S-myotrophin promotes the hypertrophy of myotube as insulin-like growth factor-I does.

We have reported previously that a novel muscle cell growth factor, having a structure of a peptide with sugar chains, was successfully purified from porcine skeletal muscle. It was named s-myotrophin. To determine the role of s-myotrophin in skeletal muscle growth, the effect of s-myotrophin on primary cultured chick skeletal muscle cells (composed almost totally of multinucleated myotubes) was investigated by comparing s-myotrophin with Insulin-like growth factor-I (IGF-I). Both s-myotrophin and IGF-I significantly increased creatine kinase activity of the cultures; both substances gave similar responses. Intracellar protein content was also increased by the addition of these factors. The content of myosin and actin in s-myotrophin treated culture in the differentiation medium was significantly higher than that of the control (unstimulated). The content of those proteins in IGF-I treated culture was also higher than that of control, but the differences were not statistically significant. Immunoblot analysis confirmed that the amounts of myosin and actin in the myocytes were greatly increased by s-myotrophin stimulation and also by IGF-I stimulation. Morphological observations using an anti-desmin antibody staining procedure demonstrated that the size of both s-myotrophin and IGF-I treated myotubes was appreciably larger than that of control myotubes. These results suggest that s-myotrophin is a potent mediator of skeletal muscle cell hypertrophy thorough the accumulations of muscle structural proteins.

Influence of turkey meat on residual nitrite in cured meat products.

A response surface experimental design was employed to estimate residual nitrite level at various initial nitrite concentrations, percent turkey meat in the formula, and heat quantity (F) values using a typical wiener as the test system. Pork and mechanically separated turkey were used as the meat ingredients. Residual nitrite and pH were measured at day 1, 7 days, 14 days, and 49 days after processing. Protein, fat, salt, moisture, and CIE (L*a*b*) color values were also determined. Results showed that the effect of turkey meat on residual nitrite level was significant (P < 0.01). An increased amount of turkey meat in the formula resulted in lower residual nitrite levels at a fixed pH. The residual nitrite level was initially proportional to initial nitrite concentration, but it became a nonsignificant factor during longer storage time. Differences in heat quantity had a significant effect (P < 0.05) on residual nitrite level initially. Greater heat quantity decreased residual nitrite level in finished cured meat products at a fixed pH. However, this effect became nonsignificant during longer storage. Reduction of residual nitrite in wieners because of turkey meat addition at a fixed pH was due to characteristics of the turkey tissue, but the mechanism of action remains unknown. It was also established that commercial wieners had a higher pH if poultry meat was included in the formulation.

Myonuclear accretion is a major determinant of avian skeletal muscle growth.

The role of satellite cells and DNA unit size in determining skeletal muscle growth was studied after mitotic activity was inhibited in the left pectoralis thoracicus of 2-wk-old tom turkeys by means of a 25-Gy dose of irradiation. Toms were killed and muscle weights were obtained 1 (n = 5), 4 (n = 6), 7 (n = 6), and 15 (n = 4) wk after irradiation. Satellite cell mitotic activity and DNA unit size were determined using enzymatically isolated myofiber segments and image analysis. Irradiated and nonirradiated muscle weights increased (P < 0.01) between all ages examined, but irradiated muscle weights were significantly (P < 0.01) lower than nonirradiated muscle weights at 4, 7, and 15 wk after irradiation. Satellite cell mitotic activity was lower (P < 0.01) in irradiated than in nonirradiated muscles at 1 and 4 wk after irradiation and resulted in a significant reduction (P < 0.05) in the number of myofiber nuclei per millimeter at 4 and 7 wk after irradiation. Satellite cell mitotic activity was higher (P < 0.05) in irradiated than in nonirradiated muscles at 7 wk after irradiation, but at 15 wk after irradiation it had fallen to low levels in both muscles. There was no significant (P > 0.10) difference in DNA unit size between muscles at any time, but there was an age-related increase (P < 0.01) for both muscles. Irradiation reduced muscle growth through a transient reduction in myonuclear production at a critical time (3-6 wk of age) in posthatch skeletal muscle development. The age-related increase in DNA unit size was not accelerated to compensate for the reduction in myonuclear accretion. Thus it appears that muscle growth potential is governed mostly by myonuclear accretion and to a lesser extent by DNA unit size.

