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AOYAMA Mineyoshi

    Graduate School of Pharmaceutical Sciences Department of Pathobiology 　Professor
Last Updated :2024/04/26

Researcher Information

URL

J-Global ID

Research Interests

  • 包括脳ネットワーク   マイクロアレイ   神経芽腫   腫瘍幹細胞   AKT   ERas   神経芽細胞腫   神経幹細胞   薬剤耐性   アストロサイト   サイトカイン   GMFB   血液脳関門   EIA   GMFG   血管内皮細胞   部位特異性   急性脳症   グリア   

Research Areas

  • Life sciences / Fetal medicine/Pediatrics

Academic & Professional Experience

  • 2015 - Today  Nagoya City UniversityGraduate School of Pharmaceutical Sciences教授
  • 2009 - 2015  Nagoya City UniversityGraduate School of Medical Sciences講師
  • 2007 - 2009  Nagoya City UniversityGraduate School of Medical Sciences助教
  • 2003 - 2007  Nagoya City UniversityGraduate School of Medical Sciences助手

Education

  •        - 2000  Nagoya City University  Graduate School of Medical Sciences
  •        - 1993  Nagoya City University  医学部

Association Memberships

  • 日本骨代謝学会   THE PHARMACEUTICAL SOCIETY OF JAPAN   THE JAPANESE ASSOCIATION OF ANATOMISTS   THE JAPANESE PHARMACOLOGICAL SOCIETY   THE PHYSIOLOGICAL SOCIETY OF JAPAN   THE JAPAN NEUROSCIENCE SOCIETY   THE JAPANESE SOCIETY FOR REGENERATIVE MEDICINE   JAPAN COLLEGE OF RHEUMATOLOGY   THE JAPANESE CANCER ASSOCIATION   THE JAPANESE SOCIETY FOR NEUROCHEMISTRY   北米神経科学会   JAPAN PEDIATRIC SOCIETY   

Published Papers

MISC

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/04 -2026/03 
    Author : 永谷 祐子; 青山 峰芳; 川口 洋平
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2022/04 -2026/03 
    Author : 千田 博也; 上用 祐士; 青山 峰芳; 永谷 祐子
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 森 義徳; 久保田 英嗣; 片岡 洋望; 青山 峰芳
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 田村 哲也; 青山 峰芳; 青木 啓将; 祖父江 和哉
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 垣田 博樹; 竹下 覚; 山田 恭聖; 青山 峰芳
     
    新生児脳障害の原因としては、大きく正期産児にみられる新生児低酸素性虚血性脳症(HIE)と早産児にみられる脳室周囲白質軟化症（PVL）が挙げられる。これらの疾患は脳性マヒをはじめとする重篤な後遺症を残し、その克服は現在の新生児医療における大きな課題である。新生児脳障害の治療法として、現在までに確立しているのはHIEに対する低体温療法のみである。しかしながら低体温療法を施行しても、依然として30-60%の症例で予後不良であるのが現状であり、さらに早産児に白質傷害を起こすPVLにおいては、確立された有効な治療法はない。 申請者らは、培養グリアを用い、低温状態がアストロサイトのEPO分泌を促進し、ニューロンの傷害を抑制することを明らかにした（J Neuroinflammation 2020）。さらに、アストロサイトを低酸素および無糖状態にすることで亢進するEPOの発現にAMPKが関与していることを明らかにした（未発表）。また申請者らは低温状態がミクログリアの活性化を包括的に抑制し、神経傷害を抑制することを明らかにした(Cell Mol Neurobiol ２０２１)。さらに、LPSで活性化したミクログリアは低温状態で活性型AMPKの発現を促進することを明らかにした（未発表） これらの研究成果はグリアに注目した基礎研究で、低体温療法の脳保護メカニズムのさらなる解明、新規補完治療法の開発につながる可能性がある。さらにグリアの機能を制御することで低体温療法が施行困難な早産児の脳室周囲白質軟化症も含めた包括的な新生児脳障害の治療成績を向上させることが期待できる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2020/04 -2024/03 
    Author : 青山 峰芳; 垣田 博樹; 山田 恭聖
     
