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The examination was performed whether aquaporin (AQP) 9 is expressed in normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of human normal muscles by oligonucleotide primers for human AQP9 showed a band of 221 basepairs, which corresponded to the basepair length between two primers of AQP9. The nucleotide sequence of RT-PCR product coincided with that of human AQP9. Immunoblot analysis revealed that the rabbit and sheep antibodies against the synthetic peptide of the C-terminal cytoplasmic domain of human AQP9 molecule reacted with a protein of approximately 30 kDa molecular weight in extracts of human normal skeletal muscles. Immunohistochemistry with our anti-AQP9 antibodies showed an immunoreaction at the myofiber surface of both type 1 and type 2 fibers with almost equal staining intensity in human skeletal muscles. The implication of AQP9 expression in skeletal myofibers was discussed.

Dysbindin was first identified by the yeast two hybrid assay as a binding partner of dystrobrevin which is a cytoplasmic member of dystrophin glycoprotein complex. Immunolocalization of dystrobrevin in the astrocyte endfeet and endothelial cells in the rat cerebellum was reported. Therefore, we were interested in the expression and localization of dystrobrevin binding protein dysbindin in the mouse brain capillary wall and its surrounding astroglial endfeet. We examined whether the dysbindin expression is present in astroglial endfeet and/or capillary endothelial cells at light and electron microscopic levels. Using brain samples from five normal mice (C57BL/6ScSn), we prepared the anti-dysbindin antibody stained brain samples with immunoperoxidase method at light microscopic level and with immunogold method at ultrastructural level. Immunohistochemistry showed that dysbindin was located in the brain capillary at light microscopic level. Immunogold electron microscopy revealed that dysbindin signal was observed at the inside surface of plasma membrane of glial endfeet which surrounded the brain capillary endothelial cells and pericytes.

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an (L)111(F) substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS.

We generated the muscle aquaporin 4 (AQP4) overexpressing transgenic mice in order to investigate the skeletal muscle pathology at RNA and protein levels. At RNA level, the AQP4 mRNA expression of soleus, EDL and cardiac muscles in Tg mice was statistically significantly higher than that in wild mice by the real-time reverse transcription polymerase chain reaction method. At protein level examinations, we used the immunoblot, immunohistochemistry and freeze-fracture electron microscopy. The immunoblot showed the single band of 31kDa with anti-AQP4 antibody in the extracts of soleus and EDL muscles of wild mice but not in extract of wild cardiac muscle; while the reaction band was noted in cardiac muscle of Tg mice and the reaction band was stronger in the extracts of soleus and EDL muscles of Tg mice. The immunohistochemistry showed that the expression of AQP4 at the myofiber surface of soleus and EDL muscles of Tg mice was more marked than that of wild mice and, interestingly, the AQP4 expression of these muscles of Tg mice appeared to be more remarkable in type 1 slow twitch myofibers as judged by the positive slow myosin immunostaining of adjacent serial sections. The immunofluorescence staining with anti-AQP4 antibody of cardiac muscles of wild mice revealed the scarcely immunopositive myofibers; whereas the immunostaining cardiac muscles of Tg mice contained the numerous AQP4 immunopositive myofibers. The freeze-fracture electron microscopy demonstrated that the orthogonal array densities in soleus and EDL muscle plasma membranes of Tg mice were significantly higher than those of wild mice and that the orthogonal array like particle density of cardiac muscle plasma membranes of Tg mice appeared to be more numerous than that of cardiac myofibers of wild mice. Finally the clinical phenotype of Tg mice appeared to be similar to that of wild mice. Further physiological examination with devices may suggest some about the physiological difference.

To examine aquaporin 1 (AQP1) expression in skeletal muscle tissue precisely, we performed reverse transcription-polymerase chain reaction (RT-PCR) at RNA level and immunoblot analysis, immunohistochemistry and immunoelectron microscopy at protein level. The RT-PCR study of total RNA from normal human skeletal muscle showed a strong single band of AQP1. At the protein level we used two commercially available antibodies, both of which recognize the cytoplasmic domain of the AQP1 molecule. One antibody gave positive results. Immunoblot of muscle extract showed a 30-kDa band protein, the molecular weight of which corresponded to that of AQP1. Immunohistochemically, AQP1 was immunostained at the myofiber surface both in type 1 and type 2 myofibers with almost the same intensity, and its staining pattern was rather diffuse and irregular compared with that of the anti-dystrophin antibody. The endomysial endothelial cells were also immunolabeled. Immunoelectron microscopy revealed that the immunogold particles indicating the presence of the AQP1 molecule were present along the inside surface of the muscle plasma membrane. However, another antibody showed negative results except for the endomysial endothelial cells which were positively stained. We drew the conclusion that AQP1 is expressed at the endomysial capillary endothelial cell and further AQP1 may be expressed at the human skeletal myofiber plasma membrane.