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

The effect of in vivo and in vitro irradiation (25 Gy) on the subsequent in vitro growth of satellite cells.

The effect of in vivo and in vitro irradiation on subsequent satellite cell growth, in vitro, was investigated to ascertain the ability of a 25 Gy dose to inhibit satellite cell proliferation. Satellite cells were isolated from the left (irradiated) and right (non-irradiated) Pectoralis thoracicus of two-week-old tom turkeys 16 h (n=3) and seven weeks (n=2) after the left Pectoralis thoracicus had been irradiated (25 Gy). Satellite cells isolated from the irradiated and non-irradiated muscles exhibited similar (P>0.10) in vitro proliferation indicating that a population of satellite cells survived an in vivo dose of 25 Gy. In additional experiments, satellite cell cultures derived from tom turkey Pectoralis thoracicus were irradiated (25 Gy) in vitro. The number of satellite cells did not (P>0.05) increase in irradiated cultures for 134 h following irradiation, while satellite cells in non-irradiated cultures proliferated (P<0.05) over this time. At later time periods, satellite cell number increased (P<0.05) in irradiated cultures indicating that a population of satellite cells survived irradiation. The results of these in vitro experiments suggest that a 25 Gy dose of irradiation does not abolish satellite cell divisions in the turkey Pectoralis thoracicus.

Dietary vitamin E supplementation shifted weight loss from drip to cooking loss in fresh beef longissimus during display.

Effects of dietary vitamin E supplementation on drip loss, cooking loss, and muscle fiber disruption in fresh beef loin steaks from Holstein and crossbred beef steers were studied. Nine Holstein steers and nine beef steers were fed a control diet and nine Holstein steers and eight beef steers were supplemented daily with 298 IU of vitamin E/kg of diet for 211, 232, or 252 d. Drip loss, cooking loss, cooking yield, and shear value were measured in each longissimus lumborum sample displayed in PVC film for 2, 6, 10, or 14 d. Dietary vitamin E supplementation produced meat that had smaller (P < .001) increases in drip loss during 14 d of display but higher (P < .01) cooking losses. Cooking yield was reduced (P < .05) by vitamin E supplementation. Vitamin E supplementation reduced (P < .01) muscle cell disruption in beef steak displayed for 14 d. These results indicated that dietary vitamin E treatment stabilized cell integrity and enhanced the ability of beef steak to hold sarcoplasmic components during display, although subsequent losses due to cooking were greater.

Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b(5) reductase) system components in bovine skeletal muscle.

Localization of metmyoglobin-reducing enzyme system components (NADH-cytochrome b(5) reductase, cytochrome b(5) outer mitochondrial membrane cytochrome b) was demonstrated in bovine skeletal muscle by immunohistochemical techniques. Both NADH-cytochrome b(5) reductase and OM cytochrome b were located in red fibers. However, localization of cytochrome b(5) did not show a definite difference between fiber types. Using an immunoblotting technique. NADH-cytochrome b(5) reductase was found predominantly in the mitochondrial fraction, but it was also detected at lower levels in the microsomal fraction. OM cytochrome b was found predominantly in the mitochondrial fraction, but cytochrome b(5) was detected only in the microsomal fraction. The results from this study, along with previous work about the localization of myoglobin in muscle, suggest that NADH-cytochrome b(5) reductase reduces metmyoglobin by using OM cytochrome b at the mitochondrial surface and, in part, by using cytochrome b(5) at the sarcoplasmic reticulum.

Atmosphere and blooming time affect color and lipid stability of frozen beef from steers supplemented with Vitamin E.

The influence of blooming time (1, 6 or 48 h) and atmosphere (air or 100% oxygen) on color and lipid stability of frozen Longissimus lumborum (LL) from control and vitamin E supplemented steers was studied. Samples were stored at -20°C with or without illumination. Blooming control LL for 48 h in air followed by dark storage increased discoloration. Supplementation and blooming in oxygen, separately or combined, increased color display life. Illumination increased discoloration. Supplementation coupled with blooming for 6 or 48 h in 100% oxygen provided the highest color stability in both dark and illuminated storage. Color display lives for supplemented LL bloomed for 6 and 48 h in oxygen were 182 and 212 days for dark storage, and 21 and 73 days for illuminated display. Supplementation decreased lipid oxidation in illuminated LL, but blooming for 48 h in oxygen minimized this effect.

