．V．loading of Mb：
0

1

2

3

4

5

6

hours

Urinary excretion of Mb was notobservedinmostcasesafter
i．V．loadingoflOmg／60kgofMb．

Fig・・DisappearancecurveofserummyO・
globininahealthyvolunteer（M・l・）ー94−DiscussionandSummary
WiththerecentdevelopmentofaradioimmunoassayforhumanMbinJapanl）andabroad，2）re−POrtSOfserumandurinaryMbvaluesinmyocardial，neurOmuSCularandrenaldisordershaveincreased．
PreviousreportsonMbturnoverincludestudiesindogsbyAmakoeta13）andthoseinhumanswith
i．V．injectionofwhaleMbbyKoskeloetal．りResultsofMbturnOVerinhumanswithhumanMbwere
reportedonlybySylv占n5）in1978．Heinjected125Ⅰ−Mbintravenouslyintohealthyindividualsandde−

terminedTl／2，Whichwasreportedly3h20mininphase2・HesupposedthatMbwasmetabolizedin
thekidney，basedontheresultthatlabelledMbwasnotdemonstratedintheurine・However，Catabolism
OfMbinorgansotherthanthekidneyremainedunclear．
WeinvestigatedTl／20fserumMbinvariousclinicalcaseswithhypermyoglobinemiaandMbturn−
OVerafteri・V・loadingofMbinvariousdisorders・Theresultscanbesummarizedasfollows；1）Tl／20f
SerumMbinsomeclinicalcaseswithhypermyoglobinemiawereclosetothatofhealthyindividualsas
determinedwithasinglei・V・injectionofMb，Whileinmostcasestheywereprolonged．Theprolongation
WaSprObablyduetocontinuousemuxofMbfromthemuscleandsomeotherfactors・2）Tl／2inrenal
insufficiencyandliverdisordersasdeterminedbyMbloadingwasprolonged，Whichsuggestedtherole
OfthekidneyandliverinMbmetabolism・Tl／2inDMDwassupposedtobeprolongedmainlybecause
OfcontinuousleakageofMbfromthemuscle．
Referemcesl）Miyoshi，K・，Saito，S・，Kawai，H・，etal：Radioimmunoassayforhumanmyoglobin＝methodsandresultsinpatients

WithskeletalmuscleormyocaJdialdisorders．J．Lab．Clin．Med．，92：341，1978．
2）Stone，MJ・，Willerson，J・T・，Gomez−Sanchez，C．E．etal：Radioimmunoassayofmyoglobininhumanserum．Results
inpatientswithacutemyocardialinfarction．J．Clin．Invest．，56：133，1975．

3）Amako，T・，Koga，J・，Kobayashi，A・etal：Experimentalinvestigationofthemetabolismofmyoglobin．Kyushu
J．Med．Sci．，14：277，1963．

4）Koskelo，P・，Kekki，M・andWager，0・‥Kineticbehaviourof131I−Iabelledmyoglobininhumanbeings．Clin．Chim．

Acta，17：339，1967．
5）Sylvin，C・：Thekineticsofmyoglobinin01dvolunteersandinpatientswithacutemyocardialinfarction．Scand．J．
q血．hb．InYeSt．，38；561，1978．

ー95−

肋∫C〟kr砂∫〟叩力yGr ）叩GOTISOZYMESINMUSCLESOFPATIENTSWITHOPHTHALMOPLEGIAPLUSSYNDROMEANDEXPERIMENTALMITOCHONDRIALMYOPATHY
SaburoOgasahara，JinKang，MitsuoTakahashi，Hir0−OWatanabe，ShiroYorifltii，SatoshiUenoand SeiichiroTarui¶leSecondDepartmentOflntemalMedicine，OsakaUniversityMedicalSchool，Osaka
It has beenreportedthat morphologiCalabnormalityofmitochondriaisinvoIvedinmusclesof
patientswiththeophthalmoplegiaplussyndromel）・However，littleisknownaboutthemitochondrial
enzymesinmuscles of patients with the ophthalmoplegia plus syndrome・Inthepresentstudy，WedeterminedtheactivitiesofGOTisozymeslocatedinthemitochondrialmatrix（m−GOT）andcytoplasm（S−GOT）inmusclesoftwopatientswiththeophthalmoplegiaplussyndromeandofratswithexperi−mentalmitochondrialmyopathy．
MaterialSandMethods
Muscle specimens were obtained from deltoid muscles of the followlng tWOPatientswiththe
ophthalmoplegiaplussyndrome．
Casel was a24year01d female withophthalmoplegia，PtOSisandmuscleweaknessoffacialI
pharyngeal，neCk andlimb muscles・Short stature，retinaldegeneration，mildEEGabnormalityand
auditorydysfunctionwereobserved・Raggedredfibersandabnomalmitochondriawithparacrystalline
inclusionswererevealedonhistologicalexaminationofthemuscle．
Case2wasa24yearold femalewithweaknessofextraocular，facialandlimbmuscles・RetinaldegenerationandmildEEGabnormalitywereobserved・HistologiCalexaminationofamusclespecimen

didnotshowraggedredfibers．
Musclespecimensobtained from7patientswithotherneuromusculardiseaseswereusedforthe
control（polymyositis，4；amyOtrOPhiclateralsclerosis，1；FSHformmusculardystrophy，1；LGform
musculardystrophy，1）．
Productionofexperimentalmitochondrialmyopathyl）DNP（2，4，−dinitrophenol）method2）：MaleWistarratsweighingabout150gmwereaneSthetizedbyi．p．injectionofpentobarbital．Therightiliacarterywascannulatedand5mlofDNPsolution（3mg／ml，neutralized withNaOH）wasi雨ectedintra−arterially．Therightgastrocnemiusmusclewasremoved
andfrozen30minafterDNPinjection．

