Researcher Database


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ASAI Kiyofumi

FacultyGraduate School of Medical Sciences Department of Molecular Neurobiology, Executives
PositionProfessor
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Last Updated :2020/07/02

Researcher Profile and Settings

Education

  •  - 1989 , Nagoya City University
  •  - 1989 , Nagoya City University, Graduate School, Division of Medicine
  •  - 1984 , Nagoya City University
  •  - 1984 , Nagoya City University, Faculty of Medicine

Degree

  • 医学博士, 名古屋市立大学

Academic & Professional Experience

  •   1998  - 2001 , Nagoya City University
  •   1989  - 1998 , Nagoya City University

Research Activities

Research Areas

  • Life sciences, Fetal medicine/Pediatrics
  • Life sciences, Neuroscience - general

Published Papers

  • The Janus kinase inhibitor tofacitinib inhibits TNF-α-induced gliostatin expression in rheumatoid fibroblast-like synoviocytes, Kawaguchi Y, Waguri-Nagaya Y, Tatematsu N, Oguri Y, Kobayashi M, Nozaki M, Asai K, Aoyama M, Otsuka T, Clinical and experimental rheumatology, 36, (4) 559 - 567, Refereed
  • Mithramycin has inhibitory effects on gliostatin and matrix metalloproteinase expression induced by gliostatin in rheumatoid fibroblast-like synoviocytes, Tatematsu N, Waguri-Nagaya Y, Kawaguchi Y, Oguri Y, Ikuta K, Kobayashi M, Nozaki M, Asai K, Aoyama M, Otsuka T, Modern Rheumatology, 28, (3) 495 - 505, Refereed
  • Hypoxic stress upregulates Kir2.1 expression by a pathway including hypoxicinducible factor-1α and dynamin2 in brain capillary endothelial cells, Yamamura H, Suzuki Y, Yamamura H, Asai K, Giles W, Imaizumi Y, American Journal of Physiology - Cell Physiology, 315, (2) C202 - C213, Refereed
  • Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease, Inamura N, Kito M, Go S, Kishi S, Hosokawa M, Asai K, Takakura N, Takebayashi H, Matsuda J, Enokido Y, Neurobiology of Disease, 120, 51 - 62, Refereed
  • CXCR4(+) CD45(-) Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow, Yoh Goto, Mineyoshi Aoyama, Takeo Sekiya, Hiroki Kakita, Yuko Waguri-Nagaya, Ken Miyazawa, Kiyofumi Asai, Shigemi Goto, STEM CELLS, 34, (11) 2733 - 2743, 11 , Bone homeostasis comprises the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCs), with an acceleration of osteoclastic bone resorption leading to osteoporosis. OCs can be generated from bone marrow cells (BMCs) under the tightly regulated local bone environment. However, it remained difficult to identify the critical cells responsible for providing an osteoclastogenesis niche. In this study, we used a fluorescence-activated cell sorting technique to determine the cell populations important for forming an appropriate microenvironment for osteoclastogenesis and to verify the associated interactions between osteoclast precursor cells and non-OCs. We isolated and removed a small cell population specific for osteoclastogenesis (CXCR4(+)CD45(-)) from mouse BMCs and cultured the remaining cells with receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor. The resulting cultures showed significantly less large osteoclast formation. Quantitative RT-PCR analysis revealed that these CXCR4(+)CD45(-) cells expressed low levels of RANK and RANKL, but high levels of critical chemokines including stromal cell derived factor 1 (SDF-1), chemokine (C-X-C motif) ligand 7 (CXCL7), and chemokine (C-X3-C motif) ligand 1 (CX3CL1). Furthermore, an SDF-1-specific antibody strongly suppressed OC formation in RAW264.7 cells and antibodies against SDF-1, CXCL7, and CX3CL1 suppressed OC formation in BMCs. These results suggest that isolated CXCR4(+)CD45(-) cells support an appropriate microenvironment for osteoclastogenesis with a direct effect on the cells expressing SDF-1, CXCL7, and CX3CL1 receptors. The regulation of CXCR4(+)CD45(-) cell function might therefore inform therapeutic strategies for diseases involving loss of bone homeostasis.
  • Inflammatory cytokine tumor necrosis factor a suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2 alpha, Yoshiaki Nagaya, Mineyoshi Aoyama, Tetsuya Tamura, Hiroki Kakita, Shin Kato, Hideki Hida, Shinji Saitoh, Kiyofumi Asai, EUROPEAN JOURNAL OF NEUROSCIENCE, 40, (11) 3620 - 3626, 12 , Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor (TNF) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNF in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12h, TNF suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNF inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2 rather than HIF-1. The effects of TNF in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2, which is regulated by the nuclear factor (NF)-B signaling pathway. TNF treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNF signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.
  • Epidermal growth factor receptor transactivation is necessary for glucagon-like peptide-1 to protect PC12 cells from apoptosis, Ryosuke Kimura, Masahiro Okouchi, Takashi Kato, Kenro Imaeda, Naotsuka Okayama, Kiyofumi Asai, Takashi Joh, Neuroendocrinology, 97, 300 - 308, 01 01 , © 2012 S. Karger AG, Basel. Aim: Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. To identify potential treatments for diabetic encephalopathy, we focused on the protective action of glucagon-like peptide-1 (GLP-1) against neural cell apoptosis. In this study, we evaluated whether exposure of cells to GLP-1 leads to epidermal growth factor receptor (EGFR) transactivation and signaling through the PI3K/Akt/mTOR/GCLc/redox pathway, which we previously reported. Methods: We monitored the phosphorylation of EGFR and Akt in PC12 cells exposed to MG and GLP-1 that had been first incubated in the presence or absence of various inhibitors of EGFR transactivation. Results: DAPI staining revealed that pretreatment of cells with BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478 abrogated the ability of GLP-1 to rescue cells from MG-induced apoptosis. We show that exposure of PC12 cells to GLP-1 induces EGFR phosphorylation and that this effect was inhibited by prior exposure of the cells to BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478. Interestingly, these agents also diminished the capacity of GLP-1 to protect cells from MG-induced apoptosis. Moreover, these agents reduced GLP-1-induced phosphorylation of Akt. EGF itself also protected the cells from MG-induced apoptosis and induced phosphorylation of Akt, which was inhibited by LY294002. Conclusion: The neuroprotective effects of GLP-1 against MG-induced apoptosis are mediated by EGFR transactivation, which signals through the PI3K/Akt/mTOR/GCLc/redox pathway in PC12 cells.
  • Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-beta signal transduction, Motoshi Mabuchi, Hiromi Kataoka, Yutaka Miura, Tae-Sun Kim, Makoto Kawaguchi, Masahide Ebi, Mamoru Tanaka, Yoshinori Mori, Eiji Kubota, Takashi Mizushima, Takaya Shimura, Tsutomu Mizoshita, Satoshi Tanida, Takeshi Kamiya, Kiyofumi Asai, Takashi Joh, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 398, (2) 321 - 325, 07 , Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of P21(Waf1/Cip1). We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-beta signal transduction. Materials and methods: To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was. performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21(Waf1/Cip1) reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-beta signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-beta 1 using confocal laser scanning microscopy. Results: Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs = 0.433, p < 0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21(Waf1/Cip1) promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-beta 1 stimulation, but after 24 h of TGF-beta 1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-beta 1 signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator. (C) 2010 Elsevier Inc. All rights reserved.
  • Functions of aquaporin in the central nervous system and diseases, Kazuya Sobue, Takehiko Takayanagi, Hiroyuki Hirate, Takeshi Sugiura, Yoshihito Fujita, Kiyofumi Asai, Journal of Japanese Dental Society of Anesthesiology, 38, 145 - 148, 05 14
  • Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-kappa B signaling in cultured astrocytes, Hiroki Kakita, Mineyoshi Aoyama, Mohamed Hamed Hussein, Shin Kato, Satoshi Suzuki, Tetsuya Ito, Hajime Togari, Kiyofumi Asai, TOXICOLOGY AND APPLIED PHARMACOLOGY, 238, (1) 56 - 63, 07 , Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased, In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte Cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-kappa B inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-kappa B p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, L-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-kappa B signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF. (c) 2009 Elsevier Inc. All rights reserved.
  • Transforming growth factor-beta signaling at the tumor-bone interface promotes mammary tumor growth and osteoclast activation, Mitsuru Futakuchi, Kalyan C. Nannuru, Michelle L. Varney, Anguraj Sadanandam, Kimihisa Nakao, Kiyofumi Asai, Tomoyuki Shirai, Shin-ya Sato, Rakesh K. Singh, CANCER SCIENCE, 100, (1) 71 - 81, 01 , Understanding the cellular and molecular changes in the bone microenvironment is important for developing novel therapeutics to control breast cancer bone metastasis. Although the underlying mechanism(s) of bone metastasis has been the focus of intense investigation, relatively little is known about complex molecular interactions between malignant cells and bone stroma. Using a murine syngeneic model that mimics osteolytic changes associated with human breast cancer, we examined the role of tumor-bone interaction in tumor-induced osteolysis and malignant growth in the bone microenvironment. We identified transforming growth factor-beta receptor 1 (TGF-beta RI) as a commonly upregulated gene at the tumor-bone (TB) interface. Moreover, TGF-beta RI expression and activation, analyzed by nuclear localization of phospho-Smad2, was higher in tumor cells and osteoclasts at the TB interface as compared to the tumor-alone area. Furthermore, attenuation of TGF-beta activity by neutralizing antibody to TGF-beta or TGF-beta RI kinase inhibitor reduced mammary tumor-induced osteolysis, TGF-beta RI expression and its activation. In addition, we demonstrate a potential role of TGF-beta as an important modifier of receptor activator of NF-kappa B ligand (RANKL)-dependent osteoclast activation and osteolysis. Together, these studies demonstrate that inhibition of TGF-beta RI signaling at the TB interface will be a therapeutic target in the treatment of breast cancer-induced osteolysis. (Cancer Sci 2009; 100: 71-81).
  • Severity of virilization of external genitalia in Japanese patients with salt-wasting 21-hydroxylase deficiency, Yukari Sugiyama, Haruo Mizuno, Yutaro Hayashi, Hiroki Imamine, Tetsuya Ito, Ineko Kato, Manami Yamamoto-Tomita, Mineyoshi Aoyama, Kiyofumi Asai, Hajime Togari, Tohoku Journal of Experimental Medicine, 215, 341 - 348, 10 09 , Females with salt-wasting (SW) 21-hydroxylase deficiency (21OHD) may present with mild external genitalia virilization, despite complete or almost complete enzyme inactivation. We therefore analyzed genotype/ phenotype correlation in 13 Japanese female patients with SW 21OHD. Criteria for classification into the SW phenotype included history of a salt-losing crisis with documented hyponatremia, hyperkalemia, add markedly elevated plasma renin activity. Urologists and pediatricians determined the Prader genital stage and classified the location of the vaginal entrance into the common urogenital sinus as low, moderate, or high. CYP21A2 gene, coding for 21-hydroxylase, was analyzed with Southern blotting and direct sequencing. Genotypes were categorized into four mutation groups, based on the degree of enzymatic activity (N, complete enzyme inactivation; groups A, < 2%, B, 3-7%, and C > 30%). Basal androgen levels were available from only six out of thirteen patients, so we could not relate androgen levels with the severity of external genitalia virilization. We compa red the degree of external genitalia virilization with genotype. The severity of external genitalia virilization varied from Prader stage 1 to 4. One patient who presented with Prader 1 had a genotype consistent with Group B. In addition, discordance between Prader classification and the location of the vaginal entrance was noted; one patient classified as Prader 4 showed low vaginal entrance, while another patient classified as Prader 3'showed high vaginal entrance. The degree of the impairment of 21-hydroxylase activity does not correlate with the severity of virilization of the external genitalia in female patients with the SW type of 21OHD. © 2008 Tohoku University Medical Press.
