gPLL, LINEg

2 .2 .2 .1 NORMAL CSF LINES

The s i x norm al CSF l i n e s used in t h i s stu d y w ere o b ta in e d as grow ing m o nolayers fro m . D r. J.A , N itk o w sk i, J e r r y Lewis M uscle R esearch C e n tre , D epartm ent o f P a e d ia t r i c s and N eo n atal M ed icin e, Hammersmith H o s p ita l London, and D r. G. P r i e s t l y , D epartm ent o f D erm atology, U n iv e rs ity o f E d in b u rg h . T hese l i n e s w ere d e riv e d from norm al w ith no fa m ily h i s t o r y o f g e n e tic d i s o r d e r s . D e ta ils a r e l i s t e d in T able ( 3 ) .

2 .2 .2 .2 DMD CSF LINES

T hree c e l l l i n e s from fo re a rm p in ch s k in b io p s ie s w ere d e riv e d from th r e e in d iv id u a l s u b je c ts confirm ed by c l i n i c a l and b io c h e m ic a l c r i t e r i a to be DMD s u f f e r e r s . They w ere a ls o o b ta in e d as m onolayer from D r. J.A . N itk o w sk i, J e r r y Lewis M uscle R esearch C e n tre , D epartm ent o f P a e d ia t r i c s and N e o n a tal M ed icin e, Hammersmith H o s p ita l London. D e ta ils a r e l i s t e d i n

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2 . 2 .3 CELL CULTURE

Each CSF l i n e (no rm al and DMD l i n e s ) u sed f o r stu d y was c u l t i v a t e d u n d er c o n d itio n s o f c u lt u r e as d e s c r ib e d i n 2 .2 .1 .

S to c k c u ltu r e s o f CSFs w ere m ain ta in e d as m onolayers in 25

2 2

cm o r 75 cm p l a s t i c t i s s u e c u lt u r e f la s k s (T25 o r T75 f l a s k s , C o rn in g , New York) a t 37°C i n a STATUS In c u b a to r (N o rth e rn M edia N orth C ave, N orth H um berside, E n g la n d ). F re sh medium was added e v e ry 3 days and s u b c u ltu r in g c a r r ie d o u t e v e ry 5-6 d a y s.

■1

59 -

P m iL S . OF CULTURED SKIN FIBROBLAST CELL LINES

a b n o r m a litie s .

** A ll c e l l l i n e s w ere d e riv e d from a fo re a rm b io p sy s i t e e x c e p t f o r HSF 6 w hich was d e riv e d from f o r e s k in .

.QËLL WNE§ AGE ( v r s ) SOURCE

i

HAM 1 M 5 .2 month HAMMERSMITH

HAM 4 M _{7 .9} HAMMERSMITH

HAM 5 M 7 .0 HAMMERSMITH

CONTROL CULTURES* ■

•Î

I2ËLL..LIIÎE5 jSSL A.Œ Iy r§ ) SODRCB

HAM 2 M 32 HAMMERSMITH Ï

HSF 26 M 23 EDINBURGH

HSF 6 M 1 EDINBURGH

HSF 9 M 25 EDINBURGH

HSF 22 M 19 EDINBURGH

-i

- 60

2 .2 .4 IN VIVO LIPID PEROXIDATION STUDIES

I n v ivo l i p i d p e r o x id a tio n was s tu d ie d u sin g an a d a p ta tio n o f th e method o f Gavino eJt. n i. (1 9 8 1 ).

2 .2 .4 . 1 IN IIIA L . ÇELL-CÜLXURE

C o n tro l (HAM 2) and DMD (HAM 5) c e l l l i n e s , m atched f o r p a ssa g e number (PIG) w ere grown and s u b c u ltu re d s im u lta n e o u s ly to g iv e , f o r each l i n e , 15 T25 f l a s k s , each seed ed w ith an i d e n t i c a l num ber o f c e l l s . These c u ltu r e s w ere allo w ed to grow to c o n flu e n c y in norm al grow th medium b u t w ith FBS in s te a d o f NBS, These c o n flu e n t c u lt u r e s w ere th en used f o r in c u b a tio n in th r e e d i f f e r e n t m edia fo llo w e d by e s tim a tio n o f IB A -re a c tiv e m a t e r i a l s .

