Jouma/ o Gene al Mic obiology (1983), 129, 829-832. P in ed in G ea B i ain 829
Complex Ca bohyd a es in he Cys Wall o His iculus similis
By
P.
CA LV0 ,
1
A
. TO
R R
ES,
1
*
P.
N A V
AS
2
AN D
J.
PE
R
E Z-SI LV
A
Depa amen o de Mic obiología
1
and Depa amen o de Ci ología e His ología 2,
Facul ad de Biología , Uni e sidad de Se illa, Se illa, Spain
( Recei ed 8 Ju/y 1982; e ised 8 Sep embe 1982)
A cy ochemical and s uc u al s udy o he cys wall o His iculus similis has been ca ied ou .
Applica ion o cy ochemical me hods o complex ca bohyd a es indica ed ha he mesocys is
ich in sulpha ed glycosaminoglycans. The endocys and g anula !aye con ained neu al and
sulpha ed glycop o eins, espec i ely.
I NTRODU CTIO N
The ul as uc u e o he cys wall has been s udied in se e a! hypo ichous cilia es. In
Oxy icha allax (G imes, 1973),Pleu o icha sp. (Ma susaka, 1976), S ylonychia my ilus (Walke
e al., 1975), Gas os yla s einii (Walke e al., 1980) and Lau en iella acumina a (Gu ie ez e al.,
l 980), he cys wall is composed o ou laye s called, om ou side o inside : he ec ocys , meso-
cys , endocys and g anula laye s. In hese cilia es a eso p ion o cilia and kine osomes occu s
du ing encys men , and he ma u e cys is e med kine osome eso bing. Dioph ys scu um
(Walke & Maugel , 1980) has a h ee laye ed cys wall and he ma u e cys is non kine osome
eso bing.
In he p esen wo k a s uc u al and cy ochemical s udy o he cys wall in His iculus similis
was ca ied ou by means o ligh mic oscopy and ansmission elec on mic oscopy .
M ETHO DS
O ganism and cul u e condi ions. His ic11lus similis (a hypo ichous cilia e) was cul u ed a 20 ± 1 ºC in P ing-
sheim·s solu ion and ed on Chlo ogoni 1m sp. (Amme ma nn, 1974). The encys men was induced by placing he
cell in P ingsheim's solu ion dep i ed o nu ien s.
Elec on mic oscopy. The cys s we e ixed in 2% ( / ) glu a aldehyde in O·I M-phospha e bu e pH 7·0 and hen
pos ixed in 1% (w/ ) osmium e oxide in he same bu e and embedded in Epon. Thin sec ions we e con as
s ained wi h u anyl ace a e and lead ci a e. Thick sec ions we e s ained wi h oluidine blue (T ump e al., 1961).
Ligh mic oscopic cy ochemis y. Thick sec ions o cys s ixed in 2 % glu a aldehyde in O·I M-phospha e bu e
pH 7·0 and embedded in Epon we e subjec ed o he ollowing ea men s: he pe iodic acid-Schi es o neu al
mucosubs ance s (McManus, 1946): alcian blue 8GX pH 2·5 (Mow y, 1956); and 0·05%(w/ ) alcian blue pH 5·8 in
he p esence o g aded concen a ion s o Mgl+ o he iden i ica ion and di e en ia ion o he h ee nega i ely
cha ged acidic g oups o mucins (-COOH,
-P0
3
H
and
-S0
3
H) (Sco & Do ling, 1965).
Ul as nc 11 al cy 1ochemis y. Thin sec ionso cys s ixed in bu e ed 2% glu a aldehyde and embedded in Epon
wc e moun ed on gold g idsand s ained by he pc iodic acid- hioca bohyd azide-sil e p o eina c (PA-TCH-SP)
me hod o Thie y (1967). Oxidized con ol sec ions we e ea ed ei he wi h sil e p o eina e wi hou p e iou s
hioca bohyd azide ea men o wi h hioca bohyd azide alone. O he sec ions we e loa ed on 1% (w/ ) phos-
pho ungs ic acid in 1 M-HCI o 40 o 60 min acco ding o Fléchon (1970) and washed wi h l ·25 M-HCI.
Al e na i ely , cys s ixed in bu e ed 2% glu a aldehyde we e s ained wi h dialyzed i on (DI; We zel e al.,
1966} o high i on diamidc (H ID: Spice e al., 1978), dehyd a ed and embedded in Epon.
R ESU LTS
A ine s uc u al analysis o he cys wall o H . similis e ealed s iking simila i ies o o he
kine osome eso bing cys s. The cys wall consis ed o ou laye s. The ou e mos !aye ( he ec o-
Abb e ia ions :
DI, dialysed i on ; HID, high i on diamide; PA-TCH-SP me hod , pe iodic acid- hioca bo-
hyd azidc-sil e p o eina e me hod.
