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 :2019/10/12

Researcher Profile and Settings

Education

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

Academic & Professional Experience

  •   1989  - 1998 , Nagoya
  •   1998  - 2001 , Nagoya
  •   2001 , - Nagoya

Research Activities

Research Areas

  • Neuroscience, Neurochemistry/Neuropharmacology
  • Clinical internal medicine, Pediatrics

Published Papers

  • The consciousness survey and the effect of educational intervention of advance care planning (ACP) including post-mortem to elderly residents living in old new town of big city, Akatsu H, Manabe T, Takeo J, Kawade Y, Kimura Y, Kondo M, Ito S, Nagano K, Nozaki Y, Dhoi M, Masaki Y, Tanaka H, Kanematsu T, Kojima M, Akashi K, Iwata A, Suzuki T, Kimura K, Asai K, Ohara H., Japanese Journal of Geriatrics, 55, (3) 358 - 366, Refereed
  • 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
  • Do Mesenchymal Stem Cells Derived From Atypical Lipomatous Tumors Have Greater Differentiation Potency Than Cells From Normal Adipose Tissues?, Hiroyuki Inatani, Hiroyuki Inatani, Norio Yamamoto, Katsuhiro Hayashi, Katsuhiro Hayashi, Hiroaki Kimura, Hiroaki Kimura, Akihiko Takeuchi, Shinji Miwa, Takashi Higuchi, Kensaku Abe, Yuta Taniguchi, Satoshi Yamada, Kiyofumi Asai, Takanobu Otsuka, Hiroyuki Tsuchiya, Clinical Orthopaedics and Related Research, 475, 1693 - 1701, 06 01 , © 2017, The Association of Bone and Joint Surgeons®. Background: The p53 protein in mesenchymal stem cells (MSCs) regulates differentiation to osteogenic or adipogenic lineage. Because p53 function is depressed in most malignancies, if MSCs in malignancy also have p53 hypofunction, differentiation therapy to osteogenic or adipogenic lineage may be an effective treatment. We therefore wished to begin to explore this idea by evaluating atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) cells, because murine double minute 2 (MDM2) gene amplification, which leads to p53 hypofunction, is found in almost all ALT/WDLs. Questions/purposes: We compared osteogenic and adipogenic differentiation potency between MSCs isolated and cultured from normal adipose tissues and ALT/WDLs from the same patients. Methods: During tumor resections in six patients with ALT/WDL, we analyzed 3 mL of tumor, and for comparison, we harvested a similar amount of normal-appearing subcutaneous adipose tissue from an area remote from the tumor for comparison. Adipogenic differentiation potency was quantitatively assessed using spectrometry after oil red O staining. Osteogenic differentiation potency was semiquantitatively assessed by measuring a specific colored area after alkaline phosphatase (ALP) and alizarin red S staining. ALP is related to preosseous cellular metabolism, and alizarin red is related to calcium deposits in cell culture. There were three observers for each assessment, and each assessment (including induced-differentiation and histologic analysis) was performed in duplicate. We then analyzed the mechanism of the difference of osteogenic differentiation potency using the MDM2-specific inhibitor Nutlin-3 at various concentrations. Results: In terms of adipogenic differentiation potency, contrary to our expectations, more fatty acid droplets were observed in MSCs derived from normal fat than in MSCs derived from ALT/WDL, although we found no significant difference between MSCs derived from ALT/WDL and MSCs derived from normal fat; the mean differentiation potency values (normal adipose tissue versus ALT/WDL) (± SD) were 0.34 (SD, ± 0.13; 95% CI, 0.24–0.44) versus 0.25 (SD, ± 0.10; 95% CI, 0.18–0.33; p = 0.22). By contrast, we found greater osteogenic differentiation potency in MSCs derived from ALT/WDL than in MSCs derived from normal fat. The mean differentiation potency values (normal adipose tissue versus ALT/WDL) (±SD) based on ALP staining was 1.0 versus 17 (SD, ± 36; 95% CI, −2.8 to 38; p = 0.04). However, we found no differences based on alizarin red S staining; mean differentiation potency value (normal adipose tissue versus ALT/WDL) (± SD) was 1.0 versus 4.2 (SD, ± 4.8; 95% CI, 1.3–7.2; p = 0.58). The gap of osteogenic differentiation potency between MSCs from normal adipose tissue and ALT/WDL was decreased as MDM2-inhibitor Nutlin-3 concentration increased. Conclusions: MSCs derived from ALT/WDL had higher osteogenic differentiation potency based on ALP staining, which disappeared as Nutlin-3 concentration increased, suggesting that could be caused by amplified MDM2 in ALT/WDL. Future laboratory studies might mechanistically confirm the gene and protein expression, and based on the mechanism of the gap of differentiation potency, if p53 contrast between MSCs in tumor and normal tissue could be stimulated, less-toxic and more-effective differentiation therapy to MSCs in malignancies might be developed.
  • Neuroprotective erythropoietin attenuates microglial activation, including morphological changes, phagocytosis, and cytokine production, Tetsuya Tamura, Tetsuya Tamura, Mineyoshi Aoyama, Mineyoshi Aoyama, Seiko Ukai, Hiroki Kakita, Hiroki Kakita, Kazuya Sobue, Kiyofumi Asai, Brain Research, 1662, 65 - 74, 05 01 , © 2017 Elsevier B.V. Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. EPO receptor (EPOR) is expressed in microglia, resident immune cells in the brain. However, the effect of EPO on microglial activation is not clear. In the present study, we demonstrated that the EPOR is highly expressed in microglia, rather than in neurons or astrocytes, in in vitro experiments. Therefore, we investigated whether EPO could attenuate lipopolysaccharide (LPS)-mediated activation of microglia in vitro. The BV-2 microglial cell line was treated with LPS in the absence or presence of EPO. In the presence of EPO, microglial expression of LPS-induced inflammatory cytokine genes was significantly decreased. In addition, EPO suppressed the LPS-induced phagocytic activity of BV-2 cells towards fluorescent beads, as well as induction of inducible nitric oxide synthase. In in vivo experiments, EPO significantly decreased the LPS-induced expression of inflammatory cytokine genes in mouse brains. Furthermore, morphological analysis of cortical microglia in the brains of mice stimulated with LPS revealed that combined treatment with EPO alleviated LPS-induced morphological changes in the microglia. These data indicate that EPO attenuates microglial activation, including morphological changes in vivo, phagocytosis in vitro, and the production of inflammatory cytokines in vivo and in vitro. Further investigation of EPO modulation of LPS-induced microglial activation may contribute to the development of novel neuroprotective therapies.
  • Expression and subcellular localization of at motif binding factor 1 in colon tumours, Kataoka H, Miura Y, Kawaguchi M, Suzuki S, Okamoto Y, Ozeki K, Shimura T, Mizoshita T, Kubota E, Tanida S, Takahashi S, Asai K, Joh T., Molecular Medicine Reports, 16, (3) 3095 - 3102, Refereed
  • CXCR4+CD45Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow, Yoh Goto, Yoh Goto, Mineyoshi Aoyama, Mineyoshi Aoyama, Takeo Sekiya, Takeo Sekiya, Hiroki Kakita, Hiroki Kakita, Yuko Waguri-Nagaya, Ken Miyazawa, Kiyofumi Asai, Shigemi Goto, Stem Cells, 34, 2733 - 2743, 11 01 , © 2016 AlphaMed Press 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. Stem Cells 2016;34:2733–2743.
  • A diagnostic marker for superficial urothelial bladder carcinoma: Lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression, Makoto Kawaguchi, Makoto Kawaguchi, Noboru Hara, Vladimir Bilim, Hiroshi Koike, Mituko Suzuki, Mituko Suzuki, Tae Sun Kim, Nan Gao, Yu Dong, Sheng Zhang, Sheng Zhang, Yuji Fujinawa, Osamu Yamamoto, Hiromi Ito, Yoshihiko Tomita, Yuchi Naruse, Akira Sakamaki, Yoko Ishii, Koichi Tsuneyama, Masaaki Inoue, Johbu Itoh, Masanori Yasuda, Nobuo Sakata, Cha Gyun Jung, Satoshi Kanazawa, Hiroyasu Akatsu, Hiroshi Minato, Takayuki Nojima, Kiyofumi Asai, Yutaka Miura, BMC Cancer, 16, 10 18 , © 2016 The Author(s). Background: Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. Methods: Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. Results: ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n = 110) and ATBF1- (n = 7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008). Conclusions: Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.
  • Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells, Hideto Yamamura, Yoshiaki Suzuki, Hisao Yamamura, Kiyofumi Asai, Yuji Imaizumi, Biochemical and Biophysical Research Communications, 476, 386 - 392, 08 05 , © 2016 Elsevier Inc. The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia.
  • Mineralocorticoid receptor stimulation induces urinary storage dysfunction via upregulation of epithelial sodium channel expression in the rat urinary bladder epithelium, Seiji Yamamoto, Yuji Hotta, Kotomi Maeda, Tomoya Kataoka, Yasuhiro Maeda, Takashi Hamakawa, Shoichi Sasaki, Takahiro Yasui, Kiyofumi Asai, Kazunori Kimura, Kazunori Kimura, Journal of Pharmacological Sciences, 130, 219 - 225, 04 01 , © 2016 The Authors. We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10-12-week-old, male Sprague-Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms.
  • Stimulation of neuronal cells by culture supernatant of T lymphocytes triggered by anti-CD3 mAb followed by propagation in the presence of interleukin-2, Masae Ishiguro, Masae Ishiguro, Alan Okada, Kiyofumi Asai, Kiyohide Kojima, Hidechika Okada, Hidechika Okada, Microbiology and Immunology, 60, 47 - 55, 01 01 , © 2016 The Societies and Wiley Publishing Asia Pty Ltd. Performance status (PS) frequently improves occurs in cancer patients who have been infused with their own lymphokine-activated killer T cells (LAK-T). In the present study, a culture supernatant of LAK-T (LAK-T sup) administered to 8-week-old rats caused neurogenesis as evidenced by increased 5-ethynyl-2′-deoxyuridine staining of brain tissues. Intravenous injection of granulocyte-macrophage colony stimulating factor (GM-CSF), a major cytokine in LAK-T sup, had a similar effect. Furthermore, LAK-T sup induced Ca ++ increase in rat hippocampal brain slices that was detected in neuronal cells by emission of Fluo-8 NW at 520nm. The same effect was observed with an rGM-CSF solution. GM-CSF may activate neuronal cells by stimulating the glial cells that surround and attach to them. If so, GM-CSF and LAK-T sup may improve the motor neurons of patients with amyotrophic lateral sclerosis. The neurogenerative effect of GM-CSF in LAK-T sup may also help improve brain function in aged adults including those with dementia such as Alzheimer's disease.
  • Regulation of store-operated Ca2+entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells, Hiroaki Kito, Hiroaki Kito, Hisao Yamamura, Yoshiaki Suzuki, Hideto Yamamura, Susumu Ohya, Susumu Ohya, Kiyofumi Asai, Yuji Imaizumi, Biochemical and Biophysical Research Communications, 459, 457 - 462, 04 10 , © 2015 Elsevier Inc. All rights reserved. Store-operated Ca 2+ entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca 2+ influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 ph ase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells.
  • Oncolytic reovirus combined with trastuzumab enhances antitumor efficacy through TRAIL signaling in human HER2-positive gastric cancer cells, Shingo Hamano, Shingo Hamano, Yoshinori Mori, Mineyoshi Aoyama, Hiromi Kataoka, Mamoru Tanaka, Masahide Ebi, Eiji Kubota, Tsutomu Mizoshita, Satoshi Tanida, Randal N. Johnston, Kiyofumi Asai, Takashi Joh, Cancer Letters, 356, 846 - 854, 01 01 , © 2014 Elsevier Ireland Ltd. The human epidermal growth factor receptor 2 (HER2)-targeting agent, trastuzumab, is effective for HER2-overexpressing gastric cancer therapy. As oncolytic reovirus is currently undergoing clinical trials internationally, we wanted to explore whether combination therapy using trastuzumab and reovirus might provide a novel, more effective therapeutic option for gastric cancer. Cell proliferation and cell apoptosis were examined in vitro, while molecular analysis of pathways responsible for cell damage was examined using polymerase chain reaction array. Activation of the proteins related to apoptosis, cell growth and survival was detected by Western blotting. Mouse tumor xenograft models were used to examine antitumor activity in vivo. Reovirus sensitized HER2-overexpressing gastric cancer cells to undergo apoptosis. Both in vitro and in vivo studies provided evidence that the combination therapy is a more powerful modality against HER2-overexpressing gastric cancer cells than treatment using a single agent. Molecular analysis indicated that combination therapy induced significantly higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in cancer cells. Antibody against TRAIL strongly inhibited cell toxicity caused by the combined treatment. These data suggest that reovirus may augment trastuzumab-induced cytotoxicity in gastric cancer cells.
  • Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions, Takayuki Nomura, Takayuki Nomura, Mineyoshi Aoyama, Yuko Waguri-Nagaya, Yoh Goto, Yoh Goto, Mieko Suzuki, Ken Miyazawa, Kiyofumi Asai, Shigemi Goto, Experimental Cell Research, 321, 167 - 177, 02 15 , Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we used human bone marrow cells (BMCs) to investigate the role of hypoxic exposure on human osteoclast (OC) formation in the presence of tumor necrosis factor (TNF). Exposing the BMCs to 3%, 5%, or 10% O 2 in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, consistent with OCs. The addition of TNF under hypoxic conditions generated significantly greater numbers of mature OCs with more nuclei than OCs generated under normoxic conditions. Longer initial hypoxic exposure increased the number of OC precursor cells and facilitated the differentiation of OC precursor cells into multinucleated OCs. Quantitative RT-PCR analysis revealed that RANKL and TNFR1 were expressed at higher levels in non-OC cells from BMCs under hypoxic conditions than under normoxic conditions. Furthermore, to confirm the involvement of TNF-induced signaling, we examined the effects of blocking antibodies against TNFR1 and TNFR2 on OC formation under hypoxic conditions. The TNFR1 antibody was observed to significantly suppress OC formation. These results suggest that hypoxic exposure plays an important role in TNF-induced osteoclastogenesis from human BMCs. © 2013 Elsevier Inc.
  • Prognostic impact of microRNA-145 down-regulation in adult T-cell leukemia/lymphoma, Hongjing Xia, Seiji Yamada, Mineyoshi Aoyama, Fumihiko Sato, Ayako Masaki, Yan Ge, Masaki Ri, Takashi Ishida, Ryuzo Ueda, Ryuzo Ueda, Atae Utsunomiya, Kiyofumi Asai, Hiroshi Inagaki, Human Pathology, 45, 1192 - 1198, 01 01 , Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive tumor caused by human T-cell leukemia virus type 1. MicroRNAs (miRNAs) are closely involved in the development and progression of various tumors. Here we investigated the dysregulation of miRNAs in ATL and its clinical significance. Studies using miRNA arrays and subsequent real-time reverse transcription polymerase chain reaction showed that, in the 9 ATL cell lines examined, 1 miRNA was consistently up-regulated, whereas another 3 were consistently down-regulated, compared with normal CD4-positive lymphocytes. Next, we analyzed the prognostic impact of these 4 miRNAs in patients with aggressive-type ATL (n = 40). Of the 4 dysregulated miRNAs selected, 3 (miR-130b higher expression, miR-145 lower expression, and miR-223 lower expression) were significantly associated with a worsened overall patient survival. We found that expressions of these 3 miRNAs were correlated with each other. To clarify which of the 3 had the most significant impact on overall survival, we performed a multivariate prognostic analysis that included these 3 miRNAs, and only miR-145 lower expression was selected as an independent risk factor (P =.0005). When overexpressed in an ATL cell line in vitro, miR-145 specifically inhibited tumor cell growth. In conclusion, our study suggests that miR-145 down-regulation provides a growth advantage in ATL and is highly associated with a worsened prognosis for patients with ALT. Hence, miR-145 may be a useful prognostic marker and a potential therapeutic target for ATL. © 2014 Elsevier Inc.
  • Membrane hyperpolarization induced by endoplasmic reticulum stress facilitates Ca2+ influx to regulate cell cycle progression in brain capillary endothelial cells, Hiroaki Kito, Hisao Yamamura, Yoshiaki Suzuki, Susumu Ohya, Kiyofumi Asai, Yuji Imaizumi, Journal of Pharmacological Sciences, 125, 227 - 232, 01 01 , Upregulation of the Kir2.1 channel during endoplasmic reticulum (ER) stress in t-BBEC117, an immortalized bovine brain endothelial cell line, caused a sustained increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and a facilitation of cell death. Expressions of Ca 2+ influx channels (TRPC, Orai1, STIM1) were unchanged by ER stress. The ER stress-induced [Ca 2+ ] i increase was mainly attributed to the deeper resting membrane potential due to Kir2.1 upregulation. ER stress arrested at the G2/M phase and it was attenuated by an inhibitor of Kir2.1. These results indicate that Kir2.1 upregulation by ER stress facilitates cell death via regulation of cell cycle progression in t-BBEC117. © The Japanese Pharmacological Society.
  • Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α, Yoshiaki Nagaya, Yoshiaki Nagaya, Mineyoshi Aoyama, Tetsuya Tamura, Tetsuya Tamura, Hiroki Kakita, Hiroki Kakita, Shin Kato, Shin Kato, Hideki Hida, Shinji Saitoh, Kiyofumi Asai, European Journal of Neuroscience, 40, 3620 - 3626, 01 01 , © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd. 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 α (TNFα) 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 TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.
