研究者詳細 - 大橋　俊孝

論文 - 大橋　俊孝

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The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. 査読国際誌

James W Fawcett, Toshitaka Oohashi, Tommaso Pizzorusso

Nature reviews. Neuroscience 20 ( 8 ) 451 - 465 2019年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Perineuronal nets (PNNs) are extracellular matrix (ECM) chondroitin sulfate proteoglycan (CSPG)-containing structures that surround the soma and dendrites of various mammalian neuronal cell types. PNNs appear during development around the time that the critical periods for developmental plasticity end and are important for both their onset and closure. A similar structure - the perinodal ECM - surrounds the axonal nodes of Ranvier and appears as myelination is completed, acting as an ion-diffusion barrier that affects axonal conduction speed. Recent work has revealed the importance of PNNs in controlling plasticity in the CNS. Digestion, blocking or removal of PNNs influences functional recovery after a variety of CNS lesions. PNNs have further been shown to be involved in the regulation of memory and have been implicated in a number of psychiatric disorders.

DOI： 10.1038/s41583-019-0196-3

BMP-2 generates a functional bone marrow niche by inducing the differentiation of local mesenchymal cells into CAR cells

Mitsuaki Ono, Ikue Tosa, Anh Tuan Dang, Ziyi Wang, Maiko Kimura, Wakana Kitagawa, Kei Ishibashi, Hang Thuy Do, Kun Zhao, Emilio Satoshi Hara, Noboru Asada, Takashi Nagasawa, Toshitaka Oohashi, Naoya Ohara, Takuo Kuboki

Blood Advances 2025年3月

掲載種別：研究論文（学術雑誌）出版者・発行元：American Society of Hematology

Ectopic bone marrow (eBM) holds tremendous potential as an artificial organ, serving not only in stem cell transplantation therapies but also as a controlled experimental system for analyzing cellular dynamics and interactions between cells and the matrix during the formation, maintenance, and aging of bone marrow (BM). Although bone morphogenetic protein (BMP)-2 has been reported to induce eBM formation, it remains unproven whether BMP-2 induced eBM (BMP-eBM) can provide a functional BM niche that is comparable to native BM in long bones (LB-BM). In this study, through the use of single-cell RNA sequencing and transplantation models, we demonstrate for the first time that BMP-eBM displays a microstructure, cellular composition, and functional hematopoiesis similar to LB-BM. BMP-eBM establishes an optimized microenvironment capable of supporting hematopoietic stem cells (HSCs) and CXC-chemokine-ligand-12 (CXCL12)-abundant reticular (CAR) cells, which are critical components of the BM niche. Significantly, BMP-eBM was able to restore survival in irradiated mice. Through parabiosis and cell transplantation experiments, we identified that in situ adipose tissue-derived CD51highCxcl12-GFP- cells are the principal source of CAR cells within BMP-eBM. Furthermore, BMP-eBM can be isolated and, after preconditioning, re-transplanted as an independent, functional hematopoietic organ. In conclusion, our study confirms that BMP-eBM functions effectively as a hematopoietic organ, capable of supporting and maintaining a functional BM niche. These findings underscore BMP-2 as a crucial molecule for eBM generation and suggest its potential for addressing BM-related diseases and for use as a platform for in vitro and ex vivo biomedical applications.

DOI： 10.1182/bloodadvances.2024014062

Optimizing β-TCP with E-rhBMP-2-infused fibrin for vertical bone regeneration in a mouse calvarium model

Kun Zhao, Mitsuaki Ono, Xindi Mu, Ziyi Wang, Shichao Xie, Tomoko Yonezawa, Masahiro Okada, Takuya Matsumoto, Takuo Kuboki, Toshitaka Oohashi

Regenerative Biomaterials 12 2025年1月

掲載種別：研究論文（学術雑誌）出版者・発行元：Oxford University Press (OUP)

Abstract

Effective reconstruction of large bone defects, particularly in thickness, remains one of the major challenges in orthopedic and dental fields. We previously produced an Escherichia coli-based industrial-scale GMP-grade recombinant human bone morphogenetic protein-2 (E-rhBMP-2) and showed that the combination of E-rhBMP-2 with beta-tricalcium phosphate (β-TCP/E-rhBMP-2) can effectively promote bone reconstruction. However, the limited mechanical strength and poor morphology retention of β-TCP granules are key points that need optimization to obtain more effective grafts and further expand its clinical applications. Therefore, we combined β-TCP/E-rhBMP-2 with fibrin gel to enhance its mechanical properties and usability for vertical bone regeneration. We investigated the mechanical properties and vertical bone regeneration effects of the materials applied, with or without fibrin containing E-rhBMP-2, in a calvarial defect model in mice. Compression tests were conducted to assess the initial stability of the materials. Scanning electron microscopy and Fourier transform infrared spectroscopy were conducted to characterize the presence of fibrin on the scaffold. After 4 and 12 weeks of implantation, micro-computed tomography and histological and immunofluorescent analyses were performed to assess the morphology and volume of the newly formed bone. The fibrin-containing groups had significantly higher initial mechanical strength and higher ability to maintain their morphology in vivo compared to the counterparts without fibrin. However, fibrin gel alone suppressed the bone formation ability of β-TCP/E-rhBMP-2 whereas the presence of high doses of E-rhBMP-2 in fibrin gel resulted in material resorption and enhanced new bone formation. In conclusion, fibrin gel significantly improved the mechanical strength and surgical manageability of the β-TCP/E-rhBMP-2 scaffold, and the addition of E-rhBMP-2 to the fibrin gel further enhanced the vertical bone regeneration and initial structural integrity of the scaffold.

DOI： 10.1093/rb/rbae144

その他リンク： https://academic.oup.com/rb/article-pdf/doi/10.1093/rb/rbae144/61841803/rbae144.pdf

O‐<scp>GlcNAcylation</scp> regulates osteoblast differentiation through the morphological changes in mitochondria, cytoskeleton, and endoplasmic reticulum

Yao Weng, Ziyi Wang, Heriati Sitosari, Mitsuaki Ono, Hirohiko Okamura, Toshitaka Oohashi

BioFactors 2024年10月

掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Abstract

To explore the potential mechanisms which O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates osteogenesis, a publicly RNA‐seq dataset was re‐analyzed with literature‐mining and showed the primary targets of O‐GlcNAcylation in osteoblasts are mitochondria/cytoskeleton. Although the O‐GlcNAcylation‐regulated mitochondria/cytoskeleton has been extensively studied, its specific role during osteogenesis remains unclear. To address this, we knocked out Ogt (Ogt‐KO) in MC3T3‐E1 osteoblastic cells. Then, significantly reduced osteoblast differentiation, motility, proliferation, mitochondria–endoplasmic reticulum (Mito–ER) coupling, volume of ER, nuclear tubulins, and oxygen metabolism were observed in Ogt‐KO cells. Through artificial intelligence (AI)‐predicted cellular structures, the time‐lapse live cells imaging with reactive‐oxygen‐species/hypoxia staining showed that lower cell proliferation and altered oxygen metabolism in the Ogt‐KO cells were correlated with the Mito–ER coupling. Bioinformatics analysis, combined with correlated mRNA and protein expression, suggested that Ezh2 and its downstream targets (Opa1, Gsk3a, Wnt3a, Hif1a, and Hspa9) may be involved in O‐GlcNAcylation‐regulated Mito–ER coupling, ultimately impacting osteoblast differentiation. In conclusion, our findings indicate that O‐GlcNAcylation‐regulated osteoblast differentiation is linked to morphological changes in mitochondria, cytoskeleton, and ER, with Ezh2 potentially playing a crucial role.

DOI： 10.1002/biof.2131

The cell membrane as biofunctional material for accelerated bone repair. 国際誌

Emi Hatano, Nahid Akhter, Risa Anada, Mitsuaki Ono, Toshitaka Oohashi, Takuo Kuboki, Hiroshi Kamioka, Masahiro Okada, Takuya Matsumoto, Emilio Satoshi Hara

Acta biomaterialia 2024年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

The cell (plasma) membrane is enriched with numerous receptors, ligands, enzymes, and phospholipids that play important roles in cell-cell and cell-extracellular matrix interactions governing, for instance, tissue development and repair. We previously showed that plasma membrane nanofragments (PMNFs) act as nucleation sites for bone formation in vivo, and induce in vitro mineralization within 1 day. In this study, we optimized the methods for generating, isolating, and applying PMNFs as a cell-free therapeutic to expedite bone defect repair. The PMNFs were isolated from different mouse cell lines (chondrocytes, osteoblasts, and fibroblasts), pre-conditioned, lyophilized, and subsequently transplanted into 2 mm critical-sized calvarial defects in mice (n = 75). The PMNFs from chondrocytes, following a 3-day pre-incubation, significantly accelerated bone repair within 2 weeks, through a coordinated attraction of macrophages, endothelial cells, and osteoblasts to the healing site. In vitro experiments confirmed that PMNFs enhanced cell adhesion. Comparison of the PMNF efficacy with phosphatidylserine, amorphous calcium phosphate (ACP), and living cells confirmed the unique ability of PMNFs to promote accelerated bone repair. Importantly, PMNFs promoted nearly complete integration of the regenerated bone with native tissue after 6 weeks (% non-integrated bone area = 15.02), contrasting with the partial integration (% non-integrated bone area = 56.10; p < 0.01, Student's test) with transplantation of ACP. Vickers microhardness tests demonstrated that the regenerated bone after 6 weeks (30.10 ± 1.75) exhibited hardness similar to native bone (31.07 ± 2.46). In conclusion, this is the first study to demonstrate that cell membrane can be a promising cell-free material with multifaceted biofunctional properties that promote accelerated bone repair. STATEMENT OF SIGNIFICANCE.

DOI： 10.1016/j.actbio.2024.07.037

Local E-rhBMP-2/β-TCP Application Rescues Osteocyte Dendritic Integrity and Reduces Microstructural Damage in Alveolar Bone Post-Extraction in MRONJ-like Mouse Model

Anh Tuan Dang, Mitsuaki Ono, Ziyi Wang, Ikue Tosa, Emilio Satoshi Hara, Akihiro Mikai, Wakana Kitagawa, Tomoko Yonezawa, Takuo Kuboki, Toshitaka Oohashi

International Journal of Molecular Sciences 25 ( 12 ) 6648 - 6648 2024年6月

掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

The pathology of medication-related osteonecrosis of the jaw (MRONJ), often associated with antiresorptive therapy, is still not fully understood. Osteocyte networks are known to play a critical role in maintaining bone homeostasis and repair, but the exact condition of these networks in MRONJ is unknown. On the other hand, the local application of E-coli-derived Recombinant Human Bone Morphogenetic Protein 2/β-Tricalcium phosphate (E-rhBMP-2/β-TCP) has been shown to promote bone regeneration and mitigate osteonecrosis in MRONJ-like mouse models, indicating its potential therapeutic application for the treatment of MRONJ. However, the detailed effect of BMP-2 treatment on restoring bone integrity, including its osteocyte network, in an MRONJ condition remains unclear. Therefore, in the present study, by applying a scanning electron microscope (SEM) analysis and a 3D osteocyte network reconstruction workflow on the alveolar bone surrounding the tooth extraction socket of an MRONJ-like mouse model, we examined the effectiveness of BMP-2/β-TCP therapy on the alleviation of MRONJ-related bone necrosis with a particular focus on the osteocyte network and alveolar bone microstructure (microcrack accumulation). The 3D osteocyte dendritic analysis showed a significant decrease in osteocyte dendritic parameters along with a delay in bone remodeling in the MRONJ group compared to the healthy counterpart. The SEM analysis also revealed a notable increase in the number of microcracks in the alveolar bone surface in the MRONJ group compared to the healthy group. In contrast, all of those parameters were restored in the E-rhBMP-2/β-TCP-treated group to levels that were almost similar to those in the healthy group. In summary, our study reveals that MRONJ induces osteocyte network degradation and microcrack accumulation, while application of E-rhBMP-2/β-TCP can restore a compromised osteocyte network and abrogate microcrack accumulation in MRONJ.

DOI： 10.3390/ijms25126648

Inverse genetics tracing the differentiation pathway of human chondrocytes 査読

H T Do, M Ono, Z Wang, W Kitagawa, A T Dang, T Yonezawa, T Kuboki, T Oohashi, S Kubota

Osteoarthritis Cartilage 32 ( 11 ) 1419 - 1432 2024年6月

Exploring the Regulators of Keratinization: Role of BMP-2 in Oral Mucosa

Xindi Mu, Mitsuaki Ono, Ha Thi Thu Nguyen, Ziyi Wang, Kun Zhao, Taishi Komori, Tomoko Yonezawa, Takuo Kuboki, Toshitaka Oohashi

Cells 13 ( 10 ) 807 - 807 2024年5月

掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial–mesenchymal cell co-culture models in the air–liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.

DOI： 10.3390/cells13100807

Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP. 国際誌

Haruko Miyazaki, Saki Nishioka, Tomoyuki Yamanaka, Manabu Abe, Yukio Imamura, Tomohiro Miyasaka, Nobuto Kakuda, Toshitaka Oohashi, Tomomi Shimogori, Kazuhiro Yamakawa, Masahito Ikawa, Nobuyuki Nukina

Transgenic research 33 ( 3 ) 99 - 117 2024年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Golli-myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli-myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli-myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli-myelin basic protein knockout through the generation of conditional knockout mice (Golli-myelin basic proteinsfl/fl; E3CreN), in which Golli-myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli-myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli-myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli-myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli-myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli-myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.

DOI： 10.1007/s11248-024-00382-0

Filamin A mediates embryonical palatal fusion by linking mechanotransduction with β-Catenin/Smad2

Ziyi Wang, Satoru Hayano, Yao Weng, Xindi Mu, Mitsuaki Ono, Jeremie Oliver Piña, Rena N. D'Souza, Takashi Yamashiro, Toshitaka Oohashi, Hiroshi Kamioka

2024年2月

出版者・発行元：Cold Spring Harbor Laboratory

To decipher potential mechanisms underlying cleft palate (CP), we used advanced bioinformatic integrated with literature mining and genome-wide association study (GWAS). Re-analysis of RNA-seq data (GSE45568, GSE185279) combined with literature mining highlighted the roles of Filamin A (Flna) and Epithelial-Mesenchymal Transition (EMT) in palatal development. Immunofluorescence of in vivo palatal shelves showed increased Flna in medial edge epithelial (MEE) cells and EMT cells located in an epithelial triangle. Inhibition of TGF-β or RhoA and mechanical stimuli impacted Flna expression in ex vivo cultured palatal shelves. Re-analysis of scRNA-seq data (GSE155928) highlighted a correlation between Flna and Ctnnb1 in EMT cells. Flna knockdown affected β-catenin/Smad2 expression in cultured palatal shelves and HaCaT cells. Epithelium-specific knockout of Flna delayed palatal fusion in female mice but not males. Mendelian randomization analysis suggested that parental habitual physical activity (HPA) was causally associated with lower risk of CP in their offspring. Together, these findings suggested that parental HPA could benefit their offspring's palatal development through Flna by linking mechanotransduction with the Wnt/TGF-β/Smad signaling pathways during palatal fusion.