Quantitation of satellite cell proliferation in vivo using image analysis.

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei+satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 +/- 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 +/- 462 microns3 of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.

Satellite cell mitotic activity in posthatch turkey skeletal muscle growth.

The relationship between satellite cell mitotic activity and skeletal myofiber growth was examined in Pectoralis thoracicus and Biceps femoris muscles of Large White tom turkeys (Nicholas strain) at 3, 6, 9, 18, and 26 wk of age. Mitotically active satellite cells were labeled with 5-bromo-2'-deoxyuridine (BrdU). Labeled satellite cells were identified on enzymatically isolated myofiber segments using mouse anti-BrdU followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibodies. Myofiber nuclei (satellite cell nuclei + myonuclei) were counterstained with propidium iodide (PI). Myofiber segment diameter, myofiber segment length, and number of FITC- and PI-labeled nuclei were determined for each segment. At each age interval there was an increase in myofiber diameter, suggesting that the myofibers were growing during the entire experimental period. There was an age-related (P < .001) decrease in satellite cell mitotic activity and an age-related increase (P < .001) in the cytoplasmic volume to nucleus ratio (CNR) from 3 to 26 wk of age. An early phase of myofiber growth, between 3 and 6 wk of age, was characterized by a high level of satellite cell mitotic activity and increased CNR. Between 6 and 9 wk of age, satellite cell mitotic activity decreased, but the CNR showed no change (P > .05). During a late phase of myofiber growth, beyond 9 wk of age, satellite cell mitotic activity continued to decrease and myofiber growth occurred by an increased CNR. This study demonstrated that both Pectoralis thoracicus and Biceps femoris undergo a significant late phase of growth without appreciable production of myonuclei by satellite cell proliferation.

Effect of dietary vitamin E on pigment and lipid stability of frozen beef: A kinetic analysis.

The effect of vitamin E supplementation on pigment and lipid stability was evaluated with beef wrapped in high or low oxygen permeability films and stored in the dark or under constant illumination at -20°C. Dietary vitamin E supplementation improved pigment and lipid stability in both cases. Illumination increased metmyoglobin accumulation but did not affect lipid oxidation rate in both control and supplemented beef. A predisplay dark storage period of 30 days delayed metmyoglobin accumulation during subsequent display. Kinetic analysis showed that vitamin E supplementation stabilized the oxymyoglobin complex by enhancing the deoxymyoglobin oxygenation rate and by decreasing oxymyoglobin autoxidation rate.

Characterization of bacteriocins from Enterococcus faecium with activity against Listeria monocytogenes.

Laboratory cultures and environmental isolates of bacteria were screened for antagonism towards Listeria monocytogenes using an agar spot test. Seven of the 163 strains that were tested, one Streptococcus bovis, one Enterococcus casseliflavus, two E. avium and three E. faecium, consistently displayed antilisterial activity. Cell-free, pH-neutralized supernatants prepared from the three E. faecium strains (JBL1061, JBL1083 and JBL1351) exhibited strong antilisterial activity against L. monocytogenes, and were subjected to more detailed analyses. The antagonistic factors produced by these three strains were sensitive to chloroform and several proteolytic enzymes, resistant to heat (121 degrees C, 20 min), and stable over a wide pH range (3.0-10.0). Moreover, they were listericidal without causing cell lysis. These data suggest that a bacteriocin(s) is involved in the inhibition of L. monocytogenes by E. faecium JBL1061, JBL1083 and JBL1351.

Dietary versus postmortem supplementation of vitamin E on pigment and lipid stability in ground beef.

