Seiji Shibuya

High levels of nervous system-specific proteins in cerebrospinal fluid in patients with early stage Creutzfeldt-Jakob disease

Concentrations of several proteins that are characteristic of the nervous system were time-sequentially analyzed by radio- and enzyme-immunoassay in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). We found abnormally high levels of several proteins, such as neuron-specific enolase (NSE), S-100b protein, brain-type isozyme of creatine kinase (CK-BB) and alpha subunit of GTP binding protein G0 (G0 alpha) in the early stage of the disease. Generally, these protein levels were far higher in CJD patients than in normal controls and other neurological patients in the early stage before the typical clinical manifestations were evident. These levels increased to maxima when the disease activity was most prominent and returned to normal or mildly elevated levels in the terminal stage. The results imply that these protein levels can serve as biochemical markers for the presence of an active destructive process in CJD brain and provide us with a useful indicator for early diagnosis of CJD.

Immunocytochemical studies of aquaporin 4 in the skeletal muscle of mdx mouse

Immunostainability of anti aquaporin 4 antiserum was investigated in the muscles of dystrophin deficient mdx mice. Western blot analysis showed that the rabbit antiserum against aquaporin 4 reacted with a 28 kDa protein in extracts of normal mouse quadriceps femoris muscles but did not react with the protein in extracts of quadriceps femoris muscles of mdx mice. Immunoperoxidase staining of the muscles from normal and mdx mice revealed the positive immunoreaction at the myofiber surface of normal mice and the negative, or the faint and discontinuous immunostaining at the surface of mdx myofibers. Immunogold electron microscopy disclosed the localization of aquaporin 4 molecules at the myofiber plasma membranes of normal mice and the localization was consistent with that of orthogonal array particles in the protoplasmic face of normal muscle plasma membrane seen in freeze fracture replicas. This study demonstrated that the density of aquaporin 4 molecules was decreased in the muscle plasma membranes of mdx mice, resulting in the faulty function of mdx myofibers.

Dystrophin immunostaining and freeze-fracture studies of muscles of patients with early stage amyotrophic lateral sclerosis and Duchenne muscular dystrophy

We used polyclonal antibodies against dystrophin for the immunohistochemical localization of this protein in human skeletal muscle. Dystrophin was localized in the sarcolemma of the myofibers in 8 infantile and 11 adult normal control muscles and in 10 early stage patient muscles with amyotrophic lateral sclerosis (ALS). The protein was absent or markedly decreased in 8 early stage patients with Duchenne muscular dystrophy (DMD). Moreover the densities of sarcolemmal plasma membrane assemblies, orthogonal arrays and their pits were estimated by freeze-fracture electron microscopy studies in the same number of muscle samples in each disease and control case. The group median densities of orthogonal arrays and their pits in the ALS group and adult control group were 4.8 with a midrange of 1.1-13.5 (25-75%) and 7.5 with a midrange of 2.3-12.9, respectively (P greater than 0.1, Wilcoxon rank-sum test), whereas those of the DMD group and child control group were 0 with a midrange of 0-1.1 and 10.8 with a midrange of 5.4-16.7 respectively (P less than 0.01). The skeletal muscles of mdx mice and their controls were also investigated by the same techniques. In mdx mice, the absence or marked deficiency of dystrophin was also noted; however, the decrease of orthogonal arrays was not as severe as in DMD, which might relate to the milder clinical features in mdx mice as compared with those in DMD.

Duchenne dystrophy Reduced density of orthogonal array subunit particles in muscle plasma membrane

Using freeze-fracture, we analyzed the density of orthogonal arrays and subunit particles in muscle plasma membrane of six patients with Duchenne muscular dystrophy and six control boys. The group median density of orthogonal arrays per 1 pm2 and the group mean density of orthogonal array subunit particles per one orthogonal array were significantly lower in Duchenne plasma membrane. The results suggested the possible impairment of orthogonal array function in the muscle plasma membrane of Duchenne muscular dystrophy.

Ultrastructural localization of α1-syntrophin and neuronal nitric oxide synthase in normal skeletal myofiber, and their relation to each other and to dystrophin

Abstract We investigated the ultrastructural localization of α1-syntrophin and neuronal nitric oxide synthase (nNOS) in normal human skeletal myofibers and analyzed their relation to each other and to dystrophin using single and double immunogold-labeling electron microscopy. Single immunolabeling showed antibodies to α1-syntrophin and nNOS on the inner surface of the muscle plasma membrane, the sarcoplasmic side of plasma membrane invaginations, and the sarcoplasm near mitochondria of subsarcolemmal areas. The epitopes of α1-syntrophin and nNOS tended to be present in clusters. Double immunolabeling revealed that epitope combinations of α1-syntrophin-dystrophin, α1-syntrophin-nNOS, and nNOS-dystrophin occurred more frequently in doublet form than did other epitope combinations, such as α1-syntrophin-β-spectrin and nNOS-β-spectrin. These increased frequencies were noted both at the muscle plasma membrane undercoat and near mitochondria of subsarcolemmal areas. A significantly higher percentage of doublets comprised antibodies against α1-syntrophin and dystrophin (28.5 ± 1.5%, group mean ± SE) than those against α1-syntrophin and β-spectrin (9.2 ± 0.8%, P < 0.01). Furthermore, nNOS formed doublets significantly more frequently with dystrophin (25.2 ± 3.3%) and α1-syntrophin (26.0 ± 4.1%) than with β-spectrin (13.9 ± 2.3%; P < 0.05). These data support the association of dystrophin, α1-syntrophin, and nNOS at the inner surface of the muscle plasma membrane and near mitochondria of subsarcolemmal areas of normal human skeletal myofibers.