DOI： 10.1101/2024.02.16.580664

発達期および成熟期におけるHapln4欠損マウスCalyx-MNTBシナプス周囲のグリア細胞の分布に関する研究

北見智, 谷祐一, 野島弘二郎, 兼城一媛乃, Nguyen Duy Hoang, 宮崎晴子, 堀哲也, 大橋俊孝

日本結合組織学会学術大会プログラム・抄録集 55回 150 - 150 2023年6月

Aggrecan governs intervertebral discs development by providing critical mechanical cues of the extracellular matrix. 国際誌

Marta Empere, Xujia Wang, Carina Prein, Anders Aspberg, Markus Moser, Toshitaka Oohashi, Hauke Clausen-Schaumann, Attila Aszodi, Paolo Alberton

Frontiers in bioengineering and biotechnology 11 1128587 - 1128587 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

Aggrecan (ACAN) is localized in the intervertebral disc (IVD) in unique compartment-specific patterns where it contributes to the tissue structure and mechanical function together with collagens. The extracellular matrix (ECM) of the IVD undergoes degenerative changes during aging, misuse or trauma, which inevitably alter the biochemical and biomechanical properties of the tissue. A deeper understanding of these processes can be achieved in genetically engineered mouse models, taking into account the multifaceted aspects of IVD development. In this study, we generated aggrecan insertion mutant mice (Acan iE5/iE5 ) by interrupting exon 5 coding for the G1 domain of ACAN, and analyzed the morphological and mechanical properties of the different IVD compartments during embryonic development. Western blotting using an antibody against the total core protein failed to detect ACAN in cartilage extracts, whereas immunohistochemistry by a G1-specific antibody showed weak signals in vertebral tissues of Acan iE5/iE5 mice. Homozygous mutant mice are perinatally lethal and characterized by short snout, cleft palate and disproportionate dwarfism. Whole-mount skeletal staining and µ-CT analysis of Acan iE5/iE5 mice at embryonic day 18.5 revealed compressed vertebral bodies with accelerated mineralization compared to wild type controls. In Acan iE5/iE5 mice, histochemical staining revealed collapsed extracellular matrix with negligible sulfated glycosaminoglycan content accompanied by a high cellular density. Collagen type II deposition was not impaired in the IVD of Acan iE5/iE5 mice, as shown by immunohistochemistry. Mutant mice developed a severe IVD phenotype with deformed nucleus pulposus and thinned cartilaginous endplates accompanied by a disrupted growth plate structure in the vertebral body. Atomic force microscopy (AFM) imaging demonstrated a denser collagen network with thinner fibrils in the mutant IVD zones compared to wild type. Nanoscale AFM indentation revealed bimodal stiffness distribution attributable to the softer proteoglycan moiety and harder collagenous fibrils of the wild type IVD ECM. In Acan iE5/iE5 mice, loss of aggrecan resulted in a marked shift of the Young's modulus to higher values in all IVD zones. In conclusion, we demonstrated that aggrecan is pivotal for the determination and maintenance of the proper stiffness of IVD and vertebral tissues, which in turn could play an essential role in providing developmental biomechanical cues.

DOI： 10.3389/fbioe.2023.1128587

Ten-m4 plays a unique role in the establishment of binocular visual circuits. 国際誌

Timothy R Young, Dylan Black, Hannan Mansuri, Toshitaka Oohashi, Xiao-Hong Zhou, Atomu Sawatari, Catherine A Leamey

Developmental neurobiology 83 ( 3-4 ) 104 - 124 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

The patterning of binocular vision requires distinct molecular pathways for inputs arising from each side of the nervous system. Recent studies have demonstrated important roles for members of the Ten-m/Odz/teneurin family in the development of ipsilateral retinal projections. Here, we further highlight the significance of this gene family in visual development by identifying a role for Ten-m4 during the formation of the ipsilateral projection in the mouse. Ten-m4 was found to be expressed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) during development. Anterograde and retrograde tracing experiments in Ten-m4 knockout (KO) mice revealed a specific increase in ipsilateral retinal ganglion cells projecting to dLGN and SC. This increase was most prominent in regions corresponding to temporal retina. Consistent with this, EphB1 expression in the retina around the time of decussation was enhanced in this temporal region for KO mice, suggesting that the increased size of the ipsilateral population arises due to an increased number of retinal ganglion cells remaining ipsilaterally at the optic chiasm due to EphB1-mediated repulsion. The ectopic ipsilaterally targeted retinal ganglion cell projection observed in Ten-m4 KOs was associated with changes in response to ethologically relevant visual stimuli. Together, these data demonstrate a requirement for Ten-m4 in the establishment of ipsilateral projections from the retina, which likely acts in combination with other Ten-m members (Ten-m2 and Ten-m3) to promote the formation of functional binocular circuits.

DOI： 10.1002/dneu.22912

Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption. 国際誌

Hirofumi Fujita, Aoi Ando, Yohei Mizusawa, Mitsuaki Ono, Takako Hattori, Munenori Habuta, Toshitaka Oohashi, Satoshi Kubota, Hideyo Ohuchi

PloS one 17 ( 11 ) e0277307 2022年

記述言語：英語掲載種別：研究論文（学術雑誌）

Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

DOI： 10.1371/journal.pone.0277307

Suppression of Bone Necrosis around Tooth Extraction Socket in a MRONJ-like Mouse Model by E-rhBMP-2 Containing Artificial Bone Graft Administration. 査読国際誌

Yukie Tanaka, Kyaw Thu Aung, Mitsuaki Ono, Akihiro Mikai, Anh Tuan Dang, Emilio Satoshi Hara, Ikue Tosa, Kei Ishibashi, Aya Ono-Kimura, Kumiko Nawachi, Takuo Kuboki, Toshitaka Oohashi

International journal of molecular sciences 22 ( 23 ) 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Medication-related osteonecrosis of the jaw (MRONJ) is related to impaired bone healing conditions in the maxillomandibular bone region as a complication of bisphosphonate intake. Although there are several hypotheses for the onset of MRONJ symptoms, one of the possible causes is the inhibition of bone turnover and blood supply leading to bone necrosis. The optimal treatment strategy for MRONJ has not been established either. BMP-2, a member of the TGF-β superfamily, is well known for regulating bone remodeling and homeostasis prenatally and postnatally. Therefore, the objectives of this study were to evaluate whether cyclophosphamide/zoledronate (CY/ZA) induces necrosis of the bone surrounding the tooth extraction socket, and to examine the therapeutic potential of BMP-2 in combination with the hard osteoinductive biomaterial, β-tricalcium phosphate (β-TCP), in the prevention and treatment of alveolar bone loss around the tooth extraction socket in MRONJ-like mice models. First, CY/ZA was intraperitoneally administered for three weeks, and alveolar bone necrosis was evaluated before and after tooth extraction. Next, the effect of BMP-2/β-TCP was investigated in both MRONJ-like prevention and treatment models. In the prevention model, CY/ZA was continuously administered for four weeks after BMP-2/β-TCP transplantation. In the treatment model, CY/ZA administration was suspended after transplantation of BMP-2/β-TCP. The results showed that CY/ZA induced a significant decrease in the number of empty lacunae, a sign of bone necrosis, in the alveolar bone around the tooth extraction socket after tooth extraction. Histological analysis showed a significant decrease in the necrotic alveolar bone around tooth extraction sockets in the BMP-2/β-TCP transplantation group compared to the non-transplanted control group in both MRONJ-like prevention and treatment models. However, bone mineral density, determined by micro-CT analysis, was significantly higher in the BMP-2/β-TCP transplanted group than in the control group in the prevention model only. These results clarified that alveolar bone necrosis around tooth extraction sockets can be induced after surgical intervention under CY/ZA administration. In addition, transplantation of BMP-2/β-TCP reduced the necrotic alveolar bone around the tooth extraction socket. Therefore, a combination of BMP-2/β-TCP could be an alternative approach for both prevention and treatment of MRONJ-like symptoms.

DOI： 10.3390/ijms222312823

Imatinib has minimal effects on inflammatory and osteopenic phenotypes in a murine cherubism model. 査読国際誌

Tomoyuki Mukai, Takahiko Akagi, Sumie Hiramatsu Asano, Ikue Tosa, Mitsuaki Ono, Mizuho Kittaka, Yasuyoshi Ueki, Ayano Yahagi, Masanori Iseki, Toshitaka Oohashi, Katsuhiko Ishihara, Yoshitaka Morita

Oral diseases 29 ( 3 ) 1089 - 1101 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVE: Cherubism is a genetic disorder characterised by bilateral jawbone deformation. The associated jawbone lesions regress after puberty, whereas severe cases require surgical treatment. Although several drugs have been tested, fundamental treatment strategies for cherubism have not been established. The effectiveness of imatinib has recently been reported; however, its pharmaceutical mechanism remains unclear. In this study, we tested the effects of imatinib using a cherubism mouse model. METHODS: We used Sh3bp2 P416R cherubism mutant mice, which exhibit systemic organ inflammation and osteopenia. The effects of imatinib were determined using primary bone marrow-derived macrophages. Imatinib was administered intraperitoneally to the mice, and serum tumour necrosis factor-α (TNFα), organ inflammation and bone properties were examined. RESULTS: The cherubism mutant macrophages produced higher levels of TNFα in response to lipopolysaccharide compared to wild-type macrophages, and imatinib did not significantly suppress TNFα production. Although imatinib suppressed osteoclast formation in vitro, administering it in vivo did not suppress organ inflammation and osteopenia. CONCLUSION: The in vivo administration of imatinib had a minimal therapeutic impact in cherubism mutant mice. To establish better pharmaceutical interventions, it is necessary to integrate new findings from murine models with clinical data from patients with a definitive diagnosis of cherubism.

DOI： 10.1111/odi.14073

Assessment of Possible Contributions of Hyaluronan and Proteoglycan Binding Link Protein 4 to Differential Perineuronal Net Formation at the Calyx of Held. 査読国際誌

Kojiro Nojima, Haruko Miyazaki, Tetsuya Hori, Lydia Vargova, Toshitaka Oohashi

Frontiers in cell and developmental biology 9 730550 - 730550 2021年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.

DOI： 10.3389/fcell.2021.730550

Preclinical bioequivalence study of E.coli-derived rhBMP-2/β-TCP and autogenous bone in a canine guided-bone regeneration model. 査読

Shuji Nosho, Mitsuaki Ono, Taishi Komori, Akihiro Mikai, Ikue Tosa, Kei Ishibashi, Yukie Tanaka, Aya Kimura-Ono, Emilio S Hara, Toshitaka Oohashi, Takuo Kuboki

Journal of prosthodontic research 66 ( 1 ) 124 - 130 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

PURPOSE: Bone morphogenetic protein (BMP)-2 is a potent growth factor that is widely used in the orthopedic and dental fields for bone regeneration.However, recombinant human BMP-2 (rhBMP-2) products have not been legally approved in Japan. Recently, our research group succeeded in producing GMP-grade rhBMP-2 using the E. coli system (E-rhBMP-2) at the industrial level and developed E-rhBMP-2 adsorbed onto β-TCP (E-rhBMP-2/β-TCP) as an alternative material to autogenous bone grafts. Previous studies on the toxicity, pharmacokinetics, and optimal doses of E-rhBMP-2 have confirmed its safety and efficiency. However, comparative studies with standard treatment therapies are still necessary before clinical application in humans. Therefore, in this preclinical study, we compared the bone regeneration ability of E-rhBMP-2/β-TCP and autogenous bone grafts in a canine guided-bone regeneration model. METHODS: Following extraction of the maxillary third premolar, box-type bone defects (10 mmL × 4 mmW × 9 mmH) were created in the extraction socket area and transplanted with E-rhBMP-2/β-TCP or autogenous bone graft in a canine. After 8 weeks, micro-CT and histological analyses were performed. RESULTS: Transplantation of both E-rhBMP-2/β-TCP and autogenous bone graft significantly promoted bone formation compared to the non-transplantation control group. The bone formation ability of E-rhBMP-2/β-TCP was equal to that of the autogenous bone graft. Histological analysis showed that excessive infiltration of inflammatory cells and residual β-TCP particles mostly were not observed in the E-rhBMP-2/β-TCP transplantation group. CONCLUSIONS: This preclinical study demonstrated that E-rhBMP-2/β-TCP and autogenous bone have equal potential to promote bone regeneration.

DOI： 10.2186/jpr.JPR_D_20_00226

Lack of collagen α6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutation. 査読国際誌

Shaoying Tang, Tomoko Yonezawa, Yukihide Maeda, Mitsuaki Ono, Takahiro Maeba, Toru Miyoshi, Ryusuke Momota, Yasuko Tomono, Toshitaka Oohashi

PloS one 16 ( 4 ) e0249909 2021年4月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

Congenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen α6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen α6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner's membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the α6(IV) chain and/or α5α6α5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.

DOI： 10.1371/journal.pone.0249909

Distinct Osteogenic Potentials of BMP-2 and FGF-2 in Extramedullary and Medullary Microenvironments. 査読国際誌

Shuji Nosho, Ikue Tosa, Mitsuaki Ono, Emilio Satoshi Hara, Kei Ishibashi, Akihiro Mikai, Yukie Tanaka, Aya Kimura-Ono, Taishi Komori, Kenji Maekawa, Takuo Kuboki, Toshitaka Oohashi

International journal of molecular sciences 21 ( 21 ) 2020年10月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

Bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) have been regarded as the major cytokines promoting bone formation, however, several studies have reported unexpected results with failure of bone formation or bone resorption of these growth factors. In this study, BMP-2 and FGF-2 adsorbed into atellocollagen sponges were transplanted into bone defects in the bone marrow-scarce calvaria (extramedullary environment) and bone marrow-abundant femur (medullary environment) for analysis of their in vivo effects not only on osteoblasts, osteoclasts but also on bone marrow cells. The results showed that BMP-2 induced high bone formation in the bone marrow-scarce calvaria, but induced bone resorption in the bone marrow-abundant femurs. On the other hand, FGF-2 showed opposite effects compared to those of BMP-2. Analysis of cellular dynamics revealed numerous osteoblasts and osteoclasts present in the newly-formed bone induced by BMP-2 in calvaria, but none were seen in either control or FGF-2-transplanted groups. On the other hand, in the femur, numerous osteoclasts were observed in the vicinity of the BMP-2 pellet, while a great number of osteoblasts were seen near the FGF-2 pellets or in the control group. Of note, FCM analysis showed that both BMP-2 and FGF-2 administrated in the femur did not significantly affect the hematopoietic cell population, indicating a relatively safe application of the two growth factors. Together, these results indicate that BMP-2 could be suitable for application in extramedullary bone regeneration, whereas FGF-2 could be suitable for application in medullary bone regeneration.

DOI： 10.3390/ijms21217967

BMP-2/β-TCP Local Delivery for Bone Regeneration in MRONJ-Like Mouse Model. 査読国際誌

Mikai A, Ono M, Tosa I, Nguyen HTT, Hara ES, Nosho S, Kimura-Ono A, Nawachi K, Takarada T, Kuboki T, Oohashi T

Int J Mol Sci. 21 ( 19 ) E7028 2020年9月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

Medication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (β-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/β-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/β-TCP transplanted group. For treatment studies, BMP-2/β-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/β-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/β-TCP could become a suitable therapy for the management of MRONJ.

DOI： 10.3390/ijms21197028

The Effect of Hapln4 Link Protein Deficiency on Extracellular Space Diffusion Parameters and Perineuronal Nets in the Auditory System During Aging. 査読国際誌

Petra Sucha, Martina Chmelova, Monika Kamenicka, Marcel Bochin, Toshitaka Oohashi, Lydia Vargova

Neurochemical research 45 ( 1 ) 68 - 82 2020年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Hapln4 is a link protein which stabilizes the binding between lecticans and hyaluronan in perineuronal nets (PNNs) in specific brain regions, including the medial nucleus of the trapezoid body (MNTB). The aim of this study was: (1) to reveal possible age-related alterations in the extracellular matrix composition in the MNTB and inferior colliculus, which was devoid of Hapln4 and served as a negative control, (2) to determine the impact of the Hapln4 deletion on the values of the ECS diffusion parameters in young and aged animals and (3) to verify that PNNs moderate age-related changes in the ECS diffusion, and that Hapln4-brevican complex is indispensable for the correct protective function of the PNNs. To achieve this, we evaluated the ECS diffusion parameters using the real-time iontophoretic method in the selected region in young adult (3 to 6-months-old) and aged (12 to 18-months-old) wild type and Hapln4 knock-out (KO) mice. The results were correlated with an immunohistochemical analysis of the ECM composition and astrocyte morphology. We report that the ECM composition is altered in the aged MNTB and aging is a critical point, revealing the effect of Hapln4 deficiency on the ECS diffusion. All of our findings support the hypothesis that the ECM changes in the MNTB of aged KO animals affect the ECS parameters indirectly, via morphological changes of astrocytes, which are in direct contact with synapses and can be influenced by the ongoing synaptic transmission altered by shifts in the ECM composition.