Effects of dietary supplementation and postmortem addition of vitamin E on pigment and lipid stability in raw ground beef were examined in this study. Six Holstein steers were fed a control diet for 232 or 252 d and six Holstein steers were supplemented with 1,500 IU of vitamin E per animal daily for 232 or 252 d. Three aliquots of ground beef from each longissimus lumborum were allotted to the following postmortem treatments: no addition (NO), white mineral oil (OIL), and white mineral oil containing sufficient D-alpha-tocopherol to equal the mean difference of alpha-tocopherol concentration between beef from supplemented and control steers (OIL + E). Metmyoglobin percentages and 2-thiobarbituric acid reactive substances values were determined at d 1, 3, 5, 7, and 9 after postmortem treatment. Dietary vitamin E supplementation delayed metmyoglobin increase and highly suppressed lipid oxidation in ground beef during 9 d of display compared with the control. The postmortem addition of vitamin E (OIL + E) was slightly effective in retarding the oxidation of pigment and lipid, especially compared with the OIL treatments. Endogenous vitamin E improved pigment and lipid stability much better than exogenous vitamin E.

Effect of training on VO2max, thigh strength, and muscle morphology in septuagenarian women.

The purpose of this study was to determine the effects of a long-term (50 wk) combined aerobic-resistance training program on maximal oxygen consumption (VO2max, thigh strength, and vastus lateralis fiber morphology in healthy septuagenarian women (mean age = 72 +/- 6 yr). Subjects volunteered to be in either an exercise (Ex; N = 17) or control (Con; N = 10) group. Con subjects were 34% less active in winter than in summer, Ex subjects maintained their summer activity level on exercise days in winter. Initial, intermediate (20 wk), and final (50 wk) measurements were made for isokinetic knee extension/flexion strength; VO2max and morphological measurements from a muscle biopsy were made at the initial and final times only. Both groups gained in leg strength (Ex = +6.5%; Con = +7.8%; P less than or equal to 0.05) during the summer; in the winter the Ex group maintained leg strength and the Con group declined 12.2% (P less than or equal to 0.05). The fast-twitch muscle fiber area (Type IIb) increased 29% (P less than or equal to 0.001) in the Ex group and declined 26% (P = 0.014) in the Con group. VO2max increased only in the Ex group (16%; P less than 0.001). We conclude that healthy septuagenarian women can increase aerobic capacity, leg strength, and Type IIb muscle fiber area with a long-duration, combined aerobic-resistance exercise program.

Histopathological and ultrastructural alterations of turkey skeletal muscle.

The pectoralis thoracicus and biceps femoris muscles were obtained from 30 18-wk-old Large White turkey males at the time of slaughter. The birds had no observable mobility or postural problems, and the meat appeared normal. Samples were processed for histology, histochemistry, and electron microscopy. Degenerative features were found in muscle from 10 of the 30 birds. Four of the 10 had alterations in both muscles. The degenerative changes included scattered focal necrosis, hypercontraction of muscle fibers, infiltration by mononuclear cells, formation of fibrous tissue scars, and Z-band streaming. These structural alterations appear to be related to muscle ischemia.

Distribution of capillaries in normal and ischemic turkey skeletal muscle.

Capillary to fiber relationship was studied in musculi biceps femoris and musculi pectoralis thoracicus from 20 normal and 10 ischemic turkeys. Capillary density, the number of capillaries surrounding a fiber, capillary to fiber ratio, intercapillary distance, and fiber area were measured. The muscles from the ischemic group had significantly lower (P less than .05) values for capillary density and capillary to fiber ratio and significantly higher intercapillary distance than those from the normal group. There was no evidence of larger muscle fiber size in the ischemic versus normal birds. It is suggested that the occurrence of muscle ischemia in the domestic turkey is due to alterations in capillarity and reduced vasodilatation, resulting from lack of exercise.

The effect of cattle breed and muscle type on discoloration and various biochemical parameters in fresh beef.

The levels of nonprotein sulfhydryls (NPSH), glutathione disulfide (GSSG), nicotinamide adenine dinucleotide (NAD), hypoxanthine (Hx), xanthine (X) and the extent of lipid (TBA) and pigment oxidation (%MetMb) were determined in longissimus and gluteus medius muscles during storage at 4 degrees C. Muscles were obtained from Holstein and crossbred beef steers. Holstein beef displayed higher values for %MetMb, NAD and TBA and lower values for GSSG and Hx than crossbred beef (P less than .05). These two breed groups did not differ (P greater than .05) in X and NPSH. During storage, GSSG, Hx, X, %MetMb, GSSG/NPSH ratio and TBA value all increased, whereas NAD and NPSH decreased. Compared to the longissimus, the gluteus medius had greater %MetMb, TBA value, GSSG/NPSH ratio and Hx but lower (P less than .05) NAD and NPSH. Differences between Holstein and crossbred animals and between gluteus medius and longissimus muscles in meat color stability may be dependent on inherent metabolic differences.