  • Lactic acid increases aquaporin 4 expression on the cell membrane of cultured rat astrocytes, Tetsuro Morishima, Mineyoshi Aoyama, Yuko Iida, Naoki Yamamoto, Hiroyuki Hirate, Hajime Arima, Yoshihito Fujita, Hiroshi Sasano, Takako Tsuda, Hirotada Katsuya, Kiyofumi Asai, Kazuya Sobue, Neuroscience Research, 61, 18 - 26, 05 01 , The water channel protein aquaporin (AQP) may play roles in the homeostasis of water content in the brain and brain edema. One possible mechanism of brain edema is glial swelling due to lactic acidosis associated with ischemia. Here, we investigated the effect of lactic acid on the expression and cellular distribution of AQP 4 in cultured rat astrocytes. After 24 h of incubation, the AQP4 expression level increased maximally with 35 mM lactic acid. The AQP4 expression levels also increased with hydrochloric acid or acetic acid. In contrast, with sodium lactate, the AQP4 levels did not increase. The increase in AQP4 expression level occurred without a significant increase in AQP4 mRNA expression level by lactic acid. Under the conditions of de novo protein synthesis inhibition with cycloheximide, lactic acid increased the AQP4 expression level. Furthermore, lactic acid increased the AQP4 expression level on the cell surface of the astrocytes, as determined by a cell surface biotinylation assay and immunocytochemical examination. The increase in AQP4 expression level on the cell membrane of astrocytes induced by lactic acid may be a new regulation mechanism of AQP4 in the brain. © 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Transforming growth factor beta derived from bone matrix promotes cell proliferation of prostate cancer and osteoclast activation-associated osteolysis in the bone microenvironment, Shinya Sato, Mitsuru Futakuchi, Kumiko Ogawa, Makoto Asamoto, Kimihisa Nakao, Kiyofumi Asai, Tomoyuki Shirai, CANCER SCIENCE, 99, (2) 316 - 323, 02 , Metastatic prostate tumors in the bone microenvironment stimulate bone resorption, resulting in release of growth factors from the bone matrix that play important roles in tumor growth and osteoclast induction. Transforming growth factor beta (TGF beta) is one of the most abundantly stored cytokines in bone matrix, regulating diverse biological activities. Here we evaluate its involvement in prostate tumor growth in the bone microenvironment, comparing with tumor growth in the subcutaneous microenvironment as a control. Rat prostate tumors were transplanted onto the cranial bone and into the subcutis of F344 male rats. Tumor cell proliferation, apoptosis, and TGF beta signal transduction were compared between the tumor-bone interface and the tumor-subcutaneous interface. Effects of TGF beta on osteoclast differentiation were also evaluated in vitro. Inhibitory effects of TGF beta receptor 1 antisense oligonucleotide on TGF beta signaling, osteolysis, osteoblasts, and tumor growth were examined in vivo. Osteolytic changes were extensively observed at the tumor-bone interface, where the TGF beta level, TGF beta signal transduction, and tumor cell proliferation were higher than at the tumor-subcutaneous interface. In vitro treatment with receptor activator of nuclear factor-kappa B ligand induced osteoclast differentiation of bone marrow stromal cells, and additional exposure to TGF beta exerted promotive effects on osteoclast induction. Intratumoral injection of TGF beta receptor 1 antisense oligonucleotide significantly reduced TGF beta signal transduction, osteolysis, induction of osteoclast and osteoblast, and tumor cell proliferation. Thus, we experimentally show that TGF beta derived from bone matrix promotes cell proliferation of rat prostate cancer and osteoclast activation-associated osteolysis in the bone microenvironment.
  • IL-10 and RANTES are elevated in nasopharyngeal secretions of children with respiratory syncytial virus infection, Hiroki Murai, Hiroki Murai, Akihiko Terada, Mihoko Mizuno, Masami Asai, Yasutaka Hirabayashi, Seiki Shimizu, Takehiro Morishita, Hiroki Kakita, Mohamed Hamed Hussein, Tetsuya Ito, Ineko Kato, Kiyofumi Asai, Hajime Togari, Allergology International, 56, 157 - 163, 01 01 , Background: Respiratory syncytial virus (RSV) infection causes asthma-like symptoms in infants and young children. Although an increase in several mediators in the airway during RSV infection has been reported, the mechanisms involved in airway inflammation are not fully understood. The aim of this study was to investigate the immunological deviation associated with airway inflammation by measuring cytokine and chemokine levels in the airway during RSV infection. Methods: One hundred and ten children under 3 years of age with respiratory symptoms were enrolled in this study from November 2004 through January 2005. Nasopharyngeal secretions (NPAs) were gently aspirated and analyzed with RSV antigen, thereafter the concentrations of IL-4, IL-10, IFN-γ, and RANTES were measured using an ELISA kit. We also investigated the prognosis of each child after 1 year by reference to clinical records or by interviews and re-evaluated the cytokine and chemokine levels. Results: Of the subjects, 70 children were RSV positive and 40 were negative. Only 4 children were given a diagnosis of asthma by the pediatrician when NPAs were collected. The levels of IL-4, IL-10, and RANTES were significantly higher in the RSV-positive patients than RSV-negative patients with P values at 0.0362, 0.0007, and 0.0047, respectively. In contrast, there was no significant difference in the levels of IFN-γ. Furthermore, there was a significant positive correlation between IL-10 and RANTES. Conclusions: The increased production of IL-4, IL-10, and RANTES in the airway may play an important role in the pathophysiological mechanisms of RSV infection. ©2007 Japanese Society of Allergology.
  • Interleukin-1 beta induces the expression of aquaporin-4 through a nuclear factor-kappa B pathway in rat astrocytes, Hiroaki Ito, Naoki Yamamoto, Hajime Arima, Hiroyuki Hirate, Tetsuro Morishima, Fuminori Umenishi, Toyohiro Tada, Kiyofumi Asai, Hirotada Katsuya, Kazuya Sobue, JOURNAL OF NEUROCHEMISTRY, 99, (1) 107 - 118, 10 , Interleukin (IL)-1 beta is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL-1 beta. We found that expression of AQP4 mRNA and protein was significantly up-regulated by IL-1 beta in cultured rat astrocytes, and that intracerebroventricular administration of IL-1 beta increased the expression of AQP4 protein in rat brain. The effects of IL-1 beta on induction of AQP4 were concentration and time dependent. The effects of IL-1 beta on AQP4 were mediated through IL-1 beta receptors because they were abolished by co-incubation with IL-1 receptor antagonist. It appeared that IL-1 beta increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL-1 beta. Inhibition of the nuclear factor-kappa B (NF-kappa B) pathway blocked the induction of AQP4 by IL-1 beta in a concentration-dependent manner. These findings show that IL-1 beta induces expression of AQP4 through a NF-kappa B pathway without involvement of de novo protein synthesis in rat astrocytes.
