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Ribonucleic acid content and distribution in normal human lens and in senile cataract
E.,'p. Erie l?,'s. (196'1) 3, ll.q-117
R i b o n u c l e i c A c i d C o n t e n t and D i s t r i b u t i o n in N o r m a l H u m a n L e n s and in S e n i l e C a t a r a c t (:IIt~VANNI .M:XRAINI. Cllolt(.~lO
l)ltyr'r!
AND ~[AR10
SAN'rOIII
Ol~hth,h,ologic, l Cli~ic of Parma 3Iedical ,qchool, Pa, rma, Da.ly (Received 16 ,&arch 1964) ..\ d i s o r d e r o f p r o t e i n m o t . a b o l i s , n is a c o m m o n {'eaturc o f t h e c a t a r a c t o u s lens. In v i e w o f t h e m a j o r vole p l a y e d b y r i b o n l l e l e i e a c i d in t h e s y n t h e s i s o f s p e c i f i c p r o t e i n s , its ( ' o n t e n b a n d d i s t r i b u t i o n h a v e b e e n s t u d i e d i n n o r m M h u m a n lens a n d in s e n i l e c a t a r a c t . In n o r m a l lens. t h e h i g h e s t ] ) r o p o v t i o , l o f t o t a l r i b o n u e l e i e a c i d h a s b e e n f o u n d in t h e .~olul)le f r a c t i o n a n d in t h e fr~u:l,ion a s s o c i a t e d wit, h t h e a l b u n l i n o i ( 1 . In s e n i l e c a t a r a c t , a (leert'a.se o f 17".,, o f tot.iI I / N A p e r g r a m p r o t e i n s w a s d e t e c t e d , l i n k e d t o a. 5 0 % loss o f solnl)h, t / N A . T h e p o s s i b i l i t y is c o n s i d e r e d t h a i t h e d e c r e a s e in p r o t e i n s y n t h e s i s o f t h e c a t a r a e t o u . ~ lens m a y be., at, least in p a r t , l i n k e d to t h e d e c r e a s e o f s o l u b l e l>, N A .
1. I n t r o d u c t i o n
T h e e x i s t e n c e of a d i s o r d e r of p r o t e i n m e t a b o l i s m of t h e lens is a c o m m o n f e a t u r e o f e x p e r i m e n t . h i c a t a r a c t a n d is well k n o w n f r o m t h e s t u d i e s of D i s c h e , B o r c n f r e u n d a n d Z e l m e n i s (1956) a n d D i s c h e , Z c l m e n i s a n d Y o u l u s (1957) a n d P i r i c a n d v a n H e y n i n g e n (195(;); t,he i m p o r t a n c e of q b e t t e r k n o w l e d g e of r i b o n u c l e i e a c i d (I~,NA) c o n t e n t a n d dist.ribution in n o c m a l a n d c a t a r a c t o u s lens is t h e r e f o r e e v i d e n t , d u e to t h e c h i e f role t h a t R N A has b e e n s h o w n to p l a y in t h e s y n t h e s i s of specific p r o t e i n s . I~NA c o n t e n t ha s b e e n s t u d i e d in n o r m a l r a t lens ( D i s c h e , D e v i a n d Z e l m e n i s , 1961; L o r m a n a n d I s h i d a . 1960), in r a b b i t lens ( M e n d e l a n d Q u a r a n t a , 1957; v a n H e y n i n g e n a n d \ V a l e y , 1961) a n d in b o v i n e lens ( M a n d e l , N o r d m a n n a n d Z i m m e r , 19.19; v a n H c y n i n g e n a n d \ V a l e y , 1961 ), b u t d a t a on h u m a n lens are, on t h e c o n t r a r y , v e r y s c a n t y , a n d t h e rally a v a i l a b l e r e p o r t is, t.o o u r k n o w l e d g e , a p r e l i m i n a r y n o t e b y D e v i (1963). I t is t h e aim. of ~he p r e s e n t p a p e r to r e p o r t on t h e first result~s o b t a i n e d b y s t u d y i n g 1R.NA cent, a n t a n d d i s t r i b u t i o n in n o r m a l h u m a n lens a n d in m a t u r e senile c a t a r a c t . 2. M e t h o d s
