Distribution of calcium-activated neutral protease inhibitor in the central nervous system of the rat.

The ubiquitous existence of calcium-activated neutral protease (CANP, calpain), an enzyme whose activity is regulated by calcium ions and a specific endogenous CANP inhibitor (calpastatin), is well known. Although there has been much investigation concerning the distribution and role of CANP, investigations of the distribution of the CANP inhibitor using immunohistochemical techniques are rare. We made antiserum against a 40K fragment of cDNA corresponding to two C-terminal repeats of rat liver CANP inhibitor expressed in Escherichia coli. Using this antiserum, we examined the distribution of CANP inhibitor in the rat central nervous system by the ABC technique and compared it with the distribution of CANP. Neurons and glias were stained, with the cytosol stained diffusely and the cell membranes stained clearly and strongly. Axons and myelin were stained faintly, but nuclei and vessels were not stained. The distribution of CANP inhibitor was thus found to be similar to that of CANP.     

Immunohistochemical distribution of calcium-activated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus

Intracellular accumulation of Ca2+ after brain ischemia is regarded as one of the principal causes of neuronal death, but details of the intracellular events occurring after Ca2+ accumulation have not yet been described. We propose that a calcium-activated neutral proteinase which can degrade neuronal cytoskeletal proteins might link Ca2+ accumulation and irreversible injury of the neuronal intracellular structure. First, therefore, we examined the distribution of calcium-activated neutral proteinase in normal brains. Immunohistochemical distribution of calcium-activated neutral proteinases (CANP) with high and low sensitivity to Ca2+ (muCANP and mCANP) and of endogenous CANP inhibitor was investigated in the dorsal hippocampus of the rabbit. muCANP-immunoreactivity was detected in almost all of the pyramidal cells and granule cells and in some other neurons. A full-length staining from perikarya to dendrites was shown in muCANP-positive neurons. mCANP-immunoreactivity was found mainly in four kinds of hippocampal interneurons: 1) basket cells in the stratum oriens of Ammon's horn, 2) pyramidal basket cells at the boundary of pyramidal cell layer and stratum oriens, 3) polymorphic cells in the hilar region of dentate gyrus, and 4) pyramidal or fusiform basket cells at the inner boundary of the granule cell layer and the hilar region. The distribution of these four kinds of neurons was similar to that of parvalbumin-containing GABAergic neurons. CANP inhibitor immunoreactivity was confined to pyramidal cells in the CA3-CA3c region and some hilar neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of an endogenous 68 kD inhibitor of calcium-activated neutral proteinase (CANP) from bovine brain: immunoblot identification and characterization

A calcium-activated neutral proteinase (CANP)-specific endogenous inhibitor (calpastatin) was purified from bovine brain by successive column chromatography. The purified inhibitor exhibited a major band on sodium dodecylsulfate polyacrylamide gel electrophoresis with an approximate molecular weight of 68 kD. The polyclonal antisera raised to the inhibitor strongly reacted with the 68 kD protein band. Two lightly stained bands approximately 55-68 kD and 120-130 kD were also recognized by the inhibitor antiserum. The inhibitor specifically inhibited CANP activity and the half-maximal inhibition was found with 75 ng of calpastatin per 1 micrograms of CANP in a final volume of 125 microliters. Cathepsin B and papain were not inhibited by the inhibitor, while trypsin and chymotrypsin were inhibited to some extent. The inhibitor formed a complex with CANP and the inactive complex was dissociated into active fractions of enzyme and calpastatin in the presence of EGTA.     

Mu-type calcium-activated neutral protease in the rat peripheral nerve

Previously we reported results of an incubation experiment with neurofilaments that supported the existence of a mu-type of Ca2+-activated neutral protease in the rat peripheral nerve; it was active with microM order Ca2+ (mu-CANP). This time, we partially purified the mu-CANP from a crude CANP fraction of rat peripheral nerve by using a DE52 column and a Phenyl-Sepharose column followed again by DE52 column chromatography. The presence of mu-CANP was verified by an immunoblotting technique. The mu-CANP degraded the neurofilament triplet as previously reported; i.e., among the neurofilament triplet, the 160K component was most sensitive, and in the order of the 160K, 68K, and 200K components, respectively.     