DOI： 10.1007/s11064-019-02894-2

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1. 査読国際誌

Takashi Ohtsuki, Akira Shinaoka, Kanae Kumagishi-Shinaoka, Keiichi Asano, Omer Faruk Hatipoglu, Junko Inagaki, Ken Takahashi, Toshitaka Oohashi, Keiichiro Nishida, Keiji Naruse, Satoshi Hirohata

Experimental cell research 383 ( 2 ) 111556 - 111556 2019年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1β and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

DOI： 10.1016/j.yexcr.2019.111556

Type XVIII Collagen Modulates Keratohyalin Granule Formation and Keratinization in Oral Mucosa. 査読国際誌

Ha Thi Thu Nguyen, Mitsuaki Ono, Emilio Satoshi Hara, Taishi Komori, Midori Edamatsu, Tomoko Yonezawa, Aya Kimura-Ono, Kenji Maekawa, Takuo Kuboki, Toshitaka Oohashi

International journal of molecular sciences 20 ( 19 ) 2019年9月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.

DOI： 10.3390/ijms20194739

Postnatal Runx2 deletion leads to low bone mass and adipocyte accumulation in mice bone tissues. 査読国際誌

Ikue Tosa, Daisuke Yamada, Misa Yasumatsu, Eiichi Hinoi, Mitsuaki Ono, Toshitaka Oohashi, Takuo Kuboki, Takeshi Takarada

Biochemical and biophysical research communications 516 ( 4 ) 1229 - 1233 2019年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Global gene deletion studies have established that Runt-related transcription factor-2 (Runx2) is essential during skeletogenesis for osteoblastic differentiation in both intramembranous and endochondral ossification processes. However, the postnatal significance of Runx2 in vivo is poorly understood because a global Runx2 deletion causes perinatal lethality. In this study, we generated tamoxifen-induced Runx2 global deficient mice by crossing Runx2flox mice with ROSA26-CreERT2 mice (Rosa26-CreERT2; Runx2flox/flox). Four-week-old mice were intraperitoneally treated with tamoxifen for five consecutive days, sacrificed, and analyzed six weeks after tamoxifen administration. Deletion of Runx2 led to low bone mass, which is associated with decreased bone formation and bone resorption as well as excessive bone marrow adiposity. Collectively, postnatal Runx2 absolutely plays an important role in maintaining the homeostasis of bone tissues not only in bone mass, but also in the bone marrow environment.

DOI： 10.1016/j.bbrc.2019.07.014

Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice. 査読

Takahiro Maeba, Tomoko Yonezawa, Mitsuaki Ono, Yasuko Tomono, Ritva Heljasvaara, Taina Pihlajaniemi, Kiichi Inagawa, Toshitaka Oohashi

Acta medica Okayama 73 ( 2 ) 135 - 146 2019年4月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.

DOI： 10.18926/AMO/56649

Acidic Pre-Conditioning Enhances the Stem Cell Phenotype of Human Bone Marrow Stem/Progenitor Cells. 査読国際誌

Yuri Hazehara-Kunitomo, Emilio Satoshi Hara, Mitsuaki Ono, Kyaw Thu Aung, Keiko Komi, Hai Thanh Pham, Kentaro Akiyama, Masahiro Okada, Toshitaka Oohashi, Takuya Matsumoto, Takuo Kuboki

International journal of molecular sciences 20 ( 5 ) 2019年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

A deeper understanding of the detailed mechanism of in vivo tissue healing is necessary for the development of novel regenerative therapies. Among several external factors, environmental pH is one of the crucial parameters that greatly affects enzyme activity and cellular biochemical reactions involving tissue repair and homeostasis. In this study, in order to analyze the microenvironmental conditions during bone healing, we first measured the pH in vivo at the bone healing site using a high-resolution fiber optic pH microsensor directly in femur defects and tooth extraction sockets. The pH was shown to decrease from physiological 7.4 to 6.8 during the initial two days of healing (inflammatory phase). In the same initial stages of the inflammatory phase of the bone healing process, mesenchymal stem cells (MSCs) are known to migrate to the healing site to contribute to tissue repair. Therefore, we investigated the effect of a short-term acidic (pH 6.8) pre-treatment on the stemness of bone marrow-derived MSCs (BMSCs). Interestingly, the results showed that pre-treatment of BMSCs with acidic pH enhances the expression of stem cell markers (OCT-4, NANOG, SSEA-4), as well as cell viability and proliferation. On the other hand, acidic pH decreased BMSC migration ability. These results indicate that acidic pH during the initial stages of bone healing is important to enhance the stem cell properties of BMSCs. These findings may enable the development of novel methods for optimization of stem cell function towards tissue engineering or regenerative medicine.

DOI： 10.3390/ijms20051097

Bone Marrow Cells Inhibit BMP-2-Induced Osteoblast Activity in the Marrow Environment. 査読国際誌

Ha Thi Nguyen, Mitsuaki Ono, Yasutaka Oida, Emilio Satoshi Hara, Taishi Komori, Kentaro Akiyama, Ha Thi Thu Nguyen, Kyaw Thu Aung, Hai Thanh Pham, Ikue Tosa, Takeshi Takarada, Koichi Matsuo, Toshihide Mizoguchi, Toshitaka Oohashi, Takuo Kuboki

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 34 ( 2 ) 327 - 332 2019年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.3598

Under the ECM Dome: The Physiological Role of the Perinodal Extracellular Matrix as an Ion Diffusion Barrier. 査読国際誌

Yoko Bekku, Toshitaka Oohashi

Advances in experimental medicine and biology 1190 107 - 122 2019年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Enriched Na+ channel clustering allows for rapid saltatory conduction at a specialized structure in myelinated axons, the node of Ranvier, where cations are exchanged across the axon membrane. In the extracellular matrix (ECM), highly negatively charged molecules accumulate and wrap around the nodal gaps creating an ECM dome, called the perinodal ECM. The perinodal ECM has different molecular compositions in the central nervous system (CNS) and peripheral nervous system (PNS). Chondroitin sulfate proteoglycans are abundant in the ECM at the CNS nodes, whereas heparan sulfate proteoglycans are abundant at the PNS nodes. The proteoglycans have glycosaminoglycan chains on their core proteins, which makes them electrostatically negative. They associate with other ECM molecules and form a huge stable ECM complex at the nodal gaps. The polyanionic molecular complexes have high affinity to cations and potentially contribute to preventing cation diffusion at the nodes.In this chapter, we describe the molecular composition of the perinodal ECM in the CNS and PNS, and discuss their physiological role at the node of Ranvier.

DOI： 10.1007/978-981-32-9636-7_8

Inhibition of the glutamine transporter SNAT1 confers neuroprotection in mice by modulating the mTOR-autophagy system. 査読国際誌

Daisuke Yamada, Kenji Kawabe, Ikue Tosa, Shunpei Tsukamoto, Ryota Nakazato, Miki Kou, Koichi Fujikawa, Saki Nakamura, Mitsuaki Ono, Toshitaka Oohashi, Mari Kaneko, Shioi Go, Eiichi Hinoi, Yukio Yoneda, Takeshi Takarada

Communications biology 2 346 - 346 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1
flox/flox
and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.

DOI： 10.1038/s42003-019-0582-4

DNA Methylation-Based Regulation of Human Bone Marrow-Derived Mesenchymal Stem/Progenitor Cell Chondrogenic Differentiation. 査読国際誌

Yu Nomura, Emilio Satoshi Hara, Yuya Yoshioka, Há Thi Nguyen, Shuji Nosho, Taishi Komori, Kei Ishibashi, Toshitaka Oohashi, Mitsuaki Ono, Takuo Kuboki

Cells, tissues, organs 207 ( 3-4 ) 115 - 126 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）

Stem cells have essential applications in in vitro tissue engineering or regenerative medicine. However, there is still a need to understand more deeply the mechanisms of stem cell differentiation and to optimize the methods to control stem cell function. In this study, we first investigated the activity of DNA methyltransferases (DNMTs) during chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) and found that DNMT3A and DNMT3B were markedly upregulated during hBMSC chondrogenic differentiation. In an attempt to understand the effect of DNMT3A and DNMT3B on the chondrogenic differentiation of hBMSCs, we transiently transfected the cells with expression vectors for the two enzymes. Interestingly, DNMT3A overexpression strongly enhanced the chondrogenesis of hBMSCs, by increasing the gene expression of the mature chondrocyte marker, collagen type II, more than 200-fold. Analysis of the methylation condition in the cells revealed that DNMT3A and DNMT3B methylated the promoter sequence of early stem cell markers, NANOG and POU5F1(OCT-4). Conversely, the suppression of chondrogenic differentiation and the increase in stem cell markers of hBMSCs were obtained by chemical stimulation with the demethylating agent, 5-azacitidine. Loss-of-function assays with siRNAs targeting DNMT3A also significantly suppressed the chondrogenic differentiation of hBMSCs. Together, these results not only show the critical roles of DNMTs in regulating the chondrogenic differentiation of hBMSCs, but also suggest that manipulation of DNMT activity can be important tools to enhance the differentiation of hBMSCs towards chondrogenesis for potential application in cartilage tissue engineering or cartilage regeneration.

DOI： 10.1159/000502885

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model. 査読国際誌

Tomoko Yonezawa, Ryusuke Momota, Hideki Iwano, Steven Zhao, Tomohiro Hakozaki, Chieko Soh, Shigetoyo Sawaki, Kazumi Toyama, Toshitaka Oohashi

Journal of cosmetic dermatology 2018年12月

担当区分：最終著者記述言語：英語掲載種別：研究論文（学術雑誌）

INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

DOI： 10.1111/jocd.12841

High molecular weight hyaluronan protects cartilage from degradation by inhibiting aggrecanase expression. 査読国際誌

Takashi Ohtsuki, Keiichi Asano, Junko Inagaki, Akira Shinaoka, Kanae Kumagishi-Shinaoka, Mehmet Z Cilek, Omer F Hatipoglu, Toshitaka Oohashi, Keiichiro Nishida, Issei Komatsubara, Satoshi Hirohata

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 36 ( 12 ) 3247 - 3255 2018年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine-inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine-stimulated chondrocytes and chondrocytic cells. Pre-incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine-stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra-articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size-dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247-3255, 2018.

DOI： 10.1002/jor.24126

Hapln4/Bral2 is a selective regulator for formation and transmission of GABAergic synapses between Purkinje and deep cerebellar nuclei neurons. 査読国際誌

Midori Edamatsu, Rinako Miyano, Atsushi Fujikawa, Fuminari Fujii, Tetsuya Hori, Takeshi Sakaba, Toshitaka Oohashi

Journal of neurochemistry 147 ( 6 ) 748 - 763 2018年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post-synaptic neurons of large axo-somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild-type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC-DCN synapses in the cerebellum. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

DOI： 10.1111/jnc.14571

Host-produced ADAMTS4 Inhibits Early-Stage Tumor Growth. 査読

Keiichi Asano, Midori Edamatsu, Omer F Hatipoglu, Junko Inagaki, Mitsuaki Ono, Takashi Ohtsuki, Toshitaka Oohashi, Satoshi Hirohata

Acta medica Okayama 72 ( 3 ) 257 - 266 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Several research groups demonstrated that 'a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS)'-family proteases play roles in cancer progression. However, the origins and contributions of these proteases are not known. Here, we demonstrate an association between host-produced ADAMTS4 and early-stage tumor growth. Murine Lewis lung carcinoma (LLC) tumors showed marked expressions of Adamts4 and Adamts5. We examined the contributions and distributions of host-derived Adamts4 and Adamts5 on tumor growth, using Adamts4LacZ/LacZ and Adamts5LacZ/LacZ knockout mice. Interestingly, the Adamts4LacZ/LacZ mice showed enhanced tumor growth compared to wild-type mice at 5-, 10- and 12-days post-inoculation, whereas the Adamts5LacZ/LacZ mice did not show significant differences in tumor growth. We next examined LacZ distribution in LLC tumor-bearing Adamts4LacZ/LacZ mice by β-galactosidase (β-gal) staining. We found that the β-gal-positive signals were strictly localized at the interior areas of the tumor at 10 days post-inoculation. Multiple staining demonstrated that most of the β-gal-positive cells were localized at the tumor vasculature in Adamts4LacZ/LacZ mice. Interestingly, β-gal-positive signals were not co-localized with biglycan after 10 days post-inoculation, excluding the biglycan cleavage by host-derived ADAMTS4. Taken together, these findings illustrate that host-derived ADAMTS4 was expressed at the tumor vessels and was associated with early-stage tumor growth.

DOI： 10.18926/AMO/56071

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation. 査読国際誌

Takanori Ishikawa, Takashi Nishida, Mitsuaki Ono, Takeshi Takarada, Ha Thi Nguyen, Shinnosuke Kurihara, Takayuki Furumatsu, Yurika Murase, Masaharu Takigawa, Toshitaka Oohashi, Hiroshi Kamioka, Satoshi Kubota

Journal of cellular physiology 233 ( 6 ) 4825 - 4840 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.

DOI： 10.1002/jcp.26285

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium. 査読国際誌

Komori T, Ono M, Hara ES, Ueda J, Nguyen HTT, Nguyen HT, Yonezawa T, Maeba T, Kimura-Ono A, Takarada T, Momota R, Maekawa K, Kuboki T, Oohashi T

Scientific reports 8 ( 1 ) 2612 - 2612 2018年2月

A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice. 査読国際誌

Michal Cicanic, Midori Edamatsu, Yoko Bekku, Ivan Vorisek, Toshitaka Oohashi, Lydia Vargova

Journal of neuroscience research 96 ( 2 ) 313 - 327 2018年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Bral2 is a link protein stabilizing the binding between lecticans and hyaluronan in perineuronal nets and axonal coats (ACs) in specific brain regions. Using the real-time iontophoretic method and diffusion-weighted magnetic resonance, we determined the extracellular space (ECS) volume fraction (α), tortuosity (λ), and apparent diffusion coefficient of water (ADCW ) in the thalamic ventral posteromedial nucleus (VPM) and sensorimotor cortex of young adult (3-6 months) and aged (14-20 months) Bral2-deficient (Bral2-/- ) mice and age-matched wild-type (wt) controls. The results were correlated with an analysis of extracellular matrix composition. In the cortex, no changes between wt and Bral2-/- were detected, either in the young or aged mice. In the VPM of aged but not in young Bral2-/- mice, we observed a significant decrease in α and ADCW in comparison with age-matched controls. Bral2 deficiency led to a reduction of both aggrecan- and brevican-associated perineuronal nets and a complete disruption of brevican-based ACs in young as well as aged VPM. Our data suggest that aging is a critical point that reveals the effect of Bral2 deficiency on VPM diffusion. This effect is probably mediated through the enhanced age-related damage of neurons lacking protective ACs, or the exhausting of compensatory mechanisms maintaining unchanged diffusion parameters in young Bral2-/- animals. A decreased ECS volume in aged Bral2-/- mice may influence the diffusion of neuroactive substances, and thus extrasynaptic and also indirectly synaptic transmission in this important nucleus of the somatosensory pathway.