2）Ischemiamethod3）：RighthindlegsofmaleWistarratSWeremadeischemicbytouniquetsand
24hourslaterrightgastrocnemiusmuscleswereremovedandfrozen・I）etemlinationofGOTisozymes4）
Muscleswerehomogenizedin20vol．ofphosphatebufferedsaline（pH7・4）after4cyclesoffreez−
ingat−200candthawingatroomtemperature・ThehomogenateswerecentrifugedatlO，000xgfor15
minat40C．ThesupematantSWereuSedfortheassayoftotalGOTactivity・Theactivityofm−GOT
wasdetermined afters−GOTwasremoved by absorption ofs−GOTwithsheepredbloodcellscoatedwithanti−S−GOTantibody．ThesolutionfortheGOTassaycontainedlOmMAsp，3・3mM2−0XOglutaricacid，0．1mMNADH，0．1MTrisbuffer，PH7．4，and3Jlg／mlofMDH・Thefinalvolumeofthereaction
mixturewaslmlandthereactionwascarriedoutat370C．Activitywascalculatedbyfollowingthe
reductionatOD340nm．

−96−

A＼鞭

Resnltsl）Fig．l showstheacti−
vities of s−GOT and m−GOT（A）and ratio of m−GOTin
the total GOT activity（％m−

0
0
X
OX

GOT）（B）in

the一muSClesof

Patients withtheophthalmo−

0

Plegia plus syndrome andotherneuromusculardiseases．

mean±2S．D．

0

二一一二

Thereis no differencein theactivities of s−GOT and m−

二一

GOT．However，％m−GOTwas
decreased inthe muscles of≧毎

thepatientswiththeophthal一moplegiaplussyndrome・
2）Fig．2showstheacti−

oPM
XDMP
OALS

O PM
X DMP
◎ALS
●

OPS

●OPS

vities of GOTisozymeS and％m−GOTin the muscles of
the controlratsandratswith
experimental

sGOT mGOT

Fig・lTheactivitiesofGOTisozymesinmusclesofpatientswith
ophthalmoplegiaplussyndIOme（OPS）andotherneuromus・
culardiseases（A）andthepercentageofm−GOTinthetotal
GOTactivity（％m−GOT）．（PM，POlymyositis；ALS，amyOtrO−
phiclateralSclerosis；DMP，prOgTeSSivemusculardystrophy）

mitochondrial

myopqthies・In both experi−mentalmitochondrialmyopa−
thies，thedecreaseofm−GOT
was more marked than thatof s−GOT．The decreasein

軋ニOLluO0−0求︸＞l苫10円％m−GOT was或ight but sig−
nificant in mitochondrial

A
S−GOT

100

myopathies．
DiSCuSSion
Berenbergetal1）studied
thebiochemicalpropertiesof
mitochondria obtained from50

three patients with theOPthalmoplegia plus syn−drome．Theyreportedthatin
the mitochondria of one ofControI DNPIschemia ControI DNP rschemia V controI DNPIschemia

Fig．2TheactivitiesofGOTisozymes（A）and％m−GOT（B）inthe
musclesofratswithexperimentalmitochondrialmyopathies．

the three patients，reSPiration

Each histogram showsthemean±SDfor5samples．（＊P＜and DNP stimulated ATPase

0．05，＊＊p＜0．01）

activityweredecreased．How−

ever，inthe othertwopatients，biochemicalabnormalitywasnotrevealed．Andyetnowcommonbio−
Chemicalabnormalitiesofmusclesofpatientswiththeophthalmoplegiaplussyndromearenotknown．
GOTisozymes werelocatedinthe mitochondrial matrix and cytoplasm，and some changesin
mitochondriamayinducechangesintheactivitiesofGOTisozymes．Asrevealedinthiswork，％m−GOT
WaSdecreasedinthemusclesoftwopatientswiththeophthalmoplegiaplussyndrome．Becausethesame
Phenomenonwasobservedinthemusclesofexperimentalmitochondrialmyopathy，itissuggestedthat
thedecreased％m−GOTreflectsthedamagetomitochondria．

ー97−

肋∫αJJ灯り∫けOpカタG′0〟P

RefefenCeSl）BerenbergRA，PeuockJM，DimauroS，etal．：Lumpingand甲Iitting？

Ophthalmoplegiaplus

orKearns−Shysyn−drome7Ann．NeufOl．1：37−54，1977．
2）Melmed C．KarpatiG，CarpenteT S；ExperimentalmitochondrialmyopathyproducedbyinviYOunCOuPlingof
OXidativephosphorylation．J．Neurol．Sci．26：305−318，1974．
3）KarpatiG，CarpenterS，MelmedC，etal．；ExpeIhlentalischemicmyopathy，J・Neurol・Sci・23：129−161，1974・
4）WadaH，TeranishiH，KagamiyamaH，etal．；AsimpleimmunologiCalmethod fordifferentialdeterminationof
serumglutamicoxaloacetictransaminaseisozymes．MedicalJ．OsakaUniversity，29，16−32，1978・ー98−

肋gc〟ねr勒grrOp力yGroIJp

ANALYSISOFTHECONTRACTILEPROTEINOFASINGLEHUMANMUSCLEFIBER
AkioTakagi
DivisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMuscularDisorders，Tokyo
SinglemusclefiberswerepreparedfrombiopsyspecimensofDuchennemusculardystrophy（DMD）
andotherdiseases．Thecontractileproteinofsinglefiberswasanalyzedandcomparedtothephysiologi−
calproperty．Ourmaininterestwastostudytheintermediatefiber（typex），WhichisabundantinDMD
musclel）．Themusclefiberofexperimentalmyopathywasalsostudied・
Resultsanddiscussion（1）Theproteincompositionofsinglemusclefiberswasstudiedbyonedimensional（lD）ortwodimen−
sional（2D）gelelectrophoresis（Fig．1）．InlDelectrophoresis，theproteincompositionwasdifferent
betweentypelandtype2fibers，eSPeCiallyinthemyosinlightchain（MLC）pattern・Thetroponincomplexandtropomyosinmightalsodifferbetweenthem，butexactdifferencescouldnotbeconfirmedwith alD gelonly．2Delectrophoresisdemonstratedthatevery