    周産期医療の進歩により新生児の救命率は著しく改善した一方で、中枢神経の後遺症に苦しむ患児を救うことは本人や家族や新生児医療関係者にとって切実な課題である。低体温療法の普及により、多くの新生児低酸素性虚血性脳症（HIE）患者の予後を改善している。しかし、効果が不十分な症例や他の合併症によって適応を断念する例が存在する。申請者らはこれまで、エリスロポエチン(EPO)をはじめとする神経保護因子の研究を通じて、グリアに注目した神経保護治療の可能性拡大を基礎研究により解明してきた。本研究では、新生児のHIEに対して効果が期待されてきた低体温療法の作用メカニズムの解明と至適化に向けた基礎研究を計画する。グリアによるNeuroinflammationの制御という観点から治療メカニズムの解明および補完できる新規治療ミックスを提案することを目指す。新生児医療におけるより効果的な低体温療法を提案し、新生児期中枢神経疾患の後遺症の予防法開発を目標とする。HIEを模したIn vitroおよびIn vivoの実験により、低体温が細胞レベルおよび生体レベルでどのような効果があるか検討する。in vitroにおいて、培養グリア（アストロサイト、オリゴデンデンドロサイト、ミクログリア）、培養ニューロンに細胞傷害をあたえたのち、低温度環境下で培養を持続した結果得られる細胞の反応について解析を行った。in vivoにおいては、病態を模したモデルラットを安定的に作成できる実験系の確立を開始した。さらに脳の凍結切片を作成し免疫染色を行い、脳傷害の部位、程度、病変部でのグリアの集積、形態変化などを検討することを計画している。本研究により、新生児医療におけるよりよい低体温療法を提案し、新生児期中枢神経疾患の後遺症の予防法開発につながることが期待できる。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : 上田 博子; 垣田 博樹; 山田 恭聖; 青山 峰芳
     
    脳室周囲白質軟化症（PVL）は早産児に発症し、将来の脳性麻痺や発達障害を引き起こす重篤な疾患である。PVLの発症は感染、炎症、低酸素虚血状態などによる局所的な軸索の脱髄が原因である。和われらはこれまでにPVLを高率に合併する晩期循環不全(LCC)を発症する早産児では、副腎不全による内因性ステロイド不足が潜在することを明らかにした。またPVLモデルにおいて、グリアが疾患の増悪や神経保護において、多面的かつ主体的に関与していることを明らかにしてきた。これらの研究成果からステロイドがグリアに及ぼす影響に着目し、PVL発症メカニズムを解明する。特にこの内因性のステロイド不足が、脳虚血の前段階として、脳室周囲白質のオリゴデンドロサイトの虚血に対する脆弱性に関わっている可能性を検証することを目的とする。 本年度は副腎不全モデルラットの作成に取り組んだ。早産児に相当するP3ラットの両側副腎を外科的に結紮するモデルの作成に取り掛かった。しかしながら、外科的結紮では、著明な低ナトリウム血症により、致死率が５０％を超え、長期生存個体による検証が困難であった。そこで現在はコルチゾール合成阻害の投与により、一過性の副腎不全をきたすモデルラットの作成をおこないっている。
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2018/04 -2022/03 
    Author : 井坂 雅徳; 長谷川 忠男; 前山 順一; 立野 一郎; 青山 峰芳; 矢木 宏和
     
    A群連鎖球菌は、咽頭炎や猩紅熱を引き起こす原因菌である。劇症型への機序について様々な研究報告が出ているが、一向に完全解明に至っていない。A群連鎖球菌劇症型株は健常人でも検出され、劇症型株に感染することが発症に必須ではない疑問がある。そこで、劇症型が発症するにはA群連鎖球菌がヒトへ感染後、変化するための何らかの情報を受け取らなければならないと考えた。ここで外界情報を受け取る二成分制御因子の研究が進んでいる、Streptococcus mutansに着目した。A群連鎖球菌と同属のこの細菌は、乳酸、酪酸を産生し、歯にバイオフィルムを形成して虫歯を増悪させる。この細菌の二成分制御因子は酸感受とバイオフィルム形成に関与する。同様の仕組みがA群連鎖球菌に存在するかを調べると、A群連鎖球菌の二成分制御因子の一つであるspy1588遺伝子欠損株は、バイオフィルム産生低下、酸抵抗性の低下を示した。我々は、酸を感受する二成分制御因子と、それに関連する遺伝子群に変異が劇症型に生じていると考え、研究を現在進めている。 本年度はSPY1588の立体構造を測定すべく、部分的ペプチドをpETベクターとタンパク質発現用大腸菌で発現させ、X線構造解析の資料作製を実施した。 Spy１５８８の遺伝子組換え体作成を実施した。昨年精製法を一部改良して実施したが、タンパク質が凝集して精製困難な場合が多いため、精製段階からの見直しを行いました。すると、大腸菌由来であろう凝集物が目的産物に結合して精製を阻害していることを突き止めた。この阻害物の除去の精製法が得られたので、再度純度を上げた精製法でペプチドを精製しています。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : Hiroki Kakita
     
    Hypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. We demonstrated that hypothermia after oxygen/glucose deprivation stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. In addition, we demonstrated that, under hypothermic conditions, expression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) was suppressed. In addition, phagocytosis of latex beads was significantly suppressed in BV-2 cells under hypothermic conditions.Investigating the neuroprotective effect of EPO from astrocytes and microglia function under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : YAMADA YASUMASA
     
    The majority of infants born very preterm now survive. However their long-term neurodevelopmental outcomes, including problems with behavior, have not improved. Some researchers have reported the incidence of autism spectrum disorder (ASDs) is more 2 times in NICU survivor than in normal population. In animal study, rat pups expose to neonatal pain showed increased anxiety-mediated behavior and disturbed Hypothalamus-Pituitary-Adrenal gland (HPA) system during adulthood. We had suggested greater neonatal pain exposure was associated with future ASDs induced by altered HPA axis regulation. In present study, we have confirmed the relationship between neonatal pain and future atypical development in very preterm infants at 12mo corrected age. Furthermore, we have presented that a consideration of blood sampling method in NICU most likely prevent atypical development leading to ASDs.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2016/04 -2019/03 
    Author : Aoyama Mineyoshi
     
    Periventricular leukomalacia (PVL) is a white matter brain injury. PVL is specific for the premature infants with the greatest risk of the cerebral palsy. Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. In our present study, we investigated whether EPO could attenuate the microglia activation, developing the neuroprotection. These data indicate that EPO treatment attenuates microglial activation including morphological change, phagocytosis, and the production of inflammatory cytokines in vitro and in vivo. Further investigation of EPO modulation of the microglial activation may contribute to the development of novel neuroprotective therapies.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2016/04 -2019/03 
    Author : Tamura Tetsuya; AOYAMA Mineyoshi; ASAI Kiyofumi
     
    Erythropoietin (EPO), which is a therapeutic drug for anemia, has recently been known to have a neuroprotective effect, but the mechanism is unknown. In many brain diseases, microglia, which are one of the cells constituting the brain, are often activated. We focused on activated microglia, which damage the brain, and found that microglia have a receptor for EPO. In this study, experiments were carried out using microglia culture cells and mice, and the neuroprotective effect of EPO was achieved by inhibiting the activity of microglia in the brain, such as inflammatory cytokine production and damaging phagocytosis. EPO may be useful as a therapeutic drug in acute brain diseases in the area of emergency and intensive care.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : Mori Yoshinori
     
    We investigated whether reovirus has antitumor activity against GIST cells in the setting of imatinib sensitivity. Cell proliferation and apoptosis assays were performed using a human GIST cell line, GIST-T1, and imatinib-resistant GIST (GIST-IR) cells that we established. The molecular pathways responsible for cell damage by reovirus were explored using PCR-arrays and Western blots. Reovirus significantly induced apoptotic cell death in GIST-T1 and GIST-IR cells in vitro, despite differences in the activation of receptor tyrosine kinase pathways between GIST-T1 and GIST-IR. Molecular assays indicated the possibility that reovirus induces apoptotic cell death via Fas signaling. Furthermore, in vivo mouse tumor xenograft models demonstrated a significant anti-tumor effect of reovirus on both GIST-T1 and GIST-IR cells. Our results demonstrate the therapeutic potential of reovirus against GIST.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : Kakita Hiroki
     
    Hypothermia has been reported to be only effective therapy for neonatal hypoxic ischemic encephalopathy (HIE) . We performed the examination of microenviroment improvement of glial function control with hypothermia. We found hypothermia (32-34℃) decreased the expression of iNOS and inflammatory cytokine in cultured glia under hypoxic condition. Furthermore, erythropoietin having neuroprotective effect made clear that overexpression lasted by a low temperature state. We found that these protein expression adjustment by hypothermia decreased brain damage. This study might contribute to the development of an etiology of HIE which is still a poor prognosis and the cure.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2013/04 -2016/03 
    Author : Aoyama Mineyoshi; ASAI KIYOFUMI
     