  • Aquaporin water channels in the brain and molecular mechanisms of brain edema, Kazuya Sobue, Kiyofumi Asai, Hirotada Katsuya, Nippon rinsho. Japanese journal of clinical medicine., 64, 1181 - 1189, 06 01 , Aquaporins(AQPs) are a family of water selective channel. Transcripts of AQP1, AQP3, AQP4, AQP5, AQP8, and AQP9 are detected in the brain. Especially in astrocytes, AQP4 is abundantly expressed in end feet at the blood-brain barrier. Brain AQPs play important roles in the regulation of water homeostasis and the cerebro spinal fluid formation. Recently, AQP4 and AQP9 have been reported to involve in the brain water accumulation in the brain edema. Studies of transgenic mouse and brain injury models reveal that AQP4 may play a role not in the edema formation, but in the fluid elimination. Further study of AQPs functions in the brain may provide new insights into the brain edema and allow the design of novel anti edema medications.
  • Erratum: Molecular characterization of histidinemia: Identification of four missense mutations in the histidase gene (Human Genetics (2005) vol. 116 (340-346) 10.1007/s00439-004-1232-5), Yoko Kawai, Akihiko Moriyama, Kiyofumi Asai, Carrie M. Coleman-Campbell, Satoshi Sumi, Hideko Morishita, Mariko Suchi, Mariko Suchi, Mariko Suchi, Human Genetics, 118, 531 - 532, 12 01
  • Glia maturation factor-beta is produced by thymoma and may promote intratumoral T-cell differentiation, H Yamazaki, H Tateyama, K Asai, Fukai, I, Y Fujii, T Tada, T Eimoto, HISTOPATHOLOGY, 47, (3) 292 - 302, 09 , Aims: To investigate whether Glia maturation factor-beta (GMFB) is expressed in thymomas and is associated with T-cell development. Methods and results: We investigated the expression of GMFB by immunohistochemistry in 86 cases of thymoma classified into five type A, 35 type AB, 11 type B1, 26 type B2, and nine type B3 thymomas according to the World Health Organization classification system. Immunoblotting and in situ hybridization (ISH) studies were also performed in selected cases. The results of the immunoblot analysis were in accordance with those of immunohistochemical scoring. The ISH study ascertained the tumour cells producing the protein. Immunohistochemically, GMFB expression was observed in one (20%) of type A, 32 (80%) of type AB, all (100%) of type B1 and B2, and eight (89%) of type B3 thymoma with statistically significant differences between type A and type AB, type B1, or type B2 thymoma, and between type B3 and type AB or type B2 thymoma. There was a significant correlation between GMFB expression and the amount of accompanying non-neoplastic T cells. GMFB promoted T-cell differentiation into CD4-/CD8+ cells when analysed by two-colour flow cytometry. Conclusions: The present study suggests that T-cell development in thymoma may be maintained partly by GMFB produced by the tumour cells.
  • Production and characterization of astrocyte-derived human apolipoprotein E isoforms from interactions with amyloid-beta, M Morikawa, JD Fryer, PM Sullivan, EA Christopher, SE Wahrle, RB DeMattos, MA O'Dell, AM Fagan, HA Lashuel, T Walz, K Asai, DM Holtzman, NEUROBIOLOGY OF DISEASE, 19, (1-2) 66 - 76, 06 , The apolipoprotein E (apoE) genotype is an important genetic risk factor for Alzheimer's disease (AD). in the central nervous system (CNS), most apoE is produced by astrocytes and is present in unique high-density lipoprotein (HDL)-like particles that have distinct properties from apoE derived from other sources. To develop an efficient system to produce astrocyte-derived apoE in large quantities, we produced and characterized immortalized cell lines from primary astrocyte cultures derived from human APOE knock-in mice. APOE2, APOE3, and APOE4 expressing cell lines were established that secrete apoE in RDL-like particles at similar levels, cholesterol composition, and size as those produced by primary astrocytes. In physiological buffers, astrocyte-secreted apoE3 and E4 associated equally well with amyloid-beta. Under the same conditions, only a small fraction of A formed sodium dodecyl sulfate (SDS)-stable complexes with apoE (E3 > E4). These immortalized astrocytes will be useful for studying mechanisms underlying the isoform-speciric effects of apoE in the CNS. (c) 2004 Elsevier Inc. All rights reserved.
  • Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells, Takahiro Yasui, Keiji Fujita, Kiyofumi Asai, Kenjiro Kohri, International Journal of Urology, 9, 100 - 108, 04 22 , Background: The association of calcium crystals with renal tubular cells is an important factor during the formation of urinary stones. We previously reported the strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays a role in the crystal-cell interaction. In the present study, we examined the biological consequences of inhibiting OPN expression at the translational level on the formation and adhesion of crystals. Methods: We synthesized antisense OPN expression vector (pTet-OPNas) using the tetracycline-regulated expression system. The pTet-OPNas was constructed using a mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones (NRK-52E/ASs) were identified by transfection of pTet-OPNas into NRK-52E cells and they showed a marked reduction of OPN synthesis in the absence of tetracycline. Calcium oxalate (CaOx) crystal suspension was spread homogeneously on top of the NRK-52E cells. After incubation, the association of CaOx crystals and cells was visualized by scanning electron microscopy. Results: Intact NRK-52E cells, NRK-52E cells transfected with empty vector and tetracycline-treated antisense clones (NRK-52E/ASs), under identical conditions, were associated with CaOx crystals. In contrast, the expression of antisense OPN prevented the association of CaOx crystals with NRK-52E cells. Conclusions: Osteopontin plays a crucial role in the adhesion process of CaOx crystals to renal tubular cells in stone formation.