DOI： 10.1002/jnr.24136

Stromal Versican Regulates Tumor Growth by Promoting Angiogenesis 査読

Keiichi Asano, Courtney M. Nelson, Sumeda Nandadasa, Noriko Aramaki-Hattori, Daniel J. Lindner, Tyler Alban, Junko Inagaki, Takashi Ohtsuki, Toshitaka Oohashi, Suneel S. Apte, Satoshi Hirohata

SCIENTIFIC REPORTS 7 2017年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

The proteoglycan versican is implicated in growth and metastases of several cancers. Here we investigated a potential contribution of stromal versican to tumor growth and angiogenesis. We initially determined versican expression by several cancer cell lines. Among these, MDA-MB231 and B16F10 had none to minimal expression in contrast to Lewis lung carcinoma (LLC). Notably, tumors arising from these cell lines had higher versican levels than the cell lines themselves suggesting a contribution from the host-derived tumor stroma. In LLC-derived tumors, both the tumor and stroma expressed versican at high levels. Thus, tumor stroma can make a significant contribution to tumor versican content. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, an ADAMTS protease-generated versican fragment uniquely localized to vascular endothelium. To specifically determine the impact of host/stroma-derived versican we therefore compared growth of tumors from B16F10 cells, which produced littleversican, in Vcan(hdf/+) mice and wild-type littermates. Tumors in Vcan(hdf/+) mice had reduced growth with a lower capillary density and accumulation of capillaries at the tumor periphery. These findings illustrate the variability of tumor cell line expression of versican, and demonstrate that versican is consistently contributed by the stromal tissue, where it contributes to tumor angiogenesis.

DOI： 10.1038/s41598-017-17613-6

Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas. 査読

Yoshikazu Sado, Satoko Inoue, Yasuko Tomono, Makoto Matsuyama, Masaki Fukushima, Toshitaka Oohashi, Takamichi Jogahara, Sen-Ichi Oda

Acta histochemica et cytochemica 50 ( 2 ) 71 - 84 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

DOI： 10.1267/ahc.17007

Modifications of perineuronal nets and remodelling of excitatory and inhibitory afferents during vestibular compensation in the adult mouse. 査読国際誌

Alessio Faralli, Federico Dagna, Andrea Albera, Yoko Bekku, Toshitaka Oohashi, Roberto Albera, Ferdinando Rossi, Daniela Carulli

Brain structure & function 221 ( 6 ) 3193 - 209 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Perineuronal nets (PNNs) are aggregates of extracellular matrix molecules surrounding several types of neurons in the adult CNS, which contribute to stabilising neuronal connections. Interestingly, a reduction of PNN number and staining intensity has been observed in conditions associated with plasticity in the adult brain. However, it is not known whether spontaneous PNN changes are functional to plasticity and repair after injury. To address this issue, we investigated PNN expression in the vestibular nuclei of the adult mouse during vestibular compensation, namely the resolution of motor deficits resulting from a unilateral peripheral vestibular lesion. After unilateral labyrinthectomy, we found that PNN number and staining intensity were strongly attenuated in the lateral vestibular nucleus on both sides, in parallel with remodelling of excitatory and inhibitory afferents. Moreover, PNNs were completely restored when vestibular deficits of the mice were abated. Interestingly, in mice with genetically reduced PNNs, vestibular compensation was accelerated. Overall, these results strongly suggest that temporal tuning of PNN expression may be crucial for vestibular compensation.

DOI： 10.1007/s00429-015-1095-7

COL4A6 is dispensable for autosomal recessive Alport syndrome 査読

Tomohiro Murata, Kan Katayama, Toshitaka Oohashi, Timo Jahnukainen, Tomoko Yonezawa, Yoshikazu Sado, Eiji Ishikawa, Shinsuke Nomura, Karl Tryggvason, Masaaki Ito

SCIENTIFIC REPORTS 6 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Alport syndrome is caused by mutations in the genes encoding alpha 3, alpha 4, or alpha 5 (IV) chains. Unlike X-linked Alport mice, alpha 5 and alpha 6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both alpha 3 and alpha 6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 x 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 x 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The alpha 5 and alpha 6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although a5 and a6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role.

DOI： 10.1038/srep29450

Human collagen XV is a prominent histopathological component of sinusoidal capillarization in hepatocellular carcinogenesis. 査読

Kouji Kimura, Masaru Nakayama, Ichiro Naito, Takaaki Komiyama, Kouichi Ichimura, Hiroaki Asano, Kazunori Tsukuda, Aiji Ohtsuka, Toshitaka Oohashi, Shinichiro Miyoshi, Yoshifumi Ninomiya

International journal of clinical oncology 21 ( 2 ) 302 - 309 2016年4月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Increased expression of collagen XV has been reported in hepatocellular carcinogenesis in mice. The aim of this study was to confirm the previous murine findings in human hepatocellular carcinoma (HCC) specimens, along with the histopathological distribution of collagen XV in tumoral tissues. METHODS: Sixty-three primary HCC specimens were examined. Immunostaining of collagen XV and quantitative reverse transcriptional PCR of COL15A1, which encodes collagen XV, were performed. RESULTS: Positive staining of collagen XV was observed in all tumoral regions, regardless of differentiation level or pathological type of HCC, along the sinusoid-like endothelium, whereas collagen XV was not expressed in any non-tumoral region. The intensity score of collagen XV immunostaining and the mRNA value of COL15A1 were significantly correlated. COL15A1 expression in tumors was 3.24-fold higher than in non-tumoral regions. Multivariate analysis showed that COL15A1 expression was significantly higher in the absence of hepatitis virus and moderately differentiated HCC. CONCLUSIONS: COL15A1 mRNA was up-regulated in HCC and collagen XV was expressed along the sinusoid-like endothelium of HCC but not in non-tumoral regions, which implies that collagen XV contributes to the capillarization of HCC.

DOI： 10.1007/s10147-015-0888-2

CCN4/WISP-1 positively regulates chondrogenesis by controlling TGF-β3 function. 査読国際誌

Yuya Yoshioka, Mitsuaki Ono, Azusa Maeda, Tina M Kilts, Emilio Satoshi Hara, Hany Khattab, Junji Ueda, Eriko Aoyama, Toshitaka Oohashi, Masaharu Takigawa, Marian F Young, Takuo Kuboki

Bone 83 162 - 170 2016年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

The CCN family of proteins plays important roles in development and homeostasis of bone and cartilage. To understand the role of CCN4 in chondrogenesis, human bone marrow stromal cells (hBMSCs) were transduced with CCN4 adenovirus (adCCN4) or siRNA to CCN4 (siCCN4) in the presence or absence of transforming growth factor-β3 (TGF-β3). Overexpression of CCN4 enhanced TGF-β3-induced SMAD2/3 phosphorylation and chondrogenesis of hBMSCs in an in vitro assay using a micromass culture model. On the other hand, knockdown of CCN4 inhibited the TGF-β3-induced SMAD2/3 phosphorylation and synthesis of cartilage matrix in micromass cultures of hBMSCs. Immunoprecipitation-western blot analysis revealed that CCN4 bound to TGF-β3 and regulated the ability of TGF-β3 to bind to hBMSCs. In vivo analysis confirmed there was a significant decrease in the gene expression levels of chondrocyte markers in cartilage samples from Ccn4-knock out (KO) mice, compared to those from wild type (WT) control. In order to investigate the regenerative properties of the articular cartilage in Ccn4-KO mice, articular cartilage defects were surgically performed in the knee joints of young mice, and the results showed that the cartilage was partially repaired in WT mice, but not in Ccn4-KO mice. In conclusion, these results show, for the first time, that CCN4 has a positive influence on chondrogenic differentiation by modulating the effects of TGF-β3.

DOI： 10.1016/j.bone.2015.11.007

The hyaluronan and proteoglycan link proteins: Organizers of the brain extracellular matrix and key molecules for neuronal function and plasticity. 査読国際誌

Toshitaka Oohashi, Midori Edamatsu, Yoko Bekku, Daniela Carulli

Experimental neurology 274 ( Pt B ) 134 - 44 2015年12月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The hyaluronan and proteoglycanbinding link protein (Hapln) is a key molecule in the formation and control of hyaluronan-based condensed perineuronal matrix in the adult brain. This review summarizes the recent advances in understanding the role of Haplns in the formation and control of two distinct types of perineuronal matrices, one for "classical" PNN and the other for the specialized extracellular matrix (ECM) at the node of Ranvier in the central nervous system (CNS). We introduce the structural components of each ECM organization including the basic concept of supramolecular structure named "HLT model". We furthermore summarize the developmental and physiological role of perineuronal ECMs from the studies of Haplns and related molecules. Finally, we also discuss the potential mechanism modulating PNNs in the adult CNS. This layer of organized matrices may exert a direct effect via core protein or sugar moiety from the structure or by acting as a binding site for biologically active molecules, which are important for neuronal plasticity and saltatory conduction.

DOI： 10.1016/j.expneurol.2015.09.010

[Formation and roles of perineuronal extracellular matrices in the adult brain]. 査読

Toshitaka Oohashi, Midori Edamatsu, Yoko Bekku

Seikagaku. The Journal of Japanese Biochemical Society 87 ( 3 ) 393 - 6 2015年6月

RXR Partial Agonist Produced by Side Chain Repositioning of Alkoxy RXR Full Agonist Retains Antitype 2 Diabetes Activity without the Adverse Effects 査読

Kohei Kawata, Ken-ichi Morishita, Mariko Nakayama, Shoya Yamada, Toshiki Kobayashi, Yuki Furusawa, Sakae Arimoto-Kobayashi, Toshitaka Oohashi, Makoto Makishima, Hirotaka Naitou, Erika Ishitsubo, Hiroaki Tokiwa, Akihiro Tai, Hiroki Kakuta

JOURNAL OF MEDICINAL CHEMISTRY 58 ( 2 ) 912 - 926 2015年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid: <bold>5</bold>, EC50 = 143 nM, E-max = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: <bold>8b</bold>, EC50 = 169 nM, E-max = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: <bold>7b</bold>, EC50 = 19 nM). NEt-4IB (<bold>8b</bold>) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to <bold>5</bold>.

DOI： 10.1021/jm501863r

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3β under Oxidative Stress. 査読

Hiroko Ishii, Shigeshi Kamikawa, Satoshi Hirohata, Akifumi Mizutani, Koji Abe, Masaharu Seno, Toshitaka Oohashi, Yoshifumi Ninomiya

Acta medica Okayama 69 ( 3 ) 145 - 53 2015年

記述言語：英語掲載種別：研究論文（学術雑誌）

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3β signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3β pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.

肝細胞癌組織におけるXV型コラーゲン発現とその意義

木村紘爾, 中山勝, 小宮山高明, 市村浩一, 内藤一郎, 浅野博昭, 佃和憲, 大塚愛二, 大橋俊孝, 二宮善文

日本結合組織学会学術大会・マトリックス研究会大会合同学術集会プログラム・抄録集 46回・61回 84 - 84 2014年6月

Light and electron microscopic detection of inflammation-targeting liposomes encapsulating high-density colloidal gold in arthritic mice. 査読国際誌

Ami Maehara, Keiichiro Nishida, Masumi Furutani, Emi Matsumoto, Aiji Ohtsuka, Yoshifumi Ninomiya, Toshitaka Oohashi

Inflammation research : official journal of the European Histamine Research Society ... [et al.] 63 ( 2 ) 139 - 47 2014年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVE: We have previously demonstrated the efficient and time-dependent transvascular localization of Sialyl Lewis X (SLX)-liposomes to inflammatory sites, but the final target of the SLX-liposomes remained uncertain. The aim of this study was to identify the target cells of the liposomes within the inflamed joints of collagen antibody-induced arthritis (CAIA) model mice. METHODS: SLX-liposomes and unlabeled liposomes encapsulating high-density colloidal gold were administered intravenously into the caudal vein of CAIA mice on day 5 after induction of arthritis when the inflammatory score was maximal (n = 6 per group). Six hours or 24 h after liposome administration, animals were euthanized and hind limbs and ankles were excised without perfusion. After fixation, synovial tissues were examined by light microscopy after silver enhancement of colloidal gold or by transmission electron microscopy. RESULTS: Silver-enhanced signals were detected within the cells around E-selectin-positive blood vessels in the synovium of the SLX-liposome group. These cells were positive for the macrophage/monocyte marker F4/80 or neutrophil marker Ly-6G. Transmission electron microscopy detected the colloidal gold signals together with liposome-like structures within the phagosomes of synovial macrophages. Transmission electron microscopy and energy dispersive X-ray spectrometry could determine gold elements in the lysosomes of synovial macrophages. CONCLUSIONS: The results of the current study demonstrate that SLX-liposomes primarily targeting E-selectin in activated endothelial cells could potentially deliver their contents into inflammatory cells around synovial blood vessels in arthritic joints.

DOI： 10.1007/s00011-013-0682-4

Design, synthesis, and preliminary ex vivo and in vivo evaluation of cationic magnetic resonance contrast agent for rabbit articular cartilage imaging 査読

Takayoshi Irie, Kazuhiro Oda, Akihiko Shiino, Mitsuhiko Kubo, Shigehiro Morikawa, Noboru Urushiyama, Shuji Aonuma, Takahide Kimura, Toshiro Inubushi, Toshitaka Oohashi, Naoki Komatsu

MedChemComm 4 ( 11 ) 1508 - 1512 2013年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Since early-stage diagnosis of osteoarthritis (OA) is necessary to retard the progress of the disease, magnetic resonance (MR) and computed tomography (CT) imaging agents have been applied to evaluate the integrity of the extracellular matrix (ECM) in the articular cartilage (AC). Although several negatively charged contrast agents were employed, they provided indirect images of the AC based on the coulombic repulsion with anionic glycosaminoglycans (GAGs) in the ECM. To achieve direct imaging of the ECM, positively charged contrast agents have quite recently been designed for optical and CT imaging. In this paper, we report on a positively charged MR contrast agent, DOTA-Gd-G 2R8, which was applied preliminarily to ex vivo and in vivo imaging of rabbit AC. In the ex vivo imaging, the contrast agent was accumulated in the AC in high concentration due to the strong coulombic attraction between the negative charge of the GAGs and the positive charge of the octaarginine (R8). The thickness of the AC measured in the MR image was found to be comparable to that determined by the histological staining of the slice. The AC was also observed in vivo by MR imaging with the use of DOTA-Gd-G2R8. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3md00229b

Ten-m2 is required for the generation of binocular visual circuits. 査読国際誌

Timothy R Young, Michael Bourke, Xiaohong Zhou, Toshitaka Oohashi, Atomu Sawatari, Reinhard Fässler, Catherine A Leamey

The Journal of neuroscience : the official journal of the Society for Neuroscience 33 ( 30 ) 12490 - 509 2013年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Functional binocular vision requires that inputs arising from the two retinae are integrated and precisely organized within central visual areas. Previous studies have demonstrated an important role for one member of the Ten-m/Odz/teneurin family, Ten-m3, in the mapping of ipsilateral retinal projections. Here, we have identified a distinct role for another closely related family member, Ten-m2, in the formation of the ipsilateral projection in the mouse visual system. Ten-m2 expression was observed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) of the developing mouse. Anterograde and retrograde tracing experiments in Ten-m2 knock-out (KO) mice revealed a specific decrease in ipsilateral retinal ganglion cells projecting to dLGN and SC. This reduction was most prominent in regions corresponding to ventral retina. No change in the topography of ipsilateral or contralateral projections was observed. While expression of a critical ipsilateral fate determinant, Zic2, appeared unaltered, a notable reduction in one of its downstream targets, EphB1, was observed in ventral retina, suggesting that Ten-m2 may interact with this molecular pathway. Immunohistochemistry for c-fos, a neural activity marker, revealed that the area of V1 driven by ipsilateral inputs was reduced in KOs, while the ratio of ipsilateral-to-contralateral responses contributing to binocular activation during visually evoked potential recordings was also diminished. Finally, a novel two-alternative swim task revealed specific deficits associated with dorsal visual field. These data demonstrate a requirement for Ten-m2 in the establishment of ipsilateral projections, and thus the generation of binocular circuits, critical for mammalian visual function.