〜pel

輌檜2

musclefibercontainedtwotropomyosinsubunits，alphaandbeta

chains．ThetypelfiberhadtwoMLC・Themolecularweightof
MLCIswas27，000andthatofMLC2swas19，000・Thetype2
fiberhadMLClf，MLC2fandMLC3f，andthemolecularweight
ofeachcomponentwas26，000，18，500and16，000，reSPeCtively・（2）In DMD，tyPelfibersexhibited the sameMLCpatternas
controltypel，andtheMLCpatternoftype2wassameasthat
ofcontroltype2．TheMLCoftypeXfiberswasequaleitherto
controltypel orcontroltype2・Thus，differentiationseemedcompleteasfarasMLCwasconcerned・（3）Experimentalmuscularatrophywasproducedbyimmobiliz−
ingtheanklejointofrats．Singlemusclecellsfromtheatrophic
soleus exhibited two types of MLC・These muscle fibers aresupposed tobehalfwaylnthetransformationfromtypelto
type2．
（4）Hypothyroidism wasinducedin rats by surgical thy−roidectomy．NochangeintheMLCcompositionwasfoundinthesoleusorEDL muscle，althoughthe time course oftwitchwas
PrOlongedbythyroidectomy・
Referemcel）Takagi，A，Nonaka，Ⅰ．：Duchennemusculardystrophy：Unusualactiva−

tionofsinglefibersinvitro．Muscle＆Nerve4：10，1981

hgendtoriguIel
Thc contractile protein ofsingletypel
and type2musclefiberswasanalyzed
by St）S−gClelectrophoresis．M，myOSin
heavy chain；alpha，alpha actinin；AC，actin；TM，TN−T，trOPOmyOSinalphaandt）etaSubunitsandtroponin T；Ll，TN−I，
myosinlight chain（MLC）l and tro−
poninI；L2，MLC2；L3，MLC3・−100−

肋∫αkr砂∫〃即妙Gro岬

STUDIESONTHESTRUCTURALPROTEINSINEXPERIMENTALSEROTONINMYOPATHY
MitsuoIida，KoshiAdachi，KeisukeMizuno，TetsuyaTakayanagiandItsuroSobue
FirstDepartmentofInternalMedicine，NagoyaUniverdtySchoolofMedicineINagoya

ImtfOductiom

ManykindsofmyopathyhavebeenreportedbutthemodesofdestruCtionofthestructuralproteins
inthevariousmyopathiesarescarcelyknown．
Wel）previouslyconfirmed pathologically theproductionofexperimentalserotoninmyopathyinrats．Therefore，WeStudiedthemodeofmuscledestruCtioninexperimentalserotoninmyopathyusing
dodecylsulfatepolyacrylamidegelelectrophoresis・MaterialsandMethodS
Productionofserotoninmyopathy

Experimental serotonin myopathy was producedin male Wistar rats weighing about100g．
Serotonininisotonicsalinewasinjectedintraperitoneallyonceaweekandinjectionswererepeated24
times．
Preparationofproteinsample
Musclefromthehindlimbwashomogenizedin9VolumesofO．lMNaCLCOntaining5mMNaHCO3
with aWaring Blender．The homogenate was then centrifuged atlO，000xgfor20minutesandthe
residuewasresuspendedinthesamesolutionasabove・Thisprocedurewasrepeated30r4times・The
residuethusobtainedwasusedastheproteinsampletobesubjectedtoelectrophoresis．
Gelelectrophoresis
The method forthe electrophoresis using dodecylsulfate polyacrylamidegelwas essentiallythesameasthatofFairba。ksetal（1971）2），TheproteinsamplewasdissoIvedinasolutioncontaininglO
mM Tris−HCl buffer，PH8．0，1％SDS，1％β−merCaPtOethanol，lmMEDTA，8MureaandlO％sucrose，
andheatedfor20minutesat800C．Theconcentrationoftheproteinsamplewas1−2mg／ml．Approxi−
mately30pgofproteinwasappliedtothegel．Theconcentrationoftheacrylamidewas5・6％・Electro−
phoresiswasperfomedataconstantcurrentof4mApergel．Therunningtimeundertheseconditions
WaSabout41lr．
Tablel ComparisonofthePercentagesofBands
Results（Densitometer）

Conh011 2 3 4 5 6 7 8 9 0 1 2
1
1

1

5

1

5

5
▲

1

っ

0

MWxlO3
8

Band

（％）
22．96

Re飢llts
Patternofnormalskeletalmuscle

SefOtOnin
myopathy
（％）

26．54

Electrophoresis ofnormalskeletalmuscle gave apat−
ternin which13wellresoIved bands−arbitarilylabeled
1−13−arePredominant・Thepercentageofeachbandwas
calculatedwitha dinSitometer（Tablel）．Molecularweight

q

5

ノ

8

4．44

2．56

14．07

11．13

estimates for bands1−13

2．96

0．85

（Tablel）．

′
b

0

5

8

4．96

3

5
2

5
1

10．37

8．93

13．33

18．83

7．4

6．33

were obtained with markers

Patternofserotoninmyopathy
Obvious changes ofpatterncompared tonormalmus−
cle were observedin bands1−9

0f which the molecularweightsrangedfrom185，000to15，000．Thechangeswere
as follows（Tablel）．

1

20．03

20．87

（1）The width as wellastearing ofbandl，andin−
creasesinbandsl and8wereobserved．
−102−

（2）Decreasesinbands2，3，4，7，9，reSPeCtively，Werealsoseen．Themostremarkabledecreasewas
Showninband40fmolecularweight95，000．
（3）Fusionofband5with6wasobservedbutthepercentageoffusedbandswassubstantiallyequal
tonormal．
DiSCuSSion
Theelectrophoreticpattemofnormalskeletalmuscleobtainedunderourconditionsprobablyre−
Vealed struCturalproteinsandmanyothercomponentsofmusclebecause13bandswereclearlydis−

tinguishedandtheirmolecularWeightsranged from185，000tolessthan15，000．Inconclusion，tWO
pointsbecameclearfromthisroughanalysis・OneofthemostremarkablechangesinstruCturalproteins
Ofserotoninmyopathyis the decreaseinα−aCtinin・Anotherimportantfindingisthewidthaswellas
tearingandincreaseoftheheavychainofthemyosinband．
Itisreported3）thatthemostcharacteristicchangesinthestructuralproteinsinpatientswith
Duchennemusculardystrophyatarelativelyearlystageofthediseasearethedecreasesina−aCtininandtroponincomponentswhnethedegradationoftheheavychainofmyosinisobservedatalaterstage．
Thedecreaseinα−aCtininanddegradationoftheheavychainofmyosininserotoninmyopathysug−
gestanalysisofthemodeofmuscledestruCtioninvariousmyopathies．
Referencesl）Iida，M・，Adachi，K・，Mizuno，K・，etal：ExperimentalSerotoninMyopathy−Biochemicalstudyofskeletalmuscle
membranefragmentscontaining（Na＋K）ATPase−CurrentReseardlinMuscularDystrophy，Japan，Tokyo，1980，
p．96．

