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Biomedical subjects

A Stracher

Publications and source records attributed to A Stracher.

At least 37 records · Page 2Linked to original sources
PubMed1989

Purification and characterization of a calcium binding protein with "synexin-like" activity from human blood platelets.

A calcium binding protein of Mr = 54,000 has been isolated from human blood platelets. This protein has been shown to enhance Ca2+-induced aggregation of phosphatidylserine liposomes, suggesting that it may be a member of a recently recognized class of binding proteins known to interact with phospholipids and the membrane in a Ca2+ dependent manner. Because of the "synexin-like" activity of this protein it may be involved in Ca2+ regulated platelet secretion as well as cell motility.

Annexin A7↗
PubMed1989

Neuromuscular recovery using calcium protease inhibition after median nerve repair in primates.

Inhibition of calcium-activated neutral protease, in muscle and nerve, by the tripeptide leupeptin after median nerve transection and epineural repair in monkeys (Cebus apella) was studied. Results indicate that inhibition of the protease after nerve repair facilitates morphologic recovery in denervated thenar muscles and in distal thenar nerve branches. In addition, functional recovery was facilitated in leupeptin-treated animals after nerve repair as measured by sensory and motor conduction velocities. Toxicologic testing showed that leupeptin, administered at 18 mg/kg, intramuscularly, twice daily, for 6 months did not adversely affect hematology, clotting, or plasma complement component C3 profiles. These data indicate that leupeptin is an effective and safe adjunct to peripheral nerve repair.

Animals↗
PubMed1988

Phosphorylation of platelet actin binding protein protects against proteolysis by calcium dependent sulfhydryl protease.

When Actin Binding Protein (ABP) isolated from human blood platelets is phosphorylated in vitro with a cyclic AMP dependent kinase it becomes resistant to proteolysis by the Calcium Dependent Sulfhydryl Protease (CDSP). This protection against proteolytic cleavage is specific for CDSP since phosphorylation of ABP does not protect against proteolysis by trypsin, papain and thermolysin. Thus, there appears to be a distinct phosphorylation site on the ABP molecule which is essential for regulating the initial proteolytic degradation of ABP by CDSP.

Blood Platelets↗
PubMed1987

Myosin isoforms in normal and dystrophic chickens.

The myosin isoform content in the affected fibers of chickens with inherited muscular dystrophy has been investigated with a new high-performance liquid chromatographic procedure for separation of the tryptic fragments of myosin subfragment 1 (S-1). The results indicate that dystrophic muscle contains substantial amounts of normal adult myosin, together with various myosin species present in normal 5-day posthatch chickens. Confirmation was obtained by comparative peptide mapping of the S-1 tryptic fragments and by N-terminal sequencing of 20-kDa species. Together with data on other contractile proteins and certain metabolic enzymes [Obinata, T., Takano-Ohmura, H., & Matsuda, R. (1980) FEBS Lett. 120, 195-198; Mikasa, T., Takeda, S., Shimizu, T., & Kitaura, T. (1981) J. Biochem. (Tokyo) 89, 1951-1962; Feit, H., & Domke, R. (1982) Cell Motil. 2, 309-315; Cosmos, E. (1966) Dev. Biol. 13, 163-181; Cosmos, E., & Butler, J. (1967) in Exploratory Concepts in Muscular Dystrophy and Related Disorders (Milhorat, A. R., Ed.) pp 197-204, Excerpta Medica, Amsterdam], the results are consistent with the hypothesis that there is a general defect in muscle maturation in avian dystrophy.

Aging↗
PubMed1987

Localization and inhibition of calcium-activated neutral protease (CANP) in primate skeletal muscle and peripheral nerve.

Localization of calcium-activated neutral protease in normal monkey (Cebus apella) skeletal muscle and peripheral nerve was studied by application of the indirect immunofluorescent and peroxidase-antiperoxidase techniques for light and electron microscopy. In muscle, the protease was demonstrated in association with endomysial collagen fibrils, basal lamina, sarcolemma, and Z-bands and also at neuromuscular junctions. In nerve, the enzyme was demonstrated in association with endoneurial collagen fibrils, basal lamina, axolemma, and neurofilaments of both myelinated and unmyelinated axons. Intramuscular injections of the thiol protease inhibitor, leupeptin, abolished Ca-activated neutral protease immunoreactivity in both muscle and peripheral nerve. These data suggest that the protease is localized both intracellularly and extracellularly in normal primate muscle and peripheral nerve. Inhibition of basal lamina associated neutral protease by leupeptin after denervation may hold significance for protecting this structure against degradation and preserving it as a neurotrophic lattice for axon regeneration.

