Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

1987 ◽  
Vol 65 (2) ◽  
pp. 124-129 ◽  
Author(s):  
P. Gopalan ◽  
M. J. Dufresne ◽  
A. H. Warner
Keyword(s):  
Protease Inhibitors ◽  
Protease Activity ◽  
Cathepsin D ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Thiol Protease ◽  
Hindlimb Muscle ◽  
Protease Activation ◽  
Dystrophic Mice ◽  
Cathepsin D Activity

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 °C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.

BIOCHEMICAL CHANGES IN PROGRESSIVE MUSCULAR DYSTROPHY: VI. INCORPORATION OF URIDINE-2-14C INTO RNA OF VARIOUS TISSUE OF NORMAL AND DYSTROPHIC MICE

1967 ◽  
Vol 45 (9) ◽  
pp. 1419-1425 ◽  
Author(s):  
Uma Srivastava
Keyword(s):  
Muscular Dystrophy ◽  
Soluble Fraction ◽  
Normal Liver ◽  
Biochemical Changes ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Progressive Muscular Dystrophy ◽  
Dystrophic Mice

Normal and dystrophic mice were injected intravenously with uridine-2-14C at various stages of the disease. Radioactivity in the acid-soluble fraction of most of the tissues studied was unchanged or not significantly different in dystrophic animals. In vivo incorporation of uridine-2-14C into RNA increased in dystrophic muscle as compared to normal muscle at 30 days, remained the same at 60 days, and was reduced at 90 days. Similar results were also observed on the in vitro incorporation of uridine-2-14C catalyzed by homogenates of normal and dystrophic muscle. Dystrophic brain and pancreas showed a decrease in the incorporation at each stage investigated as compared to controls. No change in the incorporation was noted in dystrophic and normal liver, kidney, spleen, and heart. The decrease in uridine-2-14C incorporation in dystrophic muscle at 90 days could be due to an increased RNA content. Such a phenomenon was explained as due to infiltration of dystrophic muscle by invading macrophages.It is concluded that the metabolism of RNA is not decreased in the dystrophic muscle in preliminary stages of the disease as compared to the control.

PROTEOLYTIC ENZYMES IN NORMAL AND DYSTROPHIC MOUSE MUSCLE

1966 ◽  
Vol 44 (5) ◽  
pp. 613-623 ◽  
Author(s):  
L. Berlinguet ◽  
U. Srivastava
Keyword(s):  
Proteolytic Enzymes ◽  
Chemical Change ◽  
Optimum Conditions ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Muscle Enzymes ◽  
Mouse Muscle ◽  
Ph Values ◽  
Dystrophic Mouse ◽  
Dystrophic Mice

Proteolytic enzymes extracted from normal and dystrophic mouse muscle were studied, and optimum conditions for their activities were established. It was found that these enzymes were active at two pH values, 7.5 and 9. In normal and dystrophic mice, the enzymatic activity increased with age. When the activities of dystrophic muscle enzymes were compared with those of normal muscle enzymes, the increase was most significant in animals 60–90 days of age. The results obtained when the enzymes extracted from normal or dystrophic muscle were incubated with substrates from normal or dystrophic muscle indicate that the defect in the muscle is due to an increase in the activities of the proteolytic enzymes rather than to a chemical change in the muscle proteins.

Defect in regulation of membrane transport of monosaccharides in dystrophic muscle

1979 ◽  
Vol 57 (7) ◽  
pp. 695-701 ◽  
Author(s):  
J. Elbrink
Keyword(s):  
Plasma Membranes ◽  
Sugar Transport ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Variable Effect ◽  
Cardiomyopathic Hamsters ◽  
Uncoupling Of Oxidative Phosphorylation ◽  
Dystrophic Mice ◽  
Normal Controls ◽  
Glucose Analogue

The penetration of a nonmetabolized glucose analogue, 3-O-methyl-D-glucose, across the plasma membranes of tissues from dystrophic mice and cardiomyopathic (dystrophic) hamsters has been compared with that of normal controls. Under basal conditions the penetration of test sugar was similar in lens and diaphragm of normal and dystrophic 129/ReJ mice. Stimulation of sugar transport by 2,4-dinitrophenol did occur in normal but not in dystrophic diaphragm. A submaximal concentration of insulin had a more variable effect in dystrophic than in normal muscle while a supramaximal concentration of the hormone increased the uptake of the glucose analogue to an equal extent in the two tissues. In the BIO 14.6 strain of cardiomyopathic hamsters, uncoupling of oxidative phosphorylation did not increase sugar transport in extensor digitorum longus muscles, while the normal effect was observed in dystrophic soleus and in both these muscles of the random bred controls. The absence of an effect by a condition simulating anoxia suggests that in dystrophy, certain muscles are unable to accelerate the entry of glucose when this is required.