Not'real lenses were obt-~ined w i t h i n 2 hr after d e a t h from i n d i v i d u a l s aged 55-75 years; senile c a t a r a c t s were p r o c u r e d after surgical e x t r a c t i o n . The lenses were t r e a t e d i m m e d i a t e l y or stored a t -- 25°0 until used. Lenses (2 or 3 for a single e x p e r i m e n t ) were dried with filter paper, weighed and h o m o g e n i z e d with a P o t t e r h o m o g e n i z e r in an eightfold a m o u n t of ice-cold distilled water. P r o t e i n c o n t e n t was e s t i m a t e d on t h e t o t a l h o m o g e n a t e a c c o r d i n g to L o w r y , l~osebrough, F a r r and R a n d a l l (1951). The h o m o g e n a t e was t h e n f r a c t i o n a t e d in a Spineo p r e p a r a t i v e u l t r a c e n t r i f u g e a n d was s e p a r a t e d into t h r e e fractions; the first fraction was o b t a i n e d b y c c n t r i f u g a t i o n a t 600 g for 20 rain. The s u p e r n a t a n t was c e n t r i f u g e d again a t 105,000 g for 90 rain a n d a small s e d i m e n t (fraction 11) and a s u p e r n a t a n t (fraction I I I ) were o b t a i n e d . I~NA e x t r a c t i o n was a t t a i n e d with 1 0 ~ I~C104 at t-°C a c c o r d i n g to Ogur a n d R o s e n (1950) ; after an i n c u b a t i o n t i m e of 24 h r a t 4°C, u l t r a v i o l e t a b s o r p t i o n b e t w e e n 24.5 a n d 280 m/, was '~
115
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GIOVANNI
MARAINI,
(.; I t ) R (; I t )
I)IOTT!
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recorded wilat, a 13eekman sl)ectrophoto,neter model 1)U. ()ptical d e n s i t y at 260 r,,/.t was comi)arc(] wit, h t h a t o f a s t a n d a r d solution of pure yeast~ ]~NA; al)sorl)tion curves of yeast R N A and of the different RNA fractions from n o r m a l lens are shown in Fig. 1. F o u r e x p e r i m e n t s were carried out on normal lenses and four on sonile c a t , r a c t .
I
I
I
I
I000
900
800 700
600 eQJ
-5
5OO
..
°--
o 4OO 300
200 RNA f r o c t . i o n
I00 0
240
t
i
250
260
II:
i.
270 ,t (m/t)
.
280
lhc.. 1. Lrltraviolct absorption curves of pure yeast H,NA and of the different I¢,NA fl'actions from normal human
lens.
3.
Results
and
Discussion
T a b l e I gives t h e c o n c e n t r a t i o n o f . R N A in t h e f r a c t i o n s of n o r m a l an d c a t a r a c t o u s h u n3 a n
len ses.
I n n o r m a l lens t h e h i g h e s t p r o p o r t i o n of R N A is f o u n d in f r a c t i o n II.[ ( w h i c h is t h e s u p e r n a t a n t o b t a i n e d a f t e r c e n t r i f u g a l ; i o n a t ]0 5 ,0 0 0 g a n d c o n t a i n s s o l u b l e ]%NA) a n d in f r a c t i o n I (]~NA l i n k e d to t.hc a l b u m i n o i d ) ; R N A of f r a c t i o n I I r e p r e s e n t s o n l y about; 20°/o of t o t a l lens R N A . O u r d a t a on t o t a l I { N A / g ( w / w ) in n o r m a l h , l m a n lens a r e in g o o d a g r e e m e n t w i t h t h e r e s u l t s o b t a i n e d in c a t t l e a n d a d u l t r a b b i t lens l)y v a n / - I e y n i n g c n a n d "Waley(1961), w h i l e r a t lens s h o w s a d e f i n i t e l y h i g l , e r ~ N A c o n t e n b ( 2 - 5 - 3 - 0 mg/g (w/w)) a c c o r d i n g to D i s c h e e t al. (1961) a n d to L c r m a n a n d [ s h i d a (1960). I t m a y be of s o m e i n t e r e s t to p o i n t o u t t h e high p r o p o r t i o n of s o l u b l e ]{NA (s-i¢NA) in n o r m a l h u m a n lens,
](Ii~)NUCI,EIC
ACID
1N N O R M A L
AND
(.'.ATAI¢-\CT
I,I':NS
117
similar to t h a t d e t e c t e d in calf lens by v a n H e y n i n g e n and ~Valey (1961); on the contrary, in ,'at lens the main 1)report, ion of ]Z~NAhas been f o u n d to be linked to the albuminoi(l fraction, only a smaller i)ercc~tage of t o t a l R N A being recovered in t h e s u p e r n a t a n t fraction (fraction I l l ) . T h e other interesting feature of the d a t a reported in Table I is the decrease of t o t a l ] { N A in senile cat,trac~ (31°//0 if referred to wet weight a n d 17°//o if referred to proteins); this decrease is e x c l u s i v e l y due to a loss o f s - R N A (nearly 500//o) if the data referred to proteins are considered. TABLE N o r m a l leas "l'()tal I¢NA I{NA i~,N:k HNA I(N:\ RNA "l'olal Total