2）FaiTbanks，G・，Theodore，L，Wallach，S．andD．F．H．：Electrophoreticanalysisofthem再OrpOlypeptidesofthe
humanerythrocytemembranC．Biochemistry，10（13）：2906，1971．
3）Sugita，H・，Ishiura，S．，Suzuki，K．，etal．：Ca−aCtivatedneutralproteaseanditsinhibitors：invitroeffectonintact
myonbf鮎．MuscleandNeⅣe，3：335，1980．

−103−

肋∫甜ね′砂∫打OPカブGro叩CALCIUM．DEPENDENTDEGRADATIONINMUSCLEPROTEIN−EFFECTOFCaIONOPHORE（A23187）ANDACANPINHIBITOR（E・64−C）−
HideoSugita＊，＊＊，ShoichHshiura＊＊andIkuyaNonaka＊＊＊DepartmentofNeurologyIlnstituteofBrainResearchIFacultyofMedicine，UniversityofTokyo，Tokyo＊＊DiYisionofNeuromuscularResearch，NationalCenterforNervous，MentalandMuscularDisorders，Tokyo

Manydatahaveaccumulatedsuggestingthatcalciumions（Ca2＋）areinVoIvedinthedegradationof

musclestruCturalproteins．
InDuchennedystrophy，thereistheideathattheincreasedCa2＋duetoanexcessiveinfluxof
extracellularCa2＋throughacertaindefectiveplasmamembranemaystimulatecalciumactivatedneutral

protease（CANP）andinitiatethedegradationofthestruCturalproteinsl）・
Totestthishypothesis，WeeXPerimentallyincreasedtheintracellularCa2＋concentrationofexcised

intactratmusclesbyusingCaionophore，A23187，iT

itroandobservedthedegenerationofmuscle

fibersmorphologicallyandbiochemically・TheeffectofE−64−C，aPOtentinl1ibitorofCANP，WaSalso
investigated．
Twoexperimentalmediawereprepared，OneWaSKrebs−Ringersolution（lmMCa2＋）withcyclo−
heximideandA23187（SOlutionA），andtheotherhadlmMEGTAinsteadofCa2＋（solutionB）・The

excisedratsoleusandextensordigitorumlongus（EDL）muscleswerekeptintheabovesolutions，OneinsolutionAandthecontralateraloneinsolutionB，andincubatedfor3hoursat370C・
Duringincubation，the amountofproteinliberatedfromthemusclewasdetermined・Afterin−
cubation，apartOfthemusclewasusedforanelectron一microscopicstudyandtherestforanelectro−

Phoreticstudy．

The proteins released from themuscleintothemediaweremoreabundantwhenincubatedin
solutionAthaninsolutionB，23％moreinEDLand63％insoleus・Asthereleasedproteinmeasured
by creatinephosphokinasewasalmostequal，therelativeincreaseinliberatedproteininsolutionAcomparedwithsolutionBwassupposedtomainlyoriginatefromthesolubilizationofthedegenerated
StruCturalproteinS．
Slab gelelectrophoresis of myofibrillar proteins of both EDLand soleus muscles whichwere
incubatedinthesolutioncontainingCa2＋showedagreatlyredJcedbandofα−aCtinin，mOrePrOminent

inDELthansoleus．
In contrast，theincubation of both EDL and soleusin the presence ofEGTAresultedinno
detectablechangeinα−aCtinin．
ElectronmicroscopyshowedalmostselectivedegradationanddisappearanceoftheZ−bandwith
relativepreservationofotherstructures・Themitochondriawereswollenanddisintegrated・
Onthe other hand，the musclesincubatedin the presence of EGTA showed wellpreserved
myofibrilsandmitochondria・
ToshowthatthedisappearanCeOfα−aCtininfromtheZ−bandwasnotadirectcauseofexcessive
intracellularCa2＋，WeteStedtheeffectofE−64−COnthedegradationoftheZ−band・

Addition ofE−64−C tO SOlution Aresultedinaremarkablesuppressionofthereleaseofα−aCtinin

fromthemuscle，eSPeCiallyinthecaseofEDLAsimilartendencywasreportedbyKameyamaand
Etlinger2）usinganotherinhibitor，leupeptin，inratsoleus・
Itbecameclearfromourinvitroincubationexperimentthatincubationoftheintactmusclewith
ca2＋andCaionophorecausedtheselectivedegradationoftheZ−bandandpreferentialliberationof

α−aCtininfromtheZ−band．ThisresultisessentiallythesameasthatreportedbyUchinoandChdu3）・
Asthelossofα−aCtininfromthe Z−band wassuppressed byaninhibitorofCANP，E−64−C，thedegradationoftheZ−bandissupposedtobecausednotbyadirecteffectofincreasedCa2＋，butbya−104−

proteolyticactionofCANP，aCtivatedbyanexcessiveinfluxofextracellularCa2＋・

merefore，this experiment further supports thehypothesisthatCANPmay play acrucialrolein
theinitialstepofthedegradationofthestruCturalproteinsinDuchennedystrophy・
Referencesl）Ebashi，S．andSugita，H．：TneroleofcalciuminphysiologicalandpathologiCalprocessesofskeletalmuscle．Cur−
rentTopicsinNerveandMuscleResearch，1979．pp．73−84．
2）Kameyama，T．and Etlinger，J．D．：CalciumJependentregulation ofproteinsynthesisanddegradationinmllSCle．
Nature，279：344−346，1979．
3）Uchino，M．and Chou，S．M．：Effbct ofcalciumionophore，A231870n murinemuscle．UltrastruCturaland bio−chemiCalstudy．Proc．JapanAcad．，56：480−485，1980・

ー105−

肋JC〟Jαr勒∫加pカタGro叩

DEGRADATIONOFMYOFIBRILLARPROTEINSBYCATHEPSIN H
Yukihiro Sanada，TakashiNoda，KazuhideIsogaiandNobuhikoKatunuma
DepartmentofEnzymeChemistry，InstituteforEnzymeResearch，SchoolofMedicine，TokushimaUniversity，Tokushima
Itisknownthatlysosomalproteasesplayanimportantroleintheturnoverofmyofibrillarproteins．
Butlittleis known about whichlysosomalproteasesdegradewhich myofibrillarproteinsandinwhat
mannerexceptforcathepsinsB，DandLl）2）3）・Inthiswork，themodeofdegradationofmyofibrillar
PrOteinbycathepsinHwasinvestigatedusingsodiumdodecylsulfatepolyacrylamidegelelectrophoresis．
0nlytroponin−TwasdigestedbycathepsinH，trOpOnin−Iandtroponin−Cwerenot．Figurel showsthe
degradation pattern oftroponinwith
Cathepsin H．Troponin−Tshowedtwo
fnactivation
tiIne

