of connective tissue macromolecular components in

Journal of Dermalological Science 12 (1996) 232-237
Analysis of connective tissue macromolecular components in
Ishibashi rat skin: Role of collagen and elastin in cutaneous
K. Sakuraoka', S. Tajima"*, Y. Seyamab, K. Teramoto'. M. Ishibashi'
"Depa"u,1e,1! (if Den'la!o!og.1'. Kcio Unirc"si/.1' Sc'hoo! ( / Mec!ic' "le. i5 Shi,,an0'1lu('hi. S/,!/ fl!k-,!-klr. Tokyv l60. Japan
hDe!'a""I7(!'1/ ( / C!ini('u/ C/1emisi"_1'. Hoshi Cn!!ege (if Pha ',1'a(Ll'. E!,aru. Shi,1agaltrl-kl!. Tvk_1'f' l4 . Ju!'un
cS('h(,ol o/ [
!e"i,1(/'Ll' It.letii(i,,e. A:clbfl L ,1'1 (' s'!1 Fl 'hini,b('. Su.'.'u'l7iha"a. Ku'lagu]t i 119. Ju!;un
Received 1 3 April 1995; r'evision recei¥'ed 28 August 1995: accepted 28 AuguSt 1995
The Ishibashi (IS) rat. established rrom cross-breeding betv,'een Wistar and lviid rats. has a unique skin appearance.
l'ith ¥¥,'rinklings and furrows indicative oi' skin aging appearing at the age of 12 vY'eeks. To understand the underlyin_
mechanism of the formation of ¥vrinkles. macromolecular components of connective tissue. collagen and el lstin. in
the young (5-6-¥veek-old) and the aged (23-30-week-old) IS rat sklns wel e bioc hemically analyzed. Hydroxyproline
and isodesmoslne contents In the aged IS rats were reduced 22' (, (P < O.O5) and 37'1/,, (P < 0.05) compared to the
young rats. ¥vhereas no si_,・*nificant difflerences in the contents of both macromolecules in control Spr lgue-Dawley (SD)
rats vL'ere seen. The reltlti¥,'e content of type 111 colla* :en was unaltered between the yoLlng ancl a_ ed skins of both IS
and SD rats. A relative decrease in the intact elastln molecule (65 kDa) and a relative increase in the elastin rragments
vith lower molecular weights were observed in the aged IS rat skin by immunoblotting method. These results indicate
that the reduction in coliagen and elastln contents and increased degradation of elastin-molecules in the aged IS r2lt
skln could be relateci to the formdtion of wrinkles. Thus. the IS rat maly provide a useful model fol the study of skin
Ke.1'11'ords' Ishibashi rat: Elastin: Aging
l. Introduction
The major clinical manif statlons of cutaneous
aglng are readily reco_ :nized by wrinkling and
saggin_ of the skin, with reduced elasticity and
recoil. Pro_cr*ress in underst lnding the blological
and biochemical chan_ es underlying cutaneous
aging has been slow partly because of the complexity of these processes 2tt the molecular le¥'el.
Cutaneous agin_ : represents two independent. biologically divergent processes: chronolo*a*jcal agin_ :
* Corresponding tlutllor Tel.: O3 3353 Illl (Ext. 1416):
F2lx: 03 3351 6880
and sun-exposure-related aging [1]. Several evidences su* :gest that these two processes, innate
0923-181 1 96 S15 OO 'c* Ic)c)6 Elsevier Science I]-e]and Ltd. A11 rights reser¥cd
SSDI O9 3- 1 8 1 1 (95)00478-B
K SuAt!ruvk(/ C'! (/!. Jvl!'7lcl/ (,/ Der
lgin_9: and actinic dama_ :e. have different biologi-
cal. blochemical and molecular mechanisms [2].