i)r~tcins (mg,:g w¢.t wl) fra~.tion I (/Lg per I()1) mg I)l'~)tcit~) fracti(m I (iLg/g wet wt) frac:iol~ il (iLg per I(l(| m g l ) r o t c i n ) fraction 11 [l~g..'.~w(.I wt) fractiotl I l l (l~g per Ic}0 m g protein) fract.iol~ I l l (/Lg.!g w e t wt) R N : \ (l~g'lOI) mg p r o t e i n ) | ( N A (tegfg w e t w t )
5SI-7 ± 81.2 1:1.2 -t- 3-1 75.5 4- 9-2 6.8 ± 2.3 38-0 ± 8-:~ I-1-1 +_ 5.1 79-.5 4- 19-7 34.1 _ 10-5 193.0 ± 37-1
Senile c a t a r a c t 501.3 I-l-I G5-9 {i-0 27-9 7.[i 39.7 27"7 133.5
__+ 92.(} ± 2.-I _--_-_+2S.I~ ± 2"6 ± ll-I _ 3-5 4-_ l(i-0 ± 9"5 ± 55-6 .-)~
.
After the 1),'esent investigation had been t e r m i n a t e d , a p r e l i m i n a r y note by Devi (1963) al)pcarc(l and her rcsult, s arc in good a g r e e m e n t with our data" a 20-21~/o loss of tot, al ]{NA was detected in senile c a t a r a c t (and unDublished d a t a of the same , u t h e r show t h a t this loss is in the soluble fraction of the lens) which a t the same time showed a decrease of 8-50°/o of the incori)oration of [llC ]leucine into ]~NA and proteins. The possible m e a n i n g of the decrease of s-]~,NA in senile c a t a r a c t m a y be obvious if one considers the chief role played by R N A in protein synt, hcsis; a 500//0 loss of s - ] ~ N A m ight explain, at Ic~st in ])art, the decrease iu the incorporation of labelled a m i n o acids into R N A an(i proteins, observed by L e r m a n , Dcvi aud H:awes (1961) in exDerimental cat,tract and by ])cvi (1963) in senile cataract. As far as the decreased protcir~ c o n t e n t ofcatar~lctol~s lens is concerned, the possible iml)ortance of al~ a c t i v a t e d a u t o l y s i n g system has been stlggcsted b y Devi, b u t no d e m o n s t r a t i o n of this has been m a d e up to now. It, is possible, on the o t h e r hand, t h a t further studies on R N A c o n t e n t and base composition in n o r m a l and c a t a r a c t o u s lenses will provide new d a t a for t~he i n t e r p r e t a t i o n of this problem. R.EFE
R ENCES
D e v i , A. (1963). E x p . Cell /Yes. 31, 205. l ) i s e h e , Z., B o r e n h ' e u n d , E . a n d Z c l m e n i s , G. (1956). A . l l / ' . f l . z4rch. Opht]~al. 55, 63"L I ) i s c h c , Z., .l)cvi, A. a n d Z c l m c n i s , G. ( 1 9 6 1 ) . A m e r . J . Ophthal. 5 1 , 9 9 3 . l ) i s c h c , Z., Z e l r n c l l i s , G. a n d 5"oulus, J . (1957). tl ~ner. J . Ophll~al. 44, 332. L c r m a n , S.. D c v i , A. a n d 1 4 a w e s , S. (1(.)61). zlmer. J . Ophlhol. 51, 1012. L c r m a n , S. a n d I s h i d a , ]3. K . (1960). A . M . A . . 4 r c h . Opl~thal. 63, 136. L o w r y , O. H . , l ~ o s e b r o u g h , N . J . , F a r r , A. L. a n d l l a n d a l l , I I . J . (1951). J . biol. Chem. 1 9 3 , 2 6 5 . M a n d e l , P . , N o r d m a n n , J . a n d Z i m m e r , J . (1949). C. R. Acad. Sci., P a r i s 2 2 8 , 516. M a n ( l c l , P . a n d Q u a r a n t a , C. A. (1957). C . R . Soc. Biol., P a r i s 1 5 1 , 6 0 4 . O g u r , 5I. a n d R o s e n , G. ( 1 9 5 0 ) . ~4rch. Biochem. 25, 262. Pil~ie, A. a n d v a n H e y n i n g e n , R . ( 1 9 5 6 ) . Bioche~nislry of the E y e . . B l a e k w c l l , O x f o r d . v a n ] ' T c y n i n g c n , R . ~ n d ~Valey, S. G . (1961). E x p . Eye I~es. 1, 155.