Beneficial effect of new thiol protease inhibitors, epoxide derivatives, on dystrophic mice

1986 ◽  
Vol 91 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Keiji Komatsu ◽  
Kohko Inazuki ◽  
Jun Hosoya ◽  
Susumu Satoh
Keyword(s):  
Beneficial Effect ◽  
Protease Inhibitors ◽  
Thiol Protease ◽  
Dystrophic Mice

Ca2+-dependent slow action potentials in normal and dystrophic mouse skeletal muscle

1983 ◽  
Vol 245 (5) ◽  
pp. C415-C422 ◽  
Author(s):  
L. M. Kerr ◽  
N. Sperelakis
Keyword(s):  
Skeletal Muscle ◽  
Action Potentials ◽  
Osmotic Shock ◽  
Muscle Fibers ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Recording Technique ◽  
Mammalian Muscle ◽  
Dystrophic Mice ◽  
Slow Action Potentials

Slowly rising action potentials (APs), previously described in amphibian skeletal muscle, were examined in skeletal muscle of normal and dystrophic mice (129/ReJ strain). A standard two-microelectrode recording technique was used. Muscles were bathed in a solution that was Cl- free (methanesulfonate substituted), high in K+ (20 mM), and contained 15 mM tetraethylammonium. The slow APs were elicited under conditions in which the fast Na+ channels were voltage inactivated (by partial depolarization) and in which the external Na+ concentration was only 10 mM. Increases in external Ca2+ concentration produced increases in slow AP amplitude and duration. Mn2+ (4 mM), La3+ (4 mM), and detubulation with osmotic shock blocked the slow APs. When slow APs were generated at 30-s intervals, their amplitude stayed constant. When they were generated at 15-s intervals, their amplitude decreased progressively and then fell to zero by the 11th stimulus. The Ca antagonists verapamil (10(-5) M) and bepridil (10(-5) M) caused this decrease in amplitude to occur more quickly. Voltage inactivation of the slow APs occurred between -45 and -10 mV. Slow APs recorded from dystrophic muscle fibers were decreased in amplitude and duration compared with those in normal fibers, and there was a reduced incidence of occurrence; 96% of the fibers in normal muscle exhibited slow APs compared with only 46% of dystrophic muscle fibers. In summary, slow Ca2+ APs in mammalian muscle are similar to those in cardiac and amphibian skeletal muscle, and these slow APs are depressed in dystrophic skeletal muscle.

EFFECT OF VARIOUS AMINO ACIDS ON THE INCORPORATION OF 14C-L-LEUCINE IN THE TISSUE PROTEINS OF NORMAL AND DYSTROPHIC MICE

1967 ◽  
Vol 45 (12) ◽  
pp. 1985-1993 ◽  
Author(s):  
Louis Berlinguet ◽  
Uma Srivastava
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Actinomycin D ◽  
Daily Injection ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Time Intervals ◽  
Parallel Group ◽  
Continuous Injection ◽  
Dystrophic Mice

Normal and dystrophic mice received daily injections of either water, saline, glutamate, aspartate, glycine, ACPC, or actinomycin D for 5 days. One parallel group of animals received no injections and served as control. On the 6th day after the start of the experiment, all the animals received an intravenous injection of 14C-L-leucine and were killed at various time intervals, ranging from 4 h to 12 days. Daily injections of water or saline to the animals did not cause any change in the incorporation of 14C-L-leucine into various tissue proteins. Glutamate administration increased the retention of 14C-L-leucine in normal muscle but not in dystrophic muscle. A daily injection of aspartate increased the retention of the radioactive amino acid in both normal and dystrophic muscles, the higher increase being found in the dystrophic muscle. Administration of glycine reduced the turnover of proteins in both normal and dystrophic muscles. ACPC (1-amino-cyclopentanecarboxylic acid) or actinomycin D administration caused a decrease in the incorporation of 14C-L-leucine into normal and dystrophic muscles.The continuous injection of glutamate or aspartate for 8 days after the 14C-L-leucine administration caused a very large increase in the retention of the labelled amino acid into the proteins of various tissues of normal and dystrophic mice. It is concluded that variations in the amino acid pools can modify the turnover of proteins, which can be of importance in muscular dystrophy.

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 Oestradiol inhibits collagen breakdown in the involuting rat uterus

1972 ◽  
Vol 127 (4) ◽  
pp. 705-713 ◽  
Author(s):  
Janet N. Ryan ◽  
J. Frederick Woessner
Keyword(s):  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Post Partum ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Rat Uterus ◽  
Oestradiol Treatment ◽  
Cathepsin D Activity ◽  
Stimulation Of

1. The earlier observation (Woessner, 1969) of oestradiol inhibition of collagen breakdown is confirmed and extended. Administration of 100μg of oestradiol-17β/day to parturient rats strongly inhibits the loss of collagen from the involuting uterus. Three experiments show that this effect is due to an inhibition of collagen degradation rather than to a stimulation of collagen synthesis. 2. Uterine collagen was labelled with hydroxy[14C]-proline by the administration of [14C]proline near the end of pregnancy. By 3 days post partum, control uteri lost 83% of their collagen and 90% of their hydroxy[14C]proline. Uteri from oestradiol-treated rats lost only 50% of both total and labelled hydroxyproline, with no decrease in the specific radioactivity of the hydroxyproline. 3. Incorporation of [14C]proline into uterine collagen hydroxyproline in vivo was not affected by oestradiol treatment. 4. Urinary excretion of hydroxyproline was increased in post-partum control rats and decreased in oestradiol-treated rats. 5. An enzyme capable of cleaving 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucylglycyl- l-prolyl-d-arginine (a substrate for clostridial collagenase) increased in activity in the post-partum uterus and was unaffected by oestradiol treatment. 6. Uterine homogenates digested uterine collagen extensively at pH3.2. This digestion was unaffected by the oestradiol treatment. 7. Lysosomal fractions prepared by density-gradient centrifugation of uterine homogenates contained coincident peaks of cathepsin D activity and peptide-bound hydroxyproline. The cathepsin D and hydroxyproline contents of this peak were unaffected by oestradiol treatment.

scholarly journals FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Proteolytic Activity ◽  
Guinea Pigs ◽  
Protease Activity ◽  
Amino Acid Esters ◽  
Protein Substrates ◽  
Protease Activation ◽  
Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

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