Altered aquaporin 4 expression in muscles of Fukuyama-type congenital muscular dystrophy.

This study was undertaken to investigate the expression of aquaporin 4 (AQP4) in the muscle plasma membrane of children with Fukuyama-type congenital muscular dystrophy (FCMD) at protein and mRNA levels. The biopsied six muscles with FCMD, six histochemically normal muscles and eight disease control muscles were analyzed by means of immunoblots, immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR). Immunoblots showed that the band of FCMD muscle extracts stained with anti-AQP4 antibody was faint in comparison with that of normal muscle extracts. The immunohistochemistry revealed that most of the FCMD myofibers showed negative immunostaining with anti-AQP4 antibody, although the partially positive immunostaining of sporadic FCMD myofibers was noted. The immunoreactivity was positive with anti-dystrophin and anti-beta-spectrin antibodies in almost all FCMD myofibers. The quantitative RT-PCR demonstrated that the AQP4 mRNA level of the FCMD muscles was markedly reduced. On the basis of these findings, we conclude that the expression of AQP4 in FCMD myofibers is reduced and the reduced content of AQP4 mRNA in FCMD muscles may be related to the decreased expression of AQP4 at the muscle plasma membrane of FCMD myofibers.

Localization of sarcoglycan, neuronal nitric oxide synthase, beta-dystroglycan, and dystrophin molecules in normal skeletal myofiber: triple immunogold labeling electron microscopy.

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), beta-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, beta-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of alpha-, beta-, gamma- and delta-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of alpha-, beta-, gamma- and delta-sarcoglycan molecules was frequently observed. Each molecule of nNOS, beta-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with beta-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, beta-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane.

Muscle plasma membrane changes in dystrophin gene exon 52 knockout mouse.

Changes in muscle plasma membranes in mice lacking exon 52 of the dystrophin gene (mdx52 mouse) were studied using the freeze-fracture technique. The extensor digitorum longus (EDL) muscle plasma membrane of the mdx52 mouse at 8 weeks of age showed significantly increased caveola density (p < 0.05 by two-tailed t-test) and significantly decreased densities of intramembranous particles (IMPs), orthogonal arrays (OAs) and orthogonal array subunit particles (OASPs) (p < 0.05 by two-tailed t-test, p < 0.01 by Wilcoxon rank-sum test, p < 0.05 by two-tailed t-test, respectively) on the protoplasmic face when compared with those of control EDL muscles. These changes are more similar to those seen in DMD than those in the mdx mouse at the same age as reported previously. Thus, the gene abnormality in the different exon of the mouse dystrophin gene seems to induce somewhat different changes in the muscle plasma membrane.

[Juvenile onset Creutzfeldt-Jakob disease with the history of neurosurgical operation].

Juvenile onset Creutzfeldt-Jakob disease with the history of neurosurgical operation: a case report A 15-year-old boy gradually developed gait disturbance and dementia. After three months, his condition was deteriorated and he became a state of akinetic mutism. Then, he was transferred to our hospital. He had a history of neurosurgical operation for arterio-venous malformation in his right occipital lobe at the age of five. He showed myoclonus, periodic synchronous discharges in electroencephalogram, and a high cerebrospinal fluid level of neuron-specific enolase. He also revealed progressive brain atrophy by CT examination. He was diagnosed as having Creutzfeldt-Jakob disease (CJD). We could not detect gene mutation of prion protein. As far as we know, this is the youngest case of CJD in Japan. He is too young for the sporadic form of CJD. So the possibility of new variant form of CJD or iatrogenic CJD was considered, the former having been reported in England and the neighboring countries in Europe. However, new variant form of CJD was less likely because of the presence of definite PSD in EEG and the absence of the history of his stay in England. The relationship between the CJD onset and the neurosurgical operation was suggested, but no evidence such as frozen dura matter graft was proved.

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.

Aciculin and its relation to dystrophin: immunocytochemical studies in human normal and Duchenne dystrophy quadriceps muscles.

