Dp71 can restore the dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy.

Two lines of transgenic mdx mice have been generated that express a 71 kD non-muscle isoform of dystrophin (Dp71) in skeletal muscle. This isoform contains the cysteine-rich and C-terminal domains of dystrophin, but lacks the N-terminal actin-binding and central spectrin-like repeat domains. Dp71 was associated with the sarcolemma membrane, where it restored normal expression and localization of all members of the dystrophin-associated glycoprotein complex. However, the skeletal muscle pathology of the transgenic mdx mice remained severe. These results indicate that the dystrophin C terminus cannot function independently to prevent dystrophic symptoms and confirms predictions based on patient data that both the N and C-terminal domains are required for normal dystrophin function.

Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice.

Dp71 is a non-muscle product of the Duchenne muscular dystrophy gene. It consists of the cysteine-rich and C-terminal domains of dystrophin. We have generated transgenic mdx mice which do not have dystrophin but express Dp71 in their muscle. In these mice, Dp71 was localized to the plasma membrane and restored normal levels of dystrophin associated proteins (DAPs), indicating that Dp71 is capable of interacting with the DAPs in a similar manner to dystrophin. However, the presence of Dp71 and DAPs in the muscle fibres of mdx mice was not sufficient to alleviate symptoms of muscle degeneration.

Beta-sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12.

beta-Sarcoglycan, a 43 kDa dystrophin-associated glycoprotein, is an integral component of the dystrophin-glycoprotein complex. We have cloned human beta-sarcoglycan cDNA and mapped the beta-sarcoglycan gene to chromosome 4q12. Pericentromeric markers and an intragenic polymorphic CA repeat cosegregated perfectly with autosomal recessive limb-girdle muscular dystrophy in several Amish families. A Thr-to-Arg missense mutation was identified within the beta-sarcoglycan gene that leads to a dramatically reduced expression of beta-sarcoglycan in the sarcolemma and a concomitant loss of adhalin and 35 DAG, which may represent a disruption of a functional subcomplex within the dystrophin-glycoprotein complex. Thus, the beta-sarcoglycan gene is the fifth locus identified (LGMD2E) that is involved in autosomal recessive limb-girdle muscular dystrophy.

Discharge characteristics of steady-state high-density plasma source based on cascade arc discharge with hollow cathode.

We developed a steady-state high-density plasma source by applying a hollow cathode to a cascade arc discharge device. The hollow cathode is made of a thermionic material (LaB6) to facilitate plasma production inside it. The cascade arc discharge device with the hollow cathode produced a stationary plasma with an electron density of about 1016 cm-3. It was found that the plasma source produces a strong pressure gradient between the gas feed and the vacuum chamber. The plasma source separated the atmospheric pressure (100 kPa) and a vacuum (100 Pa) when the discharge was performed with an argon gas flow rate of 5.0 l/min and a discharge current of 40 A. An analysis of the pressure gradient along the plasma source showed that the pressure difference between the gas feed and the vacuum chamber can be well described by the Hagen-Poiseuille flow equation, indicating that the viscosity of the neutral gas is the dominant factor for producing this pressure gradient. A potential profile analysis suggested that the plasma was mainly heated within cylindrical channels whose inner diameter was 3 mm. This feature and the results of the pressure ratio analysis indicated that the temperature, and, thus, viscosity, of the neutral gas increased with the increasing number of intermediate electrodes. The discharge characteristics and shape of the hollow cathode are suitable for plasma window applications.

Comparison of the sensitivities of plantar nerve conduction techniques for early detection of diabetic sensory polyneuropathy.

The purpose of this study was to determine the most sensitive diagnostic test for nerve conduction study (NCS) of the foot for early detection of diabetic polyneuropathy. We compared the sensitivities for diagnosis of sensory polyneuropathy of four different nerve conduction techniques in the same nerves: nerve conduction studies of the medial plantar nerve with surface electrodes using three different techniques and a nerve conduction study of the digital and interdigital nerves of the foot using a near-nerve needle technique. In 25 patients with diabetic polyneuropathy with normal routine NCS, diagnosis of sensory neuropathy was confirmed by medial plantar NCS in 5 patients (20.0%) using Guiloff's method, in 5 patients (20.0%) using Ponsford's method and in 9 patients (36.0%) using Hemmi's method. In digital and interdigital NCS of the foot, a definite neuropathy pattern was observed in 15 patients (60.0%). The most common abnormality was low amplitude of sensory nerve action potential, indicating axonal degeneration. This study demonstrated that digital and interdigital NCS using the near-nerve needle technique is a more sensitive method for detection of early-stage diabetic polyneuropathy.