0†

0！

5−

151

30−

45−

60−

degraded bands，having molecular鮒−

Weights of 32，000 and13，000，
respectively．Cathepsin H could notdegrade other myofibrillar proteins
SuCh as myosln，aCtin，trOPOmyOSin
Orα−aCtinin．Degradedproductsfrom
troponin−Ton cathepsinHtreatment｝−一一一一TN−1have similar molecular weights to
者

−

−

■■

恥｛＋廃線

・

穿準

肇磁真

32，000

越
顔舞

東疲

索遜

those with cathepsin B treatment．

The following experiment was done
to examine the differences of the

増額

13，000Cleaving point of cathepsin B and
Cathepsin H．The hydrolyzing ac−
tivity wasdeterminedfluorometrica1−

Fig・l Degradationpatternsoftroponin oncathepsinH
treatment

PePtides releasedinto the super−
natant from the digested protein．

〇一〇CathepsinH200

1y as the amounts ofaminoacidsor

Cathepsin H waslOO timesless●一CathepsinB／／

SenSitive to E−64than cathepsin B．

■一Cathep血B↓

Figure′′／2shows the time course of

troponindigestionwiththefollowing

Cathep血H′′／

PrOteaSe SyStemS；Cathepsin H only，／／／Cathepsin

B only，Cathepsin H after

Cathepsin Bdigestionorcathepsin B
00︵−OE￡︸GnOEdp洪諾−0銘

o‥OCathepsinH＋Bo

plus cathepsin H．Cathepsin H．■「

digested troponinafterpretreatment
With cathepsin B neither faster nor
Slowerthantreatmentwithcathepsin
H only．This might suggest that
CathepsinHdeavedadifferentpoint

30
60
Incubationtime（min）
Fig・2

90

Of troponinto cathepsin B．And

Cathepsin H might not havea stron−

Releasedamountofaminoacidsorpeptidesfrom
tTOpOninoncathepsintreatment−106−

ger abnity forthβPrOduct oftropo−ninwith cathepsln B than native

Table Susceptibuitiesofmyofibrmarproteinsto
proteases

troponin．CathepsinB and H act ontroponinina
non−involutionalbutadditionalmanner．CathepsinH

MastCem
Ca仙epshB Ca仇epsnH Se血eand cathepsinBmayattackinanindependentman−

Acth

TN・C
Tropomyosin
M−Protein±inthedegradationofthesemyofibri11arproteins．

＋

一

kindofcooperationamongtheseproteasesmayoccur

日日一

noponinTN・T
TN−I

＋

︷

α一Actininand troponimCwere not degraded．Some

十＋＋一α−ActinhldegradedbycathepsinBandmastcellserineprotease．

Lchain一

TroponimT was degraded byall of these pro−teases・Myosin，trOPOmyOSin and troponin−I were

Myo虚n Hcbain

PfOteaSe
一

tiesofmyofibri11arproteinstocathepsinB，Cathepsin
Handmastcellserineprotease2）4）

榊 ＝ こ ⁚ 十

ner・TheTablepresentsacomparisonofsusceptibili−

十十Referencesl）SchwaLtZ，W・N・＆Bird・J・W・C・：DegradationofmyofibrinarproteinsbycathepsinBandD．Biochem．J．，167：
811，1977．
2）Noda，T・，Isogai，K．＆Katunuma，N．：inpreparation．
3）Okitani，A・，Matsukura，UりKato，H・＆Fttjimaki，M・：Purincationandsomepropertiesofmyofibrillarprotein−degradingprotease，CathepsinLfromrabbitskeletalmuscle，J．Biochem．，87：1133，1980．
4）Yasogawa，N・，Sanada，Y・＆Katunuma，N．：Susceptibilitiesofvariousmyofibrinarproteinsto muscleserine
proteaselJ．Biochem．，83：1355，1978．

−107−

肋∫αJ〝砂∫仇〉タカノ伽岬

IMMUNOGLOBULINANDTHEIMMUNECOMPLEXINFUKUYAMATYPECONGENITALMUSCULARDYSTROPHY
MeisenFumita，ItaruToyoshima，YasuhiroIshikawa，MichihikoHaraguchi零，andKiyotaroKondo
DepartmentofNeurology，BrainResearchInstitute，NiigataUniversity，Niigata＊TheSecondDepartmentofInternalMedicine，NiigataUnivefSitySchoolofMedicine，Niigata

WhilstthegeneticpatternsindicatedautosomalrecessivetransmissionofFukuyamatypecongenital
muscular dystrophyIPathologicalfindings suchasmeningealinfection，micropolygyrla，andanomalies
Ofthe cerebralcorticallayers suggested the etiologicalsignificanceofintrauterineinfection．Inafew

reports，immunoglObulinswereshowntobeelevatedinthisdisease．
In14patients with the diseaseaged from6monthsto16yearS，WeeStimatedserumgammagl0−
bulins G，A and M，mOnOClonalbands ofimmunoglobulinin theCSF，aSWe11ascirculatingimmune
COmPlexeswiththefollowlngreSults；
1）Ina1114cases，theamountsofIgG，AandMwerewithinage＞SPeCificnormallimitswithafew
exceptionsinrelativelyoldcases．Inallcases，IgMwasnormal．
2）Isoelectrofocusing electrophoresis of the CSFof2patientsexaminedshowednegativeresults
fortheoligoclonalbandofimmunoglobulin．

3）Thecirculatingimmunecomplexeswereslightlyelevatedin50fthe14casesexamined．
We were unable to discloseimmunologicalevidence of the prenatalinfection，Which may be
etiologicanyrelatedwiththisdisease．