DOI： 10.1523/JNEUROSCI.4708-12.2013

Three mechanisms assemble central nervous system nodes of Ranvier. 査読国際誌

Keiichiro Susuki, Kae-Jiun Chang, Daniel R Zollinger, Yanhong Liu, Yasuhiro Ogawa, Yael Eshed-Eisenbach, María T Dours-Zimmermann, Juan A Oses-Prieto, Alma L Burlingame, Constanze I Seidenbecher, Dieter R Zimmermann, Toshitaka Oohashi, Elior Peles, Matthew N Rasband

Neuron 78 ( 3 ) 469 - 82 2013年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Rapid action potential propagation in myelinated axons requires Na⁺ channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na⁺ channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na⁺ channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na⁺ channel clustering.

DOI： 10.1016/j.neuron.2013.03.005

Bral2 is indispensable for the proper localization of brevican and the structural integrity of the perineuronal net in the brainstem and cerebellum. 査読国際誌

Yoko Bekku, Mai Saito, Markus Moser, Maki Fuchigami, Ami Maehara, Masaru Nakayama, Shozo Kusachi, Yoshifumi Ninomiya, Toshitaka Oohashi

The Journal of comparative neurology 520 ( 8 ) 1721 - 36 2012年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Perineuronal nets (PNNs) are pericellular coats of condensed matrix that enwrap the cell bodies and dendrites of many adult central nervous system (CNS) neurons. These extracellular matrices (ECMs) play a structural role as well as instructive roles in the control of CNS plasticity and the termination of critical periods. The cartilage link protein Crtl1/Hapln1 was reported to be a trigger for the formation of PNNs in the visual cortex. Bral2/Hapln4 is another link protein that is expressed in PNNs, mainly in the brainstem and cerebellum. To assess the role of Bral2 in PNN formation, we examined the expression of PNN components in targeted mouse mutants lacking Bral2. We show here that Bral2-deficient mice have attenuated PNNs, but the overall levels of chondroitin sulfate proteoglycans, lecticans, are unchanged with the exception of neurocan. Bral2 deficiency markedly affected the localization of brevican in all of the nuclei tested, and neurocan concomitant with Crtl1 in some of the nuclei, whereas no effect was seen on aggrecan even with the attenuation of Crtl1. Bral2 may have a role in the organization of the PNN, in association with brevican, that is independent of aggrecan binding. There was a heterogenous attenuation of PNN components, including glycosaminoglycans, indicating the elaborate molecular organization of the PNN components. Strikingly, a slight decrease in the number of synapses in deep cerebellar nuclei neurons was found. Taken together, these results imply that Bral2-brevican interaction may play a key role in synaptic stabilization and the structural integrity of the PNN.

DOI： 10.1002/cne.23009

RXR Partial Agonist CBt-PMN Exerts Therapeutic Effects on Type 2 Diabetes without the Side Effects of RXR Full Agonists. 査読国際誌

Hiroki Kakuta, Nobumasa Yakushiji, Ryosuke Shinozaki, Fuminori Ohsawa, Shoya Yamada, Yui Ohta, Kohei Kawata, Mariko Nakayama, Manabu Hagaya, Chisa Fujiwara, Makoto Makishima, Shigeyuki Uno, Akihiro Tai, Ami Maehara, Masaru Nakayama, Toshitaka Oohashi, Hiroyuki Yasui, Yutaka Yoshikawa

ACS medicinal chemistry letters 3 ( 5 ) 427 - 32 2012年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

Treating insulin resistance and type 2 diabetes in rodents, currently known retinoid X receptor (RXR) agonists induce significant adverse effects. Here we introduce a novel RXR partial agonist CBt-PMN (11b), which shows a potent glucose-lowering effect and improvements of insulin secretion and glucose tolerance without the serious adverse effects caused by RXR full agonists. We suggest that RXR partial agonists may be a new class of antitype 2 diabetes drug candidates.

DOI： 10.1021/ml300055n

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells. 査読国際誌

Tomoko Kanazawa, Takayuki Furumatsu, Motomi Hachioji, Toshitaka Oohashi, Yoshifumi Ninomiya, Toshifumi Ozaki

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 30 ( 3 ) 468 - 74 2012年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress-related response in meniscus cells remains unclear. We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch-induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression.

DOI： 10.1002/jor.21528

[Cutting edge on research of cartilage metabolism. Recent progress in bio-molecular imaging of articular cartilage]. 査読

Toshitaka Oohashi, Keiichiro Nishida

Clinical calcium 21 ( 6 ) 896 - 902 2011年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）

In the process of cartilage degeneration seen in osteoarthritis, loss of proteoglycan from articular cartilage has been widely accepted as a critical early event, followed by collagen degradation designated as a point of no return. Recent advance in the development of targeted molecular probes and new imaging modalities enabled the detection of qualitative and functional change of articular cartilage. In this paper, we describe the recent progress of bio-molecular imaging of articular cartilage including our fluorescent-labeled glycosaminoglycan-binding octaarginine.

Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes. 査読国際誌

Toshitaka Oohashi, Ichiro Naito, Yasuyoshi Ueki, Tomoki Yamatsuji, Rattiya Permpoon, Noriaki Tanaka, Yoshio Naomoto, Yoshifumi Ninomiya

Matrix biology : journal of the International Society for Matrix Biology 30 ( 1 ) 3 - 8 2011年1月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5-COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.

DOI： 10.1016/j.matbio.2010.09.003

Development of an active targeting liposome encapsulated with high-density colloidal gold for transmission electron microscopy. 査読

Hideki Minematsu, Takayuki Otani, Toshitaka Oohashi, Masahiko Hirai, Kazunori Oie, Koichi Igarashi, Aiji Ohtsuka

Journal of electron microscopy 60 ( 1 ) 95 - 9 2011年

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

Active targeting of the liposome is an attractive strategy for drug delivery and in vivo bio-imaging. We previously reported the specific accumulation of Sialyl Lewis X (SLX) liposome to inflamed tissue in arthritic model mice or tumor-bearing mice. SLX-liposome encapsulation with fluorescent substances allows for the visualization of these liposomes by the time-dependent transvascular accumulation of fluorescent signals in the histological sections. In the present study, we developed a new SLX-liposome encapsulated with colloidal gold for transmission electron microscopic observation. We herein describe the characterization of the colloidal gold-loaded SLX-liposomes and demonstrate its specific targeting to the endothelial cells of tumor blood vessels in tumor-bearing mice.

DOI： 10.1093/jmicro/dfq071

Bral1: its role in diffusion barrier formation and conduction velocity in the CNS. 査読国際誌

Yoko Bekku, Lýdia Vargová, Yoshinobu Goto, Ivan Vorísek, Lesia Dmytrenko, Masahiro Narasaki, Aiji Ohtsuka, Reinhard Fässler, Yoshifumi Ninomiya, Eva Syková, Toshitaka Oohashi

The Journal of neuroscience : the official journal of the Society for Neuroscience 30 ( 8 ) 3113 - 23 2010年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC NEUROSCIENCE

At the nodes of Ranvier, excitable axon membranes are exposed directly to the extracellular fluid. Cations are accumulated and depleted in the local extracellular nodal region during action potential propagation, but the impact of the extranodal micromilieu on signal propagation still remains unclear. Brain-specific hyaluronan-binding link protein, Bral1, colocalizes and forms complexes with negatively charged extracellular matrix (ECM) proteins, such as versican V2 and brevican, at the nodes of Ranvier in the myelinated white matter. The link protein family, including Bral1, appears to be the linchpin of these hyaluronan-bound ECM complexes. Here we report that the hyaluronan-associated ECM no longer shows a nodal pattern and that CNS nerve conduction is markedly decreased in Bral1-deficient mice even though there were no differences between wild-type and mutant mice in the clustering or transition of ion channels at the nodes or in the tissue morphology around the nodes of Ranvier. However, changes in the extracellular space diffusion parameters, measured by the real-time iontophoretic method and diffusion-weighted magnetic resonance imaging (MRI), suggest a reduction in the diffusion hindrances in the white matter of mutant mice. These findings provide a better understanding of the mechanisms underlying the accumulation of cations due to diffusion barriers around the nodes during saltatory conduction, which further implies the importance of the Bral1-based extramilieu for neuronal conductivity.

DOI： 10.1523/JNEUROSCI.5598-09.2010

Neurocan contributes to the molecular heterogeneity of the perinodal ECM. 査読

Yoko Bekku, Toshitaka Oohashi

Archives of histology and cytology 73 ( 2 ) 95 - 102 2010年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT SOC HISTOLOGY & CYTOLOGY

Neurocan is a central nervous tissue-specific chondroitin sulfate proteoglycan of the lectican family. Mainly expressed during modeling and remodeling stages of this tissue, it is thought to play an important role via binding to various extracellular matrix and cellular components. In adults, neurocan expression is associated with the perineuronal net structures. This study shows the neurocan immunolocalization at the node of Ranvier in mouse central nervous tissues. The N-terminal fragment of neurocan (Ncan130) was the predominant form detected in the optic nerve. The expression of neurocan in the white matter of brain tissue and nerve tracts revealed differential expression profiles compared with those of versican V2 and brevican, other perinodal extracellular matrix molecules. Double immunolabeling for neurocan and a nodal marker, Bral1, or a paranodal marker, caspr, demonstrated that neurocan was localized at the node of Ranvier. Neurocan expression was found at many--not all--nodal regions, and neurocan-positive nodes outnumbered brevican-positive nodes. The nodal localization of neurocan was diminished in Bral1-deficient mice. Taken together, these findings indicate that neurocan contributes to the molecular heterogeneity of the perinodal matrix, and its nodal expression is dependent on Bral1.

DOI： 10.1679/aohc.73.95

Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine. 査読国際誌

K Inagawa, T Oohashi, K Nishida, J Minaguchi, T Tsubakishita, K O Yaykasli, A Ohtsuka, T Ozaki, T Moriguchi, Y Ninomiya

Osteoarthritis and cartilage 17 ( 9 ) 1209 - 18 2009年9月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVE: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice. METHODS: Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT). RESULTS: Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT. CONCLUSION: R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.

DOI： 10.1016/j.joca.2009.03.010

The 3’-untranslated region of ADAMTS1 regulates its mRNA stability. 査読

Hatipoglu OF, Hirohata S, Yaykasli KO, Cilek MZ, Demircan K, Shinohata R, Yonezawa T, Oohashi T, Kusachi S, Ninomiya Y

Acta Medica Okayama 63 ( 2 ) 79 - 85 2009年4月

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS. 査読国際誌

Yoko Bekku, Uwe Rauch, Yoshifumi Ninomiya, Toshitaka Oohashi

Journal of neurochemistry 108 ( 5 ) 1266 - 76 2009年3月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.

DOI： 10.1111/j.1471-4159.2009.05873.x

ADAMTS9 activation by interleukin 1 beta via NFATc1 in OUMS-27 chondrosarcoma cells and in human chondrocytes. 査読国際誌

Kursat Oguz Yaykasli, Toshitaka Oohashi, Satoshi Hirohata, Omer Faruk Hatipoglu, Kiichi Inagawa, Kadir Demircan, Yoshifumi Ninomiya

Molecular and cellular biochemistry 323 ( 1-2 ) 69 - 79 2009年3月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

ADAMTS9 is a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes, with aggrecan-degrading activity. It has also been characterized to be reactive and highly activated ADAMTS by IL-1 beta in both chondrosarcoma cells and human chondrocytes (Demircan et al. Arthritis Rheum 52:1451-1460, 2005). In order to understand the regulation of ADAMTS9 gene expression a functional 3.0 kb human ADAMTS9 promoter has been cloned and characterized. A sequence analysis of the promoter revealed the presence of putative binding sites for Nuclear Factor of Activated T cells (NFAT), which is commonly found in the ADAMTS4 and ADAMTS5 promoters. NFATc1 was up-regulated in an activated form by IL-1 beta in human chondrocytes. The IL-1 beta inducible ADAMTS9 expression was inhibited by NFAT inhibitors, FK506 and 11Arg (11R)-VIVIT. Furthermore, direct binding of NFATc1 on distal and proximal promoters of ADAMTS9 was demonstrated by a chromatin immunoprecipitation assay. Promoter-reporter assays supported those results. These findings may provide a better understanding of the regulation of ADAMTS9 expression induced by inflammatory cytokines.

DOI： 10.1007/s11010-008-9965-4

THE ION-DIFFUSION BARRIER MODEL AT THE NODE OF RANVIER: FROM THE EXTRACELLULAR ION DIFFUSION ANALYSIS IN BRAL1-DEFICIENT MICE 査読

Yoko Bekku, Lydia Vargova, Yoshinobu Goto, Yoshifumi Ninomiya, Eva Sykova, Toshitaka Oohashi

JOURNAL OF PHYSIOLOGICAL SCIENCES 59 292 - 292 2009年

Target-tropic accumulation of SLX-liposome in arthritis mouse 査読

Minaguchi JA, Minematsu H, Oohashi T, Otani T, Inagawa K, Oie K, Nishida K, Ohtsuka A, Ninomiya Y, Igarashi K

Matrix Biology 27 43 - 43 2008年12月

Transvascular accumulation of Sialyl Lewis X conjugated liposome in inflamed joints of collagen antibody-induced arthritic (CAIA) mice. 査読

Jun Minaguchi, Toshitaka Oohashi, Kiichi Inagawa, Aiji Ohtsuka, Yoshifumi Ninomiya

Archives of histology and cytology 71 ( 3 ) 195 - 203 2008年11月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The aim of the current study was to investigate the specific accumulation of the Sialyl Lewis X (SLX) liposome in inflammation in the collagen-antibody induced arthritic (CAIA) model mice. The SLX-liposome encapsulating fluorescent substance (Cy5.5 or Cy3) was prepared for this study. The SLX-liposome was administered intravenously via the mouse caudal vein. After 1 to 24 h, the accumulation of SLX-liposome was observed using in vivo fluorescent imaging equipment (eXplore Optix), or the knee joints were removed for histological analysis. The in vivo fluorescent imaging showed that the signal was confined to the inflammatory site in the CAIA mice in an inflammatory dependent manner. The signal intensity was stronger at 24 h than at 1 h after injection. In the histological sections, the fluorescent signals were detected in the periarticular soft-tissue, especially in the hyperplastic synovium, including a pannus invasion with inflammatory cells in the CAIA. Intense signals were observed in vessel-like structures 1 h after injection; these were co-labeled with the vascular endothelial cell marker (CD31) and E-selectin, a ligand of the SLX-liposome expressed on activated endothelial cells. The diffused signals from the vessels increased time-dependently at 6 to 24 h after injection. This is the first report to examine the exact localization of the SLXliposome by encapsulated fluorescence in hyperplastic synovial tissue of CAIA mice. These results suggest the feasibility and potential use of SLX-liposome as a vehicle for the active targeting of drug delivery to inflammatory tissue.

