Hideo Sugita

Abnormal localization of laminin subunits in muscular dystrophies

To address potential involvement of muscle basal lamina and membrane cytoskeleton proteins in the etiology of non-dystrophinopathy muscular dystrophies, we examined the immunostaining intensity and distribution of laminin subunits (A, B1, B2 and M), type IV collagen, dystrophin and spectrin in skeletal muscle biopsies from 64 myopathic patients (17 Fukuyama congenital muscular dystrophy: FCMD, 13 congenital muscular dystrophy unrelated to FCMD: other CMD, 16 Duchenne muscular dystrophy: DMD, and 18 other neuromuscular diseases. In FCMD muscle, we found a significant reduction of laminin M (merosin; a striated muscle specific basal lamina-associated protein) with approximately 26% of levels seen in controls by quantitative immunofluorescence. Other CMD and DMD muscles showed less dramatic reductions (78%, 80%, respectively). The localization of laminin M was also abnormal in FCMD muscle. Laminin B1 and B2 showed abnormalities similar to those observed with laminin M, but were less marked. Laminin A was only detected in rare regenerating fibers in control biopsies, whereas it was seen around most muscle fibers in FCMD patients, and in dystrophin deficient muscle fibers from DMD patients and its carrier. Staining intensity of type IV collagen in FCMD muscle was not significantly different from the other diseases. These findings may implicate a primary or central role for the basal lamina in FCMD muscle.

Pathophysiology of muscle fiber necrosis induced by bupivacaine hydrochloride (Marcaine).

SummaryA single direct injection of a local anesthetic, 0.5% bupivacaine hydrochloride (BPVC) (Marcaine), into rat soleus and extensor digitorum longus (EDL) muscles produced massive fiber necrosis with extensive phagocytosis followed by rapid regeneration, predominantly in the soleus. Since the sarcoplasmic reticulum (SR) was functionally disturbed by BPVC administration as confirmed by an in vitro study, the sarcolemmal lysis seen in the early phase of degeneration was not assumed to simply results from direct damage to the plasma membrane caused by BPVC. The extracellular fluid containing a high concentration of calcium (Ca) ions then permeated into the sarcoplasm through the defective membrane resulting in hypercontracted myofibrils. Selective damage to the Z-line, an early sign of muscle degeneration, was shown by electron microscopy and SDS gel electrophoresis (preferential loss of α-actinin). Administration of leupeptin, a thiol protease inhibitor, proved to be ineffective in inhibiting the necrotic process, because the BPVC induced muscle fiber breakdown was probably too acute and fulminant to demonstrate the inhibitory effect upon the degenerative process. Well preserved satellite cells, peripheral nerves, and acetylcholinesterase activity, and the absence of fibrous tissue proliferation in this system may be responsible for the extremely rapid regeneration with complete muscle fiber type differentiation. Since the sequence of fiber breakdown induced by BPVC administration was similar to that of progressive muscular dystrophy, this chemical will be one of the most useful tools for studying the pathophysiology of fiber necrosis and regeneration in diseased muscle.

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

Abstract: In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7‐8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 μM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val‐methyl chloride, eluted at a position corresponding to a molecular mass of ∼ 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase‐like protease. This suggestion was confirmed by the finding that the 25‐kDa protein band was stained with anti‐elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopoly‐saccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.

Mosaic Expression of Dystrophin in Symptomatic Carriers of Duchenne's Muscular Dystrophy

A deficiency of the protein dystrophin is known to be the cause of Duchennes muscular dystrophy. To examine the expression of dystrophin in symptomatic female carriers of this X-linked recessive disorder, we performed immunohistochemical studies on muscle-biopsy specimens from three such carriers, using an antiserum raised against a synthetic peptide fragment of dystrophin. In all three carriers, most individual muscle fibers reacted either strongly or not at all to the antiserum for dystrophin; only 2 to 8 percent of fibers showed partial immunostaining. This mosaic staining pattern was present on both cross-sectional and longitudinal muscle specimens. Although the mosaic pattern was seen in all fiber types, more than 80 percent of type 2B and 2C fibers from two of the carriers did not react with the antiserum. Similar studies in nine normal subjects showed consistently strong staining of all muscle fibers. No muscle fibers from 31 patients with Duchennes muscular dystrophy reacted with the antiserum. We conclude that symptomatic carriers of Duchennes muscular dystrophy can be identified by a distinct mosaic pattern in the immunohistochemical staining of the surface membrane of skeletal-muscle specimens. This finding may have practical implications for genetic counseling, although it remains to be shown whether the same staining pattern will be found in muscle specimens from asymptomatic carriers of Duchennes muscular dystrophy.