Aciculin is a novel adherens junction antigen extracted from human uterine smooth muscle that is reported to associate biochemically with dystrophin. We attempted to determine (i) the immunostainability of anti-aciculin antibody for the 6 histochemically normal human muscles and seven muscles from boys with Duchenne muscular dystrophy (DMD) and 11 disease control muscles, (ii) the ultrastructural localization of aciculin in normal skeletal myofibers, (iii) aciculin's spacial relationship with dystrophin and beta-spectrin, and (iv) if the aciculin is ultrastructurally colocalized with dystrophin, the distance from the aciculin epitope to the epitope of the dystrophin N- or C-terminal domain. For this, rabbit anti-aciculin antibody was generated against the synthetic peptide of aciculin fragment D [4]. Immunohistochemical staining showed that the immunostainability of DMD muscles for anti-aciculin antibody was markedly decreased as compared with normal and disease control muscles. Single and double immunogold labeling electron microscopy of 6 histochemically normal human quadriceps femoris muscles revealed that aciculin was present along the inner surface of muscle plasma membrane and that aciculin formed doublets more frequently with dystrophin (23.5 +/- 1.8%; group mean +/- SE) than with beta-spectrin (12.8 +/- 1.1%; P < 0.01 two tailed t test). Rabbit anti-aciculin antibody frequently formed doublets with monoclonal antibodies against the N- or C-terminal domain of dystrophin at the muscle cell surface. These results suggest that aciculin is associated with dystrophin and may interact with both the N- and C-terminal domains of dystrophin.

[Respiratory function impairment in patients with Parkinson's disease--a consideration on the possible pathogenetic relation to autonomic dysfunction].

To investigate the characteristics and clinical significance of respiratory function in patients with Parkinson's disease (PD), we studied 38 patients (male, 19; female, 19: mean age, 65.5 years: mean duration of disease, 6.7 years) who had no history of respiratory disease and smoking. Fifty three non-respiratory disease subjects (male, 26; female, 27: mean age, 67.6 years) were served as age-matched control. We measured spirometry and maximal expiratory flow-volume curve in all patients, and analyzed the relations between respiratory function variables and clinical profiles. The clinical disability of PD was indicated by Hoehn-Yahr (H-Y) scale. The number of PD patients was 15 in H-Y 2, 18 in H-Y 3 and 5 in H-Y 4, respectively. The values of % VC, %FEV 1, FEV 1/FVC, %PEFR, % V50 in H-Y 4 group were significantly smaller than those in H-Y 2 and 3 groups. Small airway dysfunction (SAD) was represented by abnormality of % V25, % V50/V25. The prevalence of impairment in % V25 and % V50/V25 was detected in 13 patients (34.2%) and 15 patients (39.5%), respectively, this was significantly higher than age-matched controls. However, the mean value and prevalence of impairment in % V25, % V50/V25 were not affected by H-Y scale and duration of disease or ideal body weight (%predicted value). Twenty seven patients showed normal ventilatory function based on % VC over 80% and FEV 1/FVC over 70%. The prevalence of impairment in % V25, % V50/V25 was detected in 8 patients (29.6%), 9 patients (33.3%), respectively, among 27 patients with normal ventilatory function. These results suggest that ventilatory dysfunction is concerned with clinical disability but SAD which is independent of clinical disability seen prevalently in patients with PD. It is widely accepted that patients with PD frequently have cardiac or bowel dysfunction based on the visceral autonomic dysfunction. We hypothesize that SAD may also be caused by possible autonomic dysfunction in patients with PD.

Expression of selectin families and their ligand sialyl Lewis X in the muscles of inflammatory myopathies: an immunohistochemical study.

OBJECT: Adhesion molecules are suggested to play important roles in the pathogenesis of inflammatory diseases. We examined the expression of adhesion molecules in the muscles of human inflammatory myopathies. METHODS: We immunohistochemically studied the expression and distribution of two molecules in the selectin family (E- and P-selectin) and their common ligand sialyl Lewis X in 18 inflammatory myopathies, 13 disease controls, and 16 normal controls. RESULTS: In inflammatory myopathies, E- and P-selectin were upregulated on the surface of blood vessels, especially on the endothelial cells of the venules. Sialyl Lewis X was upregulated in the blood vessels, infiltrating leukocytes, and the surface of some atrophic myofibers. Some control muscles also showed weakly positive staining with these molecules, however, expression of these molecules was most striking in the muscles of inflammatory myopathies. CONCLUSION: The results suggested that these molecules are upregulated in inflammatory myopathies and might play a role in the pathogenesis of inflammatory myopathies.