  • Enhancement of FGF-1 release along with cytosolic proteins from rat astrocytes by hydrogen peroxide, Jin Ichi Ito, Yuko Nagayasu, Mariko Hoshikawa, Koichi H. Kato, Yutaka Miura, Kiyofumi Asai, Hideki Hayashi, Shinji Yokoyama, Shinji Yokoyama, Makoto Michikawa, Brain Research, 1522, 12 - 21, 07 19 , We previously observed that the production and relea se of fibroblast growth factor (FGF-1) are increased in rat astrocytes during in vitro long-term culture, that FGF-1 enhances the generation of apoE-containing high density lipoproteins (apoE/HDL), and that the wound healing of brain cryoinjury delays in apoE-deficient mouse. The detail mechanism underlying these phenomena remains unknown. In this study, we examined effects of oxidative stress on release of FGF-1 from cultured rat astrocytes. The treatment of rat astrocytes with 100 μM hydrogen peroxide (H 2 O 2 ) for 10 min enhanced FGF-1 release without inducing apoptosis. The conditioned medium prepared from the cells cultured in a fresh medium after the treatment with H 2 O 2 had the FGF-1-like activities, which enhanced cholesterol synthesis, signalings to phosphorylate Akt and ERK, and apoE secretion. The oxidative stress induced by H 2 O 2 enhanced the release of cytosolic proteins such as HSP70 and HSP90 in addition to FGF-1. Antioxidants such as ascorbic acid and ebselen suppressed the release of cytosolic proteins induced by H 2 O 2 treatment. The addition of lipoproteins such as low density lipoproteins (LDL), furthermore, canceled H 2 O 2 -induced release of FGF-1 and cytosolic proteins. Proteolysis of cytosolic proteins in the H 2 O 2 -treated rat astrocytes was enhanced in the presence of exogenous trypsin, which was attenuated by the pretreatment with LDL, suggesting that H 2 O 2 increases the permeability of the membrane of cells, which was prevented by the addition of lipoproteins. These findings suggest that oxidative stress is one of the candidates which triggers FGF-1 release from astrocytes in the brain, and that the lipid homeostasis in the cell membrane may regulate H 2 O 2 -induced release of FGF-1. © 2013 Elsevier B.V.
  • Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production, Hiroki Kakita, Hiroki Kakita, Mineyoshi Aoyama, Yoshiaki Nagaya, Yoshiaki Nagaya, Hayato Asai, Hayato Asai, Mohamed Hamed Hussein, Mohamed Hamed Hussein, Mieko Suzuki, Shin Kato, Shin Kato, Shinji Saitoh, Kiyofumi Asai, Toxicology and Applied Pharmacology, 268, 99 - 105, 04 05 , Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-1β, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed that cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N G -monomethyl-l-arginine (l-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF. © 2013 Elsevier Inc.
  • Diclofenac enhances proinflammatory cytokine-induced aquaporin-4 expression in cultured astrocyte, Hayato Asai, Hayato Asai, Hiroki Kakita, Hiroki Kakita, Mineyoshi Aoyama, Yoshiaki Nagaya, Yoshiaki Nagaya, Shinji Saitoh, Kiyofumi Asai, Cellular and Molecular Neurobiology, 33, 393 - 400, 04 01 , Acute encephalopathy is a generic term for acute brain dysfunction occurring after infection. Acute encephalopathy induced by influenza virus results in high mortality, and most cases of influenza-associated encephalopathy (IAE) result in brain edema. Administration of diclofenac sodium (DCF), a non-steroidal anti-inflammatory drug (NSAID), is associated with a significant increased mortality rate of IAE. These previous clinical findings proposed further investigation of DCF administration and brain edema to clarify how DCF aggravates IAE. Aquaporin-4 (AQP4) is the predominant water channel protein in the mammalian brain, and is mainly expressed in astrocytes. AQP4 plays an important role in brain water homeostasis. Therefore, we investigated a possible association between DCF and AQP4 production in astrocytes. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor α, and interferon γ, and then treated with DCF. DCF enhanced proinflammatory cytokine-induced AQP4 gene and protein expression in astrocytes, whereas DCF alone did not change the AQP4 gene expression. The addition of nuclear factor-kappa B (NF-κB) inhibitor abrogated AQP4 gene and protein expression completely in astrocytes treated with cytokines alone and in those also treated with DCF. In conclusion, this study demonstrated that AQP4 is upregulated in astrocyte by proinflammatory cytokines, and that the addition of DCF further augments AQP4 production. This effect is mediated via NF-κB signaling. The enhancement of AQP4 production by DCF may explain the significantly increased mortality rates in IAE patients treated with DCF. © 2013 Springer Science+Business Media New York.
  • CD36-related protein in Schistosoma japonicum: Candidate mediator of selective cholesteryl ester uptake from high-density lipoprotein for egg maturation, Kuniko Okumura-Noji, Kuniko Okumura-Noji, Kuniko Okumura-Noji, Yutaka Miura, Rui Lu, Rui Lu, Kiyofumi Asai, Nobuo Ohta, Nobuo Ohta, Paul J. Brindley, Shinji Yokoyama, Shinji Yokoyama, Shinji Yokoyama, FASEB Journal, 27, 1236 - 1244, 03 01 , Familial cholesteryl ester transfer protein (CETP) deficiency is more common in some East Asian populations than elsewhere, suggesting the possibility of a selective advantage of this genetic defect against regional infectious diseases. Historically, infection with the Asian blood fluke Schistosoma japonicum has been endemic in these regions, including Japan. We previously reported that eggs of S. japonicum require cholesteryl ester uptake from normal high-density lipoprotein (HDL) but not from CETP-deficient HDL for their maturation to miracidia, a critical step of the hepatic pathogenesis of schistosomiasis. Herein we show that cholesteryl ester uptake is selective from HDL, and identified CD36-related protein (CD36RP) as a candidate to mediate the reaction. CD36RP was cloned from the adult and the egg developmental stages of S. japonicum, with 1880 bp encoding 506 amino acid residues exhibiting the CD36 domains and two transmembrane regions. Using antibodies against recombinant peptides representing the potential extracellular domains of CD36RP, Western blotting detected a protein with a molecular mass of 82 kDa in the particulate fraction of the adult parasite cells, which was reduced to 62 kDa after N-glycanase treatment. The extracellular domain peptide bound human HDL, as established by immunoblots following nondenaturing gel electrophoresis. Antibodies against the extracellular domain suppressed HDL cholesteryl ester uptake and maturation of the eggs in vitro. CD36RP is a candidate receptor on eggs of S. japonicum that facilitates uptake of HDL cholesteryl ester necessary for egg embryonation and maturation. © FASEB.
  • 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.
  • Region-specific expression of a water channel protein, aquaporin 4, on brain astrocytes, M. Aoyama, H. Kakita, H. Kakita, S. Kato, S. Kato, M. Tomita, K. Asai, Journal of Neuroscience Research, 90, 2272 - 2280, 12 01 , Cellular activities within the brain display regional specificity and a neuronal and glia interdependence. Components characterizing the regional specificity of neurons have been identified. However, characterization of the astrocyte remains in question. To identify region specific features of astrocytes, we have characterized the molecular phenotype of cells derived from regions with different levels of neuronal excitability, the cortex and striatum. Astrocytes were identified in cryostat sections of adult rat brain by rapid immunostaining for glial fibrillary acidic protein (GFAP), and individual cells were collected from each region by using laser microdissection (LMD). Total RNA was isolated and subjected to DNA microarray analysis. At least eight genes showed a differential expression level. Among them, aquaporin 4 (AQP4), a water channel protein, was expressed at higher levels within the cortex compared with the striatum, as confirmed by immunohistochemistry. Primary cultured astrocytes isolated from rat cortex or striatum also showed a differential expression of AQP4. These data may reflect unique properties of astrocytes across different brain regions. However, they may also reflect the interactive demands of neurons with different activity levels. Further examination of the heterogeneous astrocyte populations within each region will lend additional support to the regional specificity of neuronal functions and neuronal-glial interactions. © 2012 Wiley Periodicals, Inc.
  • AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1, Sheng Zhang, Sheng Zhang, Sheng Zhang, Tae Sun Kim, Tae Sun Kim, Yu Dong, Satoshi Kanazawa, Makoto Kawaguchi, Makoto Kawaguchi, Nan Gao, Nan Gao, Hiroshi Minato, Tsutomu Takegami, Takayuki Nojima, Kiyofumi Asai, Yutaka Miura, Biochemical and Biophysical Research Communications, 427, 537 - 541, 10 26 , ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1. © 2012 Elsevier Inc.
  • Subventricular zone-derived oligodendrogenesis in injured neonatal white matter in mice enhanced by a nonerythropoietic erythropoietin derivative, Eisuke Kako, Eisuke Kako, Naoko Kaneko, Mineyoshi Aoyama, Hideki Hida, Hirohide Takebayashi, Kazuhiro Ikenaka, Kiyofumi Asai, Hajime Togari, Kazuya Sobue, Kazunobu Sawamoto, Stem Cells, 30, 2234 - 2247, 10 01 , Perinatal hypoxia-ischemia (HI) frequently causes white-matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI-induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell-type- and region-specific genetic labeling methods in a mouse HI model, we characterized the Olig2-expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long-term postinjury treatment with a nonerythropoietic derivative of EPO, asialo-erythropoietin, promoted the maturation of pSVZ-derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white-matter injury. © AlphaMed Press.
  • The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes, Kenji Ikuta, Yuko Waguri-Nagaya, Kae Kikuchi, Takaya Yamagami, Masahiro Nozaki, Mineyoshi Aoyama, Kiyofumi Asai, Takanobu Otsuka, Arthritis Research and Therapy, 14, 04 25 , Introduction: Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs).Methods: FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection.Results: The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin.Conclusions: Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy. © 2011 Ikuta et al.; licensee BioMed Central Ltd.
  • Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury, Shin Kato, Shin Kato, Mineyoshi Aoyama, Hiroki Kakita, Hiroki Kakita, Hideki Hida, Ineko Kato, Tetsuya Ito, Tatenobu Goto, Mohamed H. Hussein, Kazunobu Sawamoto, Hajime Togari, Kiyofumi Asai, Journal of Neuroscience Research, 89, 1566 - 1574, 10 01 , The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury. © 2011 Wiley-Liss, Inc.
  • ERas enhances resistance to CPT-11 in gastric cancer, Eiji Kubota, Hiromi Kataoka, Mamoru Tanaka, Yasuyuki Okamoto, Masahide Ebi, Yoshikazu Hirata, Kenji Murakami, Tsutomu Mizoshita, Takaya Shimura, Oshinori Mori, Satoshi Tanida, Takeshi Kamiya, Mineyoshi Aoyama, Kiyofumi Asai, Takashi Joh, Anticancer Research, 31, 3353 - 3360, 10 01 , Background/Aim: We have reported that embryonic stem cell-expressed Ras (ERas) is expressed in human gastric cancer and is associated with its tumorigenicity. Here, we asked whether ERas plays a role in resistance to chemotherapy in gastric cancer. Materials and Methods: To assess the cytotoxicity of chemotherapeutic agents, ERas-overexpressing human gastric cancer GCIY cells were exposed to anticancer agents, including CPT-11 and inhibitor of mammalian target of rapamycin (mTOR). We also investigated the mechanisms by which ERas induces chemoresistance. Results: ERas-overexpressing clones were significantly more resistant to CPT-11 than were the control (p < 0.001). Administration of rapamycin was significantly cytotoxic to the ERas-overexpressing clones compared with the control (p < 0.01). Electrophoresis mobility shift assay revealed that ERas enhanced nuclear factor (NF)-κB activity. PCR array demonstrated that ERas up-regulated several multidrug efflux transporter genes, including ABCG2. Conclusion: ERas induces chemoresistance to CPT-11 via activation of phosphatidylinositol-3 kinase-protein kinase β mTOR pathway and NF-κB, and consequently results in upregulation of ABCG2.
  • Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells, Hiroaki Kito, Daiju Yamazaki, Daiju Yamazaki, Daiju Yamazaki, Susumu Ohya, Hisao Yamamura, Kiyofumi Asai, Yuji Imaizumi, Biochemical and Biophysical Research Communications, 411, 293 - 298, 07 29 , Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K + channel (K ir 2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K ir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca 2+ concentration due to Ca 2+ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K ir 2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions. © 2011 Elsevier Inc.
  • A new enteral diet, MHN-02, which contains abundant antioxidants and whey peptide, protects against carbon tetrachloride-inducedhepatitis, Takehiko Takayanagi, Hajime Sasaki, Akihiro Kawashima, Yuichiro Mizuochi, Hiroyuki Hirate, Takeshi Sugiura, Takafumi Azami, Kiyofumi Asai, Kazuya Sobue, Journal of Parenteral and Enteral Nutrition, 35, 516 - 522, 07 01 , Background: Inflammatory or oxidative stress is related to various diseases, including not only inflammatory diseases, but also diabetes, cancer, and atherosclerosis. The aim of this study was to evaluate the anti-inflammatory effects of a new enteral diet, MHN-02, which contains abundant antioxidants and whey peptide. The study also investigated the ability of MHN-02 to attenuate lethality, liver injury, the production of inflammatory cytokines, and the production of oxidized products using a carbon tetrachloride-induced rat model of severe fulminant hepatitis. Methods: Male Sprague-Dawley rats were fed either a control diet or the MHN-02 diet for 14 days and injected with 2 mL/kg of carbon tetrachloride. Survival of rats was monitored from day 0 to day 3. To evaluate liver injury, inflammation, and oxidative stress, blood and liver samples were collected, and aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, interleukin 6, tumor necrosis factor-á, and superoxide dismutase activity as a free radical scavenger were measured. A portion of the liver was evaluated histologically. Results: Th e survival rates of rats receiving the MHN-02 diet and the control diet were 90% and 55%, respectively. In the MHN-02 diet group, levels of serum liver enzymes and serum cytokines were significantly lower than in the control group. Superoxide dismutase activity in the MHN-02 diet was significantly higher in the MHN-02 group. Pathological lesions were significantly larger in the control group. Conclusion: Supplementation of enteral diets containing whey peptide and antioxidants may protect against severe hepatitis. © 2011 American Society for Parenteral and Enteral Nutrition.
  • Contribution of Kir2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model, Daiju Yamazaki, Daiju Yamazaki, Daiju Yamazaki, Hiroaki Kito, Seiji Yamamoto, Seiji Yamamoto, Susumu Ohya, Hisao Yamamura, Kiyofumi Asai, Yuji Imaizumi, American Journal of Physiology - Cell Physiology, 300, 01 01 , Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca 2+ ] i rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca 2+ -activated K + channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ∼15% of t-BBEC 117, in which inward rectifier K + channel (K ir 2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba 2+ but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K ir 2.1 mRNAs was significantly higher and Ba 2+ -sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba 2+ . In the human embryonic kidney 293 cell model, where SK2 and K ir 2.1 were heterologously coexpressed, [Ca 2+ ] i rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K ir 2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover. Copyright © 2011 the American Physiological Society.
  • The ZFHX3 (ATBF1) transcription factor induces PDGFRB, which activates ATM in the cytoplasm to protect cerebellar neurons from oxidative stress, Tae Sun Kim, Makoto Kawaguchi, Mitsuko Suzuki, Cha Gyun Jung, Kiyofumi Asai, Yuta Shibamoto, Martin F. Lavin, Kum Kum Khanna, Yutaka Miura, DMM Disease Models and Mechanisms, 3, 752 - 762, 11 01 , Ataxia telangiectasia (A-T) is a neurodegenerative disease caused by mutations in the large serine-threonine kinase ATM. A-T patients suffer from degeneration of the cerebellum and show abnormal elevation of serum alpha-fetoprotein. Here, we report a novel signaling pathway that links ATM via cAMP-responsive-element-binding protein (CREB) to the transcription factor ZFHX3 (also known as ATBF1), which in turn promotes survival of neurons by inducing expression of platelet-derived growth factor receptor β (PDGFRB). Notably, AG1433, an inhibitor of PDGFRB, suppressed the activation of ATM under oxidative stress, whereas AG1433 did not inhibit the response of ATM to genotoxic stress by X-ray irradiation. Thus, the activity of a membrane-bound tyrosine kinase is required to trigger the activation of ATM in oxidative stress, independent of the response to genotoxic stress. Kainic acid stimulation induced activation of ATM in the cerebral cortex, hippocampus and deep cerebellar nuclei (DCN), predominately in the cytoplasm in the absence of induction of γ-H2AX (a marker of DNA double-strand breaks). The activation of ATM in the cytoplasm might play a role in autophagy in protection of neurons against oxidative stress. It is important to consider DCN of the cerebellum in the etiology of A-T, because these neurons are directly innervated by Purkinje cells, which are progressively lost in A-T.
  • Resistance to chemotherapeutic agents and promotion of transforming activity mediated by embryonic stem cell-expressed Ras (ERas) signal in neuroblastoma cells, Mineyoshi Aoyama, Hiromi Kataoka, Eiji Kubota, Toyohiro Tada, Kiyofumi Asai, International Journal of Oncology, 37, 1011 - 1016, 10 01 , Neuroblastoma is a common childhood tumor derived from neural crest precursor cells. In the present study, we investigated the expression and function of embryonic stem cell-expressed Ras (ERas), a novel Ras family protein previously reported as the specific expression gene in embryonic stem cells (ES cells), in neuroblastoma cell lines. Our results showed that the expressions of ERas were detected in neuroblastoma cell lines by RT-PCR and Western blotting. Therefore, we transfected a full length ERas expression vector into the neuroblastoma cell line SH-SY5Y, which has weak endogenous expression of ERas, and obtained clones with higher levels of expression. Overexpression of ERas did not increase the growth rate of the ERas transfectants but promoted their transforming activity. The ERas transfectants were more resistant to all the chemotherapy agents than the parental cell line. The ability of ERas to rescue cells from the toxic effect of chemotherapeutic agents was inhibited by the phosphatidylinositol 3′-kinase (PI3K) inhibitor PD294002. These results show that the ERas/PI3K pathway may provide resistance to chemotherapy and promote transforming activity in neuroblastoma.