ー108−

肋gC〟Jαrqygfmp妙Gro岬

lSOLATION OF NERVEGROWTH FACTOR FROM HUMANPLACENTALTISSUE
KyozoHayashi＊and

ShoeiFurukawa＊＊＊DepartmentofBiologicalChemistry，FacultyofPharmaCeuticalSciences，KyotoUniversity，Kyoto＊＊NationalCenterforNervous，MentalandMuscularDisorders，Tokyo

Anumberofrecentpapersl￣3）havesuggestedthattheremightbearelationshipbetweenalteredlevels of nerve growth factor（NGF）activityin human tissues and neurologic disorders that have aknowndefectivegeneticcomponent．At present，the NGFlevel良inhuman tissuesareusuallymeasured
and standardized by using mouse NGF andits antibodies，yielding many contradictory results．The
availabilityofpurifiedhumanNGFwnlallowthestandardizationandunequivocalquantitativemeasure−mentsofNGFlevelsinhumantissuesandfluidsinanumberofneuropathies．
This paper deals with theisolation ofhuman NGFfromhumanplacentaltissueaccordingto themethoddescribedbyGoldsteinetal．4）withsomemodifications．
Humantermplacentaltissuewashomogenizedincold saline（20％W／V）at20，000rpmfor2min．
Thehomogenatewasadjusted to pH13．0withlONNaOH，re−homogenizedat20，000rpmfor30sec，andcentrifugedat30，000xgfor10min．TheresultingsupernatantwasadjustedtopH3．8withglacialaceticacid，auOWedtostandfor5minatOOc，andcentrifugedat30，000×gfor30min．

ThesupernatantwasthenadjustedtopH5．Owith4NNaOH，Centrifugedat30，000xgfor50min，
and the supernatant was dialyzed against distilled water，Centrifuged at20，000x g forlO min，and
appliedtoacolumnofCM−52cellulose．ThecombinedactiveCM−52fractionswerere−Chromatographed
On a COlumn of CM−52cenulose．The active fractions were pooled，COnCentratedbypressuredialysis
INDUCTION OF NERVE FIBER OUTGROWTH BY HUMAN PLACENTANERVE GROWTH FACTOR FROM THE DORSAL ROOT GANGLIA OF
CHICKEMBRYO

Fig．l Thedorsalrootgangliawereculturedat370cfor3daysinthepresence
OfhumannerVegrOWthfactor（7ng／ml）．
−110−

andgel−filteredona SephacrylS−300column．meyieldofpurifiedmaterialwasabout10mgfromone
humanplacental．

GelfiltrationusingSephacrylS−300gaveamolecularweightofabout160，000．ThepurifiedNGF
WaSteStedbyimmunodiffusionagainstantiserumtOPurifiedmouseNGF，andtherebycomparedwith
mouse NGF．Theinteraction of the precipitinlines shows that there are antibodies that recognize
determinants commontobothhumanand mouseNGFs，butthat themouseantigenhasdeterminantS
not presentinhumanNGF（spurformation）．PurifiedNGFgaveasinglebandonpolyacrylamidediscgelelectrophoresisatpH7．5．But，aSShownonsodiumdodecylsulfatediscgelelectrophoresis，purified
high−mOlecular weight NGF showedseveralbands．Thus，high−mOlecularWeightNGFwassubjectedto
PreParative disc gel electrophoresis at pH2．3．The280nmabsorptionprofneofthecolumnfractions
revealed pluralbonds，One OfwhichshowedspecificbiologicalactivityforNGF．The NGFwasmaxi−
mally active at approximate7ng／mlwith the tissueculture method using chickembryodorsalroot
gangliaasshowninFig．1．
IsoelectricfocusingusingagradientbetweenpH3．5andpHlO．OgaveapIvaluecorrespondingto
about5．0．SodiumdodecylsulfatediscgelelectrophoresisoftheNGFobtainedgaveamolecularweight
Ofabout65，000．msNGFdiffersfromotherNGFsincludingmouse伊NGFandsnakeNGFsinhaving
a highmolecular weight and maycorrespondtothehighmolecularweight NGFisolatedfrom mousesubmandibularglandbyYoungetal．5）
Referencesl）Fabricant，R・N・，Todaro，G．J．，＆Eldridge，R．：IncreasedlevelsofanervegrowthfactoTCrOSS−reaCtingproteinin
Centralneurofibromatosis．TheLancet，i：4，1979．
2）Siggers，D．C．，Rogers，J．G．，Boyer，S．H．，Margolet，L．，Dorkin，H．，Banedeee，S．P．，＆Shooter，E．M．：Increased
nervegrowthfactorβ−Chaincross−reaCtingmaterialinfamuialdysautonomia．NewEngl．J．Med．，295：629，1976．
3）Bigazzi，M．R．，Revolteua，R．，Casciano，Sり＆Vigneti，E．：HighlevelofanervegrowthfactorintheserumOfa
Patientwithmedullarycarcinomaofthethyroidgland．Cl血．Endocrin01．6：105，1977．
4）Goldstein，LD．，Reynolds，C．P．，＆Perez−Polo，J．R．：IsolationofhumannelVegrOWtllfactorffomplacentaltissue．
Neurochem．Res．，3：175，1978．
5）Young，M．，Saide，J．D．，Murphy，R．A．，＆BlanchaLd，M．H．：Multipledissociationproductsinhomogenatesof
mousesubmandibulargland．PuLificationandmolecularpropertiesoftheintactundissociatedfromoftheprotein．
Biochemistry，17：1490，1978．

−111−

肋∫闘わ′砂∫〝op力yGr0岬

LEVELOFNERVEGROWTHFACTOR−uKEIMMUNOREACTIVITYINHUMANSERUM
KyozoHayashi＊，ShoeiFurukawa＊＊，TakekazuOhi＊＊＊andHirooImura＊＊＊＊DepaJtmentOfBiologicalChemistry，FacultyofPhamaceuticalSciences，KyotoUniversity，Kyoto＊＊NationalCenterofNervous，MentalandMusclllarDisorders，Tokyo＊＊＊TheSecondDepartmentofIntemalMedicine，FacultyofMedicine，KyotoUniversity，Kyoto