0022-1287/83/0001-0680 $02.00
©
1983 SGM
830
P. CA L VO ANO OTHE RS
En
Fig. l . Cys wall o H . similis. (a) Pho omic og aph o a hick sec ion s ained wi h oluidine blue. (b- )
Elec on mic og aphs o ul a hin sec ions: (b) es ing cys wall; (e) cys ea ed by he PA-TCH-SP
me hod; (d) cys wall s aincd wi h phospho ungs ic acid a low pH; (e) DI s aining;
( )
HID s aining.
Ec, ec ocys ; M, mesocys : En, endocys : G. g anula !aye . The ba ma ke s ep esen! 1 µm.
cys ) p esen ed an elec on dense )amella s uc u e o ming concen ic pla es, while he second
)aye ( he mesocys ) was o med by a s a um o in e wo en ib es. The endocys was an elec on
dense ib illa hin laye and he inne mos laye (g anula laye ) had an ex emely a iable
hickness and appea ed o be composed o a g anula ed and elec on dense ma e ial. (Fig. 1a,b).
Glycoconjuga es in he His icu/us cys wal/ 831
Bo h he endocys and g anula laye s had an in ense pe iodic acid-Schi eac ion which d id
no disappea a e diges ion wi h a-amylase. The mesocys exhibi ed alcianophilia a pH 2·5
ha was comple ely abolished a e me hyla ion a 60 ºC; his alcianophilia was e e sed com-
ple ely a e a me hyla ion -saponi ica ion-alcian blue (pH 2·5) sequence o ea men . The
s aining wi h alcian blue a pH 5·8 in he p esence o
O·
l M-MgCl
2
ga e in ense s aining o he
mesocys ; his in ense eac ion also occu ed in 0·5 M-MgCl 2 bu dec eased in 0·8 M- and 1 M-
MgCl2. The in ense alcianophilia o he mesocys could mask any eac ion o he endocys wi h
alcian blue; he e o e, he eac i i y o he endocys owa ds ca ionic eagen s was no de e -
mined by ligh mic oscopy. The ec ocys appea ed un eac i e in echniques used o
cha ac e izing glycoconjuga es.
In ag eemen wi h he abo e esul s, he PA-TCH-SP echnique e ealed he exis ence o
glycoconjuga es in bo h endocys and g anula laye s (Fig. 1e). T ea men wi h phospho ungs ic
acid a low pH p oduced simila esul s: p ecipi a es o phospho ungs ic acid appea ed in he
endocys and g anula !aye (Fig. 1 d). Finally, acidic mucosubs ances we e es ed by elec on
mic oscopy using he DI and HID p ocedu es. Bo h he mesocys and g anula laye s showed DI
and HID a ini y (Fig. 1 e, J).
DISCUSSIO N
The cha ac e is ics o complex ca bohyd a es de e mined a he elec on mic oscopic le e!
ag eed wi h hose ound by ligh mic oscopic cy ochemis y . Acco ding o Spice e al. (1979),
mucosubs ances which possess PA-TCH-SP eac i i y bu which lack DI and HID a ini y a e
neu al glycop o eins, while mucosubs ances which a e DI o HID posi i e bu which lack PA-
TCH-SP eac i i y can be ega ded as glycosaminoglycans. Subs ances which possess PA-
TCH-SP eac i i y and also DI and/o HID a ini y a e acidic glycop o eins. These can be
sepa a ed u he in o sulpha ed glycoconjuga es which ha e a ini y o bo h DI and HID, and
ca boxyla ed glycop o eins which ha e a ini y o DI bu no o HID.
Cy ochemical esul s ob ained om he cys wall o H .similis indica e ha he mesocys is ich
in sulpha ed glycosaminoglycans, he endocys con ains neu al glycop o eins and he g anula
!aye con ains sulpha ed glycop o eins. The ec ocys was un eac i e in he echniques used o
cha ac e izing glycoconjuga es. Howe e , he ec ocys p ecu so s a e PA-TCH-SP posi i e in
he hypo ichous cilia e Lau en iella acumina a (J. C. Gu ie ez & A. To es, unpublished da a).
The lack o PA-TCH-SP s aining in he ec ocys migh be due o a blockage o icinyl-glycol
g oups occu ing du ing he encys men .
Sulpha ed glycosaminoglycans in he mesocys o m a hyd ophilic egion ha would play an
impo an ole in he anspo o solu es and in he e en ion o wa e . Acidic glycop o eins o
he g anula !aye , in con ac wi h he cy oplasm o he cys , a e good candida es o media ing
cys -en i onmen in e ac ions.
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