Muscular fatigue and decremental response to repetitive nerve stimulation in X-linked spinobulbar muscular atrophy.

BACKGROUND AND PURPOSE: We report decremental responses to repetitive nerve stimulation (RNS) in 11 patients diagnosed with X-linked spinobulbar muscular atrophy (X-SBMA). METHODS: The compound muscle action potential (CMAP) of the right abductor digiti minimi (ADM) and trapezius (TZ) in response to a 3-Hz stimulation of the ulnar nerve at the wrist and accessory nerve at the neck were recorded by surface electrodes. RESULTS: A decremental response to RNS was observed in 90.9% of the TZ muscle and 27.2% in the ADM muscle of patients with X-SBMA. CONCLUSION: These electrophysiological features of X-SBMA are considered to be useful for diagnosis of X-SBMA. Furthermore, the waning phenomena that mostly appeared in the TZ muscle and increment of CMAP in RNS after the exercise also suggest a unique manifestation in X-SBMA.

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass.

RNA interference (RNAi) offers a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. Since it relies on small interfering RNAs (siRNAs), a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on vector delivery may be of only limited clinical use. The more desirable approach is to directly apply active siRNAs in vivo. Here, we report the effectiveness of in vivo siRNA delivery into skeletal muscles of normal or diseased mice through nanoparticle formation of chemically unmodified siRNAs with atelocollagen (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, in mouse skeletal muscles or intravenously, caused a marked increase in the muscle mass within a few weeks after application. These results imply that ATCOL-mediated application of siRNAs is a powerful tool for future therapeutic use for diseases including muscular atrophy.

Caveolin-3 regulates myostatin signaling. Mini-review.

Caveolins, components of the uncoated invaginations of plasma membrane, regulate signal transduction and vesicular trafflicking. Loss of caveolin-3, resulting from dominant negative mutations of caveolin-3 causes autosomal dominant limb-girdle muscular dystrophy (LGMD) 1C and autosomal dominant rippling muscle disease (AD-RMD). Myostatin, a member of the muscle-specific transforming growth factor (TGF)-beta superfamily, negatively regulates skeletal muscle volume. Herein we review caveolin-3 suppressing of activation of type I myostatin receptor, thereby inhibiting subsequent intracellular signaling. In addition, a mouse model of LGMD1C has shown atrophic myopathy with enhanced myostatin signaling. Myostatin inhibition ameliorates muscular phenotype in the model mouse, accompanied by normalized myostatin signaling. Enhanced myostatin signaling by caveolin-3 mutation in human may contribute to the pathogenesis of LGMD1C. Therefore, myostatin inhibition therapy may be a promising treatment for patients with LGMD1C. More recent studies concerning regulation of TGF-beta superfamily signaling by caveolins have provided new insights into the pathogenesis of several human diseases.

Inhibitors of the TGF-beta superfamily and their clinical applications.

The transforming growth factor-beta (TGF-beta) superfamily includes TGF-betas, activin, myostatin and bone morphogenetic proteins. Misregulation of the activity of TGF-beta family members is involved in pathogenesis of cancer, muscular dystrophy, obesity and bone and tooth remodeling. Natural inhibitors for the TGF-beta superfamily regulate fine-tuning of activity of TGF-beta family in vivo. In addition to natural inhibitors for the TGF-beta family, soluble forms of receptors for the TGF-beta family, blocking monoclonal antibodies and small chemical TGF-beta inhibitors have been developed. In this review, we summarize recent advances in our understanding of inhibitors for the TGF-beta superfamily and their medical applications.

Structural organization, complete genomic sequences and mutational analyses of the Fukuyama-type congenital muscular dystrophy gene, fukutin.