    Recently, it has been demonstrated that erythropoietin (EPO), a hematopoietic hormone, has a neuroprotective effect and EPO receptor (EPOR) is expressed in the central nervous system (CNS). In our previous study, brain immune cells microglia had higher expressions of EPOR than astrocytes and neurons. In vitro , we assessed the effect of EPO using microglial cell line BV-2 activated by lipopolysaccharide(LPS). LPS increased the gene expressions of cytokines in BV-2, but these increases were significantly suppressed by EPO. Next, LPS treatment increased the phagocytosis in BV-2 compared with untreated BV-2, but this increase was significantly suppressed by EPO. In vivo, LPS induced microglia to be activated types, but EPO alleviated the active morphological change. These data indicated that LPS made microglia activated and cytotoxic, but EPO-EPOR signal might attenuate LPS-induced microglial cytotoxic activation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : MORI Yoshinori; KATAOKA Hiromi; MIURA Yutaka; KASUYA Hideki; AOYAMA Mineyoshi
     
    Trastuzumab is the key drug against HER2-positive gastric cancer. However, the anti-tumor effect is not enough. Recently oncolytic virus is expected as new anticancer therapy. We examined antitumor effect by the combination of trastuzumab and oncolytic reovirus in HER2-positive gastric cancer cells this time. The combination of trastuzumab and reovirus for HER2-positive gastric cancer cells was thought to reinforce antitumor effect by apoptotic instruction through caspase 3/7.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : ASAI Kiyofumi; AOYAMA Mineyoshi; KAKITA Hiroki
     
    To understand the pathophysiological mechanism of acute encephalopathy, and analyze the aggregative action of diclofenac sodium (DCF) on acute encephalopathy, we stimulated primary cultured rat astrocytes and microglia with IL-1beta;, TNF-alpha and IFN-gamma. The expression of iNOS and AQP4 and the production of NO were upregulated in astrocytes and the expression of iNOS and the activities of phagocytosis were increased in microglia. The addition of DCF highly enhanced the expression of iNOS and AQP4, and the activities of phagocytosis. This enhancement may explain the significantly increased mortality rates in acute encephalopathy patients treated with DCF.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2010 -2012 
    Author : KUBOTA Eiji; KATAOKA Hiromi; ASAI Kiyofumi; AOYAMA Mineyoshi; SUZUKI Syugo
     
    Despite of recent advances in therapies for cancers, it is not sufficient to improve the outcome of advanced gastric cancer patients. Therefore, it is indispensable to develop novel target molecules for gastric cancer treatment. We have previously demonstrated that ES cell-expreesed Ras, ERas is expressed in gastric cancer, and is involved in gastric cancer invasiveness and metastasis. Our data also indicated the significance of ERas as a predict biomarker for gastric cancer patients. In this study, we showed the mechanisms by which ERas enhances the metastatic ability, and a utility of ERas as a target of gastric cancer treatment.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2010 -2011 
    Author : AOYAMA Mineyoshi; ASAI Kiyofumi
     
    We have made human neural stem cell from human iPS cell and investigated whether the induction of exogenous oncogene N-myc could transform the cell or not. We would prepare human or mouse neural stem cells at various step of the development. And N-myc genes were induced in the cells. The effect on cell proliferation was observed. These studies may reflect the cellular transformation from the normal cells to the cancer cells to clarify the mechanism of the carcinogenesis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2008 -2010 
    Author : ASAI Kiyofumi; AOYAMA Mineyoshi
     
    We have tried to establish immortalized mouse brain endothelial cell lines and rat astrocytes cell line for the co-culture system of both cells. Primary cultured mouse brain endothelial cells were transfected with SV40 T-antigen expressing vector and selected by zeocin and blasticidin. Primary cultured rat astrocytes were transfected with same vectors and selected by some procedures. Three colonies from endothelial cells and 10 colonies from astrocytes were obtained, but in all of the immortalized cell lines, expression of SV40 T was not controlled by tetracycline. Thus Tet-repressor expressing vector was re-transfected to the cell lines and finally 3 astrocyte cell lines were obtained in which expression of SV40 T was controlled by tetracycline. Now we are investigating whether the expression of GFAP changes or not by on/off of SV40 T, and trying to establish co-culture system of endothelial cell lines and astrocyte cell lines.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2007 -2009 
    Author : Mineyoshi AOYAMA; Kiyofumi ASAI
     