  • Tumor suppressor, AT motif binding factor 1 (ATBF1), translocates to the nucleus with runt domain transcription factor 3 (RUNX3) in response to TGF-β signal transduction, Motoshi Mabuchi, 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, 321 - 325, 07 01 , 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-β 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-β signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-β1 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-β1 stimulation, but after 24h of TGF-β1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-β signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator. © 2010 Elsevier Inc.
  • 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
  • TNF-alpha-induced aquaporin 9 in synoviocytes from patients with OA and RA., Masashizu Nagahara, Yuko Waguri-Nagaya, Takaya Yamagami, Mineyoshi Aoyama, Toyohiro Tada, Katsuhisa Inoue, Kiyofumi Asai, Takanobu Otsuka, Rheumatology (Oxford, England), 49, 898 - 906, 05 01 , To determine whether aquaporins (AQPs) are expressed in the synovial tissues of patients with OA and RA, and to examine the patterns of expression in patients with and without hydrarthrosis. AQPs were detected in synovial tissue samples from patients with OA and RA using RT-PCR and immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from patients with OA and RA were cultured and stimulated with TNF-alpha. The expression of AQPs in FLSs was examined using RT-PCR and western blot analyses and the function of aquaglyceroporins was examined by a glycerol uptake assay. AQP1, -3 and -9 mRNAs were expressed in synovial tissues from patients with OA and RA. AQP1, -3 and -9 proteins were also detected by immunohistochemistry. AQP9 mRNA was expressed more strongly in the synovial tissues of OA patients with hydrarthrosis than those without. AQP9 mRNA and protein expression were strongly induced with TNF-alpha treatment in FLSs, whereas the expression of AQP1 and -3 mRNAs was not induced with TNF-alpha treatment. AQP9 as an aquaglyceroporin was induced by TNF-alpha. AQP9 mRNA was detected in synovial tissues from OA and RA patients with hydrarthrosis. AQP9 expression was strongly induced in FLSs with TNF-alpha. Although the functions of AQP1, -3 and -9 in synovial tissues remain to be elucidated, it suggested that AQP9 might be related to the pathogenesis of hydrarthrosis and inflammatory synovitis.
  • Tranilast suppresses prostate cancer growth and osteoclast differentiation in vivo and in vitro, Shinya Sato, Satoru Takahashi, Makoto Asamoto, Taku Naiki, Taku Naiki, Aya Naiki-Ito, Kiyofumi Asai, Tomoyuki Shirai, Prostate, 70, 229 - 238, 02 15 , BACKGROUND. In bone metastatic sites, prostate cancer cells proliferate on interacting with osteoclasts. Tranilast, which is used for an antiallergic drug, has been shown to inhibit growth of several cancers and stromal cells. The present study was conducted to assess suppressive effects of Tranilast on prostate cancer growth and osteoclast differentiation in vivo and in vitro. METHODS. In vivo, rat prostate cancer tissue was transplanted onto cranial bones of F344 rats and Tranilast was given for 9 days at doses of 0, 200, or 400 mg/kg/day. In vitro, human prostate cancer cell lines, LNCaP, PC3, and DU145, the rat prostate cancer cell line, PLS-10, and rat bone marrow cells were similarly treated with the agent. RESULTS. In vivo, tumor volumes were significantly decreased in the high dose group. While cell proliferation did not appear to be affected, apoptosis was induced and tumor necrosis was apparent. Cranial bone defects were decreased in the high dose group. In vitro, cell proliferation rates of all four cell lines were reduced by Tranilast and increased apoptosis was observed in LNCaP and PLS-10. In addition, Tranilast significantly reduced osteoclast differentiation of rat bone marrow cells. Western blot analysis of PLS-10 and LNCaP revealed that phospho-GSK3b was up-regulated and phospho-Akt was down-regulated. CONCLUSIONS. Tranilast here suppressed rat prostate cancer growth and osteoclast differentiation. Growth of human prostate cancer cells was also inhibited. Thus, this agent deserves consideration as a candidate for conventional therapy of bone metastatic prostate cancer. © 2009 Wiley-Liss, Inc.
  • Role of ES cell-expressed Ras (ERas) in tumorigenicity of gastric cancer, Eiji Kubota, Hiromi Kataoka, Mineyoshi Aoyama, Tsutomu Mizoshita, Yoshinori Mori, Takaya Shimura, Mamoru Tanaka, Makoto Sasaki, Satoru Takahashi, Kiyofumi Asai, Takashi Joh, American Journal of Pathology, 177, 955 - 963, 01 01 , ERas, a unique member of the Ras family, was initially found only in embryonic stem (ES) cells, where it plays a crucial role in the transformation of transplanted ES cells to teratomas. ERas is involved in ES cell survival, and unlike other Ras family members, is constitutively active without any mutations. The aim of this study was to investigate the expression and role of ERas in human gastric cancer. To test whether ERas played a significant role in human cancer cells, we examined its expression and function in gastric cancer. ERas was expressed in gastric cancer cell lines at different levels. Induction of ERas expression activated the phosphatidylinositol 3 kinase (PI3K)/Akt axis and then enhanced anchorage-independent growth and ERas knockdown by siRNA suppressed cell invasion. Immunohistochemical analyses revealed that ERas was expressed in 38.7% (55/142) of human gastric carcinoma tissues, and its expression was significantly associated with metastasis to the liver (P < 0.0001) and lymph nodes (P < 0.05). ERas up-regulated transcription regulatory factors including ZFHX1A, ZFHX1B, and TCF3, which repress E-cadherin. These data suggest that ERas is activated in a significant population of gastric cancer, where it may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin. Copyright © American Society for Investigative Pathology.
  • Prolonged exposure to ammonia increases extracellular glutamate in cultured rat astrocytes, Kentaro Ohara, Mineyoshi Aoyama, Masataka Fujita, Kazuya Sobue, Kiyofumi Asai, Neuroscience Letters, 462, 109 - 112, 09 22 , Abnormal alteration of brain function is a characteristic complication of hepatic encephalopathy in both acute and chronic liver failure. Previous studies suggest that the pathogenesis of hepatic encephalopathy involves chronic glial edema with subsequent alteration of glioneuronal communication, N-methyl-d-aspartate (NMDA) receptor activation, and oxidative/nitrosative stress. In the present study, we investigated extracellular glutamate levels in cultured astrocytes under prolonged exposure to ammonia. Using an enzyme-linked high-performance liquid chromatography assay to detect glutamate, prolonged (48 h) exposure of cultured astrocytes to ammonia resulted in a concentration- and time-dependent increase in extracellular glutamate. Similar increases were observed when ammonia-containing medium (pH 7.8) was adjusted to the pH of control medium (pH 7.4), indicating that the effect is not due to pH. Treatment of astrocytes with an antioxidant (l-ascorbic acid), an NADPH oxidase inhibitor (apocynin), a Ca 2+ chelator (BAPTA-AM), an NMDA receptor antagonist (NK801), or a mitochondrial permeability transition inhibitor (cyclosporine A) suppressed the increase of extracellular glutamate in response to prolonged ammonia exposure. Prolonged exposure to ammonia increased extracellular glutamate through the NMDA receptor, increased intracellular Ca 2+ levels, and upregulation of excitatory amino acids. The addition of ATP further increased extracellular glutamate levels in astrocytes subjected to prolonged ammonia treatment (5 mM, 48 h) in a dose-dependent manner. These results indicate that the deregulation of glutamate release from astrocytes may contribute to the dysfunction of glutamatergic neurons in patients with acute liver failure (ALF). © 2009 Elsevier Ireland Ltd. All rights reserved.
  • Glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through the PI3K/Akt/mTOR/GCLc/redox signaling pathway, R. Kimura, M. Okouchi, H. Fujioka, A. Ichiyanagi, F. Ryuge, T. Mizuno, K. Imaeda, N. Okayama, Y. Kamiya, K. Asai, T. Joh, Neuroscience, 162, 1212 - 1219, 09 15 , Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. Oxidative stress-induced neuronal cell apoptosis is a contributing factor. Glucagon-like peptide (GLP)-1 has recently become an attractive treatment modality for patients with diabetes. It also readily enters the brain, prevents neuronal cell apoptosis, and improves the cognitive impairment characteristic of Alzheimer's disease. Therefore, we investigated whether GLP-1 could protect against oxidative stress-induced neuronal cell apoptosis in pheochromocytoma (PC12) cells. PC12 cells were exposed to 1 mM methylglyoxal (MG) or MG plus 3.30 μg/ml GLP-1. Cell apoptosis, expression and phosphorylation of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/γ-glutamylcysteine ligase catalytic subunit (GCLc), and redox balance were then determined. The data showed that MG induced PC12 apoptosis in accordance with the redox (glutathione (GSH) and GSH/glutathione disulfide [GSSG]) imbalance. GLP-1 protected against this MG-induced apoptosis, which corresponded to the phosphorylation of PI3K, Akt, and mTOR, as well as the upregulation of GCLc and the restoration of the redox imbalance. Inhibitors of PI3K (LY294002), Akt (Akt-I), and mTOR (rapamycin) reduced the GLP-1-induced GCLc upregulation and its protection against MG-induced PC12 apoptosis. The GLP-1-induced redox restoration was also attenuated by rapamycin. In conclusion, the neuroprotective effect of GLP-1 is due to an enhancement of PI3K/Akt/mTOR/GCLc/redox signaling. © 2009 IBRO.
  • Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-κB signaling in cultured astrocytes, Hiroki Kakita, Hiroki Kakita, Hiroki Kakita, Mineyoshi Aoyama, Mohamed Hamed Hussein, Mohamed Hamed Hussein, Mohamed Hamed Hussein, Shin Kato, Shin Kato, Satoshi Suzuki, Tetsuya Ito, Hajime Togari, Kiyofumi Asai, Toxicology and Applied Pharmacology, 238, 56 - 63, 07 01 , 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-1β, tumor necrosis factor-α and interferon-γ, 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-κB inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-κB 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-κB 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. © 2009 Elsevier Inc. All rights reserved.
  • Localization of reversion-induced LIM protein (RIL) in the rat central nervous system, Yuko Lida, Toshiyuki Matsuzaki, Toshiyuki Matsuzaki, Tetsuro Morishima, Hiroshi Sasano, Kiyofumi Asai, Kazuya Sobue, Kuniaki Takata, Kuniaki Takata, Acta Histochemica et Cytochemica, 42, 9 - 14, 05 05 , Reversion-induced LIM protein (RIL) is a member of the ALP (actinin-associated LIM protein) subfamily of the PDZ/LIM protein family. RIL serves as an adaptor protein and seems to regulate cytoskeletons. Immunoblotting suggested that RIL is concentrated in the astrocytes in the central nervous system. We then examined the expression and localization of RIL in the rat central nervous system and compared it with that of water channel aquaporin 4 (AQP4). RIL was concentrated in the cells of ependyma lining the ventricles in the brain and the central canal in the spinal cord. In most parts of the central nervous system, RIL was expressed in the astrocytes that expressed AQP4. Double-labeling studies showed that RIL was concentrated in the cytoplasm of astrocytes where glial fibrillary acidic protein was enriched as well as in the AQP4-enriched regions such as the endfeet or glia limitans. RIL was also present in some neurons such as Purkinje cells in the cerebellum and some neurons in the brain stem. Differential expression of RIL suggests that it may be involved in the regulation of the central nervous system. © 2009 The Japan Society of Histochemistry and Cytochemistry.
  • IGF2 modulates the microenvironment for osteoclastogenesis, Kimihisa Nakao, Kimihisa Nakao, Mineyoshi Aoyama, Hayato Fukuoka, Hayato Fukuoka, Masataka Fujita, Ken Miyazawa, Kiyofumi Asai, Shigemi Goto, Biochemical and Biophysical Research Communications, 378, 462 - 466, 01 16 , We previously reported that hypoxic stress enhanced osteoclast differentiation via increasing insulin-like growth factor 2 (IGF2) production. However, the mechanisms underlying IGF2 stimulation remains unknown. In this study, we investigated the molecular mechanisms of osteoclastogenesis by IGF2 treatment. Primary mouse bone marrow cells were cultured with IGF2. Total RNAs were applied to a DNA microarray analysis, and quantitative RT-PCR was then used to confirm the microarray data and clarify which cells expressed the relative genes. The most interesting findings were the upregulations of CXC chemokine ligand 7 (CXCL7) expression in stromal cells and stromal cell-derived factor 1 (SDF1) expression in osteoblastic cells with IGF2 treatment. The addition of exogenous SDF1 to CXCL7 increased the number of osteoclasts and promoted the formation of giant osteoclasts. These results suggest that IGF2 modulates the microenvironment around osteoclast precursor cells. SDF1 together with CXCL7 may promote the formation of giant osteoclasts. © 2008 Elsevier Inc. All rights reserved.
  • Transforming growth factor-β signaling at the tumor-bone interface promotes mammary tumor growth and osteoclast activation, Mitsuru Futakuchi, 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, 71 - 81, 01 13 , 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-β receptor 1 (TGF-βRI) as a commonly upregulated gene at the tumor-bone (TB) interface. Moreover, TGF-βRI 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-β activity by neutralizing antibody to TGF-β or TGF-βRI kinase inhibitor reduced mammary tumor-induced osteolysis, TGF-βRI expression and its activation. In addition, we demonstrate a potential role of TGF-β as an important modifier of receptor activator of NF-κB ligand (RANKL)-dependent osteoclast activation and osteolysis. Together, these studies demonstrate that inhibition of TGF-βRI signaling at the TB interface will be a therapeutic target in the treatment of breast cancer-induced osteolysis. © 2009 Japanese Cancer Association.
  • 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 β 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, 316 - 323, 02 01 , 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 β (TGFβ) 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 TGFβ signal transduction were compared between the tumor-bone interface and the tumor-subcutaneous interface. Effects of TGFβ on osteoclast differentiation were also evaluated in vitro. Inhibitory effects of TGFβ receptor 1 antisense oligonucleotide on TGFβ signaling, osteolysis, osteoblasts, and tumor growth were examined in vivo. Osteolytic changes were extensively observed at the tumor-bone interface, where the TGFβ level, TGFβ signal transduction, and tumor cell proliferation were higher than at the tumor-subcutaneous interface. In vitro treatment with receptor activator of nuclear factor-κB ligand induced osteoclast differentiation of bone marrow stromal cells, and additional exposure to TGFβ exerted promotive effects on osteoclast induction. Intratumoral injection of TGFβ receptor 1 antisense oligonucleotide significantly reduced TGFβ signal transduction, osteolysis, induction of osteoclast and osteoblast, and tumor cell proliferation. Thus, we experimentally show that TGFβ derived from bone matrix promotes cell proliferation of rat prostate cancer and osteoclast activation-associated osteolysis in the bone microenvironment. © 2008 Japanese Cancer Association.
  • Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation, Masahiro Itaya, Masahiro Itaya, Eiji Sakurai, Miho Nozaki, Kiyoshi Yamada, Satoshi Yamasaki, Kiyofumi Asai, Yuichiro Ogura, Investigative Ophthalmology and Visual Science, 48, 5677 - 5683, 12 01 , PURPOSE. This study was conducted to determine changes in the expression of vascular endothelial growth factor (VEGF) in murine retina after retinal scatter laser photocoagulation. METHODS. Photocoagulation (PHC) was performed on wild-type C57BL/6J mice using a diode laser, and the eyes were enucleated 1, 2, 3, 4, 7, and 14 days after laser treatment. VEGF and monocyte chemoattractant protein (MCP)-1 levels in the sensory retina and retinal pigmented epithelial (RPE) cells in both tissues were measured by ELISA. The VEGF mRNA was measured by real-time RT-PCR. Leukocyte infiltration into the RPE-choroid was determined by flow cytometry. VEGF comparisons between mice subjected to PHC and those treated with monocyte recruitment inhibitor (anti-MCP-1) were performed and statistically analyzed. The expression of VEGF and MCP-1 in the retina was determined by immunohistochemistry. RESULTS. VEGF protein levels significantly increased 1 day after PHC in both the RPE-choroid and the sensory retina. VEGF concentrations were maximum at day 3 after photocoagulation and stayed elevated until day 7. The number of choroid-infiltrating macrophages was markedly increased in mice with laser treatment compared with those without laser treatment. VEGF expression decreased after treatment with neutralized antibody to monocyte recruitment. We demonstrate that MCP-1 expression in the retina increased markedly after scatter laser photocoagulation by immunohistochemistry and EUSA. CONCLUSIONS. Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes. Copyright © Association for Research in Vision and Ophthalmology.
  • Mechanism for FGF-1 to regulate biogenesis of apoE-HDL in astrocytes, Jin Ichi Ito, Yuko Nagayasu, Kuniko Okumura-Noji, Rui Lu, Tomo Nishida, Yutaka Miura, Kiyofumi Asai, Alireza Kheirollah, Alireza Kheirollah, Seiichi Nakaya, Shinji Yokoyama, Journal of Lipid Research, 48, 2020 - 2027, 09 01 , Fibroblast growth factor-1 (FGF-1) is secreted by astrocytes and stimulates apolipoprotein E (apoE)-HDL biogenesis by an autocrine mechanism to help in recovery from brain injury. In apoE-deficient mouse astrocytes, FGF-1 stimulated cholesterol biosynthesis without enhancing its release, indicating a signaling pathway independent of apoE biosynthesis upregulation. SU5402, an inhibitor of FGF receptor, inhibited FGF-1-induced phosphorylation of MEK, ERK, and Akt, as well as all the apoE-HDL biogenesis-related events in rat astrocytes. LY294002, an inhibitor of phosphatidylinositide 3-OH kinase (PI3K) and of Akt phosphorylation, inhibited apoE-HDL secretion but not cholesterol biosynthesis, whereas U0126, an inhibitor of MEK and of ERK phosphorylation, inhibited cholesterol biosynthesis but not apoE-HDL secretion. Increase of apoE-mRNA by FGF-1 was not influenced by either inhibitor. When rat apoE/pcDNA3. his was transfected to transformed rat astrocyte GA-1 cells that otherwise do not synthesize apoE (GA-1/25), FGF-1 did not influence apoE-mRNA, but did increase the apoE secretion and Akt phosphorylation that were suppressed by LY294002. Lipid biosynthesis was increased by FGF-1 in GA-1/25 cells and suppressed by U0126. FGF-1 upregulates apoE-HDL biogenesis by three independent signaling pathways. The PI3K/Akt pathway upregulates secretion of apoE/apoE-HDL, the MEK/ERK pathway stimulates cholesterol biosynthesis, and an unknown pathway enhances apoE transcription. Copyright © 2007 by the American Society for Biochemistry and Molecular Biology, Inc.