Inhibitory effect of a brain derived peptide preparation on the Ca++-dependent protease, calpain

Overactivated calpain might be a key factor in destruction of cytoskeletal proteins involved in the pathophysiology of ischemia and disorders like Alzheimer's disease. Therapeutic effects imply the possible interference of Cerebrolysin (Ebewe Arzneimittel, Austria) with these molecular events. In this work several in vitro methods have been applied to investigate the interaction between Cerebrolysin and calpain [Enzyme Commission (EC) number: 3.4.22.17]. A conventional caseinolytic assay beside two flourimetric assays using a synthetic peptide substrate and a fluorescence labelled cytoskeletal protein [microtubule-associated protein 2 labelled with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (MAP2-DTAF)] respectively for a highly sensitive fluorimetric calpain activity assay were applied for kinetic analysis. The caseinolytic assay showed that the drug inhibits both mu- and m-calpain and to a significantly lower extent also trypsin [Enzyme Commission (EC) number: 3.4.21.1] and papain [Enzyme commission (EC) number: 3.4.22.6]. Dialysis experiments revealed Cerebrolysin mediated calpain inhibition to be reversible. Kinetic analysis exhibited a non-competitive, or tight-binding competitive, mode of inhibition. This latter mode, substantiated by serial dilution experiments, and the likely existence of calpastatin in a brain derivative suggests the occurrence of calpastatin fragments or calpastatin-like fragments in Cerebrolysin. The clearly competitive inhibition of trypsin by the drug indicates distinct mechanisms and active components against different proteases.     

Expression of tissue inhibitor of metalloproteinase-1 in progression muscular dystrophy

Objective Tissue inhibitor of metalloproteinase-1(TIMP-1) is a multifunctional protein that has the capacity to modify cellular activities and to modulate matrix turnover. This paper revealed the contributive role of TIMP-1 in progressive muscular dystrophy (PMD). Methods We examined the expression and cellular localization of TIMP-1 protein using biopsied frozen muscle from patients with Duchenne muscular dystrophy ( DMD) , Becker muscular dystrophy (BMD) , congenital muscular dystrophy (CMD) by immunohistochemistry, double immunofluorescence and Western blot analysis. Results The results of immunohistochemistry and double immunofluorescence showed that TIMP-1 was positive only in vascular endothelial cells of normal muscles. Immunohistochemistry and Western blot analysis showed that the staining intensity was distinctly increased in some dystrophic muscles of PMD for TIMP-1. Double immunofluorescence revealed that TIMP-1 strongly expressed in the regenerating muscle fibers, macrophages and macrophage infiltrating necrotic fibers. Some activated fibroblasts in endomysium and perimysium of DMD and CMD muscles were also positive for TIMP-1. Conclusion The functional consequence of overexpression of TIMP-1 in the dystrophic muscles is unknown, but the elevated local expression of TIMP-1 in diseased muscles of PMD and their distinct distribution pattern provide evidence that TIMP-1 may participate in the pathogenesis of PMD.     

Expression of tissue inhibitor of metalloproteinase-1 in progression muscular dystrophy

Objective Tissue inhibitor of metalloproteinase-1 （TIMP-1） is a muhifunctional protein that has thc capacity to modify cellular activities and to modulate matrix turnover. This paper revealed the contributive role of TIMP-1 in progressive muscular dystrophy （PMD）. Methods We examined the expression and cellular localization of TIMP-1 protein using biopsied frozen muscle from patients with Duchenne muscular dystrophy （DMD）, Becker muscular dystrophy （BMD） , congenital muscular dystrophy （CMD） by immunohistochemistry, double immunofluorescence and Western blot analysis. Results The results of immunohistochemistry and double immunofluorescence showed that TIMP-1 was positive only in vascular endothelial cells of normal muscles. Immunohistochemistry and Western blot analysis showed that the staining intensity was distinctly increased in some dystrophic muscles of PMD for TIMP-1. Double immunofluorescence revealed that TIMP-1 strongly expressed in the regenerating muscle fibers, macrophages and macrophage infiltrating necrotic fibers. Some activated fibroblasts in endomysium and perimysium of DMD and CMD muscles were also positive for TIMP- 1. Conclusion The functional consequence of overexpression of TIMP-1 in the dystrophic muscles is unknown, but the elevated local expression of TIMP-1 in diseased muscles of PMD and their distinct distribution pattern provide evidence that TIMP-1 may participate in the pathogenesis of PMD.     

Therapeutic trial with protease inhibitor (Leupeptin) in chicken muscular dystrophy

Summary For the purpose of observing the therapeutic benefit of protease inhibitors for progressive muscular dystrophy, a large quantity of doses of leupeptin of 10 mg/kg/day and 50 mg/kg/day were administered i.p. to male chickens afflicted with hereditary muscular dystrophy (line 413) for 4 months starting on the 7th day ex ovo. No clinical improvement was identified in physical ability as a result of the examination by flip test, and creatine kinase (CK) values. The number of necrotic fibers in the pectoralis superficialis (PS) muscle which is known to be preferentially damaged in dystrophic chicken, did not decrease significantly in the birds treated with 10 mg leupeptin/kg/day (number of necrotic fibers; 47.7/mm²) and 50 mg/kg/day (46.4/mm²) as compared to that of the untreated ones (43.2/mm²). A morphometric analysis of fiber diameter distribution also showed no statistical difference between the treated and untreated birds.In the second group, 10 mg leupeptin/kg and a combination of leupeptin and bestatin of 10 mg/kg each were injected directly into the left lower half of the PS muscle three times a week for 4 months. Necrotic fibers were still present in the injected site, remote area of the left upper PS muscle treated with leupeptin (52.7/mm²), leupeptin and bestatin (52.2/mm²), and contralateral right upper PS muscle (41.6 and 53.5/mm², respectively). The number of necrotic fibers in treated muscles was again not significantly different from that in untreated dystrophic ones (39.6/mm²). In fiber diameter analysis, no statistical difference was recognized between the treated and untreated dystrophic muscles.Partly supported by a grant-in-aid for New Drug Development Research from the Ministry of Health and Welfare, Japan     