R i b o n u c l e i c A c i d C o n t e n t and D i s t r i b u t i o n in N o r m a l H u m a n L e n s and in S e n i l e C a t a r a c t (:IIt~VANNI .M:XRAINI. Cllolt(.~lO
l)ltyr'r!
AND ~[AR10
SAN'rOIII
Ol~hth,h,ologic, l Cli~ic of Parma 3Iedical ,qchool, Pa, rma, Da.ly (Received 16 ,&arch 1964) ..\ d i s o r d e r o f p r o t e i n m o t . a b o l i s , n is a c o m m o n {'eaturc o f t h e c a t a r a c t o u s lens. In v i e w o f t h e m a j o r vole p l a y e d b y r i b o n l l e l e i e a c i d in t h e s y n t h e s i s o f s p e c i f i c p r o t e i n s , its ( ' o n t e n b a n d d i s t r i b u t i o n h a v e b e e n s t u d i e d i n n o r m M h u m a n lens a n d in s e n i l e c a t a r a c t . In n o r m a l lens. t h e h i g h e s t ] ) r o p o v t i o , l o f t o t a l r i b o n u e l e i e a c i d h a s b e e n f o u n d in t h e .~olul)le f r a c t i o n a n d in t h e fr~u:l,ion a s s o c i a t e d wit, h t h e a l b u n l i n o i ( 1 . In s e n i l e c a t a r a c t , a (leert'a.se o f 17".,, o f tot.iI I / N A p e r g r a m p r o t e i n s w a s d e t e c t e d , l i n k e d t o a. 5 0 % loss o f solnl)h, t / N A . T h e p o s s i b i l i t y is c o n s i d e r e d t h a i t h e d e c r e a s e in p r o t e i n s y n t h e s i s o f t h e c a t a r a e t o u . ~ lens m a y be., at, least in p a r t , l i n k e d to t h e d e c r e a s e o f s o l u b l e l>, N A .
1. I n t r o d u c t i o n
T h e e x i s t e n c e of a d i s o r d e r of p r o t e i n m e t a b o l i s m of t h e lens is a c o m m o n f e a t u r e o f e x p e r i m e n t . h i c a t a r a c t a n d is well k n o w n f r o m t h e s t u d i e s of D i s c h e , B o r c n f r e u n d a n d Z e l m e n i s (1956) a n d D i s c h e , Z c l m e n i s a n d Y o u l u s (1957) a n d P i r i c a n d v a n H e y n i n g e n (195(;); t,he i m p o r t a n c e of q b e t t e r k n o w l e d g e of r i b o n u c l e i e a c i d (I~,NA) c o n t e n t a n d dist.ribution in n o c m a l a n d c a t a r a c t o u s lens is t h e r e f o r e e v i d e n t , d u e to t h e c h i e f role t h a t R N A has b e e n s h o w n to p l a y in t h e s y n t h e s i s of specific p r o t e i n s . I~NA c o n t e n t ha s b e e n s t u d i e d in n o r m a l r a t lens ( D i s c h e , D e v i a n d Z e l m e n i s , 1961; L o r m a n a n d I s h i d a . 