  • Subcellular localization of ATBF1 regulates MUC5AC transcription in gastric cancer, Yoshinori Mori, Hiromi Kataoka, Hiromi Kataoka, Yutaka Miura, Makoto Kawaguchi, Eiji Kubota, Naotaka Ogasawara, Tadayuki Oshima, Satoshi Tanida, Makoto Sasaki, Hirotaka Ohara, Tsutomu Mizoshita, Masae Tatematsu, Kiyofumi Asai, Takashi Joh, International Journal of Cancer, 121, 241 - 247, 07 15 , Human gastric epithelium has a unique mucin gene expression pattern, which becomes markedly altered in gastrointestinal disorder. This alteration in mucin expression, including the mucin MUC5AC, may be related to the development and prognosis of gastric cancers, and MUC5AC-positive gastric cancer has been reported to be poor prognosis. However, the molecular mechanism of MUC5AC transcriptional regulation has not been fully elucidated. AT motif-binding factor 1 (ATBF1) is a homeotic transcriptional regulatory factor recently identified as a tumor suppressor gene, and its subcellular localization suggests a link to cell proliferation and differentiation. We investigated the mechanism of MUC5AC transcriptional regulation by ATBF1. In 123 gastric cancer lesions, ATBF1 expressed in the nucleus significantly suppressed MUC5AC expression, as determined by immunohistochemistry. In addition, analysis of the MUC5AC promoter region revealed an AT motif-like element. This element was found to be essential for ATBF1 suppression of MUC5AC promoter activity as shown in a dual luciferase-reporter assay. Over-expressed ATBF1 also significantly suppressed endogenous MUC5AC protein expression in gastric cancer cells. Chromatin immunoprecipitation demonstrated that ATBF1 binds to the AT motif-like element in the MUC5AC promoter. These results indicate that ATBF1 in the nucleus negatively regulates the MUC5AC gene in gastric cancer by binding to an AT motif-like element in the MUC5AC promoter. © 2007 Wiley-Liss, Inc.
  • Characteristics of the ATP-induced Ca2+-entry pathway in the t-BBEC 117 cell line derived from bovine brain endothelial cells, Daiju Yamazaki, Susumu Ohya, Kiyofumi Asai, Yuji Imaizumi, Journal of Pharmacological Sciences, 104, 103 - 107, 05 28 , ATP-receptor (P2Y) stimulation induced sustained Ca 2+ -entry, which was essential for the enhanced cell-proliferation in t-BBEC117, an immortalized cell-line derived from bovine brain endothelial cells. Application of Ca 2+ following store-depletion with thapsigargin in Ca 2+ -free solution induced Ca 2+ -entry through store-operated channels (SOCs). Ca 2+ -entry induced by ATP or 1-oleoyl-2-acetyl-sn- glycerol (OAG) together with Ca 2+ was significantly larger than that by Ca 2+ alone, suggesting the involvement of receptor-operated channels (ROCs) in the Ca 2+ -entry. Results obtained using pharmacological tools suggest that the contribution of Ca 2+ sources to ATP-induced [Ca 2 +] i rise in t-BBEC117 is estimated as approximately 2:1:2 for Ca 2+ -release and Ca 2+ -entry though SOCs and ROCs, respectively. ©2007 The Japanese Pharmacological Society.
  • Gliostatin/thymidine phosphorylase-regulated vascular endothelial growth-factor production in human fibroblast-like synoviocytes, Tomohiro Tanikawa, Yuko Waguri-Nagaya, Takuma Kusabe, Takuma Kusabe, Mineyoshi Aoyama, Kiyofumi Asai, Takanobu Otsuka, Rheumatology International, 27, 553 - 559, 04 01 , Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to elucidate whether GLS/TP is involved in the regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs) from patients with RA were cultured and stimulated with recombinant human GLS (rHuGLS) and interleukin (IL)-1β. Immunohistochemistry showed that GLS/TP and VEGF were detectable in the synovial lining cells. In cultured FLSs, both VEGF mRNA and protein levels were markedly increased by rHuIL-1β treatment. rHuGLS increased VEGF mRNA expression in a dose-dependent manner. We detected high concentrations of VEGF165 protein in culture supernatants from FLSs treated with rHuGLS (300 ng/ml), which were comparable to GLS levels found in synovial fluid of RA patients. These findings indicate that GLS/TP and VEGF have synergistic effects on angiogenesis in rheumatoid synovitis, and that GLS/TP has a role in regulating VEGF. © 2006 Springer-Verlag.
  • Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation, Hiroaki Wake, Hiroaki Wake, Miho Watanabe, Andrew J. Moorhouse, Takashi Kanematsu, Shoko Horibe, Shoko Horibe, Noriyuki Matsukawa, Kiyofumi Asai, Kosei Ojika, Masato Hirata, Junichi Nabekura, Junichi Nabekura, Junichi Nabekura, Junichi Nabekura, Journal of Neuroscience, 27, 1642 - 1650, 02 14 , The K + Cl - cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H 2 O 2 ) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg 2+ ) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl - ] i , as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors. Copyright © 2007 Society for Neuroscience.
  • 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.
  • Novel functions of small conductance Ca2+-activated K + channel in enhanced cell proliferation by ATP in brain endothelial cells, Daiju Yamazaki, Mineyoshi Aoyama, Susumu Ohya, Katsuhiko Muraki, Kiyofumi Asai, Yuji Imaizumi, Journal of Biological Chemistry, 281, 38430 - 38439, 12 15 , Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 μM ATP to t-BBEC 117 concentration-dependently increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca 2+ ] i rise were mainly due to Ca 2+ release and sustained Ca 2+ influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca 2+ -activated K + channels (SK), significantly reduced both the [Ca 2+ ] i increase and K + current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca 2+ -activated K + channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca 2+ ] i rise and the K + current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPγS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca 2+ ] i rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca 2+ entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Interleukin-1β induces the expression of aquaporin-4 through a nuclear factor-κB pathway in rat astrocytes, Hiroaki Ito, Naoki Yamamoto, Hajime Arima, Hiroyuki Hirate, Tetsuro Morishima, Fuminori Umenishi, Toyohiro Tada, Kiyofumi Asai, Hirotada Katsuya, Kazuya Sobue, Kazuya Sobue, Journal of Neurochemistry, 99, 107 - 118, 10 01 , Interleukin (IL)-1β 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-1β. We found that expression of AQP4 mRNA and protein was significantly up-regulated by IL-1β in cultured rat astrocytes, and that intracerebroventricular administration of IL-1β increased the expression of AQP4 protein in rat brain. The effects of IL-1β on induction of AQP4 were concentration and time dependent. The effects of IL-1β on AQP4 were mediated through IL-1β receptors because they were abolished by co-incubation with IL-1 receptor antagonist. It appeared that IL-1β 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-1β. Inhibition of the nuclear factor-κB (NF-κB) pathway blocked the induction of AQP4 by IL-1β in a concentration-dependent manner. These findings show that IL-1β induces expression of AQP4 through a NF-κB pathway without involvement of de novo protein synthesis in rat astrocytes. © 2006 The Authors.
  • 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
  • Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation, Cha Gyun Jung, Hye Jung Kim, Makoto Kawaguchi, Kum Kum Khanna, Hideki Hida, Kiyofumi Asai, Hitoo Nishino, Yutaka Miura, Development, 132, 5137 - 5145, 12 01 , The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers β-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.
  • The inhibitory effect of disease-modifying anti-rheumatic drugs and steroids on gliostatin/platelet-derived endothelial cell growth factor production in human fibroblast-like synoviocytes, Takuma Kusabe, Takuma Kusabe, Yuko Waguri-Nagaya, Tomohiro Tanikawa, Mineyoshi Aoyama, Muneyoshi Fukuoka, Masaaki Kobayashi, Takanobu Otsuka, Kiyofumi Asai, Rheumatology International, 25, 625 - 630, 10 01 , Gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) is known to have both angiogenic and arthritogenic activities. The purpose of this study was to investigate whether disease-modifying anti-rheumatic drugs (DMARDs) and steroids are involved in the regulation of GLS expression. Fibroblast-like synoviocytes (FLSs) obtained from patients with rheumatoid arthritis (RA) were cultured and stimulated by interleukin (IL)-1β with or without DMARDs and steroids. The expression levels of GLS were determined using the reverse transcription-polymerase chain reaction and an ELISA. In cultured rheumatoid FLSs, the expression of GLS mRNA was significantly increased by stimulation with IL-1β. By contrast, GLS mRNA levels in IL-1β-stimulated FLSs were reduced by treatment with aurothioglucose (AuTG) and dexamethasone (DEX). These findings indicate that AuTG and DEX have anti-rheumatic activity, which is mediated via the suppression of GLS production. Neither methotrexate (MTX) nor sulfasalazine (SSZ) had a significant influence on GLS levels in our study. © Springer-Verlag 2005.
  • Glia maturation factor-β is produced by thymoma and may promote intratumoral T-cell differentiation, H. Yamazaki, Hisashi Tateyama, Hisashi Tateyama, K. Asai, I. Fukai, Y. Fujii, T. Tada, T. Eimoto, Histopathology, 47, 292 - 302, 09 01 , Aims: To investigate whether Glia maturation factor-β (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 B 3 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. © 2005 Blackwell Publishing Limited.
  • Expression of myelencephalon-specific protease after cryogenic lesioning of the rat parietal cortex, Yuichi Oka, Atsushi Uchida, Mineyoshi Aoyama, Mineyoshi Aoyama, Masataka Fujita, Naokazu Hotta, Toyohiro Tada, Hiroyuki Katano, Mitsuhito Mase, Kiyofumi Asai, Kazuo Yamada, Journal of Neurotrauma, 22, 501 - 510, 04 01 , The gene for myelencephalon-specific protease (MSP) is a member of the kallikrein gene family and in rats is expressed mainly in the central nervous system. Its function and alteration in brain injury have not yet been clarified. We examined the expression of MSP after cryogenic injury (CI) using in situ hybridization, immunohistochemistry, and Western blotting. Analysis of MSP mRNA by in situ hybridization revealed a higher level of expression around the cryogenic area than on the contralateral side at 2-7 days after CI, with peak expression occurring 7 days after CI. Immunohistochemical analysis demonstrated expression of MSP protein at 1 day after CI, in the same region in which MSP mRNA was observed, with peak expression again at 7 days after CI, in the area around the lesion. Double immunohistochemical labeling revealed that MSP was expressed mainly in oligodendrocytes. These results suggest that expression of MSP may be related to the turnover of myelin-associated proteins and extracellular matrix p roteins after CI. The regulation of active MSP may be important in the physiological or pathological changes involved in remyelination or demyelination.
  • Molecular characterization of histidinemia: Identification of four missense mutations in the histidase gene, Yoko Kawai, Akihiko Moriyama, Kiyofumi Asai, Carrie M. Coleman-Campbell, Satoshi Sumi, Hideko Morishita, Mariko Suchi, Mariko Suchi, Human Genetics, 116, 340 - 346, 04 01 , Histidinemia (MIM235800) is characterized by elevated histidine in body fluids and decreased urocanic acid in blood and skin and results from histidase (histidine ammonia lyase, EC 4.3.1.3) deficiency. It is the most frequent inborn metabolic error in Japan. Although the o riginal description included mental retardation and speech impairment, neonatal screening programs have identified the majority of histidinemic patients with normal intelligence. Molecular characteristics of histidase in histidinemia have not been determined, and cytogenetically visible deletions of 12q22-24.1 in which histidase gene resides have not been identified in histidinemic patients. In order to investigate whether individuals with this disorder have small deletions, additions, or point mutations in the histidase gene, we screened genomic DNA isolated from 50 histidinemic individuals who were discovered by the neonatal screening program. The methods employed included polymerase chain reaction (PCR) amplification of exons 1-21 of the histidase gene, followed by mutation detection enhancement gel electrophoresis and sequencing of the PCR products displaying heteroduplex bands. Four missense mutations (R322P, P259L, R206T, and R208L), two exonic polymorphisms (T141T c.423A → T and P259P c.777A → G), and two intronic polymorphisms (IVS6-5T → C and IVS9+25A → G) were identified. The frequencies of each polymorphism estimated either by dot blot allele-specific oligonucleotide hybridization, restriction enzyme digestion, or direct sequencing of the PCR products amplified from 50 unrelated normal individuals were 0.28, 0.30, 0.40, and less than 0.01, respectively. Mutation analysis of one family demonstrated that the patient inherited R322P from the mother and P259L from the father. This report describes the first mutations occurring in the coding region of the histidase structural gene in patients with histidinemia. © Springer-Verlag 2005.
  • Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells, Hayato Fukuoka, Hayato Fukuoka, Mineyoshi Aoyama, Ken Miyazawa, Kiyofumi Asai, Shigemi Goto, Biochemical and Biophysical Research Communications, 328, 885 - 894, 03 25 , Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation. © 2005 Elsevier Inc. All rights reserved.
  • Expression of glia maturation factor beta after cryogenic brain injury, Naokazu Hotta, Mineyoshi Aoyama, Masaaki Inagaki, Masashi Ishihara, Yutaka Miura, Toyohiro Tada, Kiyofumi Asai, Molecular Brain Research, 133, 71 - 77, 01 05 , Glia maturation factor beta (GMFB) was identified as a growth and differentiation factor acting on neurons as well as glia. We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB-immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. These findings may provide new insight into GMFB function in pathological conditions following brain injury. © 2004 Elsevier B.V. All rights reserved.
  • Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-β, Masayuki Morikawa, John D. Fryer, Patrick M. Sullivan, Erin A. Christopher, Suzanne E. Wahrle, Ronald B. DeMattos, Mark A. O'Dell, Anne M. Fagan, Hilal A. Lashuel, Thomas Walz, Kiyofumi Asai, David M. Holtzman, David M. Holtzman, David M. Holtzman, Neurobiology of Disease, 19, 66 - 76, 01 01 , 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 HDL-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-β. Under the same conditions, only a small fraction of Aβ formed sodium dodecyl sulfate (SDS)-stable complexes with apoE (E3 > E4). These immortalized astrocytes will be useful for studying mechanisms underlying the isoform-specific effects of apoE in the CNS. © 2004 Elsevier Inc. All rights reserved.
  • Expression of myelencephalon-specific protease in transient middle cerebral artery occlusion model of rat brain, Atsushi Uchida, Yuichi Oka, Mineyoshi Aoyama, Shugo Suzuki, Takashi Yokoi, Hiroyuki Katano, Mitsuhito Mase, Toyohiro Tada, Kiyofumi Asai, Kazuo Yamada, Molecular Brain Research, 126, 129 - 136, 07 26 , Myelencephalon-specific protease (MSP) is one of the serine proteases and is expressed in the central nervous system of rats. Its function and alternation in brain injury have not yet been clarified. In this study, we investigated the expression of MSP after transient middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. In situ localization of MSP mRNA demonstrated a higher level in the corpus callosum and around the ischemic area from 12 h to 14 days after MCA reperfusion, with the peak of expression coming 3 days after reperfusion in both regions. Immunohistochemically, the expression of protein was found 1 day after reperfusion in the same brain region that was observed for mRNA. The peak was 7 days after reperfusion in both regions. Micro-autoradiography, immunostaining and double immunohistochemical labeling revealed the expression of MSP to be located mainly in the oligodendrocytes. The present results indicate that MSP may be related to the turnover of the myelin-associated proteins and the extracellular matrix proteins after transient MCAO. The activation of MSP may play a role in remodeling processes such as neurite outgrowth and remyelination. © 2004 Elsevier B.V. All rights reserved.
  • Sensitive immunoassays for human and rat GMFB and GMFG, tissue distribution and age-related changes, Masaaki Inagaki, Masaaki Inagaki, Mineyoshi Aoyama, Kazuya Sobue, Naoki Yamamoto, Tetsuro Morishima, Akihiko Moriyama, Hirotada Katsuya, Kiyofumi Asai, Biochimica et Biophysica Acta - General Subjects, 1670, 208 - 216, 02 24 , We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21-30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat. The present EIA systems are sufficiently sensitive for studying GMFs in human and rat organs. © 2004 Elsevier B.V. All rights reserved.
  • ATBF1 enhances the suppression of STAT3 signaling by interaction with PIAS3, Shunsuke Nojiri, Takashi Joh, Yutaka Miura, Nobuo Sakata, Tomoyuki Nomura, Haruhisa Nakao, Satoshi Sobue, Hirotaka Ohara, Kiyofumi Asai, Makoto Ito, Biochemical and Biophysical Research Communications, 314, 97 - 103, 01 30 , ATBF1 was first discovered as a suppressor of AFP expression in hepatocytes. It is present in brain, adult liver, lung, and gastro-intestinal tract. Recently, it has been reported that ATBF1 regulates myoblastic differentiation and interacts with v-Myb in regulation of its transactivation. Using the yeast two-hybrid system, we searched for protein-protein interactions to uncover new functions for ATBF1. We present here experimental evidence that ATBF1 is a new regulatory factor for STAT3-mediated signal transduction through its interaction with PIAS3. PIAS3 was thus identified as an ATBF1-binding protein. In co-transfection experiments, the full-length ATBF1 was found to form complexes with PIAS3 in Hep G2 cells. In the luciferase assay, ATBF1 was found to have no influence on STAT3 signaling induced by IL-6 stimulation, but it did synergistically enhance PIAS3 inhibition of activated STAT3. In conclusion, ATBF1 can suppress the IL-6-mediated cellular response by acting together with PIAS3. © 2003 Elsevier Inc. All rights reserved.