Previously，WerePOrtedthat，aSmeaSuredbythetissueculturemethod，thelevelofnervegrowth
factor（NGF）inthesubmaxiuaryglandsofmaleheterozygousdystrophicmiceexceededthatofmale
homozygousmicebyanOrderofmorethantwol），andalsoasmeasuredbyradioimmunoassay，thelevelofNGFJikeimmunoreactivityinthemuscleofhomozygousdystrophicmicewasabouthalfthat
ofheterozygousmice2）．Theseresultssuggestedthatadysfunctionofthenervestotheskeletalmuscles
resultedinthedevelopmentofthedisease．
In connection withthis，We tried to examine thelevelof NGF−likeimmunoreactivityin sera ofPatientswithdystrophyuslngthesolid−Phaseradioimmunoassaymethod．First，WeeXaminedtheassay
PrOCeduresforthelevelofNGFlikeimmunoreactivityinhumanserum．Theresultsrevealedthatthere
wereatleasttwo NGF−likemoleculesinhumanserum，andthatmostoftheNGFinthehumanserum

existedinacomplexformassociatedwithserumcomponentsofhighmolecularWeight3）．Unlikethe
NGF，thiscomplexpossessednobiologicalorimmunologicalactivity．
（a）Presence of Serum Components which Bind NGF．In a competitive radioimmunoassay，any
decreasein radioactivityin the bound antibodiesisinterpreted as showingthepresenceofNGF．We
examinedtheNGFlevelinhumanserumwithatwo−antibodyradioimmunoassaysystem，inwhichthe
125Ⅰ−6−NGFboundtoitsanti−rabbitantibodywasprecipitatedbyanti−gOatrabbitIgGantibody・Thelevelof NGFJikelmmunoreactivityin human serum meaSured by this method was found to be30−500
ng／mi．But human sera showed only a weak responsein the nerve fiber outgrowth test．Aftergel
filtration of human serum On a Sepharose CL−6B column，eaCh fraction was m由sured by the two−
antibodyradioimmunoassaysystep．Remarkablebindinginhibitionof125ⅠやNGFwasobservedinthe
eluate ofthe macroglobulin fraction，butthisfractionshowednoNGFactivitywhatsoever．Hence，if
the125I−β−NGFwasboundtotheserumCOmpOnentS，itsbindingtotheantibodieswouldbeprevented，
and theresult would beanoverestimationofthe NGFintheserum．WethereforetestedwhetherNGF−
bindingcomponentswerepresentinthesera．125I−β−NGFwasgelfilteredinthepresenceorabsenceof
human sera on a column of SepharoseCL−6B（Fig．1）．Aconsiderableshiftofthepeakwasobserved，
suggestingthat NGFindeedbindstotwolargemoleculesintheserum．However，Onlyonecomponent
wasfoundtointerferewiththeantigen−antibodyreaction．Thecomponentelutedinthesecondfraction
didnotshowcompetitionintheradioimmunoassay．Theseresultssuggestthattheserum COmPOnentS，
哺hichmigratedasmacroglobulin，boundcompetitivelyto125I−8−NGF，COntributingtotheapparently
highlevelsofNGFlikeimmunoreactivityfoundinhumanserum．Fabricantetal．4）havealSoreported
thesameresultsforaNGFradioimmunoassay．Therefore，atthebeginnlngOftheassay，itisnecessary
toensuretheabsenceofthesecomponentsintheoftencontaminatedserasothatreliablevaluesofNGF
inseraorothertissuescanbeobtained．
（b）Distribution of NGFlikeImmunoreactivityin Human Serumunder NeutralConditions．To
avoidtheinteraction between NGF−likeimmunoreactivity and macromoleculesinhuman serum，We
tried to use a solid−Phase radioimmunoassay system forhuman sera・The advantages ofthis method
arethatmixingofserumand125Ⅰ−6−NGFsamplesisavoidedandthecompetitivebindingcomponents，

WhenboundtoendogenousNGF，formacomplexthatdissociatesunderalkalineconditions．
（C）DistributionofNGFJikelmmunoreactivityinHumanSerumunderAlkalineConditions．On
gelfiltrationofhumanserumona SephadexG−150columnunderalkalineconditions，tWOPeakswith
NGFJikeimmunoreactivity appeared；One WaSalmostatthepositionobservedatneutralpH，andthe−112−

Other

was

a

new

peak

elutedapproxi一

mately at the column volume．With thetissueculturemethodusingchickembryo
dorsal root ganglia，the first peak gave
Only a weak response，but the second
peak produced distinct outgrowth ofnerve fibers．Theseresultssuggestedthat
there were atleast two NGF−like mole−
Culesin human serum，and thatmostof
the NGF existedin a complex form as−

SOCiated with serumcomponentsofhighmolecular weight．UnliketheNGF，how−ever，thiscomplexpossessednobiologicalOrimmunological activity．The other
NGF−like molecules obtained with gel
filtration on Sephadex G−150had mole−∩

Cularweightsof30，000to50，000．Thesefindingsindicated that the NGF−likeacti−
Vitiesin human serumWeredistributedinthesecategories．Actual1y，alargenumber
Of NGF，like molecules seemed to beelutedina complex form with nobiological activity．The serum COm−
ponents，i．e．，125I−P−NGF−binding com−POnentS，that migrated as macroglObulin
might be carrier proteins which par−ticipateintheregulationofthebiological
functionofNGFinhumanserum．

20

40

60
Tube No．

Fig．l Elutionpatternsof125ⅠサNGFinthepresenceorabsenceof
human serum On a COlumn of Sepharose CL−6B．Normal
human serum（320pl）wasmixed with125I−β−NGF（12，000Cpm）andincubatedfor severalhouTSatrOOmtemperature．
neinclubation mixture wasthen applied to a column ofSepharose CL−6B（105xl．0cm）equilibrated with O．05M
Phosphatebuffer，PH6．8，andelutedwiththesamebuffbr．
400FLlofO．05Mphosphatebuffer，PH6．8，COntainingO．5％
bovineserumalbuminand125Ⅰ−P−NGF（10，000cpm）wasap−

Plied to the same column equilibrated with O．05Mphos−
phatebuffer，PH6．8．Fractionsofl．2mleachwerecouected．
Tnesolidlineindicatesthedistributionofradioactivityinthe
PreSenCeOfserum；thedashedline，intheabsenceofserum．

Referencesl）Furukawa，S・，Nishitani，H・，＆Hayashi，K・：LevelofnervegrOWthfactoractivityinthesubmaxillaryglandsof
genetica11ydystrophicmouse（C57BL／6J）．Biochem．Biophys．Res．Commun．，76：1202，1977．
2）Furukawa，S・，Hayashi，K・，＆Ohi・T．etal．：LevelofnerYegrOWthfactorJikeimmunoreactivityinthelowerlimb
musclesofmusculardystrophicmice・Biochem・Biophys・Res・Commun．，90‥130，1979．

