15. 12. 1975

Specialia

Gradient plating of Candida lipolytica on ethionine after 10 days incubation at 30~ A few types of colony variants can be seen. Arrow indicates a rough colony variant.

I n o r d e r to s t u d y t h e effect of e t h i o n i n e alone on t h e cells, a c t i v e l y g r o w i n g culture was t r e a t e d w i t h e t h i o n i n e as follows: 4 ml of m i n i m a l salts m e d i u m (K~HPO4 0.7% ; NH4C 1 0.5/0, o/ 9 MgSO4 7H20 0.02% ; NaCt 0.01% a n d glucose 1.0%) c o n t a i n i n g d i f f e r e n t levels of e t h i o n i n e r a n g i n g f r o m zero (control) to 16 m g / m l in b r o a d t e s t t u b e s was i n o c u l a t e d w i t h 0.5 ml cell s u s p e n s i o n f r o m e x p o n e n t i a l g r o w t h p h a s e c o n t a i n i n g 10 s cells/ml. The t u b e s were fixed a t an angle of 45 ~ a n d a g i t a t e d in a r e c i p r o c a t o r y s h a k e r a t 30 ~ for 4 days. I t h a s p r e v i o u s l y b e e n o b s e r v e d t h a t in t h e p r e s e n c e of e v e n low levels of e t h i o n i n e (i.e., 1 mg/ml) t h e c u l t u r e s h o w e d a g r o w t h lag of a b o u t 4 d a y s a n d t h e cells w h i c h grew a f t e r this period were e n t i r e l y r e s i s t a n t to i n h i b i t o r y levels (i.e., 10 mg/ml) of e t h i o n i n e . Hence, a f t e r 4 d a y s of i n c u b a t i o n , w h e n t h e g r o w t h was n o t y e t e v i d e n t in t h e a b o v e tubes, t h e cells were w a s h e d twice w i t h physiological saline a n d p l a t e d o u t on m a l t agar for viable c o u n t a n d o b s e r v a t i o n of m u t a n t s . A u x o t r o p h y was scored b y p l o t t i n g t h e colonies on m i n i m a l salts a n d m a l t agar media. I t could be seen

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f r o m d a t a in T a b l e I I t h a t cell i n a c t i v a t i o n was p r o p o r t i o n a l to e t h i o n i n e c o n c e n t r a t i o n . I t could be f u r t h e r seen t h a t b o t h p e t i t e colonies a n d a u x o t r o p h s were scored o n l y for t h e e t h i o n i n e t r e a t e d cells, t h e i r f r e q u e n cies in b o t h cases b e i n g p r o p o r t i o n a l t o e t h i o n i n e conc e n t r a t i o n . A u x o t r o p h s were, h o w e v e r , f o u n d o n l y in t h o s e cultures t r e a t e d w i t h ettlionine a t c o n c e n t r a t i o n h i g h e r t h a n 8 m g / m l . A l t h o u g h a n u m b e r of v a r i o u s o t h e r t y p e s of colony v a r i a n t s w e r e o b s e r v e d on t h e e t h i o n i n e p l a t e s (Figure) or in t h e e t h i o n i n e t r e a t e d cultures, for c o n v e n i e n c e of c o u n t i n g o n l y t h e p e t i t e colonies were considered. The a b o v e o b s e r v a t i o n s i n d i c a t e d t h a t e t h i o n i n e was m u t a g e n i c to Candida lipolytica, w h e n to be in c o n t a c t w i t h t h e cells for a fairly long period of 4 days. M o s t c o n v e n t i o n a l c h e m i c a l m u t a g e n s such as m u s t a r d s , alkane sulphonic esters etc. h a v e , h o w e v e r , b e e n k n o w n to be m u t a g e n i c a t n o r m a l dose levels b y v e r y m u c h s h o r t e r d u r a t i o n s of c o n t a c t w i t h t h e m i c r o b i a l cells. Several factors s u c h as p o s s i b l e c h e m i c a l c h a n g e in ethionine, p e r m e a b i l i t y , site of i n t e r a c t i o n w i t h t h e cell, cellular c o m p o n e n t s etc., will be of i n t e r e s t for u n d e r s t a n d ing t h e m e c h a n i s m of e t h i o n i n e m u t a g e n e s i s in t h i s system.