!v!ogi('u/ S('ie"( ! 12 (l996) 132--237 '
2.3. Dete,'117ination ( / /1_T;dro_r_lpro!ine a,Id i'elutiL'e
c0'7te,7! ( /' !_1'pe 111 co!lag'e,1
The samples were hydrolyzed wlth 6 M HC1
Chronologlc agln_ is clinically characterized by
fine wrinkling, atrophy of the dermis. and reduction of subcutaneous adipose tissue. In contrast.
actinic'ally-dama .cTed skin is chru acterized by
mined according: to the method oi' Prockop [7].
coarse wrinklin_ !1 and rurrowln_ :.
acid, digested with pepsin (lOO /lglml) (Worthin_g:ton Biochemlcal Co.. New Jersey) at 4'C
Ishlbashi (IS) rats. establlshed f rom a crossbreed between WistLlr tlnd wild r2uts. is character-
ized by congenital vertebr211 mLtlformzltion and
qualltative Ind cluantitati¥"e changes oi' plasma
allkaline phosphtrtalse (ALP). The chtln_2:e of ALP
in IS rats might be considered to be related to
their vertebral malformation. Pltlsmzl ALP in the
for 18 h
tt I lO'C and hydl oxyprollne was deter-
The dermis was homogenized in 0.5 M acetic
o¥'erni_ ht, and centrifuged at 10000 >< g for l
h at 4'C. The resulting Supernatant was dialyzed
against 0.02 M dibasic sodium phosphate 'at 4'C
for 48 h. Collargen was precipitated Lind subjected to 5' , sodium dodecyl sulphate polyacry-
lamide _ el electrophoresis (SDS-PAGE) con-
rats is conb idered to originate in the intestine.
bone and liver [3-5]. Cutalneous m mifestation in
the oid IS rLlts mimics sun-ciLlmtl_ led aging skin.
taining 3.6 M urea [8] under non-reducin* : con-
w,'ith marked wrinkles tlnd sa_ :_cr*jng and i'ormLl-
destained. The bands of [ (1(lll)]3.
tlon of comedos. Here ¥ 'e htl"'e attempted to
were determined ¥vith Ll densitometer.
ditions. The polyacrylamide ge]s were stained
with Coommasie brilliant blue (CBB)-R250 and
(1(1) and
evalualte the IS rtlt skin Els an anim ll model for
cutaneous tgln_ l and have determlned the
macromolecular components (collLlgen alnd
elastin) ot' connectlve tissue in the ciermis Prellmintlry studles hLl¥'e been published elsevt"here
[6] .
7_. Materials and methods
2.4. D(!! ,7,1i,1uti0'1 ( / iso(k・,s""o,s'i,1('
The stlmples were hydrolyzed vL'ith 6 M HCl
for 18 h 2lt I lO'C tlnd isociesmosine ¥vtls deter-
mined using high performance liquid chromatograph y ( H PLC. LiChrosorb R P- 1 8 col umn .
Merck. Germany) [9]2._1' . Inll,'In,ohlo!ting u,1ul.1'si,s' on c'!us!ill nlok・t llk'
The eltlstin molecule wLlb extralcted rrom skins as
'.1. Thc' IS ,・(1!
The IS rzlt strain. ISh homo (ih,lh) used in
this study ¥,'as established by crossbreeding bet¥veen Wistar tlnd ¥vild rats. The IS rats vvere
maintLlined in indil"idual czrges in our laboratory
at F24 progeny of full-sib mating [-')]・ SprzrgueDLlvvley (SD) rats LLSed for control were obtLllned
i'rom Charles River Jtlpan. Inc.
descrlbed previously [l0,1 I]. Briefly. the dermls
( - O.1 _ wet weight) WLIS homogenlzed in phosphate-burl red saline (PBS) Llt 4"C and centrii'ugeci
at 10000 x s.' i'or 30 min. The precipit rte ¥'as
extracted twice with 0.5 M tlcetic tcid conttlining
l mM ethyleneciialminetetraalcetic tlcid (EDTA).
N-ethylmaleimide (r¥IEM) and phenylmethylSulfonyl fluoride (PMSF) t¥ 'ice tlt 4'C 2lnd centril'uged tlt 10000 x g i'or I h Llt 4'C. The
resulting superntltLlnt wtls precipittlted
". P,・L・!'u,'u!ivl, ( 1 ,,ki,?,
Balck skins of male IS tls well as male SD rats
from 5-6- veeks-olcl (young :roup) and 23-30-
¥'ith IO' ,
NatC1. The precipitate wals dissolved ¥vith 0.5 M
ammoniLm formalte. pH 5.5. containing prote lse
vere dissected. The epi-
inhibitor cocktail. alfter ¥vhich insoluble mLlterials
dermal layer ¥,'2ls peeled ol ' by tl ypsin treLltment
were remo "ed by centril ugation alt 10 OOO x ,.' i'or
(O. ';.i,) for 30 min at 370C. The dermis was
lyLtshed vith H.O ancl lyophillzed. An aliquot
¥vLls t lken r'or histolo_2:ic211 ex2lmlnLltion to tlscer-
15 min. To one Volume oi the supernatttnt. 1.5
volumes oi l-propalnol wLIS Slo vly added dropvise, follo¥ved by the dropwise tlddition ol' 2.5
volumes ot' I -butanol. After stirrin*2: o¥'ernight. the
veeks-olci (Llgecl gl Oup)
¥'hether or not the epldermal layer lvilb
mo¥'eci rrom the dermtll strips.