DOI： 10.1679/aohc.71.195

Molecular cloning and developmental expression of a hyaluronan and proteoglycan link protein gene, crtl1/hapln1, in zebrafish. 査読

Jeong Suk Kang, Yasuhiko Kawakami, Yoko Bekku, Yoshifumi Ninomiya, Juan Carlos Izpisúa Belmonte, Toshitaka Oohashi

Zoological science 25 ( 9 ) 912 - 8 2008年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The proteoglycan aggregate of the cartilage is composed of aggrecan, link protein (LP), and hyaluronan, providing resistance to compression in joints and cartilage structures. To further understand the function of LP during the process of chondrogenesis and bone formation in zebrafish, we cloned the zebrafish cDNA for hyaluronan and proteoglycan link protein 1 (crtl1/hapln1) and examined the expression of the gene during embryogenesis using in-situ hybridization. crtl1/hapln1 expression is first observed in the adaxial cells at the bud- stage. Throughout somitogenesis, crtl1/hapln1 is expressed in the sclerotomes, floor plate, and hypochord. In addition, crtl1/hapln1 is expressed in rhombomeres 3 and 5, pharyngeal arches, telecephalon, otic vesicles, and pectral fins. During chondrocranial/skull formation, crtl1/hapln1 expression is highest at around 4 dpf and is colocalized with aggrecan in the cartilaginous arches and with dermacan in the dermal bones.

DOI： 10.2108/zsj.25.912

Ten_m3 regulates eye-specific patterning in the mammalian visual pathway and is required for binocular vision. 査読国際誌

Catherine A Leamey, Sam Merlin, Paul Lattouf, Atomu Sawatari, Xiaohong Zhou, Natasha Demel, Kelly A Glendining, Toshitaka Oohashi, Mriganka Sur, Reinhard Fässler

PLoS biology 5 ( 9 ) e241 - 2092 2007年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Binocular vision requires an exquisite matching of projections from each eye to form a cohesive representation of the visual world. Eye-specific inputs are anatomically segregated, but in register in the visual thalamus, and overlap within the binocular region of primary visual cortex. Here, we show that the transmembrane protein Ten_m3 regulates the alignment of ipsilateral and contralateral projections. It is expressed in a gradient in the developing visual pathway, which is consistently highest in regions that represent dorsal visual field. Mice that lack Ten_m3 show profound abnormalities in mapping of ipsilateral, but not contralateral, projections, and exhibit pronounced deficits when performing visually mediated behavioural tasks. It is likely that the functional deficits arise from the interocular mismatch, because they are reversed by acute monocular inactivation. We conclude that Ten_m3 plays a key regulatory role in the development of aligned binocular maps, which are required for normal vision.

DOI： 10.1371/journal.pbio.0050241

ADAMTS-9 is synergistically induced by interleukin-1beta and tumor necrosis factor alpha in OUMS-27 chondrosarcoma cells and in human chondrocytes. 査読国際誌

Kadir Demircan, Satoshi Hirohata, Keiichiro Nishida, Omer F Hatipoglu, Toshitaka Oohashi, Tomoko Yonezawa, Suneel S Apte, Yoshifumi Ninomiya

Arthritis and rheumatism 52 ( 5 ) 1451 - 60 2005年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

OBJECTIVE: To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene. METHODS: OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1beta and/or TNFalpha. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1beta and/or TNFalpha. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1beta-stimulated OUMS-27 cells was investigated. RESULTS: IL-1beta increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1beta stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1beta and TNFalpha had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1beta-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up-regulation in OUMS-27 cells. CONCLUSION: ADAMTS9 is an IL-1beta- and TNFalpha-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.

DOI： 10.1002/art.21010

Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21. 査読国際誌

Zheng-Nan Shen, Keiichiro Nishida, Hideyuki Doi, Toshitaka Oohashi, Satoshi Hirohata, Toshifumi Ozaki, Aki Yoshida, Yoshifumi Ninomiya, Hajime Inoue

Biochemical and biophysical research communications 328 ( 2 ) 375 - 82 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPAR gamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ(2)-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ(2), but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ(2) was partly, but not completely, blocked by PPAR gamma antagonist (GW9662) suggesting the 15d-PGJ(2) exerted its effect by PPAR gamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ(2) in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma.

DOI： 10.1016/j.bbrc.2004.12.186

[Bral1, Bral2: the novel brain specific-hyaluronan and protoglycan link protein genes]. 査読

Toshitaka Oohashi, Yoko Bekku, Yoshifumi Ninomiya

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 49 ( 15 Suppl ) 2354 - 61 2004年11月

Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction. 査読国際誌

Keigo Nakamura, Satoshi Hirohata, Takashi Murakami, Toru Miyoshi, Kadir Demircan, Toshitaka Oohashi, Hiroko Ogawa, Kazuya Koten, Kenichi Toeda, Shozo Kusachi, Yoshifumi Ninomiya, Yasushi Shiratori

Journal of biochemistry 136 ( 4 ) 439 - 46 2004年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPANESE BIOCHEMICAL SOC

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.

DOI： 10.1093/jb/mvh138

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells. 査読国際誌

Hiroko Ogawa, Toshitaka Oohashi, Masataka Sata, Yoko Bekku, Satoshi Hirohata, Keigo Nakamura, Tomoko Yonezawa, Shozo Kusachi, Yasushi Shiratori, Yoshifumi Ninomiya

Matrix biology : journal of the International Society for Matrix Biology 23 ( 5 ) 287 - 98 2004年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells.

DOI： 10.1016/j.matbio.2004.07.001

Characterization of dermacan, a novel zebrafish lectican gene, expressed in dermal bones. 査読国際誌

Jeong Suk Kang, Toshitaka Oohashi, Yasuhiko Kawakami, Yoko Bekku, Juan Carlos Izpisúa Belmonte, Yoshifumi Ninomiya

Mechanisms of development 121 ( 3 ) 301 - 12 2004年3月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

We report here the isolation and characterization of a cDNA encoding zebrafish dermacan, a novel member of hyaluronan (HA)-binding proteoglycans, which was termed after its characteristic expression in the zebrafish dermal bones. The deduced protein sequence shares the typical modular elements of lecticans. Sequence comparison covering the C-terminal globular domain demonstrated that dermacan shows high homology with zebrafish versican but is distinct from any other identified lecticans. Genomic DNA analysis demonstrated that dermacan and versican were encoded by distinct genes in the zebrafish genome. The expression of dermacan is initiated in the sclerotome and cephalic paraxial mesoderm at 16 h postfertilization. During the pharyngular period, dermacan transcripts were detected in the sclerotome, tail fin bud, pharyngular arch primordial region, and otic vesicle. In the development of craniofacial bones, dermacan expression was detected typically in the opercle and dentary. These regions belong to the craniofacial dermal bones. aggrecan expression, in contrast, was observed in the elements of craniofacial cartilage bones. In the dermacan-morpholino-injected embryos, dermal bones, e.g. opercle, dentary, and branchiostegal rays, as well as axial skeleton in the trunk, showed decreased ossification. We conclude that dermacan is a novel lectican gene, and that zebrafish lectican genes have genetically diverged. In addition, our data suggest the involvement of dermacan in zebrafish dermal bone development.

DOI： 10.1016/j.mod.2004.01.007

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6. 査読国際誌

Uwe Rauch, Satoshi Hirakawa, Toshitaka Oohashi, Joachim Kappler, Gunnel Roos

Matrix biology : journal of the International Society for Matrix Biology 22 ( 8 ) 629 - 39 2004年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.

DOI： 10.1016/j.matbio.2003.11.007

Molecular cloning of Bral2, a novel brain-specific link protein, and immunohistochemical colocalization with brevican in perineuronal nets. 査読国際誌

Yoko Bekku, Wei-Dong Su, Satoshi Hirakawa, Reinhard Fässler, Aiji Ohtsuka, Jeong Suk Kang, Jennifer Sanders, Takuro Murakami, Yoshifumi Ninomiya, Toshitaka Oohashi

Molecular and cellular neurosciences 24 ( 1 ) 148 - 59 2003年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

The hyaluronan binding chondroitin sulphate proteoglycans, called lecticans, are the abundant extracellular matrix molecules in the developing and/or adult brain. The link proteins (LPs) are also known to be coordinately present in brain. We report here the molecular cloning and expression analysis of a novel member of LPs: Bral2, predominantly expressed in brain. The Bral2 mRNA expression is first detected at P20 and continued through adulthood, suggesting its functional importance and association with adult-type lecticans. The substantial immunoreactivity of Bral2 is found in several nuclei throughout the midbrain and hindbrain in a perineuronal net pattern. In situ hybridization revealed that Bral2 is synthesized by these neurons themselves, especially by the GABAergic neurons in the cerebellar cortex. Interestingly, the colocalization and synergic importance of Bral2 and brevican in the perineuronal nets is indicated by the comparative immunohistochemical analysis using wild-type and brevican-deficient mouse brain. Our results suggest that Bral2 is involved in the formation of extracellular matrix contributing to perineuronal nets and facilitate the understanding of a functional role of these extracellular matrices.

Molecular cloning of a novel transmembrane protein MOLT expressed by mature oligodendrocytes. 査読国際誌

Hiroyuki Nomoto, Tomoko Yonezawa, Kouichi Itoh, Katsuhiko Ono, Kiyotaka Yamamoto, Toshitaka Oohashi, Fumio Shiraga, Hiroshi Ohtsuki, Yoshifumi Ninomiya

Journal of biochemistry 134 ( 2 ) 231 - 8 2003年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

A novel oligodendrocyte (OL)-specific cDNA was isolated from brain capillary endothelial cells and characterized. The cDNA encodes a protein of 1099 amino acids that contains a signal peptide and a transmembrane domain. The protein was expressed in mature OLs in vivo and in vitro cell cultures and was thus designated as mature OL transmembrane protein (MOLT). RT-PCR analysis showed that MOLT mRNA was expressed in brain, lung, pancreas, and testis. A polyclonal antibody raised against a part of the mouse MOLT reacted specifically with multipolar OLs possessing radially oriented processes that penetrated into the gray matter. More cells were detected in the white matter, and these had longitudinally oriented processes. In a rat OL lineage culture system, oligodendrocyte precursor cells did not initially produce MOLT mRNA and protein, but when they begun to differentiate into mature OLs, they started expressing MOLT. Consequently, MOLT may function as OLs become mature and may serve as a cell-surface marker for OL differentiation.

The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain. 査読国際誌

Xiao-Hong Zhou, Oliver Brandau, Kang Feng, Toshitaka Oohashi, Yoshifumi Ninomiya, Uwe Rauch, Reinhard Fässler

Gene expression patterns : GEP 3 ( 4 ) 397 - 405 2003年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

The mouse TEN-M/ODZ proteins belong to a new family of type II transmembrane proteins with unknown function. The family consists of four members, which are expressed highly in brain and less in many other tissues. In the present study we have generated specific RNA probes and antibodies to characterize the expression of the 4 Ten-m/Odz genes in the developing and adult central nervous system (CNS) of mice. Ten-m/Odz3 and Ten-m/Odz4 mRNAs were first detectable at E7.5, Ten-m/Odz2 expression started at the 37 somite (E 10.5) stage, while Ten-m/Odz1 mRNA is not found before E15.5. In the adult mouse CNS mRNAs of the 4 Ten-m/Odzs were expressed in distinct patterns, which partially overlapped. Immunostaining and in situ hybridization localized proteins and mRNAs of Ten-m/Odzs in adjacent areas suggesting that TEN-M/ODZ proteins might be transported from the cell body along the axon or that they are shed from the cell surface and diffuse into distant regions.

Vascular endothelial growth factor principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. 査読国際誌

Takayuki Furumatsu, Zheng Nan Shen, Akira Kawai, Keiichiro Nishida, Hironori Manabe, Toshitaka Oohashi, Hajime Inoue, Yoshifumi Ninomiya

Journal of biochemistry 133 ( 5 ) 633 - 9 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is essential for bone formation. However, the effect of VEGF on osteoblastic cells during osteoblastogenesis is still controversial. The aim of this study was to clarify the relationship between osteoblastic cells derived from human mesenchymal stem cells (MSCs) and VEGF in the early stage of osteoblastic differentiation. Continuous dexamethasone treatment with a low concentration stimulated osteoblastogenesis of MSCs and the expression of VEGF121 mRNA. The VEGF secretion from osteoblastic cells also increased along with osteoblastogenesis. Neuropilin-1, which mainly binds VEGF165, was detected at all stages during early osteoblastogenesis, but VEGF receptor-1 and -2 were not detected on RT-PCR analyses. In this study, VEGF had no direct effect on the proliferation of osteoblastic cells. However, the secreted VEGF in the conditioned medium of osteoblastic cells exhibited high angiogenic power as to endothelial cell proliferation. Our findings indicated that VEGF121 principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. The present study also demonstrated the differential expression of VEGF121 during osteoblastogenesis. The increase of VEGF in the early stage might be a useful marker of induction of bone formation due to human MSCs.

All four members of the Ten-m/Odz family of transmembrane proteins form dimers. 査読国際誌

Kang Feng, Xiao-Hong Zhou, Toshitaka Oohashi, Matthias Mörgelin, Ariel Lustig, Satoshi Hirakawa, Yoshifumi Ninomiya, Jürgen Engel, Uwe Rauch, Reinhard Fässler

The Journal of biological chemistry 277 ( 29 ) 26128 - 35 2002年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes.

Human BRAL1 and BCAN genes that belong to the link-module superfamily are tandemly arranged on chromosome 1q21-23. 査読

Hiroyuki Nomoto, Toshitaka Oohashi, Satoshi Hirakawa, Yasuyoshi Ueki, Hiroshi Ohtsuki, Yoshifumi Ninomiya

Acta medica Okayama 56 ( 1 ) 25 - 9 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

We herein determined by fluorescence in situ hybridization the chromosomal localization of 2 human genes, BRAL1 and BCAN, both of which belong to the link-module superfamily, i.e. to the same band of chromosome 1q21-23. Further analysis of the genomic organization of BRAL1 and BCAN revealed that the BRAL1 gene was located 20-kb upstream of the BCAN start site. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the BCAN gene. High heterozygosity (0.79) makes this polymorphism a useful marker in the study of genetic disorders. Knowledge of the structure of the genes and the marker provides essential information for further analysis of the gene locus at chromosome 1q21-23.

Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems. 査読国際誌

Toshitaka Oohashi, Satoshi Hirakawa, Yoko Bekku, Uwe Rauch, Dieter R Zimmermann, Wei-Dong Su, Aiji Ohtsuka, Takuro Murakami, Yoshifumi Ninomiya

Molecular and cellular neurosciences 19 ( 1 ) 43 - 57 2002年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Bral1, a brain-specific hyaluronan-binding protein, has been cloned recently. To gain insight into the role of Bral1, we generated a specific antibody against this protein. We have examined the detailed localization pattern of Bral1 protein and compared it with that of other members of the lectican proteoglycan family, such as brevican and versican, with which Bral1 is predicted to interact. The immunoreactivity of Bral1 antibody was predominantly observed in myelinated fiber tracts in the adult brain and could be detected at P20 in the white matter of the developing cerebellum, suggesting that expression starts when axonal myelination takes place. Furthermore, immunostaining demonstrated that Bral1 colocalized with the versican V2 isoform at the nodes of Ranvier. The present data suggest that Bral1 may play a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes.

