Multinucleation of Skeletal Muscle in vitro

Healthy, mature, spontaneously contracting muscle was cultivated from explants of 13-day chick embryos for periods up to 4 months in the multipurpose chamber (Rose, 1954) using cellophane-strip technique (Rose et al., 1958) with silicone gaskets, Eagle's medium including 10 per cent horse serum reinforced with 300 mg-per cent of glucose, and the teased type of explant. This method provided optically ideal conditions for the study of muscle fibers with oil immersion, phase contrast time-lapse cinematography at 1 frame per minute without apparent damage for periods as long as 10 days. In no case was mitosis, amitosis, or nuclear "budding" observed in the course of muscle development. Multinuclear muscle fibers have been shown with cine technique to result from both myoblast fusion and polar extension of preformed (explanted) muscle tissue. Myoblast fusion was the only demonstrable way of giving rise to multinucleation. Nuclear membrane "wrinkling" was shown to be merely a temporary distortion that occurred during nuclear migration and rotation. It is suggested that this phenomenon may be responsible for numerous reports of amitosis in the genesis of muscle fibers. The histological development of new straps resulted from an orderly sequence of events. Included in these were polar extension, nuclear migration, rotation, and fixation. Following these events there was increased mitochondrial activity, myofibril formation, and cross-banding. Spontaneous contractions were seen throughout the entire course of differentiation in vitro but became more regular and stronger in the later stages.     

Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures.

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.     

Epithelial outgrowth from suspension cultures of human prostatic tissue

Summary The primary objective of this study was to obtain pure cultures of prostatic epithelium. Encapsulation by epithelial cells and hypocellularity in stroma occurred when explants of prostatic tissue were maintained in suspension cultures. Twenty per cent fetal bovine serum incorporated into the medium provided optimal conditions for encapsulation and preservation of epithelial cell viability and architecture. Horse serum at the same concentration was less effective. When encapsulated explants were allowed to attach to the substrate, 10 and 20% horse serum favored growth of epithelial cells while fetal bovine serum also stimulated fibroblastic growth. Mechanisms for the induction of hypocellularity, encapsulation, squamous metaplasia and central necrosis in explants were studied. Relationship between the type and concentration of serum and the nature and extent of outgrowth are discussed. This work was supported in part by the American Cancer Society Grants DT-20 and IN-5N, U. S. Public Health Service Grant RR-05357, the Parke Davis Fund, and the John U. White Fund for Urological Research.     

Effect of serum from chickens treated with clenbuterol on myosin accumulation, β-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 microM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The beta-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 microM isoproterenol, limited increases of 12-20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 microM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher betaAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.     

Experiment to study some suspension media for the lyophilization of actinomycetes]

Viability and cultural properties of 59 actinomycetes and 17 bacteria lyophilized in polyvinylpyrrolidone (PVP), sodium glutamate, their combinations and horse serum were studied after storage for 2 years at a temperature of 4-10 degrees. A 5 per cent solution of sodium glutamate had a high protective effect on viability of the above organisms. The solution containing 3 per cent of sodium glutamate and 3 per cent of PVP was somewhat less effective. The cultures lyophilized in 5 per cent solution of sodium glutamate had the same viability levels as those lyophilized in horse serum, while the latter had better growth rates on their plating out on nutrient media. A 5 per cent solution of PVP had no advantages over sodium glutamate or horse serum with respect to preservation of the organism viability. No significant differences in the cultural properties: colour of the aerial and substrate mycelium and pigment production were noted in the actinomycetes lyophilized in various protective media and the analogous control cultures maintained by means of passages on fresh nutrient media.     