Dystrophinopathy in isolated cases of myopathy in females

X-linked dystrophinopathy is the most common cause of isolated cases of myopathy in males. To investigate dystrophin abnormalities as a cause of myopathy in girls and women, we used dystrophin immunocytochemistry to study muscle biopsies from 505 girls and women with neuromuscular disease. Forty-six muscle biopsies showed a combination of fibers containing or lacking dystrophin; this mosaic immunostaining pattern denoted a carrier status. Twenty-one of 46 (45.6%) had a family history of Duchenne muscular dystrophy in males. Twenty-five of 46 (54.3%) were isolated cases, with no previous family history of neuromuscular disorder. The laboratory findings of the isolated cases were consistent with the familial cases; all showed myopathic histopathology and abnormal elevations of serum CK. The clinical presentations of the isolated cases varied but were consistent with the familial cases: 40% (10/25) of isolated cases showed proximal limb weakness before age 10, 24% (6/25) presented with myalgias or cramps, 24% (6/25) presented with incidental findings of grossly elevated CK levels, 8% (2/25) noted easy fatigue, and 4% (1/25) had slowly progressive proximal limb weakness beginning at age 45. From our data, the clinical criteria for consideration of an underlying dystrophinopathy in isolated female cases of myopathy are CK levels greater than 1,000 IU/1 and myopathic histopathology. About 10% of the isolated cases of hyperCKemic myopathy (25/210) were proven by dystrophin analysis to have a dystrophinopathy as the cause of their disease (manifesting carriers of Duchenne dystrophy). However, we feel that this may be an underestimate. The correct diagnosis in these patients is imperative for appropriate genetic counseling to the patients and their families.

Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice

The amino acid sequence of the polyclonal antibodies we developed against the carboxyl terminus of the dystrophin-related protein, the putative gene product of B3 cDNA, had no homologous sequence to the dystrophin molecule except for two amino acids located at its ends for immunization. By immunohistochemical examination in C57B1/10ScSn and C57B1/10ScSn-mdx mice we found that the DRP was expressed on the surface membrane of fetal muscle fibers, was assembled at the neuromuscular junctions of the mature muscle fibers, and reappeared on the surface membrane of muscle fibers after denervation. Its localization was similar to that of the acetylcholine receptor, suggesting that DRP is one of the cytoskeletons which organize and stabilize the cytoplasmic domain of the acetylcholine receptor.

Alzheimer's disease amyloid β-clipping enzyme (APP secretase): Identification, purification, and characterization of the enzyme

Alzheimers disease (AD) is the most frequent cause of dementia, although no genetic abnormality has been identified. Recent studies have elucidated the molecular defect in AD, including the abnormal deposition of amyloid beta peptide (beta/A4) in senile plaques of affected individuals. Normal brain contains the enzyme, APP secretase, which cleaves inside the beta/A4 portion of the precursor protein (APP); abnormal processing of APP occurs in AD brain. Until now, no evidence has been provided that APP secretase is an intracellular proteinase. We have now prepared two synthetic substrates of APP secretase, both of which contain the cleavage point and are much more sensitive than substrates previously available to identify APP secretase. Using these substrates, we found an intracellular proteinase that has APP secretase activity. This proteinase has been identified as cathepsin B.

Identification of a putative amyloid A4-generating enzyme as a prolyl endopeptidase

The A4 amyloid peptide is deposited in Alzheimers disease inside neurons as neurofibrillary tangles or extracellularly as vascular amyloid. The A4 peptide is cleaved off by an unidentified proteinase from a larger precursor protein (APP), which resembles a cell surface receptor. The proteinase, which cleaves off the membrane‐spanning domain of APP, may be important in amyloid formation. To evaluate this, a model peptide substrate, succinyl‐ isoleucyl‐alanine‐methylcoumarinamide, which is homologous to the C‐terminal portion of A4 peptide, was synthesized to screen the putative A4‐generating proteinase. On chromatographie purification, it was found that two proteinases are involved in the hydrolysis of the peptide, the major one being identified as a prolyl endopeptidase. This evidence may facilitate elucidation of the mechanism of amyloid deposition in Alzheimers disease.

Identification of Ca2+‐Activated Neutral Protease in the Peripheral Nerve and Its Effects on Neurofilament Degeneration

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca2+ and was inhibited by the addition of E‐64‐c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca2+‐activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 ± 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.

The frequency of patients with dystrophin abnormalities in a limb-girdle patient population.

Of the 3,048 diagnostic muscle biopsies processed by the National Institute of Neuroscience, Tokyo, over 12 years, 41 cases carried the clinical diagnosis of limb-girdle muscular dystrophy. We have analyzed all 41 cases for dystrophin content in muscle by both immunofluorescence and immunoblot. We identified five male patients with an abnormal dystrophin pattern diagnostic of Becker muscular dystrophy, and two female patients with dystrophin patterns consistent with a manifesting carrier of Duchenne muscular dystrophy diagnosis. Thus, 17% of our limb-girdle patients showed a dystrophinopathy, indicating that they in fact had a disorder related to Duchenne/Becker muscular dystrophy. Misclassification of isolated male limb-girdle patients was 31% (4/13), while misclassification of isolated female limb-girdle patients was 13% (2/15). Using multiplex polymerase chain reaction analyses of small amounts of muscle biopsy DNA confirmed a dystrophin gene deletion in all five male Becker dystrophy patients identified. This study emphasizes the clinical overlap between limb-girdle muscular dystrophy and dystrophinopathies, and reinforces the necessity of dystrophin protein and gene studies for the accurate clinical diagnosis of isolated cases of muscular dystrophy.