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy in combination with cerebral cortical dysplasia. Previously, we identified the gene responsible for FCMD, termed fukutin, through positional cloning. In this study, we have sequenced 131892 bp of genomic DNA in the region of the fukutin gene on chromosome 9q31 and obtained its complete genomic structure. The fukutin genomic sequence spans approximately 100 kb and is organized into 10 exons (41-6067 bp) and nine introns (1841-21460 bp). Using these sequence data, we have identified three novel fukutin mutations in FCMD patients. We have also located a putative TATA box in the flanking 5' region and identified numerous alternatively spliced fukutin mRNA transcripts. Analysis of expressed sequence tag clusters within the region revealed two novel genes upstream of the fukutin gene. These data provide fundamental information to support detailed genetic and functional analyses of the fukutin gene.

Absence of gamma-sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12.

We have partially sequenced rabbit skeletal muscle gamma-sarcoglycan, an integral component of the dystrophin-glycoprotein complex. Specific antibodies were produced against a gamma-sarcoglycan peptide and used to examine the expression of gamma-sarcoglycan in skeletal muscle of patients with severe childhood autosomal muscular dystrophy linked to chromosome 13q12 (SCARMD). We show by immunofluorescence and Western blotting that in skeletal muscle from these patients gamma-sarcoglycan is completely absent and alpha- and beta-sarcoglycan are greatly reduced in abundance, whereas other components of the DGC are preserved. In addition, we show that in normal muscle alpha-, beta-, and gamma-sarcoglycan constitute a tightly associated sarcolemma complex which cannot be disrupted by SDS treatment.

Merosin-negative congenital muscular dystrophy associated with extensive brain abnormalities.

Congenital muscular dystrophies (CMDs) are autosomal recessive, heterogeneous disorders. The most frequent form in the Caucasian population is classic (occidental) CMD, characterized by exclusive muscle involvement, although abnormal brain white matter signals are occasionally observed on MRI. Recently, deficiency of merosin, the laminin isoform in skeletal muscle, has been identified in classic CMD patients. In skeletal muscle, merosin is a native ligand for dystroglycan linking the extracellular matrix and dystrophin. Thus, merosin deficiency could disrupt the attachment of muscle cell to the extracellular matrix and lead to muscle cell necrosis. Since merosin is also expressed in the nervous system and has biologic activities on neurite outgrowth and Schwann cell migration, deficiency of merosin could affect the development of the nervous system. We report here two patients with merosin-negative CMD presenting extensive brain abnormalities characterized by cortical anomaly, polymicrogyria, and abnormal white matter signals.

Peripheral nerve involvement in merosin-deficient congenital muscular dystrophy and dy mouse.

Merosin, also called laminin-2, is an isoform of laminin comprised of the alpha 2, beta 1 and gamma 1 chains. Deficiency of merosin alpha 2 chain was recently identified as the primary cause of the classical form of congenital muscular dystrophy (CMD), an autosomal recessive neuromuscular disorder characterised by muscular dystrophy and brain white matter abnormalities. Interestingly, merosin-deficient CMD and its animal model dy mouse are also accompanied by dysmyelination of peripheral motor nerves. In peripheral nerve, merosin is expressed in the endoneurium surrounding the Schwann cell/myelin sheath, while the putative merosin receptors dystroglycan and alpha 6 beta 4 integrin are expressed in the outer membrane of Schwann cell/myelin sheath. Together with the well known fact that the deposition of laminin in the basement membrane is essential for Schwann cell myelination, these findings indicate that the interaction of merosin with dystroglycan and/or alpha 6 beta 4 integrin plays an important role in peripheral myelinogenesis and that the disturbance of this interaction leads to peripheral dysmyelination in merosin deficiency. The clinical significance of peripheral dysmyelination in merosin deficiency is also discussed.

Beta-sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate.

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.

Identification of muscle-specific calpain and beta-sarcoglycan genes in progressive autosomal recessive muscular dystrophies.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

The role of dystroglycan, a novel receptor of laminin and agrin, in cell differentiation.