2 .2 .4 .2 EXPERIMENTAL INCUBATION CONDITIONS

2 ,2 .4 ,2 ,1 EXPERIMENTAL. MEDIA

A, Growth medium (a s in TABLE 1) b u t supp lem en ted w ith 20% Newborn B ovine Serum (NBS)

B, A rach id o n ic a c id supplem ented medium- 5 ,8 ,1 1 ,1 4 - e ic o s a te tr a e n o ic a c id (2 0 :4 ; a ra c h id o n ic a c id ) was d is s o lv e d in 95?

— 6 1 “

f i n a l 1:500 d i l u t i o n , w ith e x p e rim e n ta l medium, A. ( f i n a l c o n c e n tr a tio n o f 2 0 :4 = 12()uM).

C. As B b u t c o n ta in in g o n ly 95? e th a n o l.

D. TBH -supplem ented medium. 7 ÿ i l o f TBH was added

p e r 10ml A ( f i n a l c o n c e n tr a tio n = 30QuM),

2 .2 .4 .2 .2 CONDITIONS OF INCUBATION '

The medium was. removed from c u l t u r e s which had j u s t a t t a i n e d c o n flu e n cy as d e ta i le d in 2 .2 .4 .1 , th e c e l l s washed w ith

PBS and f r e s h medium, e i t h e r B, C o r D added ( t= 0 ) . The f la s k s w ere in c u b a te d a t 37°C f o r v a ry in g p e rio d s o f tim e , a t w hich

d u p l ic a te f la s k s w ere ta k e n and t r e a te d as below f o r e s tim a tio n ^ o f T B A -reactio n m a t e r i a ls .

2 .2 .4 .3 ASSAY OF TBA^REACTIVE MATERIALS

2 .2 .4 .3 .1 UNNASHED CSFs

A f te r a s p e c if ie d tim e i n t e r v a l , c e l l s w ere k i l l e d and d is r u p te d by th e a d d itio n o f 2.0m l o f 20? t r i c h l o r o a c e t i c a c id to th e medium in th e f l a s k . Four ml o f 0 .6 7 ? t h i o b a r b i t u r i c a c id was ad d ed , and th e medium and c e l l s w ere in c u b a te d f o r 20 m in u te s a t 97^C. A f te r c o o lin g th e medium + c e l l d e b r is w ere c e n tr if u g e d a t 12,000xg a t 4°C f o r 10 m in u te s, to p r e c i p i t a t e p r o te in s and c e l l u l a r d e b r i s . The o p t i c a l d e n s ity o f th e s u p e r n a ta n t was m easured a t 532nm (G avino e t a l . .1 9 8 1 ). A b la n k c o n ta in in g a l l

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r e a g e n ts e x c e p t c e l l s was ru n in p a r a l l e l . MDA (som etim es r e f e r r e d to T B A -reactiv e m a te r ia ls ) w ere c a lc u la te d from _m f o r MDA=1.56x10^ u sin g A bsorbance was c o n v e rte d to nmol MDA p e r c u l t u r e f l a s k .

2 .2 .4 .3 .2 WASHED. CSFs

In a n o th e r s e t o f e x p e rim e n ts, washed CSFs w ere u se d . The e x p e rim e n ta l medium was d e c an te d and th e c e l l s washed tw ice w ith

PBS. And th e n th e c e l l s w ere k i l l e d and d is r u p te d by th e a d d itio n o f f iv e ml o f 8? t r i c h l o r o a c e t i c a c id to th e c e l l s in th e f l a s k s . F iv e ml o f 0 .5 4 ? t h i o b a r b i t u r i c a c id was added to th e f la s k and T B A -reactiv e m a te r ia ls assay ed as in 2 .2 .4 .3 * 1 •