1960), in r a b b i t lens ( M e n d e l a n d Q u a r a n t a , 1957; v a n H e y n i n g e n a n d \ V a l e y , 1961) a n d in b o v i n e lens ( M a n d e l , N o r d m a n n a n d Z i m m e r , 19.19; v a n H c y n i n g e n a n d \ V a l e y , 1961 ), b u t d a t a on h u m a n lens are, on t h e c o n t r a r y , v e r y s c a n t y , a n d t h e rally a v a i l a b l e r e p o r t is, t.o o u r k n o w l e d g e , a p r e l i m i n a r y n o t e b y D e v i (1963). I t is t h e aim. of ~he p r e s e n t p a p e r to r e p o r t on t h e first result~s o b t a i n e d b y s t u d y i n g 1R.NA cent, a n t a n d d i s t r i b u t i o n in n o r m a l h u m a n lens a n d in m a t u r e senile c a t a r a c t . 2. M e t h o d s
Not'real lenses were obt-~ined w i t h i n 2 hr after d e a t h from i n d i v i d u a l s aged 55-75 years; senile c a t a r a c t s were p r o c u r e d after surgical e x t r a c t i o n . The lenses were t r e a t e d i m m e d i a t e l y or stored a t -- 25°0 until used. Lenses (2 or 3 for a single e x p e r i m e n t ) were dried with filter paper, weighed and h o m o g e n i z e d with a P o t t e r h o m o g e n i z e r in an eightfold a m o u n t of ice-cold distilled water. P r o t e i n c o n t e n t was e s t i m a t e d on t h e t o t a l h o m o g e n a t e a c c o r d i n g to L o w r y , l~osebrough, F a r r and R a n d a l l (1951). The h o m o g e n a t e was t h e n f r a c t i o n a t e d in a Spineo p r e p a r a t i v e u l t r a c e n t r i f u g e a n d was s e p a r a t e d into t h r e e fractions; the first fraction was o b t a i n e d b y c c n t r i f u g a t i o n a t 600 g for 20 rain. The s u p e r n a t a n t was c e n t r i f u g e d again a t 105,000 g for 90 rain a n d a small s e d i m e n t (fraction 11) and a s u p e r n a t a n t (fraction I I I ) were o b t a i n e d . I~NA e x t r a c t i o n was a t t a i n e d with 1 0 ~ I~C104 at t-°C a c c o r d i n g to Ogur a n d R o s e n (1950) ; after an i n c u b a t i o n t i m e of 24 h r a t 4°C, u l t r a v i o l e t a b s o r p t i o n b e t w e e n 24.5 a n d 280 m/, was '~
115
llti
GIOVANNI
MARAINI,
(.; I t ) R (; I t )
I)IOTT!
AXI)
MARIO
HAN'I'()I{I
recorded wilat, a 13eekman sl)ectrophoto,neter model 1)U. ()ptical d e n s i t y at 260 r,,/.t was comi)arc(] wit, h t h a t o f a s t a n d a r d solution of pure yeast~ ]~NA; al)sorl)tion curves of yeast R N A and of the different RNA fractions from n o r m a l lens are shown in Fig. 1. F o u r e x p e r i m e n t s were carried out on normal lenses and four on sonile c a t , r a c t .
I
I
I
I
I000
900
800 700
600 eQJ
-5
5OO
..
°--
o 4OO 300
200 RNA f r o c t . i o n
I00 0
240
t
i
250
260
II:
i.
270 ,t (m/t)
.
280
lhc.. 1. Lrltraviolct absorption curves of pure yeast H,NA and of the different I¢,NA fl'actions from normal human
lens.
3.
Results
and
Discussion
T a b l e I gives t h e c o n c e n t r a t i o n o f . R N A in t h e f r a c t i o n s of n o r m a l an d c a t a r a c t o u s h u n3 a n
len ses.