  • Susceptibility to Killer T Cells of Gastric Cancer Cells Enhanced by Mitomycin-C Involves Induction of ATBF1 and Activation of p21 (Waf1/Cip1) Promoter, Yutaka Miura, Yutaka Miura, Hiromi Kataoka, Takashi Joh, Toyohiro Tada, Kiyofumi Asai, Makoto Nakanishi, Noriko Okada, Hidechika Okada, Microbiology and Immunology, 48, 137 - 145, 01 01 , Alpha-fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re-elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP-producing gastric cancer (AFP-gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin-C (MMC) and lymphokine-activated killer T (LAK-T) cells. Treatment with MMC of AFP-gastric cancer cells enhanced their susceptibility to LAK-T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP-gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4-fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 μg/ml) treatment. The MMC tr eatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose-dependent manner up to 7-fold compared with basal activity.
  • Hyperosmolar Mannitol Stimulates Expression of Aquaporins 4 and 9 through a p38 Mitogen-activated Protein Kinase-dependent Pathway in Rat Astrocytes, Hajime Arima, Hajime Arima, Naoki Yamamoto, Kazuya Sobue, Fuminori Umenishi, Toyohiro Tada, Hirotada Katsuya, Kiyofumi Asai, Journal of Biological Chemistry, 278, 44525 - 44534, 11 07 , The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitonea l infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.
  • Structure and promoter activity of the human glia maturation factor-gamma gene: A TATA-less, GC-rich and bidirectional promoter, Yoko Kawai, Yoko Kawai, Kiyofumi Asai, Yutaka Miura, Yuichiro Inoue, Manami Yamamoto, Akihiko Moriyama, Naoki Yamamoto, Taiji Kato, Biochimica et Biophysica Acta - Gene Structure and Expression, 1625, 246 - 252, 02 20 , Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5′-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5′RACE (rapid amplification of cDNA 5′ ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity. © 2003 Elsevier Science B.V. All rights reserved.
  • Effect of mild hypothermia on the expression of aquaporin family in cultured rat astrocytes under hypoxic condition, Yoshihito Fujita, Naoki Yamamoto, Kazuya Sobue, Masaaki Inagaki, Hiroaki Ito, Hajime Arima, Tetsuro Morishima, Akinori Takeuchi, Takako Tsuda, Hirotada Katsuya, Kiyofumi Asai, Neuroscience Research, 47, 437 - 444, 01 01 , Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In this study, we investigated the effect of mild hypothermia on the expression of AQP4, AQP5 and AQP9 in rat astrocytes cultured under hypoxic conditions. At 37°C, a marked decrease in the expression of AQP4, AQP5 and AQP9 mRNAs was observed. However, at 32°C (mild hypothermia), the expression of AQP5 mRNA was restored to its basal level. Interestingly, under mild hypothermia AQP4 mRNA expression transiently decreased and then increased about two-fold; while AQP9 mRNA expression decreased the same as at 37°C. The changes in the expression of A QP4 and AQP9 proteins were confirmed by Western blot analysis. The restoration of the AQP4 and AQP5 expression at 32°C from the hypoxia-induced decrease at 37°C may play an important role in the reduction of brain edema under hypothermic conditions. © 2003 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved.
  • ACTH1-24 down-regulates expression of ciliary neurotrophic factor mRNA in cultured rat astrocyte, Minoru Kokubo, Minoru Kokubo, Kiyofumi Asai, Naoki Yamamoto, Mineyoshi Aoyama, Masayuki Morikawa, Hajime Togari, Yoshiro Wada, Taiji Kato, Pediatric Research, 52, 950 - 957, 12 01 , We examined the effects on astrocytes of ACTH, which is used to treat West syndrome. We stimulated cultured rat astrocytes with ACTH 1-24 , corticotropin-releasing factor, and dexamethasone, and examined changes in neurotrophic factor mRNAs by reverse transcription-PCR. Down-regulation of ciliary neurotrophic factor mRNA expression was observed by stimulation with ACTH 1-24 , but the expression of nerve growth factor, brain-derived neurotrophic factor, and nerotrophin-3 mRNAs was unaffected. Northern blot analysis revealed that the decrease in ciliary neurotrophic factor mRNA occurred 4 h after stimulation with more than 10 nM of ACTH 1-24 . Up-regulation of nerotrophin-3 mRNA expression was found after stimulation with 1 mM dexamethasone. These results suggest that ACTH 1-24 administrated in West syndrome may influence the expression of neurotrophic factors in astrocytes in vivo.
  • Differential regulation of aquaporin-5 and -9 expression in astrocytes by protein kinase A, Naoki Yamamoto, Kazuya Sobue, Masataka Fujita, Hirotada Katsuya, Kiyofumi Asai, Molecular Brain Research, 104, 96 - 102, 07 15 , Aquaporins (AQPs) transport water through the membranes of numerous tissues, but the molecular mechanisms for regulating water balance in brain are unknown. In this study, we investigated the effects of a protein kinase A (PKA) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with dbcAMP caused decreases in AQP5 mRNA and protein and increases in AQP9 mRNA and protein in time- and concentration-dependent manners. However, AQP4 mRNA and protein were not changed by treatment with dbcAMP. The dbcAMP-induced effects on AQP5 and AQP9 mRNAs were inhibited by PKA inhibitors. In addition, pretreating the cells with an inhibitor of protein synthesis, cycloheximide, inhibited the increase in AQP9 mRNA induced by dbcAMP, but not the decrease in AQP5 mRNA. These results suggest that signal transduction via PKA may play important roles in regulating the expression of AQP5 and AQP9, and the effect on AQP9 may be mediated by some factors induced by dbcAMP. © 2002 Elsevier Science B.V. All rights reserved.
  • Developmental changes and localization of mouse brain serine proteinase mRNA and protein in mouse brain, Takashi Yokoi, Takashi Yokoi, Takashi Yokoi, Naoki Yamamoto, Toyohiro Tada, Masataka Fujita, Akihiko Moriyama, Hitoshi Matsui, Takayuki Takahashi, Hajime Togari, Taiji Kato, Kiyofumi Asai, Neuroscience Letters, 323, 133 - 136, 04 26 , Serine proteases are known to be involved in neural development and various functions in the central nervous system. Mouse brain serine proteinase (mBSP) is expressed almost exclusively in the mouse brain and it has been characterized at the molecular and biochemical levels. In this study, we analyzed the developmental changes and localization of mBSP mRNA and protein in the mouse brain, using reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Expression of mBSP was strong in the white matter and the nerve tracts after postnatal day 30, especially in the cerebellum and the medulla oblongata. These results suggest that mBSP contributes to development and sustaining the functions in the mouse brain. © 2002 Elsevier Science Ireland Ltd. 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.
  • Epidermal growth factor down-regulates connexin-43 expression in cultured rat cortical astrocytes, Takatoshi Ueki, Takatoshi Ueki, Masataka Fujita, Masataka Fujita, Kohji Sato, Kiyofumi Asai, Kazuo Yamada, Taiji Kato, Neuroscience Letters, 313, 53 - 56, 11 02 , Astrocytes are coupled via gap junction channels, predominantly formed by connexin-43 (Cx43), and contribute to neuronal function in the normal and diseased brain. In this study, we demonstrate that epidermal growth factor (EGF), applied to cortical astrocytes, results in a decrease in the expression of Cx43 mRNA and protein. We have further shown that the decrease is associated with the receptor tyrosine kinase pathway and the MEK inhibitor prevents EGF-stimulated down-regulation of Cx43 expression. These findings demonstrate a previously unknown function of EGF on cultured astrocytes, which may be relevant to the regulation of astrocytic growth and differentiation. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
  • Differential regulation of aquaporin expression in astrocytes by protein kinase C, Naoki Yamamoto, Kazuya Sobue, Taishi Miyachi, Masaaki Inagaki, Yutaka Miura, Hirotada Katsuya, Kiyofumi Asai, Molecular Brain Research, 95, 110 - 116, 11 01 , Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [6], that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9. © 2001 Elsevier Science B.V. All rights reserved.
  • Expression of glia maturation factor during retinal development in the rat, Akiko Nishiwaki, Kiyofumi Asai, Toyohiro Tada, Takashi Ueda, Shoichi Shimada, Yuichiro Ogura, Taiji Kato, Molecular Brain Research, 95, 103 - 109, 11 01 , Glia maturation factor plays important roles in the development and growth of glia and neurons. We investigated the expression and localization of Glia maturation factor-β (GMFB) and Glia maturation factor-γ (GMFG) in the rat retina. By northern blot analysis, both GMFB and GMFG mRNAs were detected in retina as early as embryonic day (E) 18 and persisted until adult. The expression of GMFB mRNA was always much greater than that of GMFG mRNA. In situ hybridization showed that the GMFB mRNA signal was positive in the retina from E14 till adult. Immunostaining revealed that GMFB protein was present in the inner layer of retina at E14 and P1, and in Müller cells in adult. GMFG immunoreactivity was observed only in the inner limiting membrane from E14 to P1 rat retina, and was not detected in the adult retina. These results show that GMFs are synthesized and localized mainly in Müller cells in the rat retina, and suggest that they may contribute to the development and growth of glia and neurons. © 2001 Elsevier Science B.V. All rights reserved.
  • Serum gliostatin levels in patients with rheumatoid factor-negative and -positive rheumatoid arthritis and changes of these levels after surgical treatments, H. Muro, Y. Waguri-Nagaya, T. Otsuka, N. Matsui, K. Asai, T. Kato, Clinical Rheumatology, 20, 331 - 336, 10 16 , Gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) has a potential for arthritogenic action. The aim of this study was to examine whether measurement of serum GLS can be used to evaluate symptomatic improvements after surgery (arthroplasty or synovectomy) as well as the aggressiveness of disease activity in rheumatoid arthritis (RA). Serum GLS levels were determined by enzyme immunoassay in rheumatoid factor (RF)-positive and -negative RA patients. In those undergoing surgery, levels were measured 3 months before and after the operations. Both RF-positive and -negative RA sera showed higher GLS levels than normal and osteoarthritis sera. Patients undergoing arthroplasty demonstrated a decrease in serum GLS levels after the operations, but patients undergoing synovectomy did not, reflecting the extent of remaining or reproliferating synovial tissues rich in GLS production. These findings suggest that the serum GLS levels is a useful indicator for evaluation of synovitis and the systemic efficacy of surgical treatment.
  • 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.
  • Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, NRK-52E, Takahiro Yasui, Takahiro Yasui, Keiji Fujita, Keiichi Tozawa, Kiyofumi Asai, Tsuyoshi Soji, Taiji Kato, Kenjiro Kohri, Urologia Internationalis, 67, 73 - 76, 08 06 , Madin-Darby canine kidney (MDCK) cells have been used in research on crystal adhesion to epithelial cells. Recently, matrix proteins were identified, and studies of the genes and proteins expressed in renal epithelial cells have become active. The present study confirms the usefulness of the NRK-52E cell line, derived from the rat, in the study of attachment with calcium oxalate crystals. The calcium oxalate crystal suspension was distributed on top of the cells. After incubation, the monolayers were rinsed to remove non-associated crystals. After fixation, the association of crystals and NRK-52E cells was visualized using scanning electron microscopy. Calcium oxalate crystals were attached to the surface of NRK-52E cells. Under high magnification, many of the microvilli of the cells had elongated towards the crystals, and microvilli projections appeared to catch the crystals. The NRK-52E cell line is useful in the study of attachment between crystals and urinary epithelial cells in the kidney, especially for the regulation and analysis of genes and proteins. Copyright © 2001 S. Karger AG, Basel.
  • Dopamine D2 receptor inhibition of adenylyl cyclase is abolished by acute ethanol but restored after chronic ethanol exposure (Tolerance), Lina Yao, Lina Yao, Kiyofumi Asai, Zhan Jiang, Akira Ishii, Peidong Fan, Adrienne S. Gordon, Adrienne S. Gordon, Adrienne S. Gordon, Adrienne S. Gordon, Ivan Diamond, Ivan Diamond, Ivan Diamond, Ivan Diamond, Ivan Diamond, Journal of Pharmacology and Experimental Therapeutics, 298, 833 - 839, 08 02 , Dopamine D2 (D2) receptors seem to mediate reinforcing responses to addicting drugs. A stably transfected NG108-15 cell line expressing the long form of the rat brain D2 receptor (D2L) was used to determine how ethanol modifies D2 receptor coupling to adenylyl cyclase. Activation of D2L receptors by the D2 receptor-specific agonist R-(-)-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) inhibits both basal and receptor-stimulated cAMP production in these Cells. Ethanol added acutely prevents D2L receptor inhibition of cAMP production. After chronic exposure to ethanol, however, D2L receptor coupling to adenylyl cyclase becomes tolerant to rechallenge with ethanol, i.e., ethanol no longer inhibits D2L receptor coupling and NPA inhibition of cAMP production is restored. Acute ethanol does not change NPA binding to D2 receptor in cell membranes but abolishes guanosine-5′-O-(3-thio)triphosphate induction of a lower-affinity state; chronic ethanol is without effect. The protein kinase A (PKA) inhibitor adenosine 3′,5′ cyclic monophosphorothioate, Rp-isomer, prevents acute ethanol inhibition of D2L receptor coupling. In contrast, the PKA activator adenosine 3′,5′ cyclic monophosphorothioate, Sp-isomer, reverses chronic ethanol-induced tolerance of D2L receptor coupling, restoring coupling to an ethanol-sensitive state. These results suggest that D2L receptor coupling to adenylyl cyclase via G i develops tolerance to ethanol inhibition, which appears to be influenced by PKA activity.
  • A metabotropic glutamate receptor antagonist, α-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells, Hiroyuki Katano, Kaori Fujita, Taiji Kato, Kiyofumi Asai, Yasuhiro Kawamura, Atsuo Masago, Kazuo Yamada, Neuroscience Letters, 306, 101 - 105, 06 22 , 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-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX) for the α-amino-3-hydroxy-5methyl-4-isoxazole propionate /kinate receptor and (S)-α-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 μM 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. © 2001 Published by Elsevier Science Ireland Ltd.
  • 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.
  • Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation, Naoki Yamamoto, Kazuhiro Yoneda, Kiyofumi Asai, Kazuya Sobue, Toyohiro Tada, Yoshihito Fujita, Hirotada Katsuya, Masataka Fujita, Noritaka Aihara, Mitsuhito Mase, Kazuo Yamada, Yutaka Miura, Taiji Kato, Molecular Brain Research, 90, 26 - 38, 05 20 , Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury. © 2001 Elsevier Science B.V.
  • Recurrent subthreshold electrical activities of rat neocortical neurons progress during long-term culture, Keiko Nakanishi, Fumio Kukita, Kiyofumi Asai, Taiji Kato, Neuroscience Letters, 304, 85 - 88, 05 18 , The properties of neocortical neurons during long-term culture on monolayers of astrocytes were examined using whole-cell recording and immunocytochemical techniques. The soma size of neocortical neurons became larger and most neurites reached neighboring neurons within 2 weeks. Recurrent subthreshold electrical activities mediated by synaptic activation were observed at 10 days of culture and became more frequent as the neurons grew. Their frequency reached to about 1 Hz at 4 weeks. While the total number of neurons decreased during culture, the ratio of γaminobutyric acid (GABA) positive to total (MAP2 positive) neurons increased. These results suggest that neurons grown on astrocytes become mature during cultivation, and that recurrent subthreshold electrical activities may be related to the development of GABAergic inputs. © 2001 Elsevier Science Ireland Ltd.
  • Interleukin-1β induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes, Taishi Miyachi, Taishi Miyachi, Kiyofumi Asai, Hideki Tsuiki, Haruo Mizuno, Naoki Yamamoto, Takashi Yokoi, Mineyoshi Aoyama, Hajime Togari, Yoshiro Wada, Yutaka Miura, Taiji Kato, Neuroscience Research, 40, 53 - 60, 05 01 , Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-1β acts as a mediator of inflammation and has been reported as a 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-1β in cultured rat astrocytes. Time-and dose-dependent experiments of IL-1β on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1β at 8 h, 10 ng/ml. In addition, IL-1β evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1β was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 μM), phospholipase A 2 inhibitor mepacrine (30 μM) and protein synthesis inhibitor cycloheximide (CHX) (10 μg/ml). This suggests that induction of LC1 by IL-1β is through a MAPKs and phospholipaseA 2 pathway and requires protein synthesis. These results indicate that IL-1β 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-1β in brain damage. Copyright © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society.
  • Regulation of aquaporin-4 expression in astrocytes, Kazuhiro Yoneda, Kazuhiro Yoneda, Naoki Yamamoto, Kiyofumi Asai, Kazuya Sobue, Yoshihito Fujita, Masataka Fujita, Mitsuhito Mase, Kazuo Yamada, Makoto Nakanishi, Toyohiro Tada, Yutaka Miura, Taiji Kato, Molecular Brain Research, 89, 94 - 102, 04 18 , Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes. © 2001 Elsevier Science B.V.
  • 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.