mtlterial ¥vLts filtered throug:h Whatmtln No. l
4 _ (It)96) .
'- _77
A S(!llt!'tlfJk(! t'! ,f/. ,l,],!'V'f// f'/ D(・,7,,(1!('h,, !,t'/ S( !t'/'('(' / 1
paper. The filtrtlte wtls dried. solubilized in 0.5 M
acetic Licid. dialyzed Llgalinst l"/*, acctic acid con-
colllaen wlS IppIOXlll rtely 61']/(1 71'1/f' ill the
l'eSpectively. whell eStillltlted by the hydl'OXypl'O-
and PMSF) Llnd lyophilized. The proteins were
lille colltellt ill the pepSlll-SOluble alld -ilISOIUble
dissolveci in 0.1 M Tris-HCl, pH 7.4. contLlinin_ 2
rr 1CtiollS. Type 111 CO1ltellt ill the tlged IS
prote2lse inhibitors ( I ITIM EDTA.
M ulel Ind l' , SDS. 2lnd resolved on gradient
2' , - 1 5' , S DS-PAGE. and electrophoretically
blotted to nitrocellulose membranes (Schleicher
and Schnell) in Ll tank blotter (Bio]'ad) at 3O V
overnight [12]. The membrLlncs were blocked vith
5' , bovine serum tlibumin (BSA) in 10 mM TrisHCI, pH 8.0. 150 mM NaCl. lnd 0.05'!/,, Tween
(TBST) for I h tlt room temperature and w'ashed
with TBST f'or 3 x lO min, after which they were
incL[btlted with ITIOnOclonal anti-bovine tropoeldstin antibody (Elastin Product Company) at If
lOOO diiution for I h ilt room temperature. After
Llnother rinse with TBST for 3 x 10 min. the
membraines were incubated with the sec*ond antibody ( tnti-mouse immuno_"*lobulins (Igs)) conju:llted wlth horseradish peroxid lse lbr I h lnd
visuallzeci by chemiluminescence (Amershilm ).
young and aged gfOups ot
IS tlnd SD rat SkinS.
lnd SD
l'tit Sl(iilS dctel'lllilled With tl dellsitO11letel' w lS
Slightly decretlSed bUt exllibited nO SttitlStic llly
SigllinC lllt difference betweell tlle yoLlll_
:rOups oi
lnd tlged
IS tllld SD l'trtS (Fi_ . Ib).
- 60
o oo o ●*■●3
P 40
o_ :'
*x ¥ 20
7. 6. S!uti. '!ica! u/'u/.1'."is
The results presentcd here dre expressed as
mean + S.D. Student'S l-test ¥A'as used to evaluate the difl rences between yoLLn._<*' and olci groups.
3. Results
_ . l. IVla/7il ・stutioil o/ clrtun(/ol./,s' uf.>i,,,.' il! IS ,'u!
IS rats trt 3-6 weeks oi'
lge showed
l normill-
C s
appearin_ skin (figures not shown). In contrast.
marked wrinklings aind f urrows on the bac k and
tlbdomlnLll area,
lnd eLlrly comedo formation
O 5 6 2324 2830
were seen on the 12-week IS rat skin. These
ch lnges were most p]'onounced in the 23 - 30-week
^●●I ^^^^
Age ( week )
S rat skin (see [6]).