3）Hayashi，K・，Furukawa，S．，＆Ohi，T．etal：Levelofnervegrowthfactorinmammaliantissues．In

Growthand

GrowthFactors （ed・byShizume，K・＆Takan0，K．），UniveristyofTokyoPress，Tokyo，1980，p．253．
4）Fabricant，R・N・，DeLarco，J．E．，＆Todaro，G．J．：Nervegrowthfactorreceptorsonhumanmelanomaceusincul−
ture，Proc・Natl．Acad．Sci．USA，74：565，1977．

−113−

MEMBERS OF THE RESEARCH GROUP ON
MUSCULAR DYSTROPHY TNJAPAN（CLINICAL RESEARCHES）
Chairman
MIYOSHI，Kazuo

OkinakaMemorialInstituteforMedicalResearch，Tokyo

Members
ARAKI，Shukuro

TheThirdDepartmentofInternalMedicine，MiyazakiMedical
College，Miyazaki

FUKtJHARA，NobllyOShi

DepartmentofNeurology，BrainResearchInstitute，Niigata
University，Niigata

FURUKAWA，Tetsuo

DepartmentofNeurology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo

FURUSHO，Toshiyuki

DepartmentofHygiene，FacultyofMedicine，KagoshimaUniversity，
Kagoshma

HAYASHI，Kyozo

DepartmentofBiologiCalChemistry，FacultyofPharmaceutical

IMURA，Hiroo

The2ndDepartmentofInternalMedicine，FacultyofMedicine，

Sciences，KyotoUniversity，Kyoto

KyotoUnlversity，Kyoto

IIDA，Mitsuo

FirstDepartmentofInternalMedicine，NagoyaUniversitySchoolof
Medicine，Nagoya

KAWAI，Hisaomi

TheFirstDepartmentofInternalMedicine，SchoolofMedicine，
TheUniversityofTokushima，Tokushima

KINOSHITA，Masao

DepartmentofMedicine，TohoUniversity，Tokyo

KONDO，Kiyotaro

DepartmentofNeurology，BrainResearchInstitute，Niigata
University，Niigata

KURIHARA，Teruyuki

TheThirdDepartmentofMedicine，MiyazakiMedicalCo11ege，Miyazaki

KUROIWA，Yoshigoro

DepartmentofNeurology，NeurologicalInstitute，Facultyof
Medicine，KyushuUniversity，Fukuoka

MAWATARI，Shiro

HealthAdministrationCenter，KyushuInstituteofTechnology，
Kitakyushu

MIYATAKE，Tadashi

DepartmentofNeurology，JichiMedicalSchool，Tochigi

MOZAI，Tpshiji

TheFirstDepartmentofInternalMedicine，OsakaMedicalCollege，
Osaka

NAKAMURA，HaruOmi

DivisionofNeuropathology，InstituteofNeurologiCalSciences，TottoriUniversitySchoolofMedicine，Yonago

NAKANISHI，Takao

DepartmentofNeurology，InstituteofClinicalMedicine，
TheUniversityofTsukuba，Ibaraki

NATORI，Yasuo

TheDepartmentofNutritionalChemistry，SchoolofMedicine，
TheUniversityofTokushima，Tokushima−115−

MSHITANI，HiTOSu

DepartmentofNeurology，UtanONationalHospital，Kyoto

NONAKA，Ikuya

DivisiohofNeuromuscularResearch，NationalCenterforNervous，
MentalandMuscularDisorders，Tokyo

OGUCHI，Kimio

DepartmentofMedicine（Neurology），ShinshuUniversitySchoolof
Medicine，Matsumoto

OSAME，Mitsuh址oDepartmentofInternalMedicine，SchoolofMedicine，Kagoshima
University，Kagoshima

SANADA，Yukihiro

DepartmentofEnzymeChemistry，InstituteforEnzymeResearch，
SchoolofMedicine，TokushimaUmiversity，Tokushima

SATO，Takeshi

DepartmentofNeurology，JuntendoUniversitySchoolofMedicine，
Tokyo

SHOJI，Shin

iehi

DepartmentofMedicine（Neurology），ShinshuUniversitySchoolof
Medicine，Matsumoto

SUGITA，Hideo

DepartmentOfNeurology，InstituteofBrainResearch，Facultyof
Medicine，UniversityofTokyo，Tokyo
DivisionofNeuromuscularResearch，NationalCenterforNervOuS，MentalandMuscularDisorders，Tokyo

TAKAGI，Akio

DivisionofNeuromuscularResearch，NationalCenterforNervous，
MentalandMuscularDisorders，Tokyo

TAKAHASHI，Keiichi

The3rdDivision，DepartmentofMedicine，KobeUniversitySchoolof
Medicine，Kobe

TAKAMORI，Masaharu

FirstDepartmentofInternalMedicine，NagasakiUniversitySchoolof
Medicine，Nagasaki

TAKAOKA，Yoshito

FirstDepartmentofInternalMedicine，NagasakiUniversitySchoolof
Medicine，Nagasaki

TARtJI，Seiichiro

The2ndDepartmentofInternalMedicine，OsakaUniversityMedical
School，Osaka

TOYOKURA，Yasuo

DepartmentofNeurology，InstituteofBrainResearch，Facultyof
Medicine，UniversityofTokyo，Tokyo

TSUKAGOSHI，Hiroshi

DepartmentofNeurology，FacultyofMedicine，TokyoMedicalandDentalUniversity，Tokyo

UEDA，Keishi

HealthAdministrationCenter，UniversityofEhime，Matsuyama

UONO，Masanori

DepartmentofNeurology，TokyoMetropolitanHospitalofFuchu，
Tokyo

YAGITA，Masatoshi

TheFirstDepartmentofInternalMedicine，SchoolofMedicine，
TheUnlversityofTokushima，Tokusldma

YOSHIDA，Mizuko

NationalCenterforNervous，MentalandMuscularDisorders，Tokyo

WAKAYAMA，Yoshihiro

DivisionofNeurology，DepartmentofMedicine，ShowaUniversity，
FldigaokaHospital，Yokohama−116−