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Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.

Objective: To detect and characterise enterovirus RNA in skeletal muscle from patients with chronic fatigue syndrome (CFS) and to compare efficiency of muscle energy metabolism in enterovirus positive and negative CFS patients. Methods: Quadriceps muscle biopsy samples from 48 patients with CFS were processed to detect enterovirus RNA by two stage, reverse transcription, nested polymerase chain reaction (RT-NPCR), using enterovirus group specific primer sets. Direct nucleotide sequencing of PCR products was used to characterise the enterovirus. Controls were 29 subjects with normal muscles. On the day of biopsy, each CFS patient undertook a subanaerobic threshold exercise test (SATET). Venous plasma lactate was measured immediately before and after exercise, and 30 minutes after testing. An abnormal lactate response to exercise (SATET+) was defined as an exercise test in which plasma lactate exceeded the upper 99% confidence limits for normal sedentary controls at two or more time points. Results: Muscle biopsy samples from 20.8% of the CFS patients were positive for enterovirus sequences by RT-NPCR, while all the 29 control samples were negative; 58.3% of the CFS patients had a SATET+ response. Nine of the 10 enterovirus positive cases were among the 28 SATET+ patients (32.1%), compared with only one (5%) of the 20 SATET- patients. PCR products were most closely related to coxsackie B virus. Conclusions: There is an association between abnormal lactate response to exercise, reflecting impaired muscle energy metabolism, and the presence of enterovirus sequences in muscle in a proportion of CFS patients.

Twelve biopsied thymuses taken from 4 cases with myasthenia gravis (MG group) and 8 cases without myasthenia gravis (control group) including 2 thymoma cases in each group were immunopathologically investigated in relation to myoglobin (Mb). Mb positive cells of various degrees were detected in all thymuses of both groups, and immunoelectron microscopical examination disclosed that Mb positive cells corresponded to interdigitating reticulum cells and myoid cells in non-neoplastic thymuses, and neoplastic epithelial reticular cells in thymomas. Anti-Mb antibody staining by direct immunoperoxidase technique revealed positive localization to the lymphoid cells in the thymuses of 2 cases of MG group with thymoma. In addition, indirect immunofluorescent study with the serum of each case which was applied to the normal human skeletal muscle, showed positive staining of the sarcoplasm in 3 cases of MG group, including 2 thymoma cases, and using peroxidase labeled serum IgG F (ab')2 of the same patients this anti-muscle antibodies were proved to be against both postsynaptic cytosol and sarcoplasm of the extraocular muscle of the guinea pig. From these results, it was suggested that Mb may conduct itself as a homologous antigen between the thymus and the skeletal muscle in the myasthenic patient with thymoma, and in the thymus the interdigitating reticulum cell, the myoid cell, or the neoplastic epithelial reticular cell may retain or produce Mb as an antigen-presenting cell.

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.