EDL MUSCLES IN MyoD DEFICIENT MICE. P.M. T i i h , E. Bombardier, J. Xeni, N. Bestic, R. Vandenboom,. M. Rudnicki and M.E. Houston. Wilfrid Laurier ...
Biochemical SocietyTransactions(1 996) 24 525s C9 INDUCTION OF INDUCIBLE NO-SYNTHASE IS AN ESSENTIAL PART OF TNFa-INDUCED APOPTOSIS IN MCF-7 CELLS. M. Schulz‘, B. FUR’, W. Hiddemann’, M. Oellerich2 Deps. of Hematol./Oncol.‘ and Clin. Chemist$, GeorgAugust-Universky, D37075 Gottingen, Germany. The TNFa-induced cytotoxic signal is mediated by the intracellular ‘death domain’ of the 55kD-TNF-receptor. In various cell types the death signal has been demonstrated to be coupled with induction of the inducible NO-Synthase (iNOS). However, it is still widely unknown, whether iNOSinduction is essential for TNF-cytotoxicity, especially in cells derived from sold t u r n . NO-producbjon and iNOS expression in relation to TNFa-induced cytotoxicity were therefore investigated in the human breast cancer cell line MCF-7 and a TNFa-resistent variant. Nogeneration was evaluated by photometric detection of the stable end product NO2in the supernatant Differential RT-PCR was used for semiquantitative measurement of iNOS-mRNA. Incubation with TNFa (max 3600 IUlml) for 24 hrs led to induction of iNOS-mRNA after 2-4 hrs as well as generation of N O (min 0.46f 0.18, max 3.23 f 0.24 nmoU1O’ cells124hrs) and was followed by cell death (max 34%) in a concentrationdependent way. Dying celb showed the characteristic features of apoptosis and wem quantitated by flow cytometry with propidium iodide. NO-produdion and cell death were further enhanced (5.39 f 0.57 nmoU10’ calls124hn. 65%) by addition of cycloheximide (CX, 10 pg/ ml), while CX alone had no such effect. Addition of the competitive iNOSinhibitor L-NAME (100 pM) greatly diminished Nogeneration and apoptosis. In MCF-7 variants iNOS-mRNA was still induced by TNFa, however. NO-production was much lowar than in sensitive cells. TNFa-sensitivity and NO-production were restored by CX. No difference was seen in basal expression of bcl-2-mRNA in sensitive and resistent cell lines as measured by differential RT-PCR. Taken together, iNOS-induction plays an essential role in TNFa-triggered apoptotic cell death of MCF-7cells. Prolongation of iNOSmRNA half l i by CX may partly account for its additive effect on NOproduction and cytotoxicity. The mechanisms leading to decreased NO-generation in resistent variants - e. g. reduced iNOS-activity and/ or upregulationof scavenger molecules - still need further clarification.
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C11 ANTIOXIDANT ENZYME STATUS OF SOLEUS AND EDL MUSCLES IN MyoD DEFICIENT MICE. P.M. T i i h , E. Bombardier, J. Xeni, N. Bestic, R. Vandenboom, M. Rudnicki and M.E. Houston. Wilfrid Laurier, Waterloo and McMaster Universities, Waterloo CANADA N2L 3C5. MyoD is a myogenic transcription factor which can activate muscle specific genes and induce skeletal muscle differentiation during fetal development. Previous studies have reported contractile and biochemical differences between genetically altered, MyoD deficient (MyoD-’.) and normal (MyoD”’) mouse muscle. This study determined superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) enzyme activities in homogenates of soleus (SOL) and extensor digitorum longus (EDL) muscles from MyoD-” and MyoD+’+mice. Animals were sacrificed between ages 12-24 wk, and muscle samples immediately frozen in liquid nitrogen. Between 10-15 SOL and EDL muscle samples were used for each group. Compared to MyoD”’, CAT activity 6at.f x lO”.mgprotein”) were significantly (p < 0.05) elevated in both SOL (13.7k0.5 vs. 11.8k0.6) and EDL (3.2k0.2 vs. 2.4k0.2) muscles from MyoD” mice. SOD and GPX activities were not significantly different (p > 0.05) between SOL or EDL muscles from MyoD“’ and MyoD” mice. Increased antioxidant enzyme activities are generally indicative of elevated oxidative stress, suggesting that muscles from MyoD” mice may be more prone to oxidative stress than MyoD”’ mouse muscles. The physiological reasons for increased oxidative stress in MyoD” muscles are unclear. However, elevations in CAT activity in MyoD-’. mouse muscles, without changes to SOD or GPX suggest that alternative factors may be responsible for the observed differences. Most muscle CAT enzymes are located in peroxisomes. Therefore any differentiation between CAT activities in MyoD-’. and MyoD”’ muscles could also be accounted for by factors which induce differential peroxisome development during or after muscle cell differentiation. Supported by grants from NSERC Canada & Wilfrid Laurier University