  • Human neuroblastomas with unfavorable biologies express high levels of brain-derived neurotrophic factor mRNA and a variety of its variants, Mineyoshi Aoyama, Mineyoshi Aoyama, Kiyofumi Asai, Tomotane Shishikura, Takemasa Kawamoto, Taishi Miyachi, Takashi Yokoi, Hajime Togari, Yoshiro Wada, Taiji Kato, Akira Nakagawara, Cancer Letters, 164, 51 - 60, 03 10 , The expression of human brain-derived neurotrophic factor (BDNF) was investigated in 16 primary human neuroblastomas with favorable biologies, 15 with unfavorable biologies, and in human neuroblastoma cell lines. We demonstrated higher expressions of human BDNF mRNA in neuroblastomas with unfavorable biologies and with N-myc amplification than in those with favorable biologies. For the first time we revealed the composition of splice variants of human BDNF mRNA and analyzed their expression in neuroblastomas by reverse transcription polymerase chain reaction (RT-PCR). Interestingly, human BDNF mRNA consisted of at least six isoforms, four isoforms resembling those of rat BDNF mRNA, a human-specific isoform and a new isoform. The expression of four isoforms were more prominent in tumors with unfavorable biologies than in those with favorable biologies (P < 0.05). As previously we had reported, over 80% of the primary tumors expressed either the full-length form of BDNF receptor, TRKB, or a truncated form of TRKB lacking the tyrosine kinase domain. The full-length TRKB was predominantly detected in tumors with unfavorable biologies, and the truncated one in those with favorable biologies. These results suggest that an autocrine and/or paracrine mechanism involving BDNF may stimulate signal transduction via TRKB receptors rich in neuroblastomas with unfavorable biologies, resulting in an aberrant survival of tumor cells. © 2001 Elsevier Science Ireland Ltd.
  • Isolation and characterization of a new immortal rat astrocyte with a high expression of NGF mRNA, Masayuki Morikawa, Masayuki Morikawa, Kiyofumi Asai, Minoru Kokubo, Kaori Fujita, Kazuhiro Yoneda, Naoki Yamamoto, Yuichiro Inoue, Yuichiro Inoue, Junzo Iida, Toshifumi Kishimoto, Taiji Kato, Neuroscience Research, 39, 205 - 212, 02 26 , We have established a new line of immortalized rat astrocytes through transfection of plasmid pSV3-neo encoding the large T antigen of simian virus 40 into normal astrocytes. One of these immortalized astrocytes (ACT-57) with a flat and polygonal cell shape, exhibited stable growth in a chemically defined medium (modified N-2 medium) as well as in medium containing ordinary serum. ACT-57, retained a detectable level of expression of glial fibrillary acidic protein (GFAP) and its mRNA, and exhibited a stronger expression of nerve growth factor (NGF) mRNA than that of normal rat astrocytes or C6 glioma cells. NGF mRNA was significantly up-regulated by phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and γ-amino-n-butyric acid (GABA) but not by hydrocortisone. None of stimulants (TPA, dibutyryl cyclic AMP (db-cAMP), hydrocortisone, L-glutamate, carbacol, GABA, dopamine, or isoproterenol) changed the expression level of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). There was a discrete difference between ACT-57 and normal astrocytes in the response to GABA and isoproterenol. These findings imply that normal cortical astrocytes possess a functional heterogeneity whereas the clonal astrocyte, ACT-57, does not, indicating that ACT-57 cells may be useful for in vitro studies of neuron-astrocyte interactions involving the induction of neurotrophic factors such as NGF. Copyright © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society.
  • Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1, Hiromi Kataoka, Yutaka Miura, Takashi Joh, Kyoji Seno, Toyohiro Tada, Taiki Tamaoki, Hidekazu Nakabayashi, Makoto Kawaguchi, Kiyofumi Asai, Taiji Kato, Makoto Itoh, Oncogene, 20, 869 - 873, 02 15 , Alpha-fetoprotein (AFP) producing gastric cancer (AFP-GC) is very malignant and highly metastatic compared with common gastric cancer. However, the causal relationship between AFP production and the high malignancy of AFP-GC is unclear. We investigated AFP gene regulation in AFP-GC by an active transcription factor, HNF1 (hapatocyte nuclear factor 1) and a repressive transcription factor, ATBF1 (AT motif binding factor 1). RNase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Methylation analysis of the AFP promoter region in gastric cancer cells suggested that methylation itself could not explain the silencing of the AFP gene. Immunohistochemistry of resected clinical samples revealed that AFP producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-GC and might be important for its highly malignant nature.
  • 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.
  • Regulation of rat hippocampal neural cadherin in the kainic acid induced seizures, Masataka Fujita, Noritaka Aihara, Noritaka Aihara, Manami Yamamoto, Takatoshi Ueki, Kiyofumi Asai, Toyohiro Tada, Taiji Kato, Kazuo Yamada, Neuroscience Letters, 297, 13 - 16, 01 05 , Regulation of neural (N-) cadherin expression in the hippocampus was examined by in situ hybridization and immunohistochemistry methods in the rat model of kainic acid (KA) induced seizures. After 12 and 24 h of KA administration, mRNA expression level of N-cadherin decreased in the hippocampal CA1 and CA3 area in parallel with decrease of the number of neural cells. In contrast, after 48 h and 7 days, mRNA expression level recovered partially, although the number of neural cells remained small. In addition, immunohistochemical staining indicated that N-cadherin protein expression of survived neurons increased significantly after 48 h of KA administration. These results indicated that N-cadherin might be involved in neuronal reconstruction at the hippocampus. Copyright (C) 2001 Elsevier Science Ireland Ltd.
  • Synovial inflammation and hyperplasia induced by gliostatin/platelet-derived endothelial cell growth factor in rabbit knees, Y. Waguri-Nagaya, T. Otsuka, I. Sugimura, N. Matsui, K. Asai, K. Nakajima, T. Tada, S. Akiyama, T. Kato, Rheumatology International, 20, 13 - 19, 12 28 , Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA). The objective of this study was to examine synovial inflammation in rabbit knees induced by intra-articular administration of human gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF), which shares a high degree of chemical homology with thymidine phosphorylase (dThdPase) and is known to have angiogenic activity. Purified recombinant human gliostatin (rHuGLS) and its mutant protein, which was prepared by site-directed mutagenesis and which lacks dThdPase activity, were administered at various doses to rabbit knee joints. The effects of rHuGLS and the mutant were examined histologically. Intra-articular injection of rHuGLS resulted in the development of diffuse synovitis resembling RA. The mutant protein also brought about the same effect. These findings suggest that human GLS can cause RA-like synovitis in rabbit knee joints via a mechanism other than its dThdPase activity.
  • Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture, Taeko Hotta, Kiyofumi Asai, Kaori Fujita, Taiji Kato, Taiji Kato, Haruhiro Higashida, Journal of Neurochemistry, 74, 669 - 675, 01 29 , ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [ 3 H]cyclic ADP-ribose production converted from [ 3 H]NAD + by ADP-ribosyl cyclase (2.015 ± 0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained ~50% of the total cyclase activity in astrocytes. The formation rate of [ 3 H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD + by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD + in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [ 3 H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol- induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of -adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.
  • AT motif binding factor 1-A (ATBF1-A) negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine, Hiromi Kataoka, Takashi Joh, Yutaka Miura, Taiki Tamaoki, Kyoji Senoo, Hirotaka Ohara, Tomoyuki Nomura, Toyohiro Tada, Kiyofumi Asai, Taiji Kato, Makoto Itoh, Biochemical and Biophysical Research Communications, 267, 91 - 95, 01 07 , This is the first study to demonstrate that the AT motif binding factor 1-A (ATBF1-A) is expressed in the crypts and the bases of villi of the small intestine and negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N (APN). In situ hybridization visualized a limited ATBF1-A mRNA expression in the crypts and the bases of villi. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1-A suppressed the activity of APN promoter, but did not that of AT motif deleted promoter. These results imply that ATBF1-A inhibits the transcription of APN gene through its direct binding to the AT motif element. Furthermore, butyrate-induced differentiation of Caco-2 cells, retaining the enterocytic phenotypes such as a villus structure and the expression of brush-border enzymes, leads to a reduced expression of ATBF1-A mRNA. We proposed that ATBF1-A regulating APN gene expression in the crypt-villus axis of the small intestine is a landmark of enterocyte differentiation and maturation. (C) 2000 Academic Press.
  • 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.
  • Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells, Kazuya Sobue, Kazuya Sobue, Naoki Yamamoto, Naoki Yamamoto, Kazuhiro Yoneda, Kazuhiro Yoneda, Kaori Fujita, Yutaka Miura, Kiyofumi Asai, Takako Tsuda, Hirotada Katsuya, Taiji Kato, Biochimica et Biophysica Acta - Gene Structure and Expression, 1489, 393 - 398, 12 23 , Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5'-genomic sequence analysis showed that the 5'-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung. Copyright (C) 1999 Elsevier Science B.V.
  • Autocrine induction of gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) and GLS-induced expression of matrix metalloproteinases in rheumatoid arthritis synoviocytes, H. Muro, Y. Waguri-Nagaya, Y. Waguri-Nagaya, Y. Mukofujiwara, T. Iwahashi, T. Otsuka, N. Matsui, A. Moriyama, K. Asai, T. Kato, Rheumatology, 38, 1195 - 1202, 12 01 , Objective. The purpose of this study was to examine how gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) is involved in the molecular mechanism of cartilage degradation in rheumatoid arthritis (RA) with special reference to the GLS-induced gene expression and protein synthesis of matrix metalloproteinase (MMP)-1 (collagenase-1) and MMP-3 (stromelysin-1). Methods. Fibroblast-like synoviocytes (FLSs) obtained from RA patients were cultured and stimulated by GLS. Changes in the expression levels of GLS, MMP-1 and MMP-3 were assessed by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) for GLS, and by RT-PCR and enzyme-linked immunosorbent assay for MMPs and tissue inhibitor of metalloproteinase 1. Results. GLS demonstrated a self-induction of mRNA in cultured RA FLSs. GLS evoked a dose-dependent induction of MMP-1 and MMP-3 mRNAs, and subsequently their extracellular secretion. Conclusion. These findings suggest that GLS is a plausible pathogenic factor causing the extensive joint destruction in RA mediated via MMPs.
  • Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors, Kazuya Sobue, Naoki Yamamoto, Kazuhiro Yoneda, Mark E. Hodgson, Kyoko Yamashiro, Nobuo Tsuruoka, Takako Tsuda, Hirotada Katsuya, Yutaka Miura, Kiyofumi Asai, Taiji Kato, Neuroscience Research, 35, 155 - 164, 11 01 , The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t- BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.
  • Traumatic injury in vitro induces IEG mRNAs in cultured glial cells, suppressed by co-culture with neurons, Hiroyuki Katano, Kaori Fujita, Taiji Kato, Kiyofumi Asai, Yasuhiro Kawamura, Atsuo Masago, Kazuo Yamada, NeuroReport, 10, 2439 - 2448, 08 20 , Glial changes following traumatic injury to glial monolayers as well as to neuronal-glial co-culture systems in vitro were examined with a focus on the expression of mRNAs coding for the immediate early genes (IEG) c-fos, c- jun and zif/268, demonstrated using in situ hybridization. Glial cells along scratch wound lines extended cytoplasmic processes as early as 10 min post- injury and the whole wound was covered with gliosis by 24h. For complete restoration in the case of glial cells co-cultured with neurons, this required 48h. Induction of the threw IEG mRNAs was eminent along the edges of scratch wound, peaking at 30-60 min post-injury and subsiding by 3 h. The peak expression of IEG mRNAs was delayed to 1-3h post-injury and became undetectable at 6 h in neuronal-glial co-cultures. The data suggest that mechanical injury to glial cells causes gliosis and the expression of IEG mRNAs, which are suppressed by co-culture with neurons, indicating some influence of neuronal-glial interactions.
  • Experimental implication of celiac ganglionotropic invasion of pancreatic-cancer cells bearing c-ret proto-oncogene with reference to glial- cell-line-derived neurotrophic factor (GDNF), Yuji Okada, Yuji Okada, Hiromitsu Takeyama, Mikinori Sato, Masayuki Morikawa, Kazuya Sobue, Kiyofumi Asai, Toyohiro Tada, Taiji Kato, Tadao Manabe, International Journal of Cancer, 81, 67 - 73, 03 22 , Perineural invasion is a prominent clinical feature of pancreatic cancer which causes difficulty in curative resection. In the present study, the human pancreatic cancer cell lines, PaCa-2, AsPC-1, SW1990 and Capan-2, were all found to express abundant c-ret proto-oncogene mRNA and RET protein, a member of the receptor-tyrosine-kinase superfamily, identified as being a receptor for glial-cell-line-derived neurotrophic factor (GDNF). In an invasion assay, the migration of pancreatic cancer cells was markedly induced by co-cultivation with human glioma cells, T98G or A172, capable of producing and secreting Gdnf. Anti-GDNF antibody in conditioned media of glioma cells suppressed much of the migratory activity. Checkerboard analysis of the migration showed both chemotactic and chemokinetic activity of GDNF. There was no detectable expression of another GDNF receptor component, a glycosyl- phosphatidylinositol-linked receptor (GFRα-I), in pancreatic-cancer cell lines, suggesting that the neural invasion of pancreatic-cancer cells spreads along a concentration gradient of GDNF produced from peripheral ganglions through direct interaction of GDNF with its receptor, the c-ret proto- oncogene product. Immunochemical localization of GDNF in human celiac ganglionic tissue supported this contention.
  • Neurotrophic action of lipocortin 1 derived from astrocytes on cultured rat cortical neurons, Haruo Mizuno, Kiyofumi Asai, Kaori Fujita, Kenji Uemura, Yoshiro Wada, Akihiko Moriyama, Hisamitsu Ogawa, Shigeki Kimura, Taiji Kato, Molecular Brain Research, 60, 28 - 39, 09 18 , The lipocortins are a family of structurally related proteins, namely an annexin family, that exerts a variety of cellular functions through Ca 2+ -dependent binding to phospholipase A 2 [EC 3.1.1.4], including a crucial role in the central nervous system (CNS) such as antipyrogenic, thermoregulatory and neuroprotective agents in vivo. To elucidate the paradigm of lipocortin 1 functions in the CNS, we have first demonstrated (1) the induction and subsequent extracellular secretion of LC1 by glucocorticoid in cultured rat astrocytes, and (2) neurotrophic activities (survival-promoting, neuritogenic and synaptogenic actions on rat cortical neurons) of recombinant LC1. Time-and dose-dependent experiments of a synthetic glucocorticoid, dexamethasone (DEX), on rat cortical astrocytes in culture revealed that the expression of the intracellular LC1 mRNA and protein were significantly augmented by DEX (1 μM). In addition, DEX evoked an extracellular secretion of LC1 without its cytotoxic effects. Furthermore, the recombinant LC1 appeared to promote not only the survival and neurite outgrowth but also the synaptogenesis of embryonal rat cortical neurons. These results suggest that LC1 induced and selectively released from astrocytes by either endogenously or exogenously introduced glucocorticoids may play a specific and essential role on development and regeneration of the central nervous system.
  • Expression of mRNA for heregulin and its receptor, ErbB-3 and ErbB-4, in human upper gastrointestinal mucosa, Hiromi Kataoka, Takashi Joh, Kunio Kasugai, Naotsuka Okayama, Akihiko Moriyama, Kiyofumi Asai, Taiji Kato, Life Sciences, 63, 553 - 564, 07 10 , Expression of mRNA for heregulin (HRG), a member of the epidermal growth factor (EGF) family and its receptors, ErbB-3 and ErbB-4, were evaluated in human upper gastrointestinal (GI) mucosa. Multi -target reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using capillary electrophoresis and laser-induced fluorescence allowed us to quantify the minute amounts of mRNA from one biopsy specimen with high sensitivity. HRG, ErbB-3 and ErbB-4 mRNA were detected in esophagus, stomach and duodenum and the highest expression was found in duodenum. In gastric cancer, mRNA for ErbB-4 was significantly overexpressed. Immunoreactivity of ErbB-4 in carcinoma cell membrane was also confirmed. These findings suggest that HRG and its receptors, ErbB-3 and ErbB-4 may be physiologically significant in the human upper GI mucosa, especially in duodenum, and that ErbB-4 may contribute to the growth of gastric cancer.
  • 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-γ) homologous to Glia Maturation Factor-β gene, Kiyofumi Asai, Kaori Fujita, Manami Yamamoto, Taeko Hotta, Masayuki Morikawa, Minoru Kokubo, Akihiko Moriyama, Taiji Kato, Biochimica et Biophysica Acta - Gene Structure and Expression, 1396, 242 - 244, 03 13 , A novel full-length human cDNA homologous to Glia Maturation Factor-β (GMF-β) 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-β (82% identity) and named for GMF-γ. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-β, is predominantly expressed in human lung, heart, and placenta.
  • Varying effects of ethanol on transfected cell lines, Kiyofumi Asai, Akira Ishii, Lina Yao, Ivan Diamond, Adrienne S. Gordon, Adrienne S. Gordon, Alcoholism: Clinical and Experimental Research, 22, 163 - 166, 02 01 , The use of transfected cell lines has provided a powerful approach to study the functions of transfected proteins. This approach has also proven useful to determine the effects of acute and chronic ethanol exposure on particular proteins. We show here, however, that the effects of ethanol on transfected proteins vary between transiently transfected cells and stably trensfected cell lines. Moreover, we found that the effect of ethanol on a particular protein depends on the plasmid vectors used for the transfection.
  • Molecular biology of blood-brain barrier, K. Sobue, H. Katsuya, K. Asai, T. Kato, Neurological Surgery, 26, 561 - 569, 01 01
