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

1987 ◽  
Vol 65 (9) ◽  
pp. 833-841 ◽  
Author(s):  
U. S. Srivastava ◽  
E. A. Sugden ◽  
P. K. Majumdar ◽  
M. L. Thakur ◽  
G. M. Bhatnagar
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cdna Probe ◽  
Sodium Dodecyl ◽  
Mouse Muscle ◽  
Skeletal Muscle Myosin ◽  
Dystrophic Mouse ◽  
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 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. In conclusion, it appears that murine muscular dystrophy produces a shorter species of pre-M mRNA via decreased polynucleotide elongation.

Biochemical Changes in Progressive Muscular Dystrophy. IX. Synthesis of Native Myosin, Actin, and Tropomyosin in the Skeletal Muscle of Mouse as a Function of Muscular Dystrophy

1972 ◽  
Vol 50 (4) ◽  
pp. 409-415 ◽  
Author(s):  
Uma Srivastava
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Protein Purification ◽  
Gel Electrophoresis ◽  
Dystrophic Muscle ◽  
Mouse Muscle ◽  
Progressive Muscular Dystrophy ◽  
Dystrophic Mice

The synthesis of native myosin, actin, and tropomyosin in the skeletal muscle of normal and hereditary dystrophic mice was studied with the help of direct counting as well as acrylamide-gel electrophoresis and protein purification procedures.Labelling of the nascent protein indicated that heavier polysomes from the normal muscle were able to incorporate more radioactivity into the protein than the heavier polysomes from the dystrophic muscles. Contrary to this, lighter polysomes in the dystrophic muscle demonstrated higher incorporation as compared to the normal.Results of in vivo and in vitro incorporation as well as those of acrylamide-gel electrophoresis and protein purification procedures indicated that synthesis of myosin decreased in the dystrophic muscle. The synthesis of actin did not show a significant change either in normal or dystrophic muscle, whereas that of tropomyosin increased sharply in the dystrophic mouse muscle.

Enzyme studies of skeletal muscle in mice with hereditary muscular dystrophy

1963 ◽  
Vol 205 (5) ◽  
pp. 897-901 ◽  
Author(s):  
Marilyn W. McCaman
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Glutathione Reductase ◽  
Enzyme Activities ◽  
Lactic Dehydrogenase ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Nerve Section ◽  
Mouse Muscle ◽  
Dystrophic Mouse

The activities of 20 enzymes in normal, heterozygous, and dystrophic mouse muscle were studied by means of quantitative microchemical methods. Enzyme activities in normal and heterozygous muscle were essentially the same. In dystrophic muscle glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, glutathione reductase, peptidase, ß-glucuronidase, and glucokinase activities were significantly higher than in normal muscle, while α-glycero-P dehydrogenase and lactic dehydrogenase activities were significantly lower. The pattern of enzyme activities found in normal gastrocnemius denervated by nerve section was strikingly similar to that in dystrophic muscle.

scholarly journals Degradation of myofibrillar proteins by trypsin-like serine proteinases.

1982 ◽  
Vol 201 (2) ◽  
pp. 279-285 ◽  
Author(s):  
J Kay ◽  
L M Siemankowski ◽  
R F Siemankowski ◽  
J A Greweling ◽  
D E Goll
Keyword(s):  
Skeletal Muscle ◽  
Gel Electrophoresis ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Myofibrillar Proteins ◽  
Serine Proteinases ◽  
Bovine Trypsin

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

scholarly journals Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

1979 ◽  
Vol 183 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Jean-Louis Azanza ◽  
Jacques Raymond ◽  
Jean-Michel Robin ◽  
Patrick Cottin ◽  
André Ducastaing
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Rabbit Skeletal Muscle ◽  
Neutral Proteinase

Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase.

CREATINE KINASE IN NORMAL AND DYSTROPHIC MOUSE MUSCLE

1962 ◽  
Vol 40 (4) ◽  
pp. 443-447 ◽  
Author(s):  
C. Nichol ◽  
P. L. McGeer ◽  
J. R. Miller
Keyword(s):  
Creatine Kinase ◽  
Muscular Dystrophy ◽  
Kinase Activity ◽  
Creatine Kinase Activity ◽  
Mouse Muscle ◽  
Dystrophic Mouse ◽  
Leg Muscle ◽  
The Difference ◽  
Dystrophic Mice

The hind leg muscle of normal mice and mice with hereditary muscular dystrophy was taken for individual analyses of the creatine-phosphorylating enzyme, creatine kinase. Creatine kinase activity was always found to be higher in the muscle of normal mice than in that of dystrophic mice, and the values in each group decreased with increasing age of the mice. The difference was greatest in mice of 2 weeks of age, and in the light of these findings some suggestions are made about the pathogenesis of muscular dystrophy.