Neurocan is dispensable for brain development. 査読国際誌

X H Zhou, C Brakebusch, H Matthies, T Oohashi, E Hirsch, M Moser, M Krug, C I Seidenbecher, T M Boeckers, U Rauch, R Buettner, E D Gundelfinger, R Fässler

Molecular and cellular biology 21 ( 17 ) 5970 - 8 2001年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain. 査読国際誌

S Hirakawa, T Oohashi, W D Su, H Yoshioka, T Murakami, J Arata, Y Ninomiya

Biochemical and biophysical research communications 276 ( 3 ) 982 - 9 2000年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

We report here molecular cloning and expression analysis of the gene for a novel human brain link protein-1 (BRAL1) which is predominantly expressed in brain. The predicted open reading frame of human brain link protein-1 encoded a polypeptide of 340 amino acids containing three protein modules, the immunoglobulin-like fold and proteoglycan tandem repeat 1 and 2 domains, with an estimated mass of 38 kDa. The brain link protein-1 mRNA was exclusively present in brain. When analyzed during mouse development, it was detected solely in the adult brain. Concomitant expression pattern of mRNAs for brain link protein-1 and various lectican proteoglycans in brain suggests a possibility that brain link protein-1 functions to stabilize the binding between hyaluronan and brevican. The human BRAL1 gene contained 7 exons and spanned approximately 6 kb. The entire immunoglobulin-like fold was encoded by a single exon and the proteoglycan tandem repeat 1 and 2 domains were encoded by a single and two exons, respectively. The deduced amino acid sequence of human brain link protein-1 exhibited 45% identity with human cartilage link protein-1 (CRTL1), previously reported as link protein to stabilize aggregates of aggrecan and hyaluronan in cartilage. These results suggest that brain link protein-1 may have distinct function from cartilage link protein-1 and play specific roles, especially in the adult brain.

Differential expression of mouse alpha5(IV) and alpha6(IV) collagen genes in epithelial basement membranes. 査読国際誌

K Saito, I Naito, T Seki, T Oohashi, E Kimura, R Momota, Y Kishiro, Y Sado, H Yoshioka, Y Ninomiya

Journal of biochemistry 128 ( 3 ) 427 - 34 2000年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.

Peri-implantation lethality in mice lacking the Sm motif-containing protein Lsm4. 査読国際誌

E Hirsch, T Oohashi, M Ahmad, S Stamm, R Fässler

Molecular and cellular biology 20 ( 3 ) 1055 - 62 2000年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Small nuclear ribonucleoproteins (snRNPs) are particles present only in eukaryotic cells. They are involved in a large variety of RNA maturation processes, most notably in pre-mRNA splicing. Several of the proteins typically found in snRNPs contain a sequence signature, the Sm domain, conserved from yeast to mammals. By using a promoter trap strategy to target actively transcribed loci in murine embryonic stem cells, a new murine gene encoding an Sm motif-containing protein was identified. Database searches revealed that it is the mouse orthologue of Lsm4p, a protein found in yeast and human cells and putatively associated with U6 snRNA. Introduction of the geo reporter gene cassette under the control of the murine Lsm4 (mLsm4) endogenous promoter showed that the gene was ubiquitously transcribed in embryonic and adult tissues. The insertion of the geo cassette disrupted the mLsm4 allele, and homozygosity for the mutation led to a recessive embryonic lethal phenotype. mLsm4-null zygotes survived to the blastocyst stages, implanted into the uterus, but died shortly thereafter. The early death of mLsm4p-null mice suggests that the role of mLsm4p in splicing is essential and cannot be compensated by other Lsm proteins.

The extracellular matrix in the mouse brain: its reactions to endo-alpha-N-acetylgalactosaminidase and certain other enzymes. 査読

T Murakami, A Ohtsuka, W D Su, T Taguchi, T Oohashi, T Murakami, K Abe, Y Ninomiya

Archives of histology and cytology 62 ( 3 ) 273 - 81 1999年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

As our previous studies have indicated, the cingulate cortex of the adult mouse brain contains many neurons with rich cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The present study demonstrated that exclusive incubation with endo-alpha-N-acetylgalactosaminidase abolished the lectin Vicia villosa or Wisteria floribunda agglutinin (VVA or WFA) labeling of the nerve cell surface glycoproteins, while it neither interfered with the cationic iron colloid or aldehyde fuchsin stainings of the perineuronal proteoglycans nor abolished the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Double incubations with endo-alpha-N-acetylgalactosaminidase and collagenase did not eliminate the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, though they did eliminate the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans as well as the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Triple incubations with endo-alpha-N-acetylgalactosaminidase, collagenase, and endo-alpha-N-acetylgalactosaminidase abolished the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, and also eliminated the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans and the Gömöri's ammoniacal silver impregnation of the collagenous ligands. These findings indicate that: the nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase; these galactosamines associated with the collagenous ligands or perineuronal proteoglycans are not digested by endo-alpha-N-acetylgalactosaminidase; and the terminal N-acetylgalactosamines newly exposed by collagenase incubation are digested by this galactosaminidase. It was further demonstrated that hyaluronidase incubation neither digests the collagenous ligands nor revives the lectin VVA or WFA labeling of the nerve cell surface proteoglycans.

Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues. 査読国際誌

T Oohashi, X H Zhou, K Feng, B Richter, M Mörgelin, M T Perez, W D Su, R Chiquet-Ehrismann, U Rauch, R Fässler

The Journal of cell biology 145 ( 3 ) 563 - 77 1999年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

Differential tissular expression and localization of type IV collagen alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains and their mRNA in normal breast and in benign and malignant breast tumors. 査読国際誌

S Nakano, K Iyama, M Ogawa, H Yoshioka, Y Sado, T Oohashi, Y Ninomiya

Laboratory investigation; a journal of technical methods and pathology 79 ( 3 ) 281 - 92 1999年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha chains. We investigated the cellular regulation and origin of these alpha(IV) chains in normal and neoplastic breast tissues by immunohistochemistry by using alpha(IV) chain-specific antibodies and by in situ hybridization. In normal breast, alpha1(IV) and alpha2(IV) chains were stained in all BM, whereas alpha5(IV) and alpha6(IV) chains were restrictively localized in a linear pattern in the BM of the mammary gland. Similar immunostaining profiles were observed in benign breast tumors and in the intraductal components of invasive ductal carcinoma. However, in invasive ductal carcinoma, alpha1(IV) and alpha2(1V) chains were discontinuously or negatively stained in the cancer cell nests, and the assembly of alpha5(IV) and alpha6(IV) chains into the BM was completely inhibited. Coexpression of alpha5(IV) and alpha6(IV) chains was related to the localization of alpha-smooth muscle actin (alpha-SMA)-positive myoepithelial cells. By in situ hybridization, in fibroadenoma and invasive ductal carcinoma, the signals for alpha1(IV) and alpha2(IV) mRNA were abundant in stromal cells. However, the signals for alpha5(IV) and alpha6(IV) mRNA were not seen in any of these cells. In contrast, in intraductal papilloma, coexpression of alpha1 (IV)/alpha2(IV) mRNA and alpha5(IV)/alpha6(IV) mRNA was identified in epithelial cells. The results indicate that the mammary gland forms a second network of BM composed of alpha5(IV)/alpha6(IV) chains, in addition to the classic network of alpha1(IV)/alpha2(IV) chains. The expression of type IV collagen alpha chains seems to be differentially regulated by the epithelial-myoepithelial interaction and to be associated with the invasive potential of breast cancer.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene. 査読国際誌

A J Coffey, R A Brooksbank, O Brandau, T Oohashi, G R Howell, J M Bye, A P Cahn, J Durham, P Heath, P Wray, R Pavitt, J Wilkinson, M Leversha, E Huckle, C J Shaw-Smith, A Dunham, S Rhodes, V Schuster, G Porta, L Yin, P Serafini, B Sylla, M Zollo, B Franco, A Bolino, M Seri, A Lanyi, J R Davis, D Webster, A Harris, G Lenoir, G de St Basile, A Jones, B H Behloradsky, H Achatz, J Murken, R Fassler, J Sumegi, G Romeo, M Vaudin, M T Ross, A Meindl, D R Bentley

Nature genetics 20 ( 2 ) 129 - 35 1998年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Differential expression of type IV collagen isoforms, alpha5(IV) and alpha6(IV) chains, in basement membranes surrounding smooth muscle cells. 査読国際誌

T Seki, I Naito, T Oohashi, Y Sado, Y Ninomiya

Histochemistry and cell biology 110 ( 4 ) 359 - 66 1998年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using alpha(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the alpha chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [alpha1(IV)]2alpha2(IV),alpha3(IV)alpha4(IV)alpha5+ ++(IV), and alpha5(IV)/alpha6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of alpha1, alpha2, alpha5, and alpha6 chains. The same alpha chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly alphal and alpha2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for alpha1 and alpha2 chains; however, alpha5 and alpha6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by alpha5/alpha6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs.

Differential expression of type IV collagen isoforms, alpha 5(IV) and alpha 6(IV) chains, in basement membranes surrounding smooth muscle cells 査読

T Seki, Naito, I, T Oohashi, Y Sado, Y Ninomiya

HISTOCHEMISTRY AND CELL BIOLOGY 110 ( 4 ) 359 - 366 1998年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER VERLAG

Smooth muscle is composed of cigar-shaped, non-striated cells, each of which is encapsulated by a basement membrane and forms the contractile portion of tubular organs such as the gastrointestinal tract, pulmonary tract, genitourinary tract, and vasculature, in which slow and sustained contractions are needed. We examined basement membranes produced by smooth muscle cells and, using alpha(IV) chain-specific monoclonal antibodies, analyzed type IV collagens in these organs. Detailed distribution analysis of the alpha chains in normal and Alport cases by use of specific antibodies indicated that there are at least three molecular forms of type IV collagen, [alpha 1(IV)](2)alpha 2(IV), alpha 3(IV)alpha 4(IV)alpha 5(IV), and alpha 5(IV)/alpha 6(IV). Smooth muscle cells in the urinary bladder and uterus were enclosed by basement membranes composed of alpha 1, alpha 2, alpha 5, and alpha 6 chains. The same alpha chains were present around smooth muscle cells in the muscular layer of the fundus of the stomach, whereas those in the antrum and further distal side of the gastrointestinal tract expressed mostly alpha 1 and alpha 2 chains. In addition, immunostaining analysis of the vasculature also showed that most of the smooth muscle cells were positive for alpha 1 and alpha 2 chains; however, alpha 5 and alpha 6 chains were also expressed by smooth muscle cells in the aorta and some arteries where blood pressure changes significantly. These results suggest that the smooth muscle cells enclosed by alpha 5/alpha 6-containing basement membranes might have some particular function related to mechanical stress or tensile strength during the characteristic contractile activity of tubular organs.

Organization and Expression of Basement Membrane Collagen IV Genes and Their Roles in Human Disorders.(共著) 査読国際誌

Journal of Biochemistry 123 ( 5 ) 767 - 76 1998年5月

Two genes, COL4A3 and COL4A4 coding for the human alpha3(IV) and alpha4(IV) collagen chains are arranged head-to-head on chromosome 2q36. 査読国際誌

R Momota, M Sugimoto, T Oohashi, K Kigasawa, H Yoshioka, Y Ninomiya

FEBS letters 424 ( 1-2 ) 11 - 6 1998年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

We first isolated and characterized genomic DNA fragments that cover the 5' flanking sequences of COL4A3 and COL4A4 encoding the human basement membrane alpha3(IV) and alpha4(IV) collagen chains, respectively. Nucleotide sequence analysis indicated that the two genes are arranged head-to-head. To determine transcription start site for COL4A4 gene, we performed RACE and RNase protection assays, indicating that there are two alternative transcripts presumably derived from two different promoters. Interestingly, one transcription start site (from exon 1') of COL4A4 is only 5 bp away from the reported transcription start site of COL4A3, whereas the other transcript (from exon 1) starts 373 nucleotides downstream from the first one, generating the two kinds of transcripts that differ in the 5' UTR regions. Expression of these two transcripts appears tissue-specific; exon 1 transcript was expressed predominantly in epithelial cells, while exon 1' transcript showed rather ubiquitous and low expression. The nucleotide sequence of the promoter region is composed of dense CpG dinucleotides, GC boxes, CTC boxes and a CCAAT box but no TATA box. These results provide information to delineate the promoter activity for the tissue-specific expression of the six type IV collagen genes and basement membrane assembly in different tissues and organs.

DOI： 10.1016/S0014-5793(98)00128-8

Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart. 査読

J Kondo, S Kusachi, Y Ninomiya, H Yoshioka, T Oohashi, M Doi, T Murakami, H Moritani, H Kumashiro, T Tsuji

Japanese heart journal 39 ( 2 ) 211 - 20 1998年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.

DOI： 10.1536/ihj.39.211

Topoisomerase I and II consensus sequences in a 17-kb deletion junction of the COL4A5 and COL4A6 genes and immunohistochemical analysis of esophageal leiomyomatosis associated with Alport syndrome. 査読国際誌

Y Ueki, I Naito, T Oohashi, M Sugimoto, T Seki, H Yoshioka, Y Sado, H Sato, T Sawai, F Sasaki, M Matsuoka, S Fukuda, Y Ninomiya

American journal of human genetics 62 ( 2 ) 253 - 61 1998年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Diffuse esophageal leiomyomatosis (DL), a benign smooth-muscle-cell tumor, is characterized by abnormal cell proliferation. DL is sometimes associated with X-linked Alport syndrome (AS), an inherited nephropathy caused by COL4A5 gene mutations. COL4A5 is tightly linked, in a head-to-head fashion, to the functionally related and coordinately regulated COL4A6 gene. No X-linked AS cases are due to COL4A6 mutations, but all DL/AS cases are always associated with deletions spanning the 5' regions of the COL4A5/COL4A6 cluster. Unlike the COL4A5 breakpoints, those of COL4A6 are clustered within intron 2 of the gene. We identified a DL/AS deletion and the first characterization of the breakpoint sequences. We show that a deletion eliminates the first coding exon of COL4A5 and the first two coding exons of COL4A6. The breakpoints share the same sequence, which, in turn, is closely homologous to the consensus sequences of topoisomerases I and II. Additional DNA evidence suggested that the male patient is a somatic mosaic for the mutation. Immunohistochemical analysis using alpha-chain-specific monoclonal antibodies supported this conclusion, since it revealed the absence of the alpha5(IV) and alpha6(IV) collagen chains in most but not all of the basement membranes of the smooth-muscle-cell tumor. We also documented a similar segmental staining pattern in the glomerular basement membranes of the patient's kidney. This study is particularly relevant to the understanding of DL pathogenesis and its etiology.

DOI： 10.1086/301703

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model. 査読国際誌

R Fleischmajer, K Kühn, Y Sato, E D MacDonald 2nd, J S Perlish, T C Pan, M L Chu, Y Kishiro, T Oohashi, S M Bernier, Y Yamada, Y Ninomiya

The Journal of investigative dermatology 109 ( 4 ) 527 - 33 1997年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Differential expression of alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) collagen chains in the basement membrane of basal cell carcinoma. 査読国際誌

K Tanaka, K Iyama, M Kitaoka, Y Ninomiya, T Oohashi, Y Sado, T Ono

The Histochemical journal 29 ( 7 ) 563 - 70 1997年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Type IV collagen, the major component of basement membrane, consists primarily of alpha 1(IV) and alpha 2(IV) chains. Recently, other types of collagen IV chains, i.e. alpha 3(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of alpha (IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six alpha (IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, alpha 1(IV) and alpha 2(IV) chains were discontinuously stained, and alpha 5(IV) and alpha 6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, alpha 1(IV) and alpha 2(IV) chains were continuously stained, and alpha 5(IV) and alpha 6(IV) chains were discontinuous or absent. The assembly of alpha 5(IV) and alpha 6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma.