  • Abnormal glycosylation of IgG as a clinical parameter in patients with rheumatoid arthritis: Its constitutional analysis by HPLC, Yuka Mukofujiwara, Takanobu Otsuka, Yuko Waguri, Nobuo Matsui, Kiyofumi Asai, Taiji Kato, Hiroko Araki, Yoshinori Tsukamoto, Noriko Takahashi, Journal of Clinical Biochemistry and Nutrition, 25, 131 - 142, 01 01 , To determine the abnormal glycosylation patterns of IgG in patients with rheumatoid arthritis (RA), we analyzed these oligosaccharide profiles using a recently established high-performance liquid chromatography (HPLC) method. Oligosaccharides of IgG proteins purified from sera of RA patients were labeled with a fluorescent reagent, 2-aminopyridine. The oligosaccharide derivatives were separated into 12 peaks by HPLC, and compared with those of age-matched controls. Serum IgG from patients with RA (RA-IgG) contained a higher content of oligosaccharides with bisecting GlcNAc than normal IgG, which was accompanied by an increase in the glycosylation of the bisected oligosaccharides. The ratio of the glycosylation of bisected to nonbisected oligosaccharides correlated with RA disease activity as well as with its clinical markers. This ratio reflecting the balance of glycosylation between bisected and nonbisected oligosaccharides may be a useful clinical parameter to monitor RA activity.
  • 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.
  • Evidence for participation of gliostatin/platelet-derived endothelial cell growth factor in gastric ulcer healing, Kunio Kasugai, Takashi Joh, Takashi Joh, Hiromi Kataoka, Makoto Sasaki, Toyohiro Tada, Kiyofumi Asai, Taiji Kato, Makoto Itoh, Life Sciences, 61, 1899 - 1906, 10 03 , Gliostatin (GLS)/Platelet-derived endothelial cell growth factor (PD-ECGF) is a protein factor that has angiogenic and thymidine phosphorylase activity. It has been recently demonstrated to be related to disease activity in rheumatoid arthritis. However, its physiological role in the gastric mucosa is unknown. In the present study, concentrations of this protein in human gastric mucosa and plasma were evaluated. Further, the effect of purified human GLS/PD-ECGF on experimental ulcer healing was investigated in the rat. The human plasma concentration of GLS/PD-ECGF was significantly higher in patients with intractable gastric ulcer than in patients with significant resolution. The tissue content was significantly higher at the gastric ulcer edge than in either the fundic or pyloric region. GLS/PD-ECGF infusion delayed ulcer healing in a dose-dependent manner. These results suggest that gastric tissue and/or circulating GLS/PD-ECGF may participate in pathology and etiology of gastric ulcers and that this mechanism may relate to the pathogenesis of RA.
  • Specific detection of κ light chain in uric acid stones, Yoshihiro Hashimoto, Kenjiro Kohri, Yutaro Hayashi, Akihiko Moriyama, Masanori Iguchi, Kiyofumi Asai, Taiji Kato, Life Sciences, 61, 249 - 253, 06 13 , 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 α heavy and κ light chains. Although immunoglobulin heavy chain (γ and μ, as well as α) and a κ light chain were clearly detected in uric acid stones by Western blotting using their specific antibodies, no λ chains whatsoever could be detected. The results suggest that immunoglobulins selectively containing κ light chain might have specific functions in uric acid stone formation as stone matrices.
  • Gliostatin/platelet-derived endothelial cell growth factor as a clinical marker of rheumatoid arthritis and its regulation in fibroblast-like synoviocytes, Y. Waguri, T. Otsuka, I. Sugimura, N. Matsui, K. Asai, A. Moriyama, T. Kato, British Journal of Rheumatology, 36, 315 - 321, 03 01 , The objective was to assess the congruity of gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF) with other clinical markers of rheumatoid arthritis (RA) and to define its molecular mechanism of action in the complicated cytokine network during RA pathogenesis. Immunoassay systems were used to quantify GLS or cytokine levels in laboratory and clinical samples. Expression levels of GLS were determined by reverse transcription-polymerase chain reaction methods. The GLS levels in synovial fluid were correlated with interleukin-1 (IL-1) and IL-8. The serial data of serum GLS levels reflected well changes in the disease activity during the clinical course of four representative patients with RA. In cultured fibroblast-like synoviocytes, tumour necrosis factor-α (TNF-α), IL-1, IL-6 and IL-8 induced GLS expression. In conclusion, our results suggest that the serum GLS level, mostly derived from cytokine-stimulated synoviocytes, was a useful clinical marker of RA.
  • Glucocorticoid induced the expression of mRNA and the secretion of lipocortin 1 in rat astrocytoma cells, Haruo Mizuno, Kenji Uemura, Akihiko Moriyama, Yoshiro Wada, Kiyofumi Asai, Shigeki Kimura, Taiji Kato, Brain Research, 746, 256 - 264, 01 23 , The lipocortins area family of structurally related proteins that have been shown to be implicated in multiple aspects of cell biology. Subsequent research has shown that lipocortin 1 (LC1) participates in the physiological and pathological functioning of the CNS and neuroendocrine system. In the present study, the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), dibutyryl cyclic AMP (Bt 2 cAMP) or dexamethasone (DEX) on expression of LC1 were investigated by a sandwich enzyme-immunoassay and reverse transcription polymerase chain reaction (RT-PCR) in rat astrocytoma (C6) cells time-dependent experiments revealed that the intracellular protein content and the mRNA of rat LC1 increased significantly 4 h after TPA (10 nM) or DEX (1 μM) addition. TPA and DEX elicited a prominent induction of LC1 at 10 -8 M and 10 -6 M, respectively. Bt 2 cAMP (0.5 mM) also appeared to induce, but the induction was not statistically significant. In addition, DEX increased the extracellular secretion of LC1 without cytotoxicity. These results suggest that LC1 synthesis is chemically induced and selectively released from C6 cells by dexamethasone.
  • Tissue distribution of human gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) and its drug-induced expression, Kohei Matsukawa, Akihiko Moriyama, Yoko Kawai, Kiyofumi Asai, Taiji Kato, Biochimica et Biophysica Acta - Molecular Cell Research, 1314, 71 - 82, 11 08 , Human tissue contents of gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF) and its drug-induced expression in tumor cells were currently examined by a sandwich enzyme immunoassay (EIA) system and a reverse transcription-polymerase chain reaction (RT-PCR) method. Gliostatin/PD-ECGF was found to distribute in rather ubiquitous than specific human tissues and organs, with a relatively high levels in the tissues of digestive system (esophagus and rectum), brain, spleen, bladder and lung, but not in gall bladder, aorta, muscle, fat and kidney. Most of examined human tumor cell fines showed 4- or 5-fold higher contents (21.5 ± 3.9 ng/mg protein) than normal tissue contents (4.4 ± 1.1 ng/mg protein) on the average. While gliostatin/PD-ECGF is known to lack a signal sequence, some tumor cells (A431 and MKN74) appeared to release it into the conditioned medium. Expression of gliostatin/PD-ECGF in epidermoid carcinoma cell (A431) and stomach cancer cell (MKN45) was induced by dibutyryl cyclic AMP and phorbol ester, and uniquely in MKN45 by hydrocortisone. In particular, this hydrocortisone specifically caused an increase of the apparent secretion of MKN74 without its cytotoxic effects, suggesting a possible secretion of gliostatin/PD-ECGF in the restricted but not universal cell line. Biological significance on the chemical induction of gliostatin/PD-ECGF in tumor cells and on its extracellular secretion are discussed.
  • Heat shock-mediated cell cycle arrest is accompanied by induction of p21 CKI, Takahisa Fuse, Takahisa Fuse, Kazuo Yamada, Kiyofumi Asai, Taiji Kato, Makoto Nakanishi, Biochemical and Biophysical Research Communications, 225, 759 - 763, 08 23 , Heat shock induces several events in cells including a series of gene expressions and cell cycle arrest. Recently, several types of cell cycle arrest have been related to the function of cyclin-dependent kinase inhibitors (CKI). Here we show that heat shock treatment up-regulates p21 CKI mRNA and protein in A172 glioma cells and arrests the cell cycle at the G1 phase. p53-deficient cell lines, MDAH041 and T98G, also showed a significant increase in p21 CKI expression after heat stress, indicating that the induction involves a p53-independent pathway. The kinetics of this transient induction, which is not affected by cycloheximide, demonstrate that p21 CKI is an immediate-early response gene.
  • 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.
  • Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+oscillations, Keiko Nakanishi, Yuka Okouchi, Takatoshi Ueki, Kiyofumi Asai, Ichiro Isobe, Yaman Z. Eksioglu, Taiji Kato, Yasuhiro Hasegawa, Yoichiro Kuroda, Brain Research, 659, 169 - 178, 10 03 , Glial contribution to in vitro synaptic function was investigated in a neuron-glia co-culture system by monitoring spontaneous oscillations of intracellular Ca 2+ in neurons. Rat cortical neurons, grown stably on a cortical astrocyte monolayer, extended neurites resulting in marked functional synapse formation. Little synapse formation was observed in neuronal co-culture with meaningeal fibroblasts or endothelial cells. Aged astrocytes in vitro (C35) were found to attenuate synaptic development, while young astrocytes (C5) markedly promoted synaptic function. C5 and C35 astrocyte media conditioned yielded no significant synaptogenic effect, indicating diffusible factor(s) are not responsible for our observation. Modulation of astrocytic proliferation and differentiation by gliostatin, a glial growth inhibitor, or dibutyryl cAMP affected neuronal synaptic function on the co-cultures. Site-specific analysis in homologous and heterologous neuron-astrocyte co-cultures among cortex, hippocampus, septum, and striatum revealed that homologous combinations of neurons and astrocytes derived from identical brain regions elicited the largest number of synchronizing neur̀ons. Thee results suggest that in vivo neuronal synaptic function essentially requires the participation of adjacent astrocytes, which is site-specific and age-dependent. © 1994.
  • Human neuroblastoma growth inhibitory factor (h-NGIF), derived from human astrocytoma conditioned meduim, has neurotrophic properties, Yaman Z. Eksioglu, Junko Iida, Kiyofumi Asai, Takatoshi Ueki, Keiko Nakanishi, Ichiro Isobe, Kazuo Yamagata, Taiiji Kato, Brain Research, 644, 282 - 290, 05 02 , Investigations on the general characteristics of human astrocytoma cell line NAC-1 revealed neuroblastoma growth inhibitory activity in conditioned medium. Neuroblastoma growth inhibitory factor (NGIF) was partially purified by Econo Q, Econo CM, and Superose 12 column chromatography. The protein is weakly basic with an estimated M r of 120,000, possibly having an M r 60,000 dimeric structure. NGIF inhibits the growth of neurobalstoma cell lines but has no effect on morphology nor does it produce any change in the growth of human glioblastoma cell lines. Interstingly, NGIF appears to promote survival and neurite outgrowth of embryonal rat cortical neurons. These neurotrophic properties suggest a role for NGIF in the development of the nervous system. © 1994.
  • 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
  • Neurotrophic action of gliostatin on cocultured neurons with glial cells, Takatoshi Ueki, Keiko Nakanishi, Kiyofumi Asai, Yuka Okouchi, Ichiro Isobe, Yaman Z. Eksioglu, Taiji Kato, Kunio Kohno, Brain Research, 622, 299 - 302, 09 17 , Gliostatin is a polypeptide factor (apparent M r = 100 k with a homodimeric structure comprising two 50 kDa subunits) acting on cortical neurons (neurotrophic action) as well as astrocytic cells (growth inhibition). Under the coculture system of cerebral cortical neurons and astrocytes from fetal rats (E15 or E16), the neurotrophic action of gliostatin was examined immunocytochemically. Immunostaining by an anti-neurofilament (NF) monoclonal antibody visualized a marked neurite-outgrowth and interconnecting bundles of neuritic processes induced by gliostatin in the coculture system. Neurons stimulated by gliostatin formed dense aggregates in clumps, while neurons in control coculture spread out. Gliostatin has also shown survival-promoting effects on neurons. Furthermore, it was shown that gliostatin induced the differentiation of protoplasmic astrocytes to fibrous astrocytes. These results further support our previous contention that gliostatin plays physiological roles on neuronal and glial development. © 1993.
  • High concentrations of immunoreactive gliostatin/platelet-derived endothelial cell growth factor in synovial fluid and serum of rheumatoid arthritis, Kiyofumi Asai, Takayoshi Hirano, Kohei Matsukawa, Jun ichi Kusada, Masanori Takeuchi, Takanobu Otsuka, Nobuo Matsui, Taiji Kato, Clinica Chimica Acta, 218, 1 - 4, 09 17 , Since neovascularization plays an important role in the propagation of rheumatoid synovitis, we analyzed the concentration of gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF), a potent angiogenic and chemotactic factor, in the synovial fluid and serum of rheumatoid arthritis (RA) patients. The immunoreactive GLS/PD-ECGF concentrations (mean value ±S.D.) in synovial fluid, measured by a sandwich enzyme immunoassay, were significantly higher in RA patients than in osteoarthritis (OA) patients (233.02±219.40 vs 9.09 ± 14.86 ng/g, P < 0.001), and the serum concentrations were also higher in RA patients than in age-matched controls (8.77 ± 7.60 vs. 3.74 ±2.61 g/ml, P > 0.005). These results suggest that GLS/PD-ECGF may participate in the endothelial proliferation resulting in initiation of the extensive emigration of mononuclear cells and proliferation of the synovial tissues in rheumatoid arthritis, and that the immunoreactive GLS/PD-ECGF in serum as well as synovial fluids may be a useful diagnostic marker of RA. © 1993.
  • 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.
  • Growth‐Promoting Action of Adenosine‐Containing Dinucleotide on Neuroblastoma Cells: Detection of Adenosine‐Cytidine Dinucleotide (ApCp) in Neurofibroma (NF1) Extracts, Taeko Hotta, Kiyofumi Asai, Naohito Takeda, Hideo Yoshizumi, Akira Tatematsu, Keiko Nakanishi, Yaman Z. Eksioglu, Ichiro Isobe, Taiji Kato, Journal of Neurochemistry, 61, 1430 - 1437, 01 01 , Abstract: 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. Copyright © 1993, Wiley Blackwell. All rights reserved
  • 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
  • Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth, Taeko Hotta, Kiyofumi Asai, Naohito Takeda, Akira Tatematsu, Keiko Nakanishi, Yaman Z. Eksioglu, Ichiro Isobe, Taiji Kato, Journal of Neurochemistry, 60, 312 - 319, 01 01 , Abstract: Human glioma cell extracts were found to elicit a marked growth‐promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography‐mass spectrometry. The authentic uridine showed a strong growth‐promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses. Copyright © 1993, Wiley Blackwell. All rights reserved
  • 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
  • von Recklinghausen neurofibroma produces neuronal and glial growth-modulating factors, Kiyofumi Asai, Taeko Hotta, Keiko Nakanishi, Jin ichi Ito, Ryo Tanaka, Takanobu Otsuka, Nobuo Matsui, Taiji Kato, Brain Research, 556, 344 - 348, 08 16 , Two kinds of novel neural trophic factors were currently detected in von Recklinghausen neurofibroma (NF1) extracts. One of the two was a growth factor, neuroblastoma growth factor (M r < 5kDa), which promotes the proliferation of human neuroblastoma cell and survival and neurite-extension of rat cortical neurons, but differently from nerve growth factor (NGF) of NGF-like factors. The other one was a glial growth inhibitor (M r =100 kDa), which suppresses the growth of glioma cell lines, astrocytoma, glioblastoma, oligodendroglioma and Schwannoma. These factors do not appear to be previously identified cytokines or growth factors such as interleukins, granulocyte colony-stimulating factor, NGF and fibroblast growth factor. There was also detectable ciliary neurotrophic factor-like activity in the extracts. The primary cause of high contents of these factors in NF1 is not known, but may relate to fundamental mechanisms controlling growth and differentiation of neurons and glias during development of nervous system. © 1991.
  • Neural trophic factors and neuron-glia interaction, K. Asai, T. Kato, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 36, 1220 - 1226, 05 01
  • Probable Assignment of the Dihydropteridine Reductase Gene to 4p15, Satoshi Sumi, Tatsuya Ishikawa, Yasuhiko Ito, Hidetsune Oishi, Kiyofumi Asai, Yoshiro Wada, Tohoku Journal of Experimental Medicine, 160, 93 - 94, 01 01 , SUMI, S., ISHIKAWA, T., ITO, Y., OISHI, H., ASAI, K. and WADA, Y. Probable Assignment of the Dihydropteridine Reductase Gene to 4p15.31. Tohoku J. Exp. Med., 1990, 160 (1), 93-94—We report the dihydropteridine reductase (DHPR) activity in cases with interstitial deletion of the short arm of chromosome 4. Case 1 with the segment 4p15.32-4p16 deleted has normal DHPR activity. Case 2 with 4p15.2-4p15.32 deleted has reduced DHPR activity, less than 50% of normal.—dihydropteridine reductase ; chromosome 4. © 1990, Tohoku University Medical Press. All rights reserved.
  • 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
  • Urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency, quantified by a carboxylic acid analyzer with a reversed-phase column, Kiyoshi Kidouchi, Toshimitsu Niwa, Daisuke Nohara, Kiyofumi Asai, Naruji Sugiyama, Hideko Morishita, Masanori Kobayashi, Yoshiro Wada, Clinica Chimica Acta, 173, 263 - 272, 04 29 , A quantitative analysis for urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency is described. This method (liquid chromatography) can quantify twelve acylcarnitines including glutarylcarnitine and 3 isomeric acylcarnitines (butyryl-1, valeryl- and octanoylisomer) in urine. Before and up to the 15th hour of DL-carnitine therapy, isovalerylcarnitine was the largest single component existing in urinary acylcarnitines. Its excretion increased approximately 10 times within 1 day of DL-carnitine therapy. However, the acetyl-, the isobutyryl- and the butyrylcamitine values increased gradually. From the 8th day of the therapy, the isobutyrylcarnitine value exceeded the isovalerylcarnitine. The patient's dominant urinary specific acylcarnitine derived from amino acids oxidation deficiency was changed from isovalerylcarnitine(leucine) to isobutyrylcarnitine (valine) during the early period of DL-carnitine therapy. Glutarylcarnitine was a minor component in the urine. Its degree of increase was as small as that of octanoylcarnitine. 2-Methylbutyrylcarnitine and propionylcarnitine were not detected. © 1988.