2 . 2 .4 . 4 .PROTEIN..ESTIMATION

The p r o te in c o n te n t o f CSFs was d e te rm in e d a c c o rd in g to th e m ethod o f Lowry (1 9 5 1 ). ( i ) REAGENTS ( a ) 1? c u p ric s u lp h a te ( o r c u p ric s u lp h a te 5 - w a te r ). (b ) 2? p o tassiu m sodium t a r t r a t e (w /v ). (c ) 2? sodium c a rb o n a te in 0.1N sodium h y d ro x id e . (d ) IN F o lin - C io c a lte a u ^ s r e a g e n t (S to c k 2N ). (e ) S to c k b o v in e album in s ta n d a rd s o lu t i o n , 100mg/ml. ( f ) S o lu tio n A

1? c u p ric s u lp h a te and 2 |? sodium t a r t r a t e were mixed 'titl th e n made up to 100 ml w ith 2? sodium c a rb o n a te in 0.1N sodium

■ ^1^

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h y d ro x id e . S o lu tio n A i s found to be s t a b l e f o r o n ly a few h o u r s ,

(g ) S o lu tio n B

2N F o l in - C i o c a l t e a u 's R eagent (s to c k ) was d i lu t e d 1:1 w ith d i s t i l l e d w a te r. S o lu tio n B was p re p a re d f r e s h l y each d ay .

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( i l ) PROCEDURE

■STANDARD. CURVE

The s to c k album in s o lu tio n was d i lu t e d to g iv e a p r o te i n d i l u t i o n s e r i e s ra n g in g from 0-10 (Jug, The s to c k (lOOmg/ml) was d i lu t e d 1:100 w ith PBS. T o ta l volume o f album in p lu s PBS was 0 .2 m l.

MEIËQD

To 0.1 ml sam ple ( in 5.0m l t e s t - t u b e s ) was added 2.5m l s o lu t i o n A. T his was in c u b a te d f o r 10 m in u tes a t room te m p e ra tu re and 0.5m l o f s o lu t i o n B added. The tu b e s w ere m ixed and ab so rb ap ce a t 540nm was re a d a g a in s t a r e a g e n t b lan k a f t e r 30 m in u te s.

2 .2 .5 MEMBRANE STUDIES

2 .2 .5 .1 MEMBRANE PREPARATION

U ltr a s o n ic a tio n was chosen f o r c e l l d i s i n t e g r a t i o n a s i t was f a s t , e f f i c i e n t and co u ld be used w ith a s m a ll volume (a p p ro x im a te ly 1.0 ml s a m p le ), p ro d u cin g a " c o n c e n tra te d c e l l s u s p e n s io n " . In f a c t , th e r e a r e two o th e r m ethods w hich can be c o n s id e re d f o r r u p tu r in g th e CSF. These a re : r e p e a te d f r e e z in g and thaw ing and h o m o g en iz atio n . N e ith e r o f th e s e p ro c e s s w ere s u i t a b l e f o r t h i s p a r t i c u l a r work owing p a r t i c u l a r l y to th e s m a ll sam ple volum es used b u t a ls o th e lo n g e r tim e n e c e s s a ry to a c h ie v e co m p lete d i s i n t e g r a t i o n .

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2 . 2 . 5 . 1 . 1 MElfiQP

F ro zen sam ples ( s t o r e d a t -YO^C ) w ere d e f r o s te d and suspended in a minimum volume (1 ,0 m l) o f PBS in a g l a s s

I

s c i n t i l l a t i o n v i a l . To av o id l o c a l h e a t p ro d u c tio n by th e f? u l t r a s o n i c a t i o n p r o c e s s , which can in d u ce l i p i d p e r o x id a tio n as

w e ll a s d e n a tu re membrane p r o te i n , sam ples w ere alw ays k e p t on i c e . The u l t r a s o n i c a t o r probe was i n s e r te d j u s t below th e l iq u i d s u r f a c e . Haying ta k e n a d e q u a te n o is e p r o te c tio n p r e c a u tio n s , th e sam ple was s o n ic a te d a t an am p litu d e o f 8 -9 m icro n s peak f o r 1 m in u te, w ith a b re a k f o r 30 seco n d s to a llo w a d e q u a te c o o lin g . T his was re p e a te d to g iv e each sam ple a t o t a l s o n ic a tio n tim e o f