Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.     

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.     

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.     

Synthesis of apolipoprotein A1 in skeletal muscles of normal and dystrophic chickens

We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A1 (apo-A1) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following surgical denervation (Shackelford, J. E., and Lebherz, H. G. (1983) J. Biol. Chem. 258, 7175-7180; 14829-14833). In the present work we investigate the effect of avian muscular dystrophy on the synthesis of apo-A1 in chicken muscles. The relative rate of synthesis of apo-A1 and levels of apo-A1 RNA in mature dystrophic breast (fast-twitch) muscle were about 6-fold higher than normal, while synthesis of apo-A1 in breast muscles derived from 2-day-old dystrophic chicks was close to normal. These observations suggest that the elevated apo-A1 synthetic rate in mature dystrophic breast muscle results from a failure of the diseased tissue to "shut down" apo-A1 synthesis to the normal level during postembryonic maturation. Apo-A1 synthesis in the "slow-twitch" lateral adductor muscle of dystrophic chickens was found to be normal. Our work is discussed in terms of the apparent similarities between the effects of surgical denervation and muscular dystrophy on the protein synthetic programs expressed by chicken skeletal muscles.     

There is selective accumulation of a growth factor in chicken skeletal muscle. II. Transferrin accumulation in dystrophic fast muscle

Transferrin or a transferrin-like protein, with ability to stimulate myogenesis and terminal differentiation in vitro, is found in fast chicken muscle during embryonic development. After hatching, however, transferrin is no longer accumulated or is only weakly accumulated by fast muscles like the pectoralis major and the posterior latissimus dorsi but continues to be accumulated by slow muscles like the anterior latissimus dorsi. In congenic lines of chickens bearing the gene for muscular dystrophy, however, adult fast muscles do not lose the ability to accumulate transferrin. While transferrin is found selectively in adult normal and dystrophic muscle it does not appear to be synthesized by muscle cells. Immunocytochemical localization shows that transferrin is accumulated not so much by muscle fibers as it is by single cells in the muscle interstitial space. The relationship between transferrin presence and growth patterns in adult skeletal muscle is not currently understood but evidence suggests that transferrin stimulation of myogenesis observed in vitro may be mediated in vivo by non-muscle cells dwelling within the muscle interstitial space. These cells may act as transferrin-uptake sources for subsequent satellite cell stimulation.     

Calcium- and calmodulin-dependent phosphorylase kinase activity in porcine uterine smooth muscle

Porcine uterine smooth muscle phosphorylase kinase has been partially purified. The enzyme was activated about 1.5-2.0-fold by exogenous calmodulin. Half maximal stimulation was observed at about 100 nM calmodulin. The activation was dependent on calcium and was maximum at pH 7.5 in the range of pH from 6 to 9. This activation was completely abolished by 100 microM trifluoperazine. The result suggested that unlike slow and cardiac muscles, phosphorylase kinase of uterine smooth muscle showed similar response to calmodulin with that of fast muscle. The physiological role of the calcium and calmodulin-dependent activation of myometrium phosphorylase kinase is briefly discussed.     

Avian muscular dystrophy: Thyroidal influence on pectoralis muscle growth and glucose-6-phosphate dehydrogenase activity

The pectoralis muscles of dystrophic chickens (line 413) were hypertrophic on the basis of fresh weight and fat-free dry weight. They also had greater DNA content and greater glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. Of the parameters measured, the largest differences between pectoralis muscles from dystrophic and normal (line 412) chickens were for DNA content and G6PD activity. These parameters were 4.3- and 6.7-fold, respectively, the values for control pectoralis at 5 wk of age. The average number of nuclei per unit length of isolated muscle fiber was also greater (approximately 3-fold) for the dystrophic pectoralis. Body weight and pectoralis fresh weight, fat-free dry weight, DNA content, G6PD activity and 6PGD activity were reduced significantly in propylthiouracil (PTU)-treated normal and dystrophic chickens. Moreover, the effects of PTU were more pronounced in the dystrophic strain. Thyroid deprivation significantly improved the righting ability of the dystrophic chickens, in addition to its influence on muscle hypertrophy and body growth. Thyroxine (T₄) replacement reversed the PTU effects in both strains. Of all the variables measured, total G6PD activity was the most affected by PTU treatment of dystrophic chickens and was only 16% of the control dystrophic value.In addition to the effects of thyroid deprivation on the expression of avian muscular dystrophy, we observed significant differences in thyroid-related variables in the two strains. The average thyroid weight at 4 wk and serum triiodothyronine level at 5 wk for dystrophic chickens were 65 and 76%, respectively, of the normal values. The results that we report here indicate that altered thyroid function affects the expression of avian muscular dystrophy.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Calcium-related defects in cardiac and skeletal muscles of dystrophic mice

Ca2+ ATPase and calcium binding proteins were studied in cardiac and skeletal muscles of normal and dystrophic mice. In normal and dystrophic mice, Ca2+ ATPase was quite reduced in cardiac muscle compared to skeletal muscle and was, unlike skeletal muscle, insensitive to orthovanadate. Ca2+ ATPase in skeletal muscle of dystrophic mice was reduced as compared to normal mice. In both cases (normal and dystrophic), calcium binding proteins were the same (identical molecular weight). The effect of 2 drugs (Polymixine B and Bepridil) which decrease protein bound calcium was studied: the muscle proteins of dystrophic mice did not present the same sensitivity to Bepridil as controls. These findings suggest the existence of a calcium-related defect in skeletal and cardiac muscle of dystrophic mice.     

