Kazunori Yoshimura

Rho modulates hepatic sinusoidal endothelial fenestrae via regulation of the actin cytoskeleton in rat endothelial cells

The presence of actin-like microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. Rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The present study aimed to examine how a Rho stimulator; lysophosphatidic acid (LPA), and a Rho inhibitor; bacterial toxin C3 transferase (C3-transferase), affect the morphology of SEF. Monolayers of SEC culture were established by infusing a rat liver with collagenase for 30u2009min and then culturing in RMPI medium for 24u2009h. The cells were separated into three groups; control, LPA-treated (15u2009μM), and C3-transferase-treated (15u2009μg/ml) groups. SEF morphology was observed by scanning electron microscopy. Formation of F-actin stress fibers was observed by confocal microscopy. Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. Active Rho was measured by Rens modification. Treatment of SECs with LPA contracted the SEF, concomitant with increases in F-actin stress fiber and actin microfilament, and high expression of phosphorylated myosin light-chain kinase. Following treatment with C3-transferase, SEF dilated and fused, concomitant with a loss of F-actin and microfilament, and low expression of phosphorylated myosin light chain. Rho A expression does not change by both treatments. In conclusion, these results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.

Structure of a Major Oligosaccharide of PASII/PMP22 Glycoprotein in Bovine Peripheral Nerve Myelin

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln‐Asn‐Cys‐Ser‐Thr, where the asparagine was glycosylated. To eliminate all the contaminated Po glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N‐[2‐(2‐pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2‐aminopyridine (PA) as a fluorescent tag. One major PA‐oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and 1H‐NMR studies and comparing the derivatives of PA‐OPPE1 and PA‐oligosaccharides of γ‐globulin on HPLC. The structure, SO4‐3GlcAβ1‐3Galβ1‐4GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐4) (GlcNAcβ1‐2Manα1‐3)Manβ1‐4GlcNAcβ1‐4(Fucα1‐6)GlcNAc‐PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine Po previously reported.

Antigens of monoclonal antibody NB3C4 are novel markers for oligodendrocytes.

We produced NB3C4, a novel monoclonal antibody specific for oligodendrocytes, using human neuroblastoma IMR-32 cells. NB3C4 specifically recognized oligodendrocytes in the CNS, although it bound to neuroblastoma IMR-32 cells and oligodendrocytes in vitro. Double immunofluorescence staining of rat brain using NB3C4 and anti-GST-π, anti-glial fibrillary acidic protein (GFAP), or anti-neurofilament 200 (NF) antibody revealed that anti-GST-π antibody identified an oligodendro- cyte marker recognizing NB3C4-positive cells, while both anti-GFAP and anti-NF antibody did not. Western blotting of rat brain homogenates showed that NB3C4 bound three proteins of 22–28 kDa, while the anti-GST-π recognized a 27 kDa protein. Therefore, antigens recognized by NB3C4 could be novel markers for oligodendrocytes.

A new monoclonal antibody, 4F2, specific for the oligodendroglial cell lineage, recognizes ATP‐dependent RNA helicase Ddx54: Possible association with myelin basic protein

Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2+, Sox10+, A2B5+, NG2+, Olig2+, O4+, and myelin basic protein (MBP)+ cells but did not label any GFAP+ or NeuN+ cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2‐positive cells were located initially in the ventricular zone as Musashi1+ Tuj1− populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5‐kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination. Journal of Neuroscience Research (2011)

Relation between ultrastructural localization, changes in caveolin-1, and capillarization of liver sinusoidal endothelial cells in human hepatitis C-related cirrhotic liver.