    We had a plan to isolate putative cancer stem cells from mouse neuroblastoma cell lines. And also we would isolate mouse neural stem cells from mouse embryonal and adult brain. Following the isolation of both stem cells, we would find the similarity and the difference between neuroblastoma cancer stem cells and neural stem cells. We have isolated mouse neural stem cells. However, we have failed to isolate neuroblastoma cancer stem cells using the sorting method by the specific cell surface marker or the neurosphere method. We suggest that after frequent passages, cancer cell lines could lose the cancer stem cells, which can be found in human primary tumor.
  • 文部科学省：科学研究費補助金(特定領域研究)
    Date (from‐to) : 2006 -2007 
    Author : 浅井清文; 三浦裕; 青山峰芳
     
    申請者らは,アストロサイトが様々な脳内部位において異なる機能を有することを想定し,その相違を分子レベルで証明することを目的に解析を行ってきた。昨年までに,凍結ラット脳切片を迅速免疫組織染色法によって抗GFAP抗体にて染色し,laser microdissection(LMD)法を用いて各脳内部位からGFAP陽性細胞のみを採取し,遺伝子発現の違いをDNA microarray(Agilent社製)を用いて比較した。その中で大脳皮質と線条体において有意に発現の違いを認めた遺伝子,12個を見いだした。そこで,今年度は詳細な観察を行うため,大脳皮質で発現が高かった水の輸送に関与するアクアポリン4(AQP4)の発現について注目し解析を行った。空間的,時間的多様性を調べるために年齢別,部位別に凍結ラット脳切片を用いた免疫組織染色を行った。また,年齢別,部位別のアストロサイト培養細胞を用いた免疫細胞染色を行い,細胞培養系においても脳内部位特異性が保持されるか検討した。その結果,少なくとも大脳皮質と線条体においてアストロサイトにおけるAQP4の染色性が異なることがわかり,それは部位特異的に均一な細胞集団であるというより,部位特異的にヘテロな細胞集団の構成が異なることが考えられた。
  • 文部科学省：科学研究費補助金(若手研究(B))
    Date (from‐to) : 2004 -2005 
    Author : 青山峰芳
     
    本年度は、ES細胞特異的に発現している新規遺伝子ERasの発現を実際の神経芽腫摘出標本で確認し、腫瘍細胞株を用いてERas発現による腫瘍形成能、薬剤感受性の変化について解析した。 1,神経芽腫摘出標本でのERasタンパクの発現 患者または患者家族から遺伝子およびタンパク発現の解析について同意を得た神経芽腫患者摘出腫瘍切片を用いて免疫染色によりタンパクレベルでの発現を確認した。抗体は研究協力者の山中らがすでに作成し、特異性を確認できている抗ERas抗体を用いた。今回用いたほとんどすべての神経芽腫切片で発現を確認したが、特に未分化な腫瘍細胞ほど強い染色性を示した。また、ERasタンパクは細胞内の細胞膜に局在していると考えられた。 2,神経芽腫細胞株でのERasの発現 神経芽腫細胞株では複数の細胞株でERas遺伝子の発現をRT-PCRによって確認していたが、Western blottingによってタンパクレベルでの発現も同時に確認できた。 3,神経芽腫ERas遺伝子強制発現株の解析 ERas発現が確認できている中でも低発現である神経芽腫細胞株SH-SY5Yを用いてERas遺伝子を導入し強制発現株を作成した。これらのクローン細胞株は細胞増殖能に影響は与えない一方で、soft agarを用いた足場非依存性の増殖を亢進し、シスプラチン、ドキソルビシン、エトポシド、ビンブラスチンといった神経芽腫への化学療法剤による細胞死を抑制した。この薬剤耐性のメカニズムにはERas発現による下流シグナル分子のリン酸化を介したERas/Akt pathwayの活性化が重要な働きをしている可能性が示唆された。
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2003 -2004 
    Author : Kiyofumi ASAI; 青山峰芳; 三浦裕
     
    1, Analysis of promoter activity of the human glia maturation factor-gamma gene 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 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. 2, Development of two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) 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. 3, Analysis of expression of glia maturation factor beta after cryogenic injury 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.

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