Ultrastructural localization of alpha-, beta- and gamma-sarcoglycan and their mutual relation, and their relation to dystrophin, beta-dystroglycan and beta-spectrin in normal skeletal myofiber.

Ultrastructural localization of alpha-, beta- and gamma-sarcoglycan and their mutual relation, and their relation to dystrophin, beta-dystroglycan and beta-spectrin were investigated in normal skeletal myofibers. Single-immunogold labeling electron microscopy showed that the signals of rabbit and sheep polyclonal antibodies against the synthetic peptide of the cytoplasmic domain of alpha-, beta or gamma-sarcoglycan were present along the inside surface of muscle plasma membrane and at the sarcoplasmic side of plasma membrane invaginations and vesicular structures in subsarcolemmal areas. These localizations were similar to that of dystrophin, beta-dystroglycan and beta-spectrin. Double-immunogold labeling disclosed the close association of alpha-, beta- and gamma-sarcoglycan each other and alpha-, beta-, gamma-sarcoglycan with dystrophin or beta-dystroglycan, and this was confirmed by statistical analysis. Monoclonal antibody against the extracellular domain of alpha-sarcoglycan was used with above-mentioned polyclonal anti-beta- and -gamma-sarcoglycan antibodies for triple-immunogold labeling, in which signals of alpha-sarcoglycan localized at the outer surface of muscle plasmalemma and those of beta- and gamma-sarcoglycans were present at the inside surface of plasma membrane. The triple immunolabeling showed an occasional closely associated presence of the three signals for alpha-, beta-and gamma-sarcoglycans, and a more frequent association for two signals out of alpha-, beta- and gamma-sarcoglycans. This study demonstrated that alpha-, beta- and gamma-sarcoglycan are closely located to one another and to dystrophin and beta-dystroglycan at the muscle plasma membrane.

[A management for severe acquired stuttering in a case of pure akinesia syndrome].

We reported a 73-year-old man with pure akinesia syndrome who showed severe acquired stuttering and paradoxical kinesia on speech. He was evaluated in another hospital for bradykinesia and frozen gait at age of 67 when his cranial MRI disclosed ischemic changes in bilateral basal ganglia and periventricular deep white matter. The treatment with L-dopa and L-threo DOPS was not effective. His symptoms were slowly progressive and got worse gradually. At age of 72, he began to have difficulty in speech due to severe acquired stuttering, and one year later, he visited our hospital. The diagnosis of pure akinesia syndrome was made because of akinesia, micrographia, marked frozen gait with paradoxical kinesia and disturbance of postural reflex without tremor and rigidity. His speech showed severe acquired stuttering with marked blocking and repetition of initial phonemes at the beginning of speech. But intelligible speech recurred with pointing the characters by his finger or with writing an initial letter of word, although his speech was small and monotonous. Surface EMG findings of muscles participating speech in acquired stuttering showed the similar tonic discharge to those of muscles of lower extremity in frozen gait. These results implied that freezing phenomenon and festination of muscles participating speech in our patient may result in acquired stuttering.

Ultrastructural localization of alpha 1-syntrophin and neuronal nitric oxide synthase in normal skeletal myofiber, and their relation to each other and to dystrophin.

We investigated the ultrastructural localization of alpha 1-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 alpha 1-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 alpha 1-syntrophin and nNOS tended to be present in clusters. Double immunolabeling revealed that epitope combinations of alpha 1-syntrophin-dystrophin, alpha 1-syntrophin-nNOS, and nNOS-dystrophin occurred more frequently in doublet form than did other epitope combinations, such as alpha 1-syntrophin-beta- spectrin and nNOS-beta-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 alpha 1-syntrophin and dystrophin (28.5 +/- 1.5%, group mean +/- SE) than those against alpha 1-syntrophin and beta-spectrin (9.2 +/- 0.8%, P < 0.01). Furthermore, nNOS formed doublets significantly more frequently with dystrophin (25.2 +/- 3.3%) and alpha 1-syntrophin (26.0 +/- 4.1%) than with beta-spectrin (13.9 +/- 2.3%; P < 0.05). These data support the association of dystrophin, alpha 1-syntrophin, and nNOS at the inner surface of the muscle plasma membrane and near mitochondria of subsarcolemmal areas of normal human skeletal myofibers.