Myoferlin is a novel protein of unknown function with high homology to dysferlin, the gene mutations of which cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. The myoferlin gene seems to be a candidate for the modifier, and because of the high homology to dysferlin myoferlin may work as a compensator for the absence of dysferlin in dysferlinopathy. This hypothesis is based on the observation that utrophin, which has 80% homology with dystrophin, is overexpressing in the dystrophin deficient myofibers. To test this hypothesis, we investigated the myoferlin expression by immunoblot and immunohistochemical analysis in muscles of five patients with dysferlinopathy. For this aim, we generated a myoferlin specific antibody that does not cross react with dysferlin, and performed the immunoblot, immunohistochemical and immunoelectron microscopic studies. Immunohistochemical analysis showed that the antibodies against myoferlin and dysferlin clearly stained the normal human myofiber surface membranes. The electron microscopy of single immunogold labeled samples for myoferlin showed the presence of the molecular signal along the normal muscle cell membrane. Immunoblot analysis showed that the intensity of 230-kDa myoferlin band of dysferlinopathy muscle extracts was similar to that of normal muscle extracts. The immunostaining of dysferlinopathy muscles with anti-myoferlin antibody revealed a weak immunoreactivity along the muscle cell surface. Thus, the compensatory overexpression of myoferlin was not detected in muscles with dysferlinopathy.

It is likely that orthogonal arrays (OAs) and caveolae seen on the replicas of freeze-fractured muscle plasma membranes are involved in maintaining osmotic homeostasis. Therefore, using the freeze-fracture technique, we examined the ultrastructural changes in OAs and caveolae of the skeletal muscle plasma membrane of dehydrated and water-loaded mice. In the muscle plasma membranes of 6 dehydrated and 6 water-loaded mice, caveolar distribution was not altered, and the densities of caveolae and OAs did not show statistically significant differences when compared with those in 6 control mice, although the skeletal muscles of water-loaded mice sometimes had muscle plasma membranes with extremely numerous OAs. In contrast, the muscle plasma membranes of dehydrated mice often revealed changes in the distribution of OAs, which existed in a group at the confined area of the muscle plasma membranes and were frequently accompanied by the aggregations of intramembranous particles (IMPs) around OAs. Thus, on the basis of the present study, we suggest that OAs in skeletal muscles as well as those in brain may play an important role in maintaining osmotic homeostasis of these organs under abnormal water balance.

Our previous freeze-fracture study demonstrated the decreased density of intramembranous particles (IMPs) on the protoplasmic (P) face of muscle plasma membranes in mdx mice. However, the molecular mechanism is unknown. In the present freeze-fracture study, we examined whether the reduced P-face IMP density in mdx mice would be caused by depletion of the rosette-like IMP clusters, which are IMP aggregations differing from crystal-like orthogonal arrays (OAs). By comparison with control mice, the P-face plasma membranes of extensor digitorum longus (EDL) and soleus (SOL) muscles of mdx mice demonstrated the following findings: (1) decreased IMP density with subunit particles of OAs and IMP clusters, (2) decreased IMP density without subunit particles of OAs, (3) normal IMP density without subunit particles of OAs and IMP clusters, (4) decreased OA density in EDL muscles and normal OA density in SOL muscles, and (5) decreased IMP cluster densities in both muscles. Thus, the reduced IMP density of P-face muscle plasma membranes in mdx mice may result from the decreased IMP clusters, suggesting the relationship between IMP clusters and the integral membrane proteins is influenced by dystrophin deficiency such as that of dystrophin-associated glycoproteins or other membrane proteins.

To analyze the molecular mechanism of the increased caveolin 3 activities in dystrophin-deficient muscles, we investigated three-dimensionally the changes in caveolin 3 molecular distribution and density at the sarcolemma of mdx mice by the fracture-label electron microscopic technique. At the sarcolemma of skeletal muscles from mdx mice, the densities of gold particles associated with caveolae, non-associated with caveolae and arranged circularly without caveolae were higher than those in control mice (P

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.

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%; P0.01 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.

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.

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.

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.