Isolation and characterization of transverse tubule from normal and dystrophic mice

I have recently reported the isolation and characterization of sarcoplasmic reticulum from normal and dystrophic mice. These sarcoplasmic reticulum fractions were similar in calcium pump function, calcium release properties, and lipid composition. In this report, I describe the isolation of mouse muscle transverse tubule membranes using a calcium phosphate-loading technique. When the relative purity of normal and dystrophic preparations was considered, transverse tubule from normal and dystrophic mice were similar in calcium-insensitive ATPase activity, cholesterol content, and membrane microviscosity (as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene); transverse tubule yield from dystrophic muscle, however, was twice that from normal muscle, while sarcoplasmic reticulum yield from these same dystrophic muscles was only 60% that from normal muscle. This result may reflect a difference in the relative quantities of these membranes in situ.     

An effect of avian hereditary muscular dystrophy on the reaction of troponin-C with its antibody

Antibody prepared against troponin-C, the calcium binding component of the troponin complex, was reacted with I band segments, and the distribution of antibody binding was assessed by immuno-electron microscopy. The I segments were isolated from glycerinated pectoral muscle which was prepared from normal adult chickens and from dystrophic chickens of strain 308. The antibody was deposited at 384 Å ± 7 Å intervals along the thin filaments of the normal muscle. In contrast to the normal controls the dystrophic muscle did not exhibit a distinct periodicity when reacted with anti-troponin-C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that although protein bands corresponding to troponin-C could be observed in the gels of the dystrophic preparations, the troponin-C band had migrated slower than that from normal thin filaments. It is concluded that avian muscular dystrophy produces an alteration of the structure of troponin-C resulting in (1) an inability of the protein to combine with its specific antibody and (2) a change in its electrophoretic behavior.     

Comparison of the Molecular Forms of the Cholinesterases in Tissues of Normal and Dystrophic Chickens

Abstract: The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (ΦChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (H_c) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic, oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and ΦChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or ΦChE in a slow-tonic muscle. In the pectoral or PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the ΦChE pattern remains abnormal. Muscle ΦChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of ΦChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and H_c. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or ΦChE were found in cardiac or neural tissues from dystrophic chickens.     

Thyroidal involvement in the expression of avian muscular dystrophy

We showed previously that propylthiouracil (PTU), a thyroid inhibitor, could alleviate several major signs of hereditary muscular dystrophy in chickens. The goals of the present investigation were to: (1) determine whether a nearly athyroid condition (achieved within two days after hatching by surgical thyroidectomy plus PTU) during an 11-day period beneficially affects the dystrophic condition when followed by triiodothyronine (T3) replacement to 33 days of age; (2) determine the beneficial effects on the expression of avian dystrophy when the thyroidectomized-PTU-treated chickens received a wide range of moderate to low T3 replacement doses beginning by two days after thyroidectomy; and (3) examine the thyroid hormone receptor system in dystrophic muscle for a possible abnormality. Thyroid deprivation increased muscle function (righting ability) and reduced plasma creatine kinase activity in dystrophic chickens. The major thyroid-related abnormality in dystrophic pectoralis muscles was an increased maximum binding capacity of solubilized nuclear T3 receptors.     

Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90-95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2-3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15-20-fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast-twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60-80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H₂, 19-20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast-twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.     

Differentiation of slow and fast muscles in chickens

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.     

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Muscle glycolipids in inherited muscular dystrophy of chickens

Gangliosides and neutral glycolipids of muscles from normal and dystrophic chickens were studied. Total glycolipid content of the degenerating muscles was higher than the normal muscles. In addition, the myopathic muscles contained a ganglioside which was absent in the unaffected muscles from normal and dystrophic chickens. Based on the thin-layer chromatographic mobility, treatment with neuraminidases from Vibrio cholerae and Arthrobacter ureafaciens, and reactivity of the asialo-derivative towards anti-ganglio-N-triaosylceramide antibody, the dystrophic-specific ganglioside was tentatively identified as G_M2. Data obtained from young and old dystrophic chickens suggested a direct relationship of this ganglioside to muscular dystrophy.