Dystroglycan was originally identified as the extracellular and transmembrane constituents of a large oligomeric complex of sarcolemmal proteins associated with dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene. During the last few years, dystroglycan has been demonstrated to be a novel receptor of not only laminin but also agrin, two major proteins of the extracellular matrix having distinct biological effects. The fact that the drastic reduction of dystroglycan in the sarcolemma, caused by the absence of dystrophin, leads to muscle cell death in DMD patients and mdx mice indicates that, as a laminin receptor, dystroglycan contributes to sarcolemmal stabilization during contraction and stretch of striated muscle cells. Dystroglycan is also expressed in the neuromuscular junction and non-muscle tissues such as kidney, brain and peripheral nerve, and, as a receptor of laminin/agrin, has been implicated in such diverse and specific developmental processes as epithelial morphogenesis, synaptogenesis and myelinogenesis. These findings point to the fundamental role of dystroglycan in the cellular differentiation process shared by many different cell types. In this paper, we review the recent publications on the biological functions of dystroglycan and discuss its roles in cell differentiation.

Differential expression of the parkin gene in the human brain and peripheral leukocytes.

Molecular cloning of the responsible gene on chromosome 6q25.2-27 for autosomal recessive juvenile parkinsonism (AR-JP) identified a novel protein of unknown function, named parkin. In patients with AR-JP, deletions most commonly involve exons 3-5 in the parkin gene. For mutation screening we tried to analyze the parkin transcript amplified by RT-PCR. Based on the assumption that illegitimate transcription of the parkin gene may occur in every cell type, we successfully amplified the parkin message from human peripheral leukocytes using RT-PCR. The parkin transcript in leukocytes was smaller in size than the full-length transcript in the brain. DNA sequencing determined that exons 3-5 were spliced out in the normal human leukocyte transcript. Our results demonstrate that alternative splicing produces distinct parkin transcripts in different tissues. Moreover, physiological splicing of deletion-prone exons may provide an important clue to understanding the pathogenesis of AR-JP.

Cerebellar ataxia and polyneuropathy in a patient with IgM M-protein specific to the Gal(beta 1-3)GalNAc epitope.

A 79-year-old man with sensory dominant polyneuropathy, cerebellar ataxia, and palatal myoclonus had serum IgM M-protein that specifically bound to GM1, GD1b, and asialo-GM1. IgM with the same specificity was detected in his cerebrospinal fluid. Results of immunohistochemical studies showed specific binding of this monoclonal IgM to the cerebellar granular layer, dentate nucleus, inferior olive, and gray matter of the cerebrum and spinal cord. Monoclonal antibody GGR12, monospecific to GD1b, had an immunostaining distribution similar to that of the patient's IgM M-protein. The binding of M-protein may be associated with the development of cerebellar ataxia and palatal myoclonus in this patient.

Degradation of connectin (titin) in Fukuyama type congenital muscular dystrophy: immunochemical study with monoclonal antibodies.

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elasticity of myofibrils. In a previous study, we demonstrated by Western blot analysis of the biopsied skeletal muscles using an anti-connectin monoclonal antibody that connectin was degraded extensively after 5 years of age in Duchenne muscular dystrophy (DMD), while it was degraded mildly in Becker muscular dystrophy and only minimally in myotonic dystrophy, limb girdle dystrophy, amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. In the present study, we investigated the degradation state of connectin in Fukuyama type congenital muscular dystrophy (FCMD) by a similar method using 2 distinct anti-connectin monoclonal antibodies. In FCMD, connectin degradation began much earlier than in DMD: Definite degradation was already observed in 5-8-month-old patients. It was presumed that connectin degradation would play an important role in the myofibrillar degeneration in the early stage of FCMD.

Lattice corneal dystrophy type II with familial amyloid polyneuropathy type IV.

Lattice corneal dystrophy type II with familial amyloid polyneuropathy type IV (Finnish type, Meretoja's syndrome, FAP-IV) has not been reported in Japan to date. In this study we report on 7 cases in a Japanese family which we recently examined. The proband, a 64-year-old man, suffering from itching in his limbs, impaired lip movement and dysarthria, consulted the Department of Neurology, University of Tokyo. Neurological examinations revealed bilateral facial, glossopharyngeal, vagal and hypoglossal nerve palsies, and also impaired distal vibratory perception. Immunohistological and biochemical studies confirmed the diagnosis of FAP-IV. Ophthalmological examinations showed his vision was 1.2 with fine lattice corneal dystrophy in both eyes. The lattice dystrophy was randomly scattered with short glassy lines. Corneal sensation was normal and there was no evidence of recurrent corneal erosion. Six family members with similar lattice corneal dystrophies also were suspected to be affected neurologically by FAP-IV. The family pedigree suggested an autosomal dominant trait of inheritance.