Muscle plasma membrane undercoats were investigated by conventional electron microscopy in both Duchenne muscular dystrophy (DMD) and Fukuyama congenital muscular dystrophy (FCMD). The densities of the plasma membrane undercoats were rarefied in the parts of the plasma membranes overlying the degenerating focus in both DMD and FCMD myofibers. The degree of rarefaction tended to be parallel to the degree of degeneration in the myofibers. It was hard to distinguish the undercoat densities of normal-looking myofibers of DMD and FCMD muscles from those of control myofibers from histochemically-normal muscles. On the other hand, the undercoats of regenerating myofibers in DMD and FCMD muscles were denser than normal.

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities in skeletal muscle plasma membranes from eight patients with Becker's muscular dystrophy (BMD) were analysed by the freeze-fracture technique. The results showed almost normal IMP density with the significant decrease of OA and OASP densities in BMD. The group mean densities +/- SE of IMPs on the protoplasmic faces with and without OASPs, and on extracellular faces/microns 2 were 2137 +/- 207, 1839 +/- 68 and 895 +/- 108, respectively in controls; whereas those of BMD were 1989 +/- 259, 1837 +/- 203 and 900 +/- 239, respectively (P > 0.1 by two-tailed t-test). The group median density of OAs and their pits/microns 2 was 4.89 with mid-ranges (25-75% values of the counts) of 2.66-10.18 in controls; whereas that in BMD was 2.15 with mid-ranges of 1.14-4.31 (P < 0.01 by Wilcoxon rank-sum test). The group mean density +/- SE of OASPs in controls was 15.99 +/- 1.83; whereas that in BMD was 13.47 +/- 1.07 (P < 0.01 by two-tailed t-test). However, the diminution of OA and OASP densities in BMD muscle plasma membranes was not as severe as in Duchenne's muscular dystrophy. There was a relationship between OA density and clinical severity in BMD patients; the decrease of OA density in a severe BMD patient was more marked than that in mildly affected BMD patients. Therefore, it seems that marked depletion of OA density may lead to the severe disability in muscular dystrophies.

To evaluate the detrition of clear corneal incisions (CCIs) after intraocular lens (IOL) implantation using an injector system in porcine eyes.Group A: after CCIs were performed with 1.8, 2.0, 2.2, 2.4, and 2.65 mm wide slit knives, a Y-60 H (HOYA) IOL was implanted in the anterior chamber using an injector system. Group B: after CCIs were performed with 2.4, 2.65, 2.8, 3.0, and 3.2 mm wide slit knives, a PY-60 R (HOYA) IOL was implanted in the anterior chamber using an injector system. Group C: after CCIs were performed with 2.8, 3.0, 3.2, 3.4 mm wide slit knives, a SN 60 AT (Alcon) IOL was implanted in the anterior chamber using an injector system.CCIs were performed with 3.0 mm wide slit knives. Each group used five porcine eyes for each slit knife (Group A 25 eyes; Group B 25 eyes; Group C 20 eyes; CONTROL 5 eyes). The detrition of the CCIs was evaluated on three different aspects using a scanning electron microscope: a) external expansion at both edges of CCIs; b) rupture of the collagen fibers; c) expansion between the collagen fibers. Aspects a, b and c were given a score of 0, 1, and 2, respectively, and the total points were compared statistically between test and control groups.The degree of CCIs detrition was significantly reduced in CCIs with a width of more than 2.4 mm of CCIs width in Group A, more than 3.0 mm in Group B, and more than 3.2 mm in Group C.Minimizing the detrition of corneal incisions after IOL implantation needs a larger than the recommended width of corneal incision.

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously. This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.

The plasma membrane of skeletal muscles contains water channels such as aquaporin 4 (AQP4), aquaporin 3 (AQP3) and aquaporin 7 (AQP7). In dehydrated mice, we have recently reported the altered distribution of the aggregations of intramembranous particles (IMPs), such as orthogonal array (a crystal-like structure) and IMP cluster (a rosette-like structure) on the freeze-fractured skeletal muscle plasma membranes. In this fracture-label study, we first tested whether the orthogonal arrays (OAs) were composed of AQP4 in skeletal muscles and further analyzed the relationship between IMPs including IMP clusters and AQP3 molecules. As a result, many of the gold particles indicating AQP4 was associated with OAs (79%) by our fracture-label technique. On the other hand, approximately 50% of gold particles indicating AQP3 were associated with IMP clusters. Thus we confirmed that the OAs are composed of AQP4 in skeletal muscles, and further demonstrated that some of the IMP clusters are composed of AQP3 and may participate in maintaining osmotic homeostasis in skeletal myofibers. The fracture-label method is useful in investigating the molecular identification of membrane proteins such as AQP3 and AQP4.