Animals↗
PubMed1987

Treatment of experimental autoimmune myasthenia gravis in rabbits with leupeptin, a protease inhibitor.

We injected 12 New Zealand white rabbits intraperitoneally with 15 mg/kg Leupeptin on alternative days for about 4 months. After 1 week of Leupeptin treatment, they were challenged with purified acetylcholine receptor (AChR) from Torpedo californica in Freund's complete adjuvant. All control animals died within 60 days. Six animals treated with Leupeptin did not develop EAMG in spite of repeated AChR injections. Three animals developed clinical signs of EAMG after 65 days. The clinical course was short in the one that survived and prolonged in the 2 that finally died. All animals (Leupeptin-treated and controls) had circulating anti-AChR antibodies. Among the survivors, titers were slightly lower and EMG repetitive stimulation tests were normal. Leupeptin (0.02-200 mM) did not prevent curaremimetic [3H]toxin binding to AChR in membranes or in solution, nor dissociate AChR-toxin-antibody complexes. Immune response to antigens other than receptor remained intact in Leupeptin-treated animals. Leupeptin was not toxic at the doses given. The mechanism of this protection is not well understood. Leupeptin seems to decelerate the turnover rate of AChR induced by anti-AChR antibodies and/or to decrease the complement-mediated immune attack against the muscle end-plate.

Animals↗
PubMed1987

Enhancement of neuromuscular recovery after nerve repair in primates.

This investigation describes the use of the calcium-activated protease inhibitor, leupeptin, as an adjunctive therapy to the microsurgical repair of median nerves in a primate model. Our results indicate that leupeptin facilitates morphological recovery in denervated thenar muscles and in distal sensory and mixed motor-sensory nerve trunks and functional recovery measured by motor nerve conduction velocity. Toxicological testing of leupeptin showed that, when administered at a dose of 12 mg/kg, intramuscularly, once daily, haematological and clotting profiles were not adversely affected.

Animals↗
PubMed1986

Calcium-induced degeneration of the cytoskeleton in monkey and human peripheral nerves.

Biopsy specimens of human and monkey peripheral nerves, when incubated in calcium containing media, showed a loss of neurofilaments and microtubules with replacement by granular debris. Cytoskeletal structures remained intact when incubated in calcium-free media. Disruption of neurofilaments and microtubules in calcium containing media was inhibited by the thiol protease inhibitor, leupeptin. Similar incubations of excised Pacinian corpuscles revealed evidence of early terminal axon degeneration in the presence of calcium. These data substantiate the hypothesis that neural cytoskeletal degradation in primates and in man is calcium-mediated.

Animals↗
PubMed1985

Platelet cytoskeleton--membrane interactions.

The order of attachment of the purified platelet cytoskeletal proteins, F-actin, alpha-actinin and Actin Binding Protein (ABP) to the isolated platelet membrane has been investigated. Of the three proteins, only F-actin would directly interact with a membrane preparation that had been extensively washed to remove associated cytoskeletal proteins. alpha-Actinin would only add to the membrane to which F-actin had been re-attached and ABP only if both F-actin and alpha-actinin were present on the membrane. These studies provide some insight into the nature of the attachment of the platelet cytoskeleton to the cytoplasmic side of the membrane.

Actinin↗
PubMed1985

Partial reconstruction of the platelet membrane-cytoskeleton complex.

The order of attachment of the purified platelet cytoskeletal proteins to the isolated platelet membrane has recently been determined and it has been shown that only F-actin would interact directly with the membrane. Subsequently alpha-actinin and only then, ABP, would attach in that sequence. More recently the role of the platelet membrane glycoproteins Ib, IIb and IIIa in the binding of the cytoskeleton to the membrane has been investigated. The evidence suggests that F-actin attaches to the membrane via IIb and IIIa and the association of ABP with the cytoskeleton appears to be related to both its ability to interact with the F-actin-alpha-actinin complex as well as the glycoprotein Ib.

Blood Platelets↗
PubMed1984

Role of actin binding protein phosphorylation in platelet cytoskeleton assembly.

Actin binding protein from human blood platelets is shown to exist in the resting platelet as a phosphorylated protein and contains two residues of phosphate per 260,000 kd. Removal of one-half of these residues with E. coli alkaline phosphatase results in the loss of its ability to crosslink F-actin into a low speed sedimentable complex (its cytoskeleton) and to bind to an F-actin affinity column. Thus, phosphorylation-dephosphorylation of ABP may be an important regulatory mechanism by which the platelet regulates its shape via its cytoskeletal structure.