scholarly journals Skeletal-muscle sarcolemma from normal and dystrophic mice. Isolation, characterization and lipid composition

1977 ◽  
Vol 168 (2) ◽  
pp. 229-237 ◽  
Author(s):  
T A de Kretser ◽  
B G Livett
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Neutral Lipid ◽  
Lipid Composition ◽  
Free Cholesterol ◽  
Total Lipid Content ◽  
Dystrophic Muscle ◽  
Mouse Muscle ◽  
Membrane Function ◽  
Dystrophic Mice

1. Mouse skeletal-muscle sarcolemma was isolated, and the preparations obtained from normal mouse muscle and from muscle of mice with hereditary muscular dystrophy were characterized with respect to appearance under the optical and electron microscopes, distribution of marker enzymes, histochemical properties and biochemical composition. 2. The sarcolemmal membranes from normal and dystrophic muscle were subjected to detailed lipied analysis. Total lipid content was shown to increase in sarcolemma from dystrophic mice as a result of a large increase in neutral lipid and a smaller increase in total phospholipids. Further analysis of the neutral-lipid fraction showed that total acylglycerols increased 6-fold, non-esterified fatty acid 4-fold and cholesterol esters 2-fold, whereas the amount of free cholesterol remained unchanged in sarcolemma from dystrophic muscle. Significant increases were found in lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in dystrophic-muscle sarcolemma; however, the relative composition of the phospholipid fraction remained essentially the same as in the normal case. 3. The overall result of alterations in lipid composition of the sarcolemma in mouse muscular dystrophy was an increase in neutral lipid compared with total phospholipid, and a 4-fold decrease in the relative amount of free cholesterol in the membrane. The possible impact of these changes on membrane function is discussed.

scholarly journals Fiber typing human skeletal muscle with fluorescent immunohistochemistry

2019 ◽  
Vol 127 (6) ◽  
pp. 1632-1639 ◽  
Author(s):  
Kevin A. Murach ◽  
Cory M. Dungan ◽  
Kate Kosmac ◽  
Thomas B. Voigt ◽  
Timothy W. Tourville ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Accurate Determination ◽  
Sodium Dodecyl ◽  
Healthy Human ◽  
Critical Determinant ◽  
Skeletal Muscle Myosin ◽  
Fluorescent Immunohistochemistry

Skeletal muscle myosin heavy chain (MyHC) fiber type composition is a critical determinant of overall muscle function and health. Various approaches interrogate fiber type at the single cell, but the two most commonly utilized are single-muscle fiber sodium dodecyl sulfate-polyacrylamide gel electrophoresis (smfSDS-PAGE) and fluorescent immunohistochemistry (IHC). Although smfSDS-PAGE is generally considered the “gold standard,” IHC is more commonly used because of its time-effectiveness and relative ease. Unfortunately, there is lingering inconsistency on how best to accurately and quickly determine fiber type via IHC and an overall misunderstanding regarding pure fiber type proportions, specifically the abundance of fibers exclusively expressing highly glycolytic MyHC IIX in humans. We therefore 1) present information and data showing the low abundance of pure MyHC IIX muscle fibers in healthy human skeletal muscle and 2) leverage this information to provide straightforward protocols that are informed by human biology and employ inexpensive, easily attainable antibodies for the accurate determination of fiber type.

scholarly journals Ca2+ Regulation of Bovine Thrombosthenin

1977 ◽  
Author(s):  
L. Muszbek ◽  
J. Kuźnicki ◽  
W. Drabikowski
Keyword(s):  
Skeletal Muscle ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Regulatory Proteins ◽  
Skeletal Muscle Myosin ◽  
Myosin Binding ◽  
Moving Band ◽  
Low Ionic Strength ◽  
Muscle Myosin

In order to reveal the type of its Ca2+-regulation bovine thrombosthenin - natural platelet actomyosin - was investigated by competitive actin and myosin binding assay and by urea gel electrophoresis. The Ca2+-sensitivity of low ionic strength Mg-ATPase activity of platelet actomyosin was not influenced by the addition of excess skeletal muscle actin free of regulatory proteins. In contrast, the replacement of platelet myosin by skeletal muscle myosin resulted in a hybrid actomyosin insensitive to Ca2+. If actomyosin was reconstructed from crude platelet actin and skeletal muscle myosin again no regulatory effect of Ca2+ could be observed. In the presence of EGTA a fast moving band with the mobility corresponding to muscle troponin C (TN-C) was detected by alkaline urea Polyacrylamide gel electrophoresis. However, if Ca2+ was added this protein, unlike TN-C, neither changed its mobility nor formed a complex even if muscle troponin I was included into the system. The experimental results indicate that our Ca2+ sensitive bovine thrombosthenin preparation does not contain TN-C like component and is not regulated by an actin -, but rather by a myosin-connected system.

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