Sarcospan is a 25 kDa transmembrane component of dystrophin-associated glycoprotein. We generated a rabbit polyclonal antibody against synthetic peptide of the N-terminal domain of human sarcospan. Using this antibody we investigated the localization of sarcospan and its spacial relation to the components of sarcoglycan subcomplex in normal human skeletal myofibers by immunofluorescent microscopy and immunogold electron microscopy. In immunofluorescence the reaction was observed continuously at the myofiber surface. Ultrastructurally the gold signals of rabbit anti sarcospan antibody were present along the muscle plasma membrane, mainly at its inside surface. The triple immunogold labeled muscle samples showed that the signals of rabbit or sheep polyclonal anti alpha-, beta-, gamma- and delta-sarcoglycan antibodies and/or mouse monoclonal anti beta-, gamma- and delta-sarcoglycan antibodies were located along the muscle plasma membrane, and the cluster formation of different two or three sarcoglycan molecules was observed. The triple immunogold labeling also revealed that the signal of sarcospan molecules are present frequently in doublets and/or triplets with the components of sarcoglycan subcomplex, resulting in the cluster formation of signals of sarcoglycan and sarcospan molecules. The result of this study showed that sarcospan was expressed at the myofiber surface and that sarcospan was present in close association with alpha-, beta-, gamma- and delta-sarcoglycans and formed a functional unit with sarcoglycan subcomplex.

The initial contamination of heparin-saline solution (HS) in multiple-dose vials (MDVs) by Serratia marcescens was experimentally investigated using various isolates. Isolates I2 and S1 were from blood specimens from patients with a hospital-acquired infection (HAI). Isolates I13 and FHSM9043 were from urine and blood specimens, respectively, from patients without HAI. Isolate I124, with a pulsed-field get electrophoresis pattern identical to that of isolate I2, was from the hospital environment. Viable cells of isolate I2 were carried over into the HS of MDVs when the contaminated rubber septum was pierced with a syringe needle. When the outside surface of the septum was contaminated by inoculating it with wet-cell suspensions in HS or Müller-Hinton broth, the viable cells carried over were detected at a minimum inoculum size (MIS) of 10(3) s cfu/ml. However, when the surface was contaminated by inoculating it with dry-cell suspensions, the viable cells carried over were detected at an MIS of 10(7) s cfu/ml. The viable cells in the internal lumen of the needle much more than those on its outside surface spread to the HS of MDVs. For exposures of 24 h and 72 h at 4 degrees C to HS with 1% benzyl alcohol as a preservative in MDVs, viable cells of all isolates tested were detected at MIS values of 1 s and 10 s cfu/ml, respectively, increases three orders of magnitude smaller than those of reference strain IFO3736. These results suggest that S. marcescens isolates are readily carried over into the HS of MDVs by piercing a wet, contaminated rubber septum with a syringe needle. Also, despite the sterilization action of 1% benzyl alcohol, the organism persistently survived at 4 degrees C, even when initial contamination was with a small amount of inoculum.

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of Alpha-dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via Beta-dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.

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.

We examined whether AQP7 molecules are expressed in the normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of normal human or mouse muscles by using oligonucleotide primers for human or mouse AQP7 showed a band of 328 or 369 basepairs, which corresponded to the basepair length between two primers of AQP7. The nucleotide sequence of these RT-PCR products coincided with those of human and mouse AQP7. Immunoblot, immunohistochemical and immunoelectron-microscopic studies of the protein were done by using the rabbit antibody against the synthetic peptide of the N-terminal cytoplasmic domain of the human AQP7 molecule. Immunoblot analysis showed that the rabbit antibody against human AQP7 reacted with a protein of approximately 30 kDa molecular weight in extracts of normal human and mouse skeletal muscles, and normal mouse liver. Immunohistochemistry with our anti-AQP7 antibody showed an immunoreaction at the myofiber surface of type 1 and type 2 fibers in human muscles and of type 2 fibers in mouse muscles.