Fig I Delerminalion ol'colltlgen contcnt in lhe IS tlnd SD rtlt
3.2. Co!luS.'e,1 cont(・,1r in IS culd SD rur '
Collagen content as measured by hydroxypro-
skin (il) Colltl*',_'en conlent tls meLlsLtred by hydroxyp.roline
v2ts determined in the young und aged IS (clos'ed
cii'cles) tlnd SD (op-en circles) rilt skins. Dalil ilre pres'ented il _'
line content in the aged (23-30 week-old) IS rat
skin ¥vas decreased compared with the young (5or 6-week-old) rat (36.2 5.6 /lg vs. 46.2 3.9
c2lnce 2lt P < O 05 ( <) (b) Type lll collLlgen content rel rtivc lo
/t_ :. P < 0.05). In contr2lst, there were no slgnlfi-
totzll collLl._'*.'en wLIS determined by scilnnin_
ctlnt differences in collagen content between the
youn_"* and old SD rat skins (40.2 3.1 vs. 41.5
4.6) (Fi_"* [a). The yield oi pepsin-soiubie
pg m_ : dry weigllt dermis. Indic lted ilre meLln valucs i S.D
l'rom young (/1 = 5) ilnd old (,, = 7) rLlts ilnd stLllistical s'ignifi-
the gels Data
reprcscnt percenlil..'-.'e of' t),'pe lll collzL_ en of tolal coll lgen ( I +
lll) Indiciited ilre meun values i S.D. I'ro]n the young
(,, = I ) ilnd old (/i = 12) h・kius dcri¥'ed I rom IS (closed circles)
tlnd SD (open circles) rals
A: Sf!Al!f(!t'A(! C'! ill
C )
significantly decreased in the a_ :ed (2_')--30-week-
old) IS rats, where elastin fra_ ments with the
lower molecular weights were predominalntly detected (Fi_ . 3c).
4. Discussron
'Oo :
/i fl')96)・S' ' 7
tropoelastin molecule with a molecultlr weight of
65 kDa was predomintlntly detected in the youn_a,_
(5-6-week-old) IS r'ats. It's re]ative content was
,h,t!'1'tl/ !'/ D(.1""fl!(,h,.,.・i(,(1/ S( i("I'('t'
A significtlnt difference in colla_2:en content and
o. 1
the proportion of type 111 collagen between immature (embryonic or fetal) and mtlture tissues llaS
been demonstrated [l_'1- 16]. However, when these
data were compared between matured and
2324 28 30
Age (week)
Fi_ :'. I Dete] mintltion ol' isodesnlosine content in the IS ilnd
tissues, no si_ :nificant dilTerences were found
[13,16,17]. In our studies, there were no differences in both colla_ en content and proportion of'
type 111 colla_ en in SD rat skins durlng chronoiogical a_*(;jn_ ;. There were sli_ :ht decreases In colla-
SD rLlt skins Elil .'tin conlcnt as measured by i odcsmosinc wils
dctermined in the young ilnd old IS (clob'ed circles) tlnd SD
(opcn circics) rilts Dilla rel i csenl nmoi isodesmosine!mg dry
wei_ hl cie]'mis Indicated alre mean ¥*LlILLe '
S.D r'rom young
('/ = 3) tlnd old (,1 = 3) rilts. and st21li . llcill h.'ignificance Llt P <
O 05 (= }.
3._?. Jsodesl'losine ('ontcnt und (.!astin li・agn,e,1Ls in
!S und SD ra!s
A signlfictrnt decrease in isodesmosine content
between the young and old IS rats was found
(0.27 + 0.03 vs. 0.17
0.04 l/g. P < O.O5). In
contrast, no si_ *.nificant differences in Isodesmosine
65 k - -
45k ->
30k --'
content between the youn_ : and old SD rats were
seen (0.24
0.03 vs. 0.21 ll 0.04 l!g) (Fig 2).
Elastin rr'a_('*ments isolated i rom youn_ * and aged
1 2 5 6 25 30 5 6 23 24 28
t;'kins were analyzed by an Immunoblotting proce-
dure usin* : antl-tropoelastin monoclon ll antibody.