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid

Two lines of genetically involved and control chickens were compared with regard to the onset of muscle dystrophy during the early stages of growth ex ovo. Definite structural and functional involvement of pectoralis muscle developed within the first 4-5 weeks. In parallel experiments, microsomal membranes were obtained weekly from pectoralis muscle during the first 14 weeks ex ovo. The microsomes were studied with respect to ultrastructural features, protein composition, Ca2+ uptake and ATPase activity. Microsomal preparations obtained from all newborn chickens contain two types of vesicles: one type reveals an asymmetric distribution and 'high density' of particles on freeze-fracture faces which is characteristic of sarcoplasmic reticulum (SR) membrane; the other type reveals a symmetric distribution and 'low density' of particles. The yield of 'low density' microsomes from muscle of normal birds is very much reduced as the chicks grow from 1 to 4-5 weeks ex ovo. On the contrary, it remains high in chicks developing muscle dystrophy. Ca2+ uptake and coupled ATPase activity are found to be of nearly identical specific activity in control and genetically involved newborn chicks. The specific activity of the control birds, however, increases as the chicks grow from 1 to 4-5 weeks of age, while the specific activity of the dystrophic birds remains low. Such a difference appears to be related to the relative representation of sarcoplasmic reticulum and 'low density' vesicles in the microsomal preparations. It is concluded that failure to obtain a normal differentiation of muscle cell membranes is a basic defect noted in the early growth of genetically involved chickens. This defect appears along with the earliest signs of the dystrophic process.     

Effect of potassium deficiency on carbon dioxide,cation, and phosphate content of muscle,with a note on the carbon dioxide content of human muscle

Albino rats weighing 160 to 175 gm. were fed a complete synthetic diet containing 0.003 per cent potassium and 0.7 per cent sodium for 40 days. Controls were given the same diet plus adequate added potassium. 1. Data from analyses of serum and skeletal muscle showed (a) a fall in serum chloride concentration and an increase in serum carbon dioxide concentration and pH in the potassium-deficient rats; (b) increases of sodium, magnesium, and calcium and a decrease of potassium in the muscle of the potassium-deficient rats; (c) no change of muscle chloride or carbon dioxide concentrations in the potassium-deficient rats. (2) Application of the Wallace-Hastings calculations to these data revealed (a) intracellular pH of the skeletal muscle of the normal rat to be 6.98 +/- 0.08; (b) an increase in serum partial pressure of carbon dioxide (pCO(2)) in potassium deficiency, together with increases in concentrations of [H(2)CO(2)] and [HCO(3) (-)] per kg. extracellular water and [H(2)CO(3)] per kg. cell water; (c) a decrease in values for [CO(2)] and [HCO(3) (-)] per kg. intracellular water; (d) a fall of intracellular pH in potassium deficiency to 6.42 +/- 0.05. (3) Analyses of sacrospinalis muscle from five men undergoing operation for ruptured intervertebral disc showed a mean value of 9.46 +/- 1.31 mM carbon dioxide per kg. blood-free tissue. Some problems of interpretation of data are briefly discussed.     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A₁₂ form was however higher in the defined medium (25–30%) than in FCS supplemented medium (about 5–6%). In HS supplemented medium the A₁₂ form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A₁₂. In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A₁₂ in a drastic manner. FCS components therefore seem to repress the biosynthesis of A₁₂ AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G₁ form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A₁₂ and G₄ seemed to be located preferentially in the external compartment, whereas G₂ was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

Thalidomide may inhibit proliferation of mesenchyme in human limb buds

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.     

Development of sarcoplasmic reticulum in cultured chicken muscle.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Effect of sex, age and cultivation time on number of satellites and acrocentric associations in man.

Lymphocyte cultures of 120 normal persons evenly distributed in relation to sex and age groups were studied. A total of 3900 cells were examined, 3600 in 3-day cultures and 300 in cultivations maintained for 2 days. In both age groups (10--13; 62--96 years) men showed higher numbers of satellites per cell but less cells with D/G associations. Older subjects presented a lower number of cells with D satellites and acrocentric associations; the pattern of these associations, however, did not seem to vary with age. The mean number of associations per cell decreased significantly in 3-day as opposed to 2-day cultures.     