I n n o r m a l lens t h e h i g h e s t p r o p o r t i o n of R N A is f o u n d in f r a c t i o n II.[ ( w h i c h is t h e s u p e r n a t a n t o b t a i n e d a f t e r c e n t r i f u g a l ; i o n a t ]0 5 ,0 0 0 g a n d c o n t a i n s s o l u b l e ]%NA) a n d in f r a c t i o n I (]~NA l i n k e d to t.hc a l b u m i n o i d ) ; R N A of f r a c t i o n I I r e p r e s e n t s o n l y about; 20°/o of t o t a l lens R N A . O u r d a t a on t o t a l I { N A / g ( w / w ) in n o r m a l h , l m a n lens a r e in g o o d a g r e e m e n t w i t h t h e r e s u l t s o b t a i n e d in c a t t l e a n d a d u l t r a b b i t lens l)y v a n / - I e y n i n g c n a n d "Waley(1961), w h i l e r a t lens s h o w s a d e f i n i t e l y h i g l , e r ~ N A c o n t e n b ( 2 - 5 - 3 - 0 mg/g (w/w)) a c c o r d i n g to D i s c h e e t al. (1961) a n d to L c r m a n a n d [ s h i d a (1960). I t m a y be of s o m e i n t e r e s t to p o i n t o u t t h e high p r o p o r t i o n of s o l u b l e ]{NA (s-i¢NA) in n o r m a l h u m a n lens,
](Ii~)NUCI,EIC
ACID
1N N O R M A L
AND
(.'.ATAI¢-\CT
I,I':NS
117
similar to t h a t d e t e c t e d in calf lens by v a n H e y n i n g e n and ~Valey (1961); on the contrary, in ,'at lens the main 1)report, ion of ]Z~NAhas been f o u n d to be linked to the albuminoi(l fraction, only a smaller i)ercc~tage of t o t a l R N A being recovered in t h e s u p e r n a t a n t fraction (fraction I l l ) . T h e other interesting feature of the d a t a reported in Table I is the decrease of t o t a l ] { N A in senile cat,trac~ (31°//0 if referred to wet weight a n d 17°//o if referred to proteins); this decrease is e x c l u s i v e l y due to a loss o f s - R N A (nearly 500//o) if the data referred to proteins are considered. TABLE N o r m a l leas "l'()tal I¢NA I{NA i~,N:k HNA I(N:\ RNA "l'olal Total
i)r~tcins (mg,:g w¢.t wl) fra~.tion I (/Lg per I()1) mg I)l'~)tcit~) fracti(m I (iLg/g wet wt) frac:iol~ il (iLg per I(l(| m g l ) r o t c i n ) fraction 11 [l~g..'.~w(.I wt) fractiotl I l l (l~g per Ic}0 m g protein) fract.iol~ I l l (/Lg.!g w e t wt) R N : \ (l~g'lOI) mg p r o t e i n ) | ( N A (tegfg w e t w t )
5SI-7 ± 81.2 1:1.2 -t- 3-1 75.5 4- 9-2 6.8 ± 2.3 38-0 ± 8-:~ I-1-1 +_ 5.1 79-.5 4- 19-7 34.1 _ 10-5 193.0 ± 37-1
Senile c a t a r a c t 501.3 I-l-I G5-9 {i-0 27-9 7.[i 39.7 27"7 133.5
__+ 92.(} ± 2.-I _--_-_+2S.I~ ± 2"6 ± ll-I _ 3-5 4-_ l(i-0 ± 9"5 ± 55-6 .-)~
.
After the 1),'esent investigation had been t e r m i n a t e d , a p r e l i m i n a r y note by Devi (1963) al)pcarc(l and her rcsult, s arc in good a g r e e m e n t with our data" a 20-21~/o loss of tot, al ]{NA was detected in senile c a t a r a c t (and unDublished d a t a of the same , u t h e r show t h a t this loss is in the soluble fraction of the lens) which a t the same time showed a decrease of 8-50°/o of the incori)oration of [llC ]leucine into ]~NA and proteins. The possible m e a n i n g of the decrease of s-]~,NA in senile c a t a r a c t m a y be obvious if one considers the chief role played by R N A in protein synt, hcsis; a 500//0 loss of s - ] ~ N A m ight explain, at Ic~st in ])art, the decrease iu the incorporation of labelled a m i n o acids into R N A an(i proteins, observed by L e r m a n , Dcvi aud H:awes (1961) in exDerimental cat,tract and by ])cvi (1963) in senile cataract. As far as the decreased protcir~ c o n t e n t ofcatar~lctol~s lens is concerned, the possible iml)ortance of al~ a c t i v a t e d a u t o l y s i n g system has been stlggcsted b y Devi, b u t no d e m o n s t r a t i o n of this has been m a d e up to now. It, is possible, on the o t h e r hand, t h a t further studies on R N A c o n t e n t and base composition in n o r m a l and c a t a r a c t o u s lenses will provide new d a t a for t~he i n t e r p r e t a t i o n of this problem. R.EFE
R ENCES
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