Actins↗
PubMed1984

Inhibition of neural and muscle degeneration after epineural neurorrhaphy.

Investigations were undertaken on the regeneration of transected rat sciatic nerves. The ability of the protease inhibitor leupeptin to inhibit wallerian degeneration and muscle atrophy was evaluated. After transection of a sciatic nerve and immediate neurorrhaphy, animals were treated with leupeptin for a period of 1 week to 6 months. Our results indicate a significant increase in the numbers of myelinated and unmyelinated neurofibers in the distal segment of treated nerves. Peroxidase tracer, injected intramuscularly, was transported by retrograde axonal flow and was observed to label increased numbers of treated axons both distal and proximal to the repair site. This finding suggests that treated neurofibers are functionally viable. Evaluation of muscle showed that secondary muscular atrophy is also significantly inhibited by leupeptin.

Animals↗
PubMed1982

Effect of various extraction solutions and thrombin activation on the composition of the platelet cytoskeleton.

When human blood platelets were immersed in an ice-cold solution containing 1% Triton X-1200, 40 mM KCl, 10 mM EGTA, 10 mM imidazole-HCl, and 2 mM NaN3 pH 7.0, a flocculent precipitate appeared immediately in the tube. This precipitate was collected at 3,000g and SDS-polyacrylamide gel analysis showed it to consist mainly of actin, alpha-actinin, actin-binding protein (ABP), and varying amounts of myosin. Any modifications of this solution used to isolate the platelets' Triton-insoluble cytoskeleton caused profound changes in the nature of the cytoskeleton isolated. Increasing the KCl concentration resulted in a lower yield of cytoskeletal actin and ABP. Inclusion of EDTA in the solution resulted in an increased amount of myosin associated with the cytoskeleton, whereas including MgATP decreased the myosin yield. Experiments with the purified proteins showed that ABP and myosin can each protect the actin from depolymerizing when dialyzed into the Triton solubilization solution. In addition, it was found that when platelets were stimulated with thrombin for 2 min prior to the addition of the Triton solution, 3-4 times more myosin was associated with the cytoskeletal precipitate. The results suggest, therefore, that any variations in solution conditions used for isolating the cytoskeleton from resting platelets, which results in alterations in the amount of ABP, may have profound effects on the state of actin polymerization. Likewise, in thrombin-activated platelets, it is suggested that the increased association of myosin with the cytoskeleton results in a greater stabilization of the F-actin associated with the cytoskeleton. These factors must be considered when interpreting the results regarding the nature of actin transformations in the resting and activated platelet.

Actinin↗
PubMed1982

Two major allelic forms of myosin light chain-1 in strains of normal and dystrophic chickens.

Evidence is presented from electrophoresis and peptide-mapping for the existence of two major allelic forms of myosin light chain-1 in the fast white muscle fibers of domestic chickens. One form predominates in birds of White Leghorn stock, the other in birds of New Hampshire Red stock. The two light chain-1 forms were invariant during development. Variability was not detected in light chains-2 or -3. The distribution of the two forms in two strains homozygous for the am gene for muscular dystrophy--Connecticut dystrophic and line 413--and their controls, White Leghorn and line 412, respectively, while clearly unrelated to avian dystrophy, emphasizes the heterogeneity in background genes of these non-inbred lines and indicates caution in their use in studies of avian dystrophy.

Alleles↗
PubMed1982

Effect of actin-binding protein on the sedimentation properties of actin.

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F-actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.

Actins↗
PubMed1981

Isolation and characterization of a calcium-sensitive alpha-actinin-like protein from human platelet cytoskeletons.

Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.

Actinin↗
PubMed1981

Abnormal myosin heavy chain variant associated with avian muscular dystrophy.

Avian muscular dystrophy is characterized by the degeneration of fast white skeletal muscle fibers, with onset during development. Using a one-dimensional peptide mapping technique, we have detected two forms of the myosin heavy chain in the fast white fibers of adult domestic chickens, one form characteristic of birds homozygous for muscular dystrophy, the other of their normal controls. Four dystrophic strains carrying the same gene for muscular dystrophy were examined. No differences were detected in the embryonic heavy chain peptide maps of normal and dystrophic chickens, consistent with the developmental onset of the condition. Differences were also absent from the peptide maps of heavy chains from slow red fibers, which are unaffected in dystrophy. No dystrophy-specific peptide map differences were detected in the three light chains. Analysis of peptide maps of rod and the heavy chain component of subfragment-1 from normal and dystrophic heavy chains indicates the presence of amino acid sequence differences in the two proteins.

Amino Acid Sequence↗