Phosphorylation of myosin regulatory light chain (R-LC) is associated with potentiated work and power during twitch afterloaded contractions in mouse extensor digitorum longus muscle [R. W. Grange, C. R. Cory, R. Vandenboom, and M. E. Houston. Am. J. Physiol. 269 (Cell Physiol. 38): C713-C724, 1995]. We now describe the association between R-LC phosphorylation and potentiated concentric work when the extensor digitorum longus muscle is rhythmically shortened and lengthened to simulate contractions in vivo. Work output (at 25 degrees C) was characterized at sine frequencies of 3, 5, 7, 10, and 15 Hz at excursions of 0.6, 1.2, and 1.6 mm (approximately 5, 9, and 13% optimal muscle length) at a low level of R-LC phosphorylation. Muscles stimulated during the sine function with a single twitch at specific times before or after the longest muscle length yielded maximal concentric work near the longest muscle length at a sine frequency of 7 Hz (e.g., excursion approximately 9% optimal muscle length = 1.6 J/kg). Power increased linearly between sine frequencies of 3 and 15 Hz at all excursions (maximum approximately 29 W). After a 5-Hz 20-s conditioning stimulus and coincident with a 3.7-fold increase in R-LC phosphate content (e.g., from 0.19 to 0.70 mol phosphate/mol R-LC), work at the three excursions and a sine frequency of 7 Hz was potentiated a mean of 25, 44, and 50% (P < 0.05), respectively. The potentiated work during rhythmic contractions is consistent with enhanced interaction between actin and myosin in the force-generating states. On the basis of observations in skinned skeletal muscle fibers (H. L. Sweeney and J. T. Stull. Proc. Natl. Acad. Sci. USA 87:414-418, 1990), this enhancement could result from increased phosphate incorporation by the myosin R-LC. Under the assumption that the predominant effect of the conditioning stimulus was to increase R-LC phosphate content, our data suggest that a similar mechanism may be evident in intact muscle.

Phosphorylation of myosin regulatory light chain (R-LC) increases the Ca2+ sensitivity of cross-bridge transitions, which determine rate of force development in skinned skeletal muscle fibers. The purpose of this study was to determine whether phosphorylation of R-LC is the molecular basis for the increased force development rates (+dF/dtmax) observed in fatigued mouse extensor digitorum longus muscle (EDL) (stimulated in vitro at 25 degrees C). Parameters of twitch and tetanic force were obtained after the application of different-frequency conditioning stimuli (CS), which were used to vary R-LC phosphorylation and reduce peak tetanic force (Po). Without CS, R-LC phosphorylation (in moles phosphate per mole R-LC) was not elevated above rest (0.11 +/- 0.02 vs. 0.13 +/- 0.02, respectively), and no aspect of the twitch (Pt) Po was altered. Stimulating muscles at 2.5-20 Hz increased R-LC phosphorylation in a frequency-dependent manner, from 0.23 +/- 0.04 to 0.82 +/- 0.03, respectively. Moreover, stimulation at 2.5-20 Hz potentiated Pt (range: 4 +/- 2-28 +/- 2%), increased the +dF/dtmax of potentiated twitches (range: 5 +/- 1-28 +/- 2%), and reduced Po (range: 6 +/- 1-21 +/- 1%). Higher-frequency stimulation (40 or 100 Hz) did not phosphorylate R-LC or potentiate Pt or twitch +dF/dtmax further. Stimulation at 40 and 100 Hz did, however, have different effects on Po compared with 20-Hz data (Po reduced 27 +/- 2 and 11 +/- 2%, respectively). The increased +dF/dtmax of potentiated twitches observed after different CS procedures were graded to R-LC phosphorylation (r = 0.97, P < 0.001). It is concluded that phosphorylation of R-LC increases extent of twitch force development in mouse EDL muscle fatigued by CS.

The intent of this study was to determine if the stimulation-induced increase or "potentiation" of dynamic function of mouse extensor digitorum longus muscle (in vitro 25°C) during work cycles is graded to myosin regulatory light-chain (RLC) phosphorylation. To do this, concentric force and muscle work output during sinusoidal length changes were determined before (unpotentiated) and after (potentiated) the application of conditioning stimuli (CS) producing incremental elevations in RLC phosphorylation from rest. Sine wave excursion was from 1.09 to 0.91 of L (o) with a period of 142 ms; stimulating muscles to twitch and generate force during these cycles produced plots of force × displacement termed work loops. Stimulation at 2.5-, 5.0-, and 100-Hz elevated RLC phosphorylation from 0.16±0.02 (rest) to 0.29±0.03, 0.45±0.02 and 0.56±0.02 mol phos per mole RLC, respectively (n= 6-7, P<0.05). These CS potentiated mean concentric force (at all lengths) to 1.14±0.02, 1.26±0.04 and 1.41±0.06 of pre-stimulus, control levels (all n= 5-7, P<0.05) while work was increased to 1.07±0.02, 1.17±0.02 and 1.34±0.03 of controls, respectively. In a No CS condition that did not elevate RLC phosphorylation, neither mean concentric force nor work was altered. Thus, strong correlations between RLC phosphorylation and mean concentric force and work support the hypothesis that this molecular mechanism modulates muscle power output. No length-dependence for concentric force potentiation was observed in any condition, an outcome suggesting that interactions between instantaneous variations in muscle length and shortening velocity during work cycles modulates the potentiation response.

MyoD is a myogenic transcription factor responsible for skeletal muscle differentiation during development. Muscle antioxidant enzyme status was determined in transgenic MyoD deactivated mice. While catalase activity was significantly (P<0.05) elevated in soleus and extensor digitorum longus muscles from MyoD deactivated mice, superoxide dismutase and glutathione peroxidase activities were not. While this may imply a greater propensity for inherent oxidative stress, soleus glutathione status was similar between MyoD deactivated mouse and control soleus muscles. Catalase activity is localized primarily in peroxisomes. Therefore elevated catalase activity may also indicate the presence of factors associated with peroxisome proliferation in muscles from MyoD gene‐inactivated mice.

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 NO2 in 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 0.46 f 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 (1 00 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 iNOS-mRNA 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 variantse. g. reduced iNOS-activity and/ or upregulation of scavenger moleculesstill need further clarification. I n v i t r o s t u d i e s c a r r i e d out 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 at o c o p h e r o l b i n d i n g t o p r o t e i n i s c l o s e l y r e l a t e d t o i t s a n t i o x i d a n t e f f e c t . 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 thereby reduced a n t i o x i d a n t e f f e c t and increased l i p i d p e r o x i d a t i o n . I r r a d i a t i o n causes damage t o proteins, thus unable t o recognize and b i n d t o a-tocopherol 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 increased 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, mitochondria 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 @tocopherol binding. Therefore, a small amount o f added crtocopherol C10 pml could completely p r o t e c t mitochondria from p e r o x i d a t i o...