[Multiple solitary lesions in deep white matter in the early stage of Creutzfeldt-Jakob disease. A case report].

A 56-year-old housewife was admitted to our hospital because of involuntary movement on her left arm. Her neurological examination on admission showed mild weakness of her left arm and cerebellar ataxia. She developed periodic synchronous discharge on electroencephalogram and her cerebrospinal fluid revealed elevated level of neuron-specific enolase. Thereafter she developed dementia, followed by apallic state and diagnosed as having Creutzfeldt-Jakob disease (CJD). Interestingly, her MRI on admission revealed multiple solitary lesions in deep cerebral white matter, which were detected as high signal intensity by T2 weighed image. A few months later, these lesions tended to extend, and finally fused around the lateral ventricle in parallel with remarkable cortical atrophy. We excluded other diseases such as cerebrovascular disorders. Finally, we concluded the white matter change seen from early stage in this case may be the lesion associated with CJD, and the CJD case with early white matter changes has been seldomely described.

Immunogold and freeze etch electron microscopic studies of merosin localization in basal lamina of human skeletal muscle fibers.

Merosin is a basement-membrane-associated protein found in striated muscle, peripheral nerve and placenta, the deficiency of which causes the muscle wasting condition in C57BL/6J-dy/dy, so-called dy/dy mouse. Moreover, merosin is the binding protein of 156 kDa alpha-dystroglycan which binds dystrophin by way of 43 kDa beta-dystroglycan. Therefore, merosin is an important component of the basal lamina of normal skeletal myofibers. We investigated the ultrastructural localization of merosin antibody in normal human skeletal myofibers by using immunogold electron microscopy and freeze etch electron microscopy. The ultrastructure of the basal lamina showed the presence of the lamina lucida, lamina densa and lamina reticularis. The lamina lucida appeared electron translucent with the exception of fuzzy fibrils. The immunogold electron microscopy disclosed that the merosin was present at the innermost layer (lamina lucida) of the basal lamina of normal human skeletal myofibers. With freeze etch replica electron microscopy, short cross-bridge fine fibrils were noted in the lamina lucida, connecting the basal lamina to the outer leaflet of the muscle plasma membrane. They measured 3-13 nm in diameter, 20-90 nm in length and were distributed with a spacing of 30-40 nm. The immunogold particles showing the presence of the merosin epitope were associated with these connecting structures.

Electron microscopic observations of triple immunogold labelling for dystrophin, beta-dystroglycan and adhalin in human skeletal myofibers.

Dystrophin is the Duchenne muscular dystrophy gene product and is a membrane cytoskeletal protein present in the network of the plasma membrane undercoat. Adhalin (50 kDa dystrophin-associated glycoprotein) and beta-dystroglycan (43 kDa dystrophin-associated glycoprotein) are the transmembrane components of the normal muscle plasma membrane, and beta-dystroglycan has been demonstrated to bind dystrophin at the inside surface of normal muscle plasma membrane. This investigation was undertaken to test whether the epitopes of dystrophin, beta-dystroglycan and adhalin are closely associated with each other by using triple immunogold labelling electron microscopy on normal human skeletal myofibers. Although closely associated signals of triplet immunogold particles were observed, there were less numerous than expected. However, closely associated signals of two epitopes of dystrophin and beta-dystroglycan, dystrophin and adhalin, or adhalin and beta-dystroglycan were frequently observed. These ultrastructural findings are consistent with biochemical evidence implying that dystrophin, beta-dystroglycan and adhalin are closely associated with each other at the normal muscle plasma membrane.