Effects of prednisone in canine muscular dystrophy

Glucocorticoid use may provide short-term functional improvement in boys with Duchenne muscular dystrophy (DMD). We report functional and histopathologic changes following a 4-month course of daily oral prednisone in a canine model of DMD, termed golden retriever muscular dystrophy (GRMD). Muscle extension forces in GRMD dogs treated daily with 1 and 2 mg/kg prednisone measured 2.349 +/- 0.92 and 3.486 +/- 0.67 N/kg, respectively, compared to 1.927 +/- 0.63 N/kg in untreated GRMD controls (p < 0.05 for 2 mg/kg group); GRMD muscle flexion forces measured 0.435 +/- 0.13 and 0.303 +/- 0.08 N/kg, respectively, compared to 0.527 +/- 0.01 N/kg in untreated GRMD controls (p < 0.05 for both groups). Although cranial sartorius hypertrophy and tibiotarsal joint angles also tended to improve, myofiber calcification increased and fetal myosin expression decreased following prednisone. Thus, functional data indicate benefit but histopathologic changes following prednisone treatment in GRMD suggest possible deleterious consequences.     

A calcium-activated neutral protease in the frog nervous system which degrades rapidly transported axonal proteins

A Ca2+-activated protease (CANP) was prepared from a soluble extract of the frog nervous system and separated from an endogenous high-molecular weight inhibitor by ion exchange chromatography. Its proteolytic action was tested on rapidly transported [3H]leucine-labelled proteins from sensory axons of the sciatic nerve. The enzyme was activated at neutral pH by Ca2+ at millimolar level, and was inhibited by leupeptin and the endogenous inhibitor. Its most pronounced degradative action was exerted towards two prominent rapidly transported peaks corresponding to 82 kDa and 90 kDa proteins. Proteolysis was accompanied by the appearance of mainly 16 kDa degradation products. When newly synthesized soluble proteins of the spinal dorsal ganglia were used as substrates, the presence of CANP resulted in degradation of proteins in a broad molecular-weight range (40-200 kDa).     

Clinical,pathological, and genetic features of limb-girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families

We report on the clinical, pathological, and genetic features of 7 patients with limb-girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7 ± 3.1 years (mean ± SD), and loss of ambulance occurred at 38.5 ± 2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca²⁺-binding domain. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1493–1501, 1998     

Dopamine-stimulated changes in activated calpain I in rat hippocampal slices

Activated calpain I immunoreactivity (76 kDa band) was detected in membranes prepared from rat brain hippocampal slices using a polyclonal antiserum raised against an N-terminus peptide of the cleaved subunit of calpain I. While basal levels of activated calpain I were stable over incubation times, 1 nM dopamine (DA) produced an initial 32% increase (5 min) in the 76 kDa protein followed by a 53% decrease in this band at 20 min of incubation. The DA-induced changes in activated calpain I immunoreactivity were blocked by the calpain inhibitor peptide, N-acetyl-Leu-Leu-norleucinal (100 μM) or by EGTA. Basal levels of the 76 kDa band were not affected by the calpain inhibitor. These changes in activated calpain I, elicited by DA, are in accord with the DA-induced decreases in the levels of the calpain substrate, γ PKC (Yurko-Mauro and Friedman; J Cell Biochem [Abstr] 180:80, 1994; J Neurochem 65: 1622–1630, 1995) and suggest that DA activates this Ca⁺⁺-dependent protease in its regulation of neuronal signal transduction. © 1996 Wiley-Liss, Inc.     