DOI： 10.1023/A:1026428010104

The primary structure of the Cl(-)-translocating ATPase, b subunit of Acetabularia acetabulum, which belongs to the F-type ATPase family. 査読国際誌

C Moritani, T Ohhashi, H Kadowaki, M Tagaya, T Fukui, F Lottspeich, D Oesterhelt, M Ikeda

Archives of biochemistry and biophysics 339 ( 1 ) 115 - 24 1997年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes possibly encoding the b subunit (50 kDa) of the Cl(-)-translocating ATPase of Acetabularia acetabulum were cloned from total RNA and from poly(A)+ RNA and sequenced. The deduced amino acid sequence of the open reading frame consisted of 478 amino acids and showed high similarity to the beta subunit of chloroplast F1-ATPase. Gene fragments encoding the putative beta subunit of chloroplast F1- (273 bp) and mitochondrial F1-ATPases (332 bp) were also cloned from A. acetabulum and sequenced, respectively. The deduced amino acid sequence of the chloroplast F1-ATPase showed 92.5% identity to be primary structure of the b subunit of the Cl(-)-translocating ATPase, while the nucleotide sequences were 79.9% identical. The deduced amino acid sequence of the latter was 77.3% identical to that of the b subunit of the Cl(-)-translocating ATPase and the nucleotide sequences were 67.5% identical. By Northern analysis, these three beta-like genes were demonstrated to be transcribed with different sizes of RNA species. A putative chloroplast F1-beta fragment also hybridized with chloroplast DNA isolated from the organism.

Absence of alpha 6(IV) collagen in kidney and skin of X-linked Alport syndrome patients. 査読国際誌

S Hino, T Takemura, Y Sado, M Kagawa, T Oohashi, Y Ninomiya, K Yoshioka

Pediatric nephrology (Berlin, Germany) 10 ( 6 ) 742 - 4 1996年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

To identify the abnormalities of the type IV collagen alpha 6 chain, alpha 6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of alpha 6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear alpha 6(IV) staining in the basement membrane (BM) of Bowman's capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for alpha 6(IV) in Bowman's capsules and tubules. In these patients, alpha 1(IV) and alpha 2(IV) were normal, whereas alpha 3(IV), alpha 4(IV), and alpha 5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither alpha 5(IV) nor alpha 6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for alpha 5(IV) and alpha 6(IV). These findings indicate that, in addition to a disturbed alpha 3(IV)-alpha 4(IV)-alpha 5(IV) network, patients with X-linked Alport syndrome have abnormalities in alpha 6(IV) of the renal and epidermal BMs at the protein level.

DOI： 10.1007/s004670050206

Nephritogenicity and alpha-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane. 査読国際誌

S Rauf, M Kagawa, Y Kishiro, S Inoue, I Naito, T Oohashi, M Sugimoto, Y Ninomiya, Y Sado

Virchows Archiv : an international journal of pathology 428 ( 4-5 ) 281 - 8 1996年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

Isolation and structure of the COL4A6 gene encoding the human alpha 6(IV) collagen chain and comparison with other type IV collagen genes. 査読国際誌

T Oohashi, Y Ueki, M Sugimoto, Y Ninomiya

The Journal of biological chemistry 270 ( 45 ) 26863 - 7 1995年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes COL4A5 and COL4A6, coding for the basement membrane collagen chains, alpha 5(IV) and alpha 6(IV), respectively, are located head-to-head in close proximity on human chromosome Xq22, and COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion (Sugimoto M., Oohashi T., and Ninomiya Y. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11679-11683). Immunofluorescence studies using alpha chain-specific antibodies demonstrated that the two genes are expressed in a tissue-specific manner (Ninomiya, Y., Kagawa, M., Iyama, K., Naito, L., Kishiro, Y., Seyer, J. M., Sugimoto, M., Oohashi, T., and Sado, Y. (1995) J. Cell Biol. 130, 1219-1229). We report here for the first time the isolation and the structural organization of the human COL4A6 gene. The entire gene presumably exceeds 200 kilobase pairs and contains 46 exons. Exons 1' and 1 encode the two different 5'-UTRs and the two amino-terminal parts of of the signal peptide. The carboxyl part of the signal peptide and the 7 S domain are coded for by the following 6 different exons, 2-7, whereas the exons 7-42 encode the central COL 1 domain, which contains the Gly-X-Y repeats. The last three exons, 43-45, encode the carboxyl-terminal NC1 domain. Sizes of more than a half of the exons of the gene are the same as those of Col4a2 but quite different from those of COL4A5. Within the COL4A6 gene we found three CA repeat markers that can be used for allele detection. The detailed structure of the COL4A6 gene and the high heterozygosity microsatellite markers located within the gene will be useful for linkage analysis and familial diagnosis of diseases caused by mutations of this gene.

ESTABLISHMENT BY THE RAT LYMPH-NODE METHOD OF EPITOPE-DEFINED MONOCLONAL-ANTIBODIES RECOGNIZING THE 6 DIFFERENT ALPHA-CHAINS OF HUMAN TYPE-IV COLLAGEN 査読

Y SADO, M KAGAWA, Y KISHIRO, K SUGIHARA, NAITO, I, JM SEYER, M SUGIMOTO, T OOHASHI, Y NINOMIYA

HISTOCHEMISTRY AND CELL BIOLOGY 104 ( 4 ) 267 - 275 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER VERLAG

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen. 査読国際誌

Y Sado, M Kagawa, Y Kishiro, K Sugihara, I Naito, J M Seyer, M Sugimoto, T Oohashi, Y Ninomiya

Histochemistry and cell biology 104 ( 4 ) 267 - 75 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. 査読国際誌

Y Ninomiya, M Kagawa, K Iyama, I Naito, Y Kishiro, J M Seyer, M Sugimoto, T Oohashi, Y Sado

The Journal of cell biology 130 ( 5 ) 1219 - 29 1995年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

DOI： 10.1083/jcb.130.5.1219

The genes COL4A5 and COL4A6, coding for basement membrane collagen chains alpha 5(IV) and alpha 6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters. 査読国際誌

M Sugimoto, T Oohashi, Y Ninomiya

Proceedings of the National Academy of Sciences of the United States of America 91 ( 24 ) 11679 - 83 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.

DOI： 10.1073/pnas.91.24.11679

Purification and characterization of a membrane-bound ATPase from Acetabularia cliftonii that corresponds to a Cl(-)-translocating ATPase in Acetabularia acetabulum. 査読国際誌

C Moritani, T Ohhashi, S Satoh, D Oesterhelt, M Ikeda

Bioscience, biotechnology, and biochemistry 58 ( 11 ) 2087 - 9 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

A Mg(2+)-ATPase was solubilized from membranes of Acetabularia cliftonii using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. One active ATPase fraction after Mono Q chromatography had a specific activity of 10 units/mg of protein. Judged from subunit composition [54 (a), 50 (b) with a fainter band around 40 kDa], catalytic properties, and N-terminal amino acid sequence of the b subunit, the isolated enzyme was comparable to the Cl(-)-ATPase of Acetabularia acetabulum. Immunological characterization of both subunits showed significant similarity to the F type of ATPase. Cl(-)-transport activity was observed by reconstitution studies into liposomes.

Identification of a new collagen IV chain, alpha 6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same locus for COL4A5. 査読国際誌

T Oohashi, M Sugimoto, M G Mattei, Y Ninomiya

The Journal of biological chemistry 269 ( 10 ) 7520 - 6 1994年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

To date, five distinct alpha chains have been identified in basement membrane collagen IV. We have cloned a gene encoding a new alpha chain belonging to basement membrane collagen IV by cDNA isolation under low stringency conditions. Isolation of overlapping clones and the technique of 5' rapid amplification of cDNA ends enabled us to derive the primary structure of the entire polypeptide. The deduced collagen polypeptide contained 1678 amino acid residues, including a 21-residue signal peptide, a 24-residue amino-terminal NC domain, a central 1405-residue collagenous (COL1) domain, and a 228-residue carboxyl-terminal NC1 domain. We have designated this newly found alpha chain as the alpha 6(IV) chain. The gene was mapped to chromosome Xq22 by in situ hybridization of metaphase lymphocytes. This is the same region of chromosome X where the gene coding for the alpha 5(IV) collagen chain (COL4A5) resides. Mutations in COL4A5 have been characterized in more than 50 patients with Alport syndrome. However, some of the X-linked cases of Alport syndrome are not apparently caused by COL4A5 mutations. The gene we describe in this paper therefore seems to be a candidate for mutations in this group of Alport syndrome patients.

Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase. 査読国際誌

S Satoh, C Moritani, T Ohhashi, K Konishi, M Ikeda

Bioscience, biotechnology, and biochemistry 58 ( 3 ) 521 - 5 1994年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.

Isolation and characterization of human cDNAs and genomic DNAs encoding alpha-4(IV) and alpha-6(IV) chains reveal the presence of a distinct subclass of collagen IV genes. 査読国際誌

M Sugimoto, T Oohashi, M G Mattei, A Fukutomi, N Kashihara, N Matsuo, H Yoshioka, Y Ninomiya

Contributions to nephrology 107 29 - 35 1994年

cDNA isolation and partial gene structure of the human alpha 4(IV) collagen chain. 査読国際誌

M Sugimoto, T Oohashi, H Yoshioka, N Matsuo, Y Ninomiya

FEBS letters 330 ( 2 ) 122 - 8 1993年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

A novel collagen IV chain, alpha 4(IV), has recently been identified in basement membranes. We describe part of the primary structure of the human alpha 4(IV) polypeptide for the first time, which has been determined by cloning and sequencing of cDNAs encoding 241 amino acid residues of the COL domain and 231 residues of the NC1 domain. We also characterized a genomic DNA fragment containing 4 exons coding for the entire NC1 domain. Among five known alpha chains of collagen IV, the alpha 4(IV) chain is distinct from the other four chains. However, it is more similar to the alpha 2(IV) chain than to the alpha 1(IV), alpha 3(IV) and alpha 5(IV) chains in terms of amino acid sequence homology, domain structure of polypeptides and exon/intron structure of the genes, suggesting the presence of two phylogenetically distinct subclasses of collagen IV alpha chains; one composed of alpha 2 and alpha 4 chains and the other of alpha 1, alpha 3 and alpha 5 chains.

Improvement of reconstitution of the Cl--translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics 査読

Toshitaka Ohhashi, Takashi Katsu, Mikiko Ikeda

BBA - Biomembranes 1106 ( 1 ) 165 - 170 1992年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

The improved reconstitution of the Mono Q-III fraction, a Cl--translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ration of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl--transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-subsrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl--transport were also examined in the reconstituted system, both halide ions decrease the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. (1983) J. Membr. Biol. 75, 129-139). © 1992.

DOI： 10.1016/0005-2736(92)90235-E

Improvement of reconstitution of the Cl(-)-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics. 査読国際誌

T Ohhashi, T Katsu, M Ikeda

Biochimica et biophysica acta 1106 ( 1 ) 165 - 70 1992年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

The improved reconstitution of the Mono Q-III fraction, a Cl(-)-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ratio of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl(-)-transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-substrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl(-)-transport were also examined in the reconstituted system, both halide ions decreased the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. ((1983) J. Membr. Biol. 75, 129-139).

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies. 査読国際誌

T Ohhashi, C Moritani, H Andoh, S Satoh, S Ohmori, F Lottspeich, M Ikeda

Journal of chromatography 585 ( 1 ) 153 - 9 1991年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.

DOI： 10.1016/0021-9673(91)85069-R

Characterization of fibroblast-derived prolidase. The presence of two forms of prolidase. 査読国際誌

T Oono, H Yasutomi, T Ohhashi, H Kodama, J Arata

Journal of dermatological science 1 ( 5 ) 319 - 23 1990年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Crude enzyme solutions of prolidase were extracted from cultured human skin fibroblasts derived from control and prolidase-deficient sisters. Two forms of prolidases (prolidase-I and II) were partially purified by high performance liquid chromatography equipped with an ion exchange column. On gel filtration, the relative molecular weights of prolidase-I and II were estimated to be MW = 105,000 and 151,000, respectively. The substrate specificity of partially purified prolidase-I and II in control fibroblasts was estimated against Gly-Pro, Ala-Pro, Met-Pro. Each form of prolidase differed in its substrate specificity. In prolidase-deficient sisters, the elder with typical clinical manifestations and the younger with only slight clinical manifestations, the activity of prolidase-I was absent. However, the activity of prolidase-II was sufficiently present in both sisters. The substrate specificity of prolidase-II in the patients was similar to that of control. No difference in substrate specificity was found between these two patients.

Characterization of prolidase I and II from erythrocytes of a control, a patient with prolidase deficiency and her mother. 査読国際誌

T Ohhashi, T Ohno, J Arata, K Sugahara, H Kodama

Clinica chimica acta; international journal of clinical chemistry 187 ( 1 ) 1 - 9 1990年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Prolidase I (EC 3.4.13.9) was purified to homogeneity from the erythrocytes of a normal human (control) and the patient's mother, and prolidase II from erythrocytes of a control and the patient's mother, and prolidase from the patient's erythrocytes was also highly purified. The various properties of the patient's prolidase were compared to those of prolidase from a control and the patient's mother. Prolidase I from a control and the patient's mother had a molecular weight of about 112,000, and was composed of two subunits with an identical molecular weight of 56,000. The Km values for Gly-Pro of the control's and the patient's mother's prolidase I were 2.90 +/- 0.22 and 2.88 +/- 0.27 mM, but the Vmax values for Gly-Pro of the mother's enzyme was reduced about 30% compared to that of control enzymes (mother: 6.02 units/mg protein, control: 22.21 units/mg protein). Isoionic points of these enzymes by chromatofocusing were pH 4.6 approximately 4.7. Prolidase II from the control and the patient's mother, and the patient's prolidase had a molecular weight of about 185,000, and was composed of two subunits with an identical molecular weight of 95,000. The Km and Vmax values for various substrates of prolidase II from a control and the patient's mother, and the patient's prolidase were almost the same.

Characteristics and partial purification of prolidase and prolinase from leukocytes of a normal human and a patient with prolidase deficiency. 査読国際誌

H Kodama, T Ohhashi, C Ohba, T Ohno, J Arata, I Kubonishi, I Miyoshi

Clinica chimica acta; international journal of clinical chemistry 180 ( 1 ) 65 - 72 1989年3月

CHARACTERISTICS OF PARTIALLY PURIFIED PROLIDASE FROM ERYTHROCYTES OF NORMAL INDIVIDUALS, OF 2 PATIENTS WITH PROLIDASE DEFICIENCY, AND OF THE PATIENTS MOTHER 査読

T OHHASHI, T OHNO, J ARATA, H KODAMA

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 5 ( 3 ) 183 - 192 1988年11月

Biochemical investigations on prolidase and prolinase in erythrocytes from patients with prolidase deficiency. 査読国際誌

H Kodama, H Mikasa, T Ohhashi, T Ohno, J Arata

Clinica chimica acta; international journal of clinical chemistry 173 ( 3 ) 317 - 23 1988年4月

Biochemical studies on prolidase in sera from control, patients with prolidase deficiency and their mother. 査読国際誌

T Ohhashi, T Ohno, J Arata, H Kodama

Journal of inherited metabolic disease 11 ( 2 ) 166 - 73 1988年

記述言語：英語掲載種別：研究論文（学術雑誌）

Prolidase activity in serum from normal subjects and the mother of two patients was readily detected without adding Mn2+ to the assay, and the activity was increased by addition of Mn2+ to the assay or preincubation with Mn2+. However, the activity in serum from patients with prolidase deficiency against gly-pro, leu-pro and val-pro could not be detected irrespective of Mn2+ conditions and activity against met-pro, ala-pro and phe-pro also showed a marked reduction compared to controls. Both normal and the patients' mother's prolidase activity against gly-pro was reduced about 20% at 60 degrees C compared to the activity at 37 degrees C, but the addition of Mn2+ at 55 degrees C increased the activity about 1.8-fold, whereas prolidase activity of patients could not be increased by the addition of Mn2+. The addition of Co2+ increased prolidase activity in serum from control and the patients' mother but did not increase the heat stability. These results indicate that prolidase in serum from patients with prolidase deficiency is altered rather than markedly reduced in amount.

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