  • Clinical and biochemical findings of a patient with thanatophoric dysplasia type I: Additional finding of dicarboxylic aciduria, Kazuki Okajima, Kiyofumi Asai, Toshimitsu Niwa, Shigeru Ohki, Hisanori Sobajima, Jess Tyson, Sue Malcolm, Yoshiro Wada, Cleft Palate-Craniofacial Journal, 39, 246 - 248, 03 26 , Objective: A long-surviving thanatophoric dysplasia type I patient to age of 6 years is presented. Results and Conclusions: Molecular studies revealed a heterozygous point mutation, S249C in the fibroblast growth factor receptor 3 gene. Most of the clinical course was similar to previous reports, including hearing loss and acanthosis nigricans. Abnormal urinary excretion of dicarboxylic acids and 3-hydroxydicarboxylic acids was observed. We hypothesize that this was a consequence of the FGFR3 mutation.
  • Editorial comment, Hirotaka Asakura, Takahiro Yasui, Keiji Fujita, Kiyofumi Asai, Kenjiro Kohri, Int J Urol, 9, 108 - 109, 01 01
  • Hyperthermic induction of apoptosis in malignant fibrous histiocytoma cells: Possible involvement of a p53-independent pathway in the induction of bax gene, Masato Yonezawa, Masato Yonezawa, Takanobu Otsuka, Taiji Kato, Akihiko Moriyama, Kohichi H. Kato, Kiyofumi Asai, Nobuo Matsui, Journal of Orthopaedic Science, 7, 117 - 122, 01 01 , We have previously reported the unique heat sensitivity of a cell line of malignant fibrous histiocytoma cells, the MFH-2NR cell line. In the present study, treatment of MFH-2NR cells, at 43°C for 1 h evoked typical apoptosis in these cells, which showed characteristic morphological changes, such as internucleosomal DNA fragmentation (DNA ladders), cell shrinkage, and chromatin condensation. Under these conditions, we examined p53 and bax protein levels, and p53 and bax mRNA expression to assess the potential relationship between these two proteins for the induction of apoptosis. The p53 protein, which is usually detected in trace amounts in normal cells, was highly expressed in untreated MFH-2NR cells, and the level did not increase after heat treatment, whereas the bax protein level increased from 30min after the treatment. No change in p53 mRNA was found, but a transient increase in bax mRNA, peaking at 30min, was detected by Northern blotting. DNA sequence analysis of the p53 gene from MFH-2NR cells demonstrated a GGG → GAG homozygous point mutation in codon 242 of exon 6. These results suggest that the expression of bax protein and mRNA was augmented by a p53-independent pathway in the hyperthermia-induced apoptosis of MFH-2NR cells.
  • Effects of mechanical vibration on DNA and proteoglycan syntheses in cultured articular chondrocytes, J. Liu, I. Sekiya, K. Asai, T. Tada, T. Kato, N. Matsui, Modern Rheumatology, 11, 40 - 46, 10 16 , The objective of this study was to determine the effects of mechanical vibration loading on DNA and proteoglycan syntheses in cultured rabbit articular chondrocytes. Chondrocyte culture plates were placed in a vibratory apparatus and subjected to a mechanical vibratory load at various frequencies and periods during culture. Mechanical vibration was applied at a sinusoidal waveform of 1.4 G-acceleration with frequencies of 200, 300, 400, 800, and 1600Hz. 3 H-thymidine and H 2 35 SO 4 incorporation were used to detect radiolabeled DNA and proteoglycan syntheses, respectively. A frequency of 300Hz showed a time-dependent augmentation of DNA synthesis and gave a maximal increase on day 3 with periodic vibration (8h per day), and at 72h or longer with continuous vibration. It also promoted proteoglycan synthesis in long-term culture (from 3 to 15 days) by periodic vibration. However, frequencies above 400Hz suppressed biosynthesis. These results suggest that mechanical vibration at certain frequencies may modulate the biosynthetic response of articular chondrocytes.
  • Review of the research of glia maturation factor and cloning of human and rat glia maturation factor-γ (GMFG) cDNA, K. Asai, Japanese Journal of Psychopharmacology, 21, 15 - 20, 06 21 , Glia maturation factor-β (GMFB) is a 17-kDa protein that was initially identified as a growth and differentiation factor acting on neurons as well as glia in the vertebrate brain. We isolated human and rat glia maturation factor-γ (GMFG) cDNA and examined the tissue distribution of GMFG in human and rat by Northern blots and Western blots. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of its putative product is highly homologous to GMFB. Northern blot analysis indicated that a 0.9 kb mRNA is predominantly expressed in rat thymus, testis, and spleen. In comparison with GMFB, the current study demonstrated that the tissue distribution of GMFG is not the same as that of GMFB, and GMFG is predominantly in proliferative and differentiative organs.
  • A metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells, H Katano, K Fujita, T Kato, K Asai, Y Kawamura, A Masago, K Yamada, NEUROSCIENCE LETTERS, 306, (1-2) 101 - 105, 06 , The effects of three glutamate receptor antagonists, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) for the N-methyl-D-aspartate receptor, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f] quinoxaline-7-sulfonamide (NBOX) for the alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionate /kinate receptor and (S)-alpha -methyl-4-carboxyphenylglycine (MCPG) for the metabotropic receptor, on c-fos and c-jun mRNA expression were investigated in cultured cortical glial cells following traumatic scratch injury. Expression of the two genes along the edges of wounds detected by in situ hybridization was not affected by MK-801 and NBQX. However, 100 and 500 muM of MCPG remarkably reduced the hybridization signals for both c-fos and c-jun mRNAs. The present results suggest that group I metabotropic glutamate receptors might have some association with immediate early gene induction after in vitro traumatic injury in glial cells. (C) 2001 Published by Elsevier Science Ireland Ltd.