Experlments usin_ bovine tropoelastin and type l
collagen Isolated from aorta and skin revealed
thttt the tmtibody specifically reco_ *nized the tropoelLlstin and its fragments consistin_g: ot 65-. 45-
and 30 kDa polypepticles (Fig- 3a). The elastin
Fi_ * 3. Detection ol elLlstin-re]illed l'ra* :ments isolated ,rom lhe
IS tllld SD riit skin. (it) Purified t]'opoelil 'tin preptl]'ed i ]'om pi
tlorlat (lLlne I ) ilnd typc I collu'*'en pl epilred l rom bovine skin
(l lne
) wcrc separ lled on 4"';,-- 1 5'} , SDS-PAGE Llnd slained
with zlnti-tropoel2lstill tlnlibody (b) Skin
O 5 M ilcelic
v ls' extr2lcted vL,'ith
lcid ct)nlilinin'_.' 1lrotease inhibitor cocktail l'or 24
f ra_ ments were exclusively detected in 0.5 M am-
h twice E11 4'C Soiuble elastin r -al'*.'menls
monium formate-soluble rraction and were not
described in the lcxl i rom SD (b) Llnd IS (c) rtrt skins and
detected In PBS-soluble or 10' , NaCl-Supern'atant
f'raction (data not shown). The patterns oi' elastin-
vere isolated ns'
resolved on 4'*/,, 15".{, _ :raldienl SDS-PAGE The proleinb' wcre
electrophorelic211ly' blotled to the filte] s Llnd stLllncd with ilnli-
tropoelastn monoclonal ilntibody. then visutllized by chemilu-
related f'ragments in the SD rat sklns were mostly
minescence. Indicilted are tlle moleculilr
simllnr irrespective of a_"*ing (Flg. -3b). An Intact
polyp- eptides.
vei !1lt
of tl]e elLlstin
A’. 、∫“ム・“’・‘〃]大“ ピ1 ‘〃、
’二 ‘’9リ6,2.弓2 2.;ア
gen content but no ch圭H1gcs in typc l H coHagcn
contcnt in lS rat skins−These l’csuhs壬Hld prcvi−
ous reports suggcst lh∼1t thc proportion or type
lH collagen n1∼ly not be llclatcd to cutnneous
We h纈ve estimated t11e elastin co11tent in lS rHt
skin lhe by isodesnlosine vHlue,which is an iso−
n1cr or c11oss−link…ng structurcs con1posed of rour
川Uitlo」.F11zio M」.Ol‘ol 1Dlミ=Molじc[■1三u’■11ビc11unisn1‘o「
じl1l1lllC(π1川gillg..1Al11 Aじ且■d Dc1’111Hlo121=6〕 622,
[2]Lup16I’じC∼1=Thc Hgcing 1じI’mi,=T11c111HiIl cHusc f、川1−11c
el ast i n n/o lcculcs. The1’cro rc, rcd uct…o n o r
uppo呈1■1Hncc ol’’old’skil1 BI’」Dc11m三11ol12〕コSu1111135〕:
isodesn1osine contcnt in the aged 1S rat skil1s
5 11. 19りO、
docs no亡 甘1ways lleHect the l−educlion or e1齪stin
contcnt,since the possibHity still exists thHt thc
[3]丁三■kHh州hi M.Nug生isc S Nlibu,11i M=CliIlico−bioc11ビnli一
content or cl・oss−links alone l・educed without H
川1二11.ii虻11.Niドhikll“’1lS Y・koyllm・H.Kob・}・ll・lliK.
ch∼1ngc in clastin content. 1sodesn1osinc contenユ
■一11ib∼■sl1i∼11P1壬□sn1H 壬11k三11i11に 1〕■10‘Ph工■ω㌔c −ALPl in lS
in thc agcd lS skin was signi月cantly decreased by
mπs∼lnd i■Po∬iblo ol’1gi11.」11n」Vc−Sci46:607 614.
37%conlpared to the young1S skin.The signHう一
skin ,vithout any diHbrcnces in isodesn1osine in
SD mt sk1ns suggests that isodesmosine contcnt
in lS mls.」p11」Vot Sci4り137り 381.19S6,
n1ay be11e1Hted to the cutHncous Hging o「iS1’at
skin.Our results∼lre iil good Hgreement with三i
previous I’epol’t thHt isodesnlosinビ contcnt in
Hged rHt三1011t鐵w齪s decl’cased by onc11Hlr com−
Dcl’n1Htol l02=425 431.1りり二1‘in jHpH11cso〕.
pared ∼vilh the n1ntu11e rat[4].H hHs been previ_
IS」1−hly州11i T.N三1gui Y=SopHI’日正i0110「1110三11p11HじhHil“or
0usly(ienlonstrated that the content or e1壬1stin圭1s
lylrc l ul1d1H col1日gc11ドb}’SDS−poly生1c1’y1三1I1litlc皇el doじ一
nleasured by hot一三一1kaH−inso1ub1e nlateriHls wus
I7]P1’oじkop D」、UdcI1「11iビnd S=A speci∩じ111cl110d ゴor t11じ
Bioc11cI1l l=二2S 239.1り60,
111oPhorc一ト..1Biochenl S61453 45工)1 197り.