Freeze‐fracture studies of muscle plasma membrane in Fukuyama‐type congenital muscular dystrophy

We used freeze-fracture to study muscle plasma membrane in six patients with Fukuyama-type congenital muscular dystrophy and six control children. In the patients, there was significantly fewer intramembranous particles (IMPs) and orthogonal arrays in the P face, with less conspicuous depletion of IMPs in E face. However, the density of caveolae was not affected.

Expression of aquaporin 3 and its localization in normal skeletal myofibres

The question whether aquaporin 3 (AQP3) is expressed in normal human skeletal muscle at mRNA and protein levels has been examined, since AQP3 has been reported to be coexpressed with AQP4 in various kinds of tissues other than skeletal muscle. The gel electrophoresis of the reverse transcription polymerase chain reaction (RT-PCR) product of total RNA samples extracted from normal human muscle specimens by using the oligonucleotide primers for AQP3 contained a band of 629 base pairs which corresponded to the base pair length between two primers of AQP3. The nucleotide sequence of this RT-PCR product coincided with that of AQP3. At the protein level, immunoblot, immunohistochemical and immunoelectron microscopical studies were done by using rabbit antibody against the synthetic peptide of the cytoplasmic domain of the human AQP3 molecule. Immunoblot analysis showed that rabbit antibody against the human AQP3 reacted with a protein of approximately 30 kDa molecular weight in extracts of normal human skeletal muscles. The immunoreaction for the anti-AQP3 antibody with normal human muscle was noted at the myofibre surface. Immunogold labelling electron microscopy revealed that the gold particles indicating the presence of AQP3 molecules were located mainly at the inside surface of muscle plasma membrane.

Gold-labelled dystrophin molecule in muscle plasmalemma of mdx control mice as seen by electron microscopy of deep etching replica.

SummaryThe Duchenne muscular dystrophy product ‘dystrophin’ has been shown to be located at the inner surface of normal muscle plasma membrane. This study was undertaken to visualize the shape of dystrophin molecules and their topographical distribution at the inner surface of murine skeletal muscle plasma membrane. The immunogold electron microscopy of plastic-embedded quadriceps femoris muscles of six mdx mice and six control mice showed the presence of gold particles along the muscle plasma membrane undercoat of all muscle samples from the control mice without any antibody reaction in the mdx mice muscles. The gold-labelled muscles of six mdx and six control mice were quickly frozen by liquid helium in a rapid-freeze apparatus. High magnification electron microscopy of the quick-freeze, deep-etch, rotary-shadow replicas of the gold-labelled muscles demonstrated the presence of dystrophin molecules associated with gold particles at the cytoplasmic surface of mdx control mice.The dystrophin molecules displayed a variety of shapes, such as rods with a reduction in diameter from one end to the other end and/or with the enlargement of their end(s). These dystrophin molecules were incorporated in the meshowork of muscle plasma membrane-associated cytoskeletons.

Immunoreactivity of antibodies raised against synthetic peptide fragments predicted from mid portions of dystrophin cDNA

We synthesized 3 peptide fragments predicted by residues 2354-2368 (peptide I), 2310-2324 (peptide II) and 2255-2269 (peptide III) on the mid-portion of the human dystrophin cDNA map where the most frequent intragenic deletions occurred in Duchenne muscular dystrophy. Rabbit antibodies against these peptides were raised and cryosections of 47 biopsied muscles were studied immunohistochemically. The 47 biopsied muscles included the quadriceps femoris muscles of 8 Duchenne muscular dystrophy patients, 8 child and 5 adult normal controls, 1 facioscapulohumeral dystrophy, 2 limb girdle dystrophy, 3 myotonic dystrophy, 3 polymyositis, 1 mitochondrial myopathy, 1 nemaline myopathy, 3 amyotrophic lateral sclerosis and the extensor digitorum longus muscles of 6 mdx mice (C57BL/10ScSn-mdx) and 6 normal control mice (C57BL/10ScSn). The peptide I antiserum continuously stained the myofiber surface membranes in 8 child and 5 adult normal control muscles, and in 14 other muscles from various neuromuscular diseases, but failed to stain the surface membranes in normal control mice. The surface membranes of 8 Duchenne muscles were not stained by the peptide I antiserum except for a few myofibers. Although the ELISA titers of peptide I, II and III antibodies were high, immunostaining by peptide II antiserum showed no reaction in the myofibers of any of the biopsied muscles, and immunostaining by peptide III antiserum revealed faint reactions on the myofiber surface membranes of all biopsied muscles, including the mdx control mouse muscles except for the Duchenne and mdx myofibers.

Aquaporin 4: lack of mRNA expression in the rat regenerating muscle fiber under denervation

The recently identified water channel aquaporin 4 is a major component of the orthogonal arrays observed with freeze-fracture electron microscopy. We examined the expression of aquaporin 4 mRNA and protein in rat regenerating muscle under innervated and denervated conditions. We found decreased sarcolemmal immunostaining of aquaporin 4 in denervated regenerating muscle as opposed to innervated muscle. Quantitative reverse transcription-polymerase chain reaction revealed that aquaporin 4 mRNA was expressed in the innervated regenerating muscle; whereas it was not expressed in denervated muscle. Thus, lack of aquaporin 4 protein may be due to lack of aquaporin 4 mRNA in the denervated regenerating muscle. We conclude that the nerve supply influences expression of aquaporin 4 at the mRNA level in regenerating muscle.