Structure of myosin heavy chain in avian muscular dystrophy

We have studied the structure of myosin heavy chain (MHC) in the pectoralis muscle of genetically dystrophic (Connecticut Strain) and White Leghorn chicks. MHC was alkylated with N-ethylmaleimide, purified by Sepharose-4B chromatography, and cleaved with cyanogen bromide. The MHC CNBr peptides were analyzed by one-dimensional and two-dimensional isoelectric focusing/sodium dodecyl sulfate gradient gels and by amino acid sequencing. Specific changes were detected in the gel patterns which could be correlated with the loss of muscle function as measured by the exhaustion score (the ability of chicks to rise from a reclining position) in three experimental groups (exhaustion scores: less than 3, 10-20, greater than 30). We have also examined the amino acid sequence of a 3-methyl-histidine-containing peptide which originates from the 20-kDa fragment of pectoralis muscle MHC in dystrophic chicks: Val-Leu-Asn-Ala-Ser-Ala-Ile-Pro-Glu-Gly-*Gln-Phe-*Ile-Asp-Ser-Lys-Lys- Ala-Ser-Leu-Gln-Lys-Leu-Gly-Ser-Ile-Asp-Val-(Asp, 3-methylhistidine, Gln). Comparison of the homologous MHC sequences shows two positions at which MHC from dystrophic chicks differs from that of the White Leghorn chicks *(Glu----Gln and Met----Ile). Thus, both the peptide map and sequence analyses demonstrate that in avian muscular dystrophy an abnormal pectoralis MHC is synthesized. It is not yet clear whether the "dystrophic" MHC is a variant MHC or if it arises from the abnormal expression of an earlier developmental form (embryonic or neonatal) of pectoralis muscle MHC.     

Effects of Dietary Selenium, Vitamin E, and Their Combination on Growth, Serum Metabolites, and Antioxidant Defense System in Skeletal Muscle of Broilers Under Heat Stress

This experiment was conducted to evaluate the effects of dietary vitamin E, selenium (Se), and a combination of the two, on the performance, serum metabolites and oxidative stability of skeletal muscle of broilers during heat stress. The broilers raised in either a thermoneutral (23.9°C constant) or heat stress (23.9°C to 37°C cycling) environment were assigned to 6 dietary treatments (0, 0.5, or 1 mg/kg Se; 125 and 250 mg/kg vitamin E; or 0.5 mg/kg Se plus 125 mg/kg vitamin E) from 1 to 49 days of age. At the end of the experiment, blood samples were collected from chicks, the chicks sacrificed, and pectoralis superficialis muscle was used for measurement of malondialdehyde (MDA) concentration and enzyme activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). The heat-stressed chicks consumed less feed, gained less weight, and had higher feed conversion ratio when compared to thermoneutral chicks (P<0.05). Serum concentrations of iron (Fe) and zinc (Zn) were decreased by heat stress (P<0.05), whereas the serum concentrations of copper (Cu), glucose, and uric acid were significantly increased under heat stress (P<0.05). The chicks that received supplemental of vitamin E exhibited significantly higher serum concentrations of Zn (P<0.05) and significantly lower concentrations of Cu, glucose, and uric acid (P<0.05) when exposed to heat stress. Dietary Se also caused a significant decrease in serum glucose, uric acid, and Cu concentrations of heat-stressed broilers (P<0.05), but had no significant effect on Zn concentration (P>0.05). The GPx activity remained relatively constant (P>0.05), though SOD activity and MDA levels in skeletal muscle were enhanced on exposure to heat stress (P<0.05). The heat-stressed chicks that received the combined supplementary level of vitamin E and Se had the lowest concentration of MDA and the highest activity of SOD in the skeletal muscle (P<0.05). Dietary Se also caused a significant increase in enzyme activity of GPx in the skeletal muscle (P<0.05). These results indicate that the derangement of blood parameters and oxidative stability in broilers under heat stress are improved by supplemental vitamin E and Se.     

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.