エり]Sc}」呈■m呈1)’.lno[■c l−1.1w圭1m呈1M.Wut日I1∼lbc H.1r1じM.
decreHse(一、 but isodesn1osine contcnt was in_
Orin1o H−Yun1三■‘hi一;l S=1m,11’ovcd1’呈1pid生1nd’implo do−
cl’e三1sed,during raエskin Hging [18].Tl]c (lis−
cl.ep聰ncy betwecn their d…lta 三uld oul’s n1ay bc
「ol’11川11cc liし1工1idじ11ro111Htogruphy.」pn」Clin Clrじm 161
due to thcir nleasu1・en1ent oゴisodesmosine con_
3H 47. 1りS7、
エenユas isodesnlosine per insolublc n1ateri三11s,not
as isodcsmosine per dry wcigh士skin.Thc pre−
donlinant Io,v n1oleculHr㌔veight el≡一stin rrHgn]cnts
川〕1(「1ipm三■一1 SD, FHri− B. Buronc LM. PI’呈111 (1一へ.
1一’mn■blHu(’=Proce∬in!o「‘olublcd州ill incul1エI1’cd
11con呈1正;lI ml,I1looti1 11uIsclc cell‘、」 Biol C1101112ω=
1二7SO コ27S5. 19S5、
ヨn thc aged 亘S r齪t ski11s suggest th日t Hccelerated
[l11Fi’1ulzblHn c.Pl’llll c〈,Fll11i‘B.colllnnino NM.olrncl’
e1三1sti11 degrudation is involved in the cut圭1neous
CD−Mog阯}’zd P」.TI’oxlel’Rト=Rolじol’tropエ〕d壬NiIl
aging or iS l・at skin.
These results suggest that wl.inklings圭1nd rur−
[1二三]To“・’bin H, Sωohelin T. C01’do,1 」= 日cαI’o,rlro1’ctic
じlosely rclatcd lo a decl’casc in e一三1stin cOntcnt,
the・・ti・lltcddeg1111d・tio・oゴclasti… dll・light
dcc11cuse in coHugen content.Thus,the iS rHt w川
Null Aじ日d Sd USA 76=4350 4354,197り.
Chcm 249=32二≡5 3⊇31.1976.
Although theclinica1壬ippe聰r∼一nce orlS l’Ht skin
1収cn duI’i11! miltuI’呈11i011,Cell Mol Biol〕7:677−685.
llcl・c 壬1re not con1pHtible with those of sun_dan1_
agcd skin 口9].This suggests 言hat the mech圭一nisn1
19S l.
□5]Lous MD.〈l1日i11」一C.Cohcn−SolHl L.MHroteHux P1
TI1ビ ]’呈1ユo o‘’coIlH巨じ11illHturnlio11 in 1・H1三1nd I1u1羽un ski11.
Con■1ecl Ti“Rビ、9=253 26二1.1982.
κ、、∫・1人・1・1・1ん1川・’..■ノ1〃川“川’〃1・・π〃・^191ピ1lグ∫ピl1仙一2μ9州2一;2一一237 237
[161M日ys PK,Bis110p,lE,Luu1.o11t G.1=Age−1’dHtcdc11H11gosin
the pl’ol]o11ion ol’typo1三1nd lll col1日goI1,Mcch Agei11豊
[■s]B…111os F.LedviI1日M=Agech圭111gosorcllo−s−li11ks in r呈■t
ski■1el∼lsli11.A11ch Dcrm FoI’ドc11241=134_140.1971.
Uitto」,∼1日tsuoku LY.1〈or11bc■’g RL:1三1三1s−ic nbcIls in
い7」Mille■’1三」:Bioc11en1iculdlHr圭1じtc1’istiじs齪nd biologic壬11sig−
nihcHncじor t11e genelicnllv−distinct col1齪旦ens,Mo1Cell
B’oル』φ1・‘〃C““.w.一“〃〃(1〃〃∫ピ‘〃∫〃“〃o〃、サ.CV Mosby,St