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Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl ␤-Dthiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

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1. Rumen microorganisms convert trans-aconitate to tricarballylate. The following experiments describe factors affecting the yield of tricarballylate, its absorption from the rumen into blood and its effect on mammalian citric acid cycle activity in vitro.2. When mixed rumen microorganisms were incubated in vitro with Timothy hay (Phleum praiense L.) and 6.7 mM-trans-aconitate, 64 % of the trans-aconitate was converted to tricarballylate. Chloroform and nirate treatments inhibited methane production and increased the yield of tricarballylate to 82 and 75% respectively. 3. Sheep given gelatin capsules filled with 20 g trans-aconitate absorbed tricarballylate and the plasma concentration ranged from 0.3 to 0.5 mM 9 h after administration. Feeding an additional 40 g potassium chloride had little effect on plasma tricarballylate concentrations. Between 9 and 36 h there was a nearly linear decline in plasma tricarballylate.4. Tricarballylate was a competitive inhibitor of the enzyme, aconitate hydratase (aconitase; EC 4.2.1 .3), and the inhibitor constant, K I , was 0.52 mM. This KI value was similar to the Michaelis-Menten constant (K,) of the enzyme for citrate.5. When liver slices from sheep were incubated with increasing concentrations of tricarballylate, [I4C]acetate oxidation decreased. However, even at relatively high concentrations (8 mM), oxidation was still greater than 80% of the maximum. Oxidation of [I4C]acetate by isolated rat liver cells was inhibited to a greater extent by tricarballylate. Concentrations as low as 0.5 mM caused a 30% inhibition of citric acid cycle activity.

Although the essentiality of dietary Se for sheep has been known for decades, the chemical source and Se dosage for optimal health remain unclear. In the United States, the Food and Drug Administration (FDA) regulates Se supplementation, regardless of the source of Se, at 0.3 mg of Se/kg of diet (as fed), which is equivalent to 0.7 mg of Se/d or 4.9 mg of Se/wk per sheep. The objectives of this study were to evaluate the effects of Se source (inorganic vs. organic) and supplementation rate (FDA vs. supranutritional rates of 14.7 and 24.5 mg of Se/wk) on whole-blood (WB) and serum-Se concentrations. Mature ewes (n = 240) were randomly assigned to 8 treatment groups (n = 30 each) based on Se supplementation rate (4.9, 14.7, and 24.5 mg of Se•wk(-1)•sheep(-1)) and source [Na-selenite, Na-selenate (4.9 mg/wk only), and organic Se-yeast] with a no-Se control group (0 mg of Se/wk). Treatment groups were balanced for healthy and footrot-affected ewes. For 1 yr, ewes were individually dosed once weekly with 0, 4.9, 14.7, or 24.5 mg of Se, quantities equivalent to their summed daily supplementation rates. Serum- and WB-Se concentrations were measured every 3 mo in all ewes; additionally, WB-Se concentrations were measured once monthly in one-half of the ewes receiving 0 or 4.9 mg of Se/wk. Ewes receiving no Se showed a 78.8 and 58.8% decrease (P < 0.001) in WB- (250 to 53 ng/mL) and serum- (97 to 40 ng/mL) Se concentrations, respectively, over the duration of the study. Whole-blood Se decreased primarily during pregnancy (-57%; 258 to 111 ng/mL) and again during peak lactation (-44%; 109 to 61 ng/mL; P < 0.001). At 4.9 mg of Se/wk, Se-yeast (364 ng/mL, final Se concentration) was more effective than Na-selenite (269 ng/mL) at increasing WB-Se concentrations (P < 0.001). Supranutritional Se-yeast dosages increased WB-Se concentrations in a dose-dependent manner (563 ng/mL, 14.7 mg of Se/wk; 748 ng/mL, 24.5 mg of Se/wk; P < 0.001), whereas WB-Se concentrations were not different for the Na-selenite groups (350 ng/mL, 14.7 mg of Se/wk; 363 ng/mL, 24.5 mg of Se/wk) or the 4.9 mg of Se/wk Se-yeast group (364 ng/mL). In summary, the dose range whereby Se supplementation increased blood Se concentrations was more limited for inorganic Na-selenite than for organic Se-yeast. The smallest rate (FDA-recommended quantity) of organic Se supplementation was equally effective as supranutritional rates of Na-selenite supplementation in increasing WB-Se concentrations, demonstrating the greater oral bioavailability of organic Se.

Because selenium increases the levels of other selenoproteins, the influence of this element on selenoprotein W was examined in wether sheep fed either a low selenium diet (0.02 mg/kg) or the same diet supplemented with 3 mg selenium as selenite per kilogram diet. Muscle biopsies were taken initially and at 3.5, 7.0 and 10.5 wk. The sheep were killed after the last muscle biopsy and samples from nine tissues were taken. Selenoprotein W was determined in tissues by Western blots with a polyclonal antibody against a synthetic peptide based on the protein sequence of the homologous rat selenoprotein W. In supplemented sheep, muscle selenoprotein W was significantly increased over initial levels (P < 0.05) at 7 wk and afterwards, whereas in sheep consuming the low selenium diet, muscle selenoprotein W levels declined significantly (P < 0.05) after 10.5 wk. This selenoprotein was found in various amounts in all tissues examined. The highest levels of selenoprotein W were found in skeletal muscles and heart and the lowest was found in liver. Except for selenoprotein W in brain, the concentrations of selenoprotein W, selenium and glutathione peroxidase activity were significantly higher (P < 0.05) in all tissues from supplemented sheep than in those from unsupplemented sheep. The selenoprotein W levels in brains of the two groups were not significantly different. Thus, selenoprotein W levels in all tissues of sheep except the brain are sensitive to selenium status.

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