Immunocytochemical studies of aquaporin 4, Kir4.1, and α1-syntrophin in the astrocyte endfeet of mouse brain capillaries.

One of the most important physiological roles of brain astrocytes is the maintenance of extracellular K(+) concentration by adjusting the K(+) influx and K(+) efflux. The inwardly rectifying K(+) channel Kir4.1 has been identified as an important member of K(+) channels and is highly concentrated in glial endfeet membranes. Aquaporin (AQP) 4 is another abundantly expressed molecule in astrocyte endfeet membranes. We examined the ultrastructural localization of Kir4.1, AQP4, α1-syntrophin, and ß-spectrin molecules to understand the functional role(s) of Kir4.1 and AQP4. Immunogold electron microscopy of these molecules showed that the signals of these molecules were present along the plasma membranes of astrocyte endfeet. Double immunogold electron microscopy showed frequent co-localization in the combination of molecules of Kir4.1 and AQP4, Kir4.1 and α1-syntrophin, and AQP4 and α1-syntrophin, but not those of AQP4 and ß-spectrin. Our results support biochemical evidence that both Kir4.1 and AQP4 are associated with α1-syntrophin by way of postsynaptic density-95, Drosophila disc large protein, and the Zona occludens protein I protein-interaction domain. Co-localization of AQP4 and Kir4.1 may indicate that water flux mediated by AQP4 is associated with K(+) siphoning.

[Evaluation of post intraocular lens implantation detrition of clear corneal incisions using an injector system in porcine eyes].

PURPOSE: To evaluate the detrition of clear corneal incisions (CCIs) after intraocular lens (IOL) implantation using an injector system in porcine eyes. METHODS: 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. CONTROL: 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. RESULTS: 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. CONCLUSIONS: Minimizing the detrition of corneal incisions after IOL implantation needs a larger than the recommended width of corneal incision.

Aquaporin 9 expression and its localization in normal skeletal myofiber.

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.

Immunohistochemical detection of dysbindin at the astroglial endfeet around the capillaries of mouse brain.

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.

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

Actin -related protein 3 (Arp3) is mutated in proteinuric BUF/Mna rats.

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.

Generation of muscle aquaporin 4 overexpressing transgenic mouse: its characterization at RNA and protein levels including freeze-fracture study.

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.

The relationship between intramembranous particles and aquaporin molecules in the plasma membranes of normal rat skeletal muscles: a fracture-label study.

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.

Expression of myoferlin in skeletal muscles of patients with dysferlinopathy.

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.

Sarcospan: ultrastructural localization and its relation to the sarcoglycan subcomplex.

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.

Aquaporin 1: examination of its expression and localization in normal human skeletal muscle tissue.

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.

Alterations of molecular architectures in skeletal muscle plasma membranes of dehydrated and water-loaded mice.

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.

Expression and localization of aquaporin 7 in normal skeletal myofiber.

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.

Experimental confirmation of Serratia marcescens contamination in multiple-dose vials of heparin-saline solution.

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.

Reduced density of intramembranous particle clusters: freeze-fracture study of mdx mouse muscle plasma membrane.

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.

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers.

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.

Ultrastructural localization of aquaporin 4 and alpha1-syntrophin in the vascular feet of brain astrocytes.

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.

Changes in the distribution and density of caveolin 3 molecules at the plasma membrane of mdx mouse skeletal muscles: a fracture-label electron microscopic study.

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<0.01, P<0.01 and P<0.05 by two-tailed t-test), although in mdx mice, the overall arrangement of gold particles appeared to be irregular. These findings may reflect the active process of caveolar formation and the results of the disrupted protein-protein interaction in dystrophin-deficient muscle plasma membrane.

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.