Spinal epidural abscess associated with epidural anesthesia: gadolinium-enhanced magnetic resonance imaging and its usefulness in diagnosis and treatment.

A case of spinal epidural abscess following epidural anesthesia is described. Gadolinium-enhanced magnetic resonance images were essential in diagnosis of the abscess without frank pus formation, in defining the extension of the infection, and in assessing the therapeutic effect. The patient was successfully treated non-operatively before neurological symptoms developed and full recovery was achieved.

Mechanism of inhibition of H+, K(+)-ATPase by sodium 2-[[4-(3- methoxypropoxy)-3-methylpyridin-2-yl]methylsulfinyl]-1H-benzimidazole (E3810).

Sodium 2-[[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulfinyl ]- 1H-benzimidazole (E3810) and omeprazole inhibit gastric acid secretion through inhibition of the activity of H+, K(+)-ATPase present in parietal cell membrane vesicles, by chemical modification of SH groups in the enzyme molecule. In order to clarify the mechanism of the chemical modification, reaction products of E3810 and omeprazole with 2-mercaptoethanol under acidic conditions (pH 3, 4, 5, 6) were isolated by HPLC, and subjected to structural analysis by UV, 1H-NMR and mass spectrometry. E3810 and omeprazole appeared to undergo two kinds of reactions, affording disulfide-type products (type I reaction) and sulfide-type products (type II reaction). The rates of these reactions were determined by HPLC, and the stability of the products in the presence and absence of glutathione was investigated. In the case of E3810, type I reaction was found to proceed faster than type II reaction at every pH value studied. The type I reaction of E3810 was faster than that of omeprazole. The rate of type I reaction decreased at pH 5 and 6, especially for omeprazole, and the contribution of type II reaction increased as the pH of the reaction mixture was increased. The sulfide-type modification products were stable, whereas the formation of the disulfide-type modification products was reversed by the action of endogenous SH compounds such as glutathione. These results suggest that higher inhibitory activity of E3810 against gastric acid secretion and faster recovery of the enzyme activity after inhibition by E3810 can be expected, as compared with those of omeprazole.

Ultrastructural localization of adhalin in normal murine skeletal myofiber.

The ultrastructural localization of adhalin and its relations to dystrophin, beta-dystroglycan, and beta-spectrin were studied in normal murine skeletal myofibers. The C-terminal peptides of adhalin and beta-dystroglycan were synthesized based on their cDNAs, and the affinity-purified antibodies against these peptides were produced. Single-immunolabeling electron microscopy showed that the adhalin was located just inside the muscle plasma membrane or inside the myofiber a short distance from the plasma membrane. The adhalin signal was also noted at the sarcoplasmic side of plasmalemmal invaginations or at vesicular structures in subsarcolemmal areas. Double-immunogold-labeling electron microscopy disclosed a similar localization of dystrophin, beta-dystroglycan, and beta-spectrin. The close association of adhalin with dystrophin or beta-dystroglycan was demonstrated by formation of doublets by signals of antibodies of adhalin with those of dystrophin or beta-dystroglycan and was confirmed by statistical analyses. This study demonstrated that the location of adhalin is close to that of dystrophin and beta-dystroglycan at the muscle plasma membrane.

Motor neuron disease with involvement of the pallido-luysio-nigral system and mesencephalic tegmentum.

A rare case of motor neuron disease and involvement of the pallido-luysio-nigral system and brainstem tegmentum is presented. A 51-year-old man developed progressive muscle atrophy with fasciculation predominantly in the shoulder girdle, upper arms, upper back, and neck in addition to hyperreflexia and a positive Chaddock reflex. He also had retinitis pigmentosa, high arched palate, and mild hand tremor. He eventually developed bulbar palsy and died of paralysis of the respiratory muscles 11 years after the onset of his illness. Neuropathological examinations showed prominent neuronal loss and gliosis in the pallido-luysio-nigral system and the tegmentum of the brainstem in addition to the simultaneous involvement of the upper and lower motor neurons. This patient and 6 similar patients are discussed in relation to pallido-luysio-nigral atrophy and the topographic distribution of degeneration in amyotrophic lateral sclerosis.