Most vascular endothelial cells are continuously exposed to shear stress in vivo. Caveolae are omega-shaped membrane invaginations in endothelial cells (ECs) and are enriched in cholesterol, caveolins, and signaling molecules. This study was designed to elucidate the ultrastructural localization and change in caveolin-1 expression within human liver sinusoidal endothelial cells (LSECs) during the progression of cirrhosis caused by hepatitis C, using tissue sections prepared via perfusion-fixation. Normal control liver specimens and hepatitis C–related Child-Pugh A and C cirrhotic liver specimens were studied. Caveolin-1 in the liver sinusoids was examined via immunohistochemistry, Western blotting, and immunoelectron microscopy. In control liver tissue, caveolin-1 was localized on caveolae mainly in arterial and portal endothelial cells of the portal tract and was also found on vesicles and some fenestrae in LSECs around the central vein. In cirrhotic liver tissue, aberrant caveolin-1 expression was observed on caveolae-like structures in LSECs. Caveolin-1 was especially overexpressed in late-stage cirrhosis. This study demonstrates that caveolin-1 is strongly expressed within caveolae-like structures and associated vesicles within LSECs of the hepatitis C–related cirrhotic liver. These findings suggest a direct association of caveolin-1 in the process of differentiation of LSECs in cirrhosis-mediated capillarization.

Complete amino acid sequence of a unique protein related to the variable domain of lambda light chain from a case with fanconi syndrome

The complete amino acid sequence has been determined of a unique protein from a 55-years-old female with multiple myeloma associated with Fanconi syndrome. It existed in a monomer form with an apparent molecular weight of 10K daltons, and was consisted of 106 amino acid residues. The sequence was characteristic of the V-region of lambda light chains and was highly homologous with that of the first 106 residues of V lambda III subgroup. The presence of an intact light chain as well as a 13K daltons fragment, corresponding to the entire C-region, strongly suggests that the unique component is a catabolic product from the intact light chain rather than an aberrant product of synthesis.

A new immunoglobulin marker

In an examination of 16 human immunoglobulin lambda-light chains, one was found to have amino acid substitutions which have not previously been reported for the constant region of the lambda-chains. It involved in two positions: an arginine instead of lysine at position 130, and a leucine for glutamine at position 195 (Sh numbering (1) ). The structural variation is tentatively designated as Is marker.

Nuclear membrane antigen specific to nerve and muscle tissues.

A monoclonal antibody, 2F7, raised against a nuclear protein subfraction recognized the nuclear membrane of nervous and muscular tissues of guinea pig, rat and rabbit, but no other tissue was stained. In the nervous system, both neurons and glial cells were labelled. An electron microscopic immunohistochemical study demonstrated that 2F7 antibody labelled the inner surface of the nuclear membrane. Western blot analysis on the nuclear envelope fraction containing nuclear lamina revealed that this antibody reacted to two minor component proteins of 80 and 82KDa. These 2F7 antigens expressed preferentially in the nervous and muscular tissues were distinct from the major nuclear envelope proteins reported so far and might be related to neuronal or muscular tissue-specific functions.

Proteomic analysis of macrophages temporarily stimulated with albumin, oxidized or reduced galectin-1

