Biomedical subjects
Publications and source records attributed to A Stracher.
Mice with genetic muscular dystrophy were treated with intraperitoneal injections of the proteinase inhibitor leupeptin, beginning before the onset of weakness. A significant number of the treated animals failed to develop histological evidence of dystrophy, compared with controls. Leupeptin treatment prevented (or delayed) the onset of muscular dystrophy in this experiment.
Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.
A study has been undertaken to determine the rate at which stored platelets lose their ability to respond to stimuli and to establish whether this decrease in function could be ascribed to the storage-induced proteolysis of prominent platelet proteins observed by others. Platelet concentrates were stored at 4 degrees C and 25 degrees C for up to 14 days, and their ability to secrete and aggregate in response to appropriate stimuli was determined at 6, 96, and 192 hr after venipuncture. At each time point the protein complement of the platelets was also monitored by SDS-polyacrylamide gel electrophoresis to assess the extent of intracellular protein degradation. Platelets from concentrates stored at either temperature exhibited a decreased ability to respond to stimuli as storage time increased. After 8 days of storage at 4 degrees C and up to 9 days at 25 degrees C, no proteolysis of major platelet proteins was observed; however, complete loss of platelet function was observed. This strongly indicates that a decrease in platelet function should not be causally linked to degraded contractile-structural proteins and that extending the functional life of platelets during storage is still an attainable goal since proteolysis is not the inevitable result of short-term storage.
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Chicken myosin heavy chains from adult fast white muscle fibers (both normal and dystrophic), adult slow red fibers, and embryonic presumptive fast white fibers were compared by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by peptide mapping. The heavy chain of slow red myosin migrated electrophoretically more slowly than the heavy chains of the other myosins and differed markedly from them in its peptide maps. The heavy chain of dystrophic fast white myosin was similar to its normal counterpart by peptide mapping but showed slight differences. The peptide map of the heavy chain of embryonic presumptive fast white myosin had the general features of that of the heavy chain of fast white, not slow red, fibers but contained definite differences from the former. The results are consistent with the existence of a separate gene for the heavy chain of embryonic presumptive fast white myosin.
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The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular dystrophy.
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Electron microscopic x-ray microprobe analysis of pyroantimonate precipitates in platelets fixed in osmium tetroxide-pyroantimonate revealed calcium localization in the nucleoids of alpha-granules. This pool of calcium had largely disappeared within 10 sec after stimulation of platelets by thrombin. Such a rapid change suggests that this calcium pool may have a regulatory role in stimulus-response coupling.
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Physical properties of actomyosin from either fresh or stored platelets have been compared. Actomyosin obtained from platelets after 3 days of storage contained myosin that was 60-80% degraded to myosin rod. No myosin rod was detected in fresh platelets. The platelet myosin rod is similar to the rod produced by limited proteolysis of skeletal muscle myosin.
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