Incidence of microscopically detectable degenerative characteristics in skeletal muscle of turkey.

1. The incidence of microscopically detectable degenerative characteristics in 5 skeletal muscles (m. pectoralis thoracicus, m. supracoracoideus, m. biceps femoris, m. semitendinosus, m. femorotibialis medius) of turkeys was investigated. 2. Samples were obtained from 30 Large White turkey males 14, 16 and 18 weeks old. Hyaline degeneration, infiltration of mononuclear cells and necrotic fibres were observed. 3. Individual fibres varied greatly in size and muscle fibre nuclei were often shrunken and pyknotic. 4. Weak and/or uniform reaction for Ca++-ATPase and SDH in all types of muscle fibres and loss of alkaline phosphatase activity in cell membranes were noted. A positive reaction for acid phosphatase occurred in regions of perivascular infiltration and in necrotic muscle fibres. The majority of muscle fibres possessed high activity for phosphorylase a and b. 5. Based on the use of fluorescein alpha-bungarotoxin conjugate, motor end-plates appeared to be morphologically intact. Direct immunofluorescence with anti-chicken IgG showed positive reaction in muscle fibres undergoing necrosis and in the involved connective tissue. 6. Degenerative changes varied with age and were most marked in the oldest birds. 7. Because gross degenerative symptoms were absent from both the birds and the meat from them, the condition appears to be either different from or a precursor to the degenerative myopathy characterised by other authors.

Reaction of nitrite with ascorbic acid and its significant role in nitrite-cured food.

The decrease in concentration of free nitrite in an aqueous reaction solution made by mixing nitrite and ascorbic acid was studied. This decrease resulted information of a reaction product which transferred NO to ferricytochrome c to form ferrocytochrome c nitroso compound. From the solution containing the reaction product, nitrite could be regenerated spontaneously and suddenly by physical agitation. Oxygen was apparently not necessary for regeneration nitrite from the reaction solution. It does not seem that the regeneration of nitrite was responsible for a bimolecular dismutation of nitrosoascorbic acid in a reverse direction proposed by Fox and Thomson (Biochemistry2, 465; 1963). Heating of the reaction solution at less than 100°C resulted in a loss of nitrite from the solution, which was greater with increasing concentration of ascorbic acid. The reaction product formed between nitrite and ascorbic acid is probably responsible not only for nitrosation reactions, but also for the loss of nitrite observed during curing of meat.

Determination of fiber types of chicken skeletal muscles based on reaction for actomyosin, calcium+2, magnesium+2-dependent adenosine triphosphatase.

Muscle fiber subtypes, determined with the actomyosin Ca+2,Mg+2-adenosine triphosphatase (ATPase) reaction in chicken anterior latissimus dorsi and posterior latissimus dorsi muscles, were demonstrated only after acid or alkaline preincubation followed by a 60-min enzyme incubation. In contrast, subtypes were demonstrated in the sartorius muscle either with or without preincubation. A single-step procedure was therefore possible with this muscle. The results were generally similar to those obtained previously with the mycosin Ca+2-ATPase procedure. Both methods revealed corresponding muscle fiber subtypes, with the exceptions noted below. The actomyosin Ca+2,Mg+2-ATPase procedure, following preincubation at pH 9.4 and 10.3, resulted in a similar reaction intensity in all fiber types. With the myosin Ca+2-ATPase procedure, the IRA (slow) type in anterior latissimus dorsi and sartorius muscles and the I (slow), IIR (fast oxidative-glycolytic), and IIW (fast glycolytic) types in posterior latissimus dorsi muscle had a higher reaction intensity following preincubation at pH 9.4 than at pH 10.3. Fiber Types IIR and IIW in sartorius muscle were easily distinguished with the actomyosin Ca+2,Mg+2-ATPase procedure.