Dystrophin and dystrophin-related protein in the central nervous system of normal controls and Duchenne muscular dystrophy

To clarify the localization and characterization of dystrophin and dystrophin-related protein (DRP) in the human central nervous system (CNS), we carried out immunoblotting and immunostaining studies using three region-specific anti-dystrophin and one anti-DRP antibodies. With immunostaining, punctate immuno-reactivity of dystrophin was seen along the cell bodies and dendrites of the cerebral cortical neurons and cerebellar Purkinje cells in the normal controls autopsied. By contrast, dystrophin was not detected at all in the CNS of Duchenne muscular dystrophy (DMD) patients with intellectual disturbance. Immunoreactivity of DRP was observed in the vascular walls of both normal and DMD brains, but not in the neuronal cells. Compensatory increase of DRP was not noted in DMD brains. This study suggests that in DMD the brain-type dystrophin originally present in neurons is absent and may be related to the intellectual disturbance.Supported by a grant (2-A) from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan     

Immunoblot analysis of sarcoplasmic calcium binding proteins in Duchenne muscular dystrophy

The Western blotting technique was used to detect parvalbumin and S-100 protein in muscles from 10 Duchenne muscular dystrophy (DD) patients, 13 patients with other muscle diseases and 5 age-matched healthy subjects. DD muscles were found to contain decreased amounts of parvalbumin and the S-100 protein. The parvalbumin level did not relate to the age of the patients and the stage of the disease. The S-100 protein decreased progressively with the age of the patients. In a very advanced DD case the S-100 protein was present in trace amounts. In other primary myopathies, including Becker dystrophy, and neurogenic muscular atrophy both parvalbumin and S-100 protein levels were similar to that observed in healthy subjects. The decrease in the amount of both calcium binding proteins may contribute to the elevation of free intracellular Ca²⁺ level in the sarcoplasm of dystrophic muscle and would result in abnormalities in processes regulated by these proteins. The mechanism(s) responsible for the decrease of parvalbumin and S-100 protein in DD muscles are discussed.     

A study of platelet protein phosphorylation in duchenne muscular dystrophy: Further evidence against the generalised membrane defect theory

As a test of the generalised defect theory for Duchenne muscular dystrophy (DMD), basal and calcium-dependent platelet protein phosphorylation was examined in order to determine if the increased concentration of calcium in DMD skeletal muscle is reflected in DMD platelets.Protein phosphorylation was quantitated by gradient slab gel electrophoresis and autoradiography. The number of phosphoproteins in each phosphoprotein peak was determined by comparison with two-dimensional gel electrophoresis. Many phosphoprotein peaks were present in unstimulated platelet preparations both in whole platelet homogenates and in intact platelets. Two of these phosphoprotein peaks were calciumdependent, one was a single phosphoprotein, the other consisted of 4 phosphoproteins. No disease-related differences were observed in either basal or calcium-stimulated phosphoproteins.These results do not support previous reports of platelet abnormalities in DMD, and provide further evidence that the biochemical defect in Duchenne muscular dystrophy is neither generalised nor a membrane defect. The biochemical defect in DMD should be regarded as a skeletal muscle abnormality until proved otherwise.     

Unusual expression of emerin in a patient with X-linked Emery-Dreifuss muscular dystrophy

We report on a patient with the typical clinical findings of Emery–Dreifuss muscular dystrophy due to a mutation in the emerin gene that should have produced a higher molecular weight protein. Immunohistochemical analysis showed emerin localized only in the cytoplasm of muscle fibres and lymphoblastoid cells. The emerin molecule contained the nucleoplasmic domain and the transmembrane domain responsible for nuclear membrane targeting, so its incorrect localization and lack of function could be due to abnormal folding resulting in rapid degradation or inability to bind other nuclear proteins.     

Purification and characterization of Ca2+-activated neutral protease inhibitor from human platelets

An endogenous inhibitor of Ca²⁺-activated neutral protease (CANP) was purified to homogeneity from the soluble fraction of human platelets by the combination of heat treatment, ammonium sulfate fractionation, ion exchange chromatography and gel filtration. The purified inhibitor was found to be a tetramer composed of identical subunits and each subunit has a molecular weight of 63 K. The purified protein exerted specific inhibition against the low Ca²⁺-requiring form of CANP (μ-CANP) purified from human platelets in the presence of micromolar concentration of Ca²⁺. The kinetic study revealed that the inhibition is non-competitive with Ki value of 3.2 × 10^?8 M.     

Lack of anionic phospholipid calcium binding sites in Duchenne muscular dystrophy.

We studied membrane ultrastructural localization of anionic phospholipids (AP) and sialic acid (SA) calcium binding sites in muscle biopsies from Duchenne muscular dystrophy (DMD) and 3 Becker's muscular dystrophy (BMD) patients using polymyxin B (PXB) and limulus polyphemus (LP) as cytochemical markers. We found that AP calcium binding sites are lacking at muscle cell surface in all DMD muscle tissues, in both intact and degenerating muscle fibers. In BMD, AP have an unusual distribution along plasma membrane. Sialic acid calcium binding sites have the same localization along plasma membrane and basal lamina in DMD, BMD, and control muscles. The absence or alterations of structures involved in calcium binding in DMD and BMD may alter membrane calcium permeability, leading to abnormal Ca2+ influx into cells causing muscle necrosis.