Summary. E t h i o n i n e , was f o u n d t o induce a u x o t r o p h i c a n d p e t i t e colony v a r i a n t s in Candida lipolytica a f t e r p r o l o n g e d c o n t a c t w i t h t h e cells. t~. JOSEPH 5 a n d B. SIKYTA

Department o~ Technical Microbiology, Czechoslovak Academy o/ Sciences, Institute o/ Microbiology, Budd]ovickd 270, Praha d-Krd (Czechoslovakia), 24 July 7974.

5 Present Address: Central Food Technological Research Institute, Mysore-570013, India.

Effects of M a g n e t i c Field on I n f l a m m a t i o n T r e a t m e n t of s o m e r h e u m a t i c a n d i n f l a m m a t o r y diseases b y m a g n e t i c fields is c o m m o n in J a p a n as a h o m e t h e r a p y . A series of e x p e r i m e n t s on t h e biological effects of m a g n e t i c fields h a v e b e e n done m a i n l y in t h e Soviet Union. T h e s e were s u m m a r i z e d in t w o r e c e n t m o n o g r a p h s 1,2. I t was p o i n t e d o u t t h a t m a g n e t i c fields h a d s o m e influence on i n f l a m m a t i o n . B u t t h e a n t i i n f l a m m a t o r y effects of m a g n e t i c fields h a s n o t b e e n assessed in t h e usual e x p e r i m e n t a l m o d e s w i d e l y used. W e

Table I. Effects of magnetic field on carrageenan edema in rats Time after earrageenan injection (h)

1 2 4 6

Increase ill paw volume (% • SE) 9 Control

hi magnetic field

37.0 i 2.8 42.5 :j- 3.6 66.4 • 4.1 65.1 • 3.0

24.5 • 34.6 • 48.9 i 43.8 •

2.6 b 2.4 2.1 b 2.7 b

.~Average values for 14 animals, bp < 0.01 in relation to control.

t e s t e d t h e effects of a m a g n e t i c field on c a r r a g e e n a n e d e m a a n d a d j u v a n t - i n d u c e d a r t h r i t i s in rats. C a r r a g e e n a n e d e m a t e s t : r a n d o m - b r e d m a l e r a t s of t h e W i s t a r s t r a i n w e i g h i n g a b o u t 160 g were used. T h e y were h o u s e d in 2 cages, a n d fed w i t h t h e s a m e food a n d r u n n i n g water, in a l a b o r a t o r y a t a t e m p e r a t u r e of 20 ~ T h e a n i m a l s in each cage w e r e d i v i d e d j u s t before t h e e x p e r i m e n t s into 2 groups, one for t h e m a g n e t i c t r e a t m e n t a n d t h e o t h e r for controls. A 0.1 m l of 1 ~o c a r r a g e e n a n (Viscarine 402) s u s p e n s i o n in saline was i n j e c t e d in t h e s u b p l a n t a r region of t h e left h i n d p a w . The v o l u m e of t h e left h i n d p a w was m e a s u r e d before a n d 1, 2, 4 a n d 6 h a f t e r t h e c a r r a g e e n a n i n j e c t i o n according to VAN ARMAN et al. 3. A d j u v a n t - i n d u c e d a r t h r i t i s : r a n d o m - b r e d male r a t s of t h e S p r a g u e - D a w l e y s t r a i n aged f r o m 8 t o 10 weeks a n d w e i g h i n g a b o u t 220 g were used. T h e m e t h o d of feeding a n d g r o u p i n g of t h e a n i m a l s were as d e s c r i b e d 1 ~. F. BARNOTHY, Biological EHects o/ Magnetic Fields (Plenum Press, New York, Loudon 1969), vol. 2. 2 A. S. PRESSMAN,Electromagnetic Fields and Life (Plenmn Press, New York, London 1970). 3 C. G. VAN ARMAN,J. Pharln. exp. Ther, 150, 328 (1965).

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Speeialia

EXPERIENTIA 31/12

Table II. Effects of magnetic field on adjuvant-induced arthritis in rats Hindpaw volume ii1 ml b (% increase ~= SE) o

Arthritis score of forepaw (score -4- SE) a

Body weight gain (O/o).

Days after adjuvant injection

Treatment days

Control

M.1~. ~

Control

M.F. ~

Control

M.F.