Misc

  • Identification of Benzoylcarnitine in the urine of a patient of hyperammonemia., Tohoku J. Exp. Med., 159, (2) 147 - 151,   1989
  • Molecular cloning of human UMP synthetase., Purine and pyrimidine metabolism in man VI,   1989
  • Identification of immuno-reactive lipocortine 1-like molecules in serum and plasma by an enzyme immunoassay for lipocortine 1., Biochim. Biophys. Acta, 1119,   1992
  • Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+ oscillations., Brain Res, 659,   1994
  • Autocrine induction of gliostatin/platelet-derived endothelial cell growth factor(GLS/PD-ECGF)and GLS-induced expression of matrix metalloproteases in rheumatoid arthritis synoviocytes., Rheumatology, 38,   1999
  • An experimental implication of celiac ganglionotropic invasion of pancreatic cancer cells bearing c-ret prote-oncogene with reference to glial cell line-derived neurotrophic factor(GDNF)., Int. J. Cancer, 81, (1) 67 - 73,   1999
  • Traumatic injury in vitro induces IEG mRNA in cultured glial cells, suppressed by coculture with neurons., Neuroreport, 10,   1999

Books etc

  • Differential regulation of aquaporin expression in astrocytes, Brain Res Mol Brain Res,   2001
  • Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, nrk-52e, Urol Int,   2001
  • Regulation of aquaporin-4 expression in astrocytes, Brain Res Mol Brain Res,   2001
  • Growth-promoting action of adenosine-containing dinucleotide on neuroblastoma cells : detection of adenosine-cytidine dinucleotide (ApCp) in neurofibroma (NF1) extracts., J Neurochem,   1993
  • Aberrant production of gliostatin/platelet-derived endothelial cell growth factor in rheumatoid synovium, Arthritis Rheum,   1994
  • Heat shock-mediated cell cycle arrest is accompanied by induction of p21 CKI, Biochem Biophys Res Commun,   1996
  • Astrocytic contributions to blood-brain barrier(BBB)formation by endothelial cells : a possible use of aortic endothelial cell for in vitro BBB model, Neurochem Int, Neurochem Int,   1996
  • Tissue distribution of human gliostatin/platelet-derived endothelial cell growth factor(PD-ECGF)and its drug-induced expression, Biochim Biophys Acta,   1996
  • Specific detection of kappa light chain in uric acid stones, Life Sci,   1997
  • Evidence for participation of gliostatin/platelet-derived endothelial cell growth factor in gastric ulcer healing, Life Sci,   1997
  • Glucocorticoid induced the expression of mRNA and the secretion of lipocortin 1 in rat astrocytoma cells, Brain Res,   1997
  • Gliostatin/platelet-derived endothelial cell growth factor as a clinical marker of rheumatoid arthritis and its regulation in fibroblast-like synoviocytes, Br J Rheumatol,   1997
  • Abnormal glycosylation of IgG as a clinical parameter in patients with rheumatoid arthritis : Its constitutional analysis by HPLC, J Clin Biocehm Nutr,   1998
  • p27Kip1 expression by contact inhibition as a prognostic index of human glioma, J Neurochem,   2000
  • AT motif binding factor 1-A(ATBF1-A)negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine, Biochem Biophys Res Commun,   2000
  • Cloning of a rat glia maturation factor-gamma(rGMFG)cDNA and expression of its mRNA and protein in rat organs, J Biochem(Tokyo),   2000
  • Synovial inflammation and hyperplasia induced by gliostatin/platelet-derived endothelial cell growth factor in rabbit knees, Rheumatol Int,   2000
  • Human neuroblastomas with unfavorable biologies express high levels of brain-derived neurotrophic factor mRNA and a variety of its variants, Cancer Lett,   2001
  • Regulation of rat hippocampal neural cadherin in the kainic acid induced seizures, Neurosci Lett,   2001
  • Cyclic ADP-ribose as a potential second messenger for neuronal Ca(2+)signaling, J Neurochem,   2001
  • A metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells, Neurosci Lett,   2001
  • Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1, Oncogene,   2001
  • Effects of mechanical vibration on DNA and proteoglycan synthesis in cultured artcular chondrocytes., Mod Pheumatol,   2001
  • Biosynthetic response of cultured articular chondrocytes to mechanical vibration, Res Exp Med(Berl),   2001
  • Interleukin-1beta induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes, Neurosci Res,   2001
  • Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium, Eur J Gastroenterol Hepatol,   2001
  • Isolation and characterization of a new immortal rat astrocyte with a high expression of NGF mRNA, Neurosci Res,   2001
  • Recurrent subthreshold electrical activities of rat neocortical neurons progress during long-term culture, Neurosci Lett,   2001
  • Expression of glia maturation factor during retinal development in the rat, Brain Res Mol Brain Res,   2001
  • Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation, Brain Res Mol Brain Res,   2001

Research Grants & Projects

  • Neuron-Glia Interaction


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