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PROTEIN BINDING A PREREQUISITE FOR THE A N T I OXIDATION EFFECT OF a-TOCOPHEROL [ V I T A M I N E l I N CELLULAR MEMBRANES C.K. PUSHPENDRAN Radiation Biology & Biochemistry D i v i s i o n Bhabha Atomic Research Centre Trombay, Bombay 400 085, I N D I A
I n v i t r o s t u d i e s c a r r i e d o u t i n r a t l i v e r homogenate and c e l l u l a r membranes c l e a r l y i n d i c a t e t h a t atocopherol binding t o p r o t e i n i s closely related t o i t s antioxidant effect. Ascorbate/FeSo4/Heparin b l o c k s o r p r e v e n t s p r o t e i n b i n d i n g t o a-tocopherol and t h e r e b y reduced a n t i o x i d a n t e f f e c t and i n c r e a s e d l i p i d peroxidation. I r r a d i a t i o n causes damage t o proteins, t h u s unable t o r e c o g n i z e and b i n d t o a - t o c o p h e r o l r e s u l t i n g i n reduced a n t i o x i d a n t e f f e c t and i n c r e a s e d l i p i d p e r o x i d a t i o n . Among t h e c e l l u l a r membranes studied, m i t o c h o n d r i a possess s i g n i f i c a n t l y l a r g e amount o f p r o t e i n s f o r @ t o c o p h e r o l b i n d i n g . Therefore, a s m a l l amount o f added c r t o c o p h e r o l C10 pml could completely protect mitochondria from peroxidation and also protect microsomes from peroxidation. Thus m i t o c h o n d r i a c o u l d be t h e source o f p r o t e i n t h a t recognizes and b i n d s a-tocopherol. The d a t a p r o v i d e several i m p o r t a n t conclusions. F i r s t p r o t e i n binding i s a prerequisite f o r the antioxidant effect of a-tocopherol, thus just feeding/adding w i l l not increase i t s ant iox idant effect. Secondly t h e r e s u l t p r o v i d e a methodology t o e v a l u a t e a block/damage o f p r o t e i n t h a t recognizes and b i n d s t o a-tocopherol, thus leading t o the antioxidant effect. F i n a l l y the r e s u l t s provide an o p p o r t u n i t y t o examine whether t h e r a d i a t i o n e f f e c t on cancer p a t i e n t s causes damage t o s p e c i f i c p r o t e i n s t h a t b i n d t o crtocopherol, thus a f f e c t i n g t h e i r antioxidant a b i l i t y .
C12 THE USE OF MILD SALT STRESS AND NIACINAMIDE TO MEASURE THE FITNESS OF PLANT POPULTATIONS AND TISSUE CULTURES Marc De Block Plant Genetic Systems, Jozef Plateaustraat 22,9000 Gent, Belgium An assay which allows the identification of plant lines or tissue cultures with an altered metabolism versus the control lines, will be described. The principles of the fitness assay are: Explants (calli, leaf disks, ...) are put in media containing niacinamide and ascending salt concentrations. Niacinamide functions as an inhibitor of poly(ADP-ribose)polymerase,and his addition changes the reactions of the tissues to the salt stress. After incubation of the explants in the salt media for about 20 hours, the total capacity to reduce 2,3,5triphenyltetrazoliumchloride(TTC) is determined. The TTCreducing capacity is a measure for the intensity of the mitochondrial electron transport (cytochromaland alternative respiration pathways). Small sample sizes were found to represent the whole population: f.ex. 35 leaf disks (of lcm diameter) derived from 8 Brussica nupus plants, 25 three-week old rootless shoots of Arubidopsis thuliunu, 600mg fresh weight of wheat callus. Plant lines with an altered TTC-reducing capacity under the different conditions versus the control lines, contain mutations or perform less in field trials.