14 18 21

0 4 7

2.45 (0) 3.84 (60.0 • 8.2) 3.67 (52.6 :t_ 7.4)

2.56 (0) 3.72 (47.0 • 5.0) 3.38 (33.7 ~= 4.4)r

0 3.3 =E 0.39 3.4 4- 0.43

0 2.4 4- 0.24 2.8 ~ 0.48

0 2.0 4.1

0 4.5 f 8.8 ~

~Average values for 10 rats. bAverage values ior both paws. ~ relative to those of treatment day 0. a 0 None; 1 mild; 2 moderate; 3 severe; 4 very severe, eM.F. Magnetic field. ~p < 0.05 in relation to control.

in t h e c a r r a g e e n a n e d e m a t e s t . A dose of 0.1 m l of liquid p a r a f f i n (Merck, G e r m a n y ) c o n t a i n i n g 0.6 m g of h e a t killed Mycobacterium bu/yricum (Difco) was i n j e c t e d i n t r a d e r m a l l y i n t o t h e b a s a l p a r t of t h e tail. The v o l u m e of t h e h i n d p a w s was m e a s u r e d as d e s c r i b e d above. T h e a r b i t r a r y a r t h r i t i s score in t h e f o r e p a w s was also recorded. T r e a t m e n t b y a m a g n e t i c field: A n a p p a r a t u s t o p r o d u c e a 50 H z a l t e r n a t i n g m a g n e t i c field was p r o v i d e d b y K a w a s a k i E l e c t r i c I n d u s t r y Co., Ltd., Y u s h i m a 3, B u n k y o - k u , Tokyo. The i n t e n s i t y of t h e m a g n e t i c field was a b o u t 1,200 gauss in a w o o d e n cage w h e r e t h e a n i m a l s were placed. T h e c o n t r o l a n i m a l s were also p l a c e d in t h e box, b u t t h e c u r r e n t was n o t s w i t c h e d on. T h e t e m p e r a t u r e in b o t h t h e b o x e s was a d j u s t e d to a b o u t 20~ In the c a r r a g e e n a n e d e m a test, t h e r a t s were placed in t h e m a g n e t i c field for 3 h f r o m j u s t a f t e r the c a r r a g e e n a n injection. I n t h e e x p e r i m e n t of a d j u v a n t a r t h r i t i s , t h e r a t s were t r e a t e d in t h e m a g n e t i c field for 3 h e v e r y d a y f r o m 14 d a y s a f t e r t h e a d j u v a n t injection. As s h o w n in T a b l e I, t h e c a r r a g e e n a n e d e m a in t h e t r e a t e d g r o u p w a s s i g n i f i c a n t l y s u p p r e s s e d as c o m p a r e d w i t h t h a t in t h e c o n t r o l group. T h e b e h a v i o r of t h e t r e a t e d r a t s was like t h a t of c o n t r o l animals. 14 d a y s a f t e r t h e a d j u v a n t injection, t h e r a t s ' h i n d p a w s h a d swollen m o d e r a t e l y (about 2.5 ml). As s h o w n in T a b l e II, t h e swelling i n c r e a s e d f u r t h e r for t h e n e x t 4 d a y s a n d t h e n d e c r e a s e d g r a d u a l l y . The effects of t h e m a g n e t i c field on a d j u v a n t - i n d u c e d a r t h r i t i s are s u m m a r i z e d in T a b l e II. The a r t h r i t i s in f o r e p a w s was n o t seen a f t e r 14 days, b u t was i n t e n s e a f t e r 18 a n d 21 d a y s in t h e c o n t r o l animals. As s u m m a r i z e d in Table II, t h e a r t h r i t i s in t h e h i n d - a n d f o r e - p a w s in t h e r a t s e x p o s e d t o t h e m a g n e t i c field was w e a k e r t h a n t h a t in t h e controls. No c h a n g e s in b e h a v i o r were o b s e r v e d in e i t h e r group, a n d

t h e b o d y w e i g h t gain was g r e a t e r in t h e t r e a t e d g r o u p t h a n in t h e controls. I t s h o u l d be clarified w h e t h e r or n o t t h e effects of t h e m a g n e t i c field on a d j u v a n t a r t h r i t i s are o n l y due t o its a n t i - i n f l a m m a t o r y effects. As d e s c r i b e d above, t h e a l t e r n a t i n g m a g n e t i c field used in t h i s s t u d y h a s a n a n t i - i n f l a m m a t o r y effect in rats. LIKHACHEV 4 r e p o r t e d t h a t t h e a c t i o n of a c o n s t a n t m a g n e tic field o n c e n t r a l n e r v o u s s y s t e m led to a r a p i d n o r m a I ization of e r y t h r o c y t e s e d i m e n t a t i o n r a t e in e x p e r i m e n t s w i t h t u r p e n t i n e - i n d u c e d i n f l a m m a t i o n in r a b b i t s . DEGAN 5 u s e d m a g n e t i c fields in t h e t r e a t m e n t of t r a u m a t i c e d e m a in h u m a n s . The m e c h a n i s m s of t h e biological effects of m a g n e t i c field were discussed also b y LIKHACHEV a n d o t h e r i n v e s t i g a t o r s , b u t t h e m e c h a n i s m of a n t i - i n f l a m m a t o r y effects o b s e r v e d in t h i s s t u d y is as y e t u n k n o w n .