  • Interleukin-1 beta induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes, T Miyachi, K Asai, H Tsuiki, H Mizuno, N Yamamoto, T Yokoi, M Aoyama, H Togari, Y Wada, Y Miura, T Kato, NEUROSCIENCE RESEARCH, 40, (1) 53 - 60, 05 , Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-I beta acts as a mediator of inflammation and has been reported as ii potent inducer of various neurotrophic factors including nerve growth factor and fibroblast growth factor. In this study. we investigated the relationship between LC1 and IL-1 beta in cultured rat astrocytes. Time- and dose-dependent experiments of IL-IP on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1 beta at 8 h, 10 ng/ml. In addition, IL-1 beta evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1 beta was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 muM), phospholipase A(2) inhibitor mepacrine (30 muM) and protein synthesis inhibitor cycloheximide (CHX) (10 mug/ml). This suggests that induction of LC1 by IL-1 beta is through a MAPKs and phospholipaseA(2) pathway and requires protein synthesis. These results indicate that IL-1 beta released in the central nervous system (CNS) injury can stimulate the transcription of the LC1 gene. Subsequent synthesis and release of LC1 may provide trophic support to neurons and modulate the action of IL-1 beta in brain damage. (C) 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Biosynthetic response of cultured articular chondrocytes to mechanical vibration, J. Liu, I. Sekiya, K. Asai, T. Tada, T. Kato, N. Matsui, Research in Experimental Medicine, 200, 183 - 193, 04 10 , The objective of this study was to determine the effects of mechanical vibration loading on DNA and proteoglycan syntheses in cultured rabbit articular chondrocytes. Chondrocyte culture plates were placed in a vibratory apparatus and subjected to a mechanical vibratory load at various frequencies and periods in culture. Mechanical vibration was applied at a sinusoidal waveform of 1.4 g acceleration with frequencies of 200, 300, 400, 800, and 1600 Hz. 3 H-Thymidine and H 2 35 SO 4 incorporation were used to detect radiolabeled DNA and proteoglycan syntheses, respectively. A frequency of 300 Hz showed a time-dependent augmentation of DNA synthesis and gave a maximal increase at day 3 with periodic vibration (8 h per day) and at 72 h or longer with continuous vibration. It also promoted proteoglycan synthesis in long-term culture (from 3 to 15 days) by periodic vibration. However, frequencies above 400 Hz suppressed biosynthesis. These results suggest that mechanical vibration at certain frequencies may modulate biosynthetic response of articular chondrocytes.
  • Cyclic ADP-ribose as a potential second messenger for neuronal CA2+ signaling, Haruhiro Higashida, Haruhiro Higashida, Minako Hashii, Shigeru Yokoyama, Naoto Hoshi, Kiyofumi Asai, Taiji Kato, Journal of Neurochemistry, 76, 321 - 331, 02 05 , Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca 2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ), and/or secondarily potentiates [Ca 2+ ] i increases that are the result of depolarization-induced Ca 2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR, cADPR modifies K + currents or elicits Ca 2+ -dependent inward currents, cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells, cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and β-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca 2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.
  • p27(Kip1) expression by contact inhibition as a prognostic index of human glioma, Takahisa Fuse, Motoki Tanikawa, Motoki Tanikawa, Makoto Nakanishi, Makoto Nakanishi, Makoto Nakanishi, Kyoji Ikeda, Toyohiro Tada, Hiroshi Inagaki, Kiyofumi Asai, Taiji Kato, Kazuo Yamada, Journal of Neurochemistry, 74, 1393 - 1399, 01 01 , The clinical manifestations of human glioma are known to be diverse, ranging from aggressive growth and invasion to apparent dormancy; however, the molecular mechanism underlying this diversity has been largely unexplored. In the present study, we characterized four human glioma cell lines, T98G, A172, U251, and NAC6, each of which has distinct growth properties. A172 and U251 cells continue to grow after confluency, whereas the growth of T98G and NAC6 cells is contact inhibited. Northern and western blot analyses revealed that at high cell de nsity, the expression of p27(Kip1) cyclin-dependent kinase inhibitor was dramatically enhanced at both the RNA and the protein levels in T98G and NAC6 cells but not in A172 or U251. These facts together with the finding that overexpression of p27(Kip1) caused G1 arrest in A172 and T98G cells suggest that the induction of p27(Kip1) represents an important determinant of growth at high cell density. Immunohistochemical analyses of 42 primary gliomas revealed an inverse correlation between the level of p27 protein and the Ki-67 proliferative index. Kaplan-Meier plots demonstrated that a low level of p27 in tumors is associated with decreased overall survival. Thus, disrupted regulation of p27 expression at high cell density may play an important role in determining the clinical behavior of human gliomas as well as the prognosis for glioma patients.
  • Cloning of a rat glia maturation factor-γ (rGMFG) cDNA and expression of its mRNA and protein in rat organs, Hideki Tsuiki, Hideki Tsuiki, Kiyofumi Asai, Manami Yamamoto, Kaori Fujita, Yuichiro Inoue, Yoko Kawai, Toyohiro Tada, Akihiko Moriyama, Yoshiro Wada, Taiji Kato, Journal of Biochemistry, 127, 517 - 523, 01 01 , We isolated a rat glia maturation factor-γ (rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-β (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs.
  • Astrocytic gap junction blockage and neuronal Ca2+ oscillation in neuron-astrocyte cocultures in vitro, K. Fujita, K. Nakanishi, K. Sobue, T. Ueki, K. Asai, T. Kato, Neurochemistry International, 33, 41 - 49, 06 01 , We have investigated the effects of gap junction inhibitors, octanol, halothane, sodium propionate and lindane, on neuronal periodic Ca 2+ transients in neuron-astrocyte coculture systems. Octanol reduced the amplitude and frequency of Ca 2+ oscillations in dose-dependent manner. One mM octanol caused a complete disappearance of Ca 2+ oscillations. Similar suppressions were obtained by halothane (1 mM) and sodium propionate (25 mM). In contrast, lindane (300 nM) uniquely raised the basal level of [Ca 2+ ](i) in oscillating neurons as well as the height of apparent amplitude without changes in the frequency. The current results imply that octanol, halothane and sodium propionate might lower the frequency of spontaneous Ca 2+ oscillations by blocking the gap junctional communication of neighboring astrocytes and that lindane, though also blocking the gap junctions, might not affect the frequency but reversely increase both the basal [Ca 2+ ](i) and the amplitude, probably due to an increase of neuronal [Ins (1.4.5)P 3 ](i). These findings strongly suggest that astrocytes contribute to the generation of periodic neuronal Ca 2+ oscillations through astrocytic gap junctional communications and/or other signaling components between astrocytes and neurons.
  • Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene, K Asai, K Fujita, M Yamamoto, T Hotta, M Morikawa, M Kokubo, A Moriyama, T Kato, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1396, (3) 242 - 244, 03 , A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta. (C) 1998 Elsevier Science B.V.
  • Molecular biology of blood-brain barrier, K. Sobue, H. Katsuya, K. Asai, T. Kato, Neurological Surgery, 26, 561 - 569, 01 01
  • Isolation of a rat histidase cDNA sequence and expression in Escherichia coli. Evidence of extrahepatic/epidermal distribution, Hirofumi Sano, Toyohiro Tada, Akihiko Moriyama, Hisamitsu Ogawa, Kiyofumi Asai, Yoko Kawai, Mark Emory Hodgson, Taiji Kato, Yoshiro Wada, Mariko Suchi, Mariko Suchi, European Journal of Biochemistry, 250, 212 - 221, 12 08 , Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid. Full-length cDNAs encoding rat histidase have been isolated from a λZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.
  • Specific detection of kappa light chain in uric acid stones, Y Hashimoto, K Kohri, Y Hayashi, A Moriyama, M Iguchi, K Asai, T Kato, LIFE SCIENCES, 61, (3) 249 - 253, 06 , Proteins were extracted from uric acid stones with 6M guanidine chloride (pH 8.5), which were successively developed by 12% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Amino acid sequence analysis of each band on SDS-PAGE revealed that major components in uric acid stones were immunoglobulin alpha heavy and kappa light chains. Although immunoglobulin heavy chain (gamma and mu, as well as alpha) and a kappa light chain were clearly detected in uric acid stones by Western blotting using their specific antibodies, no lambda chains whatsoever could be detected. The results suggest that immunoglobulins selectively containing kappa light chain might have specific functions in uric acid stone formation as stone matrices.
  • The blood-brain barrier and astrocytes, Ichiro Isobe, Kiyofumi Asai, Taiji Kato, Kazuya Sobue, Takafumi Koyano, Drug Delivery System, 11, 375 - 383, 01 01 , The basis for the blood-brain barrier in mammals is the selective transport properties of brain capillary endothelium, including the elaborate system of tight intercellular occluding junctions that occur between apposed membrane faces of these cells. This unique specialization of brain capillary endothelial cells appears late in development and has been postulated to be under the inductive influence of astrocytes in the central nervous system. To examine this astrocytic contribution to endothelial cell monolayer permeability, we employed the cocultures of bovine endothelial cells (aortic or brain capillary endothelial cells, BBEC or BAEC) with astrocytes in a double chamber system. In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes. The obtained results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro. © 1996, THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM. All rights reserved.
  • Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for in vitro BBB model, Ichiro Isobe, Takao Watanabe, Toshihisa Yotsuyanagi, Norio Hazemoto, Kazuo Yamagata, Takatoshi Ueki, Keiko Nakanishi, Kiyofumi Asai, Taiji Kato, Neurochemistry International, 28, 523 - 533, 01 01 , Astrocytic contribution of endothelial cell monolayer permeability was examined in two bloodbrain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the L-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.
  • Aberrant production of gliostatin/platelet‐derived endothelial cell growth factor in rheumatoid synovium, Masanori Takeuchi, Takanobu Otsuka, Nobuo Matsui, Kiyofumi Asai, Takayoshi Hirano, Akihiko Moriyama, Ichiro Isobe, Yaman, Z. Eksioglu, Kohei Matsukawa, Taiji Kato, Toyohiro Tada, Arthritis & Rheumatism, 37, 662 - 672, 01 01 , Objective. To purify a protein inhibitor from rheumatoid arthritis (RA) synovial fluids which suppresses the apparent incorporation of 3 H‐thymidine into fibroblasts and synovial cells, and to define its biochemical features that have clinical relevance to the pathogenesis of RA. Methods. Several standard chromatographic techniques were employed for the purification of the protein. Immunochemical methods with monoclonal antibody were used to quantify and visualize the protein in sera, synovial fluids, and tissues from RA patients. Results. The chemical properties of purified inhibitor from RA synovial fluids confirmed its identity as gliostatin/platelet‐derived endothelial cell growth factor (PD‐ECGF), a potent angiogenic factor. The gliostatin/ PD‐ECGF level in synovial fluid and serum was higher in RA patients than in osteoarthritis controls. Conclusion. These findings strongly suggest that gliostatin/PD‐ECGF might play an important role in the aberrant neovascularization of rheumatoid synovium. Copyright © 1994 American College of Rheumatology
  • GROWTH-PROMOTING ACTION OF ADENOSINE-CONTAINING DINUCLEOTIDE ON NEUROBLASTOMA-CELLS - DETECTION OF ADENOSINE-CYTIDINE DINUCLEOTIDE (APCP) IN NEUROFIBROMA (NF1) EXTRACTS, T HOTTA, K ASAI, N TAKEDA, H YOSHIZUMI, A TATEMATSU, K NAKANISHI, YZ EKSIOGLU, ISOBE, I, T KATO, JOURNAL OF NEUROCHEMISTRY, 61, (4) 1430 - 1437, 10 , Neurofibroma type 1 tissue was investigated for the presence of growth-promoting activity on human neuroblastoma cells. The activity was isolated by gel filtration and reversed-phase column chromatographs from neurofibroma type 1 extracts. An adenosine-containing dinucleotide (adenylyl(3'-5')cytidine-3'-phosphate) was identified as one of the major components of the activities by its enzymatic fragmentation and liquid chromatography/mass spectrometry. Synthetic adenosine-containing dinucleotide derivatives such as cytidyl(3'-5')adenosine, cytidyl(2'-5')adenosine, adenylyl(3'-5')cytidine, and adenylyl(2'-5')cytidine showed a similar action. Cytidyl(3-5')adenosine, cytidyl(2'-5')adenosine, and adenylyl(2'-5')cytidine, which are able to release a free adenosine through enzymatic hydrolysis, in particular elicited a strong activity corresponding to that of adenosine with the highest action. These results suggest that neuroblastoma cells are able to use adenosine-containing dinucleotides as well as mononucleotides for their survival and proliferation.