s / Neuroscience Research 68S (2010) e223–e334 e259 P2-f06 Analysis of IP3 signaling for alpha-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) induced-nuclear Ca2+ increase and the appearance of nuclear granulation in hippocampal neurons Kyoko Ibaraki 1 , Seiji Yamamoto 1, Takashi Tsuboi 2, Susumu Terakawa 1 1 Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan 2 Laboratory of Cell Imaging, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan Under the video enhanced contrast-differential interference contrast microscope, continuous exposure to AMPA dose-dependently induced the nuclear granulation and the cellular swelling in hippocampal neurons. In this process, AMPA significantly increased the percentage of severer DNA damage after nuclear Ca2+ increase and the appearance of nuclear granulation. In this study, we investigated whether IP3 signaling is involved in the nuclear Ca2+ increase and the appearance of the nuclear granulation in hippocampal neuron. AMPA induced IP3 production in hippocampal neuron, since green fluorescent protein-tagged pleckstrin homology domain of PLC1 (GFP-PHD) was translocated from the plasmamembrane to the cytoplasm in response to increased concentration of IP3. A blocker of IP3-induced Ca2+ release, Xestospongin C (1 M), partially blocked the increase of Ca2+ concentration in the nucleus, however, it did not block the appearance of nuclear granulation. These results suggested that IP3-induced Ca2+ increase in the nucleus may not contribute to the induction of nuclear granulation although AMPA induced IP3 production in hippocampal neuron. doi:10.1016/j.neures.2010.07.1148 P2-f07 Apoptosis-inducing factor deficiency decreases the proliferation rate and protects the subventricular zone against ionizing radiation Yoshiaki Sato 1,2 , Kazuhiro Osato 1,3, Tomoyo Ochiishi 1,4, Akari Osato 1,3, Changlian Zhu 1,9, Machiko Sato 1,6, Janos Swanpalmer 5, Nazanine Modjtahedi 8, Guido Kroemer 8, Georg H. Kuhn 1, Klas Blomgren 1,7 1 Center for Brain Repair and Rehabilitation, University of Gothenburg, Gothenburg, Sweden 2 Maternity & Perinatal Care Center, Nagoya University Hospital, Nagoya, Japan 3 Department of Obstetrics and Gynecology, Miyazaki Medical College, University of Miyazaki, Kiyotake, Japan 4 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan 5 Department of Radiation Physics, Sahlgrenska University Hospital, Gothenburg, Sweden 6 Department of Obstetrics and Gynecology, Narita Hospital, Nagoya, Japan 7 Department of Pediat. Oncology, Queen Silvia Children’s Hospital, Gothenburg, Sweden 8 Inst. National de la Santé et de la Recherche Médicale, U848, Villejuif, France 9 Department of Pediat., Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China Cranial radiotherapy in children often leads to progressive cognitive decline. We have established a rodent model of irradiation-induced injury to the young brain. A single dose of 8 Gy was administered to the left hemisphere of postnatal day 10 (P10) mice. Harlequin (Hq) mice, carrying the hypomorphic apoptosis-inducing factor AIFHq mutation, express 60% less AIF at P10 and displayed significantly fewer dying cells in the subventricular zone (SVZ) 6 h after IR, compared with wild type (Wt) littermates. Irradiated cyclophilin A-deficient (CypA−/−) mice confirmed that CypA plays an essential role in AIF-induced apoptosis after IR. Hq mice displayed no reduction in SVZ size 7 days after IR, whereas 48% of the SVZ was lost in Wt mice. The proliferation rate was lower in the SVZ of Hq mice. Cultured neural precursor cells from the SVZ of Hq mice displayed a slower proliferation rate and were more resistant to IR. IR preferentially kills proliferating cells, and the slower proliferation rate in the SVZ of Hq mice may, at least partly, explain the protective effect of the Hq mutation. Together, these results indicate that targeting AIF may provide a fruitful strategy for protection of normal brain tissue against the detrimental side effects of IR. doi:10.1016/j.neures.2010.07.1149 P2-f08 Proteomic analysis of macrophages temporarily stimulated with albumin, oxidized or reduced galectin-1 Kazunori Yoshimura 1 , Fuyuki Kametani 2, Takashi Miyazaki 3, Hiromichi Suzuki 3, Yasushi Sakaoto 4, Mayumi Kato 1, Masami Nishina 5, Hidenori Horie 6, Toshihiko Kadoya 7 1 Dept Rehab, Nihon Inst Med