Aquaporin 4 (AQP4) is a recently discovered membrane bound water-selective channel and has been described at the light microscopic level to be predominantly expressed in the astrocytes of the brain, especially at the perivascular astrocyte endfoot processes. Alpha1-syntrophin, a member of dystrophin-associated protein, has also been reported at the light microscopic to be expressed level in the same site of astrocytes as AQP4 and interacts with other molecules through its PDZ domain. AQP4 expression has been reported to be absent at the sarcolemma and the perivascular astrocyte endfoot processes of alpha1-syntrophin knockout mice. Based on these observations, the molecular association between AQP4 and alpha1-syntrophin could be speculated. To test this hypothesis, we investigated the ultrasturctural localization of AQP4 and alpha1-syntrophin in the brain astrocytes by using double immunogold labeled electron microscopy. The results showed that AQP4 and alpha1-syntrophin colocalized frequently at the astrocyte membrane, especially at the perivascular astrocyte endfoot processes and suggested the presence of linkage between AQP4 and alpha1-syntrophin at the astrocyte plasma membrane.

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), β-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, β-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of α-, β-, γ- and δ-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 α-, β-, γ- and δ-sarcoglycan molecules was frequently observed. Each molecule of nNOS, β-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with β-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, β-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. Microsc. Res. Tech. 55:154–163, 2001. © 2001 Wiley-Liss, Inc.

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.

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.

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.

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%, P0.01). 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%; P0.05). 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.

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.

Ultrastructural localization of the C-terminus of a 43-kDa dystrophin-associated glycoprotein (43-DAG) and its relation to dystrophin were investigated in normal human skeletal myofibers by using single and double immunogold labelling methods. The C-terminal end of 43-DAG antibody was localized ultrastructurally at the inside surface of muscle plasma membrane and the sarcoplasmic side of the plasma membrane invaginations. Double immunolabelling electron microscopy disclosed that signals of the C-terminal end of 43-DAG and dystrophin were often associated with each other and formed a doublet at the inside surface of plasma membranes in normal human skeletal myofibers.

The ultrastructure and mode of existence of the dystrophin molecule and its relations to actin filaments were examined in murine skeletal myofibers. Electron microscopy of freeze-etched replicas of gold-labelled dystrophin molecules in quick-freeze, deep-etch, rotary-shadow preparations revealed rod-like structures 108.2 +/- 16.3 nm long and 3.1 +/- 1.5 nm thick. Some dystrophin molecules appeared to link their ends to form anastomosing networks; others were separate from each other. The dystrophin molecules were parallel or nearly parallel to the inner surface of the muscle plasma membrane. Double immuno-labelling transmission electron microscopy using N- and C-terminal dystrophin antibodies showed that the group mean distances of the N- and C-terminal signals from the muscle plasma membrane were 52.7 +/- 8.1 nm and 45.9 +/- 11.3 nm, respectively, which were not significantly different. Histograms of the distribution of the N- and C-terminal distances from the muscle plasma membrane had similar patterns with peaks 10-20 nm from the membrane. This was consistent with the findings of the mode of existence of dystrophin molecules seen in freeze-etched replicas. Finally, the dystrophin molecules were linked with the most peripheral sarcoplasmic actin like filaments, end to side as well as end to end.

The cytoskeleton of lens fiber cells in rats with hereditary cataract (ICR/f) was studied by quick-freezing and deep-etching. Two kinds of filamentous structure with diameters of 5 and 10 approximately 15 nm were observed in the cortical fiber cells. They showed meshwork structures which were buried in globular particles. These filamentous structures were mostly absent in the nucleus fiber cells, but aggregated and fused globular particles with diameters of 10 approximately 20 nm were observed. Small cavities were sometimes observed in their cytoplasm. Between lens fiber cells, many globular structures were also seen. These changes might represent the degeneration of the lens fiber cells in cataract lenses.

As anionic sites of glomerular basement membrane (GBM) stained by intravenous injection of PEI became obscure 2 hours after administration, we studied reappearance of the anionic sites upon re-injection of PEI.The following two experimental groups were prepared: In group A; 10 rats were injected with 0.3ml of 0.5% PEI (MW=1,800) solution, and left nephrectomy was performed after 3 hours (group A-1, N=5) and 5 hours (group A-2, N=5). Each tissue samples was subjected to contrasting/fixation immersion, washing, post-fixation and staining. Immediately after nephrectomy, re-administration of 0.3ml of the same PEI solution was performed on each animal. Right nephrectomy was done 15 minutes after re-injection of PEI, and renal tissue was treated the same way as that of the left kidney (group A-1', group A-2'). In group B; PEI with a molecular weight of 70,000 was used. Injection of PEI solution, nephrectomy and treatment of renal tissues were carried out in the same way as for group A (group B-l and B-l', N=6; group B-2 and B-2', N=8).