  • Establishment of an enzyme immunoassay system for gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF), Takayoshi Hirano, Kiyofumi Asai, Kohei Matsukawa, Taiji Kato, Masanori Takeuchi, Masato Yonezawa, Takanobu Otsuka, Nobuo Matsui, BBA - Molecular Cell Research, 1176, 299 - 304, 04 16 , A two-site enzyme immunoassay for gliostatin (GLS)/platelet-derived endothelial cell growth factor (PD-ECGF) has been developed. The detection limit of gliostatin/PD-ECGF was 30 pg/well, and the optimal assay range was 0.1 to 10 ng/well. This assay system enabled us to confirm the immunochemical identity of both factors and to detect immunoreactive gliostatin/PD-ECGF (IR-GLS/PD-ECGF) in human biological body fluids. The age-related analysis from newborn to 69 years revealed that the serum IR-GLS/PD-ECGF level was high in infants younger than 1 year old (1.8 ng/ml) and in the 20-year-old age group (1.8 ng/ml), and highest in the umbilical cord blood (2.1 ng/ml). Curiously high concentrations were detected in saliva with a significant sex difference (11.3 ng/ml for males and 48.7 ng/ml for females), and in synovial fluids (3.7 ng/ml). A number of human tumor cells, gastric cancer cells, MKN-74, neuroblastoma cells, GOTO, as well as epidermoid carcinoma cells, A431, were found to produce a significant amount of IR-GLS/PD-ECGF (0.2 to 21.8 ng/mg protein), and some of them secreted the IR-GLS/PD-ECGF in the conditioned medium (∼0.5 ng/ml). The enzyme immunoassay system is sufficiently sensitive for the basic and clinical study of gliostatin/PD-ECGF in human body fluids, tissues and organs. © 1993.
  • Immuno-reactive human epidermal growth factor (h-EGF) in rheumatoid synovial fluids, J. Kusada, T. Otsuka, N. Matsui, T. Hirano, K. Asai, T. Kato, Journal of the Japanese Orthopaedic Association, 67, 859 - 865, 01 01
  • Identification of immuno-reactive lipocortin 1-like molecules in serum and plasma by an enzyme immunoassay for lipocortin 1, Kenji Uemura, Kenji Uemura, Hiroshi Inagaki, Yoshiro Wada, Keiko Nakanishi, Kiyofumi Asai, Taiji Kato, Yoshihiro Ando, Reiji Kannagi, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, 1119, 250 - 255, 03 12 , A two-site enzyme immunoassay for lipocortin 1 (LC1) has been developed. The detection limit of LC1 was 0.2 ng/tube and the optimal assay range was 1 to 100 ng/tube. The assay system enabled us to identify immunoreactive lipocortin 1-like molecules (IR-LC1) in human serum and plasma. Normal human serum and plasma IR-LC1 concentrations were 44.6 ng/ml for males and 43.1 ng/ml for females with no significant difference between both sexes. The age-related analysis among nine age groups from newborn to 69 years old revealed that the serum or plasma level was high in infants (77.5 ng/ml for newborn and 75.6 ng/ml for 1 month-1 year group), and the 40-year-old (52.2 ng/ml) and 50-year-old (51.3 ng/ml) groups. The major population of plasma IR-LC1 was of 70 kDa in size corresponding to that of the LC1 homodimer. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of LC1 in human body fluids, tissues and organs. © 1992.
  • A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma, Kiyofumi Asai, Takayoshi Hirano, Shuji Kaneko, Akihiko Moriyama, Keiko Nakanishi, Ichiro Isobe, Yaman, Z. Eksioglu, Taiji Kato, Journal of Neurochemistry, 59, 307 - 317, 01 01 , Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion‐exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross‐reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemi‐cally visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M r of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M r of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50‐kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor‐stimulated astroblasts, but not neuronal cells. Copyright © 1992, Wiley Blackwell. All rights reserved
  • Neurotrophic action of gliostatin on cortical neurons. Identity of gliostatin and platelet-derived endothelial cell growth factor, K. Asai, K. Nakanishi, I. Isobe, Y. Z. Eksioglu, A. Hirano, K. Hama, T. Miyamoto, T. Kato, Journal of Biological Chemistry, 267, 20311 - 20316, 01 01 , Gliostatin is a polypeptide growth inhibitor of apparent M(r) = 100,000 with a homodimeric structure comprising two 50-kDa subunits, acting on astrocyte as well as astrocytoma cells (Asai, K., Hirano, T., Kaneko, S., Moriyama, A., Nakanishi, K., Isobe, I., Eksioglu, Y. Z., and Kato, T. (1992) J. Neurochem., 59, 307-317). The amino acid sequences of 13 tryptic peptides including the amino terminus were completely identical to those of platelet- derived endothelial cell growth factor (PD-ECGF) (Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C.-H. (1989) Nature 338, 557-562). Gliostatin and PD- ECGF, purified from human placenta, shared growth inhibition on glial cells and growth promotion on endothelial cells, and exhibited similar values for half-maximal dose of glial growth inhibition (ID 50 = 1.3 nM) and the half- maximal concentration of endothelial growth promotion (EC 50 = 1.0 nM), suggesting that both factors evoke the biological actions through an identical receptor on each cell surface. We have further demonstrated evidence of a novel neurotrophic action of gliostatin/PD-ECGF toward embryonic rat cortical neurons in culture. The half-maximal concentration of gliostatin/PD-ECGF for neurotrophic action was 0.3 nM. All actions on glial, endothelial, and neuronal cells, were abolished by a monoclonal antibody against gliostatin. These data indicate that gliostatin/PD-ECGF may play important roles on development and regeneration of the central nervous system and may also involve the induction of angiogenesis for the formation of blood brain barrier.
  • Antitumor effect of thermosensitive CDDP-liposomes on human osteosarcoma cells in culture, M. Hattori, N. Matsui, H. Ohta, T. Otsuka, K. Yamada, K. Asai, T. Kato, Journal of the Japanese Orthopaedic Association, 66, 476 - 484, 01 01
  • Plasma lactoferrin levels after bone marrow transplantation monitored by a two-site enzyme immunoassay, Tatsuhito Suzuki, Mihoko Takizawa-Mizuno, Makoto Yazaki, Yoshiro Wada, Kiyofumi Asai, Taiji Kato, Clinica Chimica Acta, 202, 111 - 117, 10 14
  • Neural trophic factors and neuron-glia interaction, K. Asai, T. Kato, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 36, 1220 - 1226, 05 01
  • Molecular cloning of human UMP synthase., M. Suchi, N. Harada, T. Tsuboi, K. Asai, K. Okajima, Y. Wada, Y. Takagi, Advances in experimental medicine and biology, 253 A, 511 - 518, 12 01


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