Sci, Saitama, Japan 2 Tokyo Inst Psychia, Tokyo Metro Org Med Res, Tokyo, Japan 3 Commun Health Sci Ctr, Saitama Med Univ, Saitama, Japan 4 Bio Med Res Ctr, Saitama Med Univ, Saitama, Japan 5 Med Res Ctr, Saitama Med Univ, Saitama, Japan 6 Res Ctr Brain Oral Sci, Kanagawa Dent Col, Kanagawa, Japan 7 Dept Biotechnol, Facul Eng, Maebashi Inst Technol, Gunma, Japan Oxidized galectin-1 was discovered as a factor that regulates initial axonal growth in the peripheral nerve after axotomy (Horie et al., 1999; Inagaki et al., 2000). Galectin-1 is well known as a lectin which binds beta-galactoside when it is in a reduced form. Once galctin-1 has been oxidized, it exhibits marked peripheral nerve regeneration-promoting activity though it loses the lectin activity. Furthermore, we have demonstrated that the oxidized galectin-1 promotes the axonal regeneration via macrophages (Horie et al., 2004). At the present study, to explore the proteins secreted from macrophages and the intracellular proteins of macrophages stimulated with oxidized galectin-1 (Ox-Gal), we performed 2-dimensional electrophoresis (2-D) following Sypro Rubby and LC–MS/MS, and 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE) of macrophages treated with bovine serum albumin, Ox-Gal or galectin-1 mutant, in which all six cysteine residues were replaced by serine (CS-Gal), or with bovine serum albumin. As a result, 2-D showed that macrophages secreted several proteins by stimulation of Ox-Gal. Proteomic analysis including both 2D-DIGE and LC–MS/MS identified several proteins such as an arginase1 and GRP78 as the changed proteins after the treatment of Ox-Gal. Moreover, Real-Time RT-PCR analysis of the macrophages showed that different stimulants gave expression of different mRNAs. Therefore these results suggest that the signaling of Ox-Gal is different from that of reduced galectin-1. doi:10.1016/j.neures.2010.07.1150 P2-f09 Combining extensive treadmill training with a selective semaphorin3A inhibitor treatment enhances locomotor functional recovery by wiring regenerated axons in adult spinal cord-transected rats Liang Zhang 1,2,3 , Shinjiro Kaneko 3,6, Akihiko Sano 5, Miho Maeda 5, Akiyoshi Kishino 5, Masahiko Mukaino 1,4, Yoshiaki Toyama 3, Meigen Liu 1, Masaya Nakamura 3, Hideyuki Okano 2 1 Dept Rehabil Med, Univ Keio, Tokyo 2 Dept Physiol, Univ Keio, Tokyo 3 Dept Orthop, Univ Keio, Tokyo 4 Rehabil Center, Univ Keio, Tokyo 5 Dainippon Sumitomo Pharma Co., Ltd., Otsuka 6 Murayama Med Center, Tokyo It has been known that completely spinal cord-transected (SCT) adult rats exhibit extremely limited functional recovery.We previously reported the efficacy of semaphorin3A (Sema3A) inhibitor treatment in this model. One of the remaining questions is whether combining rehabilitation, such as treadmill training, with Sema3A inhibitor treatment can enhance the effect of Sema3A inhibitor. In this study we employed the combinatorial therapy of selective Sema3A inhibitor (SM-34543) and treadmill training for adult SCT rats to examine its combinatorial effect. For SM-345431 administration, we employed the newly developed drug delivery system to secure the continuous drug delivery during the period of the experiment. By anatomical and kinematic analysis we found that, being consistent with our previous study rats treated with Sema3A inhibitor showed significantly enhanced axonal regeneration, but limited functional recovery. Enhanced axonal regeneration was also observed in the combinatorial group, and interestingly, significantly enhanced locomotor functional recovery was observed in this combinatorial group (compared with Sema3A inhibitor alone group). Moreover, we found that this combinatorial intervention could recruit specific populations of spinal circuits, enhancing motor function presumably via increased sensory input and functional remodeling of locomotor pathways. Conceivably, although axonal regeneration is very important in spinal cord injury (SCI) treatment, it alone cannot result in substantial locomotor functional recovery. It is speculated that treadmill training might be able to recruit regenerated axons and make them orient to their target more accurately (wiring effect), as a result, lead to improved locomotor functional recovery after SCT. These results provide a new prospect for this combinatorial ther-