Summary. The effects of a 50 H z m a g n e t i c field on e x p e r i m e n t a l l y - i n d u c e d i n f l a m m a t i o n in r a t s were studied. C a r r a g e e n a n e d e m a was i n h i b i t e d s i g n i f i c a n t l y b y e x p o s u r e to m a g n e t i c field for 3 h. A d j u v a n t - i n d u c e d a r t h r i t i s in r a t s was also s u p p r e s s e d b y t h e m a g n e t i c field. Y. MIZUSHIMA, I. AKAOKA a n d 5xz. NISHIDA 6 Department o/Medicine and Physical Therapy, Faculty o/ Medicine, University o/ Tokyo, Bunkyo-ku, Tokyo (Japan 713), 30 June 1975 4 A. I. LIKHACHEV,Biological EJ/ects o] Magnetic Fields (Plenum Press, New York, London 1969), vol. 2, p. 137. I. L. DEGEN, Orotop, Travm. Protez. 71, 47 (1970). The authors wish to thank the Kawasaki Electric Industry Co., Ltd., for providing tile magnetic field apparatus.

E n e r g y Linked Ion A c c u m u l a t i o n in M i t o c h o n d r i a f r o m Alloxan Diabetic Rats W e r e p o r t e d earlier 1 t h a t t h e m a x i m a l r e s p i r a t i o n r a t e , s t i m u l a t e d b y A D P or Ca +s, is lower in m i t o c h o n d r i a f r o m d i a b e t i c a n i m a l s (diabetic m i t o c h o n d r i a ) t h a n in n o r m a l m i t o c h o n d r i a , b u t t h e t o t a l a m o u n t of A T P p r o d u c e d or Ca +~ a c c u m u l a t e d will e v e n t u a l l y equal t h a t for n o r m a l m i t o c h o n d r i a . T h e s e r e s u l t s a p p e a r e d to be a n ion r e l a t e d p h e n o m e n o n . I t is k n o w n t h a t v a r i a t i o n s ill cellular K + a n d Ca +2 c o n t e n t s do affect m a n y cellular processes ~-4. The p u r p o s e of t h i s s t u d y was to c o m p a r e ion a c c u m u l a t i o n in n o r m a l a n d d i a b e t i c m i t o c h o n d r i a for t h e first time. M i t o c h o n d r i a were isolated f r o m livers of n o r m a l a n d alloxan d i a b e t i c r a t s as d e s c r i b e d p r e v i o u s l y i. ionic con-

t e n t s were d e t e r m i n e d as d e s c r i b e d b y MALBICA a n d HALL 5. 1. I o n c o n t e n t s in a r e s t i n g s t a t e . A f t e r isolation in 0.25 M sucrose t h e n o r m a l a n d d i a b e t i c m i t o c h o n d r i a cont a i n e d similar a m o u n t s of N a +, 6.7 a n d 7.8 n m o l e s / m g prot e i n ; K+, 82.0 and 85.7 n m o l e s / m g p r o t e i n ; Mg4~, 63.0 a n d 51.3 n m o l e s / m g p r o t e i n ; a n d Ca+2, 10.0 a n d 8.6 n m o l e s / m g p r o t e i n , respectively. T h e s e are similar to l i t e r a t u r e values, u n d e r similar isolation c o n d i t i o n s , for n o r m a l m i t o c h o n d r i a 5, 6. 2. Ionic c o n t e n t s a f t e r 3.0 m M Ca +2 i n c u b a t i o n w i t h r e s p i r a t o r y s u b s t r a t e a n d A T P p r e s e n t . A f t e r a 20 m i n i n c u b a t i o n w i t h 3.0 m M Ca +